Electrophoresis isotope separation

The optimum pH for separating cations is pK + 0.30 K. K.-C. Yeung and C. A. Lucy, Isotopic Separation of [14N]- and fI5N] Aniline by Capillary Electrophoresis Using Surfactant-Controlled Reversed Electroosmotic Flow, Anal. Chem 1998, 70. 3286. 

Yeung, K. K. C. and Lucy, C. A., Ultrahigh-resolution capillary electrophoretic separation with indirect ultraviolet detection Isotopic separation of [14N]-and [15N]ammonium. Electrophoresis 1999, 20, 2554-2559. 

Zhao, J. and Jorgenson, J. W., Application of synchronous cyclic capillary electrophoresis Isotopic and chiral separations. Journal of Microcolumn Separations 1999,11, 439-449. 

Column electrophoresis is carried out in an apparatus such as that shown in Figure 4. Glass powder, alumina, splinters, or quartz powder is used as a support. The support is first impregnated with the ionic liquid, then a solution of the sample is applied at the center of the separation column and the electric field applied. Column electrophoresis is commonly used for isotope separations. 

See also Electrophoresis Oven/iew. Liquid Chromatography Isotope Separations. 

Zhao 1, lorgenson IW (1999) Application of synchronous cyclic capillary electrophoresis isotopic and chiral separations. 1 Microcolumn Sep 11 439-449... 

Peschanski, using the isotopic method ( ° Hg), has found complete exchange (0 °C) in methanol and various other non-aqueous media. The separation methods used were, (a) paper and column chromatography, (b) paper electrophoresis, and (c) precipitation of Hg(I) with chloride. In the presence of cyanide ions, however, less than complete exchange could be observed. Zero-time exchange was again found to vary in the same manner as for aqueous media. Similar effects were observed in the presence of chloride ions. 

Terabe, S., Yashima, T., Tanaka, N., and Araki, M., Separation of oxygen isotopic benzoic acids by capillary zone electrophoresis based on isotope effects on the dissociation of the carboxyl group, Anal. Chem., 60,1673, 1988. 

Figure 3.2. Stable isotope labeling for quantifying differential protein expression. Cell populations are grown in either 14N or 15N containing medium. Protein lysates are fractionated and separated by 2D gel electrophoresis. Protein spots are excised, digested with trypsin and the mass of the resulting peptides is determined by mass spectrometry. The presence of 15N results in a shift and creates two peaks for each peptide. The ratio of intensities of the peaks is indicative of the relative expression levels of the proteins. Spot A contains a protein that is expressed at similar levels in both cell pools. Spot B contains a protein that is expressed at higher levels in cell pool 2. Figure adapted from Oda et al. (1999).

For reproducible expression analysis and protein quantification MS methods based on isotopic labeling are available. They were designed in conjunction with two or more dimensional chromatographic peptide separation coupled online to MS and require advanced bioinformatics input to analyze the complex data sets in a reasonable time frame. This is also true for the alternative fluorescence-based technology of differential gel electrophoresis (DIGE Fig. 10.6) with tailor-made software which allows statistical validation of multiple data sets. 

The separation of isotopes can be used to show the efficiency of separation by capillary electrophoresis. Moreover, the interfacing of a mass spectrometer to the capillary can be used for the study of biological substances (cf. Fig. 16.7). 

Figure 26-31 Separation of natural isotopes of 0.56 mM Cl by capillary electrophoresis with indirect spectrophotometrlc detection at 254 nm. Background electrolyte contains 5 mM CrOJ to provide absorbance at 254 nm and 2 mM borate buffer, pH 9.2. The capillary had a diameter of 75 m, a total length of 47 cm (length to detector = 40 cm), and an applied voltage of 20 kV. The difference in electrophoretic mobility of 36C and 37CI is just 0.12%. Conditions were adjusted so that electroosmotlc flow was nearly equal to and opposite electrophoretic flow. The resulting near-zero net velocity gave the two isotopes maximum time to be separated by their slightly different mobilties. [From C. A Lucy and T. L McDonald, "Separation of Chloride Isotopes by Capillary 35 40 45 Electrophoresis Based on the Isotope Effect on Ion Mobility"Anal.

Labeling with 2H, 13C, 15N and/or lsO can be introduced via peptide synthesis, cell culture, or hydrolysis in labeled water [88]. The heavy isotope-labeled peptide can be used as an IS to obtain quantitative measurements of the protein concentration. Typically, the protein sample of interest is digested with trypsin, and the isotope-labeled control peptides are added to the mixture. The signature peptides in the digest can be separated and quantified by HPLC-ESI-MS/MS. Alternatively, MALDI-MS can also be used for tryptic peptide determinations after some separation steps such as gel electrophoresis [89]. 

EDTA was determined in human plasma and urine by capillary electrophoresis/MS [85]. Using a BC stable labile isotope, the detection and quantitation limits were found to be 7.3 and 14.6 ng/mL, respectively. The running buffer was pH 3.5 ammonium formate/formic acid buffer, at an inlet pressure 50 mb and a separation potential of -30 KV. The same authors [86] utilized this technique for the determination of EDTA as the nickel chelate in environmental water. 

Gel electrophoresis (GE) is a common separation technique in protein analysis and it has also been used for the speciation of metals bound to proteins [86]. In most applications, metals have been detected by autoradiography, limiting the studies to those elements for which a relatively stable radionuclide exists [87]. As an example, 75Se radiotracer allowed Se to be detected after two-dimensional GE (2-DE) separation [88]. Owing to the high sensitivity and isotopic capability of ICP-MS, this technique has been proposed as the detection tool of choice for elements in gel. The efbcient transport of the sample from the protein spot on gel to plasma has been achieved by laser ablation (LA) [89, 90] and electrothermal (ET) atomization [62, 91] techniques. The... 

In past years, on line chromatographic coupling techniques such as HPLC and CE coupled to ICP-MS with the isotope dilution technique have been used for element quantification in speciation analysis. An interesting application of the isotope dilution technique in medical research was proposed recently by Prange and co-workers, who added highly enriched " S, Cu, Zn and Cd spikes to the interface of the CE-ICP-MS system. The authors separated isoforms of metallothionein (e.g., of rabbit liver) by capillary electrophoresis and quantified S, Cd, Cu and Zn concentrations in isoforms by ICP-SFMS using the isotope dilution technique. A new selenized yeast reference material (SELM-1) for methionine, selenomethionine (SeMet) and total selenium content has also certified by an intercomparison exercise. ... 

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