Chem.

230 – 11/25 Lecture

 Announcements I
• Homework Set 4 Solutions Posted (short
answer + long answer coming soon)
• Turn in Set 4 long problems (last graded set)
• Schedule for presentations on the internet
– Posted link to first presentation review article
– Will post homework and presentations as they become
available
• Exam 4
– Will cover HPLC detectors, Quantitation and MS
– Capillary Electrophoresis will only be on Final
 Announcements II
• Today’s Lecture
– Mass Spectrometry
• Interpretation
• Other Topics
– Capillary Electrophoresis
• Theory
• Equipment
• Summary of Main Methods
– First Special Topics Presentations (Cheng and
Clarke – MEKC)
 Mass Spectrometery
Interpretation
• Fragmentation Analysis
– Covered (briefly except for questions)
• Isotopic Analysis
– Covered (one more question)
• Determination of Charge
– Important for interpreting MALDI and ESI peaks where multiple
charges are possible
 Mass Spectrometry
Other Topics – Multiple Charges in ESI
• In ESI analysis of large
molecules, multiple charges (M+n)/n
are common due to extra
(+) or missing (-) Hs (or m/z

Ion current
e.g. Na+)
• The number of charges can
be determined by looking at
distribution of big peaks
• For + ions m/z = (M+n)/n m/z (M+n+1)/(n+1)
(most common)
Example: m/z peaks =711.2, 569.3,
• For – ions m/z = (M–n)/n 474.8, 407.1

m/z = (M+n)/n – (M+n+1)/(n+1) = (M+n)(n+1)/[n(n+1)] – (Mn+n 2+n)/[n(n+1)] =

M/[n(n+1)] = 141.9, (94.5, 67.7)
Do rest on board
 Mass Spectrometry
Other Topics – Multiple Charges in ESI
• Another way to find charge on ions is to examine the
gap in m/z between isotope peaks (0 13C vs. 1 13C)
• The +1 mass difference will be ½ if charge is +2 or 1/3
if charge is +3 gap = 405.73 – 405.23 = 0.50

Glycodendrimer core Glycodendrimer core

 Mass Spectrometry
Other Topics - MS-MS
• In LC-ESI-MS, little fragmentation occurs making
determination of unknowns difficult
• In LC-ESI-MS on complicated samples, peak
overlap is common, with interferants with the
same mass possible (e.g. PBDPs)
• In both of above samples, using MS-MS is useful
• This involves multiple passes through mass
analyzers (either separate MSs or reinjection in
ion-trap MS) and is termed MS-MS
• Between travels through MS, ions are collided
with reagent gas to cause fragmentation
 Mass Spectrometery
Questions I
1. Which ionization method can be achieved on
solid samples (without changing phase)
2. If one is using GC and concerned about
detecting the “parent” ion of a compound that
can fragment easily, which ionization method
should be used?
3. For a large, polar non-volatile molecule being
separated by HPLC, which ionization method
should be used?
 Mass Spectrometery
Interpretation Questions
1. Determine the m/z Abundance
identity of the (% of biggest)
compound giving 25 14
the following 26 34
distribution:
27 100

35 9

62 77

64 24
 Mass Spectrometery
Interpretation Questions
2. Determine the m/z Abundance
identity of the (% of biggest)
compound giving 29 9.2
the following 50 30.5
distribution:
51 84.7

77 100

93 16

123 39
 Mass Spectrometery
Interpretation Questions
3. From the following m/z Abundance
M, M+n ions, (% of biggest)
determine the 117 100
number of Cs, Brs 118 1.4
and Cls:
119 98

121 31.1

123 3
 Capillary Electrophoresis
Overview
• Basis of Electrophoresis
• Electroosmotic Flow in Capillaries
• Equipment
• Summary of Main Methods
 Capillary Electrophoresis
Basis for Separation
• Transport in electrophoresis is -
high voltage
based on electric forces on ions: +
– The electrostatic force
accelerates the ion toward the electric
electrode of opposite charge force -
X
– But “drag” in the opposite anode drag
cathode
direction soon becomes equal to
the electrostatic force leading to
constant velocity Electric Field
– velocity = v = zE/(6r)
where z = charge, E = electric
field,  = viscosity, and r = ion
radius (missing in text 13.3)
Note: for -1 anion, z = -1, so
direction is opposite to electric
field (as in example)
 Capillary Electrophoresis
Basis for Separation
• Ion velocity depends on:
– Electric field = V/L where V = voltage and L = capillary length
– Ion charge (z)
– Ion size (r)
– fastest migration for small, highly charged ions
• Complications in capillary electrophoresis
– Electroosmotic flow (EOF): bulk flow through the capillary
– EOF results from negatively charged capillary wall (for silica tubing at pH > 2)
– Positively charged counter ions are needed and migrate to cathode
– They also drag solvent toward cathode
– Because EOF originates from capillary wall, flow profile is nearly uniform
– Whereas pressure-driven flow is slow at walls
– This results in less band broadening than in chromatography

