Preparative Chromatography - The Preparative Separation of the Enantiomers of Chlorokynurenine > Page 70

The preparative column that was employed was 25 cm long, 2.21 cm in diameter and operated at a flow rate of 18 ml/min. The mobile phase (the polar organic solvent) was a mixture of methanol, trifluoroacetic acid and ammonium hydroxide (100/0.005/0.005). The initial sample size was 25 mg and the separation obtained is shown in figure 39. The first fraction was taken between the beginning of the first peak and the minimum between the peaks. As the peak were somewhat asymmetrical due to the column overload the first fraction was 100 % pure peak 1. The second fraction (mostly peak 2), however, was significantly contaminated with peak 1. The second fraction was then run again, under exactly the sample conditions. Now, however, the first peak is not overloaded and is thus symmetrical. The second peak, however, is still overloaded and asymmetrical but will have a sharp front. Consequently if the second fraction is taken between the minimum between peak 1 and peak 2 and the end of peak 2 the product will be almost pure peak 2.


Courtesy of ASTEC Inc.


Figure 39. The Primary Preparative Separation of the Isomers of Nicardipine.