HPTL (High Performance Thin Layer Chromatography)

ABSTRACT: The differentiation of inks on a questioned document can highlight a fraudulent insertion and is usually carried out by optical comparison and thin-layer chromatography (TLC). Laser desorption ionization mass spectrometry (LDI-MS) may also be used for the analysis of dyes from ink. This analytical technique was compared with a standard method of high-performance TLC (HPTLC) according to their capacity to differentiate blue ballpoint inks. Ink entries on paper from 31 blue ballpoint pens have been analyzed and their dye ink formulations compared. The pens were classified into 26 classes by LDI-MS against 18 for HPTLC. LDI-MS proved to be a more powerful method for differentiating ink formulations because it provides information about dye structures (molecular weights) and relative quantification of dye classes (peak areas). Sample preparation was minimal and analysis time was short in contrast to the more complex extraction, application, and development steps of the HPTLC method. However, only basic dyes and pigments were identified using positive mode LDI-MS, while HPTLC did yield additional information about acid dyes.

High performance thin layer chromatography is an emerging alternative analytical technique in comparison with conventional column chromatography because of its simplicity, rapidity, accuracy, robustness, and cost effectiveness. Choice of vast array of supporting matrices and solvent systems resulted in separation of almost all types of analytes except volatiles. First step of a robust method development for routine quantification is to check the stability of analyte during various steps of chromatographic development followed by preparation of calibration curves. Thereafter, various validation aspects of analysis namely peak purity, linearity and range, precision, limit of detection, limit of quantification, robustness, and accuracy have to be measured.

For the past few decades, compounds from natural sources have been gaining importance because of the vast chemical diversity they offer. This has led to phenomenal increase in the demand for herbal medicines in the last two decades and need has been felt for ensuring the quality, safety, and efficacy of herbal drugs. Phytochemical evaluation is one of the tools for the quality assessment, which include preliminary phytochemical screening, chemoprofiling, and marker compound analysis using modern analytical techniques. High-performance thin-layer chromatography (HPTLC) has been emerged as an important tool for the qualitative, semiquantitative, and quantitative phytochemical analysis of the herbal drugs and formulations. This includes developing TLC fingerprinting profiles and estimation of biomarkers. This review has an attempt to focus on the theoretical considerations of HPTLC and some examples of herbal drugs and formulations analyzed by HPTLC.

A wide range of methods exist for the on-plate detection of lipids resolved by thin layer chromatography. Fluorescence generally offers improvements in sensitivity over methods that use colorimetric or simple densitometric detection. In this paper, we report that a classic cupric sulfate charring protocol produces a fluorescent signal that sensitively and quantitatively detects a wide range of phospholipids, neutral lipids, and sterols after automated, multi-development high performance thin layer chromatography. The measured lower limits of detection and quantification, respectively, were, on average, 80 and 210 pmol for phospholipids and 43 fmol and 8.7 pmol for sterols. The simple, inexpensive, and highly sensitive approach described here was used to quantitatively analyze the lipid and sterol composition of sea urchin cortical vesicles, a stage-specific model system used to study the mechanism of regulated membrane fusion.

Context: A high-performance thin layer chromatography (HPTLC) is an analytical technique, which can be used for the determination of constituents or marker components in various parts of the plants. Earlier studies have estimated phytoconstituents from the stem and other aerial plant parts of Cissus quadrangularis Linn. Estimation of hydrocarbons can also be successfully done using HPTLC technique using suitable derivatization.

Aims: To develop and validate a simple and rapid method for the estimation of long chain aliphatic hydrocarbons from the leaves of C. quadrangularis using HPTLC technique.

Methods: Precoated silica gel 60 F254 plates were used as stationary phase. The mobile phase used was hexane (100 %). The detection of spots was carried out using berberine sulphate as detecting reagent.

Results: The method was validated in terms of linearity, sensitivity, accuracy, and precision. Linearity range was found to be 2-10 µg/mL, limit of detection 0.127 µg/mL, and limit of quantification 0.384 µg/mL.

