Capillary electrophoresis reverse mode

Identification and quantification of natural dyes need high performance analytical techniques, appropriate for the analysis of materials of complicated matrices containing a small amount of coloured substances. This requirement perfectly fits coupling of modern separation modules (usually high performance liquid chromatography in reversed phase mode, RPLC, but also capillary electrophoresis, CE) with selective detection units (mainly mass spectrometer). 

Reversed-phase HPLC can be compared in terms of its utility with other modes of chromatography and with other separation techniques such as gel electrophoresis, capillary electrophoresis (CE), and capillary electrochromatography. 

Separation is performed using free-zone electrophoresis, where the capillary is filled with a separating buffer at a defined pH and molarity. This buffer is also called a BGE. During separation, the polarity is set to cathodic or anodic mode, also called normal and reverse mode, depending on the charge of the molecule cation or anion. For anions, the capillary is usually dynamically coated with an electroosmotic flow (EOF) modifier to reverse the EOF and separate the analytes in the co-electroosmotic mode. 

Micellar electrokinetic capillary chromatography (MECC) is a mode of CE similar to CZE, in which surfactants (micelles) are added to the buffer system. Micellar solutions can be used to solubilize hydrophobic compounds that would otherwise be insoluble in water. In MECC the micelles are used to provide a reversed-phase character to the separation mechanism. Although MECC was originally developed for the separation of neutral species by capillary electrophoresis, it has also been shown to enhance resolution in the analysis of a variety of charged species.16... 

Capillary electrophoresis of PCR-amplified products is usually performed in the reverse polarity mode (negative potential at the injection end of the capillary). A coated capillary (100 mm i.d., 37-57 cm total length) is filled with a gel buffer system. PCR samples are introduced hydrodynamically or, after desalting, electrokinetically. The PCR sample and a DNA marker of known size may be injected sequentially and allowed to comigrate in the capillary. With a capillary temperature set at 20 to 30°C, separation of PCR products is accomplished at field strengths of 200 to 500 V/cm. Detection is on-line, measuring either UV absorbance at 260 nm, or LIF. 

The buffer system is a combination of buffer for electrophoresis and eluent for the particular chromatographic mode being employed. Figure 5.14 shows the separation of a group of neutral molecules using a capillary packed with a reversed-phase material.38 The buffer was a mixture of 4 mM sodium tetraborate (pH 9.1) and acetonitrile (20 80, v/v). The separation was compared with a micro-HPLC separation in which the same capillary was used but the eluent was pressure driven. As can be seen in Figure 5.14, sharper peaks were obtained with the EOF-driven system. 

The separation scientist with experience gained from a LC background may tend to limit the modes of electrochromatography to reversed phase (RP), normal phase, ion-exchange and, maybe, size-exclusion. Analysts from an electrophoretic background typically use the term "CE" in a much broader sense to include the main modes of capillary zone electrophoresis, micellar electrokinetic chromatography, capillary gel electrophoresis, isoelectric focusing and isotachophoresis. 

Wu, R., et ah. Separation of peptides on mixed mode of reversed-phase and ion-exchange capillary electrochromatography with a monolithic column. Electrophoresis, 23, 1239, 2002. 

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