Advanced Analytical Chemistry

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• Chapter 9 Capillary Electrophoresis (CE)
• References
• Dale R. Baker, Capillary Electrophoresis, John
  Wiley Sons, 1995.
• M.G. Khaledi, Ed., High-Performance Capillary
  Electrophoresis, John Wiley Sons, 1998.

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

1. An Overview of capillary electrophoresis
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CAI Florida International UniversityUpdated on
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• Column offers
• Ease of quantitation
• Automation
• Fraction collection
• On-line coupling to structure specific detectors,
  including MS, NMR
• Jorgenson and Lukacs (Anal. Chem., 1981, 53,
  1298) were the first to produce an operational
  capillary electrophoresis unit and demonstrate
  its high resolving power.

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• 1.1 Some concepts
• Electrophoresis is the movement of electrically
  charged particles or molecules in a conductive
  liquid medium, usually aqueous, under the
  influence of an electric field.

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• Electroosmotic flow Under the influence of an
  electric field, the buffer and the neutral
  molecules also move through the tube, due to
  Electroosmotic flow (well discuss the
  Electroosmotic flow in more details later).

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis


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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• Zone Spreading
• Theoretically, in electrophoresis, the compounds
  will travel through the conductive medium as zone
  that do not diffusion or spread out in the
  absence of any other influences except for the
  electric field.

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• Zone electrophoresis refers to the migration of
  molecules as zones which do not undergo zone
  spreading due to diffusion.
• Longitudinal and radical diffusion
• Do not contribute much zone spreading
• Molecular diffusion rate in a liquid is
  relatively small compared to the rate at which
  they migrate through the liquid

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• Thermal or convective diffusion
• Contributes significantly to zone spreading
• Electric current causes Joule heating
• Molecules in the warmer, center of a tube migrate
  faster than those near the cooler wall, leading
  to zone spreading
• Minimize the amount of heat generated and
  dissipate that heat.
• In addition to causing zone spreading, high T may
  also cause thermal degradation of some molecules

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

Influence of tube diameter on convective
diffusion In a round tube, the temperature
difference between the center and the wall of the
tube, ?T, can be calculated from ?T
(0.239Q/4k)r2 1 where Q is the power
density in watts/m3, k is thermal conductivity
of the solution, and r is tube radius.
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/21/2008 Chapter 9 Capillary Electrophoresis

• Minimize convective diffusion decrease diameter
  of the capillary
• Less current is generated for a given voltage,
  and less joule heat is produced.
• Fast dissipation of the heat (increase in the
  inner surface area-to-volume ratio of the tube).
• Therefore
• Narrow capillary has been selected 50 -75 ?M
  i.d.
• 400,000 theoretical plates with 80-100 cm long
  (20,000 by HPLC)

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CE is referred to
Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/21/2008 Chapter 9 Capillary Electrophoresis

• Capillary electrophoresis is sometimes referred
  to as Capillary zone Electrophoresis (CZE),
• Free solution Capillary Electrophoresis (FSCE)
• High-performance Capillary Electrophoresis (HPCE)
• Capillary Electrophoresis (CE).

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

1.2 CE system overview
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

CE Electropherograms and HPLC chromatograms Assum
ing equal solute concentrations and detector
responses
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• HPLC chromatograms
• In isocratic HPLC, the longer the retention time,
  the broader and the shorter the peaks. This is
  because solutes are diluted more as they spend
  more time inside the column.
• Area of all the peaks are approximately the same
  since all solutes through the detector at the
  same rate.

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• CE Electropherograms
• Peak height remain constant as retention gets
  longer because the solutes move through the
  detector in zones of approximately the same
  length and, therefore, the same concentration.
• The peak get wider with time because the later
  eluting solutes move through the detector more
  slowly, and consequently, reside in the detector
  cell longer. For equal concentrations and
  detector responses, the peak areas increase with
  time.

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• Sample injection
• Hydrodynamic by pressure or siphoning
  (gravity).
• Electrokinetic an electric field is applied to
  the sample vial, causing the sample components to
  migrate into the capillary.

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/21/2008 Chapter 9 Capillary Electrophoresis

Capillaries Fused silica capillaries (30-100 cm
long with inner diameters of 50-75 ?m and outer
diameters of 375 ?m). Detectors UV/Vis,
Fluorescence, conductivity, MS, ICP/MS, NMR
(new)...... Power supply Voltages up to 30 kV,
currents up to 300 ?A, and power up to 6 W.
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• 1.3 Comparison of CE to other separation
  techniques
• Efficiency
• Efficiency, N, expressed as the number of the
  theoretical plates, is related to how narrow the
  peaks are in a chromatogram or electropherogram.
• N 16(t/w)2
• The narrower the peak, the higher the
  efficiency, and the better the separation.
• CE has very high efficiency compared to HPLC and
  GC.

