Thin-layer Chromatographic Procedure Research Articles

Quantification of isoniazid in tablets for tuberculosis treatment by thin layer chromatography with smartphone image capture and ImageJ analysis

Isoniazid (INH) oral dosage tablets are an antibacterial medication commonly used to treat tuberculosis (TB). While this medication can be highly effective in managing and curing TB, manufacturing of poor-quality INH products can lead to the proliferation of drug resistant TB strains – particularly in low- and middle-income countries (LMICs). It is therefore imperative that reliable methods of quality screening of INH tablets are available for areas that have limited access to traditional testing resources. In this study, a thin layer chromatography (TLC) procedure for INH identification is validated in conjunction with smartphone image capture and open-source image analysis software to provide an alternative quantitative quality screening method for INH tablets, primarily intended for applications in LMICs. The method assessed two individual tablet formulations: 100 mg and 300 mg INH, each from different manufacturers. The modified TLC procedure used for the study was based on a validated high-performance thin layer chromatography method for the determination of rifampicin, INH, and pyrazinamide in a fixed dosage tablet. Quantitation was performed by visualizing and photographing TLC plates using a UV lamp, 3D-printed light box, and Apple iPhone SE 2nd Generation (2020) and Google Pixel 4a (5G) smartphones, and images were then analyzed using ImageJ Fiji software. The processed images’ pixel data were used to create a calibration curve from the INH standard spots to determine the concentration of an INH sample spot. Linearity, range, accuracy, repeatability and intermediate precision, specificity, and robustness were evaluated. The method was linear in the range of 0.150–0.450 mg mL−1, with coefficients of determination ≥0.98. The overall accuracy of the method was 99.2 % with an RSD of 2.4 % for image sets captured using the Apple iPhone, and 99.7 % with an RSD of 3.3 % for image sets captured using the Google Pixel. The pooled standard deviations for repeatability and intermediate precision were 2.27 % and 3.25 %, respectively, for the iPhone and 2.62 % and 3.74 %, respectively, for the Pixel. The method demonstrated sufficient specificity and robustness. The results of this method validation suggest that this procedure may provide a feasible alternative to traditional testing as a low-cost means of quality screening in LMICs.

Screening method for the analysis of Rhodamine B in chili powder

Rhodamine B is a synthetic dye known to enhance the visual appearance of chili powder. Due to its toxicity and carcinogenicity, chromatographic methods have been developed to monitor its presence in adulterated chili powder, but their assays are laborious, time consuming and expensive for screening purposes. The present studies propose an alternative for screening Rhodamine B in chili powder samples. The method combines thin layer chromatography (TLC) to solid surface room-temperature fluorescence spectroscopy. The scrape-dissolution procedure common to the instrumental analysis of TLC procedures was replaced with a fiber optic probe coupled to a commercial spectrofluorometer. The determination of Rhodamine B on the chromatographic plate is based on its retardation factor and maximum excitation and emission wavelengths. The limit of detection (1.9 ng.mL−1) and the limit of quantitation (5.2 ng.mL−1) are well below the usual contamination of Rhodamine B in adulterated foods.

A Thin-Layer Chromatography Method for the Determination of Essential Oil Components in Anise and Fennel Fruits

Scientific relevance. The State Pharmacopoeia of the Russian Federation, edition XIV, requires the identification of aniseed and fennel fruits by the same thin-layer chromatography (TLC) procedure using Sudan III and menthol. These markers are neither specific nor related to the therapeutic effects of these herbal drug products. The visual interpretation of chromatograms is complicated because of the low intensity of adsorption zones. Moreover, the corresponding compendial monographs do not characterise the adsorption zones specific to each of the plants. The most abundant component in aniseed and fennel essential oils, trans-anethole, would make a better reference standard from a methodological point of view.Aim. This study aimed to optimise the TLC procedure for essential oil determination in herbal drugs and herbal medicinal products of aniseed and fennel fruits and subsequently recommend it for inclusion in the relevant compendial monographs.Materials and methods. The study examined samples from several batches of herbal drugs, including aniseed and common fennel fruits, sourced from Russian manufacturers. The reference standards comprised commercial reagents, including trans-anethole, anise oil, and linalool, as well as fresh essential oils that had been steam distilled from the test samples in a Clevenger apparatus. The study was conducted by TLC. Sample preparation involved using Merck aluminium TLC plates, an IKA KS-501 digital orbital shaker, and a CAMAG Linomat 5 semi-automatic sample application system. The authors heated the plates in either a Binder ED53 drying oven or a CAMAG TLC plate heater. For visualisation, the authors selected a CAMAG TLC VISUALIZER 2 UV imaging and documentation system.Results. The informational and experimental research showed the feasibility of using trans-anethole as an identification reference standard for common anise and fennel fruits. The authors selected the optimal solvent for extracting active substances from the test products (hexane) and a detection reagent for improving visual evaluation of the chromatograms (1% sulfovanillin). The authors established characteristic adsorption zones for differentiating between aniseed and fennel fruits.Conclusions. The optimised procedure identifies the main components in the essential oils of aniseed and fennel fruits with a specific reference standard. This procedure may be recommended for inclusion in draft monographs for the corresponding herbal medicinal products.

