Sample Injection in CZE

Miniature amperometric or conductometric detectors can be introduced into the capillary, by plating the appropriate electrodes in the inside of the capillary walls. It is often necessary to employ a porous glass or graphite joint between the capillary containing the detector electrodes and the end of the capillary in the detector end electrode reservoir, in order to isolate the detector from the high voltage used to power the electrophoretic separation. 

Because CZE is a method for separating and measuring ions, like IC, considerations of charge balance make it particularly suitable for use of the method of indirect detection, as described at the end of Section 13.2.1. 

There are several different modes of conducting electrophoresis in capillary columns. We have just discussed at length the basic one, CZE, which is conceptually the simplest. It is conducted with a liquid buffer of uniform composition of electrolyte concentration and pH level. There are more complex versions, in which the buffer liquid is enmeshed in a porous gel of hydrophilic polymer, or the concentration or the pH of the separation buffer is not constant along the length of the column, leading to variations in the migration velocities of individual ions as the separation proceeds. Several of these will be discussed subsequently. 

CZE has already been discussed in Sections 13.6.1-13.6.3. To summarize for purposes of comparison, it is performed using a separation buffer of constant composition. The only exception is the sample plug, which is usually introduced in a buffer of lower concentration to enable faster migration velocities within it to achieve the desirable effect of sample 

Two big questions remain How do we establish and maintain a pH gradient across the column while the amphoteric analytes are migrating to their focused halt points, and how then do we move the separated and focused bands past a detector while maintaining the separation which was achieved in the first step  

AP is the pressure difference between the ends of the capillary d is its inner diameter L, is its total length 

If the capillary is both too narrow and/or too long, it may be necessary to apply additional pressure at the injection end or suction at the detector end in order to introduce a sufficient volume in a reasonable time. For electrokinetic injection, one places the injector end of the column into the sample solution and applies a voltage across the column (for this, we will need to introduce an electrode into the sample solution). The sample ions will enter the column both by migration (at rates which will vary with their electrophoretic mobilities) and by entrainment in the EOF (at a constant rate for all ions). This difference in sample amount will cause problems in quantitative CZE work, so hydrodynamic injection is preferred in that case. In the capillary mode called capillary gel electrophoresis (CGE) (discussed in Section 13.6.4), the gel in the capillary is much too viscous to employ hydrodynamic injection, so electrokinetic injection must be used. 

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Sample injection in CZE is by either hydrodynamic or electrokinetic injection... 

Hyphenation in capillary electrophoresis is still in its infancy. Critical aspects of CE hyphenation include the minute volumes of sample injected (typically a few nL) and small flow-rates (in the order of nLmin-1). Interfaces are not commercially available. CZE-UV can be used for the analysis of higher polyamide oligomers in HF1P solution [859]. A solvent elimination design with nebuliser has been described for CE-FTIR and CEC-FTIR coupling absolute detection limits are hundreds of pg [860]. An advantage of CE-FTIR is that analytes may be detected and identified without derivatisation. CE(C)-NMR [861-863] is advancing rapidly. 

This online instrumental setup was applied for the 2D separation of fluorescein isothiocyanate-derivatized human urine the results are shown in Fig. 16.7. This separation used overlapped CZE runs, which means that two samples are injected on the column, one after the other, separated by a period of time that will allow the separation of the first-injection components from the second-injection components that are present in the capillary at any point prior to detection. This was accomplished by making an injection in a time interval of exactly half of the actual CZE run time. To overlap the CZE runs, an injection was made every 29 s. For the overlapping to work,... 

In CZE, the capillary, inlet reservoir, and outlet reservoir are filled with the same electrolyte solution. This solution is variously termed background electrolyte, analysis buffer, or run buffer. In CZE, the sample is injected at the inlet end of the capillary, and components migrate toward the detection point according to their mass-to-charge ratio by the electrophoretic mobility and separations principles outlined in the preceding text. It is the simplest form of CE and the most widely used, particularly for protein separations. CZE is described in Capillary Zone Electrophoresis. ... 

In some cases, sample preparation for CZE requires only the dilution of the sample, mostly to accommodate detection (for signal and linearity of response). However, as was previously mentioned, sample characteristics such as viscosity, buffer composition (pH and excipients), and salt content can especially affect electrophoretic injection and performance. 

Ever since CE was introduced, the desire to collect fractions of pure sample components has existed. The extremely small quantities injected into the capillary in CZE has limited the number of applications in which sufficient amount can... 

Most CZE separations are very sensitive to conductivity (e.g., salt concentration) in the run buffer. Therefore, to avoid introducing a high amount of salt from the sample injection, samples should be buffer exchanged with an appropriate low salt buffer prior to analysis. Centrifugal UF/DF devices are ideal for this purpose, as they are typically very reproducible and allow the analyst greater flexibility in controlling the final sample concentration. 

Micellar electrokinetic capillary chromatography (MEKC) is used, often, for separating neutral and hydrophobic molecules. The surfactants in MEKC have the added advantage of solubilizing proteins. This can eliminate the need for extraction or deproteinization, allowing direct sample injection. The effect of sample matrix in MEKC is less dramatic than that in CZE. A... 

Figure 3 A series of CZE electropherograms for bovine serum albumin (BSA), anti-BSA, and complexes of BSA-anti-BSA. Operating conditions uncoated capillary, 50 pm X 70 cm, 40 cm to detector, 15 kV applied voltage, UV detection at 214 nm, vacuum injection 30 kpa-s, buffer 60 mM phosphate, pH 7.8,1 M AccuPure Zl-methyl reagent (Waters), sample dissolved in phosphate buffer, pH 7.0. (a) Injection of anti-BSA purified on protein G column first, (b) Injection of BSA, monomer purified by SEC first, (c) Mixture of BSA monomer and anti-BSA, with anti-BSA in excess, (d) Mixture of BSA monomer and anti-BSA, with BSA in excess (peak assignments peak 1 = 1 1 complex peak 2 = 1 2 complex, other peaks as indicated) (59).

Capella-Peiro et al. (28) used a 3 full factorial design to optimize the capillary zone electrophoresis (CZE) separation of a group of seven antihistamines (brompheniramine, chlorpheniramine, cyproheptadine, diphenhydramine, doxylamine, hydroxyzine, and loratadine). In this case, critical parameters such as pH (a concentration of 20 mM phosphate was kept constant in all the experiments) and the applied voltage were studied to evaluate their effect on the resolution and efficiency. Maximum response was achieved at pH 2.0 and an applied voltage of 5 kV. After a repeatability study to check the precision of the electrophoretic method, as well as a suitable calibration, the usefulness of this optimized method was demonstrated through the determination of the listed histamines in pharmaceuticals, urine, and serum samples (recoveries were in agreement with the stated contents). Urine samples were diluted and directly injected in the CE system, while serum samples were previously extracted by means of a solid-phase extraction (SPE) procedure. 

There are many books, book chapters, or review articles on fundamental MEKC [4-11], In particular, online sample preconcentration techniques are becoming popular in CZE to improve concentration detection sensitivity and so are in MEKC, which will be introduced rather in detail. Although it is generally stated that CE requires minimal amounts of sample (certainly true), it should be mentioned that the volume of sample solution required is much greater than that actually introduced into the capillary because the injection end of the capillary must be dipped into the sample solution— this is typically at least a few microliters. 

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