Cyclodextrin based chiral stationary phases have also been used to separate b blocking agents in conjunction with an optically active fluorescent Edman-type reagent to enhance detection. Toyo'oka and Liu [15] used the optically active Edman type fluorescent reagents, 4-(3-isothiocyanatepyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiaz-ole [(r)-(–) and (S)-(+)-NBD-PyNCS] and 4-(3-(3-isothiocyanatepyrrol-idin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxydiazole [(R)-(–)-and (S)-(+)-DBD-PyNCS] to produce diastereomers that could be separated on a simple reversed phase column and, at the same time, provide enhanced sensitivity.

The reagents reacted quantitatively with primary and secondary amino functional groups, under mild conditions (55°C for 10 min.), in the presence of triethylamine, to produce the corresponding fluorescent thiourea derivatives. The purity of the reagents was ascertained by separation on a cyclodextrin column and the results are shown in figure 50. The separation was carried out on a derivatized cyclodextrin column (ES-PhCD) 15 cm long and 6 mm I.D., packed with 5 mm particles. Chromatogram A shows the elution of the (R)-(–)-NBD-PyNCS isomer, B, the elution of the (R)-(+)-NBD-PyNCS isomer and C, the separation of the racemic mixture. The mobile phase was a mixture of acetonitrile/methanol/water : 3/3/4 v/v/v. Chromatogram D shows the elution of the (R)-(–)-DBD-PyNCS isomer, E, the elution of the (R)-(+)-DBD-PyNCS isomer and F, the separation of the racemic mixture.

Courtesy of the Royal Society of Chemistry, [Ref. 15]

Figure 50 The Separation of Enantiomeric Pairs of NBD-PyNCS and DBD-PyNCS on Utron ES-PhCD, a Cyclodextrin Based Stationary Phase