Amino acids capillary zone electrophoresis

Capillary zone electrophoresis with microchip-based SCCE has been used to separate fluorescent dye from degradation products and to separate fluorescein isothiocyanate (FITC) labeled amino acids. Capillary zone electrophoresis (CZE) in fused-silica capillaries has been used to separate the racemic mixtures of (a-hydroxybenzyl)methyltrimethylammonium and (2-hydroxy-l-phenyl)ethyltrimethylammonium with f)-cyclodextrin as the chiral pseudo-stationary phase. L-Phenylalanine and L-phenylalanine-ring-D5 (the hydrogens in the aromatic ring were substituted with deuterium) and another separation of the closely related amino acids phenylalanine and tyrosine have been resolved using CZE with capillary-based SCCE. ... 

Biomolecule Separations. Advances in chemical separation techniques such as capillary zone electrophoresis (cze) and sedimentation field flow fractionation (sfff) allow for the isolation of nanogram quantities of amino acids and proteins, as weU as the characterization of large biomolecules (63—68) (see Biopolymers, analytical techniques). The two aforementioned techniques, as weU as chromatography and centrifugation, ate all based upon the differential migration of materials. Trends in the area of separations are toward the manipulation of smaller sample volumes, more rapid purification and analysis of materials, higher resolution of complex mixtures, milder conditions, and higher recovery (69). 

Fanali, S., Ossicini, L., Foret, F., and Bocek, R, Resolution of optical isomers by capillary zone electrophoresis study of enantiomeric and distereoisomeric cobalt (III) complexes with ethylenediamine and amino acid ligands, /. Microcol. Sep., 1, 190, 1989. 

Wu, S. and Dovichi, N. J., Capillary zone electrophoresis separation and laser-induced fluorescence detection of zeptomole quantities of fluorescein thiohy-dantoin derivatives of amino acids, Talanta, 39, 173, 1992. 

Kuhr, W. G. and Yeung, E. S., Indirect fluorescence detection of native amino acids in capillary zone electrophoresis, Anal. Chem., 60, 1832, 1988. 

Capillary zone electrophoresis, an up-to-date high resolution separation method useful for proteins and peptides, has been shown to be a useful method for determining electrophoretic mobilities and diffusion coefficients of proteins [3], Diffusion coefficients can be measured from peak widths of analyte bands. The validity of the method was demonstrated by measuring the diffusion coefficients for dansylated amino acids and myoglobin. 

Wu S, Dovichi NJ (1989) High-sensitivity fluorescence detector for fluorescein isothiocyanate derivatives of amino acids separated by capillary zone electrophoresis. J Chromatogr 480 141-155... 

Cheng YF, Dovichi NJ (1988) Subattomole amino acid analysis by capillary zone electrophoresis and laser-induced fluorescence. Science 242 562-564... 

S.M. Harrison, I. Kami, V. Prokoratova, M. Mazanek, E. Kenndler, Characterisation and identification of proteinaceous binding media (animal glues) from their amino acid profile by capillary zone electrophoresis, Anal. Bioanal. Chem., 382, 1520 1526 (2005). 

Rutherfurd, S.M., Zhang, F., Harding, D.R., Woolhouse, A.D. and Hendriks, W.H. (2004) Use of capillary (zone) electrophoresis for determining felinine and it s application to investigate the stability of felinine. Amino Acids 27, 49-55. 

Amino acids derivatized with 9-fluorenylmethyl chloroformate (90) were separated by CE and determined by LIF with a pulsed laser LOD 0.5 nM (SNR 2)322. A sensitive technique for amino acids is capillary zone electrophoresis (CZE) combined with LIF of their fluorescein isothiocyanate (133) derivatives. Not all amino acids give good resolution. LOD for proline and arginine were 0.3 and 0.5 nM, respectively323. 

FIGURE 9 Capillary zone electrophoresis of eight recombinant human anti bod ies/anti body fragments. Capillary BioCAP XL coated capillary (50 pm x 47 cm) 45 mM g-amino-n-caproic acid/ acetic acid, pH 4.5, 0.1% HPMC voltage 30kV, normal polarity capillary temperature 20°C detection, UV at 214 nm. 

Mourzina, Y., A. Steffen, D. Kalyagin, R. Carius, and A. Offenhausser. Capillary zone electrophoresis of amino acids on a hybrid poly(dimethylsiloxane)-glass chip. Electrophoresis 26, 1849-1860 (2005). 

S Cladrowa-Runge, A Rizzi. Enantioseparations of 6-aminoquinolyl-lV-hydrox-ysuccinimidyl carbamate derivatized amino acids by capillary zone electrophoresis using native and substituted /3-cyclodextnns as chiral additives II. Evaluation of complexation constants. J Chromatogr A 759 167-175, 1997. 

XX Zhang, F Hong, WB Chang, YX Ci, YH Ye. Enantiomeric separation of promethazine and D,L-a-amino-/3-[4-(l,2-dihydro-2-oxo-quinoline)] propionic acid drugs by capillary zone electrophoresis using albumins as chiral selectors. Anal Chim Acta 392 175-181, 1999. 

