Chiral x Achiral Multidimensional Liquid Chromatography. Applications to the enantioseparation of DNP amino acids in honey samples of different regions of Mendoza (Argentina)

ACQUAVIVA, AGUSTÍN; CASTELLS, CECILIA; SIANO GABRIEL; FILGUEIRA MARCELO

Milan

Congreso; HPLC2019; 2019

Biological, environmental and natural samples are generally highly complex mixtures in which compounds of interest may appear as minor constituents. As a consequence, a large excess of interferences frequently hinders the direct determination of target compounds. Most HPLC enantiomer separations are performed with columns packed with a chiral stationary phase (CSP) operated with an achiral mobile phase. One common problem encountered with this approach is the intrinsically limited chemical selectivity of CSPs to the simultaneous resolution of several pairs of enantiomers. This means that complex mixtures of diverse pairs of enantiomers cannot be analyzed in one run due to peak overlapping. Moreover, some drawbacks remain when the analyte is present in very complex samples containing other achiral compounds, which can coelute with the enantiomer peaks. Multidimensional chromatography is then an interesting option to increase peak capacity. Chiral separations are commonly used as 2D columns, especially in heart-cut or multiple heart-cut 2D-LC modes1. The aim of this work was to use an on-line comprehensive 2D-LC mode based on the combination of a chiral column in the first dimension and an achiral column in the second dimension. Enantioseparation is usually associated to a slower kinetics and columns of smaller efficiencies as compared to achiral LC columns of the same geometrical features. Thus, the CSP was used in the 1D in order to sample each 1D peak multiple times. The 2DLC system is completed with a very fast second dimension (using a short column of reduced particle diameter operated at relatively high flow-rate). Several columns were tested to search for orthogonality respect to the chiral column. The 2DLC system was built with an active flow splitter pump in order to easily adjust the volume of sample transferred to the second dimension2 and to choose independently the flow rate in the first dimension, typically a larger one than the flow necessary to fill the sample loops. The chiral column consisted in a phase based on quinine linked to mercaptopropylsilica, with a final end-capping reaction with 1-hexene. This column has been previously used to enantioseparate DNPamino acids under anion exchange mode3. The present LCxLC method was optimized with respect to the sample characteristics and taking into account all parameters that influence the bidimensional peak capacity (orthogonality, sampling frequency, etc.). Samples has been previously preconcentrated in a ion-exchange column followed by derivatization. Simultaneous enantioseparation of several amino acids present in different honey samples has been achieved under optimized conditions. 1- María Eugenia León-González, et. al., Biomed. Chromatogr. 2014; 28: 59 ?83 2- Marcelo R. Filgueira, et. al., Anal. Chem., 2011, 83 (24), 9531?9539. 3- Sonia Keunchkarian et.al., J. Chromatogr. A 1218 (2011) 3660?3668.