98 A. TISELIUS [6 



of glycols, water and salts, in which insulin and recently also y-globulins 

 have been fractionated. Among adsorbents calcium phosphate seems to 

 offer special advantages, and I will show some pictures illustrating this 

 method (Fig. 4; for details see figure text). 



This experiment illustrates a behaviour which is often observed in protein 

 chromatography, and which is markedly different from what is usually ex- 

 perienced in elution chromatography of low-molecular weight substances. 

 It is very difficult or impossible to find a buffer solution which will elute 

 both the phycoerythrin and the phycocyanin as reasonably well-defined 

 zones, migrating with different 7?/-values. It appears that the Rf of many 

 proteins is highly dependent on the concentration of the eluting buffer, so 

 that its value will shift from almost zero to almost 1 -0 in a comparatively 

 narrow interval of buffer concentration. This is to be expected, according 

 to the law of mass action, if the attachment of one protein molecule to the 

 column will lead to a liberation of several small ions. The consequence is, 

 however, that elution in the ordinary way is not suitable, but rather a step- 

 wise or gradient elution must be apphed. The step-wise elution is often very 

 efficient, but involves the risk that a slowly eluted component may appear 

 in more than one step. In the interpretation of such experiments it is essen- 

 tial to rechromatograph the fractions obtained to ascertain if their different 

 chromatographic properties can be reproduced in separate experiments. I 

 want to stress this particularly, as the results so far often indicate a com- 

 plexity of proteins which were earlier believed to be reasonably homogeneous. 

 The pronounced dependence of the partition coefficient of proteins on the 

 composition of the two-phase system is, of course, an analogous phenomenon. 



There is no doubt that chromatography of proteins already has shown its 

 usefulness both in preparative and analytical work, and that the method 

 appears to possess a high degree of specificity. I shall quote only a few 

 examples : 



Synthetic polycarboxylic acid resins: cytochrome-c ribonuclease, lyso- 

 zyme, chymotrypsinogen, chymotrypsin, hemoglobins, histones. 

 Cellulose ionic exchangers: serum proteins. 

 Synthetic anion exchange resins : serum proteins, phosphatases. 

 Partition chromatography: ribonuclease, insulin, y-globulins. 

 Adsorption chromatography: various proteins and enzymes. 



References are found in the above-mentioned review by Moore and 

 Stein (1956). 



With the 'all or none' elution behaviour of many proteins discussed above 

 one may of course ask, whether column operation has any particular ad- 

 vantage or if one could not just as well apply a batch operation. This is no 

 doubt practical in many cases. However, column operation has certain prac- 

 tical advantages, particularly with systems of unknown behaviour. It should 

 be noted also that even if the length of the column does not contribute 



