Electrophoresis capillary isotachophoresis

Timerbaev, A. R., and Hirokawa, T. (2006). Recent advances of transient isotachophoresis-capillary electrophoresis in the analysis of small ions from high-conductivity matrices. Electrophoresis 27, 323-340. 

Wu, Y. et al.. Determination of paralytic shellfish toxins in dinoflagellate Alexandrium tamarense by using isotachophoresis/capillary electrophoresis, J. Sep. Sci., 29, 399, 2006. 

Figure 11.18 Schematic diagram of an in-line SPE unit for CE using (a) polyester wool frits to hold the sorbent, or (b) a paiticle-loaded membrane. Reprinted from Journal of Capillary Electrophoresis, 2, A. J. Tomlinson and S. Naylor, Enhanced performance membrane preconcenti ation-capillary electrophoresis-mass spectiometi y (mPC-CE-MS) in conjunction with ti ansient isotachophoresis for analysis of peptide mixtures, pp 225-233, 1995, with permission from ISC Teclmical Publications Inc.

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. 

Gysler, J., Mazereeuw, M., Helk, B., Heitzmann, M., Jaehde, U., Schunack, W., Tjaden, U.R., and van der Greef, J., Utility of isotachophoresis-capillary zone electrophoresis, mass spectrometry and high-performance size-exclusion chromatography for monitoring of interleukin-6 dimer formation, /. Chromatogr. A, 841, 63, 1999. 

We therefore sought to evaluate reproducibility of shotgun proteomics in studies of archival FFPE tissue. Because FFPE samples are more complex than non-cross-linked samples, we evaluated FFPE human liver for analytical reproducibility and confidence in protein assignments.20 This complexity strengthens the argument for using high-resolution separations to maximize analyte concentration and minimize matrix effects. In this case, we used transient capillary isotachophoresis/capillary zone electrophoresis (cITP/cZE) in place of IEF to help address this effect. cITP/cZE has a resolution superior even to cIEF (90% of identified peptides in 1 fraction, 95% in 2 fractions or less for cITP/cZE, vs. 75% and 80%, respectively, for cIEF). 

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. 

Y. An, J. W. Cooper, B. M. Balgley, and C. S. Lee. Selective Enrichment and Ultrasensitive Identification of Trace Peptides in Proteome Analysis Using Transient Capillary Isotachophoresis/Zone Electrophoresis Coupled with Nano-ESI-MS. Electrophoresis, 27(2006) 3599-3608. 

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... 

Capillary procedures offer several advantages, including speed, resolution, sensitivity and technical simplicity, compared with the traditional methods on which they were based. An added advantage for iso-electric focusing in capillaries is the fact that it can be performed without a gel, but a coating on the internal surface of the capillary is usually required to reduce electroendosmosis. Similarly, isotachophoresis can be conveniently performed in capillary electrophoresis apparatus. 

The hyphenation of CE and NMR combines a powerful separation technique with an information-rich detection method. Although compared with LC-NMR, CE-NMR is still in its infancy it has the potential to impact a variety of applications in pharmaceutical, food chemistry, forensics, environmental, and natural products analysis because of the high information content and low sample requirements of this method [82-84]. In addition to standard capillary electrophoresis separations, two CE variants have become increasingly important in CE-NMR, capillary electrochromatography and capillary isotachophoresis, both of which will be described later in this section. 

Finally, when RPC methods are used in preparative studies with peptides, the opportunity routinely exists for subsequent analysis of the recovered fractions by a variety of analytical methods including high-speed RP-HPLC, HP-IEX, HP-HILIC, or HP-IMAC, zonal or micellar electrokinetic high-performance capillary electrophoresis (HP-CZE and MECK-CZE), capillary electrochromatography (CEC), or capillary isotachophoresis. The combination of the RPC information, drawn from the In k versus i > plots, with the data derived from on-line spectroscopic detection thus readily provides a comprehensive opportunity to assess the purity of an isolated peptide, many of the physicochemical features of the interaction, as well as a means to optimize the resolution in the RPC separation. 

