328 BIOCHEMICAL SYSTEMATICS 



as paper and gas chromatography, spectral analysis, and so on, are 

 now quite commonly employed by biologists, some of whom have had 

 httle formal chemical training. At present, paper chromatography is 

 perhaps the most frequently employed single technique for screening 

 purposes, comparisons of crude extracts, and tentative identification. 

 Its chief advantages are its versatility and simplicity. Since excellent 

 texts are available which describe the methods of paper chromatog- 

 raphy, no description of methods need be included at this time. 

 However, a few precautions may be inserted. 



Normally, identification solely by paper chromatography, 

 whether or not multiple Rf values and response to detecting reagents 

 are utilized, is not acceptable to the chemist, and more conservative 

 use of the technique by biologists seems justified. It is a fact, how- 

 ever, that after long experience with the chromatographic behavior 

 of a limited number of compounds one can frequently recognize 

 specific compounds by their chromatographic properties alone. It is 

 virtually impossible to place absolute reliance upon Rf values. Not 

 only do obvious factors such as temperature and equilibration time of 

 the solvent affect Rf values, but more subtle influences such as the 

 shape of the chromatographic chamber or even the number of sheets 

 hung in a chamber and the exact method of equilibration may be 

 sufficient to modify Rf values significantly. 



Extraction procedures may grossly affect Rf values, and it is 

 well known that compounds chromatographed from crude extracts 

 may be affected greatlj^ either by physical properties of the extract 

 or by actual chemical modification of the substances being studied. 

 Figure 16-1 illustrates a striking effect upon the Rf values of certain 

 pure samples of lupine alkaloids when applied so as to overlap partially 

 a crude extract from Baptisia which contains similar alkaloids. 



Conservatism is also advocated in the use of chromogenic 

 sprays especially if a qualitative aspect of the color is required for 

 identification. In the case of ninhydrin, for example, most amino 

 acids give essentially similar colors while some, such as proline, yield 

 radically different colors. In contrast cyclohexylamine yields a larger 

 variety of colors with amino acids than does the ninhydrin spray, but 

 the specific colors are hard to describe, appear differently on the two 

 sides of the paper, and are influenced by the amount of amino acid 

 present. It should also be recognized that the sensitivity of a given 

 spraying reagent may vary with different compounds. For example, 

 Dragendorff reagent, which is used for the general detection of 

 alkaloids, exhibits large differences in sensitivity to different lupine 

 alkaloids. 



The extent to which chemical artifacts occur is dependent 



