H8966ast1-6 0

Agilent 1100 Series LC/MSD Ion
Trap Techniques and Operation

Course Number H8966A
Volume 1
Software Revsion 6.0
Student Manual
Agilent 1100 Series LC/MSD Ion
Trap Techniques and Operation
H8966A
Software Revision 6.0
Volume 1
Student Manual
Printed in October, 2005

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ii
Table Of Contents
AGILENT 1100 SERIES LC/MSD ....... IAGILENT 1100 SERIES LC/MSD TRAP SYSTEM
OVERVIEW: VL, SL AND XCT MODELS ..............................................................................1
IN THIS SECTION ...........................................................................................................................2
ATMOSPHERIC PRESSURE IONIZATION MASS SPECTROMETRY (API-MS).....................................4
API-MS MODES ...........................................................................................................................6
RELATIVE APPLICABILITY OF LC/MS TECHNIQUES ......................................................................8
WHAT KIND OF DATA DO YOU OBTAIN? SINGLY CHARGED IONS IN ES OR APCI MODE...........9
WHAT KIND OF DATA DO YOU OBTAIN? MULTIPLY-CHARGED IONS IN ELECTROSPRAY MODE
...................................................................................................................................................12
MAIN COMPONENTS OF THE LC/MSD TRAP .............................................................................13
MODELS: VL, SL, XCT, XCT+, XCT ULTRA ............................................................................15
API-ELECTROSPRAY IONIZATION ...............................................................................................17
API –ELECTROSPRAY ION SOURCE .............................................................................................19
NANOELECTROSPRAY .................................................................................................................21
ELECTROSPRAY SPRAY CHAMBER SETTINGS ..............................................................................22
WHY IS IT IMPORTANT TO PROPERLY CONTROL SPRAY CHAMBER PARAMETERS? EFFECT OF
DRYING GAS SETTINGS IN ES .....................................................................................................23
EFFECT OF DRYING GAS SETTINGS IN ES ....................................................................................24
ELECTROSPRAY CONSIDERATIONS ..............................................................................................25
APCI IONIZATION PROCESS ........................................................................................................26
APCI SOURCE .............................................................................................................................27
APCI SPRAY CHAMBER SETTINGS ..............................................................................................28
APCI CONSIDERATIONS..............................................................................................................30
APPI SOURCE FOR THE LC/MSD TRAP ......................................................................................31
APPI PROCESS ............................................................................................................................32
APPI MECHANISMS ....................................................................................................................33
APPI SOURCE .............................................................................................................................34
PDF-MALDI SOURCE ................................................................................................................35
DRYING GAS AND CAPILLARY ....................................................................................................36
ION OPTICS .................................................................................................................................38
MASS ANALYSIS .........................................................................................................................39
TIME SEQUENCE OF EVENTS TO GENERATE A MASS SPECTRUM ON AN ION TRAP .....................41
MATHIEU STABILITY DIAGRAM FOR IONS IN AN ION TRAP ........................................................43
DETECTOR ..................................................................................................................................45
VACUUM SYSTEM .......................................................................................................................48
UV-VIS SPECTRA ACQUIRED WITH DIODE ARRAY DETECTOR (DAD) .......................................49
TOTAL ION CHROMATOGRAM (SCAN MODE)..............................................................................50
LC/MS/MS ON AN ION TRAP ......................................................................................................51
ION TRAP MS/MS CID MASS SPECTRUM OF A HEXAPEPTIDE ....................................................52
MS/MS, SCAN, OR UV ...............................................................................................................53
CE/MS/MS.................................................................................................................................54
UNDERSTANDING ESI AND APCI: FUNDAMENTALS OF THE IONIZATION
PROCESS......................................................................................................................................55
IN THIS SECTION .........................................................................................................................56
ATMOSPHERIC PRESSURE IONIZATION MASS SPECTROMETRY (API-MS)...................................57
KEY CHARACTERISTICS OF 4 MODES OF API-MS OPERATIONS ..................................................58
ELECTROSPRAY IONIZATION – NECESSARY STEPS FOR ION FORMATION ....................................60
iii
REGARDING STEP 1 - WHY IS THE BEST ELECTROSPRAY SENSITIVITY ACHIEVED WHEN THE
ANALYTES EXIST AS IONS IN SOLUTION?....................................................................................61
STEP 1 - HOW CAN IONS BE CREATED IN SOLUTION?.................................................................63
STEP 2 - PNEUMATIC ASSISTED NEBULIZATION – TO CREATE CHARGED DROPLETS ..................64
STEP 3 - DESOLVATION OF CHARGED DROPLETS ........................................................................66
STEP 3 CON’T - COULOMB FISSION OF DROPLETS .......................................................................67
STEP 4 - DESORBING IONS FROM SOLUTION ................................................................................68
STEP 4 - MAKING IONS – ION EVAPORATION MECHANISM (IEM) ...............................................69
STEP 4- MAKING OF IONS - CHARGE RESIDUE MECHANISM (CRM) ...........................................70
PROBLEMS IN MAKING IONS USING TYPICAL BUFFERS FOR ELECTROSPRAY – ION PAIR
FORMATION ................................................................................................................................71
PROBLEMS IN MAKING IONS USING TYPICAL BUFFERS FOR ELECTROSPRAY – ION PAIR
FORMATION ................................................................................................................................72
STEP 5 - REACTIONS OF IONS IN THE GAS PHASE ........................................................................73
ELECTROSPRAY RESULTS IN THE LOWEST ION INTERNAL ENERGY RELATIVE TO OTHER
IONIZATION TECHNIQUES FOR MS ..............................................................................................74
APCI – KEY PARAMETERS..........................................................................................................75
APCI DETAILED MECHANISM- GAS PHASE IONIZATION.............................................................77
UNDERSTANDING ION TRAP MASS SPECTROMETRY ..................................................79
IN THIS SECTION .........................................................................................................................80
HEART OF AN ION TRAP ..............................................................................................................81
MATHIEU STABILITY DIAGRAM FOR IONS IN AN ION TRAP .........................................................83
MATHIEU STABILITY DIAGRAM FOR NON-LINEAR RESONANCE EJECTION .................................85
WHY NON-LINEAR RESONANCE? ...............................................................................................87
ION TRAP RELATIONSHIP BETWEEN RESOLUTION, MASS RANGE, AND SCAN SPEED ..................88
MATHIEU STABILITY DIAGRAM FOR NON-LINEAR RESONANCE EJECTION TO INCREASE MASS
RANGE ........................................................................................................................................90
MASS RANGES, MASS RESOLUTION, AND SCAN SPEEDS FOR THE AGILENT TRAP ......................91
FUNDAMENTALS OF ACQUIRING A MASS SPECTRUM ON AN ION TRAP – TIMED EVENTS ...........92
TIME SEQUENCE OF EVENTS TO GENERATE A MASS SPECTRA ON AN ION TRAP .........................93
ADVANTAGES OF ACCUMULATING ALL IONS SIMULTANEOUSLY IN A TRAP - SENSITIVITY ........95
WHY CONTROL THE ION ACCUMULATION PROCESS ...................................................................96
EXAMPLE OF WHAT HAPPENS WHEN TOO MANY IONS ARE ACCUMULATED INTO THE TRAP ......98
REDUCING SPACE CHARGING .....................................................................................................99
API – ION TRAP METHODS TO REDUCE OR REMOVE SPACE-CHARGE EFFECTS ........................100
ICC TO REMOVE SPACE CHARGE EFFECTS ...............................................................................101
ION ISOLATION TO EXCLUDE ALL BUT THE DESIRED M/Z VALUES IN THE ION TRAP .................103
ORTHOGONAL SPRAYING REDUCES THE SAMPLING OF CHARGED SPECIES IN THE TRAP ..........104
TANDEM MS ON AND ION TRAP ...............................................................................................105
TIME SEQUENCE OF EVENTS TO GENERATE A MS/MS MASS SPECTRA ON AN ION TRAP .........106
COLLISION-INDUCED DECOMPOSITION (CID) VARIABLES FOR TRAPS .....................................107
ADJUSTING FRAGMENTATION VOLTAGE TO MAXIMIZE CID ....................................................109
WHY ADJUST THE CUTOFF MASS FOR CID?.............................................................................111
MSN CAPABILITIES ON AN ION TRAP ........................................................................................112
SCHEMATIC OF MS(3)...............................................................................................................113
EXAMPLE OF MS5.....................................................................................................................114
WHEN TO USE MSN AND WHAT SHOULD ONE USE FOR THE VALUE OF N ................................115
N
LOSS OF SIGNAL LEVEL AT HIGHER STAGES OF MS ................................................................116
ADDITIONAL SOURCES OF INFORMATION ON ION TRAPS ..........................................................117
BASICS OF LC/MS/MS.............................................................................................................119
IN THIS SECTION .......................................................................................................................120
WHAT IS COLLISION INDUCED DECOMPOSITION (CID)?...........................................................121
CID REACTIONS .......................................................................................................................122
iv
CID IN API TRANSPORT REGION ..............................................................................................123
CID IN API TRANSPORT REGIONCON’T ....................................................................................124
ADVANTAGES/DISADVANTAGES OF CID IN TRANSPORT REGION .............................................125
CID IN ES TRANSPORT FOR SULFAMETHAZINE ........................................................................126
WHAT IS TANDEM MS? ............................................................................................................127
TANDEM MS IN SPACE- TRIPLE QUADRUPOLES ........................................................................128
TANDEM MS IN TIME – ION TRAPS ...........................................................................................129
CHARACTERISTICS OF TANDEM MS..........................................................................................130
ADVANTAGES/DISADVANTAGES OF TANDEM MS ....................................................................131
INCREASE IN S/N WITH INCREASING STAGES OF ANALYSIS –REMOVAL OF CHEMICAL NOISE..132
CID ON AN ION TRAP - WHAT IS MSN .....................................................................................133
ID ON AN ION TRAP - MSN........................................................................................................134
CID ON AN ION TRAP – EXAMPLE OF MS3 ...............................................................................135
CID ON AN ION TRAP – WHY NOT MSN ...................................................................................136
CID ON AN ION TRAP – LOSSES IN SIGNAL WITH MSN .............................................................137
LC/MSD TRAP SAMPLE INTRODUCTION AND LC METHOD CONVERSION..........139
IN THIS SECTION .......................................................................................................................140
SAMPLE INFORMATION AND CONSIDERATIONS .........................................................................141
SAMPLE PREPARATION .............................................................................................................142
INLET MODES DEFINED ............................................................................................................144
INFUSION HARDWARE SETUP ....................................................................................................145
INFUSION DATA ........................................................................................................................146
FIA HARDWARE SETUP ............................................................................................................147
CONVENTIONAL HPLC HARDWARE .........................................................................................148
COLUMN SELECTION FOR LC/MS APPLICATIONS .....................................................................149
WHY µ-FLOW RATE SEPARATIONS? .........................................................................................150
CAPILLARY HPLC – ADVANTAGES/DISADVANTAGES ..............................................................151
CAPILLARY HPLC CONNECTIONS- DEAD VOLUMES MUST BE ELIMINATED! ..........................152
CAPILLARY HPLC FOR SAMPLE LIMITED ANALYSES ...............................................................153
REDUCING CAPILLARY ID TO IMPROVE SENSITIVITY FOR CONCENTRATION-LIMITED SAMPLES
.................................................................................................................................................154
NANOSPRAY .............................................................................................................................155
NANOSPRAY CONFIGURATIONS ................................................................................................156
HPLC-CHIP/MS .......................................................................................................................157
CE/MS – USING LAYER FLOW INTERFACE ...............................................................................159
CE/MS OPTIMIZATION .............................................................................................................160
CE/MS DETERMINATION OF CEFTIOFUR IN MILK .....................................................................161
COLUMN SELECTION FOR LC/MSD APPLICATIONS ..................................................................162
LC/MS-COMPATIBLE MOBILE PHASES .....................................................................................164
ADAPTING EXISTING LC METHODS TO LC/API-MS.................................................................165
COMPATIBLE SOLVENTS - API-MS...........................................................................................166
CHROMATOGRAPHIC MODES (HPLC).......................................................................................167
OPTIMIZATION SCHEME FOR API-LC/MS.................................................................................169
OPTIMIZATION SCHEME FOR API-LC/MS CON’T .....................................................................170
OPTIMIZATION ESI AND APCI FOR ON-LINE LC/MS ANALYSES - LESSONS IN
SOLUTION CHEMISTRY........................................................................................................171
IN THIS SECTION .......................................................................................................................172
SAMPLES FOR ELECTROSPRAY ..................................................................................................173
FACTORS AFFECTING ELECTROSPRAY IONIZATION...................................................................175
SOLUTION CHEMISTRY .............................................................................................................177
GENERAL RULES FOR CHOOSING POLARITY OF ION DETECTION AND PH - ESI ........................179
PH EFFECTS - LYSOZYME (MW 14306) ....................................................................................180
v
FORMATION OF ION IN SOLUTION: NEGATIVE ION DETECTION ................................................181
API-MS ADDITIVES ..................................................................................................................182
USE VOLATILE ION-PAIR REAGENTS .........................................................................................183
ION-PAIR REAGENTS IN POSITIVE MODE ESI............................................................................185
EFFECT OF VOLATILE BUFFER CONCENTRATION ON ES SIGNALS.............................................186
EFFECT OF BUFFERS ON ESI SIGNAL.........................................................................................187
ESI SIGNAL AFTER 635 INJECTIONS WITH NONVOLATILE SALT MATRIX ..................................189
ESI - RESPONSE FOR PENICILLIN G IN VARIOUS PERCENTAGES OF ACETONITRILE ..................190
CATIONIZATION IN ELECTROSPRAY ..........................................................................................191
CREATING THE OPTIMAL CHEMISTRY FOR ELECTROSPRAY THROUGH POST-COLUMN ADDITION
.................................................................................................................................................192
POST-COLUMN MODIFICATION OF LC SOLVENT TO OPTIMIZE API-MS RESPONSE ..................193
SAMPLES FOR APCI AND APPI.................................................................................................194
APCI KEY PARAMETERS ..........................................................................................................195
APCI VAPORIZER TEMPERATURE .............................................................................................196
EFFECT OF VOLATILE BUFFER CONCENTRATION ON APCI SIGNALS ........................................197
EFFECT OF NON-VOLATILE BUFFER ON APCI SIGNAL .............................................................199
FLOW INJECTION ANALYSIS OF SULFACHLOROPYRADIZINE BY APCI ......................................201
APCI SPRAY CHAMBER AFTER 600 INJECTIONS OF SALT SOLUTION ........................................202
APCI SIGNAL AFTER 600 INJECTIONS OF SALT SOLUTION ........................................................203
APCI SPRAY CHAMBER AFTER 600 INJECTIONS WITH AUTOMATIC DIVERSION .......................204
IMPACT OF ION-PAIR REAGENTS ON LC/MS ANALYSIS IN APCI MODE ...................................205
COMPARISON OF ELECTROSPRAY AND APCI ............................................................................206
REFERENCES .............................................................................................................................207
LABORATORY EXERCISE: UNDERSTANDING API PROCESSES, ION TRAP MS/MS
AND INTERPRETATION OF MASS SPECTRA ..................................................................209
IN THIS SECTION YOU WILL APPLY ..........................................................................................210
CHECK YOUR UNDERSTANDING - API ......................................................................................211
CHECK YOUR UNDERSTANDING – API CONT. ..........................................................................212
TANDEM MS QUESTIONS ..........................................................................................................213
CHECK YOUR UNDERSTANDING ................................................................................................214
CHECK YOUR UNDERSTANDING - ION TRAP MS/MS ...............................................................215
PROBLEM: INTERFERENCE IN A PESTICIDE ANALYSIS ...............................................................216
PROBLEM: IDENTIFICATION OF A DRUG IMPURITY ....................................................................217
PROBLEM: QUANTITATION OF A TARGET DRUG IN PLASMA .....................................................218
PROBLEM: IDENTIFICATION OF A PROTEIN IMPURITY................................................................219
PROBLEM: TRYPTIC PEPTIDE ANALYSIS ...................................................................................220
CHEMSTATION AND WINDOWS OVERVIEW .................................................................221
IN THIS SECTION .......................................................................................................................222
OPENING THE LC/MSD TRAP CHEMSTATION...........................................................................223
HPLC MODULE CONFIGURATION .............................................................................................224
CHEMSTATION VIEWS ..............................................................................................................225
METHOD AND RUN CONTROL VIEW..........................................................................................226
DATA ANALYSIS VIEW .............................................................................................................227
REPORT LAYOUT VIEW .............................................................................................................228
VERIFICATION VIEW .................................................................................................................229
DIAGNOSIS VIEW ......................................................................................................................230
TRAP CONTROL VIEW ...............................................................................................................231
TRAP DATA ANALYSIS VIEW ....................................................................................................232
TRAP QUANTANALYSIS VIEW ..................................................................................................233
METHODS AND SEQUENCES ......................................................................................................234
DATA AND METHOD STORAGE .................................................................................................235
PROCESS CLEANER ...................................................................................................................236
MAINTAINING THE COMPUTER SYSTEM ....................................................................................237
vi
USERMANAGEMENT .................................................................................................................239
LABORATORY EXERCISE: INTRODUCTION TO THE LC/MSD TRAP
CHEMSTATION ........................................................................................................................241
IN THIS LABORATORY EXERCISE, YOU WILL NEED: ................................................................242
ACCESSING THE CHEMSTATION ................................................................................................243
ACCESSING THE ION TRAP SOFTWARE ......................................................................................245
MAINTAINING THE WINDOWS 2000 WORKSTATION .................................................................246
MAINTAINING THE WINDOWS XP WORKSTATION ....................................................................248
WINDOWS FEATURES (WINDOWS 2000 OR XP) ........................................................................250
STARTING UP AND SHUTTING DOWN THE AGILENT 1100 SERIES LC/MSD TRAP
......................................................................................................................................................253
IN THIS SECTION .......................................................................................................................254
LC/MSD TRAP INSTRUMENT STATES: OPERATION ..................................................................255
LC/MSD TRAP INSTRUMENT STATES: STANDBY .....................................................................256
LC/MSD TRAP INSTRUMENT STATES: SHUTDOWN ..................................................................257
LC/MSD TRAP INSTRUMENT STATES: OFF ..............................................................................258
WHICH MODE SHOULD I USE?..................................................................................................259
CHANGING MODES ...................................................................................................................260
CHECKING HELIUM PRESSURE ..................................................................................................276
AGILENT 1100 SERIES HPLC/MS TRAP: CALIBRATION AND TUNING...................279
IN THIS SECTION .......................................................................................................................280
ION TRAP PRESSURE .................................................................................................................281
CALIBRATION ...........................................................................................................................282
MULTIPLIER ..............................................................................................................................293
CALIBRATION ...........................................................................................................................294
OPTIMIZATION AND TUNING .....................................................................................................298
LOADING A METHOD FILE ........................................................................................................301
OPTIMIZING ELECTROSPRAY CONDITIONS ................................................................................302
OPTIMIZING APCI CONDITIONS ................................................................................................305
TUNING .....................................................................................................................................309
ICC...........................................................................................................................................316
DATA FILE SIZE ........................................................................................................................318
OPTIMIZING MS/MS .................................................................................................................319
LABORATORY EXERCISE: LC/MSD TRAP TUNING .....................................................323
IN THIS LABORATORY EXERCISE, YOU WILL NEED: ................................................................324
TUNING THE LC/MSD TRAP .....................................................................................................325
SMART TUNING .........................................................................................................................326
EXPERT TUNING........................................................................................................................330
ADJUSTING GAS FLOWS ............................................................................................................332
AGILENT 1100 SERIES LC/MSD TRAP: FLOW INJECTION ANALYSIS .....................333
IN THIS SECTION .......................................................................................................................334
DEFINITION ...............................................................................................................................335
PURPOSE ...................................................................................................................................336
FIA WITH THE MSD TRAP ........................................................................................................337
SETTING UP THE INSTRUMENT ..................................................................................................338
SPECIFIC CONCERNS .................................................................................................................342
INJECTOR PROGRAMMING .........................................................................................................343
SPECIFIC CONCERNS .................................................................................................................344
LABORATORY EXERCISE: FLOW INJECTION ANALYSIS .........................................345
IN THIS LABORATORY EXERCISE YOU WILL: ...........................................................................346
vii
TO COMPLETE THIS LABORATORY EXERCISE YOU WILL NEED: ..............................................347
FLOW INJECTION ANALYSIS ......................................................................................................348
AGILENT 1100 SERIES LC/MSD TRAP: DATA ACQUISITION ......................................353
IN THIS SECTION .......................................................................................................................354
OVERVIEW ................................................................................................................................355
SELECTING THE OPERATING MODE...........................................................................................356
FILTERS BUTTON ......................................................................................................................357
OPTIMIZING THE SPECTRUM .....................................................................................................358
SCAN RANGE ............................................................................................................................359
AVERAGES ................................................................................................................................360
ROLLING AVERAGING ...............................................................................................................361
MS OPERATION ........................................................................................................................362
MS/MS OPERATION..................................................................................................................366
MSN OPERATION ......................................................................................................................369
MANUAL OPERATION ...............................................................................................................370
MS/MS OPERATION..................................................................................................................371
SETTING THE FRAGMENTATION CUTOFF ..................................................................................372
USING A SECOND FRAGMENTATION..........................................................................................373
SETTING THE FRAGMENTATION TIME .......................................................................................375
SMARTFRAG .............................................................................................................................376
ACQUIRING DATA FILES ...........................................................................................................377
USING AUTOMSMS..................................................................................................................381
SEGMENTS ................................................................................................................................386
AGILENT 1100 SERIES LC/MSD TRAP: DEVELOPING ACQUISITION METHODS 393
IN THIS SECTION .......................................................................................................................394
METHOD STRATEGY .................................................................................................................395
CREATING AN MS METHOD ......................................................................................................396
MS ONLY METHOD ..................................................................................................................397
LOADING AN MS METHOD........................................................................................................398
EDITING AN MS METHOD .........................................................................................................399
SAVING AN MS METHOD ..........................................................................................................401
RUN AN MS METHOD ...............................................................................................................402
LC/MS METHODS .....................................................................................................................405
OFFSETTING TIME SCALE..........................................................................................................406
LC/MS METHODS .....................................................................................................................407
LABORATORY EXERCISE: SCAN ACQUISITION ON THE AGILENT 1100 LC/MSD
TRAP ...........................................................................................................................................415
IN THIS EXERCISE YOU WILL: ..................................................................................................416
PRE-ACQUISITION PARAMETERS...............................................................................................417
ENTERING INSTRUMENT PARAMETERS – LC PARAMETERS ......................................................418
ENTERING INSTRUMENT PARAMETERS - MS PARAMETERS ......................................................421
viii
Agilent 1100 Series LC/MSD TRAP
System Overview:
VL, SL and XCT Models
Agilent 1100 Series LC/MSD TRAP System Overview:
In This Section
In This Section
In This Section, We Will Discuss:
• The difference between Electrospray, Atmospheric Pressure

Chemical Ionization, Atmospheric Pressure Photoionization,
and AP-MALDI.
• Details of the ion optics in the VL, SL, and XCT

configurations and ion trap and detector fundamentals.
• How the vacuum system functions.
• The information available from mass spectral data.
You will be taking a tour of the system from

ion source to detector.
2
The Agilent 1100 Series LC/MSD Trap is a benchtop ion trap mass spectrometer
providing atmospheric pressure ionization of liquid samples. A wide variety of
compound classes may be analyzed using either electrospray (API-ES),
atmospheric pressure chemical ionization (APCI), atmospheric pressure
photoionization (APPI), or matrix assisted laser desorption ionization (MALDI).
Molecular weight information as well as structural information through collision-
induced-dissociation (CID) and MS(n) provides useful qualitative sample
information. Quantification is accomplished even in the presence of complex
matrices using MS/MS (MS(2)).
In this section you will learn:

• The difference between electrospray, atmospheric pressure chemical
ionization, atmospheric pressure photoionization, and matrix assisted laser
desorption.
• How the ion optics, quadrupole filter, and detector function.
• About the vacuum system.
2
In This Section
• About the kind of information available from a mass spectrometer.
3
Atmospheric Pressure Ionization Mass Spectrometry (API-MS)
Atmospheric Pressure Ionization Mass

Spectrometry (API-MS)

API-MS is a detection
method for samples in the
liquid phase (HPLC, FIA,
Infusion). The sample is
desolvated, ionized,
analyzed by mass/charge
ratio and detected.
h Compatible with a broad range of compounds

h pg - fg sensitivity
h Qualitative information (MW up to 100,000 daltons or
more with 0.02% accuracy and MS/MS spectral
information for compound identification)
3
The Agilent 1100 Series LC/MSD Trap is a detector for samples in the liquid
phase. The LC/MSD Trap is easily coupled to an HPLC, particularly the Agilent
1100. Samples may be introduced after separation on a variety of columns, from
0.075 mm to 7.5 mm using a range of mobile phases. The capillary and nano LCs
can be interfaced to the Trap as well. For labs desiring rapid sample throughput
of relatively pure samples, flow injection analysis, FIA, is easily automated with
the 1100 Series Liquid Chromatograph modules. Infusion, for improved ion
statistics of samples not requiring a separation, is accomplished either through an
LC pump, by injection with the optional integrated manual injection valve, or by a
syringe pump.
Mass spectral data provides the molecular weight, structural information,

selectivity, and sensitivity. With electrospray, atmospheric pressure chemical
ionization and atmospheric pressure photoionization in positive and negative
modes available, a wide range of samples can be analyzed. Molecular weight
information is possible for both small and large molecules. Electrospray,
4
producing multiply charged ions, can accurately determine the molecular weight
of molecules, including proteins, in excess of 100,000 daltons. The MS(n)
capabilities of the trap can be used for structural elucidation, gaining specificity
and sensitivity in a quantitative determination and to gain peptide sequence
information for protein data base searching.
5
API-MS Modes
API-MS Modes
API-MS Modes
Electrospray: ionization process which uses an electrical field to
generate charged droplets and subsequent analyte ions by ion
evaporation for MS analysis. Nebulization is usually pneumatically
assisted.
Atmospheric Pressure Chemical Ionization (APCI): gas phase

chemical ionization (CI) process where the solvent acts as the CI
reagent gas to ionize the sample.
Atmospheric Pressure Photoionization (APPI): Krypton lamp

producing ultraviolet light ionizes gas phase analytes or dopants
with subsequent gas-phase reactions.
Atmospheric Pressure-Matrix Assisted Laser Desorption

Ionization (AP-MALDI): matrix absorbs laser radiation causing
matrix and sample to vaporize and ionize the sample. Additional
ions may be formed in the gas-phase.
The Agilent 1100 Series LC/MSD Trap is equipped with three modes of
Atmospheric Pressure Ionization, electrospray, atmospheric pressure chemical
ionization, atmospheric pressure photoionization, and matrix assisted laser
desorption ionization. These often-complementary techniques provide the ability
to analyze a wide range of samples
Electrospray ionization is a process whereby ions are formed from the liquid
phase by ejection from shrinking charged droplets. Sensitivity is improved when
the ions are preformed in the eluent. API-ES is pneumatically assisted so that
higher liquid chromatographic flow rates can be handled easily. API-ES is useful
for the analysis of samples that become multiply charged such as proteins,
peptides and oligonucleotides. Singly charged small molecules are analyzed as
well. Nanospray is also available when sample quantity is limited.
Atmospheric Pressure Chemical Ionization is an atmospheric pressure ionization

technique using a corona discharge to ionize the analytes and mobile phase in the
gas phase. As this technique requires some sample volatility, it is best suited for
6
API-MS Modes
moderate molecular weight species of moderate polarity. This technique is better
suited for nonpolar compounds than electrospray.
Atmospheric Pressure Photoionization is an atmospheric pressure ionization
technique using photoionization to ionize the analytes in the gas phase. As this
technique requires sample volatility, it is best suited for moderate molecular
weight species of moderate polarity. This technique is better suited for nonpolar
compounds than electrospray and complements Atmospheric Pressure Chemical
Ionization.
The AP-MALDI or Atmospheric Pressure-Matrix Assisted Laser Desorption/

Ionization source is an API source suitable for peptide and protein samples.
Mixtures of matrix and femtomole amounts of sample are spotted onto a plate and
then subjected to laser desorption/ionization. MS/MS analysis with MASCOT
search follows for protein identification.
The atmospheric pressure ionization sources are easily interchanged, requiring

only a few minutes to switch between operating modes. The software
automatically recognizes which source/spray chamber is installed and adjusts
accordingly.
7
Relative Applicability of LC/MS Techniques
The combined techniques of electrospray, APCI and APPI can solve the vast
majority of application problems, far more than GC/MS or any other LC/MS
technique. Electrospray is best suited for readily ionizable species or polar
compounds. Because of the multiple charging capabilities, the molecular weight
range is significant. APCI and APPI are well suited to compounds that cannot be
analyzed by electrospray, primarily intermediate polarity species. In APCI or
APPI mode, however, the molecular weight range is limited by the mass range of
the mass spectrometer, since the formation of multiple charged ions is rare.
8
What Kind of Data Do You Obtain?
Singly Charged Ions in ES or APCI Mode


Example: Singly Charged Ions
Ginsenoside
When a small molecule is analyzed by atmospheric pressure ionization

techniques, typically, the predominant species in the mass spectrum is a
protonated molecular ion, [M+1]+. In the example above, the most abundant peak
in the mass spectrum is the sodium adduct ion, [M+Na]+ at m/z 1131.7.
Other pseudomolecular ions may be produced and even predominate, depending

upon the mobile phase, additives, and even impurities. Common ions for positive
mode include:
[M+ H]+ = [M+1] +
[M + Na]+ = [M + 23]+
[M + K]+ = [M + 39]+
[M + NH4]+ = [M + 18]+
[M + X]+ where X is solvent buffer cation
2[M + H]+ dimer at high concentrations
9
[M + H + S]+ solvent adducts
for negative mode;

[M – H]- basic conditions
[M + X]- where X is solvent or buffer cation
[M – H + S]- solvent adduct.
Your sample storage, preparation, mobile phase, and additives will affect your
overall outcome. Be mindful of these facts when developing a method.
10
What Kind of Data Do You Obtain
What Kind of Data Do You Obtain
What Kind of Data do You Obtain?

Example: MS/MS
Full scan
product ion
(MS/MS
spectrum from
the sodium
adduct at m/z
1131.7
MSn analysis in an ion trap mass spectrometer permits multiple stages of

precursor ion isolation and fragmentation. This stepwise fragmentation permits
individual fragmentation pathways to be followed and provides a great deal of
structural information.
The spectrum above shows the direct infusion and MS/MS analysis of
ginsenodise Rb1. The MS/MS of m/z 1131.7 yields a product ion at m/z 789.7
corresponding to cleavage of a single glycosidic bond. Subsequence isolation and
fragmentation can be performed.
11
Multiply-Charged Ions in Electrospray Mode


Abundance
40000 A+21
808.60 A+20
35000
A+22 848.95 A+19
30000
A+23 771.90 893.55 A+18
25000 738.45
A+24 943.15
20000 A+17
707.65 998.55
15000 A+16
A+25 1060.85 A+15
10000 679.45 1131.50 A+14
5000 1212.30
m/z--> 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250
Abundance
280000
A Assume adduct is a
240000 16959.09
200000
proton:
160000 M1=(A+n)/n
120000 M2=(A+n+1)/(n+1)
80000 Solve for A
40000
m/z--> 16400 16600 16800 17000 17200 17400 17600

API-ES spectrum of Myoglobin
High molecular weight compounds having multiple ionization sites produce

abundant multiply-charged ions. An example would be proteins whose arginine
and lysine groups can be protonated under acidic conditions. The mass to charge
ratios analyzed fall within the mass range of the mass spectrometer.
The example presented above shows the API-electrospray spectrum of
myoglobin. The spectrum on the top contains a series or envelope of multiply
charged ions representing a number of different m/z states, each corresponding to
attachment of multiple protons. The number of adducts is represented by the
number printed above the mass peak.
Any pair of ions can be utilized to compute the molecular weight. The Bruker
software is equipped to find the multiply charged ions, compute the molecular
weight, and print a report.
12
Main Components of the LC/MSD TRAP

Ion Optics
Ion Trap
Spray Detector
Chamber
+
+
+
+
+ +
+ + + + + + + + + + + +
+
Drying Gas
Waste
VL model has two skimmers
SL, XCT models have one skimmer
A schematic of the LC/MSD Trap is shown above. Regardless of whether the

ionization method is APCI, APPI, or electrospray, the spray chamber performs
three functions:
• Aerosol generation, performed by the nebulizer.
• Ionization (subsequent to that performed in solution).
• Solvent removal, performed by the desolvation assembly.
The orthogonal nebulizer is excellent for handling salts and buffers.

The charged ions are drawn into the capillary that separates the atmospheric
pressure ion source region from the higher vacuum region. As the ions leave the
capillary, they are accelerated toward skimmer one. Skimmer one acts both as a
lens and to remove neutral excess gas and solvent molecules. Skimmer two also
acts to focus ions and remove more neutral species before the ions reach the high
vacuum region of the quadrupole. The SL and XCT versions have only one
skimmer. The octopole guide will continue to focus the ions toward the
quadrupole. This region allows a significant amount of the neutral species to be
13
pumped away by the turbomolecular pump. The octopole also serves to
homogenize the energy distributions of the ions before entering the quadrupole.
The quadruple ion trap mass filter accumulates all ions until the rf is scanned,
ejecting sequential ions to the detector. The High Energy Dynode detector counts
the ions and amplifies the signal. Once the ions have been counted and amplified,
they are recorded in the mass spectrum and sent to a data file on the ChemStation.
14
Models: VL, SL, XCT, XCT+, XCT Ultra
Models: VL, SL, XCT

XCT/XCT Plus/XCT Ultra
SL
•Mass range to 4000 Da
•MS11 capability
•MS11 capability
•Better Sensitivity
•Better Sensitivity (different
•Faster scan speeds
ion optics)
VL
•MS 2 capability
•Value Priced
•Upgradeable to SL
10
Several models of the LC/MSD Trap are now available. The VL model is value
priced. The mass range is up to 2200 Da and the instrument can perform MS(2)
experiments. The full scan MS/MS sensitivity for 25 pg reserpine at 25:1 RMS,
monitoring m/z 609.3 ion. This LC/MSD Trap is well suited for the analysis of
small-molecule drugs, drug metabolites, pesticides, herbicides, and other samples.
Furthermore the instrument can be upgraded to the SL model.
The SL model is designed for better sensitivity and flexibility. A new optical
design, one skimmer, provides full scan MS/MS sensitivity of 5 pg reserpine with
a 25:1 RMS, monitoring the m/z 609.3 ion. The mass range is up to 4000 Da for
analyses of a wide range of samples. The SL model can perform MS(11)
experiments.
The XCT model is even more sensitive. The full scan MS/MS sensitivity for the
models is shown here:
XCT - 1 pg 50:1, opened the skimmer diameter and improved the analyzer.
XCT+ 250 fg at 50:1, improved the detection
15
XCT Ultra 250 fg at 50:1, faster scanning(reduced dead time between scans
resulting in 3X as many complete scan cycles at the same nominal scan speed.
16
API-Electrospray Ionization
Electrospray Ions
Nebulizer (gas Heated nitrogen drying
shown in red) gas
-4000 V
Solvent spray
+⊕
+
⊕ ⊕
⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕
Dielectric capillary
entrance
Evaporation Rayleigh Coulomb Evaporation Analyte Ion

Limit Explosion
11
The LC/MSD Trap nebulizer uses pneumatically assisted electrospray.

