1. Chem. 230 – 11/25 Lecture

2. 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

3. 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)

4. 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

5. Mass Spectrometry Other Topics – Multiple Charges in ESI
In ESI analysis of large molecules, multiple charges are common due to extra (+) or missing (-) Hs (or e.g. Na+)
The number of charges can be determined by looking at distribution of big peaks
For + ions m/z = (M+n)/n (most common)
For – ions m/z = (M–n)/n
(M+n)/n
Dm/z
Ion current
m/z
(M+n+1)/(n+1)
Example: m/z peaks =711.2, 569.3, 474.8, 407.1
Dm/z = (M+n)/n – (M+n+1)/(n+1) = (M+n)(n+1)/[n(n+1)] – (Mn+n2+n)/[n(n+1)] = M/[n(n+1)] = 141.9, (94.5, 67.7)
Do rest on board 5

6. 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 = – = 0.50
Glycodendrimer core
Glycodendrimer core

7. 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 7

8. Mass Spectrometery Questions I
Which ionization method can be achieved on solid samples (without changing phase)
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?
For a large, polar non-volatile molecule being separated by HPLC, which ionization method should be used?

9. Mass Spectrometery Interpretation Questions
Determine the identity of the compound giving the following distribution:
m/z
Abundance
(% of biggest) 25 14 26 34 27
100 35 9 62 77 64 24

10. Mass Spectrometery Interpretation Questions
2. Determine the identity of the compound giving the following distribution:
m/z
Abundance
(% of biggest) 29
9.2 50
30.5 51
84.7 77
100 93 16
123 39

11. Mass Spectrometery Interpretation Questions
3. From the following M, M+n ions, determine the number of Cs, Brs and Cls:
m/z
Abundance
(% of biggest)
117
100
118
1.4
119 98
121
31.1
123 3

12. Capillary Electrophoresis Overview
Basis of Electrophoresis
Electroosmotic Flow in Capillaries
Equipment
Summary of Main Methods

13. Capillary Electrophoresis Basis for Separation-
Transport in electrophoresis is based on electric forces on ions:
The electrostatic force accelerates the ion toward the electrode of opposite charge
But “drag” in the opposite direction soon becomes equal to the electrostatic force leading to constant velocity
velocity = v = zE/(6phr)
where z = charge, E = electric field, h = 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)
high voltage +
electric force X -
anode
cathode
drag
Electric Field

14. 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 anode
Na+
Na+
Na+
Na+
Na+
Na+
Na+
Na+
to cathode

15. Capillary Electrophoresis Separation Efficiency
Van Deemter Equation
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
highest voltage provides fastest separation and least dispersion, but
highest voltages result in heating capillary cores and dispersion due to differential viscosity
H = A + B/u + Cu
H = A + B/u
H = B/u
hotter

16. 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

17. Capillary Electrophoresis Basis for Separation
Net velocities:
vNet = vEOF + vion
vion is negative for anions, positive for cations and 0 for neutral species
No separation of neutral species in Capillary Zone Electrophoresis
Analyte migration time
time = l(L/V)vNet
where l = length from anode to detector
time depends on ion size, charge, pH (weak acids/bases), voltage, column lengths
vEOF = vNet(neutrals)
vNet
vNet
vCations
vAnions
Weak Acid Example
vEOF
vNet A-
vNet HA
at pH ~ pKa, vNet = (vNet HA + vNet A-)/2

18. 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 most common)
Safety Equipment – to turn off high voltage when accessing equipment
high voltage +

19. 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

20. Capillary Electrophoresis Equipment (Cont.)+ -
Sample injection
Electroosmotic injection (using applied voltage) (sometimes biases sample)
Hydrostatic injection (based on raising/lowering capillaries)
Hydrodynamic injection (using applied pressure)
High V

21. 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

22. 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

23. 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

24. 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)

25. 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

26. Capillary Electrophoresis Main Methods
Micellar Electrokinetic Chromatography (MEKC)
Micelles added to buffer (from surfactants)
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
Flow driven by electrophoresis
Separation based on partitioning between phases
surfactant
micelle

27. 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

28. Capillary Electrophoresis Questions
If a polymer-based capillary has positive charges at the surface, toward which electrode will neutral molecules travel?
What capillary electrophoresis methods could be used to separate phenol from methoxyphenol?
Why are UV and Fluoresence detection especially useful in CE?
If the minimum detectable UV signal is A = 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?