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One-way separation of amino acids by electrophoresis at high voltage is now a well-established procedure (Gross, 1955). With the large number of amino acids often found in biological fluids, it is, however, very difficult to obtain satisfactory separation without overlap of spots. Several investigators have used techniques for the separation of amino acids and other substances by a combination of paper electrophoresis and chromatography. Durrum (1950, 1951) developed the so-called 'strip-transfer' method. This consisted in cutting out the strip of paper on which chromatography had previously been caxried out, blotting the spots on to a second sheet and carrying out ionophoresis at right angles. This technique was further elaborated by Honegger (1956). Consden & Glynn (1955) described another two-dimensional method in which chromatography followed by ionophoresis on the same paper was used. All these methods utilize low-voltage electrophoresis, which does not give a good separation of the neutral amino acids. Since this paper was written, it has come to the author's attention that Dixon, Kauffman & Neurath (1958) have described the two-way separation of amino acids by using a combination of electrophoresis at 1500v in one direction and chromatography in the other. They found this method more satisfactory than previously-used chromatographic methods. By modifying the apparatus described by Gross (1956) for one-way high-voltage electrophoresis in such a way that a square paper can be used, amino acids can be separated by high-voltage electrophoresis along one edge of the paper, and chromatography subsequently carried out in the other direction. The two-way separation thus obtained has several advantages over previously described chromatographic methods, especially with biological fluids.

Two-way separation of amino acids and other ninhydrin-reacting substances by high-voltage electrophoresis followed by paper chromatography