Capillary Electrophoresis Detection

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Capillary Electrophoresis Detection
Detection in CE is a significant challenge as a
result of the small dimensions of the Capillary
and the nanoliter volumes of sample. A number of
detection methods have been used in CE to meet
this challenge, many of which are similar to
those employed in HPLC
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Improving Detection Limits Through Sample Matrix
Optimization
Several techniques are described to enhance
sensitivity by on-capillary sample concentration
during or just after sample injection. These
methods are based on field strength differences
between the sample zone and the running buffer,
are called stacking. One method is to make the
conductivity of the sample signifi- cantly lower
than that of the running buffer. The simplest way
to perform a stacking experiment is to dissolve
the sample in water or low conductivity buffer
(e.g., 100- 1000 times lower than the running
buffer) and inject normally. More than a 10-fold
sample enrichment can be obtained. If the
conductivity of the sample and the running
buffer are equivalent, stacking can be induced by
injecting a short plug of water before the
sample introduction.
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Optical Design for High Sensitivity Diode Array
Detector in CE
Using the DAD over more conventional UV-Vis
detectors can greatly simplify the analysis of
electrophoretic data. Once all peaks have been
detected the absorbance maxima for each peak can
be calculated. The data can be presented in
three- dimensional format. Quantification of
non-separated peaks is possible through peak
suppression. Peak purity can be examined and
confirmation of peak identity can be
achieved through comparison with a standard
spectrum
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Extended Light Path Capillary
UV-Vis absorption is the most widely used
detection method primarily due to its
universal detection nature. With
fused-silica capillaries, detection below 200
nm up through the visible spectrum can be used.

Sensitivity and linearity usually can be improved
by increasing the capillary i.d. This approach
is limited however by increase in current and
subsequent heating within the capillary.
Because the bubble is located in the detection
region no increase in current occurs. When the
zone enters the bubble, its velocity decreases
and the zone concentrates or stacks in a manner
similar to electrophoretic stacking
during injection. The sample concentration
remains constant, but the path length increases.
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Capillary Electrophoresis-Mass Spectrometry
(CE-MS)
A very powerful detector for CE is the mass
spectrometer. Molecular weight Information from
molecules is available when coupled to an
electrospray Interface with 0.25 dalton
accuracy.
Some difficulties remain including concentration
sensitivity, buffer incompatibiliies between CE
and electrospray and the high skill level
required by the operator.
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Improving Detection Limits by Optimization of
Buffer and Wavelengths
In CE, some common buffers like borate and
phosphate have low UV absorbance and thus
sensitive detection can occur at 196-200 nm.
This improves the detection relative to 214 or
254 nm, which is commonly utilized in HPLC.
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Next several slides will show Qualitative and
Quantitative Analysis in Capillary
Electrophoresis Qualitative Analysis ---
Criteria ---Reproducibility of qualtitative
Parameters Quantitative Analysis --Factors
Affecting quantitative analysis
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Qualitative Analysis in Capillary Electrophoresis
GC ----? Retention Index (RI) used in GC (Few
exp variables)
HPLC ---? Both S.P and M.P variables (RI is
difficult to measure in HPLC)
CE-? Migration time (analagoues to retention time
in chromatography), but It is difficult to
introduce RI (because composition of carrier
electrolye varies)
Can we use migration time as a qualitative
parameter in CE?
Yes, we can but it depends on the of EOF
Can we use EOF as a qualitative parameter ? Yes,
but EOF is prone to drift and is not stable
So what parameter we should use?
We should use effective mobility (mep) because
mep is independent of field strength and
capillary length but depend on buffer composition
and temperature mep ma -mEOF
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Reproducibility of Mobility and Migration Time
FACTOR CAUSE/EFFECT SOLUTION Temp
Change Change viscosity and EOF Themostat
capillary
Adsorption to the Changes EOF
-Condition capillary capillary wall Caused
by buffer and allow sufficient
additive equilibration time
Hysteresis of Caused by
conditioning -Avoid pH difference wall
charge capillary at high (or low) -Allow
sufficient pH and employing low or
equilibration time (high) pH running buffer
Changes in buffer pH changes due to
-Replenish buffer composition electrolysis
Buffer evaporation -Cap buffer vials

and cool carousel
Conditioning waste -Use
separate reservoir
flushed into outlet
to collect wash solution
Carrying sodium -First
dip capillary in hydroxide from
separate buffer conditioning vial or
water vial
into buffer vial
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Reproducibility of Mobility and Migration Time
FACTOR CAUSE/EFFECT
SOLUTION Buffer reservoir Non-reproducible
level liquid not level laminar flow
if replenshing do

not use
inlet vial for washing
capillary
Different silanol Different wall charge
Measure EOF and content of silica and
variations normalize if
necessary batches in
EOF
Variations in Proportional changes
Not user accessible applied voltage
in migration time
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Quantitative Analysis in Capillary Electrophoresis
---Dark ages of CE have passed, the technique is
now recognized as quantitative fully automated
instrumentation, on-line detection and modern
computer power allow peak integration and
quantitation.

• --FACTORS GOVERNING QUANTITATIVE ANALYSIS
• Data Sampling Rate
• Use of Peak Area
• Methods of Quantitation
• Linear Dynamic Range/Impact of Solute
  Concentration on Peak Shape

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• Data Sampling Rate
• Sampling rate of 20 Hz or less are suitable
• For example, a signal with a 5s peak width is
  sampled with 100pts

Q1. What are the effects of oversampling? Does
not substantially improve precision, occupy large
disk storage capacity
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Q2. What are the effects of undersampling?
Increase RSD for peak area

• Use of Peak Areas or Peak Height?
• Peak areas are prefered for two reasons
• Variation in ionic strength of the sample can
  effect solute stacking and
• therefore peak height.

b. Lineaer dynamic range of separation is
enhanced. As the solute concentration is
increased, electromigration dispersion begins to
appear. Peak height and peak width are affected,
but the area is conserved as shown later in
discussion on linear dynamic range.
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Other Modes Chiral Capillary Electrophoresis
(CCE) Affinity Capillary
Electrophoresis (ACE)
Capillary Isotachophoresis
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Capillary Zone Electrophoresis (CZE)
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CZE Optimization (Method Development)
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