Chem. 230

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Chem. 230 11/25 Lecture
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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

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Announcements II

• Todays Lecture
• Mass Spectrometry
• Interpretation
• Other Topics
• Capillary Electrophoresis
• Theory
• Equipment
• Summary of Main Methods
• First Special Topics Presentations (Cheng and
  Clarke MEKC)

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Mass SpectrometeryInterpretation

• 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

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Mass SpectrometryOther 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 (Mn)/n (most common)
• For ions m/z (Mn)/n

(Mn)/n
Dm/z
Ion current
m/z
(Mn1)/(n1)
Example m/z peaks 711.2, 569.3, 474.8, 407.1
Dm/z (Mn)/n (Mn1)/(n1)
(Mn)(n1)/n(n1) (Mnn2n)/n(n1)
M/n(n1) 141.9, (94.5, 67.7)
Do rest on board
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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
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Mass SpectrometryOther 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

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Mass SpectrometeryQuestions 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?

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Mass SpectrometeryInterpretation 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
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Mass SpectrometeryInterpretation 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
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Mass SpectrometeryInterpretation Questions

• 3. From the following M, Mn 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
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Capillary ElectrophoresisOverview

• Basis of Electrophoresis
• Electroosmotic Flow in Capillaries
• Equipment
• Summary of Main Methods

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Capillary ElectrophoresisBasis 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
anode
cathode
drag
Electric Field
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Capillary ElectrophoresisBasis 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 gt 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

to anode
Na
Na
Na
Na
Na
Na
to cathode
Na
Na
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Capillary ElectrophoresisSeparation 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
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Capillary ElectrophoresisSeparation 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

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Capillary ElectrophoresisBasis 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
vCations
vNet
vAnions
Weak Acid Example
vEOF
vNet A-
vNet HA
at pH pKa, vNet (vNet HA vNet A-)/2
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Capillary ElectrophoresisEquipment
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

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Capillary ElectrophoresisEquipment (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

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Capillary ElectrophoresisEquipment (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
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Capillary ElectrophoresisEquipment (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

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Capillary ElectrophoresisEquipment (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

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Capillary ElectrophoresisEquipment (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

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Capillary ElectrophoresisMain 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)

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Capillary ElectrophoresisMain 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 (gt2) 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

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Capillary ElectrophoresisMain 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
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Capillary ElectrophoresisSummary

• 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

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Capillary ElectrophoresisQuestions

• 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
  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?