Physicochemical and In-Vitro Bioequivalence Analysis of Some Oral Solid-Dosage Metronidazole Formulations

Background: Metronidazole is a 5-nitroimidazole antimicrobial agent, widely used in the treatment of infections caused by Trichomonas vaginalis, such as trichomoniasis, pseudomembranous colitis, and symptomatic amoebiasis resulting from anaerobic bacteria. Aim: The study aimed to qualitatively determine the quality and in-vitrobioequivalence of selected brands of metronidazole tablet dosage forms used in the management of infectious diseases. Method: The British Pharmacopeia (BP) Standard methods were followed to conduct weight uniformity, disintegration time, hardness, and friability tests. For the Thin Layer Chromatographic (TLC) Fingerprinting, 0.1g of the brands were weighed and dissolved ins 50 ml methanol (50%). The stock solution was prepared using 50% methanol and 50% dichloromethane and was transferred into the Chamber and allowed to stand for about 45 minutes; the plates were viewed under UV-VIS Spectrophotometer at 365 nm. Result: All the brands complied with the acceptable friability limit, and disintegrated within the appropriate time of not more than 15 minutes as specified in BP, for uncoated tablets. The tablets were found to have a dissolution profile of at least 70% of the active ingredient taken at 45 minutes from the dissolution medium as stated in BP. The analysis showed the consistency of concentration of the selected tablet brands with the label claims. Conclusion: The TLC procedure used in the analysis was accurate and precise, no interference was observed from the excipients. Thus, this method can be used in the routine quality control of metronidazole solid dosage form.

Analyses of Bioactive Compounds of Pegagan (Centella Asiatica (L.) Urb) from Samosir – Indonesia Accession

Centella Asiatica or Pegagan is classified as one of the wild plants that has not been domesticated. The excessive usage of this plant in traditional and modern medicinal applications threatens its population and sustainability. Thus, to preserve the plant and supply the high request of this plant in agromedicinal industry, studies concerned with the growth and bioactive compounds of Pegagan cultivated under commercial field conditions are urgently needed. This study purposed to examine the bioactive components of Pegagan (especially in leaves and roots) under field conditions, including asiaticoside, madeccasoside, and Asiatic acid. The Pegagan was harvested weeks after planting (WAP). The wet and dry weights of the leaves and roots were weighted and subsequently measured for their centelloside compounds by Thin-Layer Chromatography (TLC) procedure. The results revealed that the resulting asiaticoside content in the roots (1.25%) was higher than in the leaves (0.88%). The same results were achieved for the madecassoside content where the madecasosside content in the roots was 2.23%, while the content in the leaves was 2.11%. However, contrarily, the Asiatic acid compound in the leaves was 1.10% higher than the content in the roots (0.60%). It might be attributed to a longer period of field cultivation of Pegagan that delivered adequate time for the plant to alter Asiatic acid to asiaticoside and madecasosside at a later developmental growth. Moreover, these discoveries are advantageous in defining the most proper harvest time for commercial field cultivation of Pegagan to yield the highest amount of certain centelloside compounds.