O- O- O- O- O- O- O- O-
to Na+ NaNa
+ Na Na
+ + +
Na+
Na+ Na
+
to
anode cathode
 Capillary Electrophoresis
Separation Efficiency
• Van Deemter Equation
HH==AB/u
A++B/u
B/u+ Cu
– Unlike chromatography (for CZE), no stationary
phase exists, so no mass transfer
– Wall driven flow means no multipath term
– This is somewhat “idealized”
• Optimal Separation Occurs at Highest
Possible Flow Rates hotter
– highest voltage provides fastest separation and
least dispersion, but
– highest voltages result in heating capillary cores
and dispersion due to differential viscosity
 Capillary Electrophoresis
Separation Efficiency – cont.
• Van Deemter Dispersion
– Only due to molecular dispersion
– Smallest for largest ions (they have smallest
diffusion coefficients)
• Other Sources of Dispersion
– Differential heating
• core velocity is faster
• larger for larger voltages and larger diameters
– Injection plug widths (depends on method and
volume injected)
– Detection
 Capillary Electrophoresis
Basis for Separation
• Net velocities: vEOF = vNet(neutrals)
– vNet = vEOF + vion
vNet
– vion is negative for anions, positive vNet
for cations and 0 for neutral species vAnions vCations

– No separation of neutral species in

Capillary Zone Electrophoresis
• Analyte migration time
Weak Acid Example
– time = l(L/V)vNet
where l = length from anode to vEOF
detector vNet A-
– time depends on ion size, charge, pH vNet HA
(weak acids/bases), voltage, column
lengths
at pH ~ pKa, vNet = (vNet HA + vNet A-)/2
 Capillary Electrophoresis
Equipment detector
• Mobile phase (aqueous
buffer)
• Power supply (~30kV) and
electrodes
• Capillary (25 to 75 μm
diameters)
• Some way to get sample
into capillary
• Detector (through capillary high
most common) + voltage
• Safety Equipment – to turn
off high voltage when
accessing equipment
 Capillary Electrophoresis
Equipment (Cont.)
• Mobile phase (aqueous buffer)
– Ion Concentration from Buffer
• needed to carry current
• too high causes slow migration (more dispersion)
– Modifiers
• various types including organics and surfactants
• Voltage – high value allows faster separations
and minimizes dispersion
• Capillary dimensions – need to be small to avoid
excessive joule heating
 Capillary Electrophoresis
Equipment (Cont.) + -

• Sample injection High V

– Electroosmotic injection (using

applied voltage) (sometimes biases
sample)
– Hydrostatic injection (based on
raising/lowering capillaries)
– Hydrodynamic injection (using
applied pressure)
 Capillary Electrophoresis
Equipment (Cont.)
• Detectors
– Sensitivity issues (CE usually has poor conc.
detection limits but excellent mass detection)
– Through Capillary Types
• advantage: single capillary can run from anode to
cathode without a need for any connections or possible
shorting of high voltage circuit
• this is restricted to non-evasive (optical) detectors
• UV absorption and fluorescence are most common
– Others
• These require an interface at or after cathode
• Electrochemical and MS detection are most common
 Capillary Electrophoresis
Equipment (Cont.)
• Detectors
– UV
• simple beam through capillary is simplest
• concentration sensitivity is poor due to short
path length
• “bubble” or “Z-cell” increases sensitivity
modestly
– Fluorescence
• Favored due to greater sensitivity
 Capillary Electrophoresis
Equipment (Cont.)
• Detectors
– Electrochemical Detection
• Electrodes can be made small for connection to small
flow cells in CE
• Smaller size does not decrease sensitivity much with
most electrochemical detection methods and CE
already has needed buffer
• This results in very low mass detection limits
– MS
• Ionization efficiency is good with the lower flow rates
found in CE
• Volatile buffers and additives must be chosen, which
can limit choices
 Capillary Electrophoresis
Main Methods
• Separation of Ions
– Capillary Zone Electrophoresis
– Capillary Gel Electrophoresis
• Separation of Neutral Compounds
(may also be used for ions)
– Micellar Electrokinetic Chromatography
(MEKC)
– Capillary Electrochromatography (a
hybrid of CE and HPLC)
 Capillary Electrophoresis
Main Methods
• Capillary Zone Electrophoresis (CZE)
– Most common in silica capillaries in which case
net EOF is from anode to cathode
– Fused silica operation at higher pH (>2)
needed for negatively charged silanol groups
– Silica EOF can be reversed using a positive
surface coating
• Capillary Gel Electrophoresis
– Separation based on molecular sieving (size of
molecules) in gel (like standard gel
electrophoresis)
– Has been used extensively for DNA fragment
separations
 Capillary Electrophoresis
Main Methods
• Micellar Electrokinetic Chromatography (MEKC)
– Micelles added to buffer (from surfactants) surfactant
– Allows separation of neutrals based on partitioning of
analytes between micelle interiors (hydrophobic
environment) and bulk mobile phase
– Anionic micelles will travel slower than EOF and neutrals
will elute between micelle flow and EOF flow
• Capillary Electrochromatography
– Uses packed capillary column micelle
– Flow driven by electrophoresis
– Separation based on partitioning between phases
 Capillary Electrophoresis
Summary
• Capillary electrophoresis provides high
separation efficiencies (N values) in much the
same way capillary columns do for GC
• Capillary electrophoresis also is very poor for
preparative separations
• Very small volumes are injected; concentration
sensitivity is poor vs. HPLC but mass sensitivity
is good
• Electropherograms show more variability in
elution times than HPLC
 Capillary Electrophoresis
Questions
1. If a polymer-based capillary has positive charges at the surface,
toward which electrode will neutral molecules travel?
2. What capillary electrophoresis methods could be used to separate
phenol from methoxyphenol?
3. Why are UV and Fluoresence detection especially useful in CE?
4. If the minimum detectable UV signal is A = 0.00010 AU, the
capillary is 50 μm wide, and the compound of interest has an
absorptivity coefficient of 87 M-1 cm-1, what is the minimum
detectable concentration (at the electropherogram peak)? If the
injection volume was 50 nL and the peak concentration was 1/5th
the initial concentration, what is the minimum detectable
quantity?