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• Sample type
• HPLC and GC are complementary.
• HPLC and CE are more competitive with each
  other.
• CE can use buffers that covers a wide pH range,
  whereas most silica-based HPLC column cannot be
  used with mobile phases at pH above about eight.
• Sample volume
• CE the relatively small volume (few nl) of
  sample. A capillary that is 50 cm long and 50 ?m
  i.d. has a volume of only 0.98 ?l.

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• Detection limit
• The Concentration detection limit of CE is not
  as good as HPLC and GC, roughly 100-1000 times
  higher.
• Confused about
• Concentration detection limit
• Instrument detection limit
• Sensitivity
• Reagent requirements
• Compared to HPLC, CE requires much less amount
  solvents, typically a few ml for a day of
  analysis.

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• Principles
• 2.1 Electroosmotic flow (EOF)
• In CE, in addition to the solutes, the buffer
  solution usually also moves through the capillary
  under the influence of an electric field. This
  phenomenon is termed electroosmotic flow. In
  normal operation, the direction of EOF is toward
  the negatively charged cathode.
• 2.1.1 Benefits of EOF
• Separate anions and cations in a single run.
• Neutral solutes would not move through the
  capillary tube.
• Reduces the analytical time

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/23/2008 Chapter 9 Capillary Electrophoresis

2.1.2 Formation of EOF
Silanol groups have strong affinity for polar
organic molecules.
Can be deactivated by silanization with
dimethylchlorosilane (DMCS).
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

solvated
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

solvated
The build up of ions at the capillary wall (image
courtesy of Agilent Technologies)
http//www.chemsoc.org/ExemplarChem/entries/2003/l
eeds_chromatography/chromatography/eof.htm
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• Silanol (Si-OH) groups are ionized to negatively
  charged silanoate (Si-O-) groups at pH above
  about three. This ionization can be enhanced
  first by passing a basic (KOH or NaOH) solution
  through the capillary followed by the buffer.
• The negatively charged silanoate groups then
  attract positively charged cations from the
  buffer, which form an inner layer of cations at
  the capillary wall. These cations are not of
  sufficient density to neutralize all the negative
  charges, so a second, outer layer of cations
  forms. The inner layer is tightly held by the
  Si-O- groups and is referred to as the fixed
  layer. The outer layer of cations is not tightly
  held because is it further away from the
  silanoate groups, and it is referred to as mobile
  layer. These two layers make up the diffuse
  double layer of cations.
• When an electric field is applied, the mobile,
  outer layer of cations is pulled toward the
  negatively charged cathode. Since these cations
  are solvated, they drag the bulk buffer solution
  with them, thus causing EOF.

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

Zeta potential (?) is an electrical imbalance
created at the plane of shear, which is the
potential difference across the layers. ?
4??e/? where ? is the thickness of the diffuse
double layer, e is the charge per unit surface
area, ? is the dielectric constant of the
buffer. EOF is proportional to the zeta
potential.
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

2.1.3 EOF Velocity and Mobility The velocity,
vEOF, vEOF ? ? E/4?? where E is the
applied electric field in volts/cm, and ? is the
viscosity of the buffer. The mobility, ?EOF,
?EOF ? ? /4?? Note that ?EOF is dependent
solely on buffer characteristics, and independent
of the applied electric field.
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• Significant parameters that affect EOF
• Applied Voltage
• Migration time
• Currents and Joule heating
• Zone spreading.

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/23/2008 Chapter 9 Capillary Electrophoresis

Excessive heat produced Resistance goes
down Causing increase in current
Question? Effects of capillary length and
diameter on maximum voltage selection
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• (2) Buffer pH
• Changes zeta potential. As pH increases, EOF
  increases, primarily because at higher pH, there
  more dissociation of Si-OH to Si-O- on the inner
  surface of the capillary.
• Influence the degree of ionization of the solutes
  and hence their mobility.
• Must consider the effect on separation

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/23/2008 Chapter 9 Capillary Electrophoresis

(3) Buffer concentration Effect on zeta
potential
Question? Why cannot the buffer concentration be
too low?
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/23/2008 Chapter 9 Capillary Electrophoresis

• (4) Temperature
• Effect on viscosity of the buffer. A temperature
  increase of 1 ?C, from 20 to 21?C, reduces the
  viscosity of water by 2.4.
• ? 4??e/?
• ?EOF ? ? /4??
• Temperature has effect on ?, but cancelled (see
  above equations).
• Zone spreading
• Compound decomposition

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• 2.1.4 Measurement of EOF
• Neutral marker
• Requirement of neutral markers
• Uncharged under the pH of the buffer
• Detectable by detector
• No interaction with the capillary and the buffer.