Quantitative thin layer chromatography for the determination of medroxyprogesterone acetate using a smartphone and open-source image analysis

Intramuscular medroxyprogesterone acetate (MPA) products are commonly used to treat endometriosis and are the most widely used injectable contraceptives worldwide. Therefore, dependable quality screening of MPA injectables is a crucial measure necessary for ensuring that consumers are provided with safe and effective medications. Here, a thin-layer chromatography (TLC) method for MPA identification is combined with image analysis using a smartphone, 3D-printed light box, and open-source ImageJ software. The method's validation included two brands of MPA injectables, both at 150 mg mL−1 dosage. The TLC procedure used was based on the identity test found in The International Pharmacopoeia's Medroxyprogesterone injection monograph. Spots produced on the TLC plates were then photographed using a smartphone camera and quantified using ImageJ's image analysis software. The pixel data collected from each plate's standard spots were compared to the data generated from its sample spots. Data sets collected across multiple TLC plates and numerous days of method performance were evaluated to assess linearity, accuracy, precision, specificity, and robustness. Across the range of 75–125% of the target concentration, the method was found to have linearity of standard spots (with R2 generally greater than 0.99), overall accuracy of 101.0% (4.1% RSD), repeatability pooled standard deviation of 2.44%, intermediate precision pooled standard deviation of 3.68%, and observed demonstration of specificity and robustness. In low and middle-income countries (LMICs), quality screening of pharmaceutical products like MPA injectables can be challenging when testing resources are expensive, difficult to procure, or complex to utilize. The results of the TLC/ImageJ method validation suggest that this simple procedure that requires minimal resources may serve as a viable option for reliable quality screening of MPA levels in injectable suspensions.

Comparative Hypolipidemic Evaluation of Aframomum Melegueta Seeds and Moringa Oleifera Leaves

Aims: The study was carried out to compare the lipid lowering effects of both crude drugs as well as deducing the extracts with the best lipid lowering property; and the fractions.
 Study Design: The research was conducted with an experimental design solely based on laboratory trials which involved the use of ninety-six (96) male albino wistar rats to compare the hypolipidemic effects of both crude drugs and respective fractions.
 Place and Duration of Study: Department of Pharmacognosy Laboratory, Faculty of Pharmacy, Delta State University, Abraka, Nigeria and Department of Pharmacology and Toxicology Laboratory, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Agulu, Anambra State, Nigeria. The research was carried out from March to August, 2021.
 Methodology: Ethanol extracts of both plants were prepared using soxhlet extraction. Each extract was then subjected to VLC fractionation using four solvents: n-hexane, chloroform, ethyl acetate and methanol. The fractions were bulked together after conducting thin layer chromatographic procedures and each extract was bulked into four fractions. The acute toxicity studies (LD50) of both extracts were determined in the rats using Lorke’s method. The crude extracts were screened for the presence and quantity of phytoconstituents using standard methods. The antilipidemic study was carried out using sixty-eight (68) rats randomized into seventeen (17) groups of four (4) animals each. Lipid profile was determined using spectrophotometer. Liver function tests and histology was also carried out using standard procedures.
 Results: Administration of various treatments (both crude extracts and fractions) evoked a significant (p<0.05) reduction of TC, TG, and LDL-C as well as significant (p<0.05) elevation of HDL-C when compared with the negative control. With a percentage serum lipid reduction of 45.11%TC, 48.23%TG, 63.39% LDL-C and 174.69% elevation of HDL-C, the group treated with the combination of 500 mg/kg Aframomum melegueta and 500 mg/kg Moringa oleifera produced the best hypolipidemic effect. This is closely followed by fraction MO4. Comparatively, Moringa oleifera extracts exerts a better antilipidemic effect than Aframomum melegueta seed extract. The liver function test showed that both plants has no toxic effect on the liver cells at doses of 250 mg/kg and 500 mg/kg, hence confirming the hepatoprotective effect of both crude drugs at the doses administered.
 Conclusion: In conclusion, results from this study suggests that ethanol extract of Moringa oleifera leaves is more effective than ethanol extract of Aframomum melegueta seeds as a hypolipidemic agent, however, combination of both crude drugs as a lipid lowering agent has proved to be more effective and reliable when compared to each crude drug administered independently.

