Thin-layer Chromatographic Separation Research Articles

Application of thin‐layer chromatography/infrared matrix‐assisted laser desorption/ionization orthogonal time‐of‐flight mass spectrometry to structural analysis of bacteria‐binding glycosphingolipids selected by affinity detection

Glycosphingolipids (GSLs) play key roles in the manifestation of infectious diseases as attachment sites for pathogens. The thin-layer chromatography (TLC) overlay assay represents one of the most powerful approaches for the detection of GSL receptors of microorganisms. Here we report on the direct structural characterization of microbial GSL receptors by employment of the TLC overlay assay combined with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF-MS). The procedure includes TLC separation of GSL mixtures, overlay of the chromatogram with GSL-specific bacteria, detection of bound microbes with primary antibodies against bacterial surface proteins and appropriate alkaline phosphatase labeled secondary antibodies, and in situ MS analysis of bacteria-specific GSL receptors. The combined method works on microgram scale of GSL mixtures and is advantageous in that it omits laborious and time-consuming GSL extraction from the silica gel layer. This technique was successfully applied to the compositional analysis of globo-series neutral GSLs recognized by P-fimbriated Escherichia coli bacteria, which were used as model microorganisms for infection of the human urinary tract. Thus, direct TLC/IR-MALDI-o-TOF-MS adds a novel facet to this fast and sensitive method offering a wide range of applications for the investigation of carbohydrate-specific pathogens involved in human infectious diseases.

Arginase, glutamine synthetase and glutamate dehydrogenase activities in moist chilled and warm-incubated walnut kernels

The activities of arginase, glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were studied in both moist chilled (5°C) and warm (27°C) incubated walnut (Juglans regia. L) kernels to asses whether the non-germinability of dormant kernels is associated with failure in amino acid metabolism. Warm-incubated kernels showed low germination (25%), whereas cold-stratified kernels displayed germination up to 61%. Arginase activity increased about twofold in imbibed kernels. It remained at a high level in cold-stratified kernels from mid-period of incubation onwards; however, in warm-incubated kernels the activity declined after an initial increase so that by 20 days, it was negligible. No significant differences in GS activity occurred between cold-stratified and warm-incubated kernels, but the activity of GDH was significantly more in kernels incubated at warm conditions. Thin-layer chromatographic separation of polyamines revealed greater ammonia, spermidine and an unknown polyamine accumulation in warm-incubated kernels. Thus, the declined rate of walnut kernel germination under warm conditions is mainly correlated with rapid inactivation of arginase, greater levels of ammonia and alterations in kernel polyamine composition. The enhanced activity of GDH in warm-incubated kernels implies that catabolic deamination of amino acids and their subsequent respiration is the favored pathway ongoing under warm conditions. This situation compromises germination-specific metabolism of amino acids which likely to operate better at lower temperatures during cold stratification of kernels.

Development of Validated HPLC and HPTLC Methods for Simultaneous Determination of Levocetirizine Dihydrochloride and Montelukast Sodium in Bulk Drug and Pharmaceutical Dosage Form

Two chromatographic methods have been described for the simultaneous determination of levocetirizine dihydrochloride and Montelukast sodium in tablets. The fi rst method was a high performance thin layer chromatographic (HPTLC) separation followed by densitometric measurements on normal phase silica gel 60 F 254 . The second method was a high performance liquid chromatographic (HPLC) separation on a BDS Hypersil C 18 column using disodium hydrogen phosphate buffer (0.02 M): Methanol (25: 75, v/v) pH adjusted to 7 with ortho-phosphoric acid as the mobile phase. The proposed methods were validated as per ICH guidelines and successfully applied for the determination of investigated drugs in tablets.

Phosphoinositide and inositol phosphate analysis in lymphocyte activation.

