Electrophoresis, of amino acids

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). 

G22. Gross, D., Paper electrophoresis of amino-acids and oligopeptides at very high potential gradients. Nature 176, 72 (1955). 

Seherr, G. H., Use of cellulose acetate strips for electrophoresis of amino acids. Anal. Chem. 34, 777 (1962). 

Heterocycles in derivatization reactions in capillary electrophoresis of amino acids 00CLY347. 

Experiment 4. Horizontal low voltage electrophoresis of amino acids... 

The section on organic nitrogen compounds (Chapter 27) has been expanded to include electrophoresis of amino acids. 

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

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). 

Electrophoresis is used primarily to analyze mixtures of peptides and proteins, rather than individual amino acids, but analogous principles apply. Because they incorporate different numbers of amino acids and because their side chains are different, two peptides will have slightly different acid-base properties and slightly different net charges at a particular pH. Thus, their mobilities in an electric field will be different, and electrophoresis can be used to separate them. The medium used to separate peptides and proteins is typically a polyacrylamide gel, leading to the term gel electrophoresis for this technique. 

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. 

Tao, L., Thompson, J.T., Kennedy R.T. (1998). Optically-gated capillary electrophoresis of o-phthaldehyde/P-mercaptoethanol derivatives of amino acids for chemical monitoring. Anal. Chem. 70, 4015M022. 

Heterocyclic fluorophores based on the benzoxadiazole nucleous, namely 4-nitrobenz-2-oxa-l,3-diazole (NBD) 14 derivatives/analogs, have been widely used as derivatization reagents for analysis purposes. Examples include the amino- or thiol reactive 4-fluoro-7-nitrobenz-2-oxa-l,3-diazole (NBD-F) 15 and 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (NBD-C1) 16 [45-50] and the thiol-reactive /V-((2-(iodoacetoxy)ethyl)-/V-methyl)amino-7-nitrobenz-2-oxa-1,3-dia-zole (IANBD ester) 17 [51] and 7-chlorobenz-2-oxa-l,3-diazole-4-sulfonate (SBD-C1) 18 [52], NBD-F and NBD-C1 derivatives can be excited at about 470 nm by using the relatively inexpensive and reliable argon ion lasers or newer diode pumped solid state (DPSS) lasers. NBD-F has been used as a labeling tag in various capillary electrophoresis (CE) experiments for amino acids [53-57] including the monitorization of in vivo dynamics of amino acids neurotransmitters [58]. 

Klinker CC, Bowser MT (2007) 4-Fluoro-7-nitro-2, 1, 3-benzoxadiazole as a fluorogenic labeling reagent for the in vivo analysis of amino acid neurotransmitters using online microdialysis-capillary electrophoresis. Anal Chem 79 8747-8754... 

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... 

Figure 10.17 Two-dimensional high voltage electrophoresis and chromatography of amino acids. Paper high voltage electrophoresis (4000 V) in an acetic acid-formic acid buffer at pH 2.0 in the first dimension followed by descending chromatography in the second dimension in an n-butanol-acetic acid-water solvent (12 3 5). The spots were visualized with a ninhydrin-collidine reagent.

Perrin, C., Vargas, M.G, Vander Heyden, Y., Maftonh, M., Massarf D.L. Fast development of separation methods for the chiral analysis of amino acid derivatives using capillary electrophoresis and experimental designs. J. Chromatogr. A 2000, 883, 249-265. 

Moini M. 2004. Capillary electrophoresis-electrospray ionization mass spectrometry of amino acids, peptides and proteins. Methods Mol Biol 276 253. 

Bergquist J, Vona MJ, Stiller CO, O Connor WT, Falkenberg T, et al. 1996. Capillary electrophoresis with laser-induced fluorescence detection a sensitive method for monitoring extracellular concentrations of amino acids in the periaqueductal grey matter. J Neurosci Methods 65( 1) 33-42. 

Kennedy RT, Thompson JE, Vickroy TW. 2002a. In vivo monitoring of amino acids by direct sampling of brain extracellular fluid at ultralow flow rates and capillary electrophoresis. J Neurosci Methods 114(1) 39-49. 

Zhou SY, Zuo H, Stobaugh JE, Lunte CE, Lunte SM. 1995. Continuous in vivo monitoring of amino acid neurotransmitters by microdialysis sampling with on-line derivatization and capillary electrophoresis separation. Anal Chem 67(3) 594-599. 

Lin, C.-C., and Liu, C.-Y. (2004). Proline-coated column for the capillary electrochromatographic separation of amino acids by in-column derlvatlzatlon. Electrophoresis 25, 3216-3223. 

Soga, T., Kakazu, Y., Robert, M., Tomita, M., andNishioka, T. (2004). Qualitative and quantitative analysis of amino acids by capillary electrophoresis-electrospray ionization-tandem mass spectrometry. Electrophoresis 25, 1964—1972. 

Kato, M., Gyoten, Y., Sakai-Kato, K., and Toyo oka, T. (2003). Rapid analysis of amino acids in Japanese green tea by microchip electrophoresis using plastic microchip and fluorescence detection.. Chromatogr. A 1013, 183 — 189. 

Isoelectric points are useful concepts for the separation and purification of amino acids and proteins using electrophoresis. Under the influence of an electric field, compounds migrate according to their overall charge. As we have just seen for amino acids, this very much depends upon the pH of the solution. At the isoelectric point, there will be no net charge, and, therefore, no migration towards either anode or cathode. 

Diverse analytical methods have been proposed for the analysis of amino acids, including GC, HPLC, and capillary electrophoresis. The preferred method at present is RP-HPLC with precolumn derivatization, which has the advantages of requiring short analysis time, simple instrumentation, and low cost [192]. 

The electrophoresis is run at 1000 V cvfor 30-45 min. After finishing, the cellulose sheet is dried in a stream of warm air. For visual detection of amino acids the cellulose sheet is sprayed with Soln. B and incubated in a drying oven at 110-120 °C until blue spots appear. Autoradiography or a Phospholmager detects P-labled amino acids. 

Jiang, T.-F., Wang, Y.-H., and Lv, z.-H., Dynamic coating of a capillary with room-temperature ionic liquids for the separation of amino acids and acid drugs by capillary electrophoresis, ]. Anal. Chem., 61,1108-1112,2006. 

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