Desalting by High Voltage Electrophoresis Hsiu-Ying T. Yong and M. R. Shetlar, Department of Biochemistry, University of Oklahoma School of Medicine, Oklahoma City, Okla.
Figure 1. Preparation of paper for desolting by high voltage electrophoresis Sample is applied at site Indicated. Extenrionr to right and leh dip into anode and cathode chambers, respectively
INsugan, interfering salts are formed THE PAPER CHROMATOQRAPHY Of
by hydrolysis and subsequent neutralization. To obtain salt-free samples, several techniques have been used: electrolytic desalting, , ion exchange resin, and organic solvent extraction ( 1 ) . However, these techniques require relatively large volumes of solution, and therefore are not convenient for microquantities of material. Recently we have found that desalting can be carried out by high voltage electrophoresis directly on a sample which has been previously spotted on the paper. A buffer containing only volatile constituents is used. Thii method allows the desalting of small volumes of solutions (5 to 25 pl.) and does not require reconcentration of the desalted sample as in other procedures. In this method, the inorganic ions are rapidly removed and neutral sugars are left a t the position near the origin. Amino acids, amino sugars, and sugar acids migrate in the electrical field, but are also satisfactorily dedted. EXPERIMENTAL
For the Servonuclear Model ET48 high voltage electrophoresis equipment, a sheet of filter paper is cut to the size
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ANALYTICAL CHEMISTRY
and dimensions shown in Figure 1. The sample to he desalted is spotted on the filter paper, and electrophoresis is carried out with a buffer of pH 3.8, pyridine-acetic acid-water (1:10:89), a t 3000 volts for 35 to 40 minutes. The paper is then dried carefnlly with a hair dryer to remove the buffer and is cut for chromatography as indicated in Figure 1. The standards may then he applied on the paper, and chromatography carried out with the solvent flowing at right angle to the electrophoretic run. Figure 2 shows the results with a sample desalted by this procedure followed by descending chromatography. The original sample of 25 pl. contained 20 rrg. of galactose and 30 pg. of N-acetylglucosamine in 0.125M tris buffer and 2.5M NaC1. The spots were developed by benzidine reagent after paper chromatography (solvent system,
STD.
SAMPLE 3
$10.
Figure 2. Chromatography of sugars with butanol-acetic acid-water mixture (4:1:5 by volume) after desalting by high voltage electrophoresis Sugars are indicoted b y GAL, galactore, MAN, mannoses NAGLUNHr, N-acetylglucoraminei FUC, fumre. Sample 3 i i an enzymatic hydmly$ate of (I glycoprotein prepamtion
Figure 3. High voltage electrophoresis of sugars with pyridine-acet ic acidwater mixture (1:10:89 by volume) Sigors ore GAL, galocto$e; MAN, mOmO9e:
NAGLUNH,, N-ocelylql~m~omine;FUC, fucose. Glucrran:c a d d p r e p a r o ? :(GLUAI ~ y:elds I w o spolli l ~ c l o n c01 orig:n and free odd m w h g loword onode
butanol-acetic acid-water, 4:1:5). Without preliminary desalting, no discrete spots were detected. When the sample was spotted equidistant from both electrodes, no appreciable movement of the sugar occurs. When the sample was spotted at the position near the anode, the neutral sugars moved slightly toward the cathode by electro-osmosis flow. However, this movement did not influence the result of chromatography. When only neutral sugars were involved, several samples were desalted on the same strip by this technique. Both standard and samples were applied on the paper before electrophoresis. Under the conditions given above, amino sugars moved toward the cathode and free sugar acids toward the anode (Figure 3). Dicarboxylic amino acids moved as anions; other amino acids moved as cations. Preparations containing these compounds were satisfactorily desalted. LITERATURE CITED
(1) Smith, I., "Chromato&aphic
and Electrophoretio Techniques," Vol. 1, p. 40, Interscience, New York, 1960. Study su ported in part by Public Health Service &ant CA03081.
