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Improvement of derivatized amino acid detection sensitivity in micellar electrokinetic capillary chromatography by means of acid-induced pH-mediated stacking technique.

ABSTRACT: Derivatization is a frequently used sample preparation procedure applicable to the enhancement of analyte detection sensitivity. Amino acids mostly require derivatization prior to electrophoretic or chromatographic analysis, especially if spectrophotometric detection is used. This study presents an on-line preconcentration technique for derivatized amino acids. The sensitivity of the method was improved by the utilization of the proposed acid-induced pH-mediated stacking mechanism. The method is demonstrated by preconcentration of amino acids labeled with 2,4-dinitrofluorobenzene. Use of optimized conditions for a large sample volume injection (40 s, 13.8 kPa) followed by electrokinetic injection of 0.1 M HCl (20 s, 10 kV) gave a 20- to 30-fold enhancement of sensitivity. The significance of the sweeping mechanism and pseudo-isotachophoresis for the on-line sample focusing and the influence of parameters on the preconcentration process were discussed. The applicability of the elaborated method was demonstrated using human urine samples.

SUBMITTER: Dziomba S

PROVIDER: S-EPMC4182592 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Improvement of derivatized amino acid detection sensitivity in micellar electrokinetic capillary chromatography by means of acid-induced pH-mediated stacking technique.

Dziomba Szymon S Bekasiewicz Adrian A Prahl Adam A Bączek Tomasz T Kowalski Piotr P

Analytical and bioanalytical chemistry 20140822 26

Derivatization is a frequently used sample preparation procedure applicable to the enhancement of analyte detection sensitivity. Amino acids mostly require derivatization prior to electrophoretic or chromatographic analysis, especially if spectrophotometric detection is used. This study presents an on-line preconcentration technique for derivatized amino acids. The sensitivity of the method was improved by the utilization of the proposed acid-induced pH-mediated stacking mechanism. The method is ...[more]

PMID: 25146356

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Project description:Bile salts are a category of natural chiral surfactants which have ever been used as the surfactant and chiral selector for the separation of many chiral compounds by micellar electrokinetic chromatography (MEKC). In our previous works, the application of sodium cholate (SC) in the separation of four stereoisomers of palonosetron (PALO) by MEKC has been studied systematically. In this work, the parameters of other bile salts, including sodium taurocholate (STC), sodium deoxycholate (SDC), and sodium taurodeoxycholate (STDC) in the separation of PALO stereoisomers by MEKC were measured and compared with SC. It was found that all of four bile salts provide chiral recognition for both pairs of enantiomers, as well as achiral selectivity for diastereomers of different degrees. The structure of steroidal ring of bile salts has a greater impact on the separation than the structure of the side chain. The varying separation results by different bile salts were elucidated based on the measured parameters. A model to describe the contributions of the mobility difference of solutes in the aqueous phase and the selectivity of micelles to the chiral and achiral separation of stereoisomers was introduced. Additionally, a new approach to measure the mobility of micelles without enough solubility for hydrophobic markers was proposed, which is necessary for the calculation of separation parameters in MEKC. Under the guidance of derived equations, the separation by SDC and STDC was significantly improved by using lower surfactant concentrations. The complete separation of four stereoisomers was achieved in less than 3.5 min by using 4.0 mM of SDC. In addition, 30.0 mM of STC also provided the complete resolution of four stereoisomers due to the balance of different separation mechanisms. Its applicability for the analysis of a small amount of enantiomeric impurities in the presence of a high concentration of the effective ingredient was validated by a real sample.

Dataset Information

Improvement of derivatized amino acid detection sensitivity in micellar electrokinetic capillary chromatography by means of acid-induced pH-mediated stacking technique.

Publications

Improvement of derivatized amino acid detection sensitivity in micellar electrokinetic capillary chromatography by means of acid-induced pH-mediated stacking technique.

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