Thin-layer Chromatographic Detection Research Articles

Colorimetric analysis platform based on thin layer chromatography for monitoring gluten cross-contamination in food industry

Monitoring of the accidental presence of gluten (Glu), resulting from cross-contamination, is imperative in different industries, in particular food industry. The objective of this study was the development of an analytical platform utilizing thin-layer chromatography (TLC) with colorimetric read-out for making binary (yes/no) decisions on surfaces and/or point of these industries. The composition of the extractive phase was optimized with commercial products used in cleaning processing lines. Subsequently, an exploration of TLC separation and detection was undertaken. CN-modified nanosilica plates and 30:70 acetonitrile:water were used to achieve a selective signal for Glu residues. The study of the detection performance showed that both spectroscopic measurement and image analysis were resulted in satisfactory results for quantitate analysis (RSD = 5 %, LOD = 0.12 mg). The practical application of the proposed methodology on surfaces of the food processing lines. This work demonstrated the operational feasibility in detecting gluten cross-contaminations within the food processing industry.

Detection of azadirachtin from neem kernels using a paper-based sandwich sensor.

In this work, a microfluidic paper-based analytical device (μPAD) was developed to detect the biopesticide azadirachtin (Aza) through a colorimetric assay. High precision estimation of Aza is classically carried out using high performance liquid chromatography (HPLC), which requires highly skilled personnel. Acidified vanillin is a commonly used colorimetric indicator in thin layer chromatography for detection of various phytochemicals. However, the assay involves concentrated acid, which limits the choice of paper substrates for paper-based sensors and raises safety concerns. In this work, we show how the assay can be extended from the liquid phase to a paper substrate. Glass microfiber (GMF) filter paper was found to be suitable paper as it was acid resistant; besides, its hydrophilicity enabled smooth flow of reagents. A microfluidic paper-based sensor (μPAD) was developed by sandwiching 5 mm sized GMF dots between two parafilm sheets. We demonstrate the use of colorimetric assay on the μPAD for on-site detection of Aza in neem kernels. The magenta color developed upon the reaction of acidified vanillin with Aza was captured using a smart-phone and analysed using RGB levels in the image. Calibration was established using neem kernel extract of known concentration. Linearity was seen in the concentration range of 5 to 25 mg L-1 Aza. A limit of detection of 2.3 mg L-1 was obtained using this method. The colorimetric assay showed a relative recovery of >85% when compared with the values obtained from HPLC. The stability of the reagents on the GMF sensor was investigated to understand the storage conditions and shelf life of the reagents and sensor. The present work demonstrates the development of a portable sensor for on-site detection of phytochemicals that can be an integral part of the agricultural supply chain.

An improved extraction and nonradioactive thin-layer chromatography detection method of mycolic acid

Mycolic acids (MAs), i.e. 2-alkyl, 3-hydroxy long-chain fatty acids, are the hallmark of the cell envelope of Mycobacterium tuberculosis and are related with antibiotic resistance and host immune escape. Nowadays, they've become hot target of new anti-tuberculosis drugs. There are two main methods to detect MAs, 14C metabolic labeling thin-layer chromatography (TLC) and liquid chromatograph mass spectrometer (LC-MS). However, the user qualification of 14C or the lack of standards for LC-MS hampered the easy use of this method. TLC is a common way to analyze chemical substance and can be used to analyze MAs. In this study, we used tetrabutylammonium hydroxide and methyl iodide to hydrolyze and formylate MAs from mycobacterium cell wall. Subsequently, we used diethyl ether to extract methyl mycolate. By this method, we can easily extract and analyze MA in regular biological labs. The results demonstrated that this method could be used to compare MAs of different mycobacterium in different growth phases, MAs of mycobacteria treated by anti-tuberculosis drugs or MAs of mycobacterium mutants. Therefore, we can use this method as an initial validation for the changes of MAs in researches such as new drug screening without using radioisotope or when the standards are not available.

