94 A. TISELIUS [6 



proteins (e.g. the sheep hemoglobins studied by Harris and Warren (1956; 

 see also Evans et al., 1956) and the haptoglobins in human serum studied 

 by Smithies (1955), Smithies and Walker (1955) and by Smithies and Poulik 

 (1956). 



I shall confine myself here to a discussion of some recent developments 

 of two methods, namely electrophoresis and chromatography, in their appli- 

 cation to the separation of proteins and protein-like substances. Of these 

 methods electrophoresis has long been known as a particularly efficient 

 method of studying the homogeneity of proteins. The application of chroma- 

 tography to such problems is more recent, but the last few years have given 

 us several examples demonstrating that the specificity of this method, well 

 known from its appHcation to low-molecular weight substances, is also quite 

 striking with large molecular weight material. 



In electrophoresis the most important recent advance appears to me to 

 be the development of zone methods and their application to separation 

 problems on a macro and micro scale. Fig. 1 illustrates schematically the 





BOUNDARY ZONE 



SEPARATION SEPARATION 



Fig. 1 . Schematic representation of the principal difference between boundary and zone 

 methods of separation. 



difference in principle between boundary methods (as applied in the standard 

 moving boundary procedure) and zone methods in the separation of two 

 substances, A and B. It is seen that the zone method requires a supporting 

 medium to stabilize the zones against gravity convections. Thus the experi- 

 ments are performed in columns or troughs packed with cellulose, starch, 

 glass powder or similar materials, or in filter paper strips (paper electro- 

 phoresis). In certain cases zone experiments have been performed in 

 gels (especially when serological reagents are to be apphed — 'Immuno- 

 electrophoresis') or in density gradients. The apparatus required is rather 

 simple. As an example. Fig. 2 shows a zone column electrophoresis apparatus 

 constructed by Porath (1956, 1957 a, b) in Uppsala. 



The column, which is surrounded by a cooling jacket, is packed with 

 cellulose powder. Different_^types of columns may be used, depending upon 

 the desired degree of resolution and the capacity required. Quantities of 

 up to 50 g of protein have been separated in columns of this type. Fig. 3 