Electrospray is an ionization process that uses electrical fields to generate charged
droplets and subsequent analyte ions by ion evaporation. Early electrospray
operated at very low flow rates, typically 1 – 5 µl/min. Pneumatically assisted
electrospray is the same process as electrospray, but the initial droplet formation
is the result of pneumatic nebulization. Therefore, the operational flow rate can
be increased up to 1 mL/min.
The API-electrospray process comprises three basic steps:

• Nebulization and charging,
• Desolvation,
• Ion evaporation.
Nebulization and charging occur as the HPLC effluent, with analyte ions in
solution, emerges from the tip of the nebulizing needle. The needle is at ground
potential, surrounded by a semi-cylindrical electrode to which high voltage is
17
applied. The potential difference between the nebulizer and the counter electrode
produces a strong electric field that charges the surface of the emerging liquid and
forms a fine spray of charged droplets. Concentric high-pressure gas flow assists
the nebulization.
The charged droplets are attracted toward the capillary sampling orifice through a
counter flow of heated nitrogen drying gas, which shrinks the droplets and carries
away uncharged material.
The droplets continue to shrink until the repulsive electrostatic (Coulombic)

forces exceed the droplet cohesive forces (surface tension) leading to droplet
explosions. This process is repeated until analyte ions are ultimately desorbed
into the gas phase. The ions are driven by strong electric fields on the surface of
the microdroplets. The emerging gas phase ions are then passed through the
capillary sampling orifice into the low-pressure region of the ion source, and on to
the mass analyzer.
18
API –Electrospray Ion Source
API-Electrospray Ion Source
High Voltage Mesh Assembly
API-ES Nebulizer
12
The API-electrospray spray chamber is shown above. The electrospray spray

chamber has three important functional components:
Nebulizer
The nebulizer extends into the spray chamber so the nozzle is inside the field
generated by the mesh electrode. The nebulizing gas enters the spray chamber
through a tube that surrounds the needle. The nebulizer is positioned 90° to the
inlet capillary. This design allows uncharged materials to be removed quickly via
the sloped drainage port. The cross-spray nebulizer prevents plugging or fouling
of the capillary with dirty samples. The mechanical position of the nebulizer is
fixed and does not require adjustment.
Mesh Electrode
The voltage applied to the mesh electrode, like the spray shield and end cap, is
always 500 V less than the entrance capillary.
Desolvation Assembly
The desolvation assembly blows heated nitrogen, flowing at approximately 6 to
12 SLPM, across the entrance to the capillary. The drying gas removes the rest of
the mobile phase solvent, which is pumped away. Drying gas temperature is
19
usually set to 300 degrees C; for electrospray, drying gas flow is usually set to 10
l/min. The spray chamber design contains a sloped drain port for rapid
condensate cleaning. Also, notice the hinges for rapid changeover between
electrospray and APCI.
20
Nanoelectrospray
Nanoelectrospray
Nanoelectrospray Source
•Sample limited applications
•1-2 µl
•Flow rate 10-500 nL/min
•Requires modified capillary cap
and gas diverter
•Nano columns and nano pump
13
When very small sample quantities are available, the nanoelectrospray option can
be used for their direct determination. The nano source can be coupled to a nano
LC pump and capillary columns of 0.075 mm i.d.
21
Electrospray Spray Chamber Settings
Nebulizer
Pressure
Vcap Drying Gas Flow

• high water needs higher flow
• if too low, spikes in spectra from droplets
• when in doubt, use excess
Drying Gas Temperature

• higher for low vapor pressure solvents
• start with 300 - 350°C
Vcap
Drying Gas • optimize with FIA (2000-5000)
Temperature • start with 3000 V – 3500 V
and Flow Fragmentor • in negative mode, look for high chamber current
or blue glow (indicates corona): reduce Vcap if
this happens
14
Each ion source/spray chamber has appropriate settings depending upon the
mobile phase composition, flow rate, and sample identity. The nebulizer
pressure, drying gas flow, and drying gas temperature are dependent upon the
mobile phase composition and flow rate. For instance, the higher the flow rate,
the more drying gas flow will be needed to help desolvate the droplets. Reducing
the drying gas temperature may help reduce Na adduct formation. The Vcap is
dependent on these factors as well as the sample identity and type of tip.
The settings presented above are typical settings. To determine the best setting
for your method, empirically optimize the parameters.
22
Why is it Important to Properly Control Spray Chamber Parameters?
Effect of Drying Gas Settings in ES
Why is it Important to Properly Control Spray

Chamber Parameters?
Why is it Important to Properly Control Spray

Chamber Parameters?
300000
Drying gas: 300 °C, 6 l/min
200000
100000

120000
80000
40000
5 10 15 20 25 min
Spikes caused by droplets from incomplete drying
15
The two chromatograms above illustrate how important it is to have the correct
spray chamber settings. In the top chromatogram, the drying gas was set to 300°
C and the drying gas is 6 L/min. The temperature and gas flow are too low for
effective droplet desolvation. The results are spikes in the chromatogram. The
chromatogram is improved when the drying gas temperature is raised to 350° C
and the gas flow is raised to 12 L/min.
23
Effect of Drying gas Settings in ES
Effect of Drying gas Settings in ES
8000
271.1
6000 Drying gas: 300 °C, 6 l/min
221.1
447.1
4000
2000
485.1
20000 507.1
271.1
15000
10000
485.1
5000
469.2
122.1
150.1
507.1
200 400 600 800 1000
Droplets cause noisier spectra
16
Spectra are also affected by improper spray chamber settings. The top spectrum
was taken when the drying gas settings were too low. The bottom spectrum was
collected when the drying gas temperature and flow were set properly. Notice
that the quality of the top spectrum is compromised by the presence of noise. The
bottom example is a better quality spectrum.
24
Electrospray Considerations
Samples
• Ions in solution: catecholamines, sulfate conjugates, quaternary
amines
• Compounds that can have a charge induced: menthol
• Compounds containing heteroatoms: carbamates,
benzodiazepines
• Multiply charged in solution: proteins, peptides, oligonucleotides
Solution Chemistry Parameters

• flow rate
• sample pK, solution pH
• solution conductivity
Samples to Avoid
• extremely non-polar samples: PAHs, PCBs
17
Electrospray is best suited to applications where the sample is or can be made an

ion in solution. For instance, lowering the pH can protonate basic compounds.
Once they carry a positive charge in solution, they are good candidates for
positive ion mode electrospray. Acids can become negatively charged when the
pH of the mobile phase is raised. These compounds would be analyzed in
negative ion mode electrospray.
Often, compounds can have the charge induced. Many analysts add acetic acid to
their electrospray mobile phase to induce a positive charge on sample molecules.
Because sensitivity is improved when ions are preformed in solution, the solution
pH in comparison to the sample pK is very important. Flow rate is important as
it relates to the desolvation. Solution conductivity is related to the ability of the
droplets to desorb ions. At times, the analyst may add isopropanol post-column to
improve solution conductivity of a particularly nonpolar solvent.
25
APCI Ionization Process
•Nebulize
•Evaporate Droplets
•Gas-phase ionization
[Solvent + H]+ + A
Solvent + [A + H]+
18
The complimentary atmospheric pressure ionization technique is atmospheric

pressure chemical ionization, APCI. This technique is most useful for low to
medium polarity analytes. The APCI process begins with gas-assisted
nebulization into a hot, typically 250 - 400°C, vaporizer chamber that serves to
rapidly evaporate the spray droplets. The result is gas-phase HPLC solvent and
analyte molecules. Flow rates up to 1.5 mL/min of water are tolerated.
The gas-phase solvent and analyte molecules are ionized by the discharge from a
corona needle. Similar to the processes encountered in methane positive chemical
ionization, the protonated solvent transfers a proton to the analyte if the proton
affinity of the analyte is greater than that of the solvent. The analyte ions are then
transported to the mass analyzer.
APCI may be thought of as evaporation followed by ionization. APCI is only

used for those samples that can be vaporized. The process usually results in a
singly charged ion.
26
APCI Source
APCI Source
APCI Ion Source
Vaporizer
Corona
Needle
19
Similar to the electrospray source, the APCI nebulizer is positioned orthogonal to

the inlet of the capillary. The corona discharge needle can be removed easily
without venting the vacuum system or opening the spray chamber.
27
APCI Spray Chamber Settings
HPLC Flow Rate >500µL/min

Nebulizer
Nebulizer pressure
Pressure
•60 psig
Drying Gas Temperautre
Corona Heater •start with 350° C
current
Drying gas flow
•4 L/min
Vcap Vaporizer temperature
•optimize with FIA
Vcap
•optimize with FIA (2000-6000)
•start with 2500 V
Drying gas Corona current
Temperature •optimize with FIA
Fragmentor
and Flow •start with 25 µA (neg) or 4 µA (pos)
20
The APCI spray chamber has four important functional components:

Nebulizer
The nebulizer creates an aerosol of droplets containing mobile phase and analyte.
The liquid enters the nebulizer and flows through a stainless steel needle. The
nebulizing nitrogen, at 60 psi, flows through the nebulizer on the outside of the
needle. As the liquid exits the needle, the gas breaks the liquid into droplets. The
nebulizing gas helps carry the droplets through the spray chamber.
Vaporizer
The vaporizer heats the aerosol droplets and evaporates most of the mobile phase.
The vaporizer is a heated and insulated steel tube. The temperature of the
vaporizer heater is set by the data system. It usually operates at about 350 degrees
C, but should be adjusted to match the volatility of the mobile phase.
Minimum Maximum Typical
0 500 350
In general, the higher the LC flow, the higher the vaporizer temperature required
for complete vaporization.
28
Corona discharge electrode (needle)

The corona discharge electrode generates electrons. The corona needle is located
outside the exit of the vaporizer. The electrons discharged from the needle initiate
the series of chemical reactions that result in chemical ionization of the analyte
molecules.
The field of free electrons that make up this current ionizes the mobile phase
molecules. The ionized mobile phase molecules in turn react with, and ionize, the
sample molecules.
Minimum Maximum Step Size Typical

Positive Ions 0 µA 10 0.1 4
Negative Ions 0 µA 100 1 25
Optimum corona current is highly sample dependent. A current of 4 uA works
well for the APCI calibrant.
Desolvation assembly
The desolvation assembly blows heated nitrogen across the entrance to the
capillary. The drying gas removes the rest of the mobile phase solvent, which is
pumped away. For APCI, the drying gas flow is usually set to 4 l/min.
The temperature required depends on the LC flow rate, the drying gas flow rate,
and the thermal stability of the sample.
Minimum Maximum Typical
0° C 350° C 300° C
In practice, any temperature below ambient simply turns off the drying gas heater.
29
APCI Considerations
APCI Considerations
APCI Considerations
Samples
• Compounds of intermediate MW and polarity: PAHs, PCBs, fatty
acids, phthalates.
• Compounds that don’t contain acidic or basic sites (e.g.
hydrocarbons, alcohols, aldehydes, ketones, and esters
• samples containing heteroatoms: ureas, benzodiazepines,
carbamates
• samples that exhibit a poor electrospray response
Solution Chemistry Parameters

• less sensitive to solution chemistry effects than ES
• tolerates higher flow rates than ES
• accommodates some solvents not compatible with ES
Samples to Avoid
• thermally labile compounds due to vaporization process
21
APCI provides good performance for analytes of low to medium polarity. For
instance, APCI may be applied to PNAs and lower molecular weight heterocyclic
pharmaceuticals. Since ionization occurs in the gas phase after analyte
evaporation, APCI exhibits lower sensitivity to solution chemistry than
electrospray. APCI is capable of handling increased flow rates as well.
Compounds analyzed by APCI must possess reasonable volatility. This fact rules
out larger, more polar molecules such as proteins and peptides. Thermally labile
compounds such as steroids and aminoglycosides will exhibit lower sensitivity
and degradation due to the high heat applied during the vaporization process.
30
APPI Source for the LC/MSD Trap
APPI Source for the LC/MSD Trap
APPI Source for the LC/MSD
HPLC
Nebulizer
inlet
Vaporizer
(heater)
Drying
gas
hν
+ +
+ + + + +
Capillary
UV Lamp
UV-Lamp
22
The APPI interface substitutes the corona discharge needle with a source of UV
light. The interface is used for low to medium polarity analytes. The APPI
process generates ions by first nebulizing the liquid analyte into small droplets,
followed by evaporating the droplets to produce gas phase analyte molecules.
The gas phase analyte is then ionized by photoionization to form molecular
species. Heated drying gas is still used, but at a low flow to protect the mass
detector from spurious noise.
31
APPI Process
APPI Process
APPI Process
Analyte containing +
aerosol +
+
+
+
Photon ionizes + +
hυ analyte
+
Evaporation + +
+ +
+ + + Analyte ions
hυ + ++ +
Vapor + + +
+ + + + +
+
+ ++ +
+
Dopant is
photoionized and
acts as reagent
gas
23
This shows the evaporation and ionization processes in more detail. Note that the
analyte is not ionized until after it is in the gas phase. Ions can be formed in APPI
by direct photoionization or through proton transfer reactions of photoionized
dopant gas with the analyte.
32
APPI Mechanisms
APPI Mechanisms
APPI Mechanisms
Direct APPI
M + hν → M+. + e- Analyte molecule M is ionized to molecular
ion M.+ (If analyte ionization potential is below
photon energy)
M+.+ SH → [M+H]+ + S• Molecular ion M+. may abstract a hydrogen to

form [M+H]+
Dopant APPI
D + hν → D.+ + e- Photoionizable dopant D is in excess &

yields many D.+ ions
D+.+ M → → [M+H]+ + D Analyte M ionizes by hydrogen abstraction
from dopant or solvent
D+. + M → M+. + D D.+ ionizes analyte M by electron transfer
24
The two mechanisms of APPI are presented in more detail. Direct

photoionization can yield radical cations for the analyte and or protonated
molecular ions if the proton affinity of the analyte ion is greater than the LC
solvent. The addition dopant (a species that can be easily photoionized and
transfer an electron or proton to the analyte) is used to assist in the ionization of
an analyte.
33
APPI Source
APPI Source
APPI Source
Lamp Source
instead of
Discharge Needle
25
The APPI source is identical to the APCI source except the corona discharge
needle is replaced with a high intensity UV lamp as a source of photons.
34
PDF-MALDI Source
PDF-MALDI Source
• Atmospheric Pressure-Matrix
PDF-MALDI for Ion Trap Assisted Laser Desorption
Ionization
• Well-suited for protein and peptide
analyses
• Interchangeable with other
atmospheric pressure sources
Sample 500 attomoles BSA Digest

plate
hv
Matrix ion
Analyte ion 26
Another source that is available is the Agilent Atmospheric Pressure-Matrix

Assisted Laser Desorption Ionization source for the ion trap. The PDF-MALDI
source is important for proteins, peptides, and other biological macromolecules.
Samples in the low-femtomole range are spotted with matrix on to a sample plate.
The sample plate is inserted into the source and locked into position. The laser
can target the spot in a spiral pattern to ensure uniform sample desorption. A
chromophore in the matrix absorbs the laser radiation and the sample and matrix
are subsequently volatilized and ionized. The ions are typically singly charged
species. Predominantly, searchable MS/MS data of peptides and protein digests
are analyzed and can be used to identify post-translational modifications.
Because PDF-MALDI is performed near atmospheric pressure, changing plates is
quickly accomplished. An automated spotting system is available as an 1100
module.
35
Drying Gas and Capillary
27
The drying gas heater assembly is common to all sources and resides within the
main MSD Trap frame. The desolvation assembly consists of the drying gas tube,
200 W heater, sensor, and insulation. The desolvation assembly blows hot
nitrogen (drying gas) on the droplets of eluent before they enter the capillary. The
liquid drains out the source drain tube into the waste bottle.
The insulation around the drying gas tube prevents the capillary from heating,
thereby preventing thermal degradation of the sample ions. The drying gas
system has been designed to handle a wide range of HPLC flow rates up to 1,000
µL/min without the need for splitting.
The dielectric platinum-plated capillary separates the spray chamber from the ion
optics region. Ions formed in the atmospheric spray chamber travel through the
capillary to the lower pressure ion optics region where they will be focused and
introduced into the mass analyzer. The LC/MSD capillary is operated at low
temperature, which permits the analysis of fragile non-covalent interactions (e.g.
protein interaction). The large diameter, 0.5 mm, is not prone to plugging. The
SL model has a 0.6 mm diameter.
36
The capillary will need to be cleaned periodically. In general, the capillary cap
should be cleaned as frequently as the rest of the spray chamber because residues
from solvents and samples will build up over time. The residues will start to build
an insulating layer, which reduces the electric field strength necessary for high
performance electrospray and APCI operation. The effect is lowered sensitivity
and signal stability.
Over a longer period of time, the inner bore of the capillary can become
contaminated. This will tend to decrease ion transmission, especially at lower
masses. As this contamination becomes more severe, it is possible to generate a
leakage current from the high voltage (entrance) end to the low voltage (exit) end.
This can result in increased capillary current, spikes in capillary current, and in
extreme cases, spray chamber voltage faults.
The normal values for capillary current in API-ES depend on the capillary voltage
setting, solvent flow rate, and solvent conductivity. In APCI, the corona current
and vaporizer temperature may also impact the capillary current. This makes it
difficult to specify a range for proper capillary current. For the standard calibrant
solutions, the capillary current is typically in the range of 70 to 110 nA.
You should monitor the typical capillary current for a given analysis or type of
analysis. If the current changes significantly, clean the spray chamber. If the
problem persists, clean the capillary.
37
Ion Optics
Ion Optics
Ion Optics
Skimmers
Capillary VL Ion Optics

•Two skimmers
•Split octopole
Octopole
Split Octopole
Vacuum wall
Capillary
SL, XCT, XCT +, XCT Ultra
•Single Skimmer
•Dual Octopole
•XCT skimmer aperture larger
Skimmer
Dual octopoles
28
The basic elements of the LC/MSD ion optics include a capillary, skimmer(s), RF
octopoles, and lenses. Once the ions enter the capillary, they are directed into the
low-pressure ion optics region by electric fields and pressure drop. The capillary
serves to isolate the spray chamber from the low-pressure ion optics region. The
skimmer(s) serve to sample the ions while directing the flow of neutrals to the
vacuum system. They also focus the ions. The octopole transports the ions to the
mass filter.
The VL model has two skimmers. The SL and XCT models have one skimmer.
The XCT models have a larger skimmer aperture and dual octopoles.
A picture of the octopole RF ion guide assembly is shown including the mounted
skimmers and entrance lenses. The RF octopole serves as an efficient focusing
element for the ion beam. The octopole reduces the ion energy distribution which
is important before mass analysis in the mass filter. The octopole consists of
eight rods, with RF voltage, in addition to a dc offset voltage. Adjacent rods have
voltage of opposite polarity. The octopole acts as an ion guide, helping the ions
pass into the mass filter.
38
Mass Analysis
Mass Analysis
Operation of an Ion Trap
Basic construction
Two end-caps and a ring
Single inlet aperture, "pepper
shaker" outlet
Enhanced geometry to produce
multipole non-linear fields
Ring
Focusing lens
system
Endcap
Lenses Conversion
+
+ Dynode
+ +
+ + +
+ + + +
+ + + +
+
Electron
Multiplier
Octopole
Focus
Endcap
Optimized Asymptote Angle

29
A cross-sectional display of the Bruker ion trap and an exploded photographic

image are shown. The ion source region is on the left and ions travel from left to
right. The endcaps are purple and the ring electrode is orange. On the entrance
side of the ion trap is an octopole consisting of eight rods. The octopole is an RF-
only set of electrodes used for transmission of ions over a wide m/z range. The
dual lenses following the octopole are designed to focus the ions into the trap.
The exit endcap has seven holes ("pepper shaker"), each about 2 mm in diameter.
One hole is in the center surrounded by the remaining six. When the ions exit the
ion trap they pass through lenses that focus the ions into the detector region.
The detector consists of a conversion dynode and electron multiplier. These
components are oriented at a 90 degree angle with respect to the direction of the
incoming ions. This arrangement reduces chemical noise caused by neutral
species that somehow traverse both the ion optics and the ion trap.
The conversion dynode is a parabolic surface electrode held at a very high voltage
(7-10kV), opposite in polarity to the ions of interest. The incoming ions are then
accelerated, crashing into the dynode to produce fragments of charge polarity
39
Mass Analysis
equal to that of the dynode. The repulsed fragments are accelerated toward the
electron multiplier where resulting impacts produce cascading electrons and,
finally, a signal.
40
Time Sequence of Events to Generate a Mass Spectrum on An Ion
Trap
Time Sequence of Events to Generate a Mass

Spectrum on An Ion Trap

Spectrum on An Ion Trap
1. trap clear 2. Accumulation Time 3. Scan Delay 4. Mass Analysis
30
The separate events described above, of accumulation and mass analysis when put
in succession comprise the basic elements of a scan sequence. The following
diagram summarizes the timing of the primary and auxiliary RF voltages,
indicates the oscillation and displacement as well as the resulting mass spectrum.
The trap is filled with ions from the ion source by dropping the repelling voltage
on skimmer 2 to pass the ion beam. Ions are trapped in the RF field using a low
quadrupolar amplitude (referred to as the Trap-Drive mass in the Expert or Easy
Tune page of the Bruker Control software). After a given accumulation time, the
skimmer 2 voltage is raised to prevent the ions from entering the ion trap. The
accumulated ions are”cooled” by collisions with the helium bath gas to ensure
that the ion cloud is positioned in a small packed at the center of the trap. During
the scan the quadrupolar and dipolar fields are increased to progressively eject
ions of ever increasing m/z value out of the trap by passing through the exit
endcap. At the end of the scan the quadrupolar field is dropped to zero to remove
41
Time Sequence of Events to Generate a Mass Spectrum on An Ion
Trap
the remaining ions from the trap. The cycle is repeated when the trap is set to its
initial conditions and skimmer 2 is set to allow ion accumulation.
42
Mathieu Stability Diagram for Ions
in an Ion Trap

in an Ion Trap

in an Ion Trap
M2 > M1
31
The ions trapped in the ion trap undergo periodic motions in both the radial as
well as the axial direction. The axial motion in the direction of the endcaps (z -
axis), is of primary importance because this is the direction of ion injection and
ion ejection. The quadrupolar field that produces this pseudo-potential well also
induces an oscillatory harmonic motion in the ions, the major component being
the ”secular frequency”. The actual frequency of oscillation of the ions is
principally determined by the m/z ratio of the ion and the RF drive level.
A lower m/z value results in a higher secular frequency. The range of ion masses
that can be trapped simultaneously is described by the stability diagram. The
stability diagram is a two dimensional plot that indicates under what particular
potentials (both RF drive as well as any imposed DC potential between the ring
and endcaps) ions of a particular m/z value are stable or unstable in the field. The
Agilent 1100 MSD Trap works in the RF-only mode, on the line indicated in the
above figure. As the RF drive level is increased, then the corresponding point on
43
in an Ion Trap
the plot for a given mass is shifted to the right. Higher masses are found left of
smaller masses, i.e. m2 > m1. For any given point in the stability region the ion
experiences a different pseudo-potential well depth and hence a specific secular
frequency. For example, by changing the length of a swinging pendulum the
period of oscillation is changed and such a related concept lends itself to the
visualization of the motion that an ion experiences in an ion trap.
From the stability diagram there follows an important consequence: the existence
of a cut-off mass. The diagram shows that there is a boundary of stability along
the line bz=1 (bz depends on the geometry of the ion trap; bz is proportional to the
RF voltage and inversely proportional to the mass of the ions). If an ion reaches
the borders of the stability diagram (bz = 0 or bz =1), the trajectory of the ions
becomes unstable and hence they leave the ion trap in axial direction (bz). This
means that there is always a lowest mass stable in the field, with all lower masses
unstable in the field. The range of masses that can be stored simultaneously in the
trap thus has as a lower limit, the cut-off mass. The cut-off mass is determined by
the RF level on the ring electrode, and it can also be found as the mass whose
secular frequency is close to one half of the RF drive frequency
(W/2 ~ 390.5 kHz).
Theoretically, there is no upper limit to storable mass range. However, for

practical purposes and thermal reasons, there is an upper limit as well. This upper
limit is about 20 - 30 times the cut-off mass; ions with an m/z above this limit are
not efficiently trapped by the RF field.
44
Detector
Detector
Conversion Dynode Detector XCT
1. Ion lens 2. Dynode 3. Multiplier
•Detector is orthogonal to flight path of ions.

•Protects the multiplier from uncharged particles, i.e. dust, solvent, water.
32
This is a picture of the multiplier assembly for the SL and XCT. The unit is easily
accessed, particularly the replaceable multiplier horn. The instrument must be
vented when working on the multiplier.
45
Detector
Detector
Conversion Dynode Detector Operation

• Positive Ion Detection
– Ions focused through lens to negative dynode.
– Positive ions strike dynode and generate primarily electrons.
– Dynode repels electrons towards the multiplier.
– Number of electrons multiplied by a cascading effect.
– Current produced amplified as a voltage signal by the preamplifier.
• Negative Ion Detection

– Ion focused through the lens to the positive dynode.
– Negative ion strike the dynode producing positive ions and fragments.
– Dynode repels positive ions towards the multiplier.
– Incident ions generate electrons
– Electrons are magnified by cascading effect in the multiplier.
– Current produced amplified as a voltage signal.
33
The detector operates in both positive and negative ion modes. The purpose of
this detector is to amplify and record the number of ions passed by the mass filter.
The detector is off-axis to the flight path of the ions. This approach protects the
multiplier from uncharged particles like dust and solvents.
In positive mode, a positive ion will be focused into the detector by the iris.
Positive ions strike the dynode and generate primarily electrons. The electrons are
focused and accelerated by the matching dynode into the horn of the multiplier.
Each time an electron strikes the surface of the multiplier horn, many electrons
are released. Several of these events occur within the multiplier horn, effectively
increasing the signal gain.
In the negative ion mode, the negative ion is focused into detector by the iris.
Negative ions strike the dynode producing positive ions and fragments. The
dynode repels positive ions towards the multiplier. Incident ions generate
electrons. Again, each time an electron strikes the surface of the multiplier horn,
many electrons are released increasing the signal gain.
46
Detector
Detector
XCT Plus and XCT Ultra
•Previous designs, only 25% of the ions

exiting the mass analyzer were detected.
•Design has improved electrical field

between mass analyzer exit and dynode
to improve efficiency.
•Machined part not subject to failure.
•Multiplier is separate part number G2440-

60200.
•Calibrate detector more often because

more ions collected.
4 Fold Increase Ion Detection

34
The XCT Plus and XCT Ultra models have a newly designed detector. Previous
designs only captured 25% of the ions exiting the mass analyzer. The design
improves the electric field between the mass analyzer exit and the dynode to
improve efficiency. The result is a four fold increase in ion detection. The
multiplier will still need to be replaced and the detector must be calibrated more
often than the XCT, SL, or VL.
47
Vacuum System
Vacuum System
Vacuum System
255 Hi
70 L/s
Controllers
35
The four-stage vacuum system consists of an 18 m3 per hour mechanical pump

backing two air-cooled turbomolecular pumps, which provide differential
pumping of the source an analyzer manifold at 250 and 70 l/sec respectively.
Low vacuum and high vacuum gauges are included. Forced air cooling for the
electronics and turbomolecular pumps eliminates the need for water-cooling.
48
UV-Vis Spectra Acquired with Diode Array Detector (DAD)
UV-Vis Spectra Acquired with Diode Array

Detector (DAD)
UV-Vis Spectra Acquired with Diode Array

Detector (DAD)
Chlortoluron ?
Take peak spectrum
Compare
250 300 250 300

W a v e l e n g t h (nm) W a v e l e n g t h (nm)
36
The diode array is a useful, complimentary tool to the LC/MSD. The diode array
will take a complete UV-Vis spectrum approximately every 10 ms. From this
data, a signal or chromatogram is created. The spectra are saved according to the
instructions given in the DAD acquisition parameters. Saved spectra can be used
to identify unknowns when compared to standard spectra. Peak Purity analysis is
also useful.
49
Total Ion Chromatogram (Scan Mode)
Scan 4
Scan 5
Scan 3
Scan 6
Scan 2
Scan 1 Scan 7
Scan 2 Scan 5
Scan 4 Scan 7
37
The total ion chromatogram is produced by summing the abundance of each ion
in a spectrum plotted against the time at the start of the scan. Connecting these
data points results in a total-ion-chromatogram.
50
LC/MS/MS on an Ion Trap
Ion Accumulation
Isolation
Intense. -
x107
3
2 ++ + +++ +++
++ ++
1
0
0 10 20 30 40 50 60 70 80 Time [min]
Electrospray
HPLC – nano LC
MS/MS
CID
Data Analysis
Scan
+ +
+
Peak List
Protein Identification
38
Ion trap MS/MS is performed in one analyzer in a sequenced program of events

described below:
1). Ion accumulation: Initially the trap is loaded with ions formed by API of the
LC effluent, until a maximum charge level or time has been reached.
2). Isolation: Next voltages and frequencies applied to the end caps and ring
electrode keep only one m/z ion stable inside the trap.
3). CID: The isolated ion is resonantly excited by applying the resonant frequency
to end caps for the ion to gain energy for CID with the helium bath gas inside
the trap. The resulting product ions which are of different m/z, do not have the
same resonance frequency, are stored inside the trap. [ note step 2 and 3 can be
repeated again for MS3 or n-1 times for MSn ].
4). Scan: The resulting product ions are mass analyzed by scanning the voltage on
the ring electrode.
51
Ion Trap MS/MS CID Mass Spectrum of a Hexapeptide
Ion Trap MS/MS CID Mass Spectrum of a

Hexapeptide
Ion Trap MS/MS CID Mass Spectrum of a

Hexapeptide
39
The MS/MS product ion spectrum can yield significant information about the
structure of the molecular species selected. In this example, m/z 687 of this
hexapeptide was mass selected and subject to collision induced decomposition
(CID) to yield the product ion spectrum shown. Cleavage of the amide bonds in
the polypeptide chain can produce a sequence of ions that differ by the residue
mass of the amino acids that comprise the peptide sequence. There is a
specialized nomenclature assigned to peptide ions. In this example, the dominant
ion series consists of b ions. b series ions are those produced by losses from the
C-terminus, with the charge retained on the N-terminal fragment.
52
MS/MS, Scan, or UV
MS/MS, Scan, or UV
MS/MS, Scan, or UV
• MS/MS analysis offers the most specificity for
qualitative determinations. Increases in sensitivity and
improvements in precision can be obtained in
applications that exhibit significant chemical
background.
• Scan analysis is used for qualitative applications and
for target compound quantitation when not limited by
chemical noise
• If the target compound has an appropriate
chromophore and the sample matrix gives well
resolved peaks, UV detection generally provides better
linearity and precision but may lack in specificity or
might show poorer detection limits due to chemical
noise.
40
MS/MS or MS(n) is used to gain qualitative information on a sample to aid in

structural elucidation or compound confirmation. The use of MS/MS improves
quantitative determinations by reducing chemical noise that can interfere with the
analysis, improving sensitivity and by basing the determination on not just the
molecular species but on several product ions
Scan analysis is utilized for qualitative purposes. This type of acquisition is used
to obtain molecular weight information about unknown samples.
If a UV detector is used in series with the mass spectrometer and the sample
contains chromophores, it may be more desirable to use the UV signal for
quantification because of the linearity and precision of the technique.
53
CE/MS/MS
CE/MS/MS
CE/MS/MS of Terbutaline and Salbutamol

Isomers
2.0 Base peak chromtaogram
CE buffer: 50 mM NaHPO4 Terbutaline Salbutamol

1.5
5 µg/mL 5 µg/mL
% Relative Abundance
HO
1.0 HO
OH
NH
HO
NH
OH
0.5 HO
0.0
2 4 6 8 10
Time (min)
41
Capillary electrophoresis can also serve as an introduction technique for the

Agilent 1100 Trap. In the example above, salbutamol was injected at the 5 ng/uL
level in the presence of 50 mM NaPO3 pH 2.5. We see good signal in the base
peak electropherogram in the presence of high sodium phosphate salt
concentration.
Shown here is MS/MS spectrum of m/z 240 salbutamol injected at the 10 ug/ml
level in the presence of 50 mM NaPO3 pH 2.5. Loss of the terbutyl amine group
generates the m/z 167 product ion and the water-loss peak at m/z 222 is the base
peak from the MS/MS spectrum.
54
Understanding ESI and APCI:
Fundamentals of the Ionization Process
Understanding ESI and APCI: Fundamentals of the Ionization Process
In This Section
In This Section
In This Section We Will Discuss:
• The current theories of ionization for ESI and APCI.
• How API solution chemistry, nebulization, and desolvation of

liquids can lead to ionization in electrospray.
• Why certain additives cannot be used in the LC mobile phase

for LC/MS
The current ideas for ionization are presented for API. While APPI and APCI
mechanisms are well understood, there are still many questions involving the
mechanism(s) of electrospray ion formation. This chapter presents the latest
theories on the API.
56


WHAT IS IT?
•Processes which lead to the ionization and subsequent mass analysis of
compounds introduced into the system
•API-MS can be operated in the following modes:
(1) Electrospray
(2) Pneumatically assisted electrospray
(3) Atmospheric pressure chemical ionization (APCI)
(4) Atmospheric pressure photoionization (APPI)
(5) Atmospheric pressure-matrix assisted laser desorption
ionization (AP-MALDI)
Each mode has certain advantages for analyzing various compound classes
and for handling various inlets as will be explained later
3
API, atmospheric pressure ionization, is any of the ionization techniques that

occur at or near atmospheric pressure, as opposed to in a high vacuum. The
subsets under API include electrospray, pneumatically assisted electrospray,
atmospheric chemical ionization and atmospheric pressure photoionization.
These often-complementary techniques provide the ability to analyze a wide
range of samples.
57
Key Characteristics of 4 Modes of API-MS Operations
Key Characteristics of 4 Modes of API-MS

Operations
Key Characteristics of 5 Modes of API-MS

Operations
ELECTROSPRAY:
Ionization process which uses electrical fields to generate charged droplets and
subsequent analyte ions by ion evaporation for MS analysis.
PNEUMATICALLY ASSISTED ELECTROSPRAY:

Same as electrospray (above) except the initial droplet formation is the result of
pneumatic nebulization.
APCI:
A gas phase chemical ionization (CI) process where the solvent acts as the CI reagent
gas to ionize the sample.
APPI:
Krypton lamp producing ultraviolet light ionizes gas phase analytes or dopants with
subsequent gas-phase reactions.
AP-MALDI
Analyte and matrix spotted on plate. Matrix absorbs laser radiation causing matrix
and sample to vaporize and ionize the sample. Additional ions may be formed in the
gas-phase.
4
Electrospray ionization is a process whereby ions are formed from the liquid
phase by ejection from shrinking charged droplets. The initial droplet formation
is achieved by electric fields, limiting the flow rate ( < 10 uL/min) and % water (<
90%) that can be used to form droplets that yield ions. Sensitivity is improved
when the ions are preformed in the eluent.
Pneumatically assisted electrospray is the same as electrospray except the initial

droplet formation is performed through pneumatic nebulization. This allows for
the use of higher liquid chromatographic flow rates (1-2 ml/min) and enables the
handling of 100% water. API-ES is useful for the analysis of samples that
become multiply charged such as proteins, peptides and oligonucleotides. Singly
charged small molecules are analyzed as well.
Atmospheric Pressure Chemical Ionization is an atmospheric pressure ionization

technique using a corona discharge to ionize the analytes and mobile phase in the
gas phase. As this technique requires some volatility, it is best suited for
58
Key Characteristics of 4 Modes of API-MS Operations
moderate molecular weight species of moderate polarity. This technique is better

suited for nonpolar compounds than electrospray.
Atmospheric Pressure PhotoIonization is an atmospheric pressure ionization

technique using a UV light to ionize the analytes and mobile phase in the gas
phase. As this technique requires some volatility, it is best suited for moderate
molecular weight species of moderate polarity. This technique is better suited for
nonpolar compounds than electrospray and complements APCI.
59
Electrospray Ionization – Necessary Steps for Ion Formation
Electrospray Ionization – Necessary Steps for Ion

Formation
Electrospray Ionization – Necessary Steps for

Ion Formation
•Step 1 Ionization In Solution
•pKa of sample
• pH of solution
•Step 2 Nebulization
Pneumatic
•Surface tension and viscosity nebulization and
drying on
•Pneumatic assistance
modern
•Step 3 Desolvation instruments,
minimize the
•Drying gas temperatures and flow effect
•Heat capacity, H vap of these
parameters
•Step 4 Desorption of Ions From Solution
•Solvation energy
•Step 5 Reactions of Ions in the Gas Phase
•Proton affinity
•Charge exchange
5
The five steps that will yield the best electrospray sensitivity are shown above.
The factors which have an influence on the success of each are listed in bullet
form below the step. These factors are dependent on the characteristics of the
target analyte as well as the choices the operator makes in the mobile phase and
API-MS operating conditions. As noted above, step 2 and 3 require little
attention on the 1100 LC/MSD Trap as long as the drying gas temperature and
flow rates are sufficient (e.g. 350 C at 8-10 L/Min for a flow rate of 0.25
ml/min.). The details of each step are explained in more depth in the following
slides.
60
Regarding Step 1 - Why is the Best Electrospray Sensitivity Achieved
When the Analytes Exist as Ions in Solution?
Regarding Step 1 - Why is the Best Electrospray

Sensitivity Achieved When the Analytes Exist as
Ions in Solution?
Regarding Step 1 - Why is the Best Electrospray

Sensitivity Achieved When the Analytes Exist as Ions in
Solution?
•For a given field strength generated on a charged droplet, ionic

interactions can be 103 to 104 times greater than non-ionic interactions
(e.g. Van der Waal forces, hydrogen bonding) for neutral molecules
•Therefore, analyte ions can be desorbed from the charged droplets
overcoming the solvation energy holding them in the liquid far better than
neutral species.
•This desorption process is called Ion Evaporation
Solution chemistry plays an important role in enhancing sensitivity for both

positive and negative electrospray ionization. Many compounds can be analyzed
as neutral molecules in a neutral environment. Other compounds, however, can
be analyzed with much greater sensitivity if the chemical environment is one that
favors ion formation.
When an analyte is dissolved in a polar solvent such as an acid or base, it can

either ionize or take on a strong dipole moment. For analytes that ionize, ESI is
generally simple and highly sensitive. Provided no other ion-ion interactions
interfere, ions are already present in the solution before spraying. These ions are
easily evaporated from the droplets in the spray and result in a high analyte ion
abundance.
Analytes that form strong dipole moments but do not ionize can still be analyzed.
The ionization process is driven by the strong electrostatic fields in the spray
61
Regarding Step 1 - Why is the Best Electrospray Sensitivity Achieved
When the Analytes Exist as Ions in Solution?
chamber. These fields induce a charge on the spray droplets. This can induce
ionization in analyte molecules at the surface of the droplets. These analytes can
also be ionized chemically by adduction using special chemicals
62
Step 1 - How Can Ions Be Created In Solution?
Ionic Species
NH4+ PO4-
Acid/Base Chemistry
M - NH2 + Acid → [M - NH3]+ + Acid-
M - COOH + Base → [MCOO]- + Base+
Association(1) (for neutral species like sugars)
M° + Na+ ↔ [M - Na]+
(alkali metal e.g. 20 µM sodium acetate)
Derivatization
To form an ion or acid/base product
(1) associations also explain why many common LC additives cannot be used with
electrospray-MS
Examples of solution chemistry that can be used to make ions are shown above.
Note in association reactions, low concentrations of strong ion-pair nonvolatile
buffers should be used (< 20µM) to prevent signal suppression or contamination
of the spray chamber.
63
Step 2 - Pneumatic Assisted Nebulization –
To Create Charged Droplets


Charged solvent droplets
about 2 µm in diameter containing
about 100,000 charges
Fields on the
cylinder, end plate
and capillary charge
the droplets
Pneumatic nebulization reduces droplet size variations from viscosity

and surface tension due to solvent composition or flow rate
8
Nebulization (aerosol generation) begins when the sample solution enters the
spray chamber through a grounded needle. For high flow electrospray, nebulizing
gas enters the spray chamber concentrically through a tube that surrounds the
needle. The combination of strong shear forces generated by the nebulizing gas
and the strong electrostatic field (2 kV to 6 kV) in the spray chamber draw out the
sample solution and break it into droplets. As the droplets disperse, ions of one
polarity are preferentially attracted to the droplet surface by the electrostatic field.
As a result, the sample is simultaneously charged and dispersed into a fine spray
of charged droplets- hence the name electrospray. Because the sample solution is
not heated when the aerosol is created, ESI ionization does not thermally
decompose most analytes.
Pneumatic nebulization reduces droplet size variations from viscosity and surface
tension due to changes in solvent composition or flow rate. In this way flow rates
from 0.01 to 2 ml/min of solvents from 100% organic to 100% water can be used
without changes in sensitivity due to variability in droplet formation. Without
pneumatic assistance, nebulization from charging a liquid (conventional
64
electrospray) is not effective at greater than 10 ul/min or in highly aqueous
solvents. Larger droplets which are formed at higher flow rates and in aqueous
solvents cannot be desolvated quickly enough to reach the field strengths needed
for ion evaporation.
65
Step 3 - Desolvation of Charged Droplets

Heated Nitrogen evaporates the droplets, increasing the charge to
volume ratio.
Rayleigh limit is the maximum charge a droplet can hold and while
maintaining its volume. When the charge exceeds this limit, coulomb
explosions occur.
Heat and drying gas
Before the ions can be mass analyzed, solvent must be removed to yield a bare
[M+Hn] ion where n = 1,2.... A counter flow of neutral, heated drying gas,
typically nitrogen, evaporates the solvent, decreasing the droplet diameter and
forcing the predominantly like surface charges closer together.
Then the force of the Coulomb repulsion equals that of the surface tension of the
droplet (the Rayleigh limit), the droplet explodes, producing charged daughter
droplets that are subject to further evaporation. This process repeats itself, and
droplets with a high surface-charge density are formed. When charge density
reaches approximately 10(8) V/cm, ion evaporation will occur.
66
Step 3 Con’t - Coulomb Fission of Droplets

•Rayeigh explosions release smaller droplets that will yield sample ions
50-100 nm charged droplet – containing 100’s of charges
These droplets contain 10-20% or the charge but only 2 % of the

mass of the parent droplet
10
When the force of the Coulomb repulsion equals that of the surface tension of the
droplet (the Rayleigh limit), the droplet does not break into half, rather is forms
what is termed a Rayleigh jet which produces 50-100 nm droplets which upon
further deslovation will yield ions. The teardrop shape produces a high field at the
tip, enabling the release of the smaller droplets which contain a higher charge to
volume ratio than the initial droplet.
67
Step 4 - Desorbing Ions from Solution
Step 4 - Desorbing Ions from Solution
Step 4 - Desorbing Ions From Solution
When the droplet field strength exceeds exceeds the solvation energy
of the analyte in solutions, ions are desorbed into the gas phase
Ion Evaporation
Charged Residues
11
The process of ion formation has been the subject of many scientific
investigations, yet differences of opinion still exist regarding the specific physical
process. The ion evaporation process described below is the model accepted by
Fenn and others. In the ion evaporation model (sometimes referred to as ion
desorption), ions are emitted directly from the charged droplets into the gas phase.
As solvent evaporates from the droplets in the presence of the strong electric field,
the surface of the droplet becomes highly charged. When the field created by the
ions at the surface of the droplet exceeds the surface tension, bare analytes ions
are emitted directly from the droplet. This model was first described by Iribarne
and Thomson (1). The other proposed method of ion formation is the charged
residue mechanism (2).
Iribarne J.V., and B.A. Thomson; On the evaporation of small ions from charged
droplets; J. Chem. Phys., 1976, 64,2237-2294.
Rollgen, F.W., E. Bramer-Weger, and L. Butfering; Field ion Emission liquid
solutions: Ion Evaporation against Electrohydrodynamic Disintegration; Journal
de Physique, Colloque C6, November 1987, 11, 48, 253-256.
68
Step 4 - Making Ions – Ion evaporation Mechanism (IEM)
Step 4 - Making Ions – Ion evaporation Mechanism

(IEM)
Step 4 - Making Ions –

Ion evaporation Mechanism (IEM)
• Field strength (E (∆Gsol) of Ions in Solution

(Ec) exceeds solvation energy (∆
Ec > ∆Gsol
• ∆Gsol Hydrophilic > ∆Gsol Hydrophobic ( solvent choice)
• ∆Gsol [M]+ > ∆Gsol [M+(H2O)n]+ (leads to clusters)
• ∆Gsol Large molecules > ∆Gsol Small molecules
IEM valid for Mol Wt < 3500
12
In the ion evaporation mechanism, ions are emitted directly from the charged
droplets into the gas phase. As solvent evaporates from the droplets in the
presence of the strong electric field, the surface of the droplet becomes highly
charged. When the field created by the ions at the surface of the droplet exceeds
the surface tension, bare analytes ions are emitted directly from the droplet.
The sample’s hydration energy in a solvent dictates the ease of desorption of ions
into the gas phase. In general, the more hydrophobic (less hydration) a sample is
in a solvent (yet still soluble in that solvent), the better ions can be desorbed into
the gas phase.
Clusters will have lower desolvation energies accounting for their formation,
particularly when the drying gas is kept cooler.
Finally ion evaporation favors smaller molecules which have fewer sites of
solvation. The IEM model is valid for compounds less than 3500 Daltons in
molecular weight.
69
Step 4- Making of Ions - Charge Residue Mechanism (CRM)
Step 4- Making of Ions - Charge Residue

Mechanism (CRM)
Step 4- Making of Ions -

Charge Residue Mechanism (CRM)
• Rayleigh fissions desolvate large molecules when ∆Gsol > Ec
• Ions stay in droplet and the charge on molecule determined by the charge on the droplet
• CRM valid for Mol Wt > 3500
Alternative Mechanism
• Rayleigh fissions desolvates large molecules
• Small ions ( e.g. NH4+ from solvent additives) undergo IEM prior to desolvation
• Gas Phase proton transfer reactions charge molecule
[M] + [NH4]+ [M+H]+ + [NH3]
Mechanism explains “wrong way Electrospray”
Electrospray” of proteins
(Detection of positive ions at high pH from NH4OH)
13
The charge residue mechanism may be the predominate method for ionizing
larger molecules (greater than 3500 Daltons in molecular weight), which have
solvation energies greater than can be overcome by the field strength on the small
droplets. The two possible way ions are formed are:
The charge on the droplet dictates the charge on the analyte after its desolvated.
The desolvated analyte undergoes gas phase proton transfer reactions with small
ions (buffer or solvent ions) that were desorbed by ion evaporation. This explains
why cations can be detected at high pH’s (wrong way electrospray).
70
Problems in Making Ions Using Typical Buffers for Electrospray – Ion
Pair Formation
Problems in Making Ions Using Typical Buffers for

Electrospray – Ion Pair Formation
Problems in Making Ions Using Typical Buffers

for Electrospray – Ion Pair Formation
Neutralization of ions in solution or the gas phase due to ion pair
formation for positive ion detection
[M+H]+ + A- [M+H + A]o

A = B, S, P Favors Neutral
Product
A = Formate Favors Charged

Acetate Species
Ion Pair Strength:

B,S,P > Trifluoroacetic acid > Acetate, Formate
B,S,P = Borate, Sulfate. Phosphate
14
Buffers or other additives used to optimize chromatography can sometimes

interfere with the ionization process. For example, TFA is almost always used for
the chromatography of peptides and proteins. TFA enhances the chromatographic
resolution but may actually suppress ion formation. Post-separation addition of a
weaker acid such as propionic acid can effectively counteract the TFA ion
suppression problem.
When performing ESI standard buffers such as phosphate, borate, and sulfate are
non-volatile and form ion pairs in solution. To maximize ESI sensitivity, use
buffers that are volatile and do not form ion pairs. Adjust the pH with buffers such
as formic acid, acetic acid, and ammonium hydroxide or triethylamine. Typical
pH for positive ion is neutral to pH 2 and for negative mode typical pH is neutral
to pH 10. For ion pair separations, use additives such as heptafluorobutyric acid
or tetraethylammonium hydroxide or tetrabutylammonium hydroxide.
71
Problems in Making Ions Using Typical Buffers for Electrospray – Ion
Pair Formation
Problems in Making Ions Using Typical Buffers for

Electrospray – Ion Pair Formation
Problems in Making Ions Using Typical Buffers

for Electrospray – Ion Pair Formation
Neutralization of ions in solution or the gas phase due to ion pair
formation for negative ion detection
[M-H]- + C+ [M-H + C]o
C = Na, K, Li Favors Neutral

Product
C = NH4+ Favors Charged

Species
Volatility only an issue due to atmospheric

chamber contamination and electrical shorting
15
Ion pair formation also applies for negative ion formation, where small alkali
metal cations can ion pair with the analyte anion. Ion pair formation is reduced
by using ammonium buffers.
Note buffer volatility is not as important as the buffers on pairing ability for a
given analyte when it comes to electrospray sensitivity.
72
Step 5 - Reactions of Ions in the Gas Phase
Proton transfer and charge exchange reactions can occur

from reaction in the atmospheric chamber through the ion
transport region. This high pressure region permits 1000’s
of ion/molecule reactions to occur.
•Proton Transfer
Samples with lower proton affinities than HPLC additives such
as ammonia or triethylamine (206 and 232 kcal/mole
respectively) can lose a proton and become neutralized or
form adduct ions such as [M+NH4]+
16
The reversal of solution phase basicity in the gas phase can result in the
deprotonation of the analyte ion resulting in no electrospray signal for that
analyte. The use of an additive such as triethylamine, a very high gas phase base
can result in the loss of [M+H]+ ion current. The use of the previously mentioned
additives (ammonium acetate, ammonium formate, acetic acid, formic acid and
ammonium hydroxide) should minimize these reactions. (note triethylamine is a
great additive for negative ion detection since it deprotonates in the gas phase and
in the solution phase).
73
Electrospray Results in the Lowest Ion Internal Energy Relative to
Other Ionization Techniques for MS
Electrospray Results in the Lowest Ion Internal

Energy Relative to Other Ionization Techniques for
MS
Electrospray Results in the Lowest Ion Internal

Energy Relative to Other Ionization Techniques
for MS
• Max ion energy < 0.2 eV, far less than a covalent bond
• Enables detection of non-covalent complexes
17
The electrospray ionization imparts little internal energy into the ion, far less than
a covalent bond. This minimal energy transfer into the ion and collision
stabilization in the atmospheric pressure chamber makes it possible to see non-
covalent complexes that have association energies far less than covalent bonds.
74
APCI – Key Parameters

•Ionization
•Gas Phase CI
•Protonation (e.g., H3O+) (Bases)
•Charge exchange
•Deprotonation (acids)
•Electron Capture (halogens, aromatics)
•Nebulizer Temperature
•Higher temperatures to desolvate and vaporize sample.
•Too high temperatures lead to sample decomposition.
•General: APCI less dependent on solvent choice, flow rates, or

additives compared to electrospray.
18
What is the difference between ESI and APCI?