Scientific-methodological approach for standardization of herbal raw materials and herbal preparations using high-performance thin-layer chromatography procedure

The high-performance thin-layer chromatography (HPTLC) is widely used for quality control of herbal raw materials (HRM).
 The purpose of this work were to define the problematic issues of quality control of herbal drugs in Ukraine and to develop a systematic scientific and methodological approach for the standardization of HRM and herbal drugs using the modern analytical technique of HPTLC: to optimize the existing and develop new methods of identification and determination of adulterations, and to develop alternative methods of quantitative determination, as well as studying of the stability.
 As a result of a critical evaluation of existing approaches for the standardization of HRM and herbal drugs of Ukraine’s flora, numerous problematic issues were identified, including the existence of a limited number of national monographs / national parts of monographs on the HRM of the Ukraine’s flora, which were not previously standardized by other pharmacopeias; non-consideration of Ukraine’s flora species (non-pharmacopoeial) in monographs on closely related species that introduced in the State Pharmacopoeia of Ukraine; lack of specificity of combinations of physical and chemical methods used for quality control; lack of specificity and reproducibility, the labor intensity of methods; necessity of usage of toxic solvents and precursors; use of different groups of markers and methods for the HRM and its finished product or HRM and its closely related species, etc. The proposed systematic scientific and methodological approach includes: a preliminary collection of information about the object of study, markers/groups of bioactive substances, approaches to its standardization; theoretical evaluation of existing techniques of quality control in monographs on HRM and experimental approbation using samples of domestic plants, determination of the need for optimization of existed methods by the parallel development of specific, reproducible and documented methods for identification, adulterations’ detection, quantification, and bio-detection (if necessary) by HPTLC method. The approach is illustrated with examples.
 The proposed approach can be used for comprehensive HPTLC analysis of domestic HRM and herbal drugs for research, prevention of adulteration, standardization of initial HRM and its finished product, pharmaceutical development, and stability studies.

Rapid TLC with Densitometry for Evaluation of Naproxen Stability

The purpose of the work was to develop such chromatographic conditions that allowed to separate as many naproxen degradation products as possible. In order to follow this process, thin-layer chromatography (TLC) coupled with densitometry and spectrodensitometry was used. A forced degradation study was performed using an ethanolic solution of naproxen spotted on silica gel plates, existing in the form of an aqueous solution at various pH values, and as solution prepared in saline and in hydrogen peroxide. Degradative effect of UV light on naproxen was watched in the context of naproxen spotted on plates precoated with silica gel and exposed to UV light, and also for its solution treated with UV light. However, the solution of naproxen prepared in water at pH ≈ 2.60 undergoes the largest changes as the results of its exposure to UV light during 10 h. Stressed samples of naproxen were analyzed by using a new and well validated TLC procedure including toluene (TOL)—acetone (ACE)—chloroform (CHL) (2:5:12, v/v/v) as mobile phase A and glacial acetic acid (AcOH)—n-hexane (Hex)—acetone (ACE)-(0.10:10:10, v/v/v) as mobile phase B. As the newly developed TLC-densitometric method can effectively separate the substances about pharmaceutical significance from products of its degradation, which are formed as a result of stress studies, is considered to be a good alternative and important tool in routine quality control and stability testing of naproxen in pharmaceutical formulations. These results indicate that proposed TLC-densitometric method is cost-effective, rapid, specific, accurate, and precise. This TLC procedure is comparable to HPLC and UPLC method in terms of detection the number of degradation products of naproxen. In addition, it realizes the criterion of linearity. A major advantage and novelty of proposed method is its low cost and ability to analyze examined drug and all degradation products simultaneously, including those which can be observed under intensive UV radiation exposure of naproxen solution which are not described by previous HPTLC studies available in the literature.

Chromatographic Profiles Analysis of Fruits ofCrataegusL. Genus by High-Performance Thin-Layer Chromatography

AbstractIt was known that hawthorn -CrataegusL. is a polymorphic genus. Two hawthorn species and their hybrids are included in the European Pharmacopoeia, twelve – in Ukrainian pharmacopoeia. Determination of chromatographic profiles of hawthorn fruits species native to Ukraine and other countries that are non-pharmacopoeial, but have sufficient plant raw material base, is essential for quality control of drugs.Aim.To analyze and compare the chromatographic profiles of fruits of 23CrataegusL. species on phenolic compounds, evaluated by means of high-performance thin-layer chromatography procedure (HPTLC), and determine the specific features of chromatographic fingerprints.Materials and Methods.A total of 39 samples of fruits of 23 hawthorn species that are native to Europe, Asia and North America, such asCrataegus monogyna,C. laevigata/C. oxyacantha, C. leiomonogyna,C. curvisepala,C. pseudokyrtostyla,C. fallacina,C. subrotunda,C. ambigua,C. pentagyna,C. sanguinea,C. chlorosarca,C. almaatensis,C.pseudoheterophyllasubsp.turkestanica,C. pinnatifida,C. pentagynasubsp.pseudomelanocarpa,C. punctata, C. pringlei, C. festiva,C. douglasii, C. holmesiana,C. submollis,C. flabellata,C. canadensiswere investigated. The analysis has been done following the TLC method from European Pharmacopeia modified into HPTLC, using automated HPTLC herbal system (CAMAG, Switzerland).The resultshave shown that chromatographic profiles of phenolic constituents of nineCrataegusL. species of Europe, both pharmacopoeial and non-pharmacopoeial, were quite similar, despite the significant morphological distinctions. The chromatographical profiles of three species of Asia were similar to the pharmacopoeial species; three other species looked different and had specific marker zones. In addition, eightCrataegusL. species of North America had specific markers helping for discriminative analysis from pharmacopoeial species.Conclusion.The findings could help to identify the possible adulterations and prevent the falsification of finished products. The results will be taken into consideration during revision of the Ukrainian national pharmacopoeial monograph for hawthorn fruits.

