Preparative Chromatography - The Preparative Separation of the Enantiomers of Chlorokynurenine > Page 65

 

An example of the use of the radial flow column to separate some large biomolecules is shown in figure 34. The total column volume was 500 ml (that is the space between the cylindrical frits) which was filled with a proprietary packing, IMPAQRRG2020-C18 (a basically dispersive stationary phase using octadecane bonded to silica gel). The solutes were separated in the reversed phase mode. A linear gradient was employed starting with acetonitrile/0.1% TFA in water : 20/80 v/v, and terminating with acetonitrile/0.1% TFA in water : 50/50 v/v. The gradient period was 120 minutes and the flow rate was 46 ml per min.

The Preparative Separation of the Enantiomers of Chlorokynurenine

Details of this application were obtained ASTEC Inc. who obtained it by private communication from Dr. Lester Dolak and Dr. Eric Seest of the Upjohn Pharmacia Company, Kalamazoo. The column that was employed was 50 cm x 5 cm (Chirobiotic T column from ASTEC) and the separation was carried out at 40˚C. It was a little difficult to arrive at a suitable feed solvent due to the relative insolubility of the solute in the mobile phase. It was eventually dissolved at a concentration of 2 mg per ml of chlorokynurenine in a solvent (95% aqueous ethanol/water : 50/50 v/v) by heating and stirring at 40˚C. The filtered sample solution and the column were maintained at 40˚C throughout the sampling procedure and separation. 400 ml of the solution (containing 800 mg of chlorokynurenine) were pumped onto the column at 50 ml/min. for 8 minutes. The sample pump was then stopped, the solvent pump started and the solutes eluted at a flow rate of 50 ml/min. for 20 minutes.

As soon as the second enantiomer began to emerge, the flow rate was increased to 60 ml/min. An actual separation is shown in figure 35. and it is seen that the separation that was obtained was highly satisfactory. The products were analyzed on an analytical Chirobiotic T column and indicated that the first enantiomer was >99% pure, and the second enantiomer was 98% pure. The mid fraction, that was collected between the two main peaks, was recycled. The total cycle took 49 minutes and it is seen that the system operated very effectively. The use of preparative chromatography for the separation of physiologically active enantiomers is now quite well established but, even so, it is probably still in its infancy and many improvements are likely to occur in the future. The technique of LC is probably the only practical technique for the large scale resolution of many racemic mixtures.