6] ELECTROPHORESIS AND CHROMATOGRAPHY 97 



be explored. It is of course desirable that the water content of the column 

 is as high as possible. In this respect cellulose has advantages: a column 

 packed by sedimentation contains approximately 85% water, as compared 

 with 40% for glass powder and 50% in starch, of which only 33% is free 

 (Porath, 1957 b). 



The effects mentioned represent a difficulty not only by interfering with 

 the separation but also as regards the evaluation of the results in terms of 

 mobihty. It is true, of course, that most zone electrophoresis experiments 

 are made with the separation as chief purpose, and that for accurate mobility 

 determinations the free moving boundary electrophoresis method is pre- 

 ferred. The possibility of quantitative control of the separation has, how- 

 ever, been an attractive feature of the electrophoretic methods, and it would 

 be a pity if this would have to be sacrificed in order to gain the advantages 

 offered by the zone methods. If there is no interaction between the migrating 

 substances and the supporting material except the purely mechanical hind- 

 rance (or, strictly speaking, if such interaction and mechanical hindrance 

 influence the mobilities of all substances present, including the buffer ions, 

 to the same extent) it ought to be possible to determine mobilities by measur- 

 ing the volume distances between the peaks in the elution diagrams obtained 

 from zone electrophoresis columns, and comparing them with the distance 

 of migration of a substance of known mobility. Attempts in this direction 

 have been made, but I believe it is very important to pursue such investiga- 

 tions with the more inert supporting materials now available. 



When turning now to protein chromatography I wish to say first of all 

 that this field is much more recent and considerably more difficult to discuss. 

 It appears, however, as if the application of chromatography to proteins 

 of large molecular weight has now left the 'lag phase' (to use an expression 

 from bacteriology). Whether it has definitely entered a 'log phase' remains 

 to be seen, but there are already a number of beautiful results which indi- 

 cate that the method ofifers great possibilities. It is equally clear, however, 

 that the underlying principles are not yet well understood and that the 

 methods involve certain risks of making grave mistakes. 



There is an excellent recent review of this whole field by Moore and Stein 

 (1956) and for this reason I shall limit myself to some brief remarks. The 

 particular difficulties ofifered by proteins in chromatography depend on 

 their large molecular size, which may prevent them from entering into the 

 pores of the column material or make their reactions with this material too 

 slow. Also, the instabihty of proteins and particularly the risk of denatura- 

 tion at surfaces is an obstacle. Most of the common types of chromatography 

 as applied to aqueous solutions have been used for protein separations, i.e. 

 ionic exchange, partition and adsorption. In each case, however, one has 

 found that only certain systems have the properties required. Both cationic 

 and anionic exchange resins have been used, as well as ionic exchangers pre- 

 pared from cellulose. Partition systems have been worked out, consisting 



Gps 



