Thin-layer Chromatography Conditions Research Articles

Optimization of the thin layer chromatography method conditions for the detection of related dioxidine impurities

When developing methods for analysing active pharmaceutical substances (API), it is necessary to take into account modern requirements for the content of impurities in them. At the same time, for some drugs that have been on the pharmaceutical market for a long time, analysis methods are still used that are unable to provide the necessary sensitivity and specificity. One of these API is 2,3-bis(hydroxymethyl)quinoxaline-1,4-dioxide (dioxidin), which has high bactericidal activity against a wide range of microorganisms. According to the requirements of the State Pharmacopoeia of the Russian Federation XIV edition, related impurities of this API are analysed by thin layer chromatography (TLC). However, this method is semi-quantitative and relative. The purpose of this work is to determination of impurities in the dioxydine substance using high-performance liquid and thin-layer chromatography methods. In accordance with the requirements of the monograph of the State Pharmacopoeia of the Russian Federation, the total content of all impurities in the API is assessed and only the content of quinoxidine is determined separately, which is often absent from the substance. For the first time, three typical dioxidine related impurities were revealed using HPLC. Next, we selected the optimal conditions for their detection by TLC. Methanol/acetonitrile (75/25) and dichloromethane/acetonitrile (50/50) mixtures were the most suitable as a solvent for impurities and the TLC elution system, respectively. Under the selected conditions, satisfactory separation of the chromatographic zones of all four impurities and the dioxidine substance was observed. The corresponding zones were clearly visible at a substance concentration of 1%. However, reducing their concentration to the required 0.1% made it difficult to visually detect impurities. Thus, even when optimizing TLC conditions, it is impossible to obtain reliable data on the content of related impurities, which is due to the limitations of the method. On the contrary, the analysis of dioxidine substances by HPLC allows fairly accurate quantitative determination of individual related impurities.

TLC-fingerprinting and chemometrics for identification of Curcuma xanthorrhiza from different geographical origins in Indonesia

Abstract. Kartini K, Sabatini SS, Haridsa NM, Jayani NIE, Setiawan F, Hadiyat MA. 2023. TLC-fingerprinting and chemometrics for identification of Curcuma xanthorrhiza from different geographical origins in Indonesia. Biodiversitas 24: 6557-6566. Geographical origin is an important parameter that influences the quality of herbal medicine. The fingerprint of herbal plants is expressed as chemical compounds contributing to said quality. Curcuma xanthorrhiza, popular as java turmeric, is extensively used in Indonesian traditional medicine. This study aimed to validate and develop a Thin Layer Chromatography (TLC) fingerprint of C. xanthorrhiza, followed by chemometric techniques to classify C. xanthorrhiza rhizomes from 15 regions in Indonesia. Under selected TLC conditions (i.e., stationary phase: TLC plate Silica gel 60 GF254, mobile phase: dichloromethane, chloroform, and ethanol (10: 10: 1), detection: vanillin-sulfuric acid reagent), C. xanthorrhiza produced five zones of the compound. Extracts were stable both on the TLC plate and in the extract solution during chromatography and within 60 minutes after derivatization. In terms of precision, the chromatogram meets the requirement of intraday precision. Chemometric analysis with Principal Component Analysis (PCA) and Cluster Analysis (CA) showed that the samples of C. xanthorrhiza from 15 regions in Indonesia were grouped into five clusters based on the similarity of the chemical compounds, namely cluster 1 (Tawangmangu, Bangkalan, Kediri, and Surabaya); cluster 2 (Batu, Sragen, Tulungagung, Pasuruan, Blitar, and Lombok Tengah); cluster 3 (Ngawi); cluster 4 (Gresik); and cluster 5 (Bojonegoro, Banyuwangi, and Palangkaraya). The TLC fingerprinting with chemometrics on C. xanthorrhiza rhizome is useful for quality control based on geographic origin and authenticity identification.

