Dispersive liquid–liquid microextraction combined with semi-automated in-syringe back extraction as a new approach for the sample preparation of ionizable organic compounds prior to liquid chromatography

Abstract

Dispersive liquid–liquid microextraction (DLLME) followed by a newly designed semi-automated in-syringe back extraction technique has been developed as an extraction methodology for the extraction of polar organic compounds prior to liquid chromatography (LC) measurement. The method is based on the formation of tiny droplets of the extractant in the sample solution using water-immiscible organic solvent (extractant) dissolved in a water-miscible organic dispersive solvent. Extraction of the analytes from aqueous sample into the dispersed organic droplets took place. The extracting organic phase was separated by centrifuging and the sedimented phase was withdrawn into a syringe. Then in-syringe back extraction was utilized to extract the analytes into an aqueous solution prior to LC analysis. Clenbuterol (CB), a basic organic compound used as a model, was extracted from a basified aqueous sample using 25 μL tetrachloroethylene (TCE, extraction solvent) dissolved in 500 μL acetone (as a dispersive solvent). After separation of the organic extracting phase by centrifuging, CB enriched in TCE phase was back extracted into 10 μL of 1% aqueous formic acid (FA) within the syringe. Back extraction was facilitated by repeatedly moving the plunger back and forth within the barrel of syringe, assisted by a syringe pump. Due to the plunger movement, a thin organic film is formed on the inner layer of the syringe that comes in contact with the acidic aqueous phase. Here, CB, a basic analyte, will be protonated and back extracted into FA. Various parameters affecting the extraction efficiency, viz., choice of extraction and dispersive solvent, salt effect, speed of syringe pump, back extraction time period, effect of concentration of base and acid, were evaluated. Under optimum conditions, precision, linearity (correlation coefficient, r 2 = 0.9966 over the concentration range of 10–1000 ng mL −1 CB), detection limit (4.9 ng mL −1), enrichment factor (175), relative recovery (97%) had been obtained. The applicability of this newly developed method was investigated for the analysis of CB in the water samples from river, lake and stream water.