PAPER CHROMATOGRAPHY 



383 



analogues of DDT in insect metabolism studies is given in the papers of 

 Winteringham et al. (21, 50). Mixtures of the following compounds 

 were resolved and analyzed: (Br*C6ll4)2GH-CCl3, (Br*Ccn4)2C:CCl2,and 

 (Br*C6H4)2CH-COOH. For a reversed-phase chromatogram, strips of 

 Whatman No. 1 filter paper, 100 by 2.5 cm, were drawn through a bath 

 of ether containing 2.5 per cent vaseline, then drained and dried. Mix- 

 tures of these three components in ben- 

 zene were applied at the top of the strip. 

 The mobile phase consisted of 80 per 

 cent ethanol, 15 per cent water, and 

 5 per cent ammonia, and the chromat- 

 ogram was developed in the usual way. 

 The amounts of each compound used 

 were in the range of 0.2 to 50 /xg, and 

 the recoveries as dete. mined by Geiger 

 counting were excellent. Wintering- 

 ham et al. (25) also separated an un- 

 labeled mixture of these compounds 

 and determined the amounts present 

 by exposure of the chromatogram to 

 neutron bombardment and measure- 

 ment of induced radioactivity. Recov- 

 eries ranged from 62 to 126 per cent. 



Another quantitative application is 

 given in the work of Ruliffson et al. 

 (51), who separated the hydrazones of 

 ketosteroids which had been formed 

 with I'^^-labeledacethydrazide-3-iodo- 

 pyridinium bromide. After reaction 

 between this compound and the keto- 

 steroid, a 20- to 200-^1 aliciuot was 

 placed on a spot 2 cm from the base of 

 a 1-in. strip of Whatman No. 1 paper. 

 Ascending chromatography was car- 

 ried out with 84 per cent w-butanol- 

 16 per cent water. The position of the 

 steroid hydrazones and also of unre- 

 acted labeled reagent was determined by radioactivity as well as the use 

 of iodoplatinate reagent. Rf values were reported for 18 different keto- 

 steroids. Quantitative recoveries were obtained by radioactivity meas- 

 urements, provided an excess of the labeled reagent was used. 



Williams and associates (9, 52) have pointed out that, although inter- 

 pretations of paper chromatograms are fairly straightforward when pure 



Fig. 8-8. Autoradiogram of filter- 

 paper chromatogram. indicates 

 origin; U, compound U ; Tx, thyrox- 

 ine; S, solvent front. Left, from bile 

 of rat injected with I'3'-la])eled 

 thyroxine, shows formation and bili- 

 ary excretion of unidentified product. 

 Right, shows purity of unidentified 

 product, compound U, isolated by 

 paper chromatography for study of its 

 metabolism when administered to 

 rats. [From F. N. Briggs, Alvin 

 Taurog, and I. L. Chaikoff, The En- 

 terohepatic Circulation of Thyroxine in 

 the Rat, Endocrinology, 52 : 559-567 

 (1953).] 



