Electroosmotic flow capillary electrophoresis

Capillary electrophoresis—electroosmotic flow and electrophoretic mobility ... 

While some techniques require the absence of electroosmotic flow during the separation itself (capillary gel electrophoresis and capillary isoelectric focusing), most common techniques exploit electroosmotic flow for sample introduction and detection. Electrophoresis in buffer-filled capillaries uses electroosmotic flow in an analogous manner to a chromatographic mobile phase the flow is used to transport analyte from cathode to anode and separation occurs continuously between introduction and detection. 

The electroosmotic flow profile is very different from that for a phase moving under forced pressure. Figure 12.40 compares the flow profile for electroosmosis with that for hydrodynamic pressure. The uniform, flat profile for electroosmosis helps to minimize band broadening in capillary electrophoresis, thus improving separation efficiency. 

First, solutes with larger electrophoretic mobilities (in the same direction as the electroosmotic flow) have greater efficiencies thus, smaller, more highly charged solutes are not only the first solutes to elute, but do so with greater efficiency. Second, efficiency in capillary electrophoresis is independent of the capillary s length. Typical theoretical plate counts are approximately 100,000-200,000 for capillary electrophoresis. 

Capillary zone electrophoresis also can be accomplished without an electroosmotic flow by coating the capillary s walls with a nonionic reagent. In the absence of electroosmotic flow only cations migrate from the anode to the cathode. Anions elute into the source reservoir while neutral species remain stationary. 

Electroosmotic flow in a capillary also makes it possible to analyze both cations and anions in the same sample. The only requirement is that the electroosmotic flow downstream is of a greater magnitude than electrophoresis of the oppositely charged ions upstream. Electro osmosis is the preferred method of generating flow in the capillary, because the variation in the flow profile occurs within a fraction of Kr from the wall (49). When electro osmosis is used for sample injection, differing amounts of analyte can be found between the sample in the capillary and the uninjected sample, because of different electrophoretic mobilities of analytes (50). Two other methods of generating flow are with gravity or with a pump. 

The mechanism by which analytes are transported in a non-discriminate manner (i.e. via bulk flow) in an electrophoresis capillary is termed electroosmosis. Eigure 9.1 depicts the inside of a fused silica capillary and illustrates the source that supports electroosmotic flow. Adjacent to the negatively charged capillary wall are specifically adsorbed counterions, which make up the fairly immobile Stern layer. The excess ions just outside the Stern layer form the diffuse layer, which is mobile under the influence of an electric field. The substantial frictional forces between molecules in solution allow for the movement of the diffuse layer to pull the bulk... 

Isotachophoresis. In isotachophoresis (ITP), or displacement electrophoresis or multizonal electrophoresis, the sample is inserted between two different buffers (electrolytes) without electroosmotic flow. The electrolytes are chosen so that one (the leading electrolyte) has a higher mobility and the other (the trailing electrolyte) has a lower mobility than the sample ions. An electric field is applied and the ions start to migrate towards the anode (anions) or cathode (cations). The ions separate into zones (bands) determined by their mobilities, after which each band migrates at a steady-state velocity and steady-state stacking of bands is achieved. Note that in ITP, unlike ZE, there is no electroosmotic flow and cations and anions cannot be separated simultaneously. Reference 26 provides a recent example of capillary isotachophoresis/zone electrophoresis coupled with nanoflow ESI-MS. 

CEC is often inappropriately presented as a hybrid method that combines the capillary column format and electroosmotic flow employed in high-performance capillary electrophoresis with the use of a solid stationary phase and a separation mechanism, based on specific interactions of solutes with the stationary phase, characteristic of HPLC. Therefore CEC is most commonly implemented by means typical of both HPLC (packed columns) and CE (use of electrophoretic instrumentation). To date, both columns and instrumentation developed specifically for CEC remain scarce. 

J.P. Quirino and S. Terabe, Sample stacking of fast-moving anions in capillary zone electrophoresis with pH-suppressed electroosmotic flow. J. Chromatogr.A 850 (1999) 339-344. 

FIGU RE 7.12 Representation of the diffuse double layer responsible for electroosmotic flow in capillary electrophoresis. 

Effects of buffer composition on electroosmotic flow in capillary electrophoresis. /. Microcol. Sep. 2, 176-180. 

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. 

Diress, A. G., and Lucy, C. A. (2004). Electroosmotic flow reversal for the determination of inorganic anions by capillary electrophoresis with methanol-water buffer. /. Chromatogr. A 1027, 185-191. 

Li, Y., Xiang, R., Horvath, C., and Wilkins, J. A. (2004). Capillary electrochromatography of peptides on a neutral porous monolith with annular electroosmotic flow generation. Electrophoresis 25, 545-553. 

The second parameter influencing the movement of all solutes in free-zone electrophoresis is the electroosmotic flow. It can be described as a bulk hydraulic flow of liquid in the capillary driven by the applied electric field. It is a consequence of the surface charge of the inner capillary wall. In buffer-filled capillaries, an electrical double layer is established on the inner wall due to electrostatic forces. The double layer can be quantitatively described by the zeta-potential f, and it consists of a rigid Stern layer and a movable diffuse layer. The EOF results from the movement of the diffuse layer of electrolyte ions in the vicinity of the capillary wall under the force of the electric field applied. Because of the solvated state of the layer forming ions, their movement drags the whole bulk of solution. 

FA Gomez, LZ Avila, Y-H Chu, GM Whitesides. Determination of binding constants of ligands to proteins by affinity capillary electrophoresis compensation for electroosmotic flow. Anal Chem 66 1785-1791, 1994. 

Electroosmosis Here, the liquid (an electrolyte solution) moves past a charged surface (e.g., the surface of a capillary tube or through a porous plug) under the influence of an electric field. Thus, electroosmosis is the complement of electrophoresis. The pressure needed to balance the electroosmotic flow is known as electroosmotic pressure. 

CE detection is similar to detectors in, and include absorbance, fluorescence, electrochemical, and mass spectrometric detectors. The capillary can also be filled with a gel, which eliminates the electroosmotic flow. Separation is accomplished as in conventional gel electrophoresis but the capillary allows higher resolution, greater sensitivity, and on-line detection. In CE, low picogram amounts of analytes can be detected using glass fiber optics. However, this does not mean low limits of detection since only a few nanoliters can be injected. 

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