Abstract
Thin-layer chromatography (TLC) is an often omitted analytical technique due to its lower sensitivity and separation capacity. Even in the era of high-performance liquid chromatography (HPLC), thin-layer chromatography still offers many advantages, such as simplicity, rapidity, and cost-effectiveness, which predict TLC to be the first-choice method for the laborious optimization process requiring analysis of numerous samples. In this work, a thin-layer chromatography method with chemical and densitometric detection was used to optimize a dispersive liquid-liquid microextraction (DLLME) process for the extraction and preconcentration of estradiol in human urine. The chromatographic system consisted of silica gel plates as the stationary phase and toluene-ethanol (9:1; v/v) mixture as the developing solvent. The plates were dyed with 10% phosphomolybdic acid reagent and sequentially evaluated densitometrically at λ = 430 nm. In the context of DLLME optimization, parameters including the type and volume of extraction and dispersive solvents, centrifugation, salt addition and extraction time, were studied. The proposed DLLME-TLC method was successfully applied to the determination of estradiol in real human urine samples.
Highlights
Estrogens are human hormones primarily responsible for the development and function of female gonadal system, regulation of menstrual cycle, and maintenance of pregnancy[1] which take part in various biochemical processes within the organism in both males and females.[2]
Estradiol on Thin-layer chromatography (TLC) plate was visualized with 10% phosphomolybdic acid detection reagent as dark-blue spots on greenish background[20] and it was quantified densitometrically at wavelength of λ = 430 nm
Optimized TLC method was linear within the concentration range of 0.1 – 5.0 mg mL–1 (r = 0.9979) with limit of detection (LOD) = 0.03 mg mL–1 and limit of quantification (LOQ) = 0.1 mg mL–1
Summary
Estrogens are human hormones primarily responsible for the development and function of female gonadal system, regulation of menstrual cycle, and maintenance of pregnancy[1] which take part in various biochemical processes within the organism in both males and females.[2]. A mixture of extraction and dispersive solvents is rapidly injected into the aqueous sample with a syringe resulting in the formation of tiny droplets of extraction solvent in the solution allowing fast transfer of the desired analyte into the extraction solvent. This state, called cloudy solution, disappears during centrifugation when the aqueous and organic phases are separated. This work describes optimization of a dispersive liquid-liquid microextraction for the extraction and preconcentration of estradiol in human urine samples using thin-layer chromatography (TLC) with chemical and densitometric detection which has, to the best of our knowledge, not been described yet.
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