Lymphocyte antigen receptor engagement profoundly changes the cellular content of phosphoinositide lipids and soluble inositol phosphates. Among these, the phosphoinositides phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) play key signaling roles by acting as pleckstrin homology (PH) domain ligands that recruit signaling proteins to the plasma membrane. Moreover, PIP2 acts as a precursor for the second messenger molecules diacylglycerol and soluble inositol 1,4,5-trisphosphate (IP3), essential mediators of PKC, Ras/Erk, and Ca2+ signaling in lymphocytes. IP3 phosphorylation by IP3 3-kinases generates inositol 1,3,4,5-tetrakisphosphate (IP4), an essential soluble regulator of PH domain binding to PIP3 in developing T cells. Besides PIP2, PIP3, IP3, and IP4, lymphocytes produce multiple other phosphoinositides and soluble inositol phosphates that could have important physiological functions. To aid their analysis, detailed protocols that allow one to simultaneously measure the levels of multiple different phosphoinositide or inositol phosphate isomers in lymphocytes are provided here. They are based on thin layer, conventional and high-performance liquid chromatographic separation methods followed by radiolabeling or non-radioactive metal-dye detection. Finally, less broadly applicable non-chromatographic methods for detection of specific phosphoinositide or inositol phosphate isomers are discussed. Support protocols describe how to obtain pure unstimulated CD4+CD8+ thymocyte populations for analyses of inositol phosphate turnover during positive and negative selection, key steps in T cell development.

Extraction and classification of lipids from seeds of persea Americana miller and chrysophyllum albidum g. Don.

Seed lipids of Persea americana and Chrysophyllum albidum were studied. Lipids were extracted with chloroform-methanol, analysed with silicic column chromatography and thin layer chromatography. The total lipid content of Persea americana was 10.8% while that of Chrysophyllum albidum was 7.7%. Fractionation of the total lipid fraction of Persea americana using silicic acid column chromatography showed the occurrence of neutral lipids 6.5%, glycolipids 2.1% and phospholipids 2.2% while those of Chrysophyllum albidum were 4.2%, 1.7% and 1.8% for neutral lipids, gycolipids and phospholipids, respectively. Thin layer chromatography separation showed three classes of lipids in Persea americana and Chrysophyllum albidum which were oleic acid, palmitic acid and cholesterol. The colour of these lipids was yellow and solid at room temperature. These studies suggest that oil extracted from seeds of Persea americana and Chrysophyllum albidum could be used for the manufacture of industrial products.

Validated Stability-Indicating Chromatographic Methods for the Determination of Veralipride in Presence of Its Degradation Products

Two sensitive and selective chromatographic methods were developed and validated for determination of veralipride in presence of its degradation products. Forced degradation studies were performed, using HCl, NaOH and 3% H2O2. The first method is based on thin-layer chromatographic separation of the intact drug spot from its degradation, followed by densitometric measurements. The second method is based on isocratic liquid chromatographic separation of the studied drug from its degradation on a reversed phase C18 column. The proposed LC method was utilized to investigate the kinetics of alkaline degradation process of the selected drug at different temperatures.

Separation and identification of some common isomeric plant triterpenoids by thin-layer chromatography and high-performance liquid chromatography

Chromatographic separation of 10 triterpenoids (α-amyrin, β-amyrin, δ-amyrin, lupeol, lupenon, lupeol acetate, cycloartenol, cycloartenol acetate, ursolic acid, oleanolic acid) and 2 sterols (stigmasterol and β-sitosterol) was studied. The chromatographic techniques included silica gel and reversed-phase (C 18 RP) thin-layer chromatography (TLC) and C 18 RP high-performance liquid chromatography (HPLC) using UV and mass spectrometric (MS) detection with atmospheric pressure chemical ionization (APCI). The TLC separation of the isomeric triterpenols lupeol, α-amyrin, β-amyrin and cycloartenol was achieved for the first time using C 18 RP-HPTLC plates. Cycloartenol could be separated from related compounds only on C 18 RP-TLC but not on the C 18 RP-HPLC. δ-Amyrin isolated from the tomato fruit surface extract could be separated from other amyrins only by HPLC. Tandem mass spectrometry allowed discrimination between the isomers lupeol, α-amyrin, β-amyrin, δ-amyrin, cycloartenol and between lupeol acetate and cycloartenol acetate. The combination of 3 TLC methods and 2 HPLC methods enables qualitative determination of all 12 compounds and proves to be useful for the analysis of plant extracts. It is recommended that TLC screening on silica gel and C 18 RP be performed before HPLC analysis.

