In-line coupling of a reversed-phase column to cope with limlited chemoselectivity of a quinine carbamate-based anion-exchange type chiral stationary phase

A chiral stationary phase based on tert-butylcarbamoyl quinine has shown remarkable enantiomer separation capability for the thyroid hormone thyroxine (T-3) and its structural analogue triiodothyronine (T-3) employing hydroorganic buffered mobile phases (typical RP conditions). To overcome the problem of a somehow limited chemoselectivity for the critical peak pair between adjacent L-thyroxine (L-T-4) and D-thyroxine (D-T-4) peaks on the chiral anion-exchanger CSP when all four compounds need to be analysed simultaneously like in impurity profiling of L-T-4 drug products, an RP column (Gemini C18) was serially coupled with the chiral anion-exchanger column to add a hydrophobic selectivity increment and to improve thereby the critical resolution between L-T-3 and D-T-4. Various parameters such as organic modifier content, pH, buffer concentration and type, type of achiral column as well as sequence of achiral and chiral column have been investigated with individual and tandem columns. With the optimized conditions and use of the tandem column a significantly improved separation, as compared to the chiral anion-exchanger column alone, with a critical resolution as high as 3.7 and an almost equal band spacing of the four components of the test mixture could be obtained. The sequence of the columns (achiral-chiral or chiral-achiral) had no significant effect on the separation performance.