Screening of Phytochemical Secondary Metabolites of Muntingia Calabura: a Potential as Hepatoprotector

Muntingia calabura is one of the plants employed to produce herbal-based treatments. Muntingia calabura leaves are traditionally used as an alternative medicine due to their secondary metabolites. The maceration method extracted Muntingia calabura leaves using 96% ethanol solvent for 3 x 24 hours. The fractionation process was carried out using a separating funnel method with different polarities, such as n-hexane, ethyl acetate, and ethanol-water. Thin-layer chromatography (TLC) was used to confirm the phytochemical screening. TLC conditions under UV light 254 and 366 nm using solvents, such as chloroform: methanol (alkaloids), butanol: acetic acid: water (flavonoids), chloroform:methanol: water (saponins), and chloroform: methanol (phenolic). The phytochemical screening results of extracts and Muntingia calabura fractions contained secondary metabolites, such as alkaloids, flavonoids, tannins, saponins, and phenolics. TLC results showed that n-hexane fraction contained flavonoid and saponin compounds; ethyl acetate fraction contained alkaloids, flavonoids, saponins and phenolic compounds; and ethanol-water fraction contained alkaloids, flavonoids, saponins, and phenolics. Muntingia calabura leaves indicated the potential as herbal medicine by containing secondary metabolites.

Development of a Chromatographic Method for Analysis and Standardization of the New Quinazolinone Derivative 2-Benzoylamino-N-[4-(4, 6-Dimethylpyrimidin-2-Ylsulfamoyl)Phenyl]Benzamide

The chromatographic characteristics of the new biologically active quinazolinone derivative PMFI-195 were studied in solvent systems with various polarities and dielectric constants. The optimal thin-layer chromatography conditions were found and theoretically justified. The conditions for quantitative determination of PMFI-195 were selected using the computer program Video-Densitometer Sorbfil (Krasnodar). The proposed method allows the identification and quantitative determination of synthetic impurities under laboratory and industrial conditions and will be included in the corresponding regulation.

Study of the sorption properties of solvents in thinlayer and column chromatography

Much attention is paid to the consideration of the causes of transformer oil aging under the influence of technogenic and natural factors. The paper insulation destruction mechanism is considered, as a result of which furan compounds are formed that enter the transformer oil and worsen its dielectric characteristics. The characteristics of the domestic transformer oil grade GK-1 obtained using the technology of hydrocracking in a hydrogen medium are given. Furan compounds are formed in used transformer oil, which are monitored using chromatographic analysis methods according to standard procedures. The group composition of transformer oil was determined using thin layer chromatography. As a solvent used n. Hexane. To extract furan compounds from transformer oil, various organic solvents are used, the physicochemical properties of which are given in this work. It was found that the retention time of the studied sorbates corresponds to an increase in their boiling points for ethyl acetate, methyl ethyl ketone and dodecane. In the case of isopropanol, which has a close boiling point with ethyl acetate and methyl ethyl ketone, a significant increase in retention time is observed, which is associated with the formation of an intermolecular hydrogen bond. The dependence of the spot diameter of furan substances on their concentration was established under conditions of thin-layer chromatography on Sorbfil plastics. Moreover, the most effective separation is characteristic of furfural. In this case, the chromatographic spots are small in size with good fidelity. The ascending mode of column liquid chromatography was used to determine the dependence of the retention time of standard sorbates on the length of the Silyochrome S-80 sorption layer, which is parabolic. It was found that the highest retention times are characteristic of ethoxyethanol and isopropanol, which is consistent with the known theoretical principles of liquid chromatography. Histograms of the effect of the retention time of standard sorbates on their nature and boiling point, where isopropanol and 2-ethoxyethanol are extreme, are presented. In this case, isopropanol having a lower boiling point than 2-Ethoxyethanol is retained on the sorbent more strongly, which is associated with the formation of intermolecular hydrogen bonds with surface silanol groups of the sorbent.

Modeling of chromatographic retention of the selected antiarrhythmics and structurally related compounds in the hydrophilic interactions under the TLC and HPLC conditions

