S. Ohkuma, T. Shinohara
1963
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Abstract
In previous papers"'2' from this laboratory, it was reported that the separation of N-acetylhexosamines and N-acetylneuraminic acid*' can be performed by chromatography on paper impregnated with tetraborate in n-butanol-pyridine-water solvent systems and the order of RF values of these N-acetylamino sugars was as follows: N-acetylglucosamine*' > N-acetylmannosamine*' > N-acetylgalactosamine*' > SA.3' On another occasion,4' paper electrophoresis with tetraborate was found to be a valuable method for the separation of these Nacetylamino sugars; and the order of electrophoretic mobilities of these N-acetylamino sugars was as follows: SA > ManNAc > Ga1NAc > G1cNAc. These studies''-4' show that paper electrophoresis differs essentially from paper chromatography, in efficiency on the separation of these N-acetylamino sugars. It is therefore expected that separation with higher resolving power may be obtained by a method in which electrophoresis is combined with chromatography on tetraborate-buffered paper. This paper describes a two-dimensional combination of paper electrophoresis and paper chromatography for the separation and characterization of these N-acetylamino sugars and its application to the identification of free N-acylamino sugars in a human ovariancyst fluid. Experimental. Buffers, solvents and paper strip. Potassium tetraborate and sodium tetraborate in concentration of each o.025M4' were used as buffers (electrolytes). n-Butanol-pyridine-water solvent systems (solvent A, 2 : 2: 1" and solvent B, 6 : 4 : 3," by volume) were used as solvents. Whatman No. 3 MM paper strip with dimension as shown in Fig. 1 was used. Two-dimensional paper electrophoresis-chromatography. Before being placed in an electrophoretic apparatus, the paper strip was moistened with a buffer and then pressed between two sheets of clean filter paper to remove the excess of the buffer. The strip was then