Currently, there are five major modes of operation of CE capillary zone electrophoresis (CZE), also referred to as free solution or free flow capillary electrophoresis micellar electrokinetic chromatography (MEKC) capillary gel electrophoresis (CGE) capillary isoelectric focusing (CIEF) and capillary isotachophoresis (CITP). Of these, the most commonly utilized capillary techniques are CZE and MEKC (Rabel and Stobaugh 1993 Issaq 1999 Smyth and McClean 1998). 

Among the electrophoretic methods of chiral resolution, various forms of capillary electrophoresis such as capillary zone electrophoresis (CZE), capillary isotachophoresis (CIF), capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), affinity capillary electrophoresis (ACE), and separation on microchips have been used. However, in contrast to others, the CZE model has been used frequently for this purpose [44]. On the other hand, drawbacks associated with the electrophoretic technique due to lack of development of modem chiral phases have limited the application of these methods. Moreover, the electrophoretic techniques cannot be used at the preparative scale, which represents an urgent need of chiral separation science. 

A. J. Tomlinson and S. Naylor, Enhanced performance membrane preconcentration-capillary electrophoresis-mass spectrometry (mPC-CE-MS) in conjunction with transient isotachophoresis for analysis of peptide mixtures,./. High Resolut. Chromatogr. 18 384-386(1995). 

In 1989, Yamamoto et al. developed the first technique that directly coupled chromatography to capillary electrophoresis, although again in a non-comprehensive fashion. Low-pressure gel permeation chromatography, which separates analytes based on differences in molecular size, was combined with capillary isotachophore-sis, which separates according to electrophoretic mobility. Capillary isotachophoresis... 

Miiller et al., 2000 Her et al., 2003). Emphasis has been placed on (a) the use of gel chromatography or gel permeation chromatography for the fractionation of DOM on the basis of molecular size differences and (b) the application of electrophoretic separation methods (Perminova et al., 1998,2003 Specht and Frimmel, 2000), including electrophoresis, capillary electrophoresis (CE), isotachophoresis, isolelectric focusing,polyacrylamide gel electrophoresis (PAGE), and capillary zone electrophoresis (CEZ) (De Nobili et al., 1989,1998 Schmitt-Kopplin et al., 1998). 

Capillary electrophoresis (CE) coupled to MS has the advantage of high resolution and soft ionization for biomolecules, which may be used to differentiate post-translational modifications and variants of intact proteins and oligonucleotides. Different modes of CE (capillary zone electrophoresis, capillary isoelectric focusing, capillary electrochromatography, micellar electrokinetic chromatography, nonaqueous capillary electrophoresis) to MS as well as online preconcentration techniques (transient capillary isotachophoresis, solid-phase extraction, membrane preconcentration) are used to compensate for the restricted detection sensitivity of the CE methodology [77, 78]. 

Kaniansky et al. [21] have also reported on a method for the determination of nitrate, sulphate, nitrite, fluoride and phosphate by capillary zone electrophoresis coupled with capillary isotachoelectrophoresis in the column coupling configuration. Such distributions of these anions are typical for many environmental matrices, and it is shown that capillary isotachophoresis-capillary zone electrophoresis tandem enables the capillary isotachoelectrophoresis determination of the macroconstituents, while capillary isotachophoresis-preconcentrated microconstituents cleaned up from the... 

Capillary electrophoresis is a general term that is used to describe a number of different separation techniques. Capillary zone electrophoresis (CZE) is the classic technique and is therefore usually referred to as just CE. Other techniques include micellar electrokinetic chromatography (MEKC), capillary isoelectric focusing, and capillary isotachophoresis. CZE and MEKC are the predominant techniques and are those used herein, so only they will be discussed in detail here. 

Two kinds of conductivity detector are distinguished contact detectors and contactless detectors. Both types were originally developed for isotachophoresis in 0.2-0.5-mm-inner diameter (i.d.) PTFE tubes. Contactless detectors are based on the measurement of high-frequency cell resistance and, as such, inversely proportional to the conductivity. The advantage is that electrodes do not make contact with the buffer solution and are, therefore, outside the electric field. As these types of detectors are difficult to miniaturize down to the usual 50-75-jU.m capillar inner diameter, their actual application in capillary electrophoresis (CE) is limited. 

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