CHROMATOGRAPHY 155 



"take its own picture" if clamped against a sheet of photographic film 

 according to the technique known as radioautography. 



To find the location of any particular component of a mixture, two 

 chromatograms can be run simultaneously under identical conditions. 

 The mixture is placed on one paper; a sample of the compound whose 

 location is sought is placed on the other paper. This compound should go 

 to the same location on both chromatograms. 



A numerical value, R/, is frequently used in locating certain com- 

 pounds. 



rate of movement of solute 



Rf = 



rate of movement of solvent 



On paper chromatograms, different components of a mixture move at 

 different speeds. The "front" of each compound moves at a certain frac- 

 tion of the speed at which the solvent moves. If one amino acid moves 

 very rapidly in a certain solvent mixture, its R/ value will be high, 

 perhaps 0.85. Another amino acid might move very slowly in the same 

 system and have an R/ value of 0.15. 



On columns and on paper, any moving component commonly moves 

 as a mass of molecules with a "tail" trailing out behind. This tail often 

 will be mixed with the front of the next comp>onent. The second com- 

 ponent in a mixture is thus harder to purify than the first. Sometimes 

 this difficulty can be overcome by changing solvents and reversing the 

 order of the two compounds. 



It is frequently possible to determine quantitatively the amounts of 

 various materials on a paper chromatogram. If the spots or bands are 

 colored, the absorption of light by the materials will be related to their 

 concentrations in the spot. If this method is not practical because of 

 great differences in colors of materials or because they are uncolored, they 

 may be stained with a suitable dye and then scanned with a modified 

 colorimeter or spectrophotometer. 



Fraction Collectors: When columns of adsorbents or ion exchange 

 materials are used, one means of separating components is to collect them 

 in separate containers as they drip out the bottom of the column. First 

 the most rapidly moving component appears, followed at a later time 

 by the other components. Automatic fraction collectors remove most of 

 the work from this form of chromatography. Containers, usually test 

 tubes, are held in a large wheel which rotates in steps. A small amount 

 of Hquid is drained into one test tube, and the wheel rotates to place the 

 next test tube under the column. The stepwise rotation of the wheel 



