1 (No Transcript) 2 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 3 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. 4 (No Transcript) 5 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 6 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. 7 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. 8 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. 9 Next severals will show Qualitative and Quantitative Analysis in Capillary Electrophoresis Qualitative Analysis --- Criteria ---Reproducibility of qualtitative Parameters Quantitative Analysis --Factors Affecting quantitative analysis 10 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 11 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 12 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 13 (No Transcript) 14 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
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 Q 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. 16 (No Transcript) 17 (No Transcript) 18 (No Transcript) 19 Other Modes Chiral Capillary Electrophoresis (CCE) Affinity Capillary Electrophoresis (ACE) Capillary Isotachophoresis 20 Capillary Zone Electrophoresis (CZE) 21 (No Transcript) 22 (No Transcript) 23 (No Transcript) 24 (No Transcript) 25 CZE Optimization (Method Development) 26 (No Transcript) 27 (No Transcript) 28 (No Transcript) 29 (No Transcript) 30 (No Transcript) 31 (No Transcript) 32 (No Transcript) 33 (No Transcript) 34 (No Transcript) 35 (No Transcript) 36 (No Transcript) 37 (No Transcript) 38 (No Transcript)
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