Caffeic acid N-[3,5-bis(trifluoromethyl)phenyl] amide as a non-steroidal inhibitor for steroid 5α-reductase type 1 using a human keratinocyte cell-based assay and molecular dynamics

Caffeic acid derivatives containing amide moieties similar to those of finasteride and dutasteride were synthesized. An in vitro inhibitory activity evaluation of caffeic acid (1) and its amide derivatives (2 − 4) against the steroid 5α-reductase type 1 (SRD5A1) produced by human keratinocyte cells coupled with the non-radioactive high-performance thin-layer chromatography detection revealed that caffeic acid N-[3,5-bis(trifluoromethyl)phenyl] amide (4) was a promising non-steroidal suppressor, with a half-maximal inhibitory concentration (IC50) of 1.44 ± 0.13 µM and relatively low cytotoxicity with an IC50 of 29.99 ± 8.69 µM. The regulatory role of compound 4 against SRD5A1 involved both suppression of SRD5A1 expression and mixed mode SRD5A1 inhibition. The Ki value of compound 4 was 2.382 µM based on the whole-cell kinetic studies under specific conditions. Molecular docking and molecular dynamics simulations with AlphaFold generated the human SRD5A1 structure and confirmed the stability of compound 4 at the SRD5A1 catalytic site with greater interactions, including hydrogen bonding of the key M119 amino-acid residue than those of finasteride and dutasteride. Thus, compound 4 shows the potential for further development as an SRD5A1 suppressor for androgenic alopecia treatment.

β-Cyclodextrin-based chiral nanocomposite for thin-layer chromatographic detection of enantiomers of fluoxetine

β-Cyclodextrin-based chiral nanocomposite for thin-layer chromatographic detection of enantiomers of fluoxetine

Thin Layer Chromatographic Detection of Dicyclomine Hydrochloride Extracted from Whole Blood

Dicyclomine Hydrochloride belongs to a class of synthetic anticholinergics. It is regularly used for the treatment of spasmodic pain, spasmodic dysmenorrhea, and renal, ureteric and biliary colic. This drug is easily available in the market and can be abused for suicidal purposes. Its unsupervised use can lead to abuse, accidental poisoning along with the occasional report of intentional suicidal poisoning. Hence its analysis is very important for medicolegal purposes.Biological samples of choice for routine qualitative analysis of drug is liver, stomach content, whole blood, and urine. Routine toxicological procedures employ highly sophisticated state of the art instruments like High Performance Liquid Chromatography (HPLC), Gas Liquid Chromatography (GLC) and Liquid ChromatographyMass Spectrophotometer (LC-MS) for the estimation of drug from biological and non-biological matrices but the present paper deals with an attempt to analyse the drug extracted from the biological sample of interest using a simple, feasible and optimal method with the help of Thin Layer Chromatography (TLC). The study involves the extraction of the drug from matrix (blood), using Liquid-Liquid Extraction method of alkaline plasma followed by its separation and identification by Thin Layer Chromatography. The present paper describes a simple, economical, reproducible sample extraction, clean-up and detection method that can be easily attempted in a laboratory. Keywords: Whole blood; Dicyclomine; Extraction; Analysis; Drug.

A rapid reversed-phase thin layer chromatographic detection protocol for adulteration in some edible fats and oils food formulation

The problems of adulteration in the vegetable oil and fat have been the major draw backs in the food products formulation, in spite of the various adulteration detection methods in different applications that have been reported. However, the detection tools that can be fast and reliable for the routine analysis necessitated the current work. The two groups of three different samples: vegetable fat containing sample (Blue Band, Golden Penny, La Prairie Classic) and animal fat containing samples (Kell Salad Cream, Crosse & Blackwell and Nola) was used for the purity check using the reversed phased - thin layer chromatographic (RPTLC) method of analysis were developed. The average Rf ratio of 0.95 and 0.92, found for the vegetable and animal fat groups were reported, respectively. The Rf = 0.03 difference between the two groups indicated the presence of sistosterol (plant sterol) and cholesterol (animal sterol), an improvement over color detection methods to screen oils and fats to ascertain purity.
 Keywords: Sistosterol, Cholesterol, Adulteration, Animal fat, Vegetable oil

