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

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

Milán

Congreso; 48th International Symposium on High-Performance Liquid Phase Separations and Related Techniques; 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 chiral 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, especially when considered in combination with the rather limited efficiencies of enantioselective columns. 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 as well as endogenous matter, which can coelute with the enantiomer peaks.Chiral separations are commonly used as 2D columns, especially in heart-cut mode 2D-LC (Review).  However, to improve the use of an on-line comprehensive 2D-LC system, in this work the CSP was used in the 1D base on its lower chromatographic efficiency, allowing to be sampled each peak multiple times.  And the 2DLC is completed with a very fast second dimension developed to not lose the resolution gained in the 1D, with orthogonal selectivity to get the best peak capacity for the chiral analysis of amino acids in honey samples.Another advantage of the developed method is the application of an Active Flow Splitter pump that allows to: 1) easily adjust the volume of sample (with the strong solvent) transferred to the second dimension and 2)  use a high flow rate in the first dimension and per-se a fast re-equilibration and a better usage of the first dimension separation space. In brief, splitting the flow after the 1D column should allow the two dimensions to be optimized almost independently to get a better peak capacity (Marcelo).Using the concept of a comprehensive on-line 2D-HPLC (LCxLC) setup obtained by coupling an enantioselective (CSP) system in the first dimension to a ultra-fast separation RP system in the second dimension, we were able to prove the feasibility of the enantioselective analysis of natural complex mixtures. The Chiral columns consist in Quinine linked to mercaptopropylsilica, with a final end-capping reaction with 1-hexene (SONIA). The potential enantiorecognition of these columns was studied in an anion exchange mode with racemic amino acids derivatized with the Sanger reagent (DNP Amino acids), prior to 2D-LC.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, compatibility of the dimensions, etc.).