CHROMATOGRAPHY 145 



of the green acetone extract and washing with water may be repeated 

 until the pigments from the entire original acetone solution have been 

 transferred to petroleum ether. 



After the last addition of acetone, the petroleum ether solution is 

 washed several times with water to remove the last traces of acetone. The 

 almost black solution can then be placed in a flask with a few crystals 

 of anhydrous sodium sulfate to remove any remaining water. This solu- 

 tion is now ready for the chromatographic separation. The transfer of the 

 pigments requires a certain amount of judgment during the operation. 

 The behavior of one preparation may be quite different from the be- 

 havior of another. The water content of different lots of leaves may vary 

 considerably, and this variation leads to difficulty in specifying amounts 

 of various solvents to add. Occasionally an emulsion will form in the 

 separatory funnel, and it is sometimes more economical to start over 

 than to try to break it up. 



Column Chromatography: Chromatographic separations can be per- 

 formed with a finely powdered adsorbing material in glass tubes of vari- 

 ous sizes and types. A very convenient glass tube is fitted at one end with 

 a ground glass joint and a fritted glass disk. The ground joint permits the 

 easy removal of the solid material when the separation is complete. The 

 fritted disk holds the adsorbing material in the tube while it allows 

 solvents to pass through and drip out at the bottom of the tube. 



The following description will be easier to follow by referring to 

 Fig. 11-1. This set of glass tubes represents a series of stages in a 

 chromatographic separation. 



Preparing a column requires a certain knack, and the beginner should 

 not he discouraged if the first few columns do not work well. Several 

 different methods of packing columns have been described, but the fol- 

 lowing has produced good results in our laboratories. The bottom of the 

 tube is closed off so that liquid will not leak out. Petroleum ether is 

 poured in until the tube is approximately half full. Grocery store pow- 

 dered sugar, dried in an oven if the weather is damp, is sifted into the 

 liquid and stirred with a glass rod to make a very thin paste. Alternate 

 additions of powdered sugar and petroleum ether can be continued until 

 the tube is almost full of the thin paste. When the stopper is removed 

 from the bottom of the glass tube, petroleum ether will drip out and the 

 sugar will settle into a uniform column. Usuafly the sugar settles faster 

 than the solvent escapes, so a layer of clear petroleum ether appears at 

 the top of the column. Occasionally the stirring rod is used to redis- 