Advances on the compositional analysis of glycosphingolipids combining thin-layer chromatography with mass spectrometry

Glycosphingolipids (GSLs), composed of a hydrophilic carbohydrate chain and a lipophilic ceramide anchor, play pivotal roles in countless biological processes, including infectious diseases and the development of cancer. Knowledge of the number and sequence of monosaccharides and their anomeric configuration and linkage type, which make up the principal items of the glyco code of biologically active carbohydrate chains, is essential for exploring the function of GSLs. As part of the investigation of the vertebrate glycome, GSL analysis is undergoing rapid expansion owing to the application of novel biochemical and biophysical technologies. Mass spectrometry (MS) takes part in the network of collaborations to further unravel structural and functional aspects within the fascinating world of GSLs with the ultimate aim to better define their role in human health and disease. However, a single-method analytical MS technique without supporting tools is limited yielding only partial structural information. Because of its superior resolving power, robustness, and easy handling, high-performance thin-layer chromatography (TLC) is widely used as an invaluable tool in GSL analysis. The intention of this review is to give an insight into current advances obtained by coupling supplementary techniques such as TLC and mass spectrometry. A retrospective view of the development of this concept and the recent improvements by merging (1) TLC separation of GSLs, (2) their detection with oligosaccharide-specific proteins, and (3) in situ MS analysis of protein-detected GSLs directly on the TLC plate, are provided. The procedure works on a nanogram scale and was successfully applied to the identification of cancer-associated GSLs in several types of human tumors. The combination of these two supplementary techniques opens new doors by delivering specific structural information of trace quantities of GSLs with only limited investment in sample preparation.

Host Cell P-glycoprotein Is Essential for Cholesterol Uptake and Replication of Toxoplasma gondii

P-glycoprotein (P-gp) is a membrane-bound efflux pump that actively exports a wide range of compounds from the cell and is associated with the phenomenon of multidrug resistance. However, the role of P-gp in normal physiological processes remains elusive. Using P-gp-deficient fibroblasts, we showed that P-gp was critical for the replication of the intracellular parasite Toxoplasma gondii but was not involved in invasion of host cells by the parasite. Importantly, we found that the protein participated in the transport of host-derived cholesterol to the intracellular parasite. T. gondii replication in P-gp-deficient host cells not only resulted in reduced cholesterol content in the parasite but also altered its sphingolipid metabolism. In addition, we found that different levels of P-gp expression modified the cholesterol metabolism in uninfected fibroblasts. Collectively our findings reveal a key and previously undocumented role of P-gp in host-parasite interaction and suggest a physiological role for P-gp in cholesterol trafficking in mammalian cells.

Isolation, Purification, and Characterization of a Polygalacturonase Produced in Penicillium solitum-Decayed ‘Golden Delicious’ Apple Fruit

Polygalacturonase (PG) was extracted and purified from decayed 'Golden Delicious' apple fruit inoculated with Penicillium solitum. Ammonium sulfate, gel filtration, and cation exchange chromatography were used to purify the enzyme. Both chromatographic methods revealed a single peak corresponding to PG activity. The purified PG most likely originates from the fungus because PG activity from healthy and wounded apple tissue was undetectable. Analysis of cation exchange-purified material using sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a single 50-kDa band. The enzyme was active over a broad pH range (3 to 7), with optimal activity between pH 4 and 5. PG was highly active at 20 and 37 degrees C but was also detectable at 2, 50, and 75 degrees C. Divalent cations affected PG enzyme activity; Mg and Fe increased, whereas Ca and Mn reduced activity in vitro. Thin-layer chromatographic separation of hydrolysis products and data from a PG plate activity assay based on staining with ruthenium red showed that the enzyme exhibits both exo and endo activity. Purified PG incubated with intact apple fruit tissue in vitro caused a 30% reduction in mass after 48 h, suggesting a role in P. solitum-mediated decay of apple fruit.

