CHEM 4330

Lecture 5

High Performance Liquid

Chromatography
 Instrumentation

Flash column chromatography High-Performance LC

Instrumentation

High-Performance LC
Instrumentation
 Instrumentation
• High-performance liquid chromatography (HPLC)
is a form of liquid chromatography that separate
compounds in a solution mixture
• HPLC instruments consist of a reservoir of mobile
phase, a pump, an injector, a separation column,
and a detector.
• Compounds in the sample mixture are separated
in the column.
• Components in the mixture pass through the
column at different rates due to differences in
partition between the mobile liquid phase and
the stationary phase
 Instrumentation
• Pump: deliver mobile phase of defined composition
through the chromatographic column at a specific
flow rate
• Injector: introduce the liquid sample into the
chromatographic column
• Separation column: “heart of the chromatograph”.
components in the sample mixtures are separated on
the column’s stationary phase
• Detector: detect the individual molecules elute from
the column
Instrumentation
 Pumping system
• In HPLC, particles of size range from 3 to 10 m are
used as stationary phase (1.5 and 1.7 m for UPLC)
• Strong force is needed to pass mobile phase through
the column at high pressure and at a controlled flow
rate
 Pumping system
Reciprocating pump
• Back and forth movement of a motor-driven
piston control the flow of solvent
Displacement pump / Syringe pump
• Screw-driven plunger push solvent through a
syringe-like chamber
 Pumping system
• Be able to generate pressures up to 6000 psi
(400 bar)
• Pulse-free output
• Flow-rates ranging from 0.1 to 10 mL/min (cap
LC, nano LC)
• Flow control and flow reproducibility of 0.5 %
or better
• Corrosion-resistant
 Pumping system
Reciprocating pump
• Solvent is pumped by the back and forth
motion of a motor-driven piston
• ~ 90% share in HPLC market
 Pumping system
Reciprocating pump
Closed Open

Open Closed

Mobile phase enters the pumping Mobile phase leave the pumping
chamber via an inlet valve chamber via an outlet valve
 Pumping system
Reciprocating pump

Isocratic Pump Quaternary Pump Binary Pump

 Pumping system
Reciprocating pump
 Pumping system
Reciprocating pump
Advantages:
•small internal volume (10 - 200 mL)
•high output pressure (up to 10 000 psi)
•capable of gradient elution
•provide constant flow-rates (independent of column back-
pressure and solvent viscosity)
Disadvantages:
•produce a pulse flow pulse damper
 Pumping system
Reciprocating pump
Isocratic elution
•Solvent (one or two) of constant composition is
used as mobile phase.
Gradient elution
•Two or more solvent systems that differ
significantly in polarity are employed, e.g. water
and methanol/acetonitrile.
 Pumping system
Reciprocating pump

%B %B

Courtesy of Agilent Technologies

 Pumping system
Gradient elution 100
Linear gradient

•Linear vs Step
80

% organic
60
40
20
0
0 10 20 30 40 50
Problems with gradient elution Time

•Reproducibility Step gradient

•Long reequilibrium time

% organic
•Long cycle time
Time
 Sample injection system
• For the introduction of liquid sample into the
HPLC system without depressurizing the
system
• Mostly based upon sampling loop
 Sample injection system
• Interchangeable sample loops (5 to 500 L)
• Can stand for high pressure up to 7000 psi
• Good precision < 1 % RSD
 Sample injection system
Manuel injector

Load sample Inject sample

 Sample injection system
Sample transport assembly
Auto-sampler

Metering
Device

Needle

Injection Valve

Needle seat
 Sample injection system
Auto-sampler

Sample loop

Metering
device

Injection valve
 Sample injection system
Auto-sampler
Pump Pump Pump

Column Column
Column

Prior to Injection Draw Sample Injection and Run

Valve in Mainpass Position Valve in Bypass Position Valve in Mainpass Position
 Chromatographic column
• Guard column - Protects the analytical column
~ removes particulate in injected solution
~ Short, similar packing material as the analytical
column
~ Prolongs the life of the analytical column
• Analytical column - Performs the separation
 Chromatographic column

Guard column

Guard column holder

Analytical column
Chromatographic column
Chromatographic column
Chromatographic column
Chromatographic column
Chromatographic column
 Column heater
• Maintaining a constant
temperature for
chromatographic separation
• Better reproducibility
• Lower solvent viscosity back pressure
• Longer column and/or smaller particle size
 Column heater
• T , solute solubility in mobile phase 
Retention /capacity factor (k) ,
retention 
• Diffusion coefficient of solute  ,
rate of mass transfer 
Plate height, H 
column efficiency N 
 Column heater
Higher temperature higher flow rate, higher
efficiency, faster separation