Quantification of two biomarker compounds by a validated high-performance thin-layer chromatographic method from different extracts of Pluchea dioscoridis growing in Saudi Arabia

The aim of this study was to develop a validated high-performance thin-layer chromatography (HPTLC) procedure for resolution of chemical constituents and identification and quantification of two selected natural anticancer compounds, stigmasterol (PD-1) and cinnamic acid (PD-2), in Pluchea dioscoridis chloroform fraction (PDCF). The chromatographic estimations were conducted on normal HPTLC (20 cm × 10 cm glass-backed silica gel 60 F254) plates with chloroform-methanol-acetic acid (93:5:2, V/V) used as the mobile phase. para-Anisaldehyde was used for the derivatization of the developed plate, and compact spots were scanned at λmax = 513 nm. A well resolved, compact, and intense peaks of PD-1 and PD-2 were recorded at RF = 0.57 and 0.19, respectively. The proposed analytical method for both biomarker compounds was found to be handy, simple, precise, linear (%RSD = 1.03–1.45), accurate (98.91–99.14%), reliable, and sensitive for the analysis of both biomarkers. The LOD/LOQ (ng) for PD-1 and PD-2 were recorded as 38.73/117.37 and 42.58/129.04, respectively, in the linearity range of 200–1400 ng per spot. The obtained result showed maximum quantities of PD-1 and PD-2 (5.36 and 16.98 μg mg-1, respectively). The developed HPTLC was found to be suitable for the routine analysis of these 2 biomarkers in the chloroform fraction of Pluchea dioscoridis and can be further employed in the process quality control of herbal formulations containing the said biomarkers.

A Rapid and Simple TLC-Densitometric Method for Assay of Clobetasol Propionate in Topical Solution.

A rapid, simple to use and low-cost thin-layer chromatographic procedure in normal phase system with densitometric detection at 246 nm was carefully validated according to the International Conference on Harmonisation (ICH) guidelines for assay of clobetasol propionate in topical solution containing clobetasol propionate in quantity 0.50 mg/mL. The adopted thin-layer chromatographic (TLC)-densitometric procedure could effectively separate clobetasol propionate from its related compound, namely clobetasol. It is linear for clobetasol propionate in the range of 0.188 ÷ 5 µg/spot. The limit of detection (LOD) and limit of quantification (LOQ) value is 0.061 and 0.186 µg/spot, respectively. Accuracy of proposed procedure was evaluated by recovery test. The mean recovery of studied clobetasol propionate ranges from 98.7 to 101.0%. The coefficient of variation (CV, %) obtained during intra-day and inter-day studies, which was less than 2% (0.40 ÷ 1.17%), confirms the precision of described method. The assay value of clobetasol propionate is consistent with the pharmacopoeial requirements. In conclusion, it can be suitable as a simple and economic procedure for routine quality control laboratories of clobetasol propionate in topical solution.

Development of new procedures for the detection and separation of salicylic acid and acetylsalicylic acid using thin-layer chromatography with densitometry

Salicylic acid and its derivatives, such as acetylsalicylic acid, are commonly used non-steroidal anti-inflammatory agents. In the present work, different sorbents as the stationary phases and also various manners of detection using well known and new visualizing reagents have been tested for the separation and detection of the two compounds by adsorption and also partition thin-layer chromatography (TLC) with densitometry. Densitometric and spectrodensitometric analysis was used to evaluate the detectability of the examined compounds using the newly developed TLC procedures. Of all the applied manners of detection, the most universal to distinguish acetylsalicylic acid from salicylic acid in normal- as well as in reversed-phase TLC system is the use of methanolic solution of FeCl3 and CoCl2. The densitograms obtained under these conditions show symmetric and also by adsorption well separated TLC peaks of both compounds. The shape of all absorption bands of acetylsalicylic acid (ASA) and 2-hydroxybenzoic ...