TLC Separation and Identification of the Essential Oil Constituents from Aloysia gratissima

The present work had as objective the isolation of the five compounds by thin-layer Chromatography (TLC) from the essential oil of the Aloysia gratissima. For this, a number of systems of eluents were used for its separation, indicating that through the system acetone/hexane in proportions (v/v) 1:30 it was possible to isolate guaiol, elemol, pinocanphone (trans-3-pinanone), cis-pinocarvyl, and acorenone. The isolation of the compound acorenone from the other compounds was possible with the mixture of solvents hexane/dichloromethane in proportions (v/v) (1:1,3).

Preliminary phytochemical and antimicrobial investigations of the stem bark and leaves of Psidum guajava L.

Extracts (ethanol, methanol and aqueous) of the leaves and stem barks of Psidum guajava L were studied for their phytochemical constituents. Results of the phytochemical screening revealed the presence of alkaloids (all the extracts), saponins (ethanol and methanol), cardenolides with steroided rings, and cardenolides with deoxy sugar (all the extracts). Thin layer chromatographic separation of ethanol and methanol extracts gave three spots each with Rf values ranging from 0.60 – 0.70. Only the aqueous extract inhibited Bacillus subtilis andFusarium spp. The presence of these constituents tends to support the uses of this plant medicinally. Key words: Alkaloid, cardenolides, extracts, saponin.

Óleo essencial de limão no ensino da cromatografia em camada delgada

This paper describes a simple experiment employing the essential oil of limes which can be applied in undergraduate organic chemistry laboratory classes for the teaching of thin layer chromatography (TLC). The experiment consists in submit lime peel oil to TLC separation employing hexane and dichloromethane as the eluents and five different systems for visualization of the chromatogram. In one experiment it is possible to teach the different variables of the TLC technique. This experiment may also be performed following vapor distillation and liquid-liquid extraction technique in experimental classes.

Simultaneous RP-HPLC and HPTLC estimation of fluoxetine hydrochloride and olanzapine in tablet dosage forms

A binary mixture of fluoxetine HCl and olanzapine was determined by two different methods. The first method involved determination of fluoxetine HCl and olanzapine using reversed-phase liquid chromatography using acetonitrile:methanol:0.032 M ammonium acetate buffer (45:05:50, v/v/v) as the mobile phase at a flow rate of 1.5 ml/min. Quantitation was achieved with ultraviolet detection at 235 nm over concentration ranges of 0.2-4 and 0.1-2 μg/ml; mean accuracies were 101.16±0.59 and 99.79±0.56% for fluoxetine HCL and olanzapine, respectively. The second method was based on the high performance thin layer chromatography separation of the two drugs followed by densitometric measurements of their spots at 235 nm. The separation was carried out on Merck TLC aluminium sheets of silica gel 60 F254 using acetone:methanol:triethyleamine (5:3:0.5, v/v/v), as mobile phase. The linearity was found to be in the range of 300–1000 and 150–500 ng/spot; mean accuracies were 100.95±0.52 and 99.31±0.51% for fluoxetine HCl and olanzapine, respectively. The method was successively applied to tablets because no chromatographic interferences from the tablet excipients were found. The methods retained their accuracy and precision when the standard addition technique was applied. The results obtained by applying the proposed methods were statistically analyzed.

