216 WALDO E. COHN 



b. Types of Chromatography'^ 



Two methods of exploiting these factors are recognized. In "displace- 

 ment chromatography,"^" the chemical form of the exchanger is changed 

 during the elution sequence (e.g., from a hydrogen-form cation exchanger 

 to an ammonium-form, or from a hydroxide-form anion exchanger to a 

 chloride-form), and the various components trail the progressive change in 

 form by greater or lesser distances. This method has not had as wide- 

 spread practical success as the method of "development chromatography," 

 also called "elution analysis," in which the salt form of the column remains 

 unchanged from sorption sequence to elution sequence. In the latter 

 method, the influent contains, as its competing, replacing, or bulk ion, the 

 diffusible ion of the exchanger itself. This method has been essentially the 

 only one applied to the separation of nucleic acid constituents. An innova- 

 tion, in which the eluting sequence is carried out by a gradually changing 

 concentration of eluting agent, instead of changing the solvent discontinu- 

 ously, has been termed "gradient development chromatography "^°' " 



c. Scaling Up 



It should be noted that a column separation is also a preparation. The 

 magnitude of the preparation is determined only by the cross-sectional area 

 of the column. Thus analytical separations can be scaled up by proportional 

 increase in the area of the column to prepare larger amounts of material 

 with no loss in resolution.^ Such large-scale ion-exchange chromatography 

 has been the main source of most of the new nucleotides discovered by ana- 

 lytical ion-exchange chromatography. 



The factors and principles just discussed were known in 1948 when the 

 first serious efforts were made to develop precise ion-exchange chromato- 

 graphic separations of nucleic acid derivatives. Although the attempt was 

 made to find conditions giving good separations with elution curves con- 

 forming to those derived from equilibrium considerations, no systematic 

 examination of the many possible separation conditions has as yet been 

 made. Indeed, the stimulus for most of the developments arose from the 

 desire to recover degradation products as an approach to establishing struc- 

 tural relationships in the parent nucleic acid. The separations presented, 

 therefore, do not necessarily indicate the best possible apphcations of the 

 methods. 



'" E. Glueckauf , Nature 170, 150 (1952) (as reported in Principles and Applications 



of Ion Exchange). 

 11 H. Busch, R. B. Hurlbert, and V. R. Potter, /. Biol. Cheni. 196, 717 (1952). 



