656 VI. CAROTENOIDS AND RELATED COMPOUNDS 



employed, although chloroform (b.p., 61.2°C.), carbon tetrachloride (b.p., 

 76°C.), 1,2-dichloroethane (b.p., 84°C.), ethanol, anisole, acetone, and 

 diethyl ether may be used. It is usually advisable to moisten the column 

 with the solvent before the mixture to be chromatographed is poured onto 

 it. The column should not be allowed to become dry during the procedure, 

 as this will cause the upper portion to crack. Hence, any new solution will 

 not have an opportunity to be adsorbed at the same portion of the column, 

 since it may pass into the column lower down at the base of the cracks. 

 For this reason, pressure is frequently employed, rather than suction, to 

 force the solution through the column. 



After the mixture has all passed through the tube, one usually attempts 

 to separate the zones as much as possible. This can sometimes be done 

 by washing the column with an excess of the pure solvent originally em- 

 ployed. Frequently, if the zones separate too slowly when the first solvent 

 is used, an increase in eluent activity can be brought about by the use of a 

 new solvent. One continues to wash the column until the development is 

 optimal in all zones. 



In order to obtain the desired pigment, the column can then be washed 

 with an eluent, which will dissolve the pigment and allow it to be carried 

 out in the filtrate. Methanol, ethanol, and acetone are the most useful 

 eluents, a fact which was discovered by Tswett. However, it is not neces- 

 sary to use the pure eluent; the addition of the eluents, in amounts of 0.5 

 to 2.0%, to the original solvent used will result in the rapid solution of the 

 adsorbed material. 



However, a different procedure must be employed if there are several 

 adsorption zones. This involves the removal of the adsorption column 

 intact, and separation of the desired adsorption zone from the rest of the 

 column ; the eluent is then added to the adsorbent, which has been immedi- 

 ately crushed after being separated from the rest of the column. The pig- 

 ment dissolves quickly and the adsorbent is then filtered off, leaving the 

 solution of the desired pigment. In order that the column be removed 

 intact, it should be slightly dried. This can be attained by holding the 

 hand over the mouth of the tube and continuing suction for 15 seconds 

 after the solvent has passed out of the tube. More recent tubes of uniform 

 bore containing interchangeable glass connections at the lower end make it 

 simple to force out the column with a plunger of appropriate size. 



In order to obtain the sample for extraction, one should not simply cut 

 the column at right angles to the long axis. Any unloaded portions of the 

 column should first be rejected and the areas of adsorption should be care- 

 fully separated. An easy procedure is to take the portion of the column 

 desired in the left hand and carefully trim off the white or slightly colored 

 portions at an acute angle to the outside. This is advisable, since the area 

 of penetration is usually deeper in the center and a considerable amount of 