Acid hydrolysis under standard conditions (6M HC1, 110 °C, 24 h) leads to partial decomposition of selenocystine and selenocysteine derivatives, thus making quantification of this amino acid by amino acid analysis difficult. Similarly, acid hydrolysis of 5e-[2-(4-pyr-idinyl)ethyl]selenocysteine peptides, obtained by reduction of the selenocystine peptides with NaBH4 and reaction with 4-vinylpyridine, results in partial decomposition. This de-rivatization, however, is useful for the enantiomeric resolution of the acid hydrolysates by capillary zone electrophoresis by applying host-guest complexation with crown ethers.11" 22 ... 

Yu and Dovichi [10] used capillary zone electrophoresis with thermooptical absorbance detection to determine sub pg L-1 concentrations of 18 amino acids. 

In recent years capillary electro separations were found to be useful for the analysis of a variety of small molecules. Several variants of the basic technique- such as capillary zone electrophoresis, micellar electroki-netic chromatography, and capillary gel electrophoresis - are available. However, only a handful of reports are available on flavanone analysis (Cancalon and Bryan, 1993 Desiderio et ah, 2005). Typically the separations were performed on silica fused capillaries of 70-75 pm internal diameter packed with RPig or uncoated silica (Cancalon and Bryan, 1993 Desiderio et ah, 2005). One of the major strengths of the techniques is the separation of multiclasses of compounds, ranging from amino acids to carotenoids to flavonoids (Cancalon and Bryan, 1993). 

Capillary zone electrophoresis (CZE), micellar capillary electrokinetic chromatography (MECC), capillary gel electrophoresis (CGE), and affinity capillary electrophoresis (ACE) are CE modes using continuous electrolyte solution systems. In CZE, the velocity of migration is proportional to the electrophoretic mobilities of the analytes, which depends on their effective charge-to-hydrodynamic radius ratios. CZE appears to be the simplest and, probably, the most commonly employed mode of CE for the separation of amino acids, peptides, and proteins. Nevertheless, the molecular complexity of peptides and proteins and the multifunctional character of amino acids require particular attention in selecting the capillary tube and the composition of the electrolyte solution employed for the separations of these analytes by CZE. 

The reversal of the direction of the electro-osmotic flow by the adsorption onto the capillary wall of alky-lammonium surfactants and polymeric ion-pair agents incorporated into the electrolyte solution is widely employed in capillary zone electrophoresis (CZE) of organic acids, amino acids, and metal ions. The dependence of the electro-osmotic mobility on the concentration of these additives has been interpreted on the basis of the model proposed by Fuerstenau [6] to explain the adsorption of alkylammonium salts on quartz. According to this model, the adsorption in the Stern layer as individual ions of surfactant molecules in dilute solution results from the electrostatic attraction between the head groups of the surfactant and the ionized silanol groups at the surface of the capillary wall. As the concentration of the surfactant in the solution is increased, the concentration of the adsorbed alkylammonium ions increases too and reaches a critical concentration at which the van der Waals attraction forces between the hydrocarbon chains of adsorbed and free-surfactant molecules in solution cause their association into hemimicelles (i.e., pairs of surfactant molecules with one cationic group directed toward the capillary wall and the other directed out into the solution). 

Figure 12.4. Laser-induced fluorescence detector for CZE.4 [Reprinted, with permission, from Y.-F. Cheng and N. J. Dovichi, Science 242, 1988, 562-564. Subattomole Amino Acid Analysis by Capillary Zone Electrophoresis and Laser-Induced Fluorescence . Copyright 1988 by A A AS.

Amino acids and peptides have been considered to be difficult to study by mass spectrometry (MS) except by the more sophisticated modem instrumental techniques, though derivatised amino acids and peptides are readily analysed using routine laboratory spectrometers. The spectra can also give useful information, particularly through GLC-MS analysis (and recently through capillary zone electrophoresis CZE-MS) see Section 4.17.1) of mixtures of amino acids and peptides (see also Section 4.11.1). 

Application of some of the instrumentation principles of HPLC to electrophoresis, bearing in mind the need to cause all of the components of a mixture to migrate differently, has led to the development of several related techniques that are particularly useful in the amino-acid and peptide fields (Baker, 1995). A typical electro-pherogram (Figure 4.16) indicates the salient features of the capillary zone electrophoresis (CZE) analysis protocol. 

Recently, Jalali-Heravi et al. (9,11) have developed a multivariable model in order to improve the predictive ability of the Offord model and understand the effects of further structural descriptors on electrophoretic mobility in capillary zone electrophoresis (CZE), in addition to charge and size. They generated a diverse data set based on a 125-peptide study, which ranges in size from 2 to 14 amino acids and charges of 0.743-5.843. The Pe of the peptides were measured in bare fused-silica capillaries in CZE mode using 50 mM sodium phosphate buffer at pH 2.5. The detection wavelength was 214nm and the separation temperature was 37 °C. 

The most widely appHed colorimetric assay for amino acids reHes upon ninhydrin-mediated color formation (129). Fluorescamine [38183-12-9] and o-phthalaldehyde [643-79-8] are popular as fluorescence reagents. The latter reagent, in conjunction with 2-mercaptoethanol, is most often used in post-column detection of amino acids separated by conventional automated amino acid analysis. More recentiy, deterrnination by capillary zone electrophoresis has been developed and it is possible to determine attomole quantities of amino acids (130). 

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