Heart-cutting 2D-CE with on-line preconcentration for the chiral analysis of native amino acids.

The use of transient moving chemical reaction boundary (tMCRB) was investigated for the on-line preconcentration of native amino acids in heart-cutting 2D-CE with multiple detection points using contactless conductivity detection. The tMCRB focusing was obtained by using ammonium formate (pH 8.56) as sample matrix and acetic acid (pH 2.3) as a BGE in the first dimension of the heart-cutting 2D-CE. Different experimental parameters such as the injected volume and the concentration in ammonium formate were optimized for improving the sensitivity of detection. A stacked fraction from the first dimension was selected, isolated in the capillary, and then separated in the second dimension in the presence of a chiral selector ((+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid). This on-line tMCRB preconcentration coupled with heart-cutting 2D-CE was applied with success to the chiral separation of D,L-phenylalanine, and D,L-threonine in a mixture of 22 native amino acids. The sample mixture was diluted in 0.8 M of ammonium formate, and injected at a concentration of 2.5 muM for each enantiomer with a volume corresponding to 10% of the total capillary volume. An LOD (S/N=3) of 2 muM was determined for L-threonine.

Heart-cutting 2-D CE using multiple detection points for chiral analysis of native amino acids.

A new methodology based on heart-cutting 2-D CE in a single capillary was developed for the chiral separation of a mixture of 22 underivatized amino acids. The first dimension is performed in an achiral BGE (2.3 M acetic acid, pH 2.1) allowing the separation of the analytes as a function of their charge-to-radius ratio. A selected fraction from the first dimension is then separated in the second dimension in the presence of a chiral selector (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid. Since the introduction of the second electrolyte is performed by pressure mobilization, capillaries with small id of 10 microm are used to limit the peak broadening due to Taylor dispersion. Double capacitively coupled contactless conductivity detector is used for monitoring the selection and the isolation of the fraction at the different steps of the analysis (voltage and pressure mobilization steps). This double detection allows calculating the voltage and pressure stop times in real-time analysis. This new methodology is applied with success for the chiral separation of different amino acids (D,L-Tyr, D,L-Trp and D,L-Thr) contained in a mixture of 22 native amino acids.

Heart-cutting two-dimensional capillary electrophoresis for the on-line purification and separation of derivatized amino acids.

Heart-cutting two-dimensional (2D) capillary electrophoresis (CE) in a single capillary was used for analysis of derivatized amino acids. A mixture of 12 amino acids derivatized with UV-active benzyl 4-(3-(2-chloroethyl)-3-nitrosoureido)butylcarbamate label served as a model of a moderately complex sample due to the presence of numerous derivatization byproducts. The first step of the heart-cutting 2D approach was sample cleanup by capillary zone electrophoresis (CZE) in borate electrolyte. Then, only a selected portion of the first-dimension separation was transferred into the second dimension of the separation by a specific voltage and pressure program. Finally, the zone of derivatized amino acids was separated by micellar electrokinetic chromatography in a borate-sodium dodecyl sulfate system. The whole 2D process can be performed in a conventional CE analyzer without any interface for connection of the two separation modes. Intraday repeatability of the total migration time was 2%. In general, the heart-cutting 2D-CE methodology in a single capillary can be adapted for any CE mode regardless of the direction and velocity of electroosmotic flow and position of the fraction of interest in the first dimension (i.e., first, last, or intermediate fraction).