APCI is a gas phase chemical ionization mechanism very similar to methane or
ammonia CI in GC/MS. In APCI the CI reagent gas is the HPLC mobile phase:
such as, water, methanol or isopropanol. The vaporized mobile phase (reagent
gas) reacts with electrons from the corona discharge to form various adduct ions.
These adducts, based on proton affinity, will transfer a proton, in the case of the
positive ion mode, to the analyte. Depending on the analyte and solvent system,
other reactions are possible:
• Protonation (such as H3O+ and bases)

• Charge exchange
• Deprotonation (acids)
• Electron capture (halogens, aromatics)
APCI requires that the analyte must be in the gas phase for ionization to occur.
To bring the mobile phase and analyte into the gas phase APCI is typically
operated at vaporizer temperatures of 400 °C – 500 °C. In APCI, the vaporizer
75
temperature must be carefully controlled. Most compounds work best at higher

temperatures while a few compounds work best at lower temperatures. It may be
necessary to evaluate a couple of temperatures to determine the optimal APCI
vaporizer temperature.
An APCI mobile phase is preferably an aqueous-organic solvent combination

with 2 mMol – 20 mMol of volatile organic buffer. High concentrations of
acetonitrile (ACN) should be avoided and its use has been shown to quickly
carbonize the corona needle which can lead to reduced total ion current
76
APCI Detailed Mechanism- Gas Phase Ionization

Nebulizer
Pressure
Corona Heater
current
Vcap
Drying gas
Temperature Fragmentor
and Flow
Mechanisms of Ionization
Vaporization → Solvent ionized → Charge transfer to analyte
N2 (From drying gas) + e- → N2+• + 2e-

N2+• + 2N2 → N4 +• + N2
N4+• + H2O → H2O +• + 2N2
H2O +• + H2O → H3O+ + OH•
H3O+ + M → [M+H]+ + H2O
19
The vaporized mobile phase (reagent gas) reacts with electrons from the corona
discharge to form various adduct ions. These adducts, based on proton affinity,
will transfer a proton, in the case of the positive ion mode, to the analyte. The
most likely reaction is shown above, as the reaction with nitrogen (the major
molar component in the API chamber), which then reacts with the solvent (shown
as water), followed by reaction with the analyte. The CI reaction can also proceed
by electron ionization of the solvent, followed by reaction with the analyte or by
direct electron ionization of the analyte (least probable). All these reactions are
possible since there are numerous collisions in the API chamber (mean free paths
are less than 10 nm) allowing for the reaction to occur within the sample
residence time in the API chamber.
77
78
Understanding Ion Trap Mass
Spectrometry
Understanding Ion Trap Mass Spectrometry
In This Section
In This Section
• How an Ion Trap Works.
• Fundamentals of Ion Trap Operation.
• Features of Ion Trap Compared To Other Mass Analyzers.
In this chapter we will cover the basic operation of the ion trap including:
• How an Ion Trap Works.
• Fundamentals of Ion Trap Operation.
• Features of Ion Trap Compared To Other Mass Analyzers.
80
Heart of an Ion Trap
Ring electrode: 781 KHz 0-12,000 Vpp – used for mass analysis
End Caps: 0-781 KHz 0-20 V pp – used for mass isolation, CID, non-linear
resonance ejection during mass analysis and to increase mass range
Helium inside trap to collisionally dampen ion motion for trapping, CID and
mass resolution
The ion trap consists of a ring electrode between two endcap electrodes. The
internal surface shape of these three electrodes follows a three dimensional nearly
hyperbolic profile. Holes at the center of the endcaps allow ions to pass in and
out of the trap. A high voltage RF potential (W = 781 kHz) is applied to the ring,
while the endcaps are held at ground. The oscillating potential difference
established between the ring and endcap electrodes forms a substantially
quadrupolar field. Depending on the level of the RF voltage, the field can trap
ions of a particular mass range. The quadrupolar field can be thought of as a three
dimensional trough or a pseudo-potential well. Among other things, the depth of
this well is related to the mass of the ion and the level of the RF voltage. In
practice, the range of masses that experience a trapping force from this
quadrupolar field is wide enough that the ion trap can very effectively produce
full scan spectra while still offering high sensitivity.
An auxiliary voltage is fed to the exit endcap of the ion trap. This additional
voltage is used for various purposes during the precursor ion isolation,
fragmentation, and mass analysis phases of the scan sequence.
Because the ions are not produced within the ion trap but are derived from an
external source, there needs to be a mechanism whereby they can be captured in
81
the pseudo-potential well created by the ion trap. Without other means, ions
focused from an external source would simply pass through the first endcap, ”roll
down” into the pseudo-potential well created by the quadrupolar field within the
trap and conservation of energy would dictate that they continue by ”rolling back
up” and out through the other endcap. For this reason, as well as others, it is
important that a collision gas is present in the trap to extract energy from the ion
beam and cause retention of at least a certain portion of the ions being injected
into the ion trap. Additional parameters that effect trapping efficiency include the
energy of the incoming ion beam, the m/z value and the mass of the ion, the depth
of the pseudo-potential well and the actual phase of the RF voltage for any
particular ion at the point of injection. Efficiencies vary widely, but at a nominal
operating pressure within the ion trap of approx. 3 x 10 mbar, a single charged ion
of 500 m/z can expect an overall trapping efficiency of approximately 5 %.
Because the ion trap is a storage device it is possible to accumulate weak signals
over an extended period of time. When the ion signal is strong accumulation
times may be as short as 10 ms, but increase up to approx. 1 s for infusion
experiments involving trace analytes. Typical accumulation times for LC/MS and
LC/MS/MS experiments range from 0.01 ms – 200 ms. By varying the
accumulation time, the dynamic range of the ion trap analyzer is greatly extended.
82
Mathieu Stability Diagram for Ions in an Ion Trap

Classical approach –
no voltage on end
caps, rf amplitude
ramped
qz = 4zeV/mw2ro2 Point of ion ejection qz ~ 0.91
V = Amplitude of RF field
m = mass
M2 > M1
z = charge
w = rf frequency (781 KHz)
r = ion trap radius ( ~ 0.7 cm)
The ions trapped in the ion trap undergo periodic motions in both the radial as
well as the axial direction. The axial motion in the direction of the endcaps (z -
axis), is of primary importance because this is the direction of ion injection and
ion ejection. The quadrupolar field that produces this pseudo-potential well also
induces an oscillatory harmonic motion in the ions, the major component being
the ”secular frequency”. The actual frequency of oscillation of the ions is
principally determined by the m/z ratio of the ion and the RF drive level. A lower
m/z value results in a higher secular frequency.
The range of ion masses that can be trapped simultaneously is described by the
stability diagram. The stability diagram is a two dimensional plot that indicates
under what particular potentials (both RF drive as well as any imposed DC
potential between the ring and endcaps) ions of a particular m/z value are stable or
unstable in the field. The Agilent trap works in the RF-only mode, on the line
indicated above. As the RF drive level is increased, then the corresponding point
on the plot for a given mass is shifted to the right. Higher masses are found left of
smaller masses, i.e. m2 > m1. For any given point in the stability region the ion
experiences a different pseudo-potential well depth and hence a specific secular
frequency. For example, by changing the length of a swinging pendulum the
83
period of oscillation is changed and such a related concept lends itself to the
visualization of the motion that an ion experiences in an ion trap.
From the stability diagram there follows an important consequence: the existence
of a cut-off mass. The diagram shows that there is a boundary of stability along
the line bz=1 (bz depends on the geometry of the ion trap; bz is proportional to the
RF voltage and inversely proportional to the mass of the ions). If an ion reaches
the borders of the stability diagram (bz = 0 or bz =1), the trajectory of the ions
becomes unstable and hence they leave the ion trap in axial direction (bz). This
means that there is always a lowest mass stable in the field, with all lower masses
unstable in the field. The range of masses that can be stored simultaneously in the
trap thus has as a lower limit, the cut-off mass. The cut-off mass is determined by
the RF level on the ring electrode, and it can also be found as the mass whose
secular frequency is close to one half of the RF drive frequency
(W/2 ~ 390.5 kHz).
Theoretically, there is no upper limit to storable mass range. However, for

practical purposes and thermal reasons, there is an upper limit as well. This upper
limit is about 20 - 30 times the cut-off mass; ions with a m/z above this limit are
not efficiently trapped by the RF field.
84
Mathieu Stability Diagram for Non-Linear Resonance Ejection
Mathieu Stability Diagram for Non-Linear

Resonance Ejection

Resonance Ejection
Agilent-Bruker’s
Lower qz value for ion ejection
approach – rf amplitude
ramped and a separate
rf signal is added to the
end caps at a low
amplitude, at a
frequency to eject ion at
1/3 the resonance
M2 > M1
frequency (also called
axial modulation)
The Agilent trap makes use of a "multipole-superimposed” ion trap in which the
mainly quadrupolar field has contributions from hexapolar, octopolar and even
higher-order fields. The effect is created by a slight change to the angle of the
asymptotes associated with the hyperbolic profile formed by the electrodes. A
pure quadrupolar field has a linear increase in the field as the ion moves from the
center of the trap towards the ring or the endcaps. The presence of the higher
order poles in the design means that the field increases faster than linear away
from the center. This fact induces certain non-linear resonances within the
stability diagram that cause energy to be far more quickly taken up in the ion
motion. These non-linear resonances occur if the secular frequency and the
driving frequency of the ion trap have an integer relationship.
The energy take-up of the ions’ motion is triggered by the application of an

auxiliary dipole field across the endcaps of the ion trap. Since the ions are
confined in the trap and experiencing periodic oscillatory motion it is possible to
couple additional energy into their motion, analogous to the pushing on a
pendulum at its fundamental frequency. If the frequency of this dipolar field is the
85
Mathieu Stability Diagram for Non-Linear Resonance Ejection
same as the secular frequency of the ions then energy is taken up by the ions and
the oscillatory motion increases in its displacement from the center of the trap.
Of particular interest is when the ion trap is driven to such a level that the
resonance between the secular frequency of the ions and the additional dipolar
field coincides with a non-linear resonance brought about by the higher order
multipole contributions in the ion trap design. It is at this operating point that an
ion confined within the trap is particularly suited for resonance. The ions take up
energy very quickly and leave the ion trap without becoming unstable in the field.
It is this process (multiple resonance) that is unique to the Agilent ion trap design,
which allows for superior combinations of scan speed and mass resolution.
The integer relationship between the secular frequency and the driving frequency
ensures that in successive measurements identical field interactions are
experienced between the ions and the trapping and dipolar fields. This is
accomplished by phase locking the trapping and dipolar fields. Phase locking
makes it possible to achieve reproducible ion excitation necessary for accurate
mass assignment and precise MS/MS excitation. It is of particular significance
that the additional resonance introduced by the higher order multipoles at W/3
satisfies this integer relationship.
86
Why Non-Linear Resonance?
• Faster scan time with comparable mass resolution

compared to resonance ejection
An ion's resonance absorption of energy from the ion trap field can be sped up in
time if the trap is non-linear. That is, if there are higher order multipole terms,
other than the pure quadrupole, then when the ion's frequency comes into
resonance with the frequency of an applied field, the ion will take up energy much
faster than it would have in a linear ion trap. Since the frequency of the RF field
is set at 781 kHz, and the ion’s frequency of motion can never be greater than half
this value, the applied field could come from the endcaps. The result is that faster
scan speeds can be expected when applying the endcap AC voltage at a frequency
corresponding to this resonance.
87
Ion Trap Relationship between Resolution, Mass Range, and Scan
Speed
Ion Trap Relationship between Resolution, Mass

Range, and Scan Speed
Ion Trap Relationship Between Resolution, Mass

Range, and Scan Speed
• Decreasing the speed the rf amplitude on the ring

electrode is ramped reduces peak width of an ion
signal by reducing its kinetic energy spread.
– Over 1,000,000 resolution at mass 100 has been
reported
• Lowering the axial modulation frequency on the
end caps reduces the qz for ion ejection ( e.g. 0.1 qz)
increasing the mass range of the instrument
– over 100,000 dalton singly-charged ions have been
detected
According to the Mathieu equation for stability the q value is related to the m/z,
V(RF), trap geometry, and RF drive frequency. Therefore, there are four ways to
extend the mass range of the trap. One can raise the RF voltage on the ring
electrode, lower the drive frequency, decrease the dimensions of the trap, or
reduce the q value for resonance ejection. Since q is proportional to V(RF) / m,
then increasing m to 4000, while keeping the V(RF) maximum at 20kV, requires
the q value to reduce by 1/2. The beta = 0.33 line crosses the q axis at this point.
This means that the time-varying dipolar field generated across the end-caps must
operate at Omega / 6 for resonance ejection for the higher mass range. Basically,
q(eject-new) = q(eject-old)*m/z(limit-old) / m/z (limit-new).
The effect on resolution is roughly understood when one pictures a larger mass
range having to squeeze into a smaller region of space on the stability diagram.
As a result, resolution is reduced because it becomes harder to distinguish one m/z
value from the next. In addition, the scan speed can also affect resolution.
88
Ion Trap Relationship between Resolution, Mass Range, and Scan
Speed
Imagine that the scan speed is very high. When this happens, m/z values close to
the one being resonantly ejected, do not have sufficient time to relax from the
energy effects of being so close to resonance that they may "slip" out with the
ejected higher m/z before expected. The corresponding spread in signal width
results in a lowering of resolution. Therefore, if one wants to increase resolution,
one must slow the scan speed and / or reduce the mass range.
89
Mathieu Stability Diagram for Non-Linear Resonance Ejection to
Increase Mass Range

Resonance Ejection to Increase Mass Range

Resonance Ejection to Increase Mass Range
Axial modulation to increase mass range

qz of ion ejection about 0.15
Just an illustration of points on the stability diagram corresponding to mass range

limits imposed by resonance ejection according to the endcap dipole frequency
setting.
90
Mass Ranges, Mass Resolution, and Scan Speeds for the Agilent
Trap
Mass Ranges, Mass Resolution, and Scan Speeds

for the Agilent Trap
Mass Ranges, Mass Resolution, and Scan

Speeds for the Agilent Trap
Resolution
Scan Type Range FWHM Scan Speed
Ultra Scan 50 - 2200 Normal 0.6 26,000
Standard 50 - 2200 Enhanced 0.35 8,100
Standard 50 - 2200 Maximum 0.25 800
Extended 200 - 4000 3 27,000
The operational capabilities of the Agilent ion trap making use of various
resonance ejection points and scan speeds to control the mass range and mass
resolution are shown above.
91
Fundamentals of Acquiring a Mass Spectrum on an Ion Trap – Timed
Events
Fundamentals of Acquiring a Mass Spectrum on

an Ion Trap – Timed Events
Fundamentals of Acquiring a Mass Spectrum on

an Ion Trap – Timed Events
• Ions are accumulated from the API Source in the

trap
• The trap is operated in non-linear resonance to eject
ions in a sequential fashion
API Skimmer 2 Trap Detector
10
A schematic graphic of the events occurring to acquire a full scan mass spectrum
on an ion trap is shown above.
92
Time Sequence of Events to Generate a Mass Spectra on an Ion Trap

Spectra on an Ion Trap

Spectra on an Ion Trap
1. Trap clear 2. Accumulation time 3. Scan delay 4. Mass analysis
11
The three most important voltages during the operation of the Agilent ion trap are
illustrated as a function of time for a full scan mass analysis.
Note that the skimmer is set at 300V during the time that the ion trap does not
need anymore ions. Only during the accumulation time, the time spent filling the
trap, is the voltage reduce dramatically, allowing ions generated in the spray
chamber to enter the trap.
In the normal scan range the primary RF applied to the ring electrode begins a
scan cycle at a voltage level design to reduce background chemical noise by
making ions below a cutoff mass of m/z = 50, unstable and, therefore, un-trapped.
During the first step of clearing the ion trap, the RF voltage is zeroed so that no
ions are stable. The cutoff voltage is again applied during the accumulation step
for noise reduction, followed by a ramp in amplitude for scanning the trapped ions
out. Also during this mass analysis step, a time-varying dipole voltage is applied
across the end-caps for resonance ejection of the ions while they are still stable.
The cycle begins again by dumping, or clearing out, the ion trap.
93
Time Sequence of Events to Generate a Mass Spectra on an Ion Trap
Note: The time interval for Mass Analysis is dependent on the mass range and
scan speed settings. For Normal scan speed 13,000 amu/sec, the mass analysis
time interval is equal to 80µsec/amu * mass range (amu). For example, in the
Normal scan speed mode for a mass range of 500 (eg. 100 – 600), the interval
time for Mass Analysis is 80µsec/amu * 500 amu = 400,000 sec = 400 msec.
94
Advantages of Accumulating All Ions Simultaneously in a Trap -
Sensitivity
Advantages of Accumulating All Ions

Simultaneously in a Trap - Sensitivity
Advantages of Accumulating All Ions

Simultaneously in a Trap - Sensitivity
Higher duty cycles (% of the cycle time the ion is

detected) increases the signal/noise
12
The comparison of duty cycle between a quadrupole and an ion trap is shown
above. The higher the duty cycle the better the signal/noise. However the
number of scans is the important consideration, not simply duty cycle.
Increasing the number of scans means more ions. Higher ion throughput means
shorter accumulation times (lower apparent duty cycle) but greater number of
scans.
The combination of a high duty cycle and a large number of scans permits the
detection of a high number of ions resulting in a good signal/noise.
95
Why Control the Ion Accumulation Process
• API produces numerous charged species which are

brought into the ion trap.
• The ion trap can only contain a finite number of

ions during mass analysis due to field effects
created by ions as the ion population increases.
• Exceeding this number of ions (exceeding the space

charge limit) will result in reduced mass resolution,
mass accuracy, and linear dynamic range.
13
Space charge limits are reached when too many ions are stored in the trap. As the
pseudo-potential well of the ion trap begins to fill with ions, the harmonic motion
of the ions begins to be affected. The resonance for ions of each specific m/z
value is spread over a range of frequencies giving rise to broad peaks centered at a
higher mass. For good mass accuracy and resolution the number of ions in the
trap must be controlled.
During an LC peak the number of ions generated by the electrospray source varies
widely. The use of Ion Charge Control (ICC) prevents this problem. The
accumulation time for the background signal or of low intensity peaks is
established by the user at a maximum level, commensurate with the minimum
sampling rate of the chromatographic signal. As a higher intensity begins to
present itself to the ion trap, the resulting ion charge is measured and automatic
adjustments to the accumulation time are made if the maximum allowable charge
level is exceeded. Conversely, when the ion beam current begins to drop as the
LC peak falls, then the length of the accumulation time is increased until the
maximum time is reached.
96
Even for intense LC peaks the accumulation time can be adjusted to prevent
overloading of the trap. The measured intensities using ICC are scaled by the
accumulation time factor so that in the Total Ion Current (TIC) chromatogram and
in the spectra the intensities shown are independent of the actual accumulation
time.
97
Example of What Happens When too many Ions are Accumulated
into the Trap
Example of What Happens When too many Ions

are Accumulated into the Trap
Example of What Happens When too many Ions

are Accumulated into the Trap
Accumulation time 10 ms
Exceeding the Space Charge Limit
14
If the ICC is not turned on then space charging can become a problem if the ion
accumulation time is set too high. The effect of space charging is made apparent
in this slide as the accumulation time increases from 10 to 100 ms.
98
Reducing Space Charging
• Space charging –too many ionic species inside the

trap distorting the electric fields and impairing the
trap performance.
• Most of these ionic species could be matrix or

solvent background.
• Space-charge effects can be reduced by:

– Sample clean-up
– Chromatography
– API-ion trap instrumentation.
15
The ion trap can only hold so many ions. To be more specific, it can only hold so
many charges (z's). This limiting condition is due to what is called space-
charging. If the trap overfills and the ions get too close to each other, then their
similar charges will cause mutual repulsion. Compound this effect with the
screening of one ion from the ion trap field by the presence of the electron cloud
of a nearby ion, and control of the ions will be lost. One observational effect is
loss of resolution: when the rings electrode scan the ions out of the trap, some
ions leave early while some leave late, increasing the peak width.
Space charge effects can be reduced by additional sample clean-up or by better

chromatography. However, the use of instrumental design and control of various
potentials allows the operator to limit the charge in the trap to maintain acceptable
mass spectrometry performance.
99
API – Ion Trap Methods to Reduce or Remove Space-Charge Effects
API – Ion Trap Methods to Reduce or Remove

Space-Charge Effects
API – Ion Trap Methods to Reduce or Remove

Space-Charge Effects
• Ion Charge Control (ICC) : limits the total charge in the

ion trap by adjusting the accumulation time to keep a
constant level of ions in the trap.
• Ion Isolation: allows only one m/z ion to be stored in the

trap after accumulation.
• Orthogonal Spraying: reduces the sampling or charged

droplets from the electrospray ionization process.
16
The three main ways space charging can be controlled on the ion trap are shown.
Each method limits charges that enter into the trap as explained in the following
pages.
100
ICC to Remove Space Charge Effects
ICC Time adjusted inversely to TIC

In te n s.
x 1 0 7
1 .2
1 .0
ICC
Time
0 .8
TIC
0 .6
0 2
L sb 0 0 0 0 7 .d : T IC , A ll ±
4 6 8 1 0
L sb 0 0 0 0 7 .d : V a ri a b l e
1 2
T ra c e , A c c u m u la tio n
1 4
T im e
T im e
(T ra p )
[m in ]
0 ms
17
To eliminate space-charging, a bias of 300V is applied to the second skimmer,

after a certain period of trap filling, to prevent anymore ions from entering and
overfilling the trap. This effect is known as "gating." The period of time is either
set by the user, and is known as "ion accumulation time," or is controlled
automatically through software known as "ion charge control.” In the manual
mode the user must set the ion accumulation time and for each scan the trap will
collect ions for that length of time designated. This mode is not usually
recommended because if the accumulation time is set too low, then low ion
populations in the trap result in poor ion statistics. If the accumulation time is too
high, then the trap overfills, resulting in space charging, or loss of resolution and
mass shift.
Ion charge control is a mathematical algorithm used to monitor the signal

generated from a mass scan with respect to the maximum amount of signal
obtainable from the ion trap when it is optimally full. If the signal from one scan
is low with respect to the maximum, then the accumulation time will be increased
on the subsequent scan. In this mode, the user sets both target value and the ion
accumulation time. However, the ion accumulation time is actually a maximum
and the target value corresponds to the number of ions to trap from scan to scan.
101
This automatic mode is preferable because the number of ions in the trap is kept
the same from scan to scan and the accumulation time is varied to maintain this
population. The ion accumulation time is a maximum and will not be exceeded.
While this setting can prevent space charging, it can also adversely affect linearity
or lack of data points across an LC peak.
An interesting note, and hopefully not too confusing to the reader, is the fact that,
in automatic mode, the total ion current (TIC), in it's unscaled form, is actually a
constant with respect to time. That is, the same number of ions are accumulated
in the trap from scan to scan. What differs from scan to scan is the amount of
time, or accumulation time, it takes to fill the trap. When the TIC is adjusted with
respect to this accumulation time, the TIC trace of the chromatogram is what the
operator observes.
102
Ion Isolation to Exclude all but the Desired m/z Values in the Ion Trap
Ion Isolation to Exclude all but the Desired m/z

Values in the Ion Trap
Ion Isolation to Exclude all but the Desired m/z

Values in the Ion Trap
• Ion Isolation occurs after accumulation but before mass
analysis
18
Ion traps have the ability to directly eject all ions simultaneously except for the
precursor ion of interest during ion accumulation. Because each particular mass
has its own specific resonance, it is necessary to synthesize a wideband composite
of frequencies in order to excite and eject a broad mass range of ions. When it is
necessary to isolate a particular precursor ion, the electronics system on the ion
trap generates a broadband frequency spectrum with all resonating frequencies
present except for the frequency corresponding to the resonance of the precursor
ion.
103
Orthogonal Spraying Reduces the Sampling of Charged Species in
the Trap
Orthogonal Spraying Reduces the Sampling of

Charged Species in the Trap
Orthogonal Spraying Reduces the Sampling of

Charged Species in the Trap
5000 V Atmosphere
N nebulization ++
2
+ +
+ ++ ++ ++
- Charged
++ + + Residues
LC ++
+ + +
+
++
+ +
+ +
+
Drying
+ Gas N 2
+ +
+ ++
+ +
+ +
300°C Sampling
+ + + +
+ + + + + Capillary
+ ++
+ +
+ Ion Charged Residue
(noise) Vacuum
Evaporation
19
Pneumatic nebulization off axis from the sampling capillary reduces the charged
droplets that can be pulled into the transport region and enter the trap. These
charged droplets can contain 100’s of charges that can quickly degrade trap
performance and limit analyte dynamic range. Spraying at right angles can
reduce the droplet current by over a factor of 50 while maintaining a high analyte
ion flux into the system.
104
Tandem MS On and Ion Trap
Tandem MS On and Ion Trap
Tandem MS on an Ion Trap
API Skimmer 2 Trap Detector
20
The schematic above shows the events necessary to acquire a MS(2) (MS/MS)
mass spectrum on an ion trap. The main difference from full scan operation is
that the accumulated ions are isolated by changing the RF voltage on the ring-
electrode as it is scanned to move the lower mass ions out of the stability region
of the Mathieu diagram (out of the trap). The remaining higher mass ions are
ejected by applying a notched waveform, or superposition of frequencies
excluding the one corresponding to the precursor ion’s, across the endcaps. Once
isolation of the precursor ion is achieved, the RF voltage is lowered to lower the q
value of the ion. This is necessary in order to trap the subsequent fragment ions.
The other is a step to perform collision induced decomposition (CID),

accomplished by applying a dipole voltage across the end-caps as it resonantly
excites the ion into colliding with the helium atoms to induce fragmentation.
105
Time Sequence of Events to Generate a MS/MS Mass Spectra on an
Ion Trap
Time Sequence of Events to Generate a MS/MS

Mass Spectra on an Ion Trap
Time Sequence of Events to Generate a MS/MS

Mass Spectra on an Ion Trap
1. Trap clear
2. Accumulation time
3. Isolation delay
4. Ion isolation
5. Fragmentation delay
6. CID fragmentation
7. Scan delay
8. Mass analysis
21
The scan process for MS/MS is nearly identical to that for MS. An important
exception shown in both the primary and auxiliary voltages is that before the mass
scan, isolation of the precursor ion must be performed. Note that the voltages of
both the ring electrode and the end-caps are raised to knock out all ions above the
m/z of the precursor ion. In addition, though not shown, a mixture of frequencies,
except for the one corresponding to resonance of the precursor ion, are applied to
the end-caps. This technique ejects all of the ions trapped except for the
precursor. This operation is the same for selected ion monitoring (SIM).
In addition, a voltage across the end-caps is applied during the fragmentation to
induce the ions to collide with the helium atoms also resident in the trap.
For analyses of MS higher than MS/MS, one simply repeats the isolation stage
following fragmentation as many time as needed, until the final fragmentation
products are scanned out of the trap.
106
Collision-Induced Decomposition (CID) Variables for Traps
Collision-Induced Decomposition (CID) Variables

for Traps
Collision-Induced Decomposition (CID)

Variables for Traps
• Fragmentation Voltage: Voltage applied to end caps
(usually ~ 1 V) at the resonance frequency of the isolated
ion to increase ion kinetic energy
• Fragmentation Time: Length of time the fragmentation

voltage is applied (usually ~ 20ms)
• Collision gas: Helium is the collision gas
• Cut-off mass: rf amplitude on ring electrode that traps the

ions ( usually ~ 0.3 of ion ejection point in the Mathieu
stability diagram)
22
To induce fragmentation, the energy of the ion of interest is increased by

resonance excitation with the dipole field. The resonance excitation waveform
actually comprises a small frequency band above and below the precise resonance
frequency to increase the stability of the excitation, as well as to compensate for
the frequency shift with amplitude of the ions’ motion due to the non-linear field
components. The amplitude of the excitation is less than that used for ejection
and is typically about 1 volt. The resonating precursor ions quickly take up energy
from the dipolar field and begin to collide with the helium background gas which
causes them to dissociate creating predictable and reproducible mass spectra. The
time associated with MS/MS excitation and collision induced dissociation can be
varied by the operator, but is typically 20 ms - 60 ms.
Upon fragmentation of the precursor ion, the product ions need to experience a
trapping potential from the quadrupolar field. This is determined by the primary
RF drive level during the fragmentation. If the primary RF drive level is too high
then low mass product ions will be ejected from the trap. If the RF drive level is
too low then the pseudo-potential well will be insufficient to allow for effective
107
Collision-Induced Decomposition (CID) Variables for Traps
MS/MS excitation. Typically, the low mass cutoff is set at slightly less than 1/3
the precursor m/z value (default mode).
There is one major difference in the operation of MS/MS excitation on an ion trap
and on a quadrupole instrument. In quadrupole MS/MS, ions are excited by
accelerating them and passing them through a high pressure collision cell.
Because not all momentum is necessarily lost in the first collision, subsequent
collisions with the background gas can result in further MS/MS product ions. The
fragmentation is not mass-selective. In ion trap MS/MS, only the precursor ions
are activated by several collisions with the background gas, but upon
fragmentation little energy remains in the product ions that would result in
subsequent fragmentation. Because the product ions a re not resonated by the
dipolar frequency, they do not continue to be excited. The result is that MS/MS
spectra in quadrupole instruments may in fact contain product ions that are the
result of 2 or more stages of MS/MS excitation and fragmentation, while the
MS/MS spectra from ion trap instruments are generally the result of a single stage
of excitation which explains why ion trap MS/MS spectra often are clearer.
With the trap now storing product ions the RF drive level can be ramped as
described previously to produce a full mass spectrum, or an additional stage of
MS/MS isolation and fragmentation can be initiated. Optimizing MS/MS
experiments on the ion trap is simple. The main variables are the width of the
isolation window (the Agilent 1100 LCMSD Trap can perform mono-isotopic
isolation throughout the standard mass range form 50 m/z to 3000 m/z), the depth
of the pseudo-potential well during the fragmentation (referred to in the software
as the cutoff during the fragmentation), the fragmentation amplitude and
fragmentation time.
108
Adjusting Fragmentation Voltage to Maximize CID
Adjusting Fragmentation Voltage to Maximize

CID
Adjusting Fragmentation Voltage to Maximize

CID
• Smart Fragmentor linearly ramps the fragmentation voltage across a
voltage range (e.g. 0.3 – 3 V) over the fragmentation time (e.g. 20
ms) to insure all ions undergo CID (note fragmentation is fast relative to
the voltage ramp so ions will not be ejected prior to undergoing fragmentation)
Voltage too high - ions
are ejected prior to CID
Relative intensity Voltage too low - ions

do not gain kinetic
energy to undergo
fragmentation
Fragmentation Voltage
23
The fragmentation amplitude is probably the most critical parameter to adjust for
obtaining CID mass spectra on an ion trap. If the voltage is too low, only parent
ions are observed. If the voltage is too high then the parent is ejected from the
trap prior to fragmentation. Each ions fragmentation pathway has its own
activation energy, therefore one value will not be suitable to analyze all
molecules. This limitation is overcome through the use of “smart fragmentor”
which linearly ramps the fragmentation voltage across a voltage range (e.g. 0.3 –
2 V) over the fragmentation time (e.g. 20 ms) to insure all ions undergo CID. The
CID fragmentation is fast relative to the voltage ramp so that parent ions that have
enough internal energy to overcome an activation barrier will produce product
ions and not be ejected prior to undergoing that fragmentation. This approach
also can result in a CID mass spectra that contains more product ions since it is
more likely that more activation barriers for fragmentation will be exceeded prior
to ion ejection.
If “smart Frag” does not produce product ions, than the user should adjust the
cutoff mass as described on the next page. Fragmentation time has little effect on
109
Adjusting Fragmentation Voltage to Maximize CID
the extent of fragmentation in the 20-60 ms time frame and can usually be left in
the default setting.
110
Why Adjust the Cutoff Mass For CID?
• Increasing the cutoff mass increases the trapping fields on the ions,
allowing for higher fragmentation voltages (higher kinetic energies) to
be applied enabling fragmentation of stable ions.
Fast Cal setting

(automatic choice for qz )
24
The Agilent Trap sets the cutoff mass (rf amplitude on the ring electrode) to 27%
of the parent ion mass as the default setting. However, this value can be manually
adjusted. As the cutoff mass is reduced, the lower end of the mass range is
extended and the depth of the potential well, trapping the parent ion, gets more
and more shallow. As a result, when energy is added to the precursor ion to cause
it to fragment, the precursor ion is more susceptible to being ejected from the trap
before ever fragmenting. On the other hand, as the cutoff mass is increased the
lower mass range is reduced but the potential well is stronger, allowing for more
energy to be imparted into the ions for fragmentation before the parent ion is
knocked out of the trap. This is necessary to obtain CID fragments from
resonance stabilized ring system molecules. Lower mass information can be
obtained using multiple stages of MS – MS(n).
111
MSn Capabilities on an Ion Trap
• What is MSn ?
– It’s a way to denote multiple ion isolation and
fragmentation steps to yield a product ion mass
spectrum.
– n = the number of isolation and fragmentation steps
minus 1 (the last step is a full scan).
• e.g. MS3 means

– 1) isolation of a parent ion and fragmentation of that
ion.
– 2) Isolation of a product ion from the previous
fragmentation and fragmentation of that ion.
– 3) full scan mass analysis.
25
For analyses of MS (n) where n is greater than 2 (higher than MS/MS), one
simply repeats the isolation stage following fragmentation as many time as
needed, until the final fragmentation products are scanned out of the trap. The
Agilent system can perform up to n=11.
112
Schematic of MS(3)
Schematic of MS(3)
Schematic of MS3
Accumulation
Ion isolation A+
#1
Fragmentation
#1
A 1+ A 2+
MS1
A 3+ A 4+
Ion isolation
#2 A 2+
MS2
Fragmentation A11+ A21+
#2 A31+ A41+
Mass Analysis A11+ A21+ A31+ A41+ MS3

( #3)
26
Above you will find the schematic representation of the timed events in the ion
trap for MS(3).
113
Example of MS5
Example of MS5
Example of MS5
27
An Example of MS(5) for the structural characterization of an impurity in an anti-

cancer drug by positive ion electrospray ion trap MS is shown.
114
When to use MSn and What Should One Use for the Value of n
When to use MSn and What Should One Use for

the Value of n
When to use MSn and What Should One Use for

the Value of n
• Often low energy CID in a trap leads to one rearrangement product ion
(e.g. loss of H2O), therefore, additional stages of MS/MS can offer more
structural information and specificity.
• n can be from 2-10 on the Agilent Trap (record is 17 for a trap). (what
was the product ion spectrum after MS17 ?)
• If there are numerous product ions formed after each stage of MS/MS
going beyond n=4 can result in significant loss of sensitivity. Remember
the charge will not exceed the initial parent ion. The more product ions
formed the further the initial charge is divided.
28
When applying MS(n), one must balance the desire for sensitivity and structural
information with the need for acquiring enough data points to define a
chromatographic peak. Often samples that produce single ion CID spectra (e.g.
loss of water) are good candidates for n>3. If the MS(2) shows numerous product
ion, going to higher values of n will rapidly decrease sensitivity.
When going to n>4 care must be taken to adjust the number of averages to insure
enough data points are taken across each peak. Each stage of isolation and
fragmentation can add 100ms to the cycle time. For example, if the averages
selected was 10 and accumulation time was 50ms, an MS(5) experiment will
record one spectra every 5.7 seconds which will result in too few points across a
standard 15-20 second wide chromatographic peak. The number of averages
should be reduced to about 4.
115
Loss of Signal Level at Higher Stages of MSn
n
Loss of Signal Level at Higher Stages of MS
Loss of Signal Level at Higher Stages of MSn
Assuming each fragmentation step

results in 3 ions of equal intensity
29
The example above shows the theoretical sensitivity loss as a function of n

assuming each CID step results in 3 equal intensity product ions.
116
Additional Sources of Information on Ion Traps
30
Here are some recommended readings for additional information on ion trap MS.
117
118
Basics of LC/MS/MS
Basics of LC/MS/MS
In This Section
In This Section
• Transport CID
• Tandem MS
• MSn on the Ion Trap
120
Basics of LC/MS/MS
What is Collision Induced Decomposition (CID)?
A process where energy is transferred to an ion through

collision with neutral molecules. The energy transfer is
sufficient to result in bond cleavages and rearrangements of
the selected ion.
Why is it Important?
In the early 70’s McLafferty (JACS, 95, 3886, 1973) demonstrated
the bond cleavage and rearrangements observed for the ion that
undergoes CID is representative of the molecular structure of the
neutral molecule.
The process of CID is defined and the ions observed from the CID fragmentation
are related to the original structure of the species selected for CID fragmentation.
Typically the CID collisions are with a non-reactive gas like He, Ar, N2, or Xe.
121
Basics of LC/MS/MS
CID Reactions
CID Reactions
CID Reactions
CID Reaction energies are shown below:
ε
# q’ = amount of energy converted
from translational to internal from
q' the CID process
Energy
εa
ε’ =excess energy
ε
reactants products εa = activation energy

Reaction Coordinates
Total internal energy = ε + q’
ε = reactant initial energy
The CID process is like any organic reaction where the reactants have to
overcome an activation barrier to produce product. Any collision in excess of that
activation barrier will carry that energy to the products. Every organic molecule
has its own fragmentation activation barrier. The ions that are observed in a CID
mass spectra represent the pathways that are below q’. The higher the collision
energy the more activation barriers can be overcome, thus more product ions are
observed.
122
Basics of LC/MS/MS
CID in API Transport Region

• What is it?
Using electric fields in the API-MS transport region to collisionally
activate ions at a mid-pressure region (~0.01 - 1 torr) to generate
CID mass spectra.
HARDWARE:
M1
+
+ M + + - + - +
M a M M M M
a 2 1 2 3
+
M
M 3
a
M M
b C ID
a
M
c LC C a p il l a r y Q
A P I Ch am be r S k im m e r
CID can occur in the transport region of all API-MS instruments. The CID
energy is controlled by the capillary exit voltage, the higher the voltage the higher
the collision energy. The collisions are with nitrogen, introduced in the
nebulization and drying process in the API interface. All ions that enter the
system undergo CID, therefore it is important to have good chromatographic
resolution to minimize contributions in the product ion spectrum from other
compounds.
123
Basics of LC/MS/MS
CID in API Transport RegionCon’t
CID in API Transport RegionCon’t
CID in API Transport Region Con’t
• Characteristics of API Transport CID

– N2 drying gas serves as collision gas
– Collision energy controlled by the potential difference from capillary
exit (controlled by adjusting the capillary offset voltage) to skimmer. Usually
skimmer voltage is constant, so collision energy is directly proportional to
fragmentor voltage.
– N2 pressure is set by interface design. Ions undergo multiple collisions
over the 3-4 mm distance between capillary exit to skimmer. Typically
there could be hundreds of collisions. Multiple collisions can impart
more energy and reduce losses due to scatter.
– The Capillary exit voltage also effects ion transmission. Typically the capillary
exit voltage is increased as the m/z values increases to maximize ion transmission
(without fragmentation). The capillary exit voltage that yields CID fragment ions
depends on the m/z of the ion and the ion stability. Higher m/z values and more
stable ions require higher voltages.
The characteristics of API Transport CID are discussed above.
124
Basics of LC/MS/MS
Advantages/Disadvantages of CID in Transport Region
Advantages/Disadvantages of CID in Transport

Region
Advantages/Disadvantages of CID in Transport

Region
• Advantages
– Near 100% efficiency sensitive
– Simple (do not need to know in advance what ions are present)
– Wide range of energies fragments can be produced from even the
most stable compounds
– Similar CID spectra to those obtained by tandem MS
– Cost Less expensive than tandem MS
• Disadvantages
– Impurities or coeluting peaks will result in complex spectra
– Must have chromatographic resolution Increased analysis time
– Lack of libraries manual interpretation
Several advantages of transport CID include ease of use, rapidity, and sensitivity
(few CID product ions are lost during the collision process due to the relatively
high pressure between the capillary and skimmer, minimizing losses due to
scatter).
The disadvantages include chemical noise that must be removed by the LC

separation. The separation can be time consuming and less efficient than a
separation based on both LC and m/z (leading to the technique of MS/MS).
125
Basics of LC/MS/MS
CID in ES Transport for Sulfamethazine
25
20
Peak Height
15
Sum of Parent Peaks

Sum of Product Peaks
10
0
0 80 150 180
This example shows the product ion signal increase (loss of the molecular ion
signal) as the capillary voltage is increased. Note the total ion current remains
quite high (>80%) due to minimization of ion losses due to scatter in the high
pressure region between the capillary and skimmer
126
Basics of LC/MS/MS
What is Tandem MS?
What is Tandem MS?
What is Tandem MS?

