Capillary Electrophoresis - Tim Towner

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CHEM-ENG 590E: Microfluidics and Microscale Analysis in Materials and Biology

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Background

Fig. 1 Capillary Electrophoresis general setup

Capillary Electrophoresis (CE) is a separation technique performed on the micro or nanoscale. [1] This technique analyzes analytes utilizing the movement of charged particles in an electric field through a fluid or gel. [3] This difference in the charge to mass ratio is what makes this technique different from other high throuhput techniques such as High Performance Liquid Chromatography (HPLC). The primary methods used in CE are capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC or MECC), capillary electrochromatography (CEC), capillary isoelectric focusing (CIEF), capillary gel electrophoresis (CGE) and capillary isotachophoresis (CITP). [1] The instrumentation used for CE has a general setup of an injection system, a capillary tube for separation, and a high voltage supply. There are two methods for injecting samples are hydrodynamic and electrokinetic mode. Hydrodynamic injection uses pressure or vacuum application to carry the sample to the sample solution to the capillary tube and is nonselective. Described by Poiseulle's Law

V=ΔPr^4 πCt8ηL page 33 and 34

Where: ΔP= change in pressure, r= inner radius, C= sample concentration, t= injection time, η= solution viscosity, L= capillary length

This differs from electrokinetic as this technique can select the composition of the sample solution through application of potential. While the selectivity of electrokinetic injection is greater, it is harder to control without particular care. [1]

Methods

There are two main types of methods used in CE, bulk property and specific-property detectors where bulk properties measure a physical property of a solute relative to the buffer solution and a specific-property detector measures a specific property of a solute. Bulk property methods are more universal but have lower sensitivities and dynamic ranges as the properties measured are relative to the solution. Of direct detections, UV absorbance is the most common form commercially as was able to be adapted to HPLC detectors. The main material used in UV absorbance is high-quality fused silica because silica can observe materials up to 170 nm giving it a wide range of absorbance. [5]

Fig. 2 A) Coxial Sheath Flow Configuration B)Sheathless interface C)Sheathless interface with gold wiring
CZE 

One of the main methods for CE and is gone in much greater detail on this page CZE

MECC

A direct method that uses specific-property detectors which is suited for neutral substances which move at the same rate as the electroosmotic flow (EOF). In MECC, the flow is "plug-like" as the EOF reduces the band broadening is therefore an efficient method for the separation process.

Mass Spectrometry

Combining CE with Mass Spectrometry adds the benefit of analyzing the analytes differential separation and their molecular masses and/or fragmentation patterns. One problem with CE-MS is that it requires the coupling of an ionization method that will separate the sample into a liquid phase in order to do MS. [4]

Applications

Fig. 3 CE-MS apparatus

CE may be used for many applications such as the detection and quantification of mutations in nucleic acids and characterization of proteins, carbohydrates, metabolites, and pharmaceuticals. Polymerase Chain reaction (PCR) is used in conjunction with CE in order to determine the genotype of a sample. For example, exons 1-20 in Duchenne muscular dystrophy were genotyped in nine minutes and can recognize DNA fragments with as few as five base pair differences. Regarding proteomics, CE is used primarily with mass spectrometry (MS) to detect and characterize proteins as it has high accuracy the ability for real time analysis. CE-MS peptide analysis methods have mapped a therapeutic monoclonal antibody in a tryptic digest with LC-MS methods mapping 97%. CZE-ESI-MS/MS was also used to analyze E. coli proteome fractions at a rate of 145 proteins/h which was twice as fast as previous results using E. coli proteome. [3]

Advantage

Electrophoresis benefits from transitioning to the microscale due to improved performance which is determined by:

*Analysis speed and throughput
*Reduced costs
*Integration and multiplexing capabilities.

References

[1] https://doi.org/10.1016/S0379-0738(98)00010-3 A brief introduction to capillary electrophoresis

[2] 10.1021/acs.analchem.5b04125 Capillary Electrophoresis

[3] https://doi-org.silk.library.umass.edu/10.1016/j.cbpa.2003.07.001 Microscale separation and analysis

[4] https://doi.org/10.1016/0021-9673(94)01178-H Capillary electrophoresis-mass spectrometry

[5] Grossman, P. D.; Colburn, J. C. Capillary Electrophoresis Theory & Practice; Academic Press, Inc: San Diego, Ca, 1992.