High-performance liquid chromatography (HPLC) plays an important role in testing the pharmaceutically active compounds. In despite of the advantages of HPLC, thin-layer chromatography (TLC) retains its applicability to the different experimental tasks. The experimental conditions which allow hydrophilic interactions in the chromatographic system were tested in the HPLC and TLC systems for ivabradine, its related compounds, diltiazem and verapamil. Under the TLC conditions, retention behavior of the investigated compounds was tested on silica gel modified with cyanopropyl ligands as stationary phase and acetonitrile + methanol containing 25% v/v formic acid. Under the HPLC conditions, we used silica gel modified with cyanopropyl ligands as a column packing and the acetonitrile + 0.25% aqueous solution of formic acid as mobile phase. Retention behavior of the investigated analytes depending on the changing volume fractions of the mobile phase modifier was characterized both for TLC and HPLC data sets by the Soczewiński–Wachtmeister equation. Linear relationships were established between the retention coefficients characterizing the retention mechanism (RM0/m, logk0/m) and molecular properties of the investigated compounds. The Quantitative Structure Retention Relationship (QSRR) modeling was performed with the use of the stepwise multiple linear regression, in order to select molecular properties which influence retention.

Analysis of gardenia blue in foods by thin-layer chromatography

ABSTRACTIn the present study, a thin-layer chromatography (TLC) method for the analysis of gardenia blue was described. Gardenia blue is obtained from the fruits of Gardenia augusta Merrill or Gardenia jasminoides Ellis. Recently, gardenia blue has frequently been used in Japan as a natural coloring in various foods. However, the structural characterization of gardenia blue components has not been yet clarified and even chromatographic separation of the components has not been reported. Synthetic colors in foods are generally analyzed by TLC; so, we therefore investigated the analysis of gardenia blue in foods with TLC. We established two TLC conditions; reversed phase C-18 TLC using a solvent system of aqueous 0.2% TFA–acetonitrile–ethanol (1:2:3) and TLC on cellulose plate using a solvent system consisting of acetone–3-methyl-1-butanol–water (6:5:5). Both conditions yielded three well-delineated spots with good separation. We applied these separations to the analyses of gardenia blue in coloring matter preparations from different manufacturers and in foods on the market. After the gardenia blue was extracted from the samples with water, the extract was evaporated and the residue was dissolved in water–methanol (1:1). Aliquots of the dissolved solutions were then applied to TLC and their chromatographic behaviors were observed. Each preparation showed characteristic spot patterns depending on the manufacturers. The Rf values of the separated spots when extracted from foods are slightly different from the Rf values of the spots observed for standards, and we were thus able to identify the manufacturers using the spot pattern of gardenia blue. The present study is considered to be useful for the establishment of a method of analysis for gardenia blue in coloring preparations and foods.

Optimization of Thin-layer Chromatography and High-Performance Liquid Chromatographic Method for Piper guineense Extracts

In this study, a thin-layer chromatography (TLC) and a high performance liquid chromatographic (HPLC) methods were developed for the chemical profiling, qualitative and quantitative analysis of P. guineense extracts. To obtain a chromatogram with satisfactory resolution and favorable retention time, DryLab software was used to simulate and optimize a HPLC method for the analysis of P. guineense extracts. The aim was to achieve the best possible overall resolution while keeping the analysis time and solvent consumption to a minimum. With the optimized method, a total of 16 main components in the extract were separated with favorable resolution. Optimal TLC conditions were also developed using solvents of various solvent strength (ST) and solvent selectivity (PS) values. The mobile phase composition was systematically tested using various proportions of solvents differing in ST and PS values under the same experimental conditions. During the optimization, emphasis was set on achieving the best possible overall separation of the main components of the extracts (for example piperine). In addition, the effects of the developing chamber was tested using three types of unsaturated chamber conditions: horizontal chamber in sandwich configuration, horizontal chamber in non-sandwich configuration and twin-trough vertical chamber. During the study, a TLC method was developed, and the best mobile-phase composition giving favorable resolution of the bands was toluene: ethyl acetate (PS 6-4 corresponding to 60:40 % v/v). The developing chamber conditions did not affect the TLC separation efficacy in the analysis of P. guineense extracts. The HPLC method was applied to determine the percentage content of piperine in P. guineense. The piperine content was 0.43 % w/w, linearity (0.997), interday precision (% relative standard deviation (RSD), 1.6), intraday precision (% RSD, 2.7 – 5.9), recovery (98.4%), limit of detection (0.001 μg /mL) and limit of quantification (0.003 μg /mL).