A process in which an initial mass analyzer selects a parent ion or precursor
(from all ions present in the system) for CID. The product ions formed
are then mass analyzed by the second mass analyzer. Tandem MS can
be tandem in space (triple quadrupoles, sector instruments) or tandem
in time (ion trap instruments).
Definitions:
• Experiment performed using Tandem instruments are termed MS/MS
experiments
• Product Ions
fragment ions formed after CID (same term is used for in source and
tandem CID experiments)
• Parent Ion or Precursor Ion
ion selected by initial mass analyzer for CID (this term applies only to
MS/MS experiments)
The definition of tandem MS as well as, parent and product ions is presented
above.
127
Basics of LC/MS/MS
Tandem MS in Space- Triple Quadrupoles
What is it?
A method that uses the first mass analyzer to select a particular m/z for introduction into a
collision chamber. The selected ion undergoes CID with a gas (e.g., argon) in the
chamber and product ion (fragments) are mass resolved by a second mass analyzer
HARDWARE: M1+
Ma + M a+ M 2+ M 1 + - M 2 + - M 3+
M 3+
Ma + Mb +
Mc + M d + Q1 Q2 Q3
(Collison
API Cham ber) Detector
10
The schematic operation of a triple quadrupole (tandem in space) is shown above.

In this example a mixture of components (a-d) is in the API source, but is
resolved based on m/z of the parent ion selected by quadrupole 1. The CID
product ion spectra is specific for the structure of the parent ion selected (Ma).
This approach can offer great improvements in sensitivity, specificity, and speed
(MS based separations occur in ms time compared to minutes for
chromatography) relative to conventional LC/MS, especially when the analysis is
limited by chemical noise. However, the LC separation cannot always be
removed. The separation can remove salts and matrix components that suppress
API. MS/MS does not offer any benefit to the analysis if the initial parent ion is
not formed.
128
Basics of LC/MS/MS
Tandem MS in Time – Ion Traps
11
Ion trap MS/MS is performed in one analyzer in a sequenced program of events

described below:
1). Ion accumulation: Initially the trap is loaded with ions formed by API of the
LC effluent, until a maximum charge level or time has been reached.
2). Isolation: Next voltages and frequencies applied to the end caps and ring
electrode keep only one m/z ion stable inside the trap.
3). CID: The isolated ion is resonantly excited by applying the resonant frequency
to end caps for the ion to gain energy for CID with the helium bath gas inside
the trap. The resulting product ions which are of different m/z, do not have the
same resonance frequency, are stored inside the trap. [ note step 2 and 3 can be
repeated again for MS3 or n-1 times for MSn ].
4). Scan: The resulting product ions are mass analyzed by scanning the voltage on
the ring electrode.
129
Basics of LC/MS/MS
Characteristics of Tandem MS
– Ions are mass selected prior to CID

– Operator chooses collision energy, collision gas and collision gas
pressure (3 factors which require optimizations)
– Mass analyzers can be linked to perform numerous types of
MS/MS experiments
• product ion scan
• neutral loss scan
• precursor ion scans
• multiple reaction monitoring (MRM)
– Typical complexity far exceeds CID in API transport region
– CID conditions can influence MS resolution and transmission
12
Above, A list of tandem MS’s key characteristics is provided.
Tandem in space instruments (triple quadrupoles) can perform numerous MS/MS

experiments including:
Product ion - first mass analyzer chooses a parent ion for CID while the second
scans to detect the product ions.
Neutral loss – first and second mass analyzers scan at the same speed but the mass
of analyzer two is reduced by the mass of the CID neutral loss.
Precursor ion - first mass analyzer scans while the second analyzer is fixed on a
characteristic product ion formed by CID. The spectrum will give the m/z of the
ion that produces a particular product.
MRM – The first and second mass analyzers are fixed to a specific parent –
product ion combination characteristic of the compound of interest. This mode of
operation results in the best sensitivity due to the high % of time the signal is
detected compared to a scanning experiment.
Tandem in time instruments (ion traps) can only perform product ion scans.
However the ion selection –CID process can be repeated over and over again to
allow for MS(n) operation.
130
Basics of LC/MS/MS
Advantages/Disadvantages of Tandem MS
• Advantages
– Chemical specificity (components do not need to be
chromatographically resolved)
– Structural elucidation
– Reduce analysis times
– Sensitivity (gain in S/N through noise reduction)
– Chemical background is reduced by parent ion selection
– Screening: (neutral loss, parent ion scans)
• Disadvantages
– Cost
– Interpretation of spectra
– Complexity
13
Advantages and disadvantages of tandem MS are presented. The advantages of

tandem MS center around the rapid process of isolation and CID to generate
specific product ions free from chemical noise. The disadvantages are the cost
and instrument complexity to perform the MS/MS experiment. The interpretation
of the spectra while complex, may be easier than the interpretation of spectra
generated by transport CID in the API source due to the reduction of possible
interferences from co-elution achieved through parent ion selection in MS/MS.
131
Basics of LC/MS/MS
Increase in S/N with Increasing Stages of Analysis –Removal of
Chemical Noise
Increase in S/N with Increasing Stages of Analysis

–Removal of Chemical Noise
Increase in S/N with Increasing Stages of

Analysis –Removal of Chemical Noise
14
This is a representation of the improvement in signal/noise as the number of

filters (LC, UV, or MS is considered a filter) increase, when the analysis is
chemically noise limited. If the analysis is not limited by chemical noise, then
additional filters will only reduce the signal and not the noise, resulting in a
reduction in signal/noise.
132
Basics of LC/MS/MS
CID on an Ion Trap - What is MSn
15
The definition of MS(n) processes that can occur in tandem in time instruments
(ion traps) is presented above.
133
Basics of LC/MS/MS
ID on an Ion Trap - MSn
ID on an Ion Trap - MSn
CID on an Ion Trap - MSn
Why Use It?
•Additional structural information especially when all the

ion current from MS2 forms low information product
ions from the losses of H2O, CO2, etc.
16
Often n values of 3 or more are needed since the CID process in an ion trap is
very low energy and energy input into an ion is slow (ms vs µs in a triple
quadrupole). Sometimes, only simple losses that exceed the activation are
detected (such as the loss of CO2 or H2O). Often these simple losses consume all
of the excited parent ion current before the activation barrier of a higher energy
fragmentation process is met through continued excitation in the ion trap (rate of
fragmentation far greater than the rate of internal gain from the first to second
fragmentation activation barriers. Because of this fact, it is common to detect
single ion product ion spectra on an ion trap.
134
Basics of LC/MS/MS
CID on an Ion Trap – Example of MS3
17
The above example illustrates the use of MS(3) to gain additional information
other than the loss of H2O observed in MS(2) experiment from the [M+H]+ ion
at m/z 240. The ion at m/z 148 in the MS(3) spectrum is characteristic for the
class of drugs being analyzed.
135
Basics of LC/MS/MS
CID on an Ion Trap – Why Not MSn
18
The use of n>4 in MS(n) might not be useful if multiple product ions are formed
after each stage of MS. The ion current is quickly divided after sequential stages
of MS reducing sensitivity by 10-100 fold.
136
Basics of LC/MS/MS
CID on an Ion Trap – Losses in Signal with MSn
19
The chart above shows the sensitivity loss assuming each stage of MS forms 3
ions of equal intensity.
137
Basics of LC/MS/MS
138
LC/MSD TRAP Sample Introduction and
LC Method Conversion
LC/MSD TRAP Sample Introduction and LC Method Conversion
In This Section
In This Section
• Sample preparation.
• The different sample introduction modes used in

API-MS.
• Converting HPLC methods to the LC/MS.
* More detailed information about

electrospray and APCI mobile phases
and methods appear in the module:
Solution Chemistry for ES and APCI.
Gaining an understanding of the sample introduction techniques and requirements

of the LC/MSD will improve your analyses.

• About sample preparation.
• The different sample introduction modes used with API-MS.
• Important considerations when converting HPLC methods to API detection.
Note: More details of ESI and APCI mobile phases and additives are discussed in
the module: Optimizing ESI and APCI Analyses.
140
Sample Information and Considerations

• Obtain as much information as possible about
the sample
• Origin, quantity, solubility, stability, storage
conditions
• Molecular structure, pK, pI
• Use negative ion ES mode for: acids,
hydroxyls, phosphates, sulfates
• Use positive ion ES mode for: amines, amides,
antibiotics, etc.
• Use correct introduction mode for your analysis:

infusion, FIA, chromatography
• Start with existing chromatography and modify if

necessary
3
API-MS can be a very sensitive and accurate sample evaluation and quantification
tool. The analyst, however, must understand the boundaries and considerations of
this technique in order to become successful.
First, find out as much as possible about the sample. For instance, is the sample
relatively pure and therefore amenable to flow injection analysis or infusion?
Alternatively, is the sample a mixture requiring liquid chromatography?
Knowledge of the sample structure helps the analyst decide which mode will be
suitable for the sample. Electrospray is enhanced by formation of ions in
solution. The sample pK will help determine the proper solution pH to maintain
ions in solution. APCI can handle more non-polar samples.
141
Sample Preparation
Sample Preparation
Sample Preparation
Why?
Often API-MS techniques fail due to inadequate sample preparation
resulting in signal suppression or common ion interferences. The main
issues that need to be considered prior to API-MS, that can mean the
success or failure of the analysis are:
• salt
• matrix components
• concentration
Methodology
The main sample preparation methods include:
• ultrafiltration
• solvent extraction/desalting
• liquid-liquid extraction
• solid phase extraction (SPE)
• immunoaffinity
• on-column concentration
• column switching (LC/LC)
Often, sample preparation or the lack of sample preparation can have serious
affects on the API-MS analysis.
The presence of salt in the sample can lead to the formation of adduct ions such as
[M+Na]+ or [M+K]+. Abundance of the various adducts may depend on the
concentration. The overall effect of the adducts is to dilute the abundance over
many ions. A SIM analysis could be destroyed by their presence. Think about
the difficulty you would experience trying to decide the molecular weight of an
unknown when you are not certain of the adduct species present.
Matrix components, especially mobile phase additives can suppress the signal in
both electrospray and APCI modes. In electrospray, a mobile phase additive of
opposite charge may form a neutral species with the sample causing the
electrospray ionization process to cease. Improper mobile phase or additive
selection in APCI can cause the ionization of the mobile phase or additive rather
than the sample.
Sample concentrations that are too high can cause the appearance of dimers.
Many sample preparation techniques currently exist. The list above can give you a
good idea where to start. At the very minimum, do the following. First, if the
sample is a powder or solid, dissolve in a solvent and use a micro filter and
microcentrifuge to separate any solids from the supernatant. If the sample is a
142
Sample Preparation
liquid, use a guard column, LC column, or solid phase extraction cartridge to

wash the sample with water to remove salts and other water-soluble materials
prior to eluting in to the API-MS.
The 1100 Series LC/MSD Reference Collection CD-ROM (part number 5968-
0486E), can help you with your sample preparation needs.
143
Inlet Modes Defined
Inlet Modes Defined
Inlet Modes Defined
Infusion
• Continuous stream of sample
Flow Injection Analysis (FIA)

• Sample injected but not separated
Separations
• Sample separated by HPLC
• Sample separated by capillary or nano HPLC
• Sample separated by capillary electrophoresis
There are three general sample introduction for API-MS. The choice depends
upon the sample purity and type of data required.
144
Infusion Hardware Setup
Simple Infusion with Syringe Pump
Flow <25 µL/min
API-ES Source
Syringe pump
1 2
Nebulizer
6 3
5 4
Syringe pump
or
low flow LC Pump LC inlet 2 3
1 4
Sample Volume: >10 µL 6 5
Sample Concentration: >1 pmole/ µL

Waste Needle port
Infusion with Manual Valve
Infusion is the continuous delivery of sample from a pump, particularly a syringe

pump, or from a manual injection valve. Typically, a simple, inexpensive syringe
pump is utilized.
Infusions can provide high sensitivity due to their ability to collect data with slow
scan speeds over a long period. The scans can be averaged to increase the signal-
to-noise ratio. The solvent composition can be optimized for the sample
components ionization. An infusion process is used during tuning and calibration.
Infusion is not always the answer. Some of the drawbacks include the following.
Infusion requires a pure sample or simple mixtures of less than five components.
Sometimes the components of a mixture can cause ion suppression. The sample
volume has to be large due to the long injection cycle. This type of sample
introduction is not easily automated.
145
Infusion Data
Infusion Data
Infusion Data
Infusion is a steady state introduction of sample. Useful data is present

throughout the run. Spectra can be averaged across the entire time range. If the
sample introduction is from the manual injection valve, the baseline will rise as
the sample enters the mass spectrometer. A steady state introduction occurs until
the entire sample is gone from the valve and the signal returns to baseline. Here,
depending upon the flow rate and injection loop, you will also have several
minutes of data to analyze.
146
FIA Hardware Setup
FIA Hardware Setup
FIA Hardware Setup
Flow: Manual FIA Manual Injection Valve

Internal loop for < 1 µl
200 - 1000 µL/min External loop 1 - 50 µl
Syringe API-LC/MS
Pump or LC Source
Sample Volume: Automated FIA

1 - 5 µL
LC autosampler API-LC/MS
Sample Concentration: Source
>1 nmole/µL
FIA, Flow Injection Analysis, is the analysis of a sample, injected into a flowing
stream, without performing a separation.
There are two modes of FIA, manual and automated. Both types of FIA consume
little sample and are very useful for analysts who are sample limited. Manual FIA
can be accomplished with the optional injection valve and a syringe or LC.
Automated FIA is used to run single pure samples and simple mixtures with
greater speed and automation than infusion. Quantification of a pure component
is possible because integratable peaks are produced.
Like infusion, the sample must be pure to obtain information. Complex matrices
can interfere with deconvolution and ionization. Infusion is more sensitive than
FIA because of the increased acquisition time.
147
Conventional HPLC Hardware
Most Common Introduction Method
HPLC 2.1 or 4.6 mm Nebulizer API-LC/MS

Column
Flow Sample Type

• ESI: 50 - 1000 µL/min • Volume >1µL
(concentration sensitive) • Concentration limited by
• APCI: 200 - 1500 µL/min column capacity
(mass sensitive) • High complexity
HPLC is required for the API analysis of mixtures. Because the electrospray
nebulizer can be pneumatically assisted, 2.1 mm i.d. to 4.6 mm i.d. columns are
utilized with flow rates up to 1 mL/min. APCI and APPI can handle flow rates of
up to 1.5 mL/min. The process is easily automated and conventional liquid
chromatographs can be used without splitting.
148
Column Selection for LC/MS Applications

Electrospray APCI -
Operates from 1 - 1000 µL/min Atmospheric Pressure Chemical Ionization
Compatible with:
• 1.0, 2.1, 3.0and 4.6 mm i.d. columns • Operates from 200 - 1500 µL/min
• 2.1 mm i.d. column is popular choice Compatible with:
• Concentration sensitive • 4.6, 3.0, and 2.1 mm i.d. columns
• Compatible with reversed-phase • 4.6 mm i.d. column is popular choice
solvents • Not concentration sensitive

• Use with reversed- and normal- mobile
phases
10
The LC/MSD operates within a given flow rate range:

Standard ESI – 0.005 mL/min to 1.0 mL/min
APCI – 0.2 mL/min to 1.5 mL/min.
Flow rate, required sensitivity(especially when sample limited), and sample
capacity will determine the chosen column diameter for a particular method. The
LC/MSD is well suited for 2.1 to 4.6 mm i.d. columns. As electrospray is
concentration sensitive, the 2.1 mm i.d. columns are often preferred. Assuming
two columns of different internal diameters have the same efficiency, then a peak
eluting with the same peak width in time on both columns will have a smaller
peak volume on the smaller diameter column. The concentration gain of the
smaller column is given by the square of the ratio of the column diameters. In
APCI mode, 4.6 mm i.d. columns are often chosen. This source is not
concentration sensitive. Therefore, larger injections will give more signal. The
4.6 mm i.d. column can handle at least 10 times the injection volume of a 2.1 mm
i.d. column without loosing resolution. The 4.6 mm i.d. column is also more
compatible with the higher flow rates of APCI.
149
Why -Flow Rate Separations?
Why µ-Flow Rate Separations?
Why µ-Flow Rate Separations?
•Minimize the consumption of sample and solvents

•Maximize the chromatographic peak concentration for a given sample size
•Improve peak resolution and specificity
Column (mm) Flow Rate Peak Volume Normalized (1)

(µL) Concentration
100 x 4.6 1000 360 1.0
100 x 2.1 250 75 4.8
√ 100 x 1.0 50 17 21.2

√ 100 x 0.32 4 1.7 206.6
√ CE (50 x 0.05) 0.03 0.002 180,000 (Ideal)
0.15 80 (2)
(1) Assumptions: Same volume and amount injected (1 mL x 10 NG/mL). If not sample limited peak concentration can
be increased by injecting a larger quantity, based upon column capacity.
(2) Note: Layered flow is required for electrospray, diluting sample. Also, 30 nL is typical CE injection volume.
11
When the same volume and concentration of a sample is injected onto a smaller
diameter column, the peak height increases. This narrower peak is a result of less
column dispersion. This phenomenon will occur when the detector is
concentration sensitive as in electrospray. You can now perform both microflow
and nanoflow separations with the Ion Trap.
150
Capillary HPLC – Advantages/Disadvantages
•Advantages
–Complex matrices can be handled
–High peak concentrations
–High chromatographic resolution
–Fast (perfusion chromatography)
–Ideal when sample limited
•Disadvantages
–Special considerations in HPLC
hardware and dead volumes
–Limited sample injection volumes
on-column concentration
12
The advantages and disadvantages of capillary LC are presented relative to

conventional LC separations.
The main advantages include high peak capacity, good chromatographic

resolution and excellent peak concentration for limited sample injections.
Difficulties with this technique include dead volumes due to plumbing, and
limited column capacity.
151
Capillary HPLC Connections-
Dead Volumes Must be Eliminated!


13
Examples of capillary connections
High pressure (>100 psi) connections are made with the fused silica column in a
PEEK tube sealed with a compression nut The fused silica and PEEK tubing
edges must be flush with one another to minimize dead volumes.
Low pressure (<100 psi) fittings are made by butt connections in a Teflon tube.
The two pieces of fused silica must meet with no open space between them.
In either case there should be no dead volumes due to angles or fractures in the
fused silica.
Dead volumes must also be eliminated when moving to a nano-LC/MS system.
152
Capillary HPLC for Sample Limited Analyses
Capillary HPLC for Sample Limited Analyses
Capillary or Nano HPLC for Sample

Limited Analyses
Better sensitivity for analyzing
compounds in the p-mol and
femto-mol range
nl sampling analogous to gel
electrophoresis
MSD coupling with upgrade kit
for flow rates less than
5 µL/min
Capillary columns from 180µm
to 1 mm (2-40µL/min)
Nano LC with columns from
75 to 150 µm (100-1000
nL/min)
14
Another alternative for sample introduction is capillary HPLC. This technique is

particularly useful for applications where the analysis is sample limited. Capillary
columns have i.d.’s from 180 µm to 300 µm with flow rates from 2 – 4 µL/min.
Also 800µm to 1 mm i.d. columns can be used in the 20 – 40 µL/min flow rate
range. With the Agilent 1100 Nanopump you can pump at flow rates from 100 –
1000 nl using capillaries with 75 to 150 µm i.d.
The advantage of these small diameter columns is the decrease in column

dispersion providing greater detection with smaller injections. You will either
need a capillary HPLC pump or with a conventional HPLC, the pump flow can be
split. You will need an upgrade kit to handle flow rates of 5 µL/min or less. The
concentration of the sample is limited by column capacity. Difficulties with this
technique include clogging in low internal diameter components and leaks that are
difficult to detect.
153
Reducing Capillary ID to Improve Sensitivity for Concentration-
Limited Samples
Reducing Capillary ID to Improve Sensitivity for

Concentration-Limited Samples
Reducing Capillary ID to Improve Sensitivity for

Concentration-Limited Samples
mAU
200ng Biphenyl
1400
0.3mm
1200 Column: Zorbax SB C18
1000
800
Less peak dispersion

600
0.5mm
400
1mm
200
0
4.6mm
0 2.5 5 7.5 10 12.5 15 17.5 min
15
When the same volume and concentration of a sample is injected onto a smaller
diameter column, the peak height increases. This narrower peak is a result of less
column dispersion. This phenomenon will occur when the detector is
concentration sensitive as in electrospray.
154
Nanospray
Nanospray
Nanospray
View of nanospray
Useful for Protein Identification
Nanospray needle
Inside source
Nano
Nanospray needle
Direct Injection in holder
16
Orthogonal nanospray – Agilent's orthogonal nanospray ion source combines the

extreme sensitivity obtainable at nanoliter-per-minute flow rates with Agilent's
orthogonal nebulizer technology. Use the nanoflow pump with 0.075mm i.d.
columns for direct injection into the ion trap. This source is especially useful in
conjunction with protein identification analysis.
155
Nanospray Configurations
Nanospray Configurations
2D LC with Nanospray
Load Sample on Enrichment Column: •Samples trapped
2nd Pump
Waste •Samples desalted
3
1
2 4 2
6 C18 Pre-Column
Plug
3 5 1 Nanopump
4 5 6
C18 Analytical Column
MS detection
Waste
1100 Micro Well plate Sampler Micro 2-position/6-port valve
Analyze Sample:
2nd Pump
Waste
•Separate sample
on nanoflow column
3
1 4 2
2 C18 Pre-Column
6
Plug
3 5 5 Nanopump
4 6
C18 Analytical Column
MS Detection
Waste
1100 Micro Well plate Sampler Micro 2-position/6-port valve
17
For sample enrichment followed by a nano separation, incorporate a capillary

column into the instrumentation. Trap, clean, and enrich the sample, such as
peptides, on the enrichment column. Switch the valve and separate the peptides
on the nano column. You may also incorporate an SCX column and salt step
injections.
156
HPLC-Chip/MS
HPLC-Chip/MS
The HPLC-Chip/MS
RF tag
Nano LC Column
Enrichment column,
capillaries, fittings, frits
HV ESI contact
Platinum
Grounds
Solvent
Nanospray tip, tip

assembly & fittings
Encapsulated HPLC-Chip for use with HPLC-Chip interface
18
Nano LC systems and electrospray needles are often difficult to work with due to
their fragility. To improve robustness and ease-of-use, use the HPLC-Chip/MS
system. The Nano Column, Nanospray needle, enrichment column, and
connections are all incorporated into layered wafers. Currently, two chips are
available, a chip for protein identification and an infusion chip for tuning and
calibration.
157
HPLC-Chip/MS
HPLC-Chip/MS
HPLC-Chip/MS
1 2 3
Remove nano source Install spray chamber Position chip cube
Insert HPLC-Chip
•Chip tip more robust than

nanospray needle.
•Chip tip automatically lowered
into position to spray.
•Limited delicate connections.
19
It is very easy to install the HPLC-Chip Cube interface. Remove the current
source. Install the spray chamber and then attach the Chip Cube. The chip can
only be inserted one way and is inserted into the source through the software.
Each chip lasts for approximately 2 weeks of continuous use.
158
CE/MS – Using Layer Flow Interface
CE/MS – Using Layer Flow Interface
CE/MS – Using layer flow interface

1100 LCMSD Trap
Capillary
Layer flow interface Entrance
to MS
CE fused silica
Layer flow tube
Nebulization gas
20
Above you will find a typical schematic of a CE/MS system. The CE fused silica
column has about 15-30 kV applied at the entrance reservoir and the exit reservoir
is at ground to provide the electrical field to result in migration of the ions and to
create an electro-osmic flow. The exit reservoir in CE/MS actually is inside the
triaxial nebulizer. The layer flow from the middle tube in the triaxial nebulizer
provides electrical contact for the liquid that migrates from the fused silica CE
column (inner tube in the triaxial nebulizer). The layer flow can be also used to
adjust the solvent for optimal electrospray performance. The outer tube in the
triaxial nebulizer is used for pneumatic nebulization.
It is important to keep the inlet buffer and the triaxial nebulizer at the same level
in order to avoid creating a hydrodynamic driven flow which can degrade peak
resolution.
159
CE/MS Optimization
CE/MS Optimization
CE/MS Optimization
•Considerations for layered flow approach
–Column diameter
–Electrolyte (composition/pH)
–Temperature
–Pressure (Hydrodynamic flow)
–Injection (pressure/electrokinetic)
–Layer flow (composition, pH, flow rate)

–Fused silica position
–API probe position (NaOH flush)
•Layer Flow:
–Aid in nebulization and ion evaporation with isopropanol
–Adjust pH to form ions in solution
–Provide makeup flow to get stable electrospray (2-5 µL/min)
21
The optimization of CE/MS requires that both CE and electrospray MS

parameters be optimized and compatible with each other. The CE considerations
attempt to gain the best peak concentration (resolution) for a sample using API-
MS compatible solvents and additives.
The layer flow nebulizer is adjusted to produce a stable ion current showing the
maximum sensitivity for the analyte. This is performed by:
• Careful adjustments of the position of the fused silica CE column relative to
the layer flow middle tube.
• Addition of the minimal flow rate into the layer tube to obtain a stable signal.
Higher flow rates will dilute the peak concentration reducing sensitivity.
Choose a layer flow solution that will maximize electrospray MS sensitivity.

(E.g. acetic acid in isopropanol for positive ion detection).
160
CE/MS Determination of Ceftiofur in Milk

250,000
200,000
150,000
100,000
50,000
0
0 5 10 15 20 25 30
Time (minutes)
CE Conditions:
Conditions
Column: 75 mm x 93 cm untreated fused silica
Buffer: 20% H2O 70% CAN 10% acetic acid
Voltage: 30 kV
Injection: Electrokinetic 90s x 15 kV
Pressure 500 mbar-S
Pressure: 50 mbar 5 min after injection
MS:
SIM: m/z 524 and 424 (internal standard)
Dwell Time: 100 MS
22
The example above is of a CE/MS determination of ceftiofur in a milk extract.

Note the peak width is very narrow requiring limited scan range and fewer
sampling averages to acquire a sufficient number of points across the peak.
161
Column Selection for LC/MSD Applications
Determine Particle Size

5 µm Particles 3.5 µm or < Particles
• Scan Mode • Increased Sensitivity
(Time for Instrument Cycle) (> peak heights)
• Simple Sample • Increased Resolution
(< peak widths)
• Increased Throughput (scale down)
• Narrower Chromatographic Peaks
• Complex Sample
• Isobaric Components
23
Today’s HPLC columns are available with packing material particle sizes
commonly from 3 –10 µm. Ten micron packings are not suited for LC/MS
analyses as the peak dispersion is high. Packing materials with particle sizes from
3-5 microns are ideal. The 5 micron column might be more suited to scan mode
as the peak widths will be wider than a smaller particle-sized column. The peak’s
elution will take a longer period of time allowing adequate scan speeds and scans
across the peak. If the data is acquired too quickly, the spectral quality will
suffer. The 3.5 micron columns offer the advantages of increased resolution and
sensitivity. The improved resolving power will make separation of isobaric
components more reliable. The chromatographic peak, however, will have less
dispersion and therefore require faster scans or cycles.
162
Determine Ideal Column Length
Short Column Long Column

• Increased Throughput • Increased Separation
• Selected - Ion Monitoring (SIM) • Scan Mode (Time for Instrument Cycle)
Multiple - Ion Mode • Multiple - Ion Mode
• Simple Sample • Complex Sample (Prevent Quenching)
• Easily Distinguishable M.W. • Isobaric
15 - 75 mm 150 or 250 mm
24
Select the column length based upon your individual needs. For high throughput
simple analyses, rely on short columns. The total analysis time will be much
shorter. When you find that you have a complex sample and need the separation
capacity then use a longer column.
163
LC/MS-Compatible Mobile Phases
Conditions:LC: Agilent1100
Columns:Zorbax SB-C18, 3.5 µm, 4.6 x 30 mm
mAU Mobile Phase:H2O, pH 2.5 : ACN (86:14); 1% formic
2 7
120 acid overall
Volatile UV:275 nm; Flow: 2.0 mL / min.; 70°C
4 6 Inj Vol: 1 µL
Mobile Phase 80 1 DAD1 A, Sig=275,16 Re
(LC/MS) 5
3
40
Absorbance
0 1min
mAU
120
Non-Volatile 80 Conditions: LC: Agilent 1100
Columns:Zorbax SB-C18, 3.5DAD1
µm, 4.6A,
x 30 mm
Sig=275,16 Re
Mobile Phase Mobile Phase:1 mM octane sulfonic acid, Na salt, pH
40 2.5 : ACN (80:20)
UV:275 nm; Flow: 2.0 mL / min.; 70°C
0 Inj Vol: 1 µL
0 0.5 1 min
ANALGESICS 4. Acetanilide
1. Acetaminophen (4-acetamidophenol)5. Aspirin (acetosalicyclic acid)
2. Caffeine 6. Salicylic acid
3. 2-Acetamidophenol 7. Phenacetin (acetophenetidin) 001638P1.PPT
25
One of the first considerations when switching an HPLC method to an HPLC/MS

method is to consider the mobile phase. It is best if the mobile phase constituents
are volatile. In other words, use a volatile buffer, additive, or ion-pair reagent
instead of a nonvolatile one. In the example above, formic acid is substituted for
the octane sulfonic acid sodium salt.
164
Adapting Existing LC Methods to LC/API-MS
• Replace non-volatile buffers with volatile buffers at a concentration of

<10 mM for ES or <100 mM for APCI.
Substitute phosphates, sulfates, and borates with ammonium acetate
or formate, trifluoroacetic acid (TFA), heptafluorobutric acid (HFBA),
tetrabutylammonium hydroxide (TBAH)
If a non-volatile buffer must be used, use a buffer where only the
anionic or cationic part is non-volatile, i.e. ammonium phosphate, not
sodium phosphate. The non-volatile portion of the buffer must be
ionizable in the mode used, I.e. phosphate (H2PO4) in negative mode.
Ionic strength of buffer is often more important than buffer choice to
achieve a separation
• Keep the pH the same using volatile additives:

Formic acid, acetic acid, TFA, ammonium hydroxide
• Volatile ion pair reagents should be employed
26
As said before, when adapting an existing HPLC method to the API-LC/MSD,

some important considerations need to be made. One of the most important
considerations is that all mobile phase buffers or additives must be volatile. It is
also best to keep the buffer concentration down to less than 10 mM for
electrospray and less than 100 mM for APCI.
Sulfates, phosphates, and borates need to be substituted with components that are
more volatile. Volatile buffers and additives to lower the pH include: ammonium
acetate (pH 6-7), ammonium formate (pH 5-6), acetic acid (pH 3.8-5.8), and
trifluoroacetic acid (pH 1-2). To raise the pH of a solution try ammonium
hydroxide (pH 8.2-10.2).
Volatile ion pair reagents should be used such as HFBA and TBAH.
165
Compatible Solvents - API-MS
Suitable for ES and APCI Suitable for only APCI

Methanol Toluene
Ethanol Benzene (2)
Propanol Hydrocarbons
Isopropanol (e.g., Hexane)
Butanol Styrene (2)
Acetonitrile CCl4
Water CS2 (2)
DMF(1) Cyclic Hydrocarbons
DMSO(1) (e.g., Cyclohexane)
Acetic Acid
Formic Acid
Acetone
CH2Cl2
CHCl3
(1) At lower solvent percentages (~10% or less) under ES conditions
(2) These solvents not often used due to environmental health and safety issues
27
Many typical HPLC solvents are compatible with API-MS. A good electrospray
solvent will support ions in solution. Therefore, a solvent like toluene, which
does not support ions in solution, cannot be used with electrospray. Although
water is an excellent solvent for ions, its solvation energy is high making
desolvation difficult.
166
Chromatographic Modes (HPLC)

MODE
Electrospray APCI
Reversed Phase *** **
Normal Phase * *** Main Compatibility

Problems:
Size Exclusion *** * Ions in solution are favorable
for electrospray, but may lead
Ion Pair ** ** to poor retention in reversed
phase. High ionic strength
Ion Exchange * * and/or nonvolatile buffers may
be hard to electrospray
Hydrophobic * *
Interaction
*less compatible
** more compatible
Immunoaffinity *** * *** most compatible
28
The compatibility of electrospray and APCI (and APPI which is comparable to

APCI) are ranked by the various modes of HPLC. Electrospray favors ions in
solution. In contrast, reversed-phase liquid chromatographic conditions usually
try to suppress ionization and increase the molecules hydrophobic nature. The
mobile phases however are compatible. Post-column addition can be used to
overcome this type of incompatibility. Samples usually have limited volatility.
Electrospray is not very useful for normal-phase separations. The mobile phases
do not support ions in solution. APCI, on the other hand, can handle the non-
polar mobile phases. Non-polar samples are usually more volatile and therefore
good candidates for APCI.
Size Exclusion in the form of Gel Filtration is an excellent mate to electrospray.
The aqueous mobile phases and samples such as proteins, which carry a charge,
are excellent matches. Gel Filtration is not a good match for APCI. Usually the
samples are not volatile and the buffers may cause problems. Gel Permeation,
however, is not very suitable for electrospray due to the organic nature of the
mobile phases.
Choice of ion-pair reagent determines the applicability of this technique. Reagent
ions may compete for ion evaporation process. Volatile reagents should be
employed.
167
Ion Exchange liquid chromatography cannot be coupled with electrospray easily.

Ion-pair reagents have been identified that are useful for this type of analysis. See
the next module for more information.
Immunoaffinity mobile phases are often compatible with API-MS. The sample is
usually non-volatile.
168
Optimization Scheme for API-LC/MS

Tune and Calibrate API-MS
µ
Analyze Test Sample (1-10 ng/mL)
Decrease Sample Increase Sample pH

pH (1% Acetic Acid) (0.1% NH4 OH or TEA)
ES- Positive ES-Negative APCI Positive APCI Negative

Ion Detection Ion Detection Ion Detection Ion Detection
EvaluateDif ferent
Probe Temperatures
Evaluate Data:
Poor APCI Sensitivity
Choose Optimal
Mode of
Operation
Optimize Capillary Exit Voltage to

Obtain CID Structural Information
29
This is a two part flowchart for a general optimization scheme to be used when
evaluating new samples for LC/MS. If the sample is pure flow injections can be
performed in both positive and negative ion detection using electrospray, APCI
and APPI to determine the mode of ionization and detection that yields the
maximum ion current. Note that pH should be adjusted appropriately for the mode
of ion formation in electrospray (e.g. low pH for positive ion detection) and
nebulizer temperature should be evaluated for APCI or APPI.
Once the optimal mode of operation is determined optimization of the MS/MS

parameters to yield product ion can be performed.
169
Optimization Scheme for API-LC/MS Con’t
Optimization Scheme for API-LC/MS Con’t
Optimization Scheme for API-LC/MS – cont.
LC Separation
Developed?
Yes No
Are Develop API -MS

conditions Post column No compatable LC
compat able
No addition to separat ion (use
with achieve post-column addit ion if
API -MS compat a- necessary)
bilit y
Yes
Yes
Evauate LC/API-MS Method for Sensitivity,

Specificity, Accuracy, Percision, Linearity
Method Evaluate all Aspects

Meets No
A nalysis of Method to
Goals Improve Results
Yes
Analyze Sample
30
The next stage of the method development involves the LC separation. If a

separation is developed it must be tested to see if the sensitivity and specificity
requirements for the assay can be achieved. If not, can post column addition be
used to meet the assay requirements? If not a new separation may have to be
developed.
If there is no separation developed you have the luxury of creating a separation to

give both good chromatographic resolution and API-MS sensitivity.
The final test of the method is to determine linearity, precision, accuracy,
recovery and detection limits in the matrices of interest to verify performance
criteria are suitable prior to sample analysis.
170
Optimization ESI and APCI for On-Line
LC/MS Analyses - Lessons in Solution
Chemistry
Optimization ESI and APCI for On-Line LC/MS Analyses - Lessons in Solution Chemistry
In This Section
In This Section
• What type of samples are best suited for electrospray and APCI.
• How the pH and ionic additives of the mobile phase affects

ionization in both electrospray and APCI modes.
• How to select ion-pair reagents and buffers for API.
• How to overcome ion suppression when using volatile buffers-

TFA Fix
Understanding chemistry of API will improve your ability to gain solutions to

your analytical problems. Some of the important analysis variables include
mobile phase, mobile phase pH, and additives.