MARCH 2005 LCGC ASIA PACIFIC VOLUME 8 NUMBER 1 51

 Detector
• Detect compounds eluting from the column
• Small internal volume to reduce band
broadening
Bulk property detector
• response to change involving both mobile phase +
solutes, e.g. refractive index, dielectric constant,
density, conductivity
Solute property detector
• absorption, fluorescence or diffusion current
Detector
 Detector
UV/Visible Detectors
 Detector
UV/Visible Detectors
•An ultraviolet light beam is directed through a flow cell
and a sensor measures the light passing through the
cell.
•If a compound elutes from the column that absorbs
this light energy, it will change the amount of light
energy falling on the sensor.
•The resulting change in this electrical signal is
amplified and directed to a recorder or data system.
 Detector
UV/Visible Detectors Absorbance

Flow cell for HPLC with UV/visible detection

 Detector
Increased analytical sensitivity with a longer
flow cell path
 Detector
UV/Visible Detectors
•UV absorption detectors use
mercury lamps at 254 nm or
deuterium filament lamps with
monochromators or filters
•Visible absorption detectors use
tungsten filament lamps with
monochromators or filters
 Detector
UV/Visible Detectors
•The entire absorption spectrum can be
scanned when they are used in conjunction
with a photodiode array detector (PDA)
•Qualitative
 Detector
UV/Visible Detectors
 Detector
Fluorescence detection
•One of the most sensitive detectors
•Hg lamp as excitation light source and filters to isolate
the emitted light from the background.
•or Xe lamp as excitation light source and
monochromator to isolate the fluorescence radiation.
•Emission intensity is recorded as a function of time
•Chromatogram = F.I. vs time
 Detector
Fluorescence detection
 Detector
Fluorescence detection
•Excitation wavelength, λex
•Emission wavelength, λem
Excitation/absorption spectra Emission spectra
 Detector
Fluorescence detection
 Detector
Fluorescence detection
•For compounds that fluoresce
•Non-fluorescent compounds may be
derivatized by adding a fluorescent tag to the
sample molecules

Fluorescence tag

Non-fluorescent Fluorescence
 Detector
Fluorescence detection
• Chemical derivatization of amino acids with o-
phthalaldehyde (OPA) allows sensitive
detection of amino acids by fluorescence
detector
 Detector
Fluorescence detection
• Nitroreduction and intra-molecular acetlylation of
mutagenic and nephrogenic aristolochic acids (non-
fluorescent) results in the formation of aristolactams
which allows ultra sensitive detection by FLD

Non-fluorescent Fluorescence
 Detector
Refractive Index Detector
•A measure of compound’s ability to deflect light
•Universal, responding to nearly all solutes (analogous
to FID and TCD in GC)
•Amount of deflection is concentration dependent
•Highly temperature sensitive, require constant
temperature (to a few thousandths of a oC)
•Least sensitive LC detector
•Cannot be used for gradient elution
 Detector
Refractive Index Detector
 Detector
Mass spectrometer
•Detect analytes eluting from the column by their m/z
values after ionization (m for molecular weight, z for
charge status of analyte)
•Atmospheric pressure ionization e.g. electrospray
ionization (ESI), atmospheric pressure chemical
ionization (APCI) is most common ionization techniques
for LC-MS analysis
 Detector
Mass spectrometer
Different type of MS
•Ion trap
•Quadrupole mass analyzer
•Time-of-flight MS (Q-Tof)
•Triple-quadrupole mass analyzer (QqQ)
•Hybrid quadrupole time-of-flight MS (Q-Tof)
•….
 Detector
Mass spectrometer

HPLC
 Detector
Mass spectrometer
•High accuracy MS (Tof MS) give accurate m/z
value for interpreting the molecular formula
•MS/MS analysis from Q-Tof or QqQ give
fragment information of the analyte which is
important for structural elucidation
•QqQ MS/MS systems are ideal for sensitive and
selective quantification purposes
 Detector
Mass spectrometer

434.1886

C19H32NO8S

301.1
HR-MS

MS/MS

435.1890

400 450 500 390.2

m/z, amu

217.1
235.1 330.1
302.1 345.2
286.1 434.2
203 372.1
.1
300 400
m/z, amu
 Applications of HPLC
• Qualitative analysis provides information on the
identity of sample components (what?): By Retention
time, UV absorption spectrum, mass spectrometric data
• Quantitative analysis provides information on the
amount of sample components (how much?): By
peak height or peak area (with internal standard)
• Isolation of compounds, from herbal medicines,
chemical reactions or from biological samples
(metabolites of drugs in plasma, urine)
 Applications of HPLC
For the separation of chemical and biological
compounds that are non-volatile
 Applications of HPLC
For the identification(ID) of individual
compounds in the sample mixture, qualitative
• By retention time (+ UV absorption spectrum, m/z
value, MS/MS spectra for confident ID)
15.3

System dependent
• Column
• mobile phase
• conditions and the
• instrumental settings
• instrumental performance
5.5
 Applications of HPLC
For the identification(ID) of individual
compounds in the sample mixture, qualitative
• By relative retention, system independent
• The relative retention tR'' is the relative retention
between the peak of interest (x) and the (absolute)
retention time of a selected standard peak (n).
 Applications of HPLC
For quantitative analysis
• Amount of a component in a sample mixture
• By measuring the peak area/peak height as
measured from the baseline