TLC Determination of Amitriptyline HCl, Trifluoperazine HCl, Risperidone and Alprazolam in Pharmaceutical Products

A simple, sensitive, and precise thin layer chromatographic (TLC) method for simultaneous analysis of psychopharmacological drugs like amitriptyline HCl, trifluoperazine HCl, risperidone and alprazolam in their single dosage forms has been developed, validated, and used for determination of the compounds in commercial pharmaceutical products. The TLC separation was carried out on Merck TLC aluminium sheets of silica gel 60 F254 using carbon tetrachloride:acetone:triethylamine (8:2:0.3, v/v/v), as mobile phase. Densitometric measurements of their spots were achieved at 250 nm over the concentration range for amitriptyline HCl (50–1,200 ng spot−1), trifluoperazine HCl (50–1,200 ng spot−1), risperidone (100–2,400 ng spot−1) and alprazolam (25–600 ng spot−1). Limit of detection (LOD) for amitriptyline HCl (20 ng spot−1), trifluoperazine HCl (20 ng spot−1), risperidone (40 ng spot−1) and alprazolam (5 ng spot−1) was obtained. The study showed that TLC was sensitive and selective for determination of amitriptyline HCl, trifluoperazine HCl, risperidone and alprazolam using a single mobile phase. This proposed method is able for simultaneous determination of psychopharmacological drugs and also applicable for analysis of pharmaceutical formulations.

Development of Validated Chromatographic Methods for the Simultaneous Determination of Metronidazole and Spiramycin in Tablets

Two chromatographic methods have been described for the simultaneous determination of metronidazole (MET) and spiramycin (SPY) in their mixtures. The first method was based on a high performance thin layer chromatographic (HPTLC) separation of the two drugs followed by densitometric measurements of their spots at 240 nm. The separation was carried out on Merck TLC aluminum sheets of silica gel 60 F254 using methanol: chloroform (9:1, v/v) as a mobile phase. Analysis data was used for the linear regression line in the range of 1.0–2.0 and 0.8–2.0 μg band−1 for MET and SPY, respectively. The second method was based on a reversed-phase liquid chromatographic separation of the cited drugs on a C-18 column (5 μm, 250 × 4.6 mm, i.d.). The mobile phase consisted of a mixture of phosphate buffer of pH 2.4 and acetonitrile (70:30, v/v). The separation was carried out at ambient temperature with a flow rate of 1.0 mL min−1. Quantitation was achieved with UV detection at 232 nm based on peak area with linear calibration curves at concentration ranges 0.4–50.0 and 0.5–50.0 μg mL−1 for MET and SPY, respectively. The proposed chromatographic methods were successfully applied to the determination of the investigated drugs in pharmaceutical preparations. Both methods were validated in compliance with ICH guidelines; in terms of linearity, accuracy, precision, robustness, limits of detection and quantitation and other aspects of analytical validation.

Linkage Between Separation of Cu2+, Co2+, and Ni2+ on TLC and Crystal Field Theory

M(DEDTC)2 (M = Cu, Co, or Ni) and M(PyDTC)2 (M = Cu or Co) complexes prepared by reactions of sodium diethyldithiocarbamate (NaDEDTC) and ammonium pyrrolidinedithiocarbamate (NH4PyDTC) with metal (II) nitrates are examined for qualitative analysis and separation using thin-layer chromatography (TLC) systems in the literature. Already known separability of metal cations cited and their complexes are reexamined and discussed in the context of relation to the crystal field theory (CFT) and TLC as a new approach and adaptation. Because the chromatographic data have been agreed on with assumptions of CFT, it is concluded that CFT is closely related to the TLC separation of these metal cations and their complexes. Therefore, this study may be useful in understanding of the linkage between the CFT on coordination chemistry and the chromatographic parameters [e.g., retention factor (Rf) and theoretical plate numbers (N)] of the complexes.