Identification of marker compounds for Japanese Pharmacopoeia non-conforming jujube seeds from Myanmar

Jujube seed is a crude drug defined as the seed of Ziziphus jujuba Miller var. spinosa Hu ex H.F. Chou (Rhamnaceae) in the Japanese Pharmacopoeia (JP). Most of the jujube seed in the Japanese markets is imported from China, with the rest obtained from other Asian countries. Here we confirmed the botanical origins of jujube seeds from both China and Myanmar by a DNA sequencing analysis. We found that the botanical origins of the crude drugs from China and Myanmar were Z. jujuba and Z. mauritiana, respectively. Although the jujube seed from China conforms to the JP, that from Myanmar does not. A method for discriminating jujube seeds from China and Myanmar using a chemical approach is thus desirable, and here we sought to identify a compound specific to Z. jujuba. Jujuboside A (1) was identified as a compound specific to Z. jujuba. To establish a purity test of Jujube Seed in the JP against Z. mauritiana, we fractionated the extract of Z. mauritiana seeds and identified frangufoline (2) and oleanolic acid (4) as the marker compounds specific to Z. mauritiana. Thin-layer chromatography (TLC) and gas chromatography-mass spectrometry analyses revealed that the latter compound was useful for testing by TLC analysis. The established TLC conditions were as follows: chromatographic support, silica gel; developing solvent, n-hexane:EtOAc:HCOOH = 10:5:1; developing length, 7 cm; visualization, diluted sulfuric acid; R f value, 0.43 (oleanolic acid).

Количественное определениe рутина методом хроматографии в тонком слое сорбента

Разработана экономичная и экспрессная методика идентификации и количественного определения рутина методом хроматографии в тонком слое сорбента, которая может быть рекомендована к апробации и использованию в фармацевтической, косметологической и пищевой отраслях промышленности. Экспериментально подобраны и теоретически обоснованы оптимальные условия хроматографирования рутина в тонком слое сорбента с количественной интерпретацией данных ВЭТСХ на персональном компьютере.

Enantioselective analysis of naproxen using chiral molecular imprinting polymers based thin-layer chromatography

Abstract This paper describes a rational design and testing of molecularly imprinted polymers (MIPs) as chiral stationary phases of thin-layer chromatography (TLC) for enantiomeric purity of naproxen. Using D-naproxen as template, MIPs with particle size between 10~90 μm were prepared by precipitation polymerization in acetonitrile/methanol mixed solvent. The interactions between functional monomers and template were verified by UV absorption spectrometry. The morphology, particle size distribution and specific surface area of MIPs were also observed by scanning electron microscopy, particle size distribution meter and liquid nitrogen adsorption instrument, respectively. Binding capacities of MIPs had been studied by equilibrium binding assay. Preparation conditions of TLC and impact of acetic acid content on the separation of enantiomers were investigated. The results indicated that when acetic acid content was 4%, the racemates of templates were completely separated, and the chiral separation factor α was 1.58.

大麻関連試料の理化学的検査の迅速化

We previously developed a rapid method for morphological examination of cannabis and its related herbal products. In this study, general methods for chemical examination by thin layer chromatography (TLC) and gas chromatography/mass spectrometry (GC/MS) were improved. In TLC, a development using toluene-hexane-diethylamine (25:10:3, by vol.) at 40°C allowed sufficient separation of cannabinoids and rapid analysis. It was also confirmed that the TLC conditions were applicable to the discrimination of cannabinoids and the other components in herbs. In the pretreatment for GC/MS, using a 96-well plate and a multi-channel pipette allowed to prepare many analytical samples at the same time. In GC/MS, using a short and narrow bore capillary column allowed rapid analysis (ca. 10 min/sample). Based on the chemical examination method in this study and the morphological examination method that we previously developed, a rapid procedure to examine cannabis and its related herbal products was proposed. In the morphological and chemical examination methods that we developed, the total time required for morphological examination by microscopy, color test by Fast Blue B reagent, and chemical examination by TLC was approximately 3 h for 96 samples.