Conclusions: A novel, simple, accurate, precise and sensitive HPTLC method has been developed and validated for the estimation of long chain aliphatic hydrocarbons obtained from the leaves of C. quadrangularis Linn.

High-performance thin-layer chromatography (HPTLC) is a form of thin-layer chromatography (TLC) that provides superior separation power using optimized coating material, novel procedures for mobile-phase feeding, layer conditioning, and improved sample application. It promotes for higher separation efficiencies, shorter analysis time, lower amounts of mobile phase, and efficient data acquisition and processing. The major parameters that influence separation of the constituents within a mixture are the partition coefficients, retention factor (R f ), and capacity factor of the individual constituents on the plate, selectivity of the mobile and stationary phase to the solutes, and the plate height that decide the separation efficiency as well as resolution of the individual constituents within a mixture. The partition coefficient is defined as the molar concentration of the analyte in the stationary phase to that in the mobile phase. R f , a fundamental qualitative value, is expressed as the ratio of migration distances of an individual components of a mix relative to the mobile phase. Capacity factor (k) is a fundamental characteristic of a substance that determines its qualitative chromatographic behavior. It can be expressed as the ratio of the retention time of the substance in the stationary to that in the mobile phase and is influenced by the chemical nature of the two phases. The separation number (SN) that influences separation power of HPLC is defined as the highest possible number of components that are completely separated in a mixture under a gradient-free isocratic TLC. The efficacy of separation of two components of a mixture in a chromatogram is termed as resolution and is influenced by the selectivity of the components between the stationary and the mobile phase, mobile phase flow rate influenced by particle size and solvent strength that influence capacity factors.

The process of transmembrane movement of phosphatidylcholine (PC) across the outer membrane of mitochondria was investigated in vitro in mitochondrial outer membrane vesicles (OMV) from the yeast Saccharomyces cerevisiae. Phosphatidylcholine-transfer protein (PC-TP) was used to extract radiolabeled PC from OMV, with small unilamellar vesicles serving as acceptor system. Endogenously radiolabeled PC synthesized either via the CDP-choline pathway or via methylation

A new, simple high-performance thin-layer chromatographic method has been established and validated for simultaneous determination of escitalopram oxalate and clonazepam in a combined tablet dosage form. The drugs were separated on aluminum plates precoated with silica gel 60 F254; toluene–ethyl acetate–triethylamine 7:3.5:3 (v/v) was used as mobile phase. Quantitative analysis was performed by densitometric scanning at 258 nm. The method was validated for linearity, accuracy, precision, and robustness. The calibration plot was linear over the ranges 250–2,500 and 50–500 ng band−1 for escitalopram oxalate and clonazepam, respectively. The method was successfully applied to the analysis of drugs in a pharmaceutical formulation.

A method was developed for the qualitative analysis of active ingredients in soluble concentrate (SC) formulations of chlorantraniliprole (Coragen 18.5 SC) and flubendiamide (Fame 480 SC). Known concentrations of the reference grade standards and the soluble concentrate of chlorantraniliprole and flubendiamide dissolved in acetonitrile were applied on silica gel 60 F254 HPTLC plates using a Camag-Hamilton Linomat Syringe (100μL). These plates

This study was undertaken to quantify psoralen and daidzein by high-performance thin layer chromatography (HPTLC). The methanolic extract of 10 mg mL−1 concentration solution was prepared for HPTLC quantification of psoralen and daidzein. HPTLC aluminium-backed plates coated with 0.2 mm layers of silica gel 60 F254 were used as the stationary phase. The working standard solution of psoralen and daidzein was applied along with the test sample solution by means of Camag Linomat IV sample applicator. R f values of psoralen and daidzein were found to be 0.60 and 0.88, whilst as their percentage values in methanolic extract were found to be 3.02% and 5.64% (w/w), respectively. A simple quantitative estimation method of psoralen and daidzein by HPTLC is reported that can be used for the quality control of marketed preparations containing Ficus carica. However, further study is warranted to isolate and quantify active constituents present in the leaves of F. carica by sophisticated techniques.