Extending equal-spreading criteria to two-dimensional thin layer chromatography — the points in a unit square problem revisited

Summary Optimization of two-dimensional thin-layer chromatographic separation often relies on selection of the two most orthogonal chromatographic systems which best co-operate in the separation. This is mainly achieved by investigating the correlation between RF values or scoring the distances between the spots. This paper presents and discusses another approach, based on the distances to the closest spot and to the top or bottom of the plate. The theory arises from a well-known geometrical problem about equal-spreading of the points inside a unit square. Two coefficients are proposed — sensitive and insensitive to complete separation, which are the two-dimensional version of previously proposed RU and RD criteria (retention uniformity and retention distance, describing the equal-spreading of the spots in one-dimensional chromatography). They are included in the range 0–1 and their distribution as a random variable is well defined and not affected by the number of separated compounds.

Effect of alfalfa saponins and flavonoids on pea aphid

AbstractWe studied the development and feeding behaviour of the pea aphid, Acyrthosiphon pisum (Harris) (Homoptera: Aphididae), on the Radius and Sapko alfalfa (Medicago sativa L.) (Fabaceae) cultivars. Three saponins and flavones were identified in the alfalfa cultivars after thin layer chromatography separation. Cultivar Radius differed from Sapko in that it had a higher level of saponins, including zanhic acid tridesmoside and 3‐GlcA,28‐AraRhaXyl medicagenic acid glycoside. The flavones identified, including 7‐O‐β‐D‐glucuronopyranosyl‐4′‐O‐[2′‐O‐E‐feruloyl‐O‐β‐D‐glucuronopyranosyl(1→2)‐O‐β‐D‐glucuronopyranoside] apigenin, 7‐O‐{2‐O‐E‐feruloyl‐[β‐D‐glucuronopyranosyl(1→3)]‐O‐β‐D‐glucuronopyranosyl(1→2)‐O‐β‐D‐glucuronopyranoside} apigenin, and 4′‐O‐[2′‐O‐E‐feruloyl‐O‐β‐D‐glucuronopyranosyl(1→2)‐O‐β‐D‐glucuronopyranoside] apigenin, occurred in tissues of both alfalfa cultivars. However, cv. Radius had very low mean flavonoid concentrations in comparison to cv. Sapko. Pea aphids that fed on cv. Radius plants showed a reduction in reproduction and survival. The aphid pre‐reproductive period on cv. Radius was prolonged and the reproductive and post‐reproductive periods on cv. Radius were reduced, compared to those on cv. Sapko. Cultivar Radius also negatively influenced aphid probing behaviour. This was especially the case during the initial period of the pathway phase. The results suggested that alfalfa cv. Radius, which had a higher level of saponins and a lower level of flavonoids, was less accepted by the pea aphid.

Fate of Prohexadione Calcium in Annual Bluegrass (Poa annua) and Three Turfgrasses

Prohexadione calcium is an experimental turfgrass growth regulator that selectively controls or suppresses annual bluegrass in desirable turfgrass such as creeping bentgrass, Kentucky bluegrass, and perennial ryegrass. To help explain interspecific differences in turfgrass and weed response to prohexadione calcium, two laboratory trials were conducted to measure 14C-prohexadione calcium absorption, translocation, and metabolism in these four species. Annual and Kentucky bluegrass absorbed more prohexadione calcium than creeping bentgrass and perennial ryegrass when averaged over harvest timing and trial. Radioactivity partitioning to other foliage did not differ between species but annual bluegrass and Kentucky bluegrass translocated more radioactivity to roots and evolved more radioactive CO2 than creeping bentgrass and perennial ryegrass. Thin-layer chromatographic separations indicate radioactivity was translocated predominately as prohexadione calcium. When averaged over species and trial, 25 and 16% of recovered prohexadione calcium was metabolized within 1 h after treatment in treated leaves and other foliage, respectively. The rate of metabolic degradation was 0.7% h−1 in treated leaves and 0.4% h−1 in other foliage. Previous research indicates that annual and Kentucky bluegrass growth is suppressed more by prohexadione calcium than is growth of creeping bentgrass and perennial ryegrass. Increased prohexadione calcium absorption partially explained these interspecific differences in growth suppression.