• What types of samples are best suited for electrospray and APCI.
• How the pH of the mobile phase affects ionization.
• How to select ion-pair reagents and buffers for API.
• About the TFA fix in ESI.
172
Samples for Electrospray
• Samples that contain heteroatoms
• Compounds which can accept a charge by induction
• Samples that multiply charge in solution
• Compounds which are ions in solution
• Samples that run well by thermospray
• Avoid extremely non-polar samples where charge

induction is inefficient
Electrospray favors preformed ions in solution. Even samples that contain

heteroatoms such as carbamates and benzodiazepines can carry a charge by
association on their heteroatom. They have at least one set of non-bonded
electrons. Compounds that can produce a charge because of inductive effects are
also favored. These compounds include azo dyes, dioctyl phthalates, and cocaine.
Of course, samples like proteins, peptides, and oligonucleotides that can produce
multiply-charged ions are favored. Protonation of the basic amino acids sites in
the protein, lysine and arginine, produce the multiple charging. Other samples
that are ions in solution include catecholamines, sulfate conjugates, quaternary
amines, metal ions, and amino acids.
Non-polar samples do not run as well in electrospray mode. They are more
suitable for APCI. This description includes polyaromatic hydrocarbons and
polychlorinated biphenyls. Many compounds can be analyzed as neutral
molecules in a neutral environment, however. This is true of our reserpine for
positive mode, which is run with 50:50 water:organic mobile phase (where the
organic mobile phase may consist of methanol, isopropanol, or acetonitrile).
Other compounds, however, may only be analyzed if the chemical environment is
one that favors ion formation (in positive or negative mode).
173
When an analyte is dissolved in a polar solvent such as an acid or base, it can

either ionize or take on a strong dipole moment. For analytes that ionize,
electrospray is generally simple and highly sensitive. Provided no other ion-ion
interactions interfere, ions are already present in the solution before spraying.
These ions are easily evaporated from the droplets in the spray and result in a high
analyte ion abundance.
Analytes that form strong dipole moments, but do not ionize can still be analyzed.
The ionization process is driven by the strong electrostatic fields in the spray
chamber. These fields induce a charge on the surface of the droplets. These
analytes can also be ionized chemically by adduction using selected chemicals.
174
Factors Affecting Electrospray Ionization
• Solution Chemistry
– Flow rate
– Sample pKa
– Solution pH
– Solution conductivity
• Needle Setup
– Inner needle position
– Condition of needle
– Nebulizer pressure
• High Voltage Electrodes

– Capillary and Chamber voltage settings
– Condition of Capillary and Chamber high voltage
– Condition of insulators
The primary factors affecting electrospray ionization are listed above. The flow
rate needs to be optimized along with the nebulizer gas and mobile phase in order
to allow the droplet to desolvate properly with subsequent ion desorption. The
sample pK will determine at what mobile phase pH the sample is ionized.
Electrospray relies on preformed ions in solution. The conductivity of the mobile
phase also plays a role in droplet production.
The needle setup parameters determine how effectively the mobile phase and
analyte are nebulized by the nebulizer. Factors such as symmetrical spray pattern,
droplet size, and freedom from dropouts can greatly affect the ionization process
and the quality of data.
The high voltage electrodes consist of the Capillary, Mesh, and End Cap/End
Plate. The cleanliness of these electrodes determines the effective charge density
at their surface. Contamination will effectively reduce the charge density,
emulating a lower voltage for the electrode. In addition, the Capillary is subject
to wear of the platinum plated surface, and charging of the inner bore. Wear or
damage to the platinum surface generally causes charging at the capillary
entrance. This will prevent ions from entering the capillary bore. Contamination
of the bore will also cause charging, but the capillary can generally be restored by
cleaning. The mesh assembly insulator/standoffs isolate the mesh (typically at +/-
175
3,500 V) from the spray chamber, which is at ground potential. If the insulators
become contaminated, a leakage current may develop between the mesh assembly
and the spray chamber, resulting in instrument faults.
176
Solution Chemistry
Solution Chemistry
Solution Chemistry
Positive Ion Mode (pH <7)
R1 R1
:N R + HA
+ HN R + A -
2 2
R3 R3
Base Acid Sample
[M + H] +
Negative Ion Mode (pH >7)

O O
R C OH + :B R C O- + H:B+
Acid Base Sample
[M - H]-
Electrospray requires preformed ions in solution
Positive ion mode electrospray involves the creation of [M+H]+ ions in solution.
The generation of these ions is a function of the sample pKb and the solution pH.
The sample (a base) picks up a proton from an acid in solution and becomes
positively charged. Ionization is an equilibrium process and sample sensitivity
depends on the degree of protonation. Therefore, solution pH is important. It is
desirable to have an acid in solution and lower the solution pH. Solutions
containing formic or acetic acid work best to form positive ions. Strong acids
such as trifluoroacetic acid (TFA) and hydrochloric acid work poorly because the
strong acid anion pairs with the analyte cations, reducing analyte ion abundance.
Compounds that have functional groups that protonate readily such as basic
nitrogens, show good sensitivity. Those that are polar but contain no basic
nitrogens show moderate sensitivity. Hydrocarbons are poor candidates.
Solution chemistry for negative ion analysis involves the creation of [M-H]- ions
in solution. Again, the generation of these ions in solution is a function of the
sample pKa and the solution pH.
The sample (an acid in the case of negative ions) looses a proton to a base in
solution and becomes negatively charged. Therefore, for negative ion analyses, it
is important to have a base in solution and raise the pH. Solutions containing
ammonia or other volatile bases work best. For negative ionization, analytes with
177
Solution Chemistry
functional groups that deprotonate readily, such as carboxylic acids or sulfonic

acids, show the best sensitivity. Analytes that are polar but contain no acidic
groups show less sensitivity.
178
General Rules for Choosing Polarity of Ion Detection and pH - ESI
General Rules for Choosing Polarity of Ion

Detection and pH - ESI
General Rules for Choosing Polarity of Ion

Detection and pH - ESI
Positive Ion Detection:

•Basic samples (e.g., peptides containing arginine and/or lysine)
•Decrease pH Acetic Acid pH (3-4)
Formic Acid pH (2-3)
Trifluoroacetic Acid (TFA) pH (1-2)
•pH at least 2 units below pKa of samples
Negative Ion Detection:
•Acidic samples (e.g. peptides containing aspartate and/or glutamate)
•Increase pH (ammonium hydroxide)
•pH at least 2 units above pKa of samples
Neutral Samples: cationization, charge exchange, APCI (discussed later)
General rules for basic and acidic samples are listed above. The mobile phase
additives listed are volatile. Neutral samples must be dealt with in a different
manner that will be discussed later.
Some additives may cause significant background such as TFA (m/z 113) in
negative mode, and TEA(m/z 102) in positive mode. When possible, scan above
the additive.
179
pH Effects - Lysozyme (MW 14306)

18000
16000 pH 2.5
14000
12000
Abundance
10000
8000
6000
4000
2000
800 1000 1200 1400 1600 1800 2000
12000
10000 pH 6
Abundance
8000
6000
4000
2000
800 1000 1200 1400 1600 1800 2000
800
pH 12
600
Abundance
400
200
800 1000 1200 1400 1600 1800 2000

7
Lysozyme can be analyzed with positive ion electrospray. The basics sites,
arginine, lysine, and the N-terminus, are protonated at low pH. Increasing the pH
results in decreased sensitivity and a decrease in the mean charge on the molecule.
180
Formation of Ion in Solution: Negative Ion Detection
Formation of Ion in Solution: Negative Ion

Detection
Formation of Ion in Solution: Negative Ion

Detection
100
pH 3
pH 7
pH 10
Relative Response
50
1 .5 1.2 1.1 .9
0
β-Lactams Aminoglycosides Ivermeitin Tetracyclines Sulfamides
Compound Class
For negative ion detection, compounds that have functional groups that
deprotonate readily (such as carboxylic acids or sulfonic acids) show moderate
sensitivity. Those compounds that are polar, but do not contain acidic groups,
show fair sensitivity.
181
API-MS Additives
API-MS Additives
API-MS Additives
•pH
Acetic acid, formic acid, TFA, ammonium hydroxide
•General Buffers/Ion Paired Reagents
Ammonium acetate/ammonium formate
Triethylamine (TEA)
Heptafluorobutyric acid (HFBA)
Tetraethyl or tetrabutylammonium hydroxide (TBAH)
•Cationization Reagents
Sodium or potassium acetate at the 20-50 µM level
•General Considerations
Volatility (contaminates spray chamber, plugs orifice)
Conductivity (reduces formation of small droplets for ion evaporation)
Ion pairing (neutralize precharged ion when desorbed into gas phase)
•Cannot use phosphate, sulfate, or borate buffers typically used in HPLC
The main factors that influence the choice of additives in API-MS are ion pairing
and volatility. Ion pairing neutralizes precharged ions needed for electrospray
ionization. Volatility of an additive is needed to prevent plugging of the capillary
entrance and long term reproducible operation. For these reasons, common
additives used in LC such as phosphate, sulfate, and borate cannot be used. The
adjustment of pH, buffering and ion pairing reagents must be weak ion pairs
(strong enough for the chromatographic separation but with minimal ion paring in
the electrospray droplets prior to ion ejection) that are listed above.
The main factor for choices of additives for APCI or APPI is volatility. Ion
pairing does not affect APCI or APPI sensitivity
Factors involving choice of cation reagents will be discussed shortly
182
Use Volatile Ion-pair Reagents

•Typical Ion-pairing reagents cause
200000 1. Amitrol 2 3 the ion-paired complex to become
5 mM PFPA
2. Chlormequat
non-volatile.
150000
100000 3. Cyromazine 1
50000
0
•The non-volatile materials
450000
350000
1
2
3
5 mM NFPA
do not ionize.
250000
150000
•Use volatile ion-pair
50000
0 reagents.
3
•Ionization depends on analyte, ion-
450000
2
350000
250000
5 mM TDFHA
150000
1
pair reagent, and ionization mode.
50000
0
2 4 6 8 10 12 14 min
85.1
[M+H]+
167.1
[M+H]+
125000 1 350000
H2N N NH
3
1. Amitrol N NH2 250000
450000 3 75000 N N
2. Chlormequat N
3. Cyromazine NH 150000 NH2
2
168.1
350000 25000 Amitrol Cyromazine
50000
TIC
250000
60 80 100 120 140 160 60 100 140 180 m/z
122.0
1 [M+H]+
150000
200000 2 C H3
-
C l CH 2 CH 2 N C H 3 + Cl
C H3
124.0
50000
100000
Chlormequat
2 4 6 8 10 12 14 min
60 80 100 120 140 160 m/z
10
Ion-pair reagents may be necessary for the separation of ionic sample

components. The charged sample ion forms a complex with the oppositely
charged ion-pair reagent resulting in greater retention and separation.
Unfortunately, the most common ion-pair reagents are nonvolatile. These
reagents include alkyl sulfonates or tetraalkyl ammonium salts.
In LC/MS analyses, you must consider the impact that the ion pair reagent will
have on the signal and MS hardware. API LC/MS requires that the analyte be an
ion in solution (ESI) or that the analyte accepts or donates a proton in the gas
phase, APCI. Ion-pairing reagents can interfere with the ionization process. With
proper selection of a volatile ion-pair reagent, this type of analysis can be
performed. The example above shows the separation and quantification of three
pesticides in river water. Tridecafluoroheptanoic acid (TDFHA) was the most
useful volatile ion-pairing reagent based on sensitivity and separation of the three
pesticides. The recovery in river water ranged from 88.5 to 97.5% and RSDs
ranged from 3.3 to 4.2% (0.5 ng/ml). The detection limit ranged from 10 to 50
pg/ml in river water. The sample preparation procedure requires only the addition
of 10 mM TDFHA solution to the sample.
183
Column: Supelcosil ABZ+Plus 5 µm, 2.1 mm x 250 mm (Supelco)

Column temperature: 40 °C
Mobile phase: isocratic at 25% A in B
A: Acetonitrile (MeCN)
B: 5 mM TDFHA in water
Flow: 0.2 ml/min
Injection: 500 µl
Ionization: Electrospray positive ion mode
Fragmentor: 80 V
Nebulizer: 60 psig nitrogen
Drying gas: 10 l/min nitrogen at 350 °C
184
Ion-Pair Reagents in Positive Mode ESI
Norepinephrine is improved by
the presence of volatile ion-pair
reagents (VA and PFHA)
The HSA causes significant

suppression for all analytes
because the nonvolatile
ion-pair hinders the escape
of analyte from the droplets
during ESI.
11
In the experiment shown above, weak and strong volatile ion-pair reagents
(valeric acid and perfluoroheptanoic acid respectively) were compared against a
classic nonvolatile strong ion-pair reagent (sodium heptane sulfonic acid). The
impact of the ion-pair reagent on MS response was calculated as a percent of the
control (ammonium acetate only) for each analyte using the average of the
triplicate injections. The ESI signal for norepinephrine is improved by the
presence of the volatile ion-pair reagents (VA and PFHA). Ionization of
erythromycin is also improved when PFHA is present. HSA causes significant
suppression for all analytes because the nonvolatile ion-pair hinders the escape of
analyte from the droplets during electrospray ionization.
Positively charged nonvolatile ion-pair reagents such as tetrabutylammonium

hydroxide can be replaced with tributylamine.
185
Effect of Volatile Buffer Concentration on ES Signals
Effect of Volatile Buffer Concentration on ES

Signals
Effect of Volatile Buffer Concentration on ES

Signals
ESI
1.20E+07
Area
6.00E+06
0.00E+00
0 50 100 150 200 250 300 350
Concentration (mM)
Reserpine Caffeine
• ESI: At higher buffer concentrations, the analyte has more difficulty escaping
the droplet due to ion-pairing. Smaller molecules such as caffeine are desorbed
earlier in the ionization process.
12
Salts and buffers are frequently used in HPLC mobile phases and are also often
present in the sample matrices. These buffers and salts can be either volatile or
nonvolatile. API processes can be affected by both the concentration and type of
salt or buffer. In addition, the presence of nonvolatile buffers can potentially
cause instrument failure due to plugging, electrical shorting, etc.
To test the affect of the concentration of volatile buffer, reserpine (84 ng) and
caffeine (125 ng) were injected in FIA mode at a flow rate of 0.6 mL/min and a
mobile phase of ammonium acetate in 50:50 in methanol:water. The
concentration of the buffer was varied from 0 to 350 mM. The analysis was
performed in SIM mode monitoring 195.2 and 609.3 Daltons. The drying gas was
kept at 350 degrees C at 8 l/min. The nebulizer pressure was 30 psig. Vcap was
4000V.
The results show that the signal decreases slightly with higher buffer
concentrations. At higher buffer concentrations, the analyte has more difficulty
escaping the droplet.
186
Effect of Buffers on ESI Signal
Positive Ion Mode Negative Ion Mode
7.E+06
2.E+04
6.E+06
5.E+06 2.E+04
4.E+06
Area
Area
1.E+04
3.E+06
2.E+06 5.E+03
1.E+06
0.E+00 0.E+00
B C D C D
Lincomycin Sulfachlorapyr. Caffeine Lincomycin Sulfachlorapyr.
Mobile Phase Conditions: 8% methanol in (B) 0.2% acetic acid; (C) 50 mM

ammonium phosphate; or (D) 50 mM sodium phosphate.
13
In positive mode, the presence of nonvolatile buffer hinders ion ejection from the
droplet. In negative mode, the nonvolatile phosphate is being ejected which
allows some of the analyte ion to be ejected with it. Lincomycin, which should be
desorbed later because of its larger size, is not able to escape.
LC Conditions:
Mobile phase: 8% methanol in one of the following
A: water
B: 0.2% acetic acid in water
C: 50 mM ammonium phosphate, pH 7
D: 50 mM sodium phosphate, pH 7
Flow rate: ESI – 0.3 mL/min; APCI 0.7 mL/min
Injection 1 ul of a mixture containing 10 ng/ul of each of lincomycin, caffeine and
sulfachloropyradizine.
Column: Zorbax Eclipse XDB C8 2.1 mm x 50 mm @ 30C
MS Conditions:
SIM ions:
Positive mode 195, 285, and 407 daltons
187
Negative ion mode: 193, 283, and 405 Daltons

Fragmentor: Ramped 70 V for 193/195; 50 V for 283/285; 80 V for 405/407
Vcap: ESI – 4000 V; APCI – 3000 V
Drying Gas ESI-350 C 10 l/min; APCI – 350C 5 l/min
Nebulizer: ESI –25 psig; APCI – 60 psig
Vaporizer: 400C
Corona
Positive ion mode 4 uA
Negative mode: 40 uA
188
ESI Signal after 635 Injections with Nonvolatile Salt Matrix
ESI Signal after 635 Injections with Nonvolatile

Salt Matrix
ESI Signal After 635 Injections with Nonvolatile

Salt Matrix
Abundance
60000 ESI Signal Relative to UV Signal
50000 60
MS Amount/UV Amount
40000 50
40
30000
Injection #1 30
20000
Injection #300 20
10000
Injection #500 10
0 0
0.5 1 1.5 2 2.5 min 0 200 400 600 800
• The initial decline in the signal is probably due to changes in electric field
strengths as a non-conducting layer is deposited on the electrodes. Once the
layer is formed, the signal stabilizes.
• As opposed to the white powder seen in the APCI source, a brown material was
seen on the spray shield. This is probably from the glucose in the balanced salt
solution.
14
An experiment was performed to determine how salts and buffers in the sample
matrix would affect the LC/MSD. The mobile phase contained 20 mM
ammonium acetate in 50:50 methanol:water. The flow rate was 0.6 mL/min. The
sample was 100 µL of sulfachloropyradizine (5 ng) dissolved in Hanks’s
Balanced Salt solution. Hank’s Balanced Salt Solution contains the following
components: sodium chloride(8.0 g/liter), calcium chloride (0.165 g/liter),
potassium chloride (0.4 g/liter), potassium phosphate monobasic (0.06 g/liter),
magnesium sulfate (0.1 g/liter), sodium bicarbonate (0.35 g/liter), sodium
phosphate dibasic (0.048 g/liter) and glucose (1.0 g/liter). The column was a
Zorbax Eclipse XDB C8, 2.1 mm x 50 mm. The scan was 100 to 400 Daltons in
positive ion mode. The fragmentor was set to 70 V. Other interface conditions:
Vcap –4000V, drying gas 350, 10L/min, nebulizer 25 psig.
The initial decline in the signal is probably due to changes in electric field
strengths as a non-conductions layer is deposited on the electrodes. Once the layer
is formed, the signal stabilizes.
189
ESI - Response for Penicillin G in Various Percentages of Acetonitrile
ESI - Response for Penicillin G in Various

Percentages of Acetonitrile
ESI - Response for Penicillin G in Various

Percentages of Acetonitrile
150
Unsuitable mobile phases:

Toluene
Relative Intensity (%)
100
Benzene
Styrene
Hydrocarbons
CCl4
50 CS2
Cyclic Hydrocarbons
0 20 40 60 80 100
% ACN in H2O (0.1% Formic Acid)
15
Higher percentages of organic mobile phase usually result in better electrospray

sensitivity. This is because it becomes easier to desolvate the nebulized droplets.
Notice that the response does not vary drastically over a range of organic
composition, an advantage for gradient HPLC analyses.
190
Cationization in Electrospray
Neutral molecules which have any propensity

for hydrogen bonding will form adduct ions
with ammonium or alkali metal ions
Examples:
•menthol
•carbohydrates
Add a buffer of ammonium acetate or sodium

acetate to facilitate ionization.
16
The detection of neutral compounds can be enhanced in electrospray mode by the

addition of cationization reagents. Sodium acetate or potassium acetate added in
20-50 µM amounts can significantly increase the signal intensity. Note that
additional heptafluorobutyric acid can also increase the signal. Tetraethyl or
tetrabutyl ammonium hydroxide can be used for negative ion mode.
191
Creating the Optimal Chemistry for Electrospray through Post-
Column Addition
Creating the Optimal Chemistry for Electrospray

through Post-Column Addition
Creating the Optimal Chemistry for Electrospray

Through Post-Column Addition
Mixing Tee (LC Packing ~ 2 nL dead volume)
HPLC UV ES Needle Assembly
Pump
4-400 µL/min range,

pulseless delivery
17
With post-column addition, the analyst has the ability to not only optimize the
HPLC separation, but to optimize the spray chamber ion chemistry as well. Some
typical uses include:
• pH adjustment to optimize for positive or negative ion detection independent
of chromatographic mobile phase.
• Addition of isopropanol to aid in the desolvation of aqueous solvents or to
dilute ionic buffers to achieve acceptable MS performance.
• Addition of sodium acetate to aid in cationization of samples.
Typically, the flow rate is 10-50% of the column separation flow rate.
There are some disadvantages to this technique. Post-column addition requires an

extra pump. Post-column addition can broaden chromatographic peaks.
192
Post-Column Modification of LC Solvent to Optimize API-MS
Response
Post-Column Modification of LC Solvent to

Optimize API-MS Response
Post-Column Modification of LC Solvent to

Optimize API-MS Response
(1) pH adjustment to optimize for positive or negative ion detection.

(2) Addition of isopropanol to aid in the desolvation of aqueous solvent
and to dilute ionic buffers to achieve acceptable API-MS performance.
(3) Addition of sodium acetate post-column (50 µM) to aid in cationization
of samples. (Used for samples that lack or have weak sites for
protonation). *
(4) Using the “TFA FIX” to improve sensitivity for more ionic buffers. *
(5) Increase flow rate to achieve a suitable ES current (e.g., for capillary
electrophoresis separation).
(6) Reduce flow rate to achieve suitable ES operation ( e.g. prep LC)
(7) Derivatization to improve electrospray sensitivity, i.e. cationic species,
amine.
18
List of possible uses of post column addition to improve electrospray operation

are presented.
193
Samples for APCI and APPI
Samples for APCI and APPI
Samples for APCI
• Small molecules, polar to non-polar
• Samples that contain heteroatoms
• Samples that run well by particle beam (relatively

volatile) and most that run well by thermospray
• Avoid samples that typically are charged in solution
• Avoid samples that are very thermally unstable or

photosensitive
19
APCI and APPI are gas phase ionization techniques. Therefore, the molecules
analyzed are typically small and moderately polar to non-polar. Examples of
APCI or APPI samples include PAHs, PCBs, fatty acids, phthalates, and
triglycerides. Samples that contain heteroatoms such as benzodiazepines and
carbamates also work well.
Avoid samples that are thermally labile due to the vaporization process. Also,
avoid samples that are typically charged in solution such as proteins, peptides,
amino acids, or oligonucleotides.
194
APCI Key Parameters
APCI Key Parameters
APCI
Key Parameters
Ionization
Gas Phase CI
Protonation (e.g., H3O+) (Bases)
Charge exchange
Deprotonation (acids)
Electron Capture (halogens, aromatics)
Probe Temperature
• Higher temperatures to desolvate and vaporize
sample.
• Temperatures that are too high lead to sample
decomposition.
General: Use highly purified water and organic solvents.

MeOH often better choice than acetonitrile in APCI.
20
Remember that APCI is a gas phase ionization technique. The protonation will
usually result from the solvent and is based upon relative proton affinities. Gas
phase reactions that can reduce sensitivity include:
• Acetonitrile charge exchange. Often, methanol is a better solvent choice than

acetonitrile.
• Proton transfer. Samples with lower proton affinities than NH3 or
triethylamine can lose a proton, become neutralized, or form adducts in their
presence.
Probe temperature is the most important optimization parameter. A compromise

must be achieved between volatilization and sample decomposition.
195
APCI Vaporizer Temperature

100
Sulfamethazine
400 °C
Tetracycline
Sulfamethizole
AminoChlorobenzamide
BasicViolet10
Spectinomycin
MethyleneBlue
Furosemide
DisperseOrange13
DisperseBlue3
BasicYellow2
VitaminD3
50
Cloxacillin
PenicillinG
Streptonycin
Gentamicin
Relative Intensity
APCI
100
BasicViolet10
200 °C Sulfamethazine
AminoChlorobenzamide
Spectinomycin
Tetracycline
Sulfamethizole
MethyleneBlue
DisperseOrange13
DisperseBlue3
BasicYellow2
50
Cloxacillin
PenicillinG
VitaminD3
Streptonycin
Furosemide
Gentamicin
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Compound Number
Electrospray
100
Tetracycline
Methylene Blue
Sulfamethazine
Basic Violet 10
Amino Chlorobenzamide
Spectinomycin
Basic Yellow 2
Intensity
Sulfamethizole
Disperse Blue 3
Electrospray
Penicillin G
50
Streptonycin
Relative
Vitamin D3
Furosemide
Gentamicin
Cloxacillin
Time (minutes)
21
Notice that vaporizer temperature can play a significant role in signal sensitivity.
The vitamin D3 and the furosemide have little if any signal with a vaporizer
temperature of 200 degrees C. At 400 degrees C, the signal intensity is much
improved. Remember that at a higher vaporizer temperature, however, some
samples may degrade.
Notice that compounds that are charged in solution such as penicillin, methylene
blue, basic yellow 2, and basic violet 10 or nonvolatile such as gentamicin and
streptomycin are favored for electrospray analysis.
196
Effect of Volatile Buffer Concentration on APCI Signals
Effect of Volatile Buffer Concentration on APCI

Signals
Effect of Volatile Buffer Concentration on APCI

Signals APCI
2.E+07
2.E+07
Area
1.E+07
5.E+06
0.E+00
0 50 100 150 200 250 300 350
Concentration (mM)
Reserpine Caffeine
• APCI: Ammonium acetate initially aids proton transfer for reserpine. Increasing
“volatile salt” concentration makes it more difficult to effectively volatilize the
analyte. Caffeine is a weaker base than reserpine and ammonia competes for
protonation.
22
The effect of volatile buffers was studied using reserpine and caffeine. The
concentration of ammonium acetate in the mobile phase was varied from 0 to 350
mM. Ammonium acetate initially aids proton transfer for reserpine. Increasing
volatile salt concentration makes it more difficult to effectively volatilize the
analyte. Caffeine is a weaker base than reserpine and ammonia competes for
protonation.
LC Conditions:
Mobile phase: ammonium acetate in 50:50 methanol:water
Flow rate 0.6 mL/min
Injection 1ul of reserpine (84 ng) or caffeine (125 ng)
FIA with 3 injections of each compound
MS Conditions:
SIM: 195.2 and 609.3
Drying gas: APCI – 350 C, 5 L/min
Nebulizer: APCI – 60 psig
197
Effect of Volatile Buffer Concentration on APCI Signals
Vcap: 4000V
Fragmentor: Ramped 70 V for 195.2; 120 V for 609.3
Vaporizer: 400C
198
Effect of Non-Volatile Buffer on APCI Signal
Positive Ion Mode Negative Ion Mode
4.E+06 5.E+05
4.E+05
3.E+06
3.E+05
Area
Area
2.E+06
2.E+05
1.E+06 1.E+05
0.E+00 0.E+00
A B C D A B C D
Lincomycin Sulfachlorapyr. Caffeine Lincomycin Sulfachlorapyr. Caffeine
Mobile Phase Conditions: 8% methanol in (A)water; (B) 0.2% acetic acid; (C) 50
mM ammonium phosphate; or (D) 50 mM sodium phosphate.
23
The effects of buffer on APCI signal intensity were studied. In the presence of
only water (A), proton transfer is inefficient. With the addition of acetic acid (B),
proton transfer is improved. Ammonia (C) competes for protonation and
effectively suppresses protonation of all three analytes. Sodium phosphate (D)
does not compete for charge, but affects volatility of the droplets.
LC Conditions:
Mobile phase: 8% methanol in one of the following
A: water
B: 0.2% acetic acid in water
C: 50 mM ammonium phosphate, pH 7
D: 50 mM sodium phosphate, pH 7
Flow rate: APCI 0.7 mL/min
Injection 1 ul of a mixture containing 10 ng/ul of each of lincomycin, caffeine and
sulfachloropyradizine.
Column: Zorbax Eclipse XDB C8 2.1 mm x 50 mm @ 30C
MS Conditions:
SIM ions:
Positive mode 195, 285, and 407 daltons
199
Negative ion mode: 193, 283, and 405 Daltons

Fragmentor: Ramped 70 V for 193/195; 50 V for 283/285; 80 V for 405/407
Vcap: APCI – 3000 V
Drying APCI – 350C 5 l/min
Nebulizer: APCI – 60 psig
Vaporizer: 400C
Corona
Positive ion mode 4 uA
Negative mode: 40 uA
200
Flow Injection Analysis of Sulfachloropyradizine by APCI
Flow Injection Analysis of Sulfachloropyradizine

by APCI
Flow Injection Analysis of Sulfachloropyradizine

by APCI
mAU UV te r
ate
r te r
HB
S S
HB
S S SS
wa wa HB
20 in inw in fa in
fa in
fa in
u lfa lfa lfa su
l
su
l
su
l
s su su
10
Abundance TIC
150000 Scan mode
100000
50000
EIC, m/z 285

4000 Signal suppression!
2000
0 1 2 3 4 min
The signal for sulfachloropyradizine is suppressed in the presence of

salt!
24
An experiment was performed to determine how salts and buffers in the sample
matrix would affect the LC/MSD APCI signal. The mobile phase contained 20
mM ammonium acetate in 50:50 methanol:water. The flow rate was 0.6 mL/min.
The sample was 100 µL of sulfachloropyradizine (5 ng) dissolved in Hank’s
Balanced Salt solution or in water. Hank’s Balanced Salt Solution contains the
following components: sodium chloride(8.0 g/liter), calcium chloride (0.165
g/liter), potassium chloride (0.4 g/liter), potassium phosphate monobasic (0.06
g/liter), magnesium sulfate (0.1 g/liter), sodium bicarbonate (0.35 g/liter), sodium
phosphate dibasic (0.048 g/liter) and glucose (1.0 g/liter). The column was a
Zorbax Eclipse XDB C8 2.1 mm x 50 mm. The scan was 100 to 400 Daltons in
positive ion mode. SIM ion 285.1. The fragmentor was set to 70 V. Other
interface conditions: Vcap –4000V, drying gas 350, 5L/min, nebulizer 60 psig,
Vaporizer 400C, Corona 4uA.
Notice that salt in the matrix suppressed the sulfachloropyradizne signal. It would
therefore be beneficial to remove any salts from the sample matrix prior to
injection.
201
APCI Spray Chamber after 600 Injections of Salt Solution
APCI Spray Chamber after 600 Injections of Salt

Solution
APCI Spray Chamber After 600 Injections of Salt

Solution
Flow to spray chamber for entire analysis - note that

entrance to capillary is clean
25
Six hundred injections of salt solution were monitored on the LC/MSD. Note that
the entrance to the capillary remains clean.
202
APCI Signal after 600 Injections of Salt Solution
APCI Signal after 600 Injections of Salt Solution
APCI Signal After 600 Injections of Salt Solution
Abundance
350000 APCI Signal Relative to UV Signal
300000
60
MS Amount/UV Amount
250000 50
200000 40
150000
30
Injection #1 20
100000
Injection #300 10
50000
Injection #600 0
0 0 100 200 300 400 500 600
0.5 1 1.5 2 2.5 min Injection #
• Initial instability in the signal is probably due to changing electric fields as

salt deposits in the source.
• Once a layer of salt is formed, the signal stabilizes and shows a steady rate of
decline.
26
The design of the 1100 LC/MSD with orthogonal spray allows the occasional use
of some non-volatile buffers. In some cases, slight pH modifications may be
made to optimize the signal. For best long-term results, however, the method
should be modified to use a volatile buffer.
203
APCI Spray Chamber after 600 Injections with Automatic Diversion
APCI Spray Chamber after 600 Injections with

Automatic Diversion
APCI Spray Chamber After 600 Injections with

Automatic Diversion
Flow diverted to waste for 1 minute after injection

results in a much cleaner chamber
27
When using a nonvolatile buffer, you may divert the flow to waste after the
injection has been made. This will result in a cleaner source chamber and more
consistent results.
204
Impact of Ion-Pair Reagents on LC/MS Analysis in APCI Mode
Impact of Ion-Pair Reagents on LC/MS Analysis in

APCI Mode
Impact of Ion-Pair Reagents on LC/MS Analysis

in APCI Mode
•For norepeinephrine (most volatile analyte)

VA and PFHA assist in the ionization process
by serving as strong gas-phase acids.
•HSA does not improve signals because it is
not volatile.
•MGBG strongly ion pairs and shows greatly
reduced responses.
28
The impact of ion-pair reagent on MS response was calculated as a percent of the

control (ammonium acetate only) for each analyte using the average of triplicate
injections. A high concentration, 10 mM, of each ion-pair reagent was used.
Weak and strong volatile ion-pair reagents (valeric acid and perfluoroheptanoic
acid respectively) were compared to a classic nonvolatile strong ion-pair reagent
(Sodium heptane sulfonic acid). In positive mode APCI, the signal for
norepinephrine, the most volatile analyte, increases in the presence of both
volatile ion-pair reagents (VA and PFHA) because the reagents assist in the
ionization process by serving as strong gas-phase acids. Nonvolatile HAS does
not improve the norepinephrine signal. The APCI response for erythromycin,
which has low volatility, is greatly reduced by heptane sulfonic acid (nonvolatile)
and PFHA (low volatility) because these ion-pair reagents trap the analyte in the
dried droplets. MGBG, which strongly ion-pairs, shows a greatly reduced
response in the presence of heptane sulfonic acid and PFHA.
205
Comparison of Electrospray and APCI
• Sensitivity
If a sample can be ionized by both techniques, electrospray
is generally more sensitive and has less background noise
• Matrix and Mobile Phase Effects
Electrospray is more sensitive to sample and solvent matrix
than APCI (i.e. signal suppression)
Electrospray requires a lower concentration of very volatile
buffers relative to APCI
Choice of organic solvent strongly affects ionization in APCI
• Flow Rates
Electrospray works well at low flow rates (<100 µL/min) while
APCI does not
APCI is more sensitive and has less noise than Electrospray
at high flow rates ( >750 µL/min)
29
When developing your API-LC/MS methods remember the above

recommendations. In general, remember to think about solution pH and additives
in electrospray mode. Think about vaporizer temperature and solvent choice in
APCI mode.
206
References
References
References
•Meetings
•American Society For Mass Spectrometry:
www.asms.org
•The Montreux LC/MS Symposium:
www.lcmslimited.com Click on Montreux LC/MS
•Books
• Electrospray Ionization Mass Spectrometry: Fundamentals,Instrumentation and
Applications, Edited by Richard Cole, John Wiley and Sons (ISBN #0-471-14564-5),
1997.
• A Global View of LC/MS, Ross Willoughby, Edward Sheehan, Sam Mitrovich,
Global View Publishing (ISBN# 0-9660813-0-7) Pittsburgh, PA, 1998.
• Liquid Chromatography - Mass Spectrometry (2nd Edition) W.M.A. Niessen, Marcel
Dekker Publishing (ISBN #0-8247-1936-0), 1999.
30
Sources of additional information on API LC/MS are shown above.
207
References
208
Laboratory Exercise:
Understanding API Processes, Ion Trap
MS/MS and Interpretation of Mass
Spectra
Understanding API Processes, Ion Trap MS/MS and Interpretation of Mass Spectra
In this Section You Will Apply
In this Section You Will Apply
In This Section You Will Apply:
• Solution chemistry for Electrospray
• Ion Trap fundamentals
• Developing LC/MS methods (using post column addition)
210
Check Your Understanding - API

Decide on the probable mode of API-MS operation, polarity of detection
and any mobile phase changes that should be performed to analyze the
following samples: (Choose one response in each column)
Mode Polarity Mobile Phase

Compound (Electrospray (+ or - ion) (pH, post column
or APCI) addition)
1. Peptide: APCI + Increase pH

TYR-GLY-PHE-LYS-MET ES - Decrease pH
2. Benzophenone
O
C APCI + Increase pH
ES - Decrease pH
3. Sulfamethazine
N
CH 3 APCI + Increase pH
H 2N SO 2 NH ES - Decrease pH
CH 3
211
Check Your Understanding – API Cont.
Check Your Understanding – API Cont.
Check Your Understanding - API Cont’
4. Dihydroxybenzoic Acid
APCI + Increase pH
ES - Decrease pH
OH
HO COOH
5. Penicillin G.
APCI + Increase pH
CH3 ES - Decrease pH
S
CH2 C NH
CH3
N
O COOH
6. A mixture of aromatic amines separated by

LC at a pH of 8.5 ACN/H2O gradient at 1.0 APCI + Increase pH
mL/min ES - Decrease pH
212
Tandem MS Questions
Tandem MS Questions
Tandem MS Questions
(1) Name at least three variables the operator can change to increase the extent
of CID fragmentation in triple quadrupoles?
(2) What is the main difference between electrospray transport CID and triple
quadrupole MS/MS?
(3) What does tandem in space mean?
- Give examples of tandem in space and tandem in time instruments.
(4) What is the main parameter the operator can control to vary collision energy
for CID in the electrospray transport region?
(5) Why do fragment ions observed in electron ionization (EI) differ from CID
fragment ions generated from electrospray?
(6) List three reasons why one would perform MS/MS.
213
Check your Understanding
Check your Understanding
(1) What would typically be added to an acetonitrile/water mobile phase to

increase positive ion electrospray sensitivity? To increase negative
electrospray sensitivity?
(2) Why are sulfate, phosphate or borate buffers not used in electrospray-MS?
(3) Name a compatible ion pair reagent (for cation) with electrospray.
(4) What would you substitute or do when faced with theses following LC
separation conditions:
– Potassium Phosphate ( 20mM) in the separation?
– Using HFBA for an ion pair separation?
– Coupling to a prep LC running 80% MeOH in Water at 20 ml/min.
– Trying a capillary separation on a 0.32 mm i.d. column?
– Acetic acid (1%) for the electrospray analysis of carboxylic acid
compounds?
214
Check Your Understanding - Ion Trap MS/MS
(1) Does the ion trap accumulation time go up down or stay the same ( ICC on)as the
sample concentration increases?
(2) Describe what happens in a MS3 experiment? Why would one perform such an
experiment?
(3) In the CID of ions in the trap, what is the collision gas? How are ion energies
(kinetic energies) increased for collisions with a gas?
(4) As scan speed increases does resolution mass increase, decrease or remain the
same? Why?
(5) What is the advantage of non-linear resonance ejection over operating in RF only
mode to perform a mass analysis?
(6) Does an ion trap have an upper mass limit? What is done to a trap to extend its
upper mass limit?
(7) With ICC on, are space charge effects observed in the mass spectra? Is sensitivity
for a target component a potential problem?
215
Problem: Interference in a Pesticide Analysis

A carbamate pesticide (Carbofuran) using LC/Post column fluorescence
detection is measured at 4 ppm in vegetation. This is thought to be a positive
interference and a LC/MS method for confirming
the fluorescence results should be employed.
Discuss your approaches in developing such
a LC/MS confirmatory method.
•Sample preparation required? If so list some possible approaches.

•Are separation conditions compatible with MS? If not, propose new conditions or adjustments
to given conditions to achieve optimal MS performance (post column addition)?
•Mode of ionization and detection (list operating considerations for above choices. Are they
compatible with HPLC conditions?)
•Needed and expected sensitivity. What can be done if required sensitivity is not realized?
•Sufficient specificity? If not, what can be done?
•List alternative approaches (LC/MS interface, MS/MS or sample prep LC conditions, etc.)
216
Problem: Identification of a Drug Impurity
A chemist at a pharmaceutical company is interested in identifying if there are

impurities in a drug formulation. These impurities range from 0.1 - 2% relative to
the parent drug. The chemist can provide grams of the parent drug and the HPLC
separation conditions that use a phosphate buffer.
•Is sample preparation required? If so, list some possible approaches.

•Are separation conditions compatible with MS? If not, propose new conditions or
adjustments to given conditions to achieve optimal MS performance (post column addition)?
incase primary approach fails.
217
Problem: Quantitation of a Target Drug in Plasma

A pharmacologist wishes to have a LC/MS method developed to obtain
pharmacokinetic data on a basic drug called L-Thyroyine (MW 777) in plasma
down to 1 ng/mL level. The pharmacologist wishes to determine if HPLC/MS has
the sensitivity and specificity (3 ion) to detect the drug down to 1 ng/mL and have
your input on suitable extraction and HPLC conditions for the separation of the
drug from plasma.
•Is sample preparation required? If so list some possible approaches.

•Are separation conditions compatible with MS? If not, propose new conditions or adjustments
to given conditions to achieve optimal MS performance (post column addition)?
10
218
Problem: Identification of a Protein Impurity

A biochemist discovers a 5-8% impurity in a protein that is being isolated using
capillary electrophoresis and wishes to know the molecular weight of this impurity.
Size exclusion indicates the molecular weight of the impurity is close to the known
isolated protein (~20,000 mol. wt.). There are about 500 pmoles of the protein but only
5-10% of the sample can be used for the determination. The biochemist can submit the
sample in a saline solution.

11
219
Problem: Tryptic Peptide Analysis
A biochemist is unable to sequence two tryptic fragments of a protein using Edman’s

degradation methodology. The molecular weight and sequence of these two fragments
are desired. The biochemist can provide 50 pmoles of the tryptic digest, separation
conditions using a gradient 10% ACN - 80% ACN in water (0.1% TFA) in 30 minutes
on a C18 wide pore column and some sequence information on the two tryptic
fragments. The chromatographic conditions resolve the two tryptic fragments in
question from the other hydrolysis products.

