156 CHROMATOGRAPHY 



can be controlled by a timer, allowing liquid to drip into each test tube 

 for a pre-set time interval. In other fraction collectors, a siphon arrange- 

 ment measures a certain volume of liquid and delivers this volume to 

 the test tube. The most elegant fraction collectors employ phototubes 

 to count drops. 



Amino Acids: Proteins are among the very most important biological 

 compounds, and frequently the analysis of the amino acids composing 

 a protein gives important information. The group of about twenty- 

 five amino acids that result from the hydrolysis of a protein could be 

 separated by any of the chromatographic methods, but ion exchange 

 columns and paper partition chromatography are most commonly 

 used. 



Amino acids possess both acidic and basic groups in the molecules, 

 and most have side chains that may be acidic, basic, or neutral. The 

 individual molecules may have a net acidity or basicity and thus can be 

 separated on ion exchange columns. Passage through a resin such as 

 Dowex-50 might separate the acidic from the basic amino acids. Often 

 a complete mixture of amino acids is passed through several columns in 

 succession, each column containing a different resin. Careful control of 

 the pH of the developing solvent permits the elution of virtually all the 

 various amino acids as individual bands. Several models of instruments 

 have been fabricated which will perform such a separation, determine 

 the concentration of each constituent, and plot the results on a strip- 

 chart recorder, all quite automatically! 



For filter paper partition chromatography of amino acids, Whatman 

 No. 1 seems to be among the more popular papers. Each investigator 

 has his own preference, however, and for some mixtures of amino acids 

 another paper would be superior. Of all the different solvent mixtures 

 that have been used, only a few have become popular: a mixture of 

 phenol and water, a mixture of collidine, lutidine, and water, and a 

 mixture of one of the butanols, with acetic or propionic acid, and water. 

 Changes in operating conditions, modification of the proportions of the 

 solvent materials, or use of two different mixtures makes two-dimensional 

 chromatography possible. The resolved spots usually are detected and 

 frequendy are determined quantitatively by treating the developed 

 chromatogram with ninhydrin (triketohydrindene hydrate). This treat- 

 ment produces spots in various shades of pink or lavender. 



Carbohydrates: The sugars and sugar derivatives and the more elabo- 

 rate polysaccharides are an extremely complex group. The biologist who 

 must deal with these compounds is wise to enlist the help of an organic 

 chemist. Carbohydrates often are modified chemically before chromatog- 