Application of smartphone in detection of thin-layer chromatography: Case of salvia miltiorrhiza

In this work, a smartphone-based device was constructed for thin-layer chromatography (TLC) detection and semi-quantitative analysis of the components of Salvia miltiorrhiza. The key construction and shooting parameters were investigated by the relative peak area and signal-to-noise ratio. The best conditions were as follows: shooting height, 17 cm; angle between the UV lamp and TLC plate, 58°; exposure compensation, 0~0.2 EV; and shutter speed under daylight and UV 365 nm, 1/50 s and 1/5 s, respectively. These ideal conditions could be replicated by smartphones from different brands with different versions of software. With good precision, repeatability and stability, the developed device was used for the semi-quantitative analysis of salvianolic acid B, rosmarinic acid, cryptotanshinone, tanshinone I, tanshinone IIA, and miltirone in the TLC analysis of 10 batches of S. miltiorrhiza. The results were compared with those obtained by a TLC densitometric scanner and two common types of image processing software, i.e., Gelanalyzer and ImageJ. Except for salvianolic acid B in the TLC densitometric scanner, all results were not significantly different among these methods, which suggested that smartphones might be a useful tool for the quality control of traditional Chinese medicines.

Development method of high-performance thin-layer chromatographic detection of synthetic organophosphate insecticide profenofos in visceral samples

The synthetic pesticide profenofos is a member of organophosphate insecticide compounds. It is an important synthetic insecticide and has a diversified role in agriculture. During the last decade, profenofos was frequently misused in homicidal, accidental, and suicidal poisoning cases. Since then, a large number of visceral samples have been received for toxicological chemical analysis. Thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) were the preferred methods of choice. By observing the results of toxicological analyses, the conclusion can be drawn that profenofos plays a major role in poisioning. So we developed a new reagent which was found to be a selective spray reagent for profenofos in routine analysis by HPTLC.

Aktivitas Sitotoksik Ekstrak Etanol, Fraksi Etanol-Air, Etil Asetat serta N-Heksana Buah Pare (Momordica charantia) pada Sel MCF-7 secara In-Vitro

Bitter melon is one of fruit that have pharmacological effects such as an anticancer which potentially active on breast cancer cell MCF-7. The aim of this study was to compare the potential cytotoxic activity between ethanol extract of pare and the results of ethanol, ethyl acetate and n-hexane fraction on MCF-7 breast cancer cells and to determine the class of compounds contained in each sample. Pare’s powder was extracted with maceration method in 80% ethanol solvent and then continue to fractionated in 96% ethanol, ethyl acetate and hexane solvent. Extract and fractions were continue to cytotoxic assay using the MTT assay method. Cytotoxic test results showed that the ethanolic extract had no potential cytotoxic activity. Ethyl acetate fraction with the highest concentration 16 µg/mL has the highest potential inhibition 43,87% on MCF-7 cells population. Extract and fractions than continue to phytochemical screening with Thin Layer Chromatography (TLC) method. TLC detection showed that n-hexane fraction contained more compound groups than ethanolic extract, ethanolic-water fraction and ethyl acetate fraction.

Polyphasic Assessment of Aflatoxin Production Potential in Selected Aspergilli.