12
220
ChemStation and Windows Overview
In This Section
In This Section
• How to open the LC/MSD ChemStation.
• About each ChemStation View.
• A brief introduction to Methods and Sequences.
• Some important concepts for computer maintenance.
In this section, we will discuss how to access the ChemStation and Ion Trap
Control software. Each view will be reviewed and you will learn about the
method and sequence concepts. At the end of the section, you will learn about
maintaining the computer.
222
Opening the LC/MSD Trap ChemStation
Open by Double
Clicking Desktop Icon
OR
Open from Start /All Programs/Agilent

ChemStations/Instrument 1 Online
There are two ways to open the LC/MSD Trap/ChemStation. The easiest method
is by directly clicking the desktop icon “Instrument 1 Online”. The other method
requires the use of the Start menu. By clicking on Start, find the Program menu
and then the ChemStations application. Once the third menu box appears, select
the Instrument 1 Online button to open the software for communication with the
instrument. You can also open the Ion Trap Control window. This method will
open the Ion Trap portion of the software alone.
223
HPLC Module Configuration
To set
ChemStation
to control
HPLC
modules they
must be
configured
Configure your HPLC modules:

•The first time you enter the software
•When changing the modules configured (like adding an additional pump)
In the configuration window you can set up the ChemStation to control multiple
modules that are connected to the instrument. To add a module, select from the
available modules in the left hand pane. The <Add> box will be highlighted and
clicking will move the module to the right pane and begin configuration. To
remove an active module, simply click on the desired module on the right and
then click remove. The module will now appear on the left and may be
reconfigured at any time. The ion trap configuration is established during the
software installation.
224
ChemStation Views
ChemStation Views
ChemStation Views
There are Seven ChemStation Views:
1. Method and Run Control
2. Data Analysis
3. Report Layout
4. Verification (OQ/PV)
5. Diagnosis
6. Esquire Control
7. Esquire Data Analysis
“View” drop down menu can

be shortened to display fewer
options
There are seven different views that can be accessed through the ChemStation
menus. The Ion Trap Control view will take you to the Ion Trap software.
225
Method and Run Control View

Top Tool Bar
Status Tool Bar
System
Status
System Diagram
The Method and Run Control view displays several ChemStation status windows.
The System Status window indicates the overall current status of the instrument.
This window is actively updated as the ChemStation performs various procedures.
The System Diagram shows the status of each individual component that has been
configured as a part of the overall system. Clicking on individual components
will allow you to open and set parameters for each instrument individually.
226
Data Analysis View
Data Analysis View
Data Analysis View
HPLC data only
The Data Analysis view is used to view chromatographic output from instruments
other than the LC/MSD Trap. This output would include diode array output, UV,
or fluorescence traces.
227
Report Layout View
Report Layout View
Report Layout View
HPLC Data Only
The Report Layout View allows the user to create a custom report template and
view the generated layout. The custom reports here are for HPLC data only.
228
Verification View
Verification View
Verification (OQ/PV) View
The Verification view is used in conjunction with an Agilent 1100 compliance

products. This product is delivered by qualified Agilent personnel.
229
Diagnosis View
Diagnosis View
Diagnosis View
10
The diagnosis view is used to troubleshoot problems that the HPLC instrument
may be experiencing. The images in the large box indicate the modules that are
configured and operational with the instrument. To modify/troubleshoot, click on
the instrument of interest allowing you to view detailed instrument information
such as the number of valve switches that have taken place on the HPLC pump.
230
Trap Control View
Trap Control View
MSD Trap Control View
11
The MSD Trap Control view is used to control the MSD Trap module. All of the
settings and control for the mass spectrometer are located within this window.
231
Trap Data Analysis View
Trap Data Analysis View
MSD Trap Data Analysis View
12
The ion trap Data Analysis window, much like the ChemStation Data Analysis
view serves as a place to observe the data that has been generated. Here you can
view the mass spectrometric trace of the chromatogram as well as the spectra
associated with each chromatographic peak. The Data Analysis window also
contains the deconvolution software.
232
Trap QuantAnalysis View
Trap QuantAnalysis View
MSD Trap QuantAnalysis View
13
The QuantAnalysis view in the Ion Trap software is used for quantification. Load
an acquired sequence and create a quantification method. The results are
processed and available for analysis and reporting.
233
Methods and Sequences
A method is a complete set of instructions for

the instrument and computer to acquire and
process one data file.
A sequence is a set of instructions to automate

the analysis of more than one vial possibly using
more than one method.
14
Methods describe the instrument parameters and data analysis of one injection.
This information describes everything from LC conditions to MS settings (e.g.
scan type, scan duration, etc.). Sequences allow you to enter in a list of
instructions that can utilize one or multiple methods and automate data acquisition
and processing.
234
Data and Method Storage
Data and Method Storage
Directory Structure for Data and Method

Storage
Methods
.M or .MS (old versions MS only)
Data
.D
An LC/MS Method
•Start with Def_LCMS.M in the ChemStation
•Contains LC acquisition and MS acquisition parameters
•Save this method within the ChemStation
•To use the LC only part of the method, go to the

Method menu and select Disable MS Part…
•To use the MS parameters without the LC, start the

acquisition in the MSD Trap Control software
15
Data and Methods are stored in the same name directories, usually on the D:
drive. A method can consist of both an LC part and an MS part. Simply fill in all
parameters for the LC and MS parts of the method, then save the method in the
ChemStation. The method will be saved as a .M file. You can disable the MS
part of the method temporarily to work on LC method development by selecting
the Method menu in the ChemStation then Remove MS Part…. To run the MS
part of the method only, start the acquisition from the Ion Trap Control software.
You can create an MS only method from within the Ion Trap Control Software.
When you load a method, you can view the current pieces attached to that
method.
235
Process Cleaner
Process Cleaner
Process Cleaner
•Closes all ChemStation and MSD Trap
related programs
•Restart programs cleanly without
rebooting the PC.
16
If your ChemStation or Trap Control software becomes unoperational (frozen),

you can use the Process Cleaner to cleanly close down all ChemStation and Ion
Trap related software. This action may save you time by preventing a PC reboot.
From the Start menu, selectAll Programs, LCMSD Trap, Tools, then Process
Cleaner. You can also place this tool on your desktop by clicking on the menu
item and dragging it to your desktop.
236
Maintaining the Computer System

Delete temporary files on a regular basis. Use Clean Disk for
Windows 2000 and XP.
Use Checkdisk to find and correct errors on the disk.
Defragment the hard drive.
Use Virus detection software.
Create an Emergency Repair disk.
Choose a “blank screen” screensaver.
Accessories
Right-click drive letter
17
Here, we present a few tips to maintain your computer system. If your Windows
session has been abnormally terminated, you may have left a number of
temporary files that can interfere with system performance. These files should be
deleted on a regular basis. Windows 2000 and XP have the Disk Cleanup applet
found in the following menus, Start, Programs, Accessories, System Tools, then
Disk Cleanup. This tool will clean up temporary Internet files, temp files left by
programs and other computer litter that takes up hard disk space. As with all the
utilities presented here, make certain that all programs are closed before
beginning.
Windows 2000 and XP also have a defragmentation tool. Exercising this tool
maintains your system performance by correcting data fragmentation and
reorganizing the disk so that every file is stored on the computer as a complete
unit. To access the defragmentation applet in Windows select Start, Programs,
Accessories, System Tools, then Disk Defragmenter.
Windows 2000 and XP also come with a tool to scan and repair damaged areas of
your hard drive. In Windows 2000 and XP, this feature is called Error-Checking.
You can access this tool by right clicking on the drive letter in the My Computer
237
window. Select Properties, then the Tools tab. Select Check Now… to begin
the process.
You should make an Emergency Boot disk for your computer each time you add
software or hardware to your computer. The Windows ChemStation is shipped
with an emergency repair disk and an emergency boot floppy. Store these items
in a secure place.
238
UserManagement
UserManagement
UserManagement
UserManagement is an optional tool to administrate access to Agilent Trap

applications and manage individual rights based on the operator’s ID and password.
18
The UserManagement tool is an optional application that allows you to administer

access to the Agilent Ion Trap applications and manage individual rights based on
an operator ID and password. Working with UserManagement enables you to
meet FDA 21 CFR Part 11 compliance.
The software is client-server based. The server contains the UserManagement
Administration Tool and the database. The ion trap applications are installed on
the client PC’s.
Control who can log into applications and what functions may be performed by
the user. In addition, control password access and security time outs. Audit trails
are also enabled with this softwarae.
239
UserManagement
240
LABORATORY EXERCISE:
Introduction to the LC/MSD Trap
ChemStation
In this section you will:

• Access the ChemStation software and understand what is obtainable from
each view.
• Learn how to maintain the LCMSD Trap computer.
• Become familiar with some Windows 2000 and XP features.
LABORATORY EXERCISE: Introduction to the LC/MSD Trap ChemStation
In This Laboratory Exercise, You Will Need:

• A computer that is configured with the Microsoft Windows 2000 or XP
operating software.
• The ChemStation, B.01.xx or higher, software loaded onto the computer.
• The LC/MSD Trap software, 6.0, loaded on to the computer.
242
Accessing the ChemStation

1) Find the Start button in the bottom left hand corner of your screen. Click on
the Start button. Select the menu choice All Programs. You will find a list
of the software programs installed on this computer arranged in alphabetical
order. To launch the ChemStation, find and select the Agilent ChemStation
menu item.
2) Click on Instrument 1 Offline. The software program will begin loading.
This part of the software controls the Agilent HPLC. Save combined HPLC
and MS acquisition methods in this part of the software.
Question: What is the difference between the ChemStation's Online and

Offline sessions?
3) Open the View menu and select Full menu if this item is available. If it is not
available, you are already displaying the Full menus.
4) From the View menu, select Show Top Toolbar and Show Status Toolbar.
Note that these two items may have already been selected.
5) Access the View menu and examine the entries. Notice that there are five
main views starting with Method and Run Control. List the views below
and after exploring each view, list the primary function of each.
View Function
243
Switch View Here Minimize, Maximize, and Close
6) Find the Minimize, Maximize and Close buttons located in the upper right
hand corner of the Title Bar. Minimize the HPLC ChemStation software.
Notice that the ChemStation software now appears to the right of the Start
button in the Task Bar. The ChemStation software is still running, but has
been cleared from the Desktop.
7) Click on the Instrument 1 (offline) software in the Task Bar to restore the
HPLC ChemStation window.
8) To close the HPLC ChemStation operating software you may either utilize the
Close button located in the upper right hand corner of the window; or, under
File select Exit. Leave the ChemStation software open.
244
Accessing the Ion Trap Software
Accessing the Ion Trap Software

1) Select Start, All Programs, LCMSD Trap and view the resulting menu.
The sections of the Trap software are contained here.
2) View the seven sections of the Ion Trap software and list their functions
below.
Administration This software is only present when the Ion Trap Compliance
software is installed. This software controls access and
privileges for a secure system.
Utilities
DataAnalysis
Library Editor
MSD Trap
Control
QuantAnalysis
ReportDesigner
Throughout the course, you will learn about the links between the Bruker Ion
Trap software and the HPLC ChemStation software.
There is a special utility to use when the ChemStation and/or Ion Trap software
does not respond. This tool is called the ProcessCleaner. When used, it will close
all sections of the ChemStation and Ion Trap Software immediately and clean up
any processes. Try this utility now to close all the programs.
1) From the Start menu, select All Programs > LCMSD Trap > Utilities >
ProcessCleaner.
2) All associated programs will close immediately.
245
Maintaining the Windows 2000 Workstation

(Skip to Maintaining the Windows XP Workstation if you have
Windows XP)
Cleaning up Temporary Files
After the ChemStation has been used for some time, temporary files may
accumulate in the directory specified by the TEMP Variable. These files are
generally left open when Windows is abnormally terminated. The files generated
have a .tmp extension. These files should be deleted to maintain computer
efficiency.
1) Close all programs.

2) Go to the Start button then Search and select For Files or Folders….
3) In the Search for Files or Folders named: field, type *.tmp.
4) In the Look in: field, find your hard drive, C: or Local hard drives.
5) Select Search Now.
6) After a few moments, the files will be displayed. Highlight the first file.
Using the Shift key and the left mouse button, highlight the last file. Press the
Del key to remove the files. Remember that you will have to empty them
from the Recycle bin as well.
Repairing the Disk
Windows 2000 includes a utility to check disk integrity. Among the errors that
may be fixed are lost clusters and cross-linked files. Error-checking can also
move your data from bad sectors. You must be logged on as part of the
administrator's group to run this utility.
1) Log on as the administrator. Press CTRL-ALT-DEL simultaneously. Log on

as the administrator. The password is 3000hanover; or, check with your
instructor for the correct password.
2) Double click on the My Computer icon and right-click on the drive icon for
C:. Select Properties to display a selection box.
246
3) Click on the Tools tab. Find the Error-Checking tool and click Check
Now....
4) Select to Automatically fix file system errors and Scan for and attempt
recovery of bad sectors.
5) This process can take several minutes. If you don’t wish to do this in lab,
Cancel, otherwise select Start.
Disk Defragmentation
A defragmentation utility is included with Windows 2000. Several vendors also

sell utilities such as DiskKeeper. Make certain you defragment your disk drives
on a periodic basis. You must close all programs before beginning. The process
can take a long time. We will not do this here.
Creating an Emergency Repair Disk
You should create an emergency repair disk as soon as possible. Use this disk if
your system files are damaged or your computer won't start. The disk includes
system settings such as registry files, disk partitions, and installed devices. Make
a new repair disk every time you make changes to your hardware or software.
Emergency Repair Disks are specific for each computer.
1) From the Start button, select Programs, Accessories, System Tools, then
Backup.
2) Click on the Emergency Repair Disk button.
3) Insert a floppy into drive A:.
4) Select to also back up the registry to the repair directory.
5) OK any dialog boxes that appear then close the Backup program.
247
Maintaining the Windows XP Workstation

(Skip this section if you completed Maintaining the Windows 2000
Workstation)
Cleaning up Temporary Files
After the ChemStation has been used for some time, temporary files may
accumulate in the directory specified by the TEMP Variable. These files are
generally left open when Windows is abnormally terminated. The files generated
have a .tmp extension. These files should be deleted to maintain computer
efficiency.
1) Close all programs.

2) Go to the Start button then select Search. On the left select All files and
folders.
3) In the All or part of the file name: field, type *.tmp.
4) In the Look in: field, find your hard drive, C: or Local hard drives.
5) Select Search.
6) After a few moments, the files will be displayed. Highlight the first file.
Using the Shift key and the left mouse button, highlight the last file. Press the
Del key to remove the files. Remember that you will have to empty them
from the Recycle bin as well.
Repairing the Disk
Windows XP includes a utility to check disk integrity. Among the errors that may
be fixed are lost clusters and cross-linked files. Error-checking can move your
data from bad sectors as well. You must be logged on as part of the
administrator's group to run this utility.
1) Log on as the administrator. The password is 3000hanover or, check with

your instructor for the correct password.
2) Double click on the My Computer icon and right-click on the drive icon for
C:. Select Properties to display a selection box.
248
3) Click on the Tools tab. Find the Error-Checking tool and click Check
Now....
4) Select to Automatically fix file system errors and Scan for and attempt
recovery of bad sectors.
5) This process can take several minutes. If you don’t wish to do this in lab,
Cancel, otherwise select Start.
Disk Defragmentation
A defragmentation utility is included with Windows XP. Several vendors also

sell utilities such as DiskKeeper. Make certain you defragment your disk drives
on a periodic basis. You must close all programs before beginning. The process
can take a long time. We will not do this here.
Creating an Emergency Repair Disk
You should create an emergency repair disk as soon as possible. Use this disk if
your system files are damaged or your computer won't start. The disk includes
system settings such as registry files, disk partitions, and installed devices. Make
a new repair disk every time you make changes to your hardware or software.
Emergency Repair Disks are specific for each computer. The backup process for
Windows XP goes farther than Windows 2000. It consists of two of two parts: a
backup file, and a Recovery Disk. The backup file will be large (we cannot do
this here), and the Recovery Disk will be a floppy.
1) From the Start button, select All Programs, Accessories, System Tools, then
Backup.
2) If you do not see “Welcome to the Backup Utility Advanced Mode then the
Wizard has been disabled, select Tools then Switch to Wizard Mode. Click
the Advanced Mode button found in the text.
3) Click the Automated System Recovery Wizard button.
4) Click Next>. The Wizard will start and prompt you for the media to use for
the backup file. As we don’t have the proper media here, cancel out.
You can write this file to a tape drive, a hard disk or writeable CD or DVD. After
entering the destination for the backup file, click Next> again, and finally, Finish.
The Windows XP Backup utility will copy all important system files and settings
to the backup file. An estimate and status bar are provided. After this step is
complete, you will be prompted for a blank, formatted floppy disk. Several files
are written to the disk to complete the process.
249
Windows Features (Windows 2000 or XP)
Mastering WINDOWS could take a week long class in itself. This section of the
laboratory is simply a brief familiarization of some of the important aspects
frequently utilized in conjunction with ChemStations. If you are already familiar
with Windows, skip this section.
Windows Explorer
The Windows Explorer is a powerful file manager. All files are stored in folders
(directories) on your hard disk. The Explorer allows you to copy, move, and print
entire folders and individual files.
1) To open the Windows Explorer, click on the Start button and select All
Programs, Accessories, Windows Explorer. The program will load.
2) Take a look at the information displayed on the left. These are all the objects
found in your computer. If the object has a + sign next to the name, the
folder contains sub-items such as sub-folders (subdirectories) or files that are
not currently shown. Click on the + signs until you can open Chem32
(C:\Chem32). This opens the sublevels.
3) Click on the + sign next to the 1 folder. The data folder should now be
present. Similarly, open the Data and Demo folders. Click on the Demo item
itself not the + sign. Notice that the contents of the folder appear in the right
pane as a group of folders. The individual files in the Demo folder (directory)
are displayed after the sub-folders.
4) To copy files from the data folder to a floppy drive, simply click on the folder
or file which needs to be moved and drag it to the 3 1/2 Floppy (A:) item in
the left pane. Try this now with one of the displayed data files. Make sure
that you have a formatted disk in the drive.
NOTE: When you drag files between different drives they are copied, not moved.
An original copy remains on the source drive. When you drag files between
folders on the same drive, Windows assumes you want to move the files, not copy
them.
250
NOTE: To select multiple files, click on the first file to select it. Ctrl-Click on
any additional files. To select a group of consecutive files, click on the first file to
select it. Then, use a Shift-Click on the last item to be selected.
5) Check to make certain that you have copied the chosen file into drive A: by
expanding the 3 1/2 Floppy (A:) item.
6) To delete a file or folder (when you delete a folder, all the contents of the
folder will be deleted along with the folder itself) right click on the folder or
file in the left or right pane. From the File menu select Delete. Try this now
with one of the data files in the Data\Demo folder.
7) The contents of the file will be moved to the Recycle Bin. The Recycle Bin is
found as an icon on the Windows desktop. Open this now. This is a special
folder on your disk. The Recycle Bin holds your deleted files until you empty
the bin, just in case you made a mistake. To restore a file from the Recycle
Bin, click on the item or items you accidentally deleted. From the File menu
select Restore. Once the bin has been emptied however, the only way to
retrieve a file is with a special undelete program. Try to restore the file you
deleted in part 6.
NOTE: Remember you must empty the Recycle Bin to gain space on your hard
drive. The Empty Recycle Bin option is found under the File menu in the
Recycle Bin.
8) You may open any file in the Windows Explorer to display its contents.
Simply double click on the item in the right pane. Practice your skills. From
the File menu, Close the Windows Explorer.
Clipboard
You may use the clipboard to capture the entire screen, an entire window or any
selected material within a document or graphic using the cut or copy commands
found in Windows based programs. The image is temporarily stored until you
paste it into the same or another application. For instance, say that you were
creating an SOP. You can copy the Pump Settings window from the LC/MSD
ChemStation software then paste it into a word processing program such as
Microsoft Word or WordPad.
1) Open the Instrument 1 Offline session.
251
2) Go to the Method and RunControl view. Under the Instrument menu,

select Set up Pump....
3) Press the ALT and PrintScreen keys simultaneously to copy the window to
the clipboard. Cancel the Set up Pump dialog box.
4) Minimize the LC/MSD ChemStation.
5) From the Start button, go to All Programs, Accessories. Select WordPad.
6) Click on the spot in the window where you would like to place the Pump
Parameter window.
7) From the Edit menu, select Paste.
The graphic is now part of the WordPad document.
Closing a Program that is Not Operational

Occasionally, you may find that other Windows program stop functioning or
become "hung-up". You may try to cancel the unresponsive program rather than
rebooting the entire computer. To do this, strike CTRL, ALT and DEL
simultaneously and abort the offending program.
1) Press CTRL, ALT, and DEL simultaneously. The Windows Security dialog
box will appear. For XP, select the Applications tab. Select Task Manager
for Windows 2000.
2) Select the program marked for termination. Here, select WordPad, then select
End Task. Note that WordPad is closed, while other software remains open.
If these actions do not clear your problem, you would have to reboot the
computer.
252
Starting Up and Shutting Down the
Agilent 1100 Series LC/MSD Trap
Starting Up and Shutting Down the Agilent 1100 Series LC/MSD Trap
In This Section
In This Section

• The different states of instrument operation
–Operate
–Standby
–Shutdown
–Off
• How to change modes of operation of the instrument
–Change from Off to Standby mode
–Start from Standby to Operate mode
–Change from Operate to Standby mode
–Change from Standby to Shutdown mode
–Start from Shutdown to Standby mode
–Change from Shutdown in the “Off” state
• How to check the vacuum and helium pressures of the LC/MSD Trap
In this section, we will review the various operating states of the ion trap mass
spectrometer. We will go over the steps and actions that occur as you switch from
one state to another. We will also learn how to check the ion trap vacuum and
helium pressures.
254
LC/MSD Trap Instrument States: Operation
LC/MSD Trap Instrument States: Operation
LC/MSD Trap Instrument States: Operate
• Full operational status of instrument.
• API interface is on.
• Mass spectrometer is on.
• Spectra can be generated, displayed, and acquired by the data

system.
The LC/MSD Trap has three standard states of operation to choose from. The first
state is Operate, in which the instrument is in a fully operational state. In the
Operate state, you can acquire data, generate spectra, and completely control the
instrument. The API-interface is on, the mass spectrometer is on, and spectra can
be generated, displayed and acquired by the data system.
255
LC/MSD Trap Instrument States: Standby
• The API interface remains on.

• The instrument remains under vacuum.
• The drying gas remains heated.
• Nebulizer flows are maintained.
• All high voltages are switched off .
• The mass spectrometer is not generating spectra.
• RF voltage remains set to a constant voltage.
The second mode of operation of the LC/MSD Trap is the Standby mode. In the
Standby mode, the API-interface is on, the drying gas remains heated, and
nebulizer flows are maintained. All high voltages are switched off and the mass
spectrometer is not generating spectra. This state is used when the instrument
remains idle for short or long periods of time. Usually, you should place the
LC/MSD Trap system in standby state when it is not in use. Note that in the
Standby mode, all voltages of the ion trap and detector are switched off, except
the RF voltage. The RF voltage is set to a constant value corresponding roughly to
that amplitude that is necessary for measuring the mass of 300 m/z.
256
LC/MSD Trap Instrument States: Shutdown
• The API-interface and the mass spectrometer are turned off.
• System does remain under vacuum.
• DataAnalysis software is still running.
• MSD Trap Control software no longer communicating with

instrument.
In Shutdown mode, the API-interface and the mass spectrometer are turned off.
However, the system is still under vacuum, and the software is still running. Do
not leave the instrument in Shutdown mode for extended periods of time.
257
LC/MSD Trap Instrument States: Off
• The instrument is completely shutdown.
• The system is vented and no longer under vacuum.
• Power to the instrument is off.
• NOTE: The main power switch on the LC/MSD Trap must be

moved to the “OFF” position.
The Off mode refers to the status of the instrument when the instrument is
completely shutdown. The system is no longer under vacuum and the power is off
preparing for maintenance or for long periods when the instrument will not be
used. The main switch on the side of the LC/MSD Trap must also be turned off.
258
Which Mode Should I Use?
Which Mode Should I Use?
LC/MSD Trap Instrument States: Which Mode

Should I Use?
• Operate
– Normal operation of the instrument.
• Standby
– State is used when the instrument remains idle for short or long periods.
– Should usually be used whenever instrument is not in use.
• Shutdown
– To change ion source, or to turn off the drying gas/drying gas heater.
• Off
– Used for preparation of maintenance or for long periods when the
instrument will not be used. System vented.
The above guide can be used to determine which mode you should have the
instrument in at any given time. Note that when acquiring data and generating
spectra, the Operate mode will be used and alternatively the Standby mode when
you are not actively using the LC/MSD Trap. These two modes are the most
frequently used modes of operation. The Shutdown and Off modes usually
require special cases as listed above.
259
Changing Modes
Changing Modes
Changing Modes of Operation: Off to Standby

• Verify nitrogen and
helium gas supplies.
• Flush helium line

before connecting.
• Verify the capillary is

capped with a rubber
cap.
• Verify the rough pump

is connected to the
LC/MSD Trap
This procedure will bring the instrument from Off state to Standby state. When
changing from Off to Standby modes, remember that the exhaust fumes from the
vacuum pumps and spray chamber will contain trace amounts of the chemicals
you are analyzing. Health hazards include chemical toxicity from solvents,
buffers, samples and pump fluid vapor, as well as potentially biohazardous
aerosols of biological samples. Be sure to vent all exhausts external to the
building where they cannot be recirculated by environmental control systems. Do
not under any circumstances vent exhaust directly into your laboratory. To
change from Off to Standby:
1. Verify appropriate nitrogen gas supply.

Verify that nitrogen gas is present at the nitrogen port behind the service panel in
the purity and pressure specified in the site preparation guide.
260
Changing Modes
2. Verify that a supply of helium of the purity and pressure specified in the site
preparation guide is connected via a pressure regulator to the helium port behind
the service panel.
3. Flush the helium line.

To do this, disconnect the helium line from the inlet port and let the helium flow
through the tubing for 1–2 minutes. Re-connect the helium line to the helium inlet
port while the helium is flowing.
4. Verify that the rough pump is connected to the LC/MSD Trap.

Verify that the rough pump cable is connected to the “foreline pump” plug behind
the service panel of the LC/MSD Trap. Verify that the rough pump has the
correct voltage rating (230 V or 208 V). Verify that the rough vacuum tubing is
connected to the inlet part of the rough pump. NOTE: Make sure that the pump
power switch is on and be careful not to connect the drain bottle tubing to the
rough pump.
261
Changing Modes
Changing Modes
• Verify that all pump and spray chamber exhausts are vented outside the laboratory.
• Turn on the computer.
• Switch on the main breaker

and main switch of the LC/MSD
Trap.
• Start the MSD Trap Control

by clicking the “Instrument 1
Online” icon.
• Set the software to Standby.
• Check the vacuum.
5. Verify that all pump and spray chamber exhausts are vented outside the
laboratory. The rough pump should have the oil mist filter installed.
6. Turn on the power for the personal computer and monitor.
7. Switch on the main breaker behind the service panel.

This will connect the rough pump to the power supply and inner high voltage
supply.
8. Switch on the main switch of the LC/MSD Trap.

This starts the pumping system and the electronics.
9. After starting Windows, start the MSD Trap Control software from the
262
Changing Modes
desktop by selecting Start > All Programs > LCMSD Trap > MSD Trap Control.
10. Set the software to Standby.

In the main panel, in the Status group, click [Standby]. You will not be able to
activate the Standby or Operate states if the ion source is not mounted correctly
and firmly.
11. Check the pressure.

You can monitor the pressure readings in a separate window via the Options
menu and the option Vacuum System. The instrument has four differential
pumping stages; two of the stages are monitored: the foreline vacuum (“Fore”),
and the vacuum in the final pumping stage (vacuum at the ion trap and the
detector, “High”).
263
Changing Modes
Changing Modes

• From the Smart Tune page:
– Set the drying gas flow to 4 liters/min
– Set the drying gas temperature to 350 °C
– Set the nebulizing gas pressure to 5 psi.
• Remove the rubber cap from the capillary line.
• Wait for the instrument pressure to come down (~4 hours).
• Flush the helium line three times.
• Leave the drying gas

flow, temperature, and
nebulizing gas
pressure as set above.
10
12. Adjust the source values on the Smart Tune page.

Set the drying gas flow to 4 liters/min., the drying gas temperature to 350 °C, and
the nebulizing gas pressure to 5 psi.
13. Wait for the pressure to come down.

It will take the LC/MSD Trap at least 4 hours to reach its ultimate vacuum—
waiting overnight is better. The foreline pressure (pressure at the rough pump)
should be about 2–3 mbar; the high vacuum pressure (pressure in the analyzer
region) should be in the 10 -5 mbar range.
14. Flush the helium line three times.

To this end, open the Options menu and select [Vacuum System...] and press the
[Flush Helium Line] button.
264
Changing Modes
15. Leave the drying gas flow at 4 liters/min. and at a temperature of 350 °C and
the nebulizing gas pressure at 5 psi.
The system is now in Standby mode.
265
Changing Modes
Changing Modes
Changing Modes of Operation: Standby to Operate

• Click on Operate in the main panel.
• Switch to the Smart Tune page.
• Make sure that the drying gas is set to a minimum of 3 liters/min.
• Check the pressure supply reading (both low and high vacuum regions).
• Verify that the drying gas heater is on.
• The instrument is now in full Operate state.
11
This procedure will take the LC/MSD Trap from the Standby mode to the Operate
mode. This procedure assumes that the instrument is in Standby mode, the
helium pressure has been set, and the exhaust lines are connected to the fitting at
the base of the spray chamber and to the rough pump outlet. DO NOT forget that
the lines must be vented outside the laboratory. To change from Standby to
Operate mode:
1. In the main panel, click on Operate.

In the main panel, in the Status group, click Operate. It is impossible to activate
the Standby state or Operate state if the ion source is not mounted correctly or
firmly.
2. Switch to the Smart Tune page.
3. Make sure that the drying gas is set to a minimum of 3 liters/min.
266
Changing Modes
Typical values are 5–10 liters/min. This starts the flow of nitrogen drying gas to
the spray chamber. This assumes that the main valve of your nitrogen gas cylinder
is open. Optimum flow is determined by the spray chamber and LC flow rate in
use.
4. Check the pressure supply reading.

You can monitor the pressure readings in a separate window via the Options menu
and the option Vacuum System. You will get the vacuum system window
displaying the pressures. The foreline vacuum stage pressure (at the rough pump)
should be approximately 1–2 mbar (0.75–1.5 Torr) and the high vacuum stage
pressure (in the analyzer region) should be in the low 10-5 mbar range.
5. Verify that the drying gas heater is on.

Set the drying gas temperature to 250 °C. This is a generic temperature setting.
The instrument is now in the fully operational state.
267
Changing Modes
Changing Modes
Changing Modes of Operation: Operate to Standby

• Make sure you have completed data acquisition and have
saved all data.
• Switch the MSD Trap Control Software to Standby.
• NOTE: Do not turn off the main valve on the helium tank
while the instrument is in Standby state.
12
This procedure will take the LC/MSD Trap from the Operate mode to the Standby
mode. You will normally put the LC/MSD Trap in Standby state when you have
completed your analysis of samples. This procedure assumes that you have
completed tuning and data acquisition and you have saved all data as necessary.
Please do not turn off the main valve on the helium tank while the instrument is in
Standby state. Due to the low gas flows involved here, the re-equilibration of the
helium pressure may take a long time. To change from the Operate to Standby
mode:
1. Switch the LC/MSD Trap Control Software to Standby.

The drying gas heater and flow and the nebulization flow is maintained. This
switches off the high voltages of the spray chamber, the detector and all other lens
voltages, except the RF voltage. The RF voltage is set to a constant value. This
value corresponds roughly to that amplitude that is necessary for measuring the
mass of 300 m/z. The mass spectrometer stops generating spectra. One of the last
acquired spectra is shown in the line and mass spectrum windows.
268
Changing Modes
The system is now in Standby state.
269
Changing Modes
Changing Modes
Changing Modes of Operation: Standby to Shutdown
• Switch off the solvent flow.
• From the main panel, switch the software to Shutdown .
• Allow the source to cool.
13
This procedure will take the LC/MSD Trap from the Standby mode to the
Shutdown mode. When you have completed your sample analysis, you would
normally put the LC/MSD Trap in the Standby state. The Standby state is fine for
an extended period of time ranging from minutes to days or weeks. However, if
you decide that you do not want to keep the drying gas and the drying gas heater
on, this procedure will put the LC/MSD Trap in the Shutdown state. The
Shutdown state is also required to change the ion source on the LC/MSD Trap.
This procedure assumes that you have completed tuning and data acquisition and
you have saved all data as necessary. As with the Standby mode, the main valve
on the helium tank should not be closed to shut off the helium flow to the trap.
To change from the Operate to Standby mode:
1. Switch off the solvent flow.

If you have analyte in any of the components in the sample delivery system, then
flush the delivery system with pure solvent before proceeding.
270
Changing Modes
2. Switch the software to Shutdown.

In the main panel, in the Status group, click “Shutdown.” This switches off the
spray chamber high voltages, the drying gas heater and flow, the nebulization
flow, and the detector, as well as all other lens and RF voltages. If the system is
configured for APCI, the APCI heater is turned off.
3. Allow the source to cool.
The system is now in the Shutdown state.
271
Changing Modes
Changing Modes
Changing Modes of Operation: Shutdown to Standby

• Verify that the metal cap on the capillary and spray shield are
in place.
• Verify the ion source is closed firmly.
• Switch the software to the Standby state.
• Open the Smart Tune page.
• Make sure that the drying gas is set to a minimum of 3

liters/min.
• Check the pressure readings (both high and low pressure

regions).
• Verify that the drying gas heater is on (~ 300 °C).

14
This procedure will allow you to take the instrument from Shutdown mode to
Standby mode. The procedure assumes that the LC/MSD Trap is in the Shutdown
state. To change from Shutdown to Standby mode:
Verify that the metal cap on the capillary and the spray shield are in place and
the ion source is closed firmly.
2. Switch the software to the Standby state.
3. Switch to the Smart Tune page.
4. Make sure that the drying gas is set to a minimum of 3 liters/min.

Typical values for the drying gas are 5–10 liters/min. This will start the flow of
nitrogen drying gas to the spray chamber. Make sure that the main valve of your
272
Changing Modes
nitrogen gas cylinder is open. Optimum flow is determined by the spray chamber
and LC flow rate in use.
5. Check the pressure reading.

You can monitor the pressure readings in a separate window via the Options
menu and the option Vacuum System. The vacuum system window will display
the pressures. Ideally, the fore vacuum stage pressure (at the rough pump) should
be approximately 1–2 mbar (0.75–1.5 Torr) and the high vacuum stage pressure
(in the analyzer region) should be in the low 10-5 mbar range.
6. Verify that the drying gas heater is on.

Set the drying gas temperature to 250 °C. This is a generic temperature setting. It
will be reset later to match the solvent flow rates of your analyses.
The system is now in the Standby state.
273
Changing Modes
Changing Modes
Changing Modes of Operation: Shutdown in the

Off State
• Switch off the solvent.
• Switch the software to Shutdown.
• Allow the source to cool.
• Exit/close the LC/MSD Trap software.
• Power off the instrument at the main switch.
• Power off the instrument at the main breaker located behind the service
panel.
• Power off the PC and monitor.
15
The Off state of the LC/MSD Trap should only be used when you will not be
analyzing samples for an extended time or when you must perform maintenance.
Note that just selecting Shutdown from the main menu does not turn off the
LC/MSD Trap. You have to follow the entire Shutdown procedure. Be careful if
using high drying gas temperatures as the spray shield and related components
will be hot. To change from the Shutdown mode to the Off state:
1. Switch off the solvent flow.

If you have analyte in any of the components in the sample delivery system, then
flush the delivery system with pure solvent before proceeding.
2. Switch the software to Shutdown.

This switches off the Spray chamber high voltages, the drying gas heater and
flow, the nebulization flow, and the detector, as well as all other lens and RF
voltages.
274
Changing Modes
3. Allow the source to cool.

This is especially important if you were working with high temperatures.
4. Exit the LC/MSD Trap software.
5. Power off the instrument at the main switch located in the lower left corner of
the instrument.
6. Power off the instrument at the main breaker located behind the service panel.
7. Power off the PC and monitor.

This is done by shutting down the Windows session and then powering down the
PC and monitor.
The system is now in the Off state.
275
Checking Helium Pressure
Evaluate Ion Trap Pressure
Uncheck the Helium box and

Classic and VL
calculate the change in high
Fore: 1.7-2.2 mbar (1.3 – 1.7 Torr)
High: 1.2-2.0 x 10-5mbar (9.0 –15 x 10 –6 Torr) pressure reading to determine
differential Helium pressure. The
difference should be
SL and XCT
Fore: 3.8-5.8 mbar (2.9-4.4 Torr)
6 x 10-6 mbar.
High: 1.2-2.0 x 10-5mbar (9.0 –15 x 10 –6 Torr)
16
It is important to constantly monitor the helium pressure in the high vacuum stage
of the LC/MSD Trap. This can be done with the instrument in the Shutdown,
Standby, or Operate State. Be very careful never to exceed a pressure of 5 x 10 -4
mbar in the high vacuum stage. To check the helium pressure:
1. Check the pressure readings.

Monitor the pressure readings in a separate window via the Options menu and the
option Vacuum system. The instrument has four different pumping stages, two of
them are monitored—the fore vacuum (“Fore”), and the vacuum in the final
pumping stage (vacuum at the ion trap and the detector, “High”).
2. Determine the base pressure.

Deselect the Helium check-box. Wait for 5 minutes until the pressure in the High
stage has reached its base level and note this base pressure.
276
3. Determine the normal high pressure.

Select the Helium check-box. Wait for 1 minute until the pressure in the High
stage has reached its normal level and note this pressure.
If the difference is not correct, then adjust the helium valve. Turn the valve
clockwise to increase and counter-clockwise to decrease
4. Build the difference between these two pressures.

Subtract the base pressure from the normal high pressure. The difference
pressure—helium pressure—should be 6 x 10-6 mbar.
5. Close the Vacuum system dialog.
277
278
Agilent 1100 Series HPLC/MS Trap:
Calibration and Tuning
Agilent 1100 Series HPLC/MS Trap: Calibration and Tuning
In This Section
In This Section

How to obtain high quality data by ensuring:
•Mass accuracy
•Mass isolation and fragmentation accuracy
•Signal optimization
To achieve these objectives, you will need to:

•Check pressures and differential pressure.
•Check scan calibration. If undesirable, perform new scan calibration
•Check multiplier setting and adjust if necessary.
•Perform isolation calibration, if new scan calibration.
•Perform Fragmentation Calibration, if new isolation calibration.
•Tune or optimize ion trap parameters for the method.
•Optimize MS/MS fragmentation amplitude with actual compound.
In this section, we will discuss how to calibrate, optimize, and tune the ion trap
for the best possible data production and quality.
Optimizing the performance of the LC/MSD Trap can be divided into two distinct
procedures. Tuning is associated with optimizing the signal that the mass
spectrometer produces for a given analysis. Calibration establishes the accuracy
of the mass measurements in the spectra that are generated and the accuracy of the
mass isolation and fragmentation of MS/MS experiments.
280
Ion Trap Pressure
Ion Trap Pressure
Evaluate Ion Trap Pressure
Uncheck the Helium box and

Classic and VL
calculate the change in high
Fore: 1.7-2.2 mbar (1.3 – 1.7 Torr)
High: 1.2-2.0 x 10-5mbar (9.0 –15 x 10 –6 Torr) pressure reading to determine
differential Helium pressure. The
difference should be
SL and XCT
Fore: 3.8-5.8 mbar (2.9-4.4 Torr)
6 x 10-6 mbar.
High: 1.2-2.0 x 10-5mbar (9.0 –15 x 10 –6 Torr)
Check the vacuum status by selecting Options, then Vacuum system. Typical
vacuum ranges are listed above. The differential pressure due to helium should be
6 X 10-6 mbar. This should be verified by unchecking the Helium box and
calculating the change in High pressure. If the difference is not correct, then
adjust the helium valve. Turn the valve clockwise to increase and counter-
clockwise to decrease. Improper vacuum system pressures and helium pressure
will affect trap functioning so this is a good place to begin ensuring quality data.
281
Calibration
Calibration
Calibration
•Assures correct mass assignments across a mass range.
•Should be checked on a regular basis (once per month) and performed after ion
trap maintenance or venting.
•Stored in Current Calibration file automatically. Previous calibration moved to

Backup. Default Calibration is always available if problems occur.
•Can apply to any flow conditions.
•Three types: Scan calibration, Isolation calibration, and Fragmentation calibration.
Manual calibrations Automatic calibrations

4
Calibration parameters are stored separately from the instrument methods. The
currently loaded calibration will be used during any acquisition regardless of the
method loaded. The calibration assures accurate mass assignments to ions found
during acquisition. You should check your instruments calibration on a regular
basis to make certain the mass assignments are correct. The procedure is
described later in this section.
Calibration parameters are stored in one of three files, the current, backup, or
default. The calibration is largely independent on the flow to the instrument.
There are three separate types of calibration, scan calibration, isolation
calibration, and fragmentation calibration.
The user only needs to calibrate the instrument after venting the manifold, after
maintenance, or after service. Otherwise, calibration is only done when
necessary. Agilent provides one tuning mix which can be used for both standard
and extended mass ranges as well as polarities.
282
Calibration
Calibration
Agilent Calibration and Tuning Mixture

The Agilent ES Tune Mix is used for:
•Both scan ranges (standard/extended).
•Both polarities (positive/negative).
•Calibration and tuning with the electrospray source.

