Optimization of Detection of Native Amino Acids with Evaporative Light Scattering Detector in Chiral Supercritical Fluid Chromatography

Based on previously reported chiral separation of free amino-acids (AAs) using packed-column supercritical fluid chromatography (pSFC), it was desirable to investigate parameters influencing the response of evaporative light scattering detector (ELSD). The effect of several parameters, such as SFC conditions (outlet pressure, temperature and flow rate) and ELSD settings (drift tube temperature of nebulisation and nebulizer gas pressure) on the sensitivity (response) of ELSD have been studied. Based on optimization an outlet pressure of 170 bar, a temperature of 40 °C and a flow rate of 3 mL/min were chosen, and a drift tube temperature of 60 °C and a nebulizer gas pressure of 3.0 bar were found to be optimal. Limits of detection (LOD) and limits of quantification (LOQ) were determined for studied amino acids (AAs) and were found to be lower than values determined without optimization. The enantiomeric elution order was also determined for leucine, phenylalanine, isoleucine, valine, carnitine and histidine by the injection of the L-AAs in the above-mentioned optimized conditions. The L-enantiomers eluted first for four out of the six AAs studied. Conclusions on the influence of the screened variables on ELSD sensitivity and recommendations for AAs analytical studies in SFC are given.