Types of Capillary Electrophoresis

In the absence of micelles, all neutral molecules reach the detector together at a time we designate Micelles injected with the sample reach the detector at time fnic which is longer than because micelles are negative and migrate upstream. If a neutral molecule equilibrates between free solution and the inside of the micelles, its migration time is increased, because it migrates at the slower rate of the micelle part of the time. In this case, the neutral molecule reaches the detector at a time between /q and 

---

The various types of capillary electrophoresis are performed either in free solution or in gels. The choice of method depends on the nature of the sample and the analytical objective but capillary gel electrophoresis, including iso-electric focusing and SDS electrophoresis, is particularly useful for protein applications. 

The most distinguishing phenomenon in capillary electrophoresis is whether the experiments are performed in the absence or in the presence of electroos-motic flow (EOF), (see Chapter 6 for details on EOF). Unlike other types of capillary electrophoresis, isoelectric focusing can be performed under both modes. Since the experimental and theoretical principles governing these modes of CIEF are different, they will be discussed separately. 

Because the separated analytes move past a common point in most types of capillary electrophoresis, detectors are similar in design and function to those described for HPLC. Table 33-3 lists several of the detection methods that have been reported for capillary electrophoresis. The second column of the table shows representative detection limits for these detectors. 

Electroosmosis is often desirable in certain types of capillary electrophoresis, but in other types it is not. Electroosmotic flow can be minimized by coating the inside capillary wall with a reagent like trimethylchlorosilane to eliminate the surface silanol groups. 

The last set of experiments provides examples of the application of capillary electrophoresis. These experiments encompass a variety of different types of samples and include examples of capillary zone electrophoresis and micellar electrokinetic chromatography. 

In practice isotachophoresis is usually performed in narrow tubes with electrodes at either end and is one form of capillary electrophoresis. For the separation of a particular type of ion, e.g. an anion, two buffered electrolyte solutions are selected that have different anions but a common cation with a buffering capacity. One of the anions (termed the leading electrolyte) should show a greater mobility than the other anion and occupies the anodic end of... 

Although on-column fluorescence detection can provide excellent detection limits, the technique is less versatile than UV detection because many solutes of interest do not exhibit native fluorescence and must be derivatized with some type of fluorophore. Consequently, the literature contains many examples of capillary electrophoresis separations of fluorescence-labeled solutes such as dansylated amino acids.50,51... 

This chapter introduces the basic concepts and principles of capillary electrophoresis (CE), presenting some background on electrophoresis and capillary electrophesis and describing the components of the system. The two main types of CE, capillary zone and micellar electrokinetic electrophoresis, are described, and a selection strategy, based on the two types of separation, electrophoretic migration and electroosmosis, is presented. 

Capillary electrochromatography uses electroendosmotic flow (EOF) to perform highly efficient separations in small-diameter fused-silica capillaries, packed with HPLC-type stationary phases. It can be considered as a combination of capillary electrophoresis (CE) and HPLC. The separation of solutes is based on electrophoretic mobility (for charged species) and interaction with the stationary pha.se, allowing the separation of both neutral and charged compounds. 

A variety of methods has been applied to the separation of bound and free Ag. These include precipitation, solid phase attachment, capillary electrophoresis, chromatography, andmicrofiltration. Originally, precipitation and solid-phase extraction were the most common types of separations techniques. However the ease of automation of capillary electrophoresis and flow-injection analysis (chromatography) makes these two techniques very interesting. 

Even though capillary electrophoresis is a relatively simple method, several formats exist that allow for analyses of different types of samples or to take advantage of certain solute properties. The primary modes of capillary electrophoresis are as follows ... 

Several features of capillary electrophoresis are particularly interesting for forensic scientists, namely high separation efficiency, sensitivity, and small amount of samples (nanoliters) and solvents (a few milliliters per day). Regarding different operational modes, all of them are applied, although to a different extent, depending on the type of compounds to be assayed. 

Until the appearance of capillary electrophoresis, electrophoretic separations were not canned out in columns but were performed in a flat stabilized medium such as paper or a porous semisolid gel. Remarkable separations were realized in such media, but the technique was slow, tedious, and required a good deal of operator skill. In the early 1980s,. scientists began to explore the feasibility of performing these same separations on micro amounts of sample in fused silica capillary tubes. Their results proved promising in terms of resolution, speed, and potential for automation. As a consequence, capillary electrophoresis (CE) has become an important tool for a wide variety of analytical separation problems and is the only type of electrophoresis that we will consider. 

The successful application of capillary electrophoresis technology to the genotyping of various types of polymorphisms has been well documented. The flexibility and automation of the Applied Biosystems 3100 Genetic Analyzer... 

Several types of LC and one nonchromatographic separation system for liquids have been interfaced with MS. HPLC is widely used to separate nonvolatile organic compounds of all polarities and molecular weights. Coupled to a mass spectrometer, the technique is called LC-MS. Supercritical fluid chromatography (SFC) and the nonchromatographic separation technique of capillary electrophoresis (CE) are also used with mass spectro-metric detection. The interfacing, ionization sources, operation, and applications of these hyphenated methods are covered in Chapter 13. 

Y. Zhao, R. Zhao, D. Shangguan, G. Liu, A new type of capillary column for open-tubular electrochromatography. Electrophoresis, 2002, 23, 2990-2995. 

For a given operational mode the separation efficiency N depends on the conditions of electrophoresis such as the type of capillary, background electrolyte, volume and composition of the sample, the applied voltage and temperature. 

Injecting the Sample The mechanism by which samples are introduced in capillary electrophoresis is quite different from that used in GC or HPLC. Two types of injection are commonly used hydrodynamic injection and electrokinetic injection. In both cases the capillary tube is filled with buffer solution. One end of the capillary tube is placed in the destination reservoir, and the other is placed in the sample vial. 

In addition to polymeric support media, capillaries and flowing buffers have been used as support media for electrophoresis. Although these are not used as frequendy, there are definite advantages for certain types of samples and appHcations. 

In addition to chromatography based on adsorption, ion pair chromatography (IP-HPLC) and capillary electrophoresis (CE) or capillary zone electrophoresis (CZE) are new methods that became popular and are sufficiently accurate for these types of investigations. Other methods involving electrochemical responses include differential pulse polarography, adsorptive and derived voltammetry, and more recently, electrochemical sensors. 

A variety of formats and options for different types of applications are possible in CE, such as micellar electrokinetic chromatography (MEKC), isotachophoresis (ITP), and capillary gel electrophoresis (CGE). The main applications for CE concern biochemical applications, but CE can also be useful in pesticide methods. The main problem with CE for residue analysis of small molecules has been the low sensitivity of detection in the narrow capillary used in the separation. With the development of extended detection pathlengths and special optics, absorbance detection can give reasonably low detection limits in clean samples. However, complex samples can be very difficult to analyze using capillary electrophoresis/ultraviolet detection (CE/UV). CE with laser-induced fluorescence detection can provide an extraordinarily low LOQ, but the analytes must be fluorescent with excitation peaks at common laser wavelengths for this approach to work. Derivatization of the analytes with appropriate fluorescent labels may be possible, as is done in biochemical applications, but pesticide analysis has not been such an important application to utilize such an approach. 

---