SEPARATION OF THE CAROTENOID MIXTURES 



27 



from the excellent work of Hesse', is one of decreasing adsorptive capacity of 

 the solutes : petroleum ether, carbon tetrachloride, trichloroethylene, benzene, 

 methylene dichloride, chloroform, ether, ethyl acetate, acetone, w-propyl 

 alcohol, ethanol, methanol, water, pyridine. 



-Adsorbent 



'Glass tube 



Cottonwool 



Rubber stoppers 



Experimental. A suitable adsorbent and solvent, and the size of the chromatogram 

 column, are first selected by means of preliminary small-scale experiments. The chro- 

 matogram tube is then filled with the adsorbent. A variety of more or less complicated 

 arrangements for large-scale chromatography have been de- 

 scribed. Only a very simple apparatus which is readily as- 

 sembled, inexpensive, and adequate for all purposes will be 

 described here. It consists of a suction flask, a short glass tube 

 ca. 10 mm in diameter and two good rubber stoppers. The 

 stoppers are arranged back-to-back on the glass tube; one 

 stopper is placed in the suction flask and the other in the 

 chromatogram tube, as shown in the accompanying figure. 

 The filling of the tube with the adsorbent can be carried out in 

 various ways. The usual procedure is to add a small amount of 

 the adsorbent at a time and to press it down with a half-bored 

 cork attached to a glass rod. For larger tubes, Zechmeister 

 recommends a wooden stopper, the end of which has a 

 diameter corresponding to two-thirds of that of the tube. 

 After one layer has been well pressed down, more adsorbent is 

 added and the operation is repeated until the tube is suffi- 

 ciently full. It is important that the tube should be well and 

 evenly filled, otherwise distorted colour-zones are obtained 

 which are difficult to separate. When the tube has been filled, 

 the vacuum is connected and the pressing and tapping (from 

 the outside) is continued until the adsorbent no longer moves. 

 If this is done, no shrinking should occur when solvent is added. 



WiNTERSTEiN and SxEiN^ recommend that for filling large Arrangement for chro- 

 tubes the adsorbent should be moistened with the solvent matographic analysis 

 and then poured into the tube. 



When the column is ready, the solution of carotenoids is poured on and allowed 

 to penetrate completely. More solvent is then added to "develop" the chromato- 

 gram. The development is best carried out by connecting the suction flask to the 

 vacuum and then isolating the water pump by means of a screw tap. When the rate 

 of flow of the solvent becomes too small, the flask is re-evacuated. It is important 

 that the rate of flow should be neither too great nor too small. If the rate is too 

 great, the zones tend to be blurred, while if it is too small, no sharp layers are formed 

 because the rate of diffusion of the pigments then exceeds the rate of flow. After 

 development is complete, the column is sucked dry until it has the appearance 

 of a compact mass which can be extruded without falling to pieces. After mechanical 

 separation of the coloured zones, the pigments of the individual layers are eluted 

 with the solvent previously employed, but containing a little methanol(ca. 2-5 %). 

 The eluates are evaporated to dryness in vacuum and the residue from each zone 

 is either crystallised or, if the pigment is still non-homogeneous, again subjected 

 to chromatography. 

 References p. 28. 