This study investigated the aflatoxin production potentials of selected fungi using a polyphasic approach. Internally transcribed spacer region of the fungi was amplified using the polymerase chain reaction. Forty-five Aspergillus strains were further assessed for aflatoxin production using the conventional methods such as growth on yeast extract sucrose, β-cyclodextrin neutral red desiccated coconut agar (β-CNRDCA); expression of the aflatoxin regulatory genes and the use of both thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). A large proportion (82.22%) of the isolates harbored the Nor-1 gene while 55.56%, 68.89%, and 80% possessed the ver-1, omt-A, and aflR genes, respectively. All 100% the isolates harbored the aflJ gene. Twenty-three isolates were positive for aflatoxin production based on the yeast extract sucrose medium (YES) test; ammonium vapor test (51%), yellow pigment production (75.5%), and β-CNRDCA tests; and blue/green fluorescence (57.7%). Based on TLC detection 42.2% produced aflatoxins while in the HPLC, total aflatoxin (AFTOT) production concentrations ranged from 6.77–71,453 µg/g. Detectable aflatoxin B1 (AFB1) concentrations obtained from the HPLC ranged between 3.76 and 70,288 µg/g; 6.77 and 242.50 µg/g for aflatoxin B2 (AFB2); 1.87 and 745.30 µg/g for aflatoxin G1 (AFG1); and 1.67 and 768.52 µg/g for aflatoxin G2 (AFG2). AFTOT contamination levels were higher than European Union tolerable limits (4 µg/kg). The regression coefficient was one (R2 = 1) while significant differences exist in the aflatoxin concentrations of Aspergillus (p ≤ 0.05). This study reports the potentials of Aspergillus oryzae previously known as a non-aflatoxin producer to produce AFG1, AFG2, AFB1, and AFB2 toxins. Aspergillus species in feedlots of animals reared for food are capable of producing aflatoxins which could pose hazards to health.

A Novel Chromogenic Spray Reagent for Thin-Layer Chromatographic Analysis of Paraquat and Design of an Ultra-Low-Cost Sensor for On-The-Field Detection of Viologens

A novel spot test and a novel chromogenic reagent are reported for thin-layer chromatographic detection and identification of the popular but highly toxic herbicide paraquat. Alkaline phenylhydrazine instantly reduces paraquat to an intense purple radical cation. The reaction is sensitive and highly specific to viologens, i.e., paraquat and diquat herbicide. Surprisingly, 2,4-dinitrophenylhydrazine does not have reaction in this color test. Reaction of the two drugs containing related functional groups is also studied to check whether these drugs also react similarly. Based on this novel color reaction, a simple, ultra-low-cost filter-paper-based sensor is also designed for direct field testing of suspicious forensic samples like vomits and bottle of poison, which are usually found at a crime scene. The possible applications of the reported reaction in the field of organic qualitative analysis, clinical chemistry, and forensic medicine are also discussed.

TLC Detection and Theoretical Structure Elucidation of Nitrogen Containing Compounds with Cobalt Thiocyanate

Forensic Science Laboratories of Maharashtra state, India, receives large number of poisoning cases pertaining pharmaceutical drug overdose and illicit drugs. Thin-layer chromatography (TLC) is a method of choice for chemical analysis due to large number of biological/non-biological samples received. This study reports cobalt thiocyanate reagent to be more effective in detecting nitrogen containing drugs like chloroquine, chlorphenamine, mephedrone, cocaine, ketamine in addition to pesticides like pretilachlor, thiamethoxam and tebuconazole when aided by TLC. These compounds (except for ketamine which gives purple) form blue color when reacted with cobalt thiocyanate due to the formation of tetrahedral cobalt (II) thiocyanate complex. However due to lack of experimental evidences, quantum mechanical calculations have been employed to deduce their structures. The theoretical structure elucidation methodology is based on examining crystal structures of ligands having similar structural relationship to present drug molecules and investigating reactive sites in each drug molecule via. Molecular Electrostatic Potential.