Use of a model process for transfer of minilab TLC screening methods for quinine sulfate, mefloquine, and dihydroartemisinin—piperaquine phosphate tablets to quantitative HPTLC—densitometry methods

Transfer of four thin-layer chromatography (TLC) Global Pharma Health Fund Minilab mobile kit protocols for detecting fake pharmaceutical products to quantitative high-performance TLC (HPTLC)—densitometry methods was carried out using a model process published earlier. The developed and validated methods were for the drugs quinine sulfate, mefloquine, dihydroartemisinin, and piperaquine phosphate. EMD Millipore Premium Purity silica gel 60 F254 glass plates, automated standard and sample solution application with a CAMAG Linomat 4, and automated densitometry with a CAMAG Scanner 3 for detection, identification, and quantification were used. Sample peak identity and purity validation were carried out by spectral comparison checks available in the winCATS software, and accuracy was estimated by the standard addition approach. HPTLC gives better efficiency, selectivity, and resolution than TLC, and the new methods overcome the deficiencies in technology related to manual application and visual zone comparison that do not allow the Minilab TLC procedures to support regulatory compliance actions. These new methods should be fully validated according to International Conference on Harmonization guidelines or by interlaboratory studies if required by their applications. In addition, a previously reported transferred simultaneous HPTLC–densitometry method for lumefantrine and artemether was used to analyze a new combination tablet to demonstrate its applicability.

Chromatographic approach for the evaluation of radical-scavenging activity using a new time-monitoring image analysis method

A thin-layer chromatographic (TLC) procedure combined with digital image processing analysis was developed for quantitative time-monitoring of the radical- scavenging activity. Reaction of 2,2-diphenyl-1-picrylhydrazyl (DPPH•) with reference antioxidants and selected catecholamine compounds was monitored on RP-18 chromatographic plates using dot-blot technique and different concentrations of standards. Images of the chromatographic plate were recorded after background staining every 5 min for 1 h using a TLC scanner device. ImageDecipher TLC software program was used to convert image data into chromatograms, from which spot area as a function of reaction time was easily monitored for different amounts of antioxidant in all cases. For a comparative evaluation of radical-scavenging activity, all the time-acquired images were documented as a new red, green, and blue (RGB) image after conversion to different color values. Based on the chromatogram digitization results, TLC—time-monitored radical-scavenging pr...

Thin-layer chromatographic analysis of lipids and lipophilic pigments in snails

This paper reviews the use of thin-layer chromatography (TLC) for the analysis of neutral lipids and phospholipids in medically and economically important snails (gastropod molluscs). It updates and fills in unintentionally neglected papers from earlier reviews on this topic published in 1990 and 2006 in this journal, and extends coverage to lipophilic pigments for the first time. The review discusses all steps of the TLC procedure, including sample preparation, sample and standard application, layers and mobile phases, detection of zones, and quantification. Significant findings on the effects of temperature, larval parasitism, diet, and environmental changes on the lipid and lipophilic pigment content in the snails as determined by TLC as well as the results of other miscellaneous studies and advantages and future prospects are presented.

Thin-Layer Chromatography of Metal Cations Modified with Amines and Surfactants and the Sorption-Catalytic Detection of Cobalt(II) and Copper(II)

The thin-layer chromatography (TLC) procedures of metal cations, modified with amines and cationic surfactant micelles, followed by the sorption-catalytic detection of cobalt(II) and copper(II) directly into the thin layer were presented. It has been shown that the addition of hexamethylenediamine to the mobile phase acetone—3 M HCl and the impregnation of TLC plates with cetyltrimethylammonium bromide micelles and triethylamine followed by separation in 5% NaCl (pH 2.3) improves the separation efficiency of divalent metal cations mixture. A new highly sensitive detection of copper(II) and cobalt(II) after modified TLC with sorption-catalytic method was proposed. The TLC modifiers enhance the catalytic effect of copper(II) and cobalt(II) in the indicator reactions and detection selectivity. The detection limit was 0.1 ppb for copper(II) and 0.02 ppt for cobalt(II). It should be noted that cobalt(II) does not interfere with the determination of copper( II) in a 200-fold excess. The possibility of using the developed procedures in the analysis of natural water and food was shown.

