Chem. 230 – 11/25 Lecture.

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 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

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 = (M+n)/n (most common)

• For – ions m/z = (M–n)/n

Ion

curr

ent

m/z

m/z

m/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)

(M+n)/n

(M+n+1)/(n+1)

Example: m/z peaks =711.2, 569.3, 474.8, 407.1

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

Glycodendrimer core Glycodendrimer core

gap = 405.73 – 405.23 = 0.50

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

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?

Mass SpectrometeryInterpretation Questions

1. Determine the identity of the compound giving the following distribution:

m/z Abundance(% of

biggest)25 14

26 3427 10035 962 77

64 24


2. Determine the identity of the compound giving the following distribution:

m/z Abundance(% of

biggest)29 9.2

50 30.551 84.777 10093 16

123 39


3. From the following M, M+n ions, determine the number of Cs, Brs and Cls:

m/z Abundance(% of

biggest)117 100118 1.4119 98121 31.1123 3

Capillary ElectrophoresisOverview

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

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/(6r)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)

high voltage+

-

X-

Electric Field

anode cathode

electric force

drag


• 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-

Na+ to cathode

to anode

Na+ Na+ Na+ Na+Na+ Na+Na+

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 + CuH = A + B/uH = B/u

hotter

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


• 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)vNetwhere l = length from anode to

detector– time depends on ion size, charge,

pH (weak acids/bases), voltage, column lengths

vEOF = vNet(neutrals)

vCations

vNet

vAnionsvNet

Weak Acid Example

vEOF

vNet A-

vNet HA

at pH ~ pKa, vNet = (vNet HA + vNet A-)/2

Capillary ElectrophoresisEquipment

• 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

detector

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


• Sample injection– Electroosmotic injection (using

applied voltage) (sometimes biases sample)

– Hydrostatic injection (based on raising/lowering capillaries)

– Hydrodynamic injection (using applied pressure)

High V

+ -


• 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


• 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


• 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 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)


• 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


• 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

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

Capillary ElectrophoresisQuestions

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?

Chem. 230 – 11/25 Lecture.

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