A study of a termite chemical defense fluid compound of Macrotermes carbonarius

Termite caste consists of reproductive individuals, worker, and soldier. Soldiers defend their colony by physical and chemical means. For Macrotermes carbonarius, they use mandibles as their physical defense and defense fluid for chemical defense. Their defense fluid plays a vital role to defend their caste from any harm from another insect such as ants. This can be related to the fact that M. carbonarius is an urban pest that has many threats to their colony. However, less study had been focused on their defense fluid composition and how the compounds function. This study was carried out to determine the compound present in the defense fluid of M. carbonarius. The methods used in this study were thin layer chromatography (TLC) and gas chromatography mass spectrometry (GCMS). TLC detection indicated presence of three unknown compounds and one important compound. GCMS analysis had confirmed the compound as lauric acid methyl esther that was obtained from the TLC. This information would contribute to fill up the research gap and assist in better understanding the compound composition found in M. carbonarius defense fluid to protect their colony, an update on the defense fluid research since 1979 by Prestwich.

New chromogenic reagent for high-performance thin-layer chromatographic detection of organophosphorus insecticide monocrotophos in biological materials

New chromogenic reagent for high-performance thin-layer chromatographic detection of organophosphorus insecticide monocrotophos in biological materials

Use of diphenylamine reagent for high-performance thin-layer chromatographic detection of organochloro insecticide endosulfan in biological samples

Use of diphenylamine reagent for high-performance thin-layer chromatographic detection of organochloro insecticide endosulfan in biological samples

Use of 1,2-napthoquinone-4-sulfonate (folinʼs reagent) for the thin-layer chromatographic detection of p-Phenylenediamine

Use of 1,2-napthoquinone-4-sulfonate (folinʼs reagent) for the thin-layer chromatographic detection of <i>p</i>-Phenylenediamine

Screening Urinary Methylmalonic Acid by Thin Layer chromatography in children with Suspected inborn Errors of Metabolism

Introduction: Inborn errors of metabolism (IEM) are a group of disorders due to deficiency of the enzyme. The symptoms are due to the accumulation of toxic substances or deficiency of products. Organic aciduria is a group of IEM which is due to errors in the metabolism of branched chain amino acids and lysine. Studies showed that methylmalonicaciduria (MMA), glutaric aciduria, branched chain aminoacidurias are more common organicaciduria. Many methods are used for detection of methylmalonic acid some of them are not affordable by developing countries. Objective: To study the role of thin layer chromatography in detection of methylmalonic aciduria in children with suspected inborn errors of metabolism. Method: Untimed urine samples were collected from 385 children with clinically suspected inborn errors of metabolism and analyzed for MMA by colour reaction and thin layer chromatography (TLC). Bromocresol green and Ortho dianisidine were the dye used for spotting MMA on TLC. Result: Among 385 children who presented with developmental delay, seizures, hypotonia were worked up for underlying inborn errors of metabolism. Of them 52 children showed positive result for MMA by thin layer chromatography. Discussion: Significant number of children showed positive result for MMA by thin layer chromatography hence the method may be useful for screening methylmalonicaciduria. Further studies required to confirm the diagnosis

Use of cupric ferrocyanide reagent for the thin-layer chromatographic detection of organophosphate insecticide profenophos

Use of cupric ferrocyanide reagent for the thin-layer chromatographic detection of organophosphate insecticide profenophos

Detection of 2-Alkyl-4-Quinolones Using Biosensors.

2-Alkyl-4-quinolones (AQs) such as 2-heptyl-3-hydroxy-4-quinolone (PQS) and 2-heptyl-4-hydroxyquinoline (HHQ) are quorum-sensing signal molecules. Here we describe two methods for AQ detection and quantification that employ thin-layer chromatography (TLC) and microtiter plate assays in combination with a lux-based Pseudomonas aeruginosa AQ biosensor strain. For TLC detection, organic solvent extracts of bacterial cells or spent culture supernatants are chromatographed on TLC plates, which are then dried and overlaid with the AQ biosensor. After detection by the bioreporter, AQs appear as both luminescent and green (from pyocyanin) spots. For the microtiter assay, either spent bacterial culture supernatants or extracts are added to a growth medium containing the AQ biosensor. Light output by the bioreporter correlates with the AQ content of the sample. The assays described are simple to perform, do not require sophisticated instrumentation, and are highly amenable to screening large numbers of bacterial samples.