A simple and inexpensive procedure for detection of a marker of Brazilian alecrim propolis

The detection of marker compounds is a necessary step for chemical characterization and standardization of propolis. A thin-layer chromatography procedure is described for the detection of allyl 3-prenylcinnamate, a volatile marker of Brazilian green propolis. The powdered sample is heated inside a petri dish and the material condensed on the dish cap is chromatographed on silica gel plates. The visualization may be done by spraying the plates with a solution of sodium fluorescein or a solution of sulphuric acid vanillin reagent.

Thin layer chromatography coupled with surface-enhanced Raman scattering as a facile method for on-site quantitative monitoring of chemical reactions.

By coupling surface-enhanced Raman spectroscopy (SERS) with thin layer chromatography (TLC), a facile and powerful method was developed for on-site monitoring the process of chemical reactions. Samples were preseparated on a TLC plate following a common TLC procedure, and then determined by SERS after fabricating a large-area, uniform SERS substrate on the TLC plate by spraying gold nanoparticles (AuNPs). Reproducible and strong SERS signals were obtained with substrates prepared by spraying 42-nm AuNPs at a density of 5.54 × 10(10) N/cm(2) on the TLC plate. The capacity of this TLC-SERS method was evaluated by monitoring a typical Suzuki coupling reaction of phenylboronic acid and 2-bromopyridine as a model. Results showed that this proposed method is able to identify reaction product that is invisible to the naked eye, and distinguish the reactant 2-bromopyridine and product 2-phenylpyridine, which showed almost the same retention factors (R(f)). Under the optimized conditions, the peak area of the characteristic Raman band (755 cm(-1)) of the product 2-phenylpyridine showed a good linear correlation with concentration in the range of 2-200 mg/L (R(2) = 0.9741), the estimated detection limit (1 mg/L 2-phenylpyridine) is much lower than the concentration of the chemicals in the common organic synthesis reaction system, and the product yield determined by the proposed TLC-SERS method agreed very well with that by UPLC-MS/MS. In addition, a new byproduct in the reaction system was found and identified through continuous Raman detection from the point of sample to the solvent front. This facile TLC-SERS method is quick, easy to handle, low-cost, sensitive, and can be exploited in on-site monitoring the processes of chemical reactions, as well as environmental and biological processes.

DETERMINATION OF HEMIN, PROTOPORPHYRIN IX, AND ZINC(II) PROTOPORPHYRIN IX IN PARMA HAM USING THIN LAYER CHROMATOGRAPHY

An effort was undertaken to investigate a possibility to quantify hemin, protoporphyrin IX (PPIX), and zinc(II) protoporphyrin IX (Zn(II)PPIX) by means of thin-layer chromatography (TLC). All these porphyrins naturally occur in meat and meat products, and they are known for being metabolically interrelated in a not yet fully understood fashion. In view of the results of a current research in meat chemistry, it seems most likely that Zn(II)PPIX is responsible for the red color of Parma ham (a traditional Italian dry ham). This discovery prompted a novel concept that Zn(II)PPIX might replace sodium nitrite and potassium nitrate in the function of the red color-forming pigment, the latter two compounds traditionally used for the same purpose in meat industry, yet known for the formation of the carcinogenic N-nitrosamines as well. In this study, a novel analytical system was proposed for the successful baseline separation of hemin, PPIX, and Zn(II)PPIX, and a possibility was tested of quantifying these three porphyrins in Parma ham. To this effect, the calibration curves were established for the porphyrin standards. Due to the recognized photosensitivity of hemin, PPIX, and Zn(II)PPIX, extraction of these three compounds from Parma ham and the thin-layer chromatographic procedure were carried out in the darkness, although a complete blackout was not always possible. One problem remained unsolved in this study, namely the selection of a proper (preferably a visible light resistant, hence a nonporphyrin) internal standard (IS) for the performance of the recovery procedure for hemin, PPIX, and Zn(II)PPIX contained in the meat matrix. Finally, it was concluded that the elaborated TLC procedure can successfully be implemented to quantification of hemin and PPIX in Parma ham (and in the other meat products as well).