A REVERSED-PHASE TLC/SCANNING DENSITOMETRIC METHOD FOR THE ANALYSIS OF TOMATO, ORANGE, AND MARIGOLD COLORS IN FOOD

ABSTRACT In the present study, a thin-layer chromatography (TLC) method for the analysis of tomato, orange, and marigold colors in foods was developed. The colors were extracted from food samples with ethyl ether, and after the extract was evaporated, the residue was dissolved in methanol. For the analysis of the tomato color, after the addition of water to the methanol solution, it was then purified through a C18 cartridge before being subjected to the TLC analysis. With respect to the analyses of the orange and marigold colors, after adding 2 mL of a 5% sodium hydroxide–methanol solution to the methanol solution, the mixture was occasionally stirred, kept away from light, and then allowed to stand for 24 hours at room temperature. Subsequently, the pH of the mixture was adjusted to 4.5 or less using 1 mol/L hydrochloric acid. The mixture was then purified through a C18 cartridge before being subjected to the TLC analysis. The TLC conditions were as follows: plate, RP-18F254S (Art. 15389, E. Merck); solvent system, acetonitrile–acetone–n-hexane (11 : 7 : 2) and acetone–water (9 : 1). The visible absorption spectra of the colors were measured using scanning densitometry without isolation of the colors. In order to investigate the capability of the present method, 95 commercial foods (33 for the tomato color, 38 for the orange color, and 24 for the marigold color) were analyzed, and their chromatographic behaviors and spectra were observed. The separation and obtained spectra were not affected by coexisting substances in the foods and the spots always gave the same Rf values and spectra as the standard with good reproducibility. The present method is considered to be useful for the rapid analysis of the tomato color, orange color, and marigold color in foods.

Determination of Diketopiperazine Alkaloids of the Roquefortine Group by UV Spectroscopy, Thin-Layer Chromatography, and High-Performance Liquid Chromatography

Electronic absorption spectra and the chromatographic retention of six alkaloids from the roquefortine group under conditions of thin-layer chromatography and high-performance liquid chromatography were examined. The applicability of these techniques to the identification of diketopiperazine alkaloids in culture liquid extracts was demonstrated using the fungusPenicillium chrysogenumVKM F-1987 as an example.

Behaviour of Humic Acids on Fe(III)-Impregnated Silica gel Compared with Model Substances

Abstract Behaviour of humic acids under the conditions of thin layer chromatography on Fe(lll)-impregnated silica gel with tap water as developer was investigated. Compounds having similar functional groups as the humic material were compared and conclusions extended to natural conditions were drawn.

Improved phosphotyrosine analysis by TLC and HPLC

We describe here the conditions of thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC) to improve the analytical method of phosphotyrosine (P-Tyr) in biological sample. TLC was performed on silica plate with the mixture of propanol and water (2.1∶1 v/v) as a mobile phase and R1 values were 0.42, 0.39 and 0.33 for phosphotyrosine, phosphothreonine and phosphoserine, respectively. HPLC was performed on NH2 column with a mobile phase of potassium biphosphate solution by UV detection at 192 nm. The optimum condition of HPLC was obtained at 0.01 M, pH 4.5 with a clear separation within 12 min. These procedures have been applied to the analysis of phosphotyrosine obtained from tyrosine-phosphorylated enolase. Both TLC and HPLC methods were suitable to analyze tyrosine-phosphorylated protein without being affected by contaminants from hydrolysates.

Separation and estimation of anionic surfactants by thin layer chromatography: i. Mixtures of sodium dodecyl benzenesulfonate, sodium dodecyl sulfate and sodium dodecanesulfonate

AbstractThe conditions for separation and quantitative determination of anionic surfactants such as sodium dodecylbenzenesulfonate, sodium dodecanesulfonate and sodium dodecyl sulfate by thin layer chromatography (TLC) were investigated. Analytical results for mixtures of 2 or 3 components under optimal TLC conditions were in satisfactory agreement with known values. The absolute errors and variation coefficients both were within ca. 4%.

The effect of layer thickness on RF values in thin layer chromatography

Results quoted in the literature suggest that there is generally an increase of R F value with layer thickness under ordinary conditions of thin layer chromatography. Under ideal conditions it appears that R F values are independent of layer thickness, as expected theoretically. Several factors that can operate to cause R F to vary with layer thickness are discussed.

Beobachtungen an lösungsmitteln, trägern und trennkammern

Investigation of solvensts, adsorbents and separation chambers The behaviour and the interactions of the volatile and hydrophilic solvent components, such as methanol, are investigated under various conditions of thin-layer chromatography. Some properties of adsorbents and commercial plates are also discussed.