Standard syringe is
•Typically delivered at 5 ul/min using the syringe pump 1 mL, 4.61 mm i.d.
remove the spacer under the nebulizer mount.
Use the Agilent ES Tuning Mix to calibrate your instrument. You can introduce
the tuning mix using the syringe pump. If you want to introduce the tuning mix at
a higher flow rate, tee the syringe pump flow to that of your HPLC. The tuning
mix expected masses are already saved to the automated calibration files.
To operate your syringe pump, push the select key to display Menu options. You
can scroll through menu options by pressing the corresponding arrow key
repeatedly. To prime the syringe pump press Start and the right arrow
simultaneously. The stop/run key controls the pump flow.
283
Calibration
Calibration
Infusion With or Without HPLC Flow
From HPLC
Syringe pump
Tee for
HPLC
flow
•Calibrant delivery flow rate

is 1 –10 µl/min with syringe
pump only
To nebulizer
•Calibrant delivery flow rate

is 10 µl/min to 1 ml/min
You can calibrate the ion trap either at low or high flow rates. Tuning, using
either the calibration solution or a sample standard can be done at the desired
analysis flow rate and conditions. Therefore it may be done with either the HPLC
and syringe pump or the syringe pump alone.
284
Calibration
Calibration
Performing Calibrations: Loading Calibration

Files
Two different mass range modes:

– Standard mass range (50–2200 m/z)
– Extended mass range (200–4000 m/z)
• Three different mass resolutions
• To load a mass scan calibration file

– From the Options menu select the desired Scan Calibration File
• Current
• Default
• Backup
The LC/MSD Trap has two mass range modes: Standard mass range (50-2200
m/z) and Extended mass range (200-4000 m/z). The Standard mass range can be
operated in three different mass resolution modes.
Your data system saves three different calibration files, a current file, a default
file, and a backup file. You can find out which calibration file you are currently
using by opening the Options menu and checking which of the three calibration
files has been selected.
The Default calibration was generated during the installation of your instrument.
This file cannot be overwritten. You can always return to this calibration and
restart tuning and/or calibration from the setpoints in this file.
The Current calibration is most often used as the starting point for a new
calibration. When a new calibration is generated, the previous current calibration
becomes the new Backup calibration. It is always possible to return to the
previous calibration if an error should happen during calibration.
285
Calibration
Calibration
Checking the Calibration

•Make certain the current calibration is loaded
•Set the ICC target to 30,000 counts, Averages 5, Rolling Average 2
•In the Tune tab, enter appropriate flow and temperature settings
•For 5 ul/min: Nebulizer – 15 psi
Dry Gas - 5 l/min
Dry Temp –325 degrees C
Accu Time ≈ 0.5 msec (SL &
•Spacer removed
XCT) or 5 msec (Cl and VL)
0.23
322.20
118.20 Expected
118.09
622.20 322.05
622.03
Operate
Mass assignments in standard mass range should be within 0.2 amu

If the check fails, perform automated calibration 8
To check your calibration, first make certain the current calibration is loaded.
From the Options menu, select Scan Calibration. The Current option will most
likely be selected. Load an LC/MS method. Set the Ion Charge Control, ICC, to
200,000 (XCT) in the general trap parameters area. The charge control will limit
the number of ion charges filling the trap and preserving resolution. Set the
Averages to 5 and the Rolling Averaging to 2.
Select the Tune tab and set the following parameters for 5 ul/min: nebulizer = 15
psi, Gas = 5 l/min, and Dry Temp = 325º C. Make certain that the nebulizer
spacer is removed because we are using a low flow rate for the calibrant
introduction.
Infuse the ESI tuning Mix into the source at 5 ul/min. A spectrum as shown
above should appear. Check the accumulation time which should be a few
milliseconds for the Classic and VL models or approximately 0.5 msec for the SL
and XCT models. The higher the contamination level, the lower the accumulation
time.
286
Calibration
Check the mass assignments for the tune compounds. Compare the assigned
values to those listed on the previous slide. All should be within 0.2 amu.
287
Calibration
Calibration
Performing Calibrations: Automatic Calibration
• The LC/MSD Trap has two different mass range modes:

– Standard mass range (50–2200 m/z)
– Extended mass range (200–4000 m/z)
• Automatic calibration works with mass lists and calibrates each data point in this
mass list
• Standard mass range:
– Isotopes are resolved and 12C masses are used
• Extended mass range:
– Isotopes are not resolved and lists of average masses
are used
• Automatic calibration correlates the most abundant isotopic peak with the masses in
the mass list
– User must monitor that the tune compound’s 12C
isotopic peak is clearly the most abundant peak
9
This procedure describes how to automatically calibrate the mass axis as well as
calibrate for isolation and fragmentation. The automatic calibration routines work
with mass lists and calibrate each data point in the selected mass list. The mass
lists for the ESI tune mixture are installed with the software. It is suggested not to
modify them, but, if necessary, to modify and save the list to a new name. The
ions in the mass lists depend on polarity and mass resolution chosen. When the
standard mass range is selected, isotopes are resolved and 12 C masses are used.
For the extended mass range isotopes are not resolved and lists of average masses
are used. If you would like to use different tuning compounds, the mass lists can
be edited and saved via the menu. Since the automatic calibration routine
correlates the most abundant isotopic peak with the masses in the mass list, care
must be taken that the 12C isotopic peak in the tune compound is clearly the most
abundant peak.
288
Calibration
Calibration
• A new mass axis calibration can be started from an old

calibration file, Current, Default, or Backup
• To start Automatic Calibration:
– Adjust the Accumulation Time and Trap Drive
– Open the Calibration Page and select Auto
10
From the Calibration tab, set the instrument to Auto calibrate by clicking the
Auto radio button on the left hand pane.
289
Calibration
Calibration

• Click on the Mass List folder button and select a mass list
• Select the desired calibration

• Presearch adds time to
an autocalibration because
the window is 8 amu wide
as opposed to 4 amu wide
Unless you have problems,
leave unchecked.
11
Make sure that the correct Mass List has been loaded, otherwise click on the
folder next to the mass list and select the appropriate series of masses.
Remember, the ES Tuning mixture mass list comes with the instrument and
should not need to be changed or modified. If you check the Presearch box, the
window will be 8 amu wide as opposed to 4 amu wide. Generally, the 4 amu
window will work and is less time consuming. You also should avoid improper
mass assignment.
290
Calibration
Calibration
• Click on the Start button to begin
12
Start the automatic calibration by clicking Start in the Calibration tab.
291
Calibration
Calibration
• The instrument will finish calibration and display the following

window:
• Select the respective calibration check box to save the results as

Current
• The desired calibration file must be selected before making changes
13
When the instrument has completed its automatic calibration, the new calibration
will be displayed along with any comments or messages. In order to modify any
existing calibration files, the new file must be selected before continuing.
292
Multiplier
Multiplier
Verify Multiplier Setting •There is a factory established

baseline reference for EM gain
based on the isotopic resolution of
the 922 peak of the tuning
mix when scanning in the presence
of higher mass ions.
•Ensures gain remains the same as
detector conversion efficiency degrades.
•If multiplier replaced, the baseline will
need to be re-established by CE.
Automated algorithm to
check multiplier gain.
Raises the EM voltage

Leave at 100% until the gain is restored
to 100% of level found
In factory
14
Before continuing to the next calibration, you should check the multiplier setting.
If the multiplier voltage setting is too low, the ion trap my try to compensate for it
by adjusting to a longer accumulation time. This adjustment would cause more
ions to fill the trap causing space charging. Space charging can cause poor
resolution, mass shifting, and signal loss.
At the factory, your multiplier voltage was set using the EMGain.Ms method.
This method is used to set the initial multiplier voltage properly based on the
resolution between the A and A+1 mass peaks of the tune mixes’ 922 amu
component. The gain is adjustable but should be left at 100% of the level
determined by the factory. If the multiplier is ever replaced, then your CE will
have to re-establish the baseline voltage using the EMGain.Ms method in the
Service mode.
293
Calibration
Calibration
Performing Calibrations: Loading Isol/Frag

Calibration Files
• Certain frequencies are applied to the ion trap electrodes during
ion isolation and fragmentation
• Isol/Frag calibration automatically calculates the correct
frequencies during MS/MS operation
• The same Isol/Frag calibration is used for all mass ranges
• To load an Isol/Frag Calibration file:
– From the Options menu select the desired Isol/Frag Calibration File
• Current
• Default
• Backup
15
To perform MS/MS analyses, the LC/MSD Trap system applies certain

frequencies to the ion trap electrodes during the ion isolation and fragmentation
periods. The Isol/Frag calibration (isolation and fragmentation calibration) allows
the software to automatically calculate the correct frequencies. For all mass range
modes, the same Isol/Frag calibration is used.
294
Calibration
Calibration
Perform Isolation and Fragmentation

Calibrations
Make certain to perform the calibrations in order,

scan before isolation, before fragmentation.
Perform in the same manner as the scan calibration
16
A good isolation calibration depends upon a good mass scan calibration and a
good fragmentation calibration depends upon a good isolation calibration.
The Isolation and Fragmentation calibrations are performed in much the same
way as the scan calibration. The isolation consists of scanning all lower masses
out of the trap and then applying a waveform to the endcaps to remove all high
masses. The isolation is calibrated for each mass in the mass list.
When the calibration is complete, the auto calibration results will be displayed.
Click Save and the Current entry for the isolation or fragmentation calibration will
be updated.
295
Calibration
Calibration
Performing Calibrations: Editing the Mass List
• Mass lists for the Agilent ES Tune Mix are installed with the
software
– Do not change or delete the original mass lists!
• Mass lists can be edited and saved under another name
• Mass lists must be changed before loading
17
Several mass lists installed with the software agree with the delivered tuning mix.
You can edit and save them under another name when you want to adjust the
mass lists for other tuning mixtures. This change must be done before loading the
mass list. The original mass lists cannot be overwritten.
296
Calibration
Calibration
Performing Calibrations: Editing the Mass List
• To edit the mass list:

– From the Options menu select Edit Mass List
– Select a Mass List (e.g. ES Tuning Mix Neg)
– Make changes in the right hand column by adding or deleting masses
– Save the mass list under a new name
18
The above procedure describes how to edit a mass list to be used for a calibration.
This may be useful if you wish to include a known compound that can be used for
a calibration.
297
Optimization and Tuning

Optimization and Tuning parameters are saved with your method
and are method specific
•Optimize API interface parameters for best performance.
•Adjust the ion trap parameters (octopole, trap drive, cap exit, etc) for your
method and target compounds.
•Tuning and optimization are based on the method’s flow and composition
as well as compounds of interest.
•Infuse target compounds or use the tuning mix.
•Parameters are saved with the .M method.
•A good calibration and multiplier setting are necessary.
•Don’t forget Fragmentation Amplitude (must be done with target compound).
19
Once you are certain that your instrument can deliver the correct mass
assignment, it is time to optimize and tune the instrument for the method and
compounds of interest. The calibrations can be applied to any method, but the
optimization and tuning parameters are method specific.
You can optimize while infusing the tuning mix or your compound of interest. If
you infuse your compound of interest, expect to see residual background for quite
some time. Often, it is better to infuse the tuning solution and select a mass close
to your target mass for optimization and tuning. The exception to this is the
fragmentation amplitude optimization.
If your method will have a high flow rate, tee the tuning mix or compound of
interest to the HPLC flow. Make certain the nebulizer spacer is in place if
required for the method flow rate (high flow rates).
First, optimize the interface parameters for either electropspray or APCI. The
nebulizer pressure and temperature, capillary voltage, etc. are all dependent upon
your mobile phase flow and composition. Next, tune the ion trap optics
parameters so that you maximize the signal for your target mass range.
298
The last optimization is the Fragmentation Amplitude. This parameter is

important for target MS/MS operation and must be performed using the desired
compound, not the tune mix.
299
Optimizing and Tuning the LC/MSD Trap
• Involves five basic steps:
– Preparing and introducing the tuning or calibration standard
– Starting with a known set of method parameters
– Acquiring and viewing data from the instrument
– Altering API interface and/or MS parameters to optimize performance
– Saving the optimum parameters in a method file
20
The purpose of optimization and tuning is to find the settings for the API interface
and mass spectrometer that will maximize the signal and minimize the noise
associated with your analysis. The settings are determined empirically by
introducing a tuning sample with known characteristics and ions into the
instrument. The parameters are systematically varied to observe the effect on the
resulting signal.
300
Loading a Method File
Loading a Method File
Loading and Saving a Method File
• Load a previous method from the ChemStation Method menu
• If no previous method exists, or you would like to start from

the default settings, load the default ChemStation method,
Def_LCMS.M or the Ion Trap method Default.MS.
• Or…Check that all parameters are set correctly as a starting

point.
21
It is generally easy to start with a method that has already been developed and use
that as a starting point for optimizing the performance of the LC/MSD Trap. This
method can be loaded from the Method menu in the LC/MSD Trap control
software (MS only) or from the Method menu in the ChemStation (HPLC and
MS). When you are done optimizing your parameters you can save them under
the same method name or generate a new method name.
301
Optimizing Electrospray Conditions
Optimizing Conditions: Electrospray Conditions
• Electrospray Process:
– Aerosol generation
– Ionization
– Solvent removal and ion ejection
• Generation of ions is optimized by production of a very fine
aerosol while applying the proper potential field
• Flow rates from 1 µl/min up to more than 1 ml/min are
acceptable
• Optimum nebulizer pressure is a function of flow rate
– Low flow rates: ~ 7-15 psi
– Higher flow rates: up to 40 psi
22
The electrospray ionization process involves three basic steps:

• Aerosol generation
• Ionization
• Solvent removal and ion ejection
In simple terms the generation of Electrospray ions is optimized by the efficient
production of a very fine aerosol with the proper potential field applied. The
optimum nebulizer pressure depends on the flow rate. Make sure that the flow
rate is set appropriately for your application.
302
Optimizing Conditions: Electrospray Conditions
• Fix the End Plate Offset at –500 V

• Set the Capillary at –4000 V
• The End Plate is now at –3500 V
• Observed currents are a function of both flow rate and solvent
composition
• Buffers may increase the current as high as 2 µA
• The capillary voltage is set 500 V higher than the End Plate
23
Observe that the End Plate Offset is fixed at a difference of +500 volts from the
Capillary.
303
Electrospray Conditions: Typical Operating

Values
HPLC Flow Nebulizer Drying Gas Drying Gas

Rate Pressure Flow Temp.
(µL/min) (psi) (L/min) (°C)
1-10 10-15 4 325
10-50 15-20 5 325
50-200 20-40 8 350
200-500 30-50 8-10 350
500-1000 50-70 10-12 350
24
Shown above are typical electrospray conditions for various HPLC effluent flow
rates.
304
Optimizing APCI Conditions
Optimizing Conditions: APCI Conditions
• APCI Process:
– Gas phase ionization
– Mobile phase and analyte must be quickly vaporized before the corona
discharge needle
– Uses high temperature to convert the liquid to vapor
– Basic ionization process:
• Nebulization
• Vaportization
• Ionization
– The nebulization process is similar to that in electrospray
25
Under APCI conditions, the ionization takes place in the gas phase. This fact
requires that the mobile phase and the analyte to be vaporized before they reach
the corona discharge needle. For this purpose the APCI source includes a high
power vaporizer (APCI Temp) to convert the liquid flow to vapor. The basic steps
for APCI ionization are:
• Nebulization
• Vaporization
• Ionization
In addition to an optimized nebulization process as with Electrospray, it is
necessary that the vaporization step fully vaporize the analyte and the mobile
phase. Under typical conditions this is accomplished with the APCI Temperature
set to 350 °C.
305
• Optimum conditions are a function of:

– Solvent
– pH
– Corona current (typically ~ 4000 nA)
• The End Plate Offset is fixed at –500 V
• Set the capillary at –4000 V
• The End Plate is now at –3500 V
Need new screencapture
26
Following vaporization the chemical ionization process is accomplished by means

of a corona discharge needle in the presence of gaseous mobile phase and analyte.
Optimum conditions are established by the proper choice of solvent, pH, and
corona current. The above settings are a good starting point for optimization of
APCI conditions.
306
• Typical starting values with conditions with 1:1 Water/Methanol:

– HPLC Flow Rate: 200-1500 µL/min
– Nebulizer Pressure: 60 psi
– Drying Gas Flow: 4 liters/min
– Drying Gas Temperature: 350° C
– APCI Temperature: 300-500° C
• Optimize the signal while directly infusing the target analyte
27
The table above lists typical starting conditions if the Water/MeOH is 1:1.
307
APCI Conditions: Typical Operating Values
HPLC Flow Nebulizer Drying Gas Drying Gas APCI

Rate Pressure Flow Temp. Temp
(µL/min) (psi) (L/min) (°C) (°C)
200-500 50 3-5 350 350-475
500-1000 65 3-5 350 375-499
1000-1500 70 3-5 350 400-499
28
The table above contains typical operating parameters for the nebulizer pressure,
drying gas flow, drying gas temperature, and APCI temperature for varying
HPLC flow rates ranging from 200-1500 µL/min.
308
Tuning
Tuning
Tuning
• Set the system in Operate mode
• Start method flow conditions using tuning mix or

standard
• User can now optimize in one of the following modes:
– Smart tune
– Optimize tab using Smart Ramp
– Expert tune
29
When optimizing conditions for the LC/MSD Trap, there are three basic methods:
Smart Parameters (SPS)
Optimize tab smart ramping, and
Expert Tuning.
309
Tuning
Tuning
Two Tune Modes – Smart and Expert

Parameter Ramping
Smart: lens and trap

parameters set
based on user inputs
regarding compound
to be analyzed (SPS)
Expert: complete
control over lenses and
trap parameters; for
experts and service
personnel
30
The Smart tune option is easy to use. Enter a target mass and the ion trap
parameters will be adjusted for that mass range based upon default settings.
Parameter Ramping, the next option, is actually done in the Optimize tab.
Expert tuning allows you to completely control all lenses and trap parameters.
This option should only be used if you are an expert or by service personnel.
310
Tuning
Tuning
Smart Tune
•Optimize the capillary, nebulizer pressure, drying gas flow and temperature.
•Set the ICC on, Target, Max. Acc Time, Averages, Rolling Averages and
scan range.
•Make certain you do not have any current segments set.
•If using a higher flow rate, install the nebulizer spacer.
•Type in the Target Mass
•The SPS will automatically set appropriate values for the ion optics
•Or, select the mouse maximum cursor to select an ion in the
spectrum window, then right-click and select Run SPS
Enter target mass
Start with 50% for unknown

compound stability, 100%
for very stable compounds
Usually set at 100% (screening)
Normal for target analyses

Wide for screening analyses
31
To use the Smart Tune page, first make certain that you have optimized the
interface parameters. Next, set the ICC to on. The default Target is 30000, Max.
Accu Time is 300 msec, number of Averages is 5, Rolling Averaging = 2, and
scan range from 100 to 2200. Make certain that you do not have any segments in
the current method as you don’t want acquisition parameters to change while you
tune. If you are using a high flow rate, make certain that the nebulizer spacer is in
place. Open the Tune tab and select the Smart tune radio button. Type in the
Target Mass. This can be a tune mass that is close to the desired mass of your
analyte. Next, fill in the compound stability. Typically, start at 50% unless your
analyte has good stability. Set the Trap Drive to 100%. Select Normal for target
analyses and Wide for screening analyses.
Default parameters for your settings will be chosen automatically. Alternatively,
you can select the Mouse Maximum Cursor and right-click in the line spectrum
window next to the desired tuning mass. Select Run SPS from the pop-up menu.
311
Tuning
Tuning
Smart Tune Page: Trap Drive Settings
• Trap Drive:
– Higher field = more optimal the conditions for higher m/z ions
– Lower field = more efficient for trapping low m/z ions
– The field strength is set by the Trap Drive parameter
m/z Trap Drive

Value Value
50-400 50
400-800 60
800-1200 70
1200-2000 80
32
Examples of appropriate trap drive settings for given mass ranges are shown
above.
312
Tuning
Tuning
Tuning: Optimizing Parameter Ramping
• Open the Optimize tab.
• Enter the Target Mass.
• Select the parameter to

be optimized.
• Enter the number of averages (default is 2).
• Enter the parameter Ramp Range.
• Select the Adapt Scan Range check box to arrange the mass range into
the chromatogram window.
• Click Start to begin optimizing.
33
From the Optimize menu, individual parameters that appear in the Expert tuning
menu can be optimized.
Using this optimization strategy, the strength of an ion signal is monitored while
one or more parameters are incrementally increased. This procedure may increase
optimal ion signal from that of the Smart tuning results.
313
Tuning
Tuning
Tuning: Optimizing Parameter Ramping (cont.)
• Ramping status window will close

• The optimized parameter value is set as the actual new value
• Click Undo to reject the value and replace with previous value
• Continue optimization with the next parameter
34
The user can individually ramp each parameter and thereby optimize each
parameter. This procedure should not need to used routinely, however, it should
be used if the Smart tuning procedure does not yield adequate results. In the
above example, the user has selected to ramp the Skim 1 voltage and a target mass
has been selected. Note that a default ramp range will appear for each parameter
although this can easily be modified by manually entering the value.
314
Tuning
Tuning
Tuning: Manually in Expert Tune Tab

• You can optimize the ion signal manually
• Choose the parameter to adjust and visually watch the change

in ion signal while changing the parameter
• Adjust parameters starting in the direction of ion flight:
– First optimize source parameters

– Next optimize the interface
parameters
– Finish with the trap parameters
(ICC, etc.)
35
Finally, the user can optimize the instrument parameters manually by adjusting
voltages and visually inspecting the ion signal and intensity. Since voltages will
affect the ions in the direction of ion flight, it is advisable to optimize the voltages
in this order. Do not forget that a voltage upstream from other voltages can have
significant effects on the overall ion signal.
315
ICC
ICC
Optimizing Conditions: Optimizing ICC

Parameters
• ICC automatically adjusts the accumulation time.

• To optimize ICC first switch off ICC.
• Open the Mode page and access the Filters menu
– Set Gauss Filter to On
– Set Normalize to Accu Time On
36
Ion Charge Control (ICC) automatically adjusts the accumulation time. This
setting becomes imperative if the ion concentration changes during an analysis
(for example, during an LC- or CE-run). In these cases, as well as in many others,
the ion accumulation time must be set automatically by ICC.
316
ICC
ICC
Optimizing Conditions: Optimizing ICC

Parameters
• Set the accumulation time

– Should be under constant concentration conditions
– Set as high as possible without overloading the trap
• Enter the ICC Target Value

– Defines the set value for the ICC (typically 50000)
• Enter the Maximum Accumulation Time

– Ensures the system reacts quickly when a peak elutes
• Now switch the ICC On
37
The above procedure describes how to set the ICC and optimize the settings. You
generally want to set the accumulation time as high as possible. However, be
careful not to overload the ion trap. When the ion trap is overloaded, the mass
peaks become broad and shift to higher m/z values. The first evidence of
overloading is observed when the mass difference between two isotope peaks is
slightly less than 1 amu. If a change in the signal shape or a mass shift is visible
the trap already is heavily overloaded.
317
Data File Size
Data File Size
Optimizing Conditions: Optimizing Data File Size
• The file size of profile data will be large.
• In the Mode menu, you can select only line spectra or choose
to have profile data acquired with the line spectra
• A threshold may be set by checking the threshold button and

setting the cutoff
38
Line spectra are acquired by default. To select profile spectra, you must explicitly
select the appropriate setting in MSD Trap Control software. Perform this
procedure in the Mode tab by selecting the Save profile spectra option.
318
Optimizing MS/MS
Optimizing MS/MS
Optimizing MS/MS
• Optimize MS/MS fragmentation using the actual compound of

interest.
• Infusing the compound of interest may lead to significant
compound background response – take care.
• Do not use the tuning compound as its fragmentation
amplitude may be very different from your target compound.
• Three choices
– Use SmartFrag – automatic
– Automatic Fragmentation Amplitude Ramping – requires infusion
– Manual Fragmentation Ramping- multiple injections of target
compound
39
There are different levels of MS/MS optimization. If you are performing

qualitative work where a large number of fragment ions are preferred, then use the
SmartFrag option. This option is automated and works with complete unknowns
to fragment different types of precursor ions successfully. If however, you want
to maximize your sensitivity for quantitation or maximize the intensity of just a
few ions, then SmartFrag should be turned off and optimization performed for
your method.
319
Optimizing MS/MS
Optimizing MS/MS
SmartFrag
• Automatic during acquisition
• Start Ampl and End Ampl give the starting and ending amplitude for
SmartFrag
– Relative to the fragmentation amplitude
– Default values:
• 30% Start Ampl
• 200% End Ampl
• SmartFrag makes the fragmentation amplitude an uncritical parameter
without reducing the duty cycle
• SmartFrag should be used for qualitative work where an abundant amount of
fragment ions are preferred
40
You can access SmartFrag from the MS(n) tab. Select the Fragmentation button
then turn SmartFrag on by selecting the option. The default options for
SmartFrag work well for qualitative analysis.
The values Start Ampl and End Ampl give the starting and ending amplitudefor
SmartFrag relative to the fragmentation amplitude selected in the MS(n) page.
Default values are 30% (Start Ampl) and 200% (End Ampl). Using SmartFrag
makes the fragmentation amplitude a non-critical parameter without reducing the
duty cycle of the measurement.
320
Optimizing MS/MS
Optimizing MS/MS
Automatic Fragmentation Amplitude

Optimization
1. The compound of interest must be infused.
2. Turn off SmartFrag.
3. Enter MS/MS tab. Check Isolation On and enter desired precursor mass.
4. Check Fragmentation On and enter a value for the Amplitude, 1.00.
5. Locate the product ion and enter the value into the Target mass on the Optimize tab.
6. Click Start.
41
For quantitative or target analysis, you can perform automated fragmentation

amplitude ramping. This process requires infused target compound. Infusing the
target compound may not be advisable due to the possibility of contamination.
Make certain that SmartFrag is turned off. Enter the MS/MS tab, check Isolation
On and enter the desired precursor mass. Check Fragmentation On and enter a
typical value of 1.00 for the Amplitude. Locate the product ion of interest and
enter the value into the Target Mass on the Optimize tab. Make certain you have
selected Fragmentation Amplitude for the ramp. Start the ramp and save the
result to your method.
321
Optimizing MS/MS
Optimizing MS/MS
Manual Fragmentation Amplitude Optimization
1. Setup repetitive injection of the target compound using the ChemStation

Injector program.
2. Start the acquisition and increase the Fragmentation Amplitude manually
after each peak elutes. Find the best Fragmentation Amplitude by
monitoring each injection in real-time.
42
If you cannot infuse your target compound, then you can make multiple
injections. Before each new injection, change the Amplitude. Watch the real-
time plot to see what amplitude is best. Multiple injections within one data file
can be set up in the injector parameters injector program.
322
Laboratory Exercise: LC/MSD Trap Tuning
Optimizing MS/MS
Laboratory Exercise: LC/MSD Trap

Tuning
Prior to tuning the Ion Trap you should check vacuum status and make certain
that the calibration is still adequate.
In this lab you will explore two tuning modes:
• Smart Tuning with the LC/MSD Trap
• Expert Tuning with the LC/MSD Trap
323

• HPLC ChemStation B.01.xx software.
Ion Trap 6.0 software.
To perform the lab with instrument:
• The MSD Trap with the electrospray interface in the Operate mode
• An infusion pump and syringe
• Agilent 1100 pump
• Mixing tee
• The Agilent Tuning solution
Note that when optimizing for your specific compound of interest, solvent flow
should mimic anticipated flow conditions during actual analysis. The flow rate
may require that you “T” the infusion into the appropriate LC flow.
324
Tuning the LC/MSD Trap
Tuning the LC/MSD Trap

In the example below, you will optimize the signal sensitivity and the
fragmentation amplitude. To become familiar with each mode of tuning, we will
tune the instrument in each of two modes; Smart and Expert
Remember, tuning involves five basic steps:
• Preparing and introducing the tuning standard.
• Starting with a known set of method parameters.
• Acquiring and viewing data from the instrument.
• Altering API interface and/or MS parameters to optimize performance.
• Saving the optimum parameters in a method file.
We will be tuning and optimizing the instrument conditions for the m/z 622 ion in
the Agilent tuning solution in the electrospray mode.
325
Smart Tuning
Smart Tuning
1) Set up the infusion pump to deliver the tuning solution at 5 µl/min.
2) Set up a mobile phase on the HPLC consisting of 75/25 Methanol/water with
5mM ammonium formate.
3) Start by opening a preexisting or default method for the LC/MSD Trap. Go
to the HPLC ChemStation and select Method > Load Method. Choose
DEF_LCMS.M if selecting the default method.
4) Note: The default method, DEF_LCMS.M is not available in the offline
mode. Load DEF_MS.M if you do not have an instrument to use for this
lab.
5) While in the HPLC software, set the flow rate to 400 µl/min. Tee in the ESI
tuning mix. If you are using these flow rates, greater sensitivity can be
achieved by installing the nebulizer spacer.
6) Open the MSD Trap Control software and click on the Tune tab.
7) Enter the following settings for the XCT:
• ICC = On
• Smart Target = 200,000; XCT Ultra = 500,000
• Max Acc Time = 300 sec
• Number of Averages = 5
• Rolling Averaging = 2
• Scan range: 100-2200.
326
Smart Tuning
Click Apply. The above settings do not apply to VL or SL models.

8) Select the Smart Tune radio button and adjust the source conditions to:
• Nebulizer = 40 psi
• Dry gas = 9 L/min
• Dry Temp = 350oC
Click Appply.
Note: Adjust these parameters according to your total flow rate. See the Quick
Reference Guide for details.
9) Select the Mode tab.
10) Make sure the instrument is set for the following conditions:
• ESI source
• Positive polarity
• Standard-Enhanced (select the mode for your method).
11) Save the HPLC-MS method to a unique name, Method > Save Method As.
12) In the MSD Trap Control view, select the Mode tab. Divert the valve to
source.
13) Go to the Tune tab.
14) Set the Target Mass to 622.
15) Set the Compound Stability and the Trap Drive Level to 100%.
16) Set Optimize to Wide.
17) Using the mouse cursor, click to the right of the m/z 622 peak in the Line
Spectrum window. Select Run SPS.
18) Smart Parameter Settings will now run. Notice that the Spectrum window
has changed to optimize on the m/z 622 ion (if online).
327
Smart Tuning
19) Select the Optimize tab.

20) Select Oct 1 DC from the pull-down list next to Smart Ramp. Enter a
Target Mass of 622 and click Start. (XCT and SL only). Select Octopole
Delta for the VL or Classic instead of Oct 1 DC.
21) Next, optimize the Trap Drive and Cap Exit (Offset for VL and Classic) in
the same manner.
22) Resave the HPLC-MS method.
328
Smart Tuning
Optimize the fragmentation (MS/MS)
You should use your compound of interest for fragmentation optimization. In this
example, we will use the ESI tuning mix ion at 622 with two target ions at 540
and 478.
1) Click on the MSn tab.
2) Check the Manual MS(n) radio button and check Manual MS(n).
3) Select the Fragmentation button.
4) Make certain that the SmartFrag On checkbox is cleared. Click Apply and
then Close.
5) Return to the MSn tab. Verify the following settings:
• Isolation = On
• Mass = 622.1
• Width = 4
• Fragmentation = On
• Amplitude = 1
6) Go to the Optimize tab.
7) Select Smart Ramp = Frag Ampl. Select 540 as the Target Mass and then
optimize by pressing Start.
329
Expert Tuning
Expert Tuning
1) Now select the Expert setting in the Tune tab. Notice that you can now
manually change all of the MS settings. To manually adjust all settings
can be very time consuming, here we will look at the effect of adjusting
only one parameter. Here, we are looking at the 322 ion.
2) To demonstrate the danger of setting these parameters manually we will
adjust the voltage on Lens 1. Set the voltages as shown above, making
sure you have a good signal for the m/z 322 ion.
3) Once the signal is stable, change the Lens 1 to a positive (+) 5.0.
4) You should have completely lost your ion signal (if online).
5) Now change the setting back to -5.0. Your signal should return.
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Expert Tuning
6) You can go into the Optimize tab and automatically optimize each
parameter.
7) Go ahead and ramp the Lens 1 voltage from -10.0 to -1.0.
8) In the Expert tab you can also select the Detector and Block Voltages by
clicking on the button found in the Tune tab.
9) Set the Multiplier voltage to 0 and watch the ion signal.
10) Now set the multiplier back to the original setting.
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Adjusting Gas Flows
Adjusting Gas Flows

1) Let’s select Smart tuning.
2) Manually adjust the Nebulizer, Dry Gas, and Dry Temp and watch the
effect on the ion signal.
3) Remember, it may take some time for the Dry Temp to achieve the new
setting.
4) Once you have optimized the gas flows you are finished tuning.
5) Compare the results you got from each method of tuning. You will most
likely realize that the Smart Tuning method will work very well and very
quickly for your analysis. Other Tuning methods do however allow you
flexibility to adjust each parameter of the instrument
332
Agilent 1100 Series LC/MSD Trap: Flow
Injection Analysis
Agilent 1100 Series LC/MSD Trap: Flow Injection Analysis
In This Section
In This Section
• The purpose of flow injection analysis.
• How to do flow injection analysis with the MSD Trap.
• Specific concerns when doing flow injection with the MSD

Trap.
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Definition
Definition
Flow Injection Analysis: Definition
• Definition:
– The analysis of a sample, injected into a flowing stream,
without performing a separation.
• Very simple technique when a rapid result is needed to

evaluate a specific question
Flow injection analysis is the analysis of a sample, injected into a flowing stream
of mobile phase, without performing a separation. FIA may be performed
manually with a loop based injection valve, or can be done utilizing an auto-
injector. Because no separation (i.e. chromatography) is taking place, analysis is
usually very rapid and is only limited by the amount of time it takes the mobile
phase flow to deliver the injected analyte(s) to the ion source.
335
Purpose
Purpose
Flow Injection Analysis: Purpose
• Flow injection analysis is useful for:

– Checking for an ion signal before you begin sample analysis
– Quickly Evaluating CID spectra of an analyte
– Quickly evaluating the effect of individual instrument parameters on ion
signal
– Quickly checking for sensitivity and linearity
• Samples for FIA should be completely free of any interfering

matrices
– e.g. standard stock solution in mobile phase
FIA is useful for several techniques. With the LC/MSD Trap, the most useful
purpose is to check the sensitivity and presence of signal for your analyte of
interest prior to a long analytical run involving chromatography. By injecting a
standard solution of your analyte of interest at a known concentration, a direct
comparison of day to day reproducibility can be done on the instrument in a very
short time. Be aware that since we are not separating any of the matrix
components or interferences, all of the constituents of the sample will reach the
ion source at the same time. This could result in suppressed ionization and lack of
reproducible signal. It is for that reason that you should always use the “cleanest”
solution for FIA (e.g. a stock solution in mobile phase).
336
FIA with the MSD Trap
FIA with the MSD Trap
Flow Injection Analysis: FIA With the MSD Trap
• Flow Injection Analysis can be performed with either a

manual injection valve or an Agilent 1100 autosampler.
• Set up multiple injections in separate files using the

ChemStation sequence table.
• You can perform multiple injection within one data file using
an injector program.
There are ways to perform FIA analysis with the MSD Trap through the use of
sequences or injector programs. The next few pages will review how to set up
this process.
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Setting up the Instrument
FIA With the MSD Trap: Setting up the Instrument
• Start by opening the ChemStation software from the Start

menu.
• Open the optimized method file for the compound of interest.
• Make sure that your analytical column is not connected in line

with the flow.
• Start the flow of your mobile phase.
In order to prepare the instrument for FIA you must first activate the ChemStation
and load your method file for the compound of interest. Make sure that you have
started your HPLC flow, but do not have a column connected in line. If you do
not have adequate back pressure on the solvent delivery system, you may not get
reproducible peaks. If necessary, put a resistor in line (a segment of very low I.D.
tubing) that will increase the back pressure and make the flow uniform throughout
the system.
338
• Make sure that the appropriate sample

has been placed in the autosampler.
• From the Method and Run Control

view, select the Instrument menu
and Set up Injector….
To perform an FIA run, assuming the method is open and flow has started:
Make sure that the appropriate sample is ready to be injected.

Remember this can be either by auto-injector or manual injection
2. From the Method and Run Control view of ChemStation, select Instrument
then Set up Injector….
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• This opens the Injector dialog box.

• The instrument can be set to:
– Perform a standard injection,
– Inject and then wash the needle with the contents of a specified vial,
– Perform a customized injection sequence.
Now that the injector dialog box has opened, you can select either a standard
injection (or injection with needle wash), or you can utilize an injector program.
Here you can set the injection volume and indicate the wash vial if required.
340
• After injection parameters are finalized, set up a sequence table with the vial
number to be injected.
• Enter in comments for the sample.
• Enter other samples to be injected.
• Start the sequence from ChemStation.
One way to set up the instrument to perform FIA is to utilize the sequence table
option in ChemStation. Here we are specifying our descriptors for Vial 2 which
contains our analyte of interest. Remember to put in the correct method file and
make the appropriate number of injections per sample. The Sequence Table can
be found under the Sequence menu in the Method and Run Control view.
341
Specific Concerns
Specific Concerns
FIA With the MSD Trap: Specific Concerns
• Using the sequence table to inject your sample, uses a specific

method.
• Therefore, modification of parameters would need to be done
in separate method files to see the effect.
– e.g. create two method files with one parameter changed in each; then
perform two flow injection analyses
• For optimization of ionization conditions it is much easier to
infuse your compound of interest and utilize the Optimize
window of the MSD Trap Control window.
10
When using the sequence table to run an FIA, remember that each line in the
sequence table uses one method. That would be fine for checking sensitivity or
simply for an ion signal, but if you are looking to quickly optimize a parameter
for your analyte, you will probably have to generate multiple method files and
multiple sequence table lines. For this reason it is much easier to infuse your
compound of interest to optimize voltages and instrument settings.
342
Injector Programming
Injector Programming
FIA using Injector Programming

•Create an initial method.
•Instrument then Setup Injector…
•Select Use Injector Program
•Insert the injector program defining multiple injections in the same file.
•Run the method and optimize the parameters as before.
•Open one data file to view data.
11
Note also that you may use the injector program to set up multiple FIA analyses.
Different from using the sequence table (which will save each injection into a
different data file) the injector program will do multiple injections and save them
into the same data file. You can modify parameters in the method as the
injections are being made to see an effect.
343
Specific Concerns
Specific Concerns
FIA with the MSD Trap: Specific Concerns
• The greatest utility of FIA on the LC/MSD Trap is to check your ion signal and
sensitivity before beginning analysis.
• Ion intensity with FIA should be fairly reproducible if:

– LC composition is similar
– The method file has not been altered
– LC flow conditions are similar
– Absolute amount of compound injected is identical
– The compound has not degraded since the previous injection
– There are no interfering matrix ions in the sample
(ideally the compound of interest will be dissolved in a solution identical to the mobile
phase)
12
Most of the time you will use FIA to check sensitivity before a long HPLC/MS
run. Several factors govern reproducibility of ion signal from day to day and
injection to injection. Some of these are listed above. One thing to always keep
in mind is that if the matrix is complex, the unwanted constituents of that matrix
may interfere with any analysis or quantifiable result you are trying to obtain with
FIA.
344
Laboratory Exercise: Flow Injection
Analysis
Laboratory Exercise: Flow Injection Analysis
In This Laboratory Exercise You Will:
In This Laboratory Exercise You Will:
If online:
• Prepare to set up a flow injection analysis.
• Perform a flow injection analysis to assess instrument sensitivity.
• Check the sensitivity of caffeine’s m/z 195 ion.
If offline:
Step through the process with the software.
346
To Complete This Laboratory Exercise You Will Need:
To Complete This Laboratory Exercise You Will

Need:
• Αgilent ChemStation B.01 or >.
• LC/MSD Trap software, version 6.0.
• The method, CAF_FIA.M.
If online:
• A stock solution of caffeine diluted to 50 ng/mL in 100% MeOH
• The Agilent 1100 series pump to deliver LC flow to the MSD Trap.
347
Flow Injection Analysis

1) Start by opening the Agilent ChemStation software from the Start menu.
Open the Offline system if you do not have an instrument present.
2) After ChemStation loads, open the MSD Trap Control window, open the
optimized method file for caffeine, Method > Load-MSD Trap Control
Part.... Select CAF_FIA.M.
3) Go back to the ChemStation software and make certain CAF_FIA.M is
loaded.(Load if necessary – Offline).
4) Make sure that your analytical column is not connected in line with the flow.
5) Place a vial containing a caffeine solution of 50 ng/mL into position 1 of the
autosampler, if an instrument is present.
6) Now open the LC part of the Method from the ChemStation window by
selecting Edit LC Part.… (Edit Entire Method if Offline)
7) The Edit Method dialog box will open
348
8) Select Instrument/Acquisition only. Click OK.