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• Current monitoring
• The capillary and destination vials are filled
  with buffer, and the source vial filled with the
  same buffer, but at a slightly different
  concentration.

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

2.1.5 Reverse EOF
Normal CE
Reverse EOF
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CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

Detector
a
c
Eletrophoretic mobility of formate ion 5.7 x
10-4 cm2/V EOF 4.2 x 10-4 cm2/V
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

Why and How
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

2.2 Electrophoretic mobility Electrophoretic
velocity, ?EP, in cm/s ?EP
?EPE 1 ?EP eletrophoretic
mobility E applied electric field.
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• Electrophoretic mobility ?EP
• ?EP q/6??r 2
• q the charge of the ionized solute
• ? buffer viscosity
• r solute radius
• ?EP is dependent on both mobility and electric
  field.
• ?EP is dependent only on solute and buffer
  properties
• Neutral molecules ?EP 0

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

A solutes velocity is influenced both by its
velocity of EOF, vEOF and ?EP. The observed
electrophoretic velocity, vOBS vOBS vEOF
vEP 3 In normal CE, that is the detector
is on the negatively charged side, and EOF is
from source to detector. Anions vOBS lt vEOF
Cations vOBS gt vEOF Neutals vOBS vEOF
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• The observed electrophoretic mobility, ?OBS
• ?OBS ?EOF ?EP 4

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

2.2.1 Measure electrophoretic velocity and
mobility vOBS l/tm 5 tm migration
time l the effective length of capillary, from
inlet to detector. The electroosmotic velocity,
vEOF, can be determined by measuring the
migration time of a neutral marker, tnm. Then
vEP l/tm - l/tnm 6
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

Electrophoretic mobility EV/L V is the
voltage L is the total length vEP ?EPE ?EP
vEPL/V 7 ?EP (l/tm- l/tnm)(L/V) 8
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

2.2.2 Parameters influencing ?EP ?EP
q/6??r Solute charge Solute size Buffer
viscosity
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

2.3 Effects of electrophoretic parameters on
separation
Chromatographic Parameters
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CAI Florida International UniversityUpdated on
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
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• 2.3.1 Migration Time (tm)
• tm l/ vOBS 9
• l effective capillary length
• vOBS ?OBSE 10
• tm l/(?OBSE) 11
• tm lL/(?OBSV) 12
• tm lL/(?EP ?EOF)V 13

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• 2.3.2 Efficiency

Definition of plate height in Chromatography
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
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The area of triangle is 96 of total area under
the peak. ( 2 ?) W 4?
H ?2/L 14
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

Since W 4?
16
17
Because H ?2/L, H L/N, N L2/?2 18
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

N 16(tR/w)2 19 Or N
5.54(tR/w1/2)2 20 Same for CE N
16(tm/w)2 21 Or N 5.54(tm/w1/2)2 22 E
fficiency, N, is expressed as the number of
theoretical plates. w peak width measured at
the base of the peak w1/2 peak width measured
at the half the peak height
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

The amount that a zone spreads is given by its
spatial variance, ?2, which is how much an
infinitely thin zone will diffuse over time,
t ?2 2Dt 23 D the solutes diffusion
coefficient in cm2/s.
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• Assuming that all adsorption of sample, and no
  radial diffusion, Substituting Eq 13 for t in
  23
• tm lL/(?EP ?EOF)V 13
• ?2 2DlL/(?EP ?EOF)V 24
• Because N L2/?2
• Substituting Eq 24 for ?2
• N (?EP ?EOF)V/2D 25

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

2.3.3 Selectivity (relative migration rates) In
HPLC, selectivity, ?, is given by ?
(t2-to)/(t1-to) A similar expression as that used
in Chromatography can be used for in CE. ?
(t2-tnm)/(t1-tnm) 26 where t2 and t1 are
migration times of adjacent peaks, and tnm is the
migration of a neutral marker.
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

2.3.4 Resolution The most important separation
parameter is resolution, that is, how well the
components in a mixture are separated.
Resolution, R, can be calculated from an
electropherogram using R ?t/wAVE R
2(t2-t1)/(w1w2) 27 where w1 and w2 are peak
widths of adjacent peaks.
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

Recall in chromatography, we assume WA WB
W R (tR)B (tR)A/W N 16(tR/W)2
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/17/2006 Chapter 9 Capillary Electrophoresis

• In CE?
• James W. Jorgenson et al. Anal. Chem. 1981, 53,
  1298-1302.
• James W. Jorgenson et al., Science, 1983,
  222,266-272.
• J.C. Giddings, Separation Sci. 1969, 4, 181.

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