9) Set the Flow to 0.5 mL/min with a solvent composition of 20% H2O in
reservoir A and 80% ACN in reservoir B. Set the Stop Time for 1.00 min.
10) Make sure the Min Pressure Limit is set to 0 bar and click OK to continue.
Select Standard Injection with a volume of 10.0 µL. Click OK to continue.
11) If you have a column thermostat, select Not controlled and click OK to
continue.
12) Click OK for a UV or Diode Array system that may be inline with the MSD
Trap.
13) When you finish be sure to save the method file, Method > Save Method
As.
349
14) Switch the system on from the ChemStation view (online only).
15) This will begin solvent flow and set the instrument up for analysis.
16) Once solvent flow is stable, make sure there is adequate back pressure on the
solvent delivery system.
17) Select Sequence > Sequence Table in the ChemStation Method and Run
Control view.
18) Enter the vial location as “1” and the sample name as “Caffeine Std”.
19) Select the method that you saved and indicate the Sample Type as “Sample”;
set the instrument to make two injections per location.
350
Press OK.
20) Now open the Sequence Parameters dialog box from the Sequence menu
in the ChemStation window.
21) Make sure that the Data File is being written to the correct subdirectory and
location. Set the prefix and counter accordingly. OK the dialog box.
22) Once the sequence parameters have been finished save the sequence from
the Sequence menu.
23) Return to the MSD Trap Control window and make sure that the Trap is
configured to operate in MS mode, not MS(n), and that the scan range is
from m/z 100-400.
24) Since the MS method should be optimized for caffeine analysis, we will now
proceed with the FIA analysis.
25) From the ChemStation window select the Run Control menu, then Start
Sequence. The ChemStation will load the method and sequence files and
begin to prepare for sample analysis. When the ChemStation is ready, the
autosampler will inject the sample and data will be acquired.
26) When the analysis is complete open the Ion Trap Data Analysis software to
view the FIA data. If you were offline, open Caf00005.d and Caf00006.d.
Since we made two injections, there will be two separate data files.
351
27) Open both data files and select the extracted ion chromatograms for m/z 195
with a width of +/- 0.5. This will overlay each injection in one window.
Right-click the chromatogram window and select Edit Chromatogram. In
the Type drop box, select Extracted Ion Chromatogram. In the Masses
field, type 195. Add and OK. Select only the EIC from each datafile for
display as below.
28) Chromatogram traces should overlay and be reproducible; however, it is

common to see slight variability from injection to injection.
29) In order to assess instrument performance on a daily basis, chromatograms
of identical injections should be made before each analytical run and
compared.
30) This comparison can be done by overlaying chromatograms from different
days (assuming identical instrument and LC parameters).
352
Agilent 1100 Series LC/MSD Trap: Data
Acquisition
Agilent 1100 Series LC/MSD Trap: Data Acquisition
In This Section
In This Section
• How to select the correct operating mode.
• How to optimize the spectrum.
• How to operate the LC/MSD Trap in MS, MS/MS, and MS(n)

mode.
• How to optimize MS/MS operation.
• How to acquire data files.
• How to use the AutoMSMS function of the LC/MSD Trap.
In this section, we will discuss acquisition parameters.
354
Overview
Overview
Data Acquisition: Overview
• Four basic steps:
– Configuring the instrument hardware for the analysis.
– Selecting the basic Operate state of the instrument.
– Viewing data and customizing the operating parameters for the

particular analysis.
– Opening data files and saving data to disk.
The four basic data acquisition steps are outlined above.
355
Selecting the Operating Mode
Selecting the Operating Mode
Data Acquisition: Selecting the Operating Mode
• You will need to determine the following:

XCT Values
– How you would like to save your spectra
50-2200 u
– Polarity of the ions to be analyzed
– Scan range required Speed=26,000 u/s; 0.6 FWHM
– Scan type and resolution
Speed=8,100 u/s; 0.35 FWHM
– Position of divert valve
Speed=800 u/s; 0.25 FWHM
200-4000 u
Speed=27,000 u/s; 3 FWHM
Before any data can be acquired, a set of instrumental parameters needs to be

decided upon. These include the settings in the Mode tab. For instance, you need
to decide if you need to acquire spectra in profile mode or just as line spectra.
You can save space by only saving spectra above a given threshold. The scan
mode is an important choice. If you have an XCT Ultra, there are four types of
scans available. The scan speeds and resulting peak widths are outlined above.
356
Filters Button
Filters Button
Data Acquisition: Filters Button
• Open the Filters view by clicking on the Filters button.
• Switch Gauss Filter On by checking the box.
• Set standard widths.
• Normalize to accumulation time.
The Filters button is available on the Modes tab. The Filters dialog box allows
you to set the standard widths of ions as well as to normalize the signal to the
accumulation time. Note that if ICC on the Main Panel is deselected then this
check box will be grayed out. Normalization to accumulation time simply scales
the mass spectral data based on the actual accumulation time. As a consequence,
the intensity is proportional to the sample concentration.
357
Optimizing the Spectrum
Optimizing the Spectrum
Data Acquisition: Optimizing the Spectrum

• Several spectral parameters can improve data quality:
– Start and stop masses within the scan range
– The number of individual scans averaged before being sent from
the LC/MSD Trap to the data system
– Rolling averaging for low level signals
– The use of ICC for the automatic control of accumulation time
ICC Values: Classic, VL, SL = 30,000

XCT = 125,000
XCT+, XCT Ultra = 500,000
Because the scan parameters that appear on the right side of the panel are
fundamental and apply to all scans whether MS or MS/MS this part of the control
panel is always accessible. Optimizing these parameters requires that you have a
simple understanding of how each of these parameters effects the production of
each mass spectrum.
358
Scan Range
Scan Range
Optimizing the Spectrum: Scan Range
• By minimizing your scan range to only the masses of interest

you will:
– Decrease the time needed for scanning the mass spectrometer

– Allow more time to accumulate useful ions
– Perform a greater number of averages resulting in a smoother
signal
– Use less disk space to store the spectra
• You will need to re-optimize the ICC
The start and stop masses determine exactly the range of data that will be scanned
by the LC/MSD Trap. Minimizing this range to include only the masses of
interest has the following benefits:
• Reducing the scan range reduces the time for each scan.
• More time to accumulate useful ions.
• A shorter scan time allows more averaging and less noise.
• A smaller scan range requires less disk space to store the spectra. This is
particularly important for the storage of profile data.
• A scan range change causes an adjustment of the ICC target value as the
optimal ICC value will be different.
359
Averages
Averages
Optimizing the Spectrum: Averages
• Averaging several spectra provides the following benefits:
– A higher number of averages results in a more stable signal

– A higher number of averages reduces the data storage
requirements
– A higher number of averages does not reduce the fundamental
sampling rate and enhances signal to noise
• Averages and the MS/MS data mode

– MS/MS includes isolation and fragmentation steps
– MS/MS requires longer duty cycles
– MS/MS limits the number of averages
The high scan speeds and the efficient ion optics of the LC/MSD Trap make it
possible, under most conditions, to produce several spectra per second. Because
most applications do not require this high data rate, it is very beneficial to average
these spectra into a single spectrum at a reduced reporting rate. MS/MS
experiments must generally operate with a reduced number of averages
because MS/MS analysis includes isolation and fragmentation steps which
increase the cycle time.
360
Rolling Averaging
Rolling Averaging
Optimizing the Spectrum: Rolling Averaging
• Useful for detection of low levels of analyte in samples.
• Applies a moving filter to the data.
• Filter is user specified.
• Increasing the width of the filter greatly increases the dynamic

range in the reported spectra.
• Chromatographic resolution may be reduced.
Rolling averaging goes a step beyond normal averaging of the spectra. In effect
the use of rolling averaging is applying a moving filter to the data. When the filter
is turned on, you can specify the filter width. Increasing the filter width, however
will cause a reduction in chromatographic resolution.
361
MS Operation
MS Operation
Modes of Operation: MS Operation
• Simplest analysis cycle.
• Consists of the following steps:
– Accumulate ions in the LC/MSD Trap

– Produce a spectrum by ejecting the ions from the trap to the
detector
10
The user should realize that MS operation is implied when the functions on the
MS/MS page are not activated.
362
MS Operation
MS Operation
Modes of Operation: MS Operation with

Isolation
• User can isolate a single mass or mass range.
• Analysis cycle consists of the following steps:

– Isolate a specific m/z ion or ion range
– Produce a spectrum by resonantly ejecting the ions from the trap
to the detector
11
The first step towards the production of MS/MS spectra is accumulation of ions.
Before we produce the MS spectrum in the cycle described above we can insert an
additional isolation step. This method of isolating mass before producing the MS
spectrum is analogous to selected ion monitoring (SIM) analysis with a
quadrupole mass spectrometer.
363
MS Operation
MS Operation

Isolation
• In the MS(n) page select Manual MS(n).

• Click on the Manual MS(n) box.
• Deselect all boxes in the Manual group by clicking “All Off”.
• Select a peak at a certain m/z value as the precursor ion.
• Select Isolation On (click box).
12
The above procedure describes how to operate the instrument in MS operation

with Isolation.
364
MS Operation
MS Operation

Isolation
• Isolation is analogous to selected ion monitoring (SIM)

analysis with a quadrupole mass spectrometer.
• Sensitivity of the measurement increases by emptying the trap

of background at other m/z values.
• The use of isolation usually results in:
– Greater sensitivity and dynamic range because more signal is

available at the mass of interest
– Greater insensitivity to contamination in the sample
13
When the analysis only requires the measurement of a single ion it is possible to
increase the sensitivity of the measurement by emptying the trap of background at
other m/z values. This procedure makes it possible to have the maximum number
of ions per spectrum at the mass of interest. This type of analysis is most often
associated with LC/MS analysis where the amount of a substance is being
determined by the response detected in a chromatographic peak. When only one
ion of interest is required, MS with isolation may be considered.
365
MS/MS Operation
MS/MS Operation
Modes of Operation: MS/MS Operation
• User can monitor a precursor fragment ion transition.
– Accumulate ions in the LC/MSD Trap.

– Isolate a specific m/z ion.
– Fragment the isolated m/z ion.
– Produce a spectrum by resonantly ejecting the ions from the trap
to the detector.
14
The production of MS/MS spectra requires the inclusion of two steps in the
analysis cycle. The first step is isolation as described previously. The second step
is fragmentation.
366
MS/MS Operation
MS/MS Operation
• In the MS(n) page select Manual MS(n) and click the MS(n) box
• Unselect all boxes in the Manual group by clicking “Off”.
• Select a peak at a certain m/z value as the precursor ion.
• Select Isolation On (click box).
• Select Fragmentation.
• Set the Fragmentation Amplitude.
15
The above procedure describes how to operate the instrument in the MS/MS mode
using the Manual MS(n) settings.
367
MS/MS Operation
MS/MS Operation
• The MRM page can also be used to do MS/MS.

• A similar procedure is followed as in the Manual window.
• You can also do MS(3) from the MRM window.
16
Note that the MRM page can also be used to look at MS/MS as well as
MS/MS/MS.
368
MSn Operation
MSn Operation
Modes of Operation: MS(n) Operation
• User can monitor a precursor fragment fragment ion transition.

– Isolate a specific m/z ion
– Fragment the isolated m/z ion
– Trap/isolate specific m/z fragment ion (this cycle can be repeated “n”
times)
– Produce a spectrum by resonantly ejecting the ions from the trap to the
detector
• MS(n) operation can be done manually or by using context menus
17
MS(n) operation consists of multiple fragmentation and isolation events. Once

you are producing MS/MS spectra, it is easy to begin producing MS3 spectra.
369
Manual Operation
Manual Operation
MS(n) Operation: Manual Operation
• Follow the steps for Manual MS(n) operation.

• Select a peak at a certain m/z value as parent ion for the
second stage of fragmentation.
• Select Isolation and Fragmentation.
• These settings will perform an MS3 experiment .
• For MS(n) analyses, subsequent stages can be specified.
18
See how the inclusion of MS3 allows for the additional isolation/fragmentation
step in the analysis.
370
MS/MS Operation
MS/MS Operation
Data Acquisition: Optimizing MS/MS Operation
• There are several methods to improve the results of MS/MS

analysis:
– Setting the fragmentation CutOff.
– Using a second fragmentation.
– Setting the fragmentation time.
– Using SmartFrag.
19
The results of your MS/MS analysis can be improved by the manipulation of

several instrument parameters.
371
Setting the Fragmentation CutOff
Setting the Fragmentation CutOff
Optimizing MS/MS Operation: Setting the

Fragmentation CutOff
• Allows user to control m/z range of fragments.

• Click the Fragmentation box in the corner.
• This will open the Fragmentation Options field.
• Select the appropriate cutoff.
• Perform the MS/MS experiment.
• Note that here you can also turn on and off the SmartFrag.
20
Normally, the fragmentation CutOff is set to about one-third of the parent ion
m/z. In most cases, this is a good compromise. However, there are cases where
you may want to apply different values to optimize your MS/MS results. If the
parent ion fragments easily, then you can lower the fragmentation CutOff thus
getting more low-m/z fragments. If, on the other hand, the parent ion is hard to
fragment, then you can increase the fragmentation CutOff to values higher than
one-third of the parent ion m/z. Notice that a change in the fragmentation CutOff
likely requires a re-optimization of the fragmentation amplitude.
372
Using a Second Fragmentation
Optimizing MS/MS Operation: Using a Second

Fragmentation
• User can perform two fragmentations after each other, without

an intermediate isolation.
• Example:
– Problem: 80% of the ions fragment into one peak (e.g. a water
loss), while only the other 20% give structure revealing information
– Solution: add a second fragmentation on the water loss peak
– Fragments from the water loss are added to those produced from
the isolated ion
– Result: higher signal-to-noise ratio
21
Use this procedure if you want to perform two subsequent fragmentations,

without an intermediate isolation. For example, if you have an application where
80% of the parent ion fragments into an insignificant peak such as a water loss,
you may want a second fragmentation in order to obtain useful structural
information. In this case you can add a second fragmentation on the water loss
peak, whose fragments are in many cases not so different from the fragments of
the first isolated mass. Thus this procedure adds the fragments from the water
loss peak to those coming from the isolated ion, giving a higher signal-to-noise
ratio.
373
Optimizing MS/MS Operation: Using a Second

Fragmentation
• Select a peak at a certain m/z value in the MS/MS spectrum as

parent ion for the second fragmentation.
• Enter the m/z value into the entry field Isolation Mass stage 2.
• Enter the full width of the m/z window as Isolation Width.
• Do not select Isolation.
• Select Fragmentation.
22
The above procedure details the method for utilizing a second fragmentation with
your analysis.
374
Setting the Fragmentation Time
Setting the Fragmentation Time
Optimizing MS/MS Operation: Setting the

Fragmentation Time
• Fragmentation time is the length of time the ions are subjected

to collision with the background gas
• Default time is 40 ms
• Modify fragmentation time by clicking the Fragmentation
button from the MS(n) window
23
In the ion trap, the ions are fragmented by subjecting them to collisions with the
background gas for a specified time period. This time is the fragmentation time.
The default fragmentation time is 40 ms. If necessary, this value can be altered.
375
SmartFrag
SmartFrag
Optimizing MS/MS Operation: SmartFrag
• Applies a multitude of fragmentation amplitudes during each

fragmentation step.
• Start Ampl and End Ampl give the start and end amplitude for
SmartFrag relative to the fragmentation amplitude selected in
the MS/MS page.
• Makes the fragmentation amplitude an uncritical parameter
without reducing the duty cycle of the measurement.
24
The use of SmartFrag, allows the fragmentation amplitude to become much less
dependent on the characteristics of the precursor ion. The values Start Ampl and
End Ampl give the start and end amplitude for SmartFrag relative to the
fragmentation amplitude selected in the MS(n) page. Default values are 30%
(Start Ampl) and 200% (End Ampl), respectively. The fragmentation amplitude is
not critical when using SmartFrag without reducing the duty cycle of the
measurement. It is therefore recommended to always use SmartFrag.
376
Acquiring Data Files
Acquiring Data Files: Overview
• Data acquisition is the process of performing an analysis and storing

the resulting data to hard disk.
• A single mass spectrum takes about 20 to 50 kB.
• With chromatographic applications, many spectra are recorded in a

time sequence
– Could result in large data files (>100 Mb)
25
Data acquisition is the process of performing an analysis and storing the resulting
data to hard disk. Data are stored along with other information, such as the
operator (user name), the subfolder and data file name (counter or manual) under
which the data is stored, the sample comment, the parameters of the measurement
(such as source voltages, heater temperatures, etc.…), the number of the analysis,
and more.
377
Acquiring Data Files: Single Mass Spectrum
• Select the Sample Info menu

• Choose storage manually or with Prefix and Counter
• Enter file name or Prefix/Counter
• Enter a specific subdirectory where you would like the data to be stored
• Enter a sample description in the Comment field
• Select Sample Parameters (i.e. the sample name)
• To store mass spectrum to hard disk, click Save Profile from the Acquisition menu
26
The sample info menu allows you to enter specific sample information for a given
run or sequence. This menu also allows the user to specify data file information
and the location and path where the data will be saved.
378
Acquiring Data Files: Starting the Storage of a

Series of Mass Spectra
• Setup is identical to storing a

single mass spectrum
• Instead of clicking the “Save
Profile” button (F7), select the
“Run Method” button (F5) from
either window to begin
acquisition
• Or run LC/MS method from
the ChemStation.
27
Storing a series of mass spectra is very similar to storing a single spectrum. Note
that common commands such as Run Method do have hot keys for frequent use
(e.g. F5).
379
Acquiring Data Files: Stopping the Storage of a

Series of Mass Spectra
• Storage of mass spectra can be stopped by

any the following:
– Click Stop Run from the Acquisition menu.

– Press the F8 key while data is being acquired.
– Press the Stop Run button from the toolbar.
– If running ChemStation method, press Stop.
28
Stopping the storage of data can be done by either the toolbar menu or by the drop
down menu in the Acquisition header.
380
Using AutoMSMS
Using AutoMSMS
Data Acquisition: Using AutoMS(n)
• Used if you want to perform MS/MS analyses and you do not

know the m/z values of your parent ion.
• Instrument takes MS spectra and determines the tallest peaks

in the spectrum.
• If the peaks fulfill certain criteria, the LC/MSD Trap switches

to MS/MS operation on these peaks and takes one MS/MS
spectrum for each peak.
• The instrument then switches back to MS operation and

repeats the process until AutoMS(n) is switched off.
29
AutoMS(n) can be a powerful tool to identify specific ions in your sample as well
as the related fragment ion spectra. The tool is especially useful for co-eluting
chromatographic peaks. The procedure is optimal if you want to perform MS/MS
analyses and you do not know the m/z value of your parent ion. In the
AutoMS(n) mode the instrument takes MS spectra and determines the most
abundant peaks in this spectrum. Provided these peaks fulfill certain criteria, the
LC/MSD Trap switches to MS/MS operation on these mass peaks and takes one
MS/MS spectrum for each peak. The instrument then switches back to MS
operation and repeats the process until AutoMS(n)is switched off.
381
Using AutoMSMS
Using AutoMSMS
Using AutoMSMS: Parent Ion Peak Criteria
• Fulfill a threshold condition (minimum intensity).
• Have a m/z value outside of one or more excluded m/z ranges

or a m/z value within one or more included m/z ranges.
• Be within the number of peaks selected for MS/MS activation.
30
There are some criteria that individual parent ions must meet in order to be
detected by the software.
382
Using AutoMSMS
Using AutoMSMS
Using AutoMSMS: Setup and Operation
• Open the MS(n) page.

• Select Auto MS(n) in the MS(n) page.
• Set the Threshold (can also be done graphically) and number of
precursor ions.
• Note that specific mass ranges can be included or excluded.
• Also note that active exclusion can be selected after a specific number
of spectra have been acquired.
31
The above procedure details the use of the AutoMS(n) feature. AutoMS(n)
allows the user not only to select criteria for individual peaks, but include and
exclude certain mass ranges as well. Above, the user has chosen to include the
m/z range 150-2200.
383
Using AutoMSMS
Using AutoMSMS
• Set the No. of Precursor ions to number of ions desired

• Set the threshold of the signal to be applied
• Set the Fragmentation Amplitude
• Enable the AutoMSMS by clicking the “Auto MS(x)” box
32
Here the user can select one or more precursor ions and set the threshold. To
initiate the AutoMS(n) process, simply click on the Auto MS(x) box.
384
Using AutoMSMS
Using AutoMSMS
• The above picture shows a “typical” chromatogram generated

by an Auto MS(n) acquisition.
• The jagged appearance shows where the instrument has

changed MS parameters.
33
A typical chromatogram from the AutoMS(n) run is shown.
385
Segments
Segments
Creating Segments
• Creates several “parts” in which analysis parameters can change.

• Segments are individual time ranges.
• For each segment in the method, the user can change:
– Settings of the ion trap on the Main Panel
– Settings of the Mode page
– Settings of the Tune page
– Settings of the Optimize page
– Settings of the MS(n) page
– Settings of the Chromatogram page
– Setting of the Calibration page.
• Access right-click the context menu of the
Chromatogram window.
34
In a method, it is useful to create several parts (segments) in which the parameters

of the analyses can differ. Using the Context menu of the Chromatogram
window, a method can be divided into several time ranges (segments). Each
method consists of at least one segment. The default setting is therefore one
segment running a constant specific type of analysis.
386
Segments
Segments
Creating Segments: Inserting a New Segment

Limit
• Move the mouse cursor in the Chromatogram window to the desired time
limit.
• Click the right mouse button.
• Select “Insert new Segment Limit” .
• All segments will be numbered in ascending order.
35
See above to create a new segment limit. A right click in the chromatogram
window will bring up the segment dialog box.
387
Segments
Segments
Creating Segments: Editing Segment Limits
• The selected segment limit can be edited.
• Click on the limit line with the left mouse button and drag the
line to the desired time.
36
Existing segment limits can also be modified by manipulation with the mouse.
388
Segments
Segments
Creating Segments: Deleting Segment Limits
• The selected segment limit (red) can be deleted.
• Click on the limit line with the left mouse

button and select delete segment limit from
the context menu.
37
This command will delete the specific segment limit that exists in the active box
in the chromatogram window above.
389
Segments
Segments
Creating Segments: Deleting All Segment Limits
• Deletes all segments, except the first.

• Deletes all parameter settings from all segments.
• First segment limit will default to 30 min.
38
Note that you will not be able to delete “all” segment limits. The original will
default back to 30 minutes if all are cleared/deleted.
390
Segments
Segments
Creating Segments: No Runtime Limit
• The time limit of the last segment is set to infinity and is

marked with an arrow.
• Method never stops…be careful…large data files.
39
No runtime limit is a nice feature if you are unsure when your last
chromatographic peaks may elute. The major drawback to using this on a regular
basis is that file size will increase rapidly, especially if acquiring profile spectra.
391
Segments
Segments
Apply to All Segments
When Apply to All Segments

Is selected:
•Any changes made will apply
to all segments.
•There are exceptions, i.e.
Neutral Loss Masses.
•A pop-up window will notify
you of exceptions.
40
If you select Apply to All Segments, then any changes you make will be applied
to all segments. There are certain parameters that will not automatically apply to
all segments such as neutral loss masses. In these cases, a pop-up window will
notify you of the exception.
392
Agilent 1100 Series LC/MSD Trap:
Developing Acquisition Methods
Agilent 1100 Series LC/MSD Trap: Developing Acquisition Methods
In This Section
In This Section
• The difference between LC/MS and MS methods.
• How to work with MS-methods.
• How to prepare the LC/MSD Trap to run an MS-method.
• How to actually run your MS-method.
• How to work with LC/MS methods.
In this section, we will distinguish between an MS method and an LC/MS

method. LC/MS methods are only available when you also have an Agilent 1100
HPLC and ChemStation along with the ion trap software.
394
Method Strategy
Method Strategy
LC/MSD Trap Method Strategy

.M (rev 6.0) .MS (prior revs)
Two types of methods (rev. 6.0):
•Trap Control only
.M
•Start, stop, save, and load
•Agilent HPLC Control methods from the Ion Trap
Control
•Trap Control
•No LC control
•Can disable MS part to run LC
only methods •Default is Def_MS.M
•Can ignore LC part by starting
method from within Trap Control
•Save changes to both parts of

method within the ChemStation
•Default is Def_LCMS.M
Methods serve two purposes:

• To control the actual data acquisition,
• To control the post processing of the data once it is acquired.
There are two types of methods available on the Agilent LC/MSD Trap, LCMS
methods, .M, and MS methods, .M. The MS methods are accessed via the Trap
Control software only and cover only ion trap acquisition and data analysis. The
LC/MS methods cover both the HPLC and the MS.
If you have an Agilent 1100 Series HPLC with your trap, it is best to utilize the
LC/MS methods that are saved via the ChemStation software. These methods
contain the operating and data analysis parameters for both the HPLC and the Ion
Trap. You can disable the MS part of the method while developing the HPLC
separation through the menu items in the ChemStation software, Method then
Disable MS Part… You can disable the HPLC part by starting the method from
the ion trap. When the method is started from the ChemStation both the HPLC
and the Ion Trap parameters sections are utilized.
395
Creating an MS Method
Creating an MS Method
Creating a New MS-method
• Go to Ion Trap Control.
• Use New MSD Trap Control Part to create a new MS only

method.
• Create method.
• Name the new method and save it.
• The method saves all parameters in the

actual state.
The user can create, load, edit, delete, and save MS methods from this menu. The
.M LC/MS methods for ion trap control are also available. The procedure for
creating a new MS-method is shown above. Creating a new MS method loads a
default MS file, Def_MS.M. You must then enter your desired parameters and
save the method to a unique name.
396
MS Only Method
MS Only Method
MS Only Methods
• Starting and stopping methods can be done:

– Via the toolbar buttons.
– In the drop down acquisition menu.
Starting and stopping MS methods in the Ion Trap Control software can be done
two different ways. First, the user may start and stop with the menu bar. The
other option is the pull down menu from the Ion Trap software.
397
Loading an MS Method
Loading an MS Method
Loading an MS-only Method
• Use before you review, edit, or run any MS-method.
• Loading an MS-method loads the initial state into the mass

spectrometer and activates it.
• On the Method menu, click Load-MS Trap Control Part….
• Select a method from the dialog.
• Click Open.
Use this procedure before you review, edit, or run any MS-method. Loading an
MS-method loads the initial state into the mass spectrometer and activates it, thus
preparing the system for the start of the method.
398
Editing an MS Method
Editing an MS-method
• Set the initial state by editing parameters on the screen (e.g.
tune, MS(n), etc.).
• Select Save Spectra if desired.
Be careful when including profile spectra for long chromatographic runs. This
action will take up large volumes of disk space for one file.
399
• Integrate a part of a procedure from
the Data Analysis program to the method
– Add Data Analysis Part…
• Integrate a procedure from the

Data Analysis program to the method
You can add the data analysis processing to your method as well.
400
Saving an MS Method
Saving an MS Method
Saving an MS Method
• Use to save any MS-method.
• User can save (overwrite) current method or save as and select

a new filename.
• On the Method menu, click Save Method.
• Click OK
Always make sure you save your method after making changes. You can save a
method either under its old name by overwriting the old method or save it under a
new name.
401
Run an MS Method
Run an MS Method
Preparations to Run an MS Method
• Choose the Sample Info tab.

• Enter the subdirectory and the file name for the data file.
• Enter the operator (from the Acquisition menu) and a comment*.
• The new values are used for subsequent runs but can be changed.
*These parameters are optional
10
Before starting your MS only method, fill in the required sample information.
Your data will be saved to the data file name provided.
402
Run an MS Method
Run an MS Method
Running an MS Method
• General considerations:
– During the run of a method, all mass spectra are written into a data file.
– When the method stops, data file storage ends.
– Profile spectra vs. line spectra.
– Organization of data files.
• Starting an MS-Method
– Manually.
– As part of an LC/MS-method.
11
While running a method, all mass spectra are written into a data file. Data file
storage ends at the end of the method. It is advisable to always have a finite stop
time in each of your methods. Profile spectra can be stored in addition to line
spectra. This choice is a part of the method. Data files should be organized by
means of subdirectories.
403
Run an MS Method
Run an MS Method
Running an MS-Method: Starting Manually
• Use to manually start the method for the first time.

• To start the method manually:
– In the Acquisition menu choose “Run method” OR,
– Choose the [Run Method] button in the toolbar OR,
– Choose “Save Profile Now” OR,
– Choose the [Save Profile] button to save a single profile spectrum,
– Use the [Stop Run] button to stop the run manually before the
stop time.
12
Use the above procedure if you want to manually start the method for the first
time. There are several ways to actually start your method manually.
404
LC/MS Methods
LC/MS Methods
LC/MS Methods
LC/MS methods are preferred when you have both the Agilent
1100 and the Ion Trap
Use when performing HPLC

only method development
Start and Stop

methods
Loads default starting method
Always load method
from ChemStation
Save in ChemStation or
save MS part in Ion trap Control
13
An MS-method can be part of a larger LC/MS-method. LC/MS-methods are

edited and started from the ChemStation and would be activated by selecting Run
Method from the ChemStation: Method and Run Control view. You can also use
the Start and Stop buttons.
Always load an LC/MS method from the ChemStation. Loading the .M method
in the Ion Trap Control software will only load the mass spectrometer parameters.
Loading the method from the ChemStation will load both the LC and the MS
parameters.
You can disable the mass spectrometer portion of the method if you are working
solely on HPLC method development with an alternative detector.
You can save the MS parameters of an LC/MS method in the Ion Trap Control
software but not the HPLC parameters.
405
Offsetting Time Scale

• Compensates for the time delay
between the UV detector and the
mass spectrometer
• Enter the following values for the

tubing and flow between the UV
detector and LC/MSD Trap:
– Internal diameter of the tubing
– Tubing length
– LC flow rate
• Establishes a delay in the start of

the MS part of the method with
respect to the LC part of the
method
ChemStation
14
The LC/MSD Trap can compensate for the time delay between the liquid
chromatograph’s UV detector and the mass spectrometer. This is done by
entering the values for the inner diameter and length of connective tubing as well
as the flow rate. The settings will cause a delay in the start of the MS part of the
method with respect to the LC part of the method.
406
LC/MS Methods
LC/MS Methods
LC/MS Methods
• LC/MS methods are created from the ChemStation software and

saved in conjunction with MS methods.
15
LC methods will incorporate the active method in the Ion trap software and in
essence save them as LC/MS methods.
407
LC/MS Methods
LC/MS Methods
LC/MS Methods: A Brief Overview
• From the ChemStation, select the Method

and Run Control view.
• Under the Method menu select Edit LC

Part.
• The Edit Method dialog box will open.
16
Editing of LC methods is no different from modifying the LC method in

ChemStation regardless of detector (in this case LC/MSD Trap).
408
LC/MS Methods
LC/MS Methods
• When the dialog box opens, you can

select different sections of the LC
method to modify.
• The most important section will be the

Instrument/Acquisition section.
17
It is easier for this brief overview to simply discuss the portions of the method
that will directly impact acquisition methods. Therefore, the Data Analysis box
and Run Time Checklist will not be addressed today.
409
LC/MS Methods
LC/MS Methods
• Here you can enter the

LC information for your
run.
• Note that a timetable can

be used for gradient
applications.
18
After clicking OK. the ChemStation software will begin taking you through
various screens where parameters for the non-MS detector settings can be made.
Here we can enter HPLC information as necessary. Note that a gradient table can
be used by accessing the timetable from this window.
Set the maximum pressure according to column specifications provided by your
manufacturer. The minimum pressure setting is useful to shutdown a method if
the pressure falls below the given value as may happen if you run out of solvent.
410
LC/MS Methods
LC/MS Methods
• The injector dialog box allows you to set the injection volume.
• By clicking More, you can adjust the draw and eject speeds.
19
The above window allows modification of injection volume and can be expanded
by clicking More to allow manipulation of the autoinjector draw and inject
speeds.
411
LC/MS Methods
LC/MS Methods
• This shows the full

menu of options for
the injector setup.
• Draw position may

also be modified or
offset.
20
Other autoinjector parameters include the draw and eject speed. You may want to
set slower draw and eject speeds for large injection volumes or viscous samples.
The draw position can be adjusted to sample material at a given level within the
vial.
412
LC/MS Methods
LC/MS Methods
• The column thermostat view allows you to set the thermostat to

maintain your column at a set temperature.
• Settings for the switching valve can also be made, for example
by passing the column for a direct injection.
21
The above column thermostat view allows modifications to be made to the

column temperature and settings for the switching valve. The column switching
valve is useful for direct injections while bypassing the column or for column
pretreatment methods.
413
LC/MS Methods
LC/MS Methods
• Finally, UV and Diode array detector setup can be done.
• A timetable can also be used here.
• Clicking OK will complete the LC part of the Instrument/Acquisition

settings for LC methods.
22
Finally, the settings for the UV/Diode array can be placed into the method. Any
other type of detector such as a fluorescence detector could be set up here as well
as long as the software can communicate with the instrument.
414
Laboratory Exercise: Scan Acquisition
on the Agilent 1100 LC/MSD Trap
Laboratory Exercise: Scan Acquisition on the Agilent 1100 LC/MSD Trap
In This Exercise You Will:
In This Exercise You Will:

• Make preparations for an acquisition method.
• Enter instrument parameters for an acquisition method.
• Create an MS/MS acquisition method with 4 segments.
In This Exercise You Will Need:

• Agilent Ion Trap software revision 6.0.
• HPLC ChemStation software revision B.01.xx.
Online
• The sulfa test mix.
416
Pre-Acquisition Parameters
Pre-Acquisition Parameters
• Before setting up any acquisition method:
• Make sure your sample is filtered and free of interfering matrix.
• Make certain the UV lamp is warmed up (~20 minutes).
• Filter and degas your mobile phase.
• Prime the HPLC pump and connect your column.
• Verify the instrument is under proper vacuum.
• Make sure instrument temperatures are stable.
• Set the instrument to operate under optimized parameters for your
compound of interest.
Note that if you are working in the “offline” mode, you will not actually be
able to perform the examples physically; however, you will be able to access
the menus and follow the procedures. All of the recommendations above
would apply if actually performing the experiments.
417
Entering Instrument Parameters – LC Parameters

1) First we will edit the LC part of the method. Go to the Method and Run
Control view of the HPLC ChemStation.
2) Select Method > Load Method. Load the method, DEF_LCMS.M, the
combined LC and MS default method. (note: in offline mode, you will not
find DEF_LCMS.M. Load DEF_MS.M instead.)
3) Under the Method menu select Edit LC Part…. (in offline mode, select
Method > Edit Entire Method.
The Edit Method dialog box will open. Select only the Method Information and
Instrument/Acquisition sections to edit.
4) Click OK to begin.
418
5) The Method Information dialog box will open. Enter in any comments that
are relevant to the method. Click OK to continue.
6) Now enter the LC information as it appears below. To enter the composition
programming, first select the Display: Timetable. Click Insert and enter
the first line. Click Append to enter the second line and all subsequent
lines.
7) Now the Injector dialog box appears. Set the instrument for Standard
Injection and enter 1.0 µL as the injection volume
Click More to adjust the draw and eject speeds. Set the draw speed to 200
µL/min. Set the Eject Speed to 200 µL/min. Click OK to continue.
8) In the Column Thermostat dialog box set the temperature equal to 40˚C.
Select Column 1 in the Column Switching Valve section. Click OK to continue.
419
9) Now set the DAD signal to store signal A at 254 nm and click OK.
10) In the Run Time Checklist, select Data Acquisition. Leave other options
deselected.
420
Entering Instrument Parameters - MS Parameters

1) Now we will edit the MS part of the method. Click the Ion Trap icon if you
are online. This will open the MSD Trap Control window. (If you are
offline, open the MSD Trap Control view and make certain the default MS
method is loaded.
The following are XCT parameters. Classic, VL, and SL parameters would
be slightly different in some cases.
2) Select the Mode tab and enter the following parameters then Apply.
3) Set the following parameters:

• ICC – On
• Smart Target – 200,000, 500,000 XCT Ultra
• Scan Range – 50-400 m/z
• Positive polarity.
4) Click the Tune tab and enter the parameters below. Click Apply.
421
5) Click the Chromatogram tab. Make certain TIC,All MS/MS is available.
6) Now let’s set up four segments to optimize peak height for the analysis in
question.
7) Click File > Open. Select the data file SULFMS01.D and click Open.
422
8) Go to the MS(n) tab. Select the Manual MS(n) radio button. Mark the
Manual MS(n) checkbox.
9) Right-click the chromatogram and click on Insert new Segment Limit.
10) Select the number 1 time segment. Click on the line indicating the end of
the segment and adjust its position after the first peak.
11) Go to the MS(n) tab and mark the On check boxes for MS/MS Isolation and
Fragmentation.
423
12) Enter 271 as the MS/MS mass. Click Apply.
13) Now create the following segments for the remaining peaks.
14) Segment 2, MS/MS mass 285,
15) Segment 3, MS/MS mass 279,
16) Segment 4, MS/MS mass 311.
The result should look like the segments below.
424
17) Go to the HPLC ChemStation and save the LC/MS method. Method > Save
Method As.
18) Make certain that all modules are turned on. Monitor the signal from the
Trap Control window.
19) Click the Sample Info tab found in the Ion Trap Control window.
20) Give your file a unique name and subdirectory. Click OK.
21) From the HPLC ChemStation Method and Run Control view, select Start.
22) Switch to the MSD Trap Control window and watch the acquisition.
425

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Agilent 1100 Series LC/MSD Ion

Trap Techniques and Operation

Printed in October, 2005

Agilent Technologies, Inc

 2005 by Agilent Technologies, Inc.

In This Section, We Will Discuss:

• The difference between Electrospray, Atmospheric Pressure

• Details of the ion optics in the VL, SL, and XCT

• How the vacuum system functions.

• The information available from mass spectral data.

You will be taking a tour of the system from

In this section you will learn:

Atmospheric Pressure Ionization Mass

Atmospheric Pressure Ionization Mass

h Compatible with a broad range of compounds

Mass spectral data provides the molecular weight, structural information,

Atmospheric Pressure Chemical Ionization (APCI): gas phase

Atmospheric Pressure Photoionization (APPI): Krypton lamp

Atmospheric Pressure-Matrix Assisted Laser Desorption

Atmospheric Pressure Chemical Ionization is an atmospheric pressure ionization

The AP-MALDI or Atmospheric Pressure-Matrix Assisted Laser Desorption/

The atmospheric pressure ionization sources are easily interchanged, requiring

Relative Applicability of LC/MS Techniques

Relative Applicability of LC/MS Techniques

What Kind of Data Do You Obtain?

What Kind of Data Do You Obtain?

When a small molecule is analyzed by atmospheric pressure ionization

Other pseudomolecular ions may be produced and even predominate, depending

[M + H + S]+ solvent adducts

for negative mode;

What Kind of Data Do You Obtain

What Kind of Data do You Obtain?

MSn analysis in an ion trap mass spectrometer permits multiple stages of

What Kind of Data Do You Obtain?

What Kind of Data Do You Obtain?

m/z--> 16400 16600 16800 17000 17200 17400 17600

High molecular weight compounds having multiple ionization sites produce

Main Components of the LC/MSD TRAP

Main Components of the LC/MSD TRAP

A schematic of the LC/MSD Trap is shown above. Regardless of whether the

The orthogonal nebulizer is excellent for handling salts and buffers.

Models: VL, SL, XCT, XCT+, XCT Ultra

Models: VL, SL, XCT

Evaporation Rayleigh Coulomb Evaporation Analyte Ion

The LC/MSD Trap nebulizer uses pneumatically assisted electrospray.

The API-electrospray process comprises three basic steps:

The droplets continue to shrink until the repulsive electrostatic (Coulombic)

API –Electrospray Ion Source

API-Electrospray Ion Source

High Voltage Mesh Assembly

The API-electrospray spray chamber is shown above. The electrospray spray

Electrospray Spray Chamber Settings

Electrospray Spray Chamber Settings

Vcap Drying Gas Flow

Drying Gas Temperature

Why is it Important to Properly Control Spray

Why is it Important to Properly Control Spray

Drying gas: 350 °C, 12 l/min

Spikes caused by droplets from incomplete drying

Effect of Drying gas Settings in ES

Effect of Drying Gas Settings in ES

200 400 600 800 1000

Droplets cause noisier spectra