Improved separation and quantitative determination of hydrocarbon types in gas oil by normal phase high‐performance TLC with UV and fluorescence scanning densitometry

Improved methods for separation and quantitative determination of hydrocarbon types from gas oil have been developed, which were based on high‐performance thin‐layer chromatography with ultraviolet and fluorescence scanning densitometry using horizontal elution. One of the methods allows the separation, detection, and determination of alkanes and naphthenes to be carried out, using berberine‐impregnated silica gel HPTLC plates, elution with n‐hexane, and berberine‐induced fluorescence detection at 365 nm. Another developed method allows total aromatics to be determined using silica gel HPTLC plates by elution with n‐hexane and acetone, and UV detection. In turn, PACs over three aromatic rings can be determined on either silica gel or caffeine‐impregnated silica gel HPTLC plates, elution with n‐hexane, and selective detection using native fluorescence at 365 nm. Concentrations lower than 5 wt% can be determined using this technique. In addition, a technique for an efficient, baseline‐resolved separation of a gas oil according to the number of aromatics rings (mono + di‐, tri‐, and polyaromatic compounds with more than three rings) is presented here. This technique involves a multistep elution on a mixed (silica gel and caffeine‐impregnated silica gel HPTLC plate) using a counter‐elution device, and UV detection.     

Critical evaluation of experimental conditions influencing the surface-enhanced Raman spectroscopic (SERS) detection of substances separated by layer liquid chromatographic techniques

Summary Surface-enhanced Raman spectra (SERS) ofp-dimethylaminobenzylidenerhodanine have been recorded on silica gel 60 F₂₅₄ and Si60 F₂₅₄ Raman TLC plates. Spectra were enhanced by use of a silver sol prepared according to the modified Lee-Meisel procedure. The standard deviations of the intensities and the band ratios for the seven most intense peaks were calculated for 30 parallel measurements. Although the Raman plate gives more reproducible results, several experimental difficulties are encountered in the development of chromatograms. SERS detection of ascorbigen and 1′-methylascorbigen was performed after chromatography on silica gel 60 F₂₅₄ TLC and HPTLC plates and on Si60 F₂₅₄ Raman TLC plates. Traditional development was used for the silica gel 60 F₂₅₄ TLC plates and Si60 F₂₅₄ Raman plates, and the personal OPLC technique for the silica gel 60 F₂₅₄ HPTLC plates. It was found that the SERS spectrum gave information about the indole ring only. Because bonding of the analyte to the stationary phase results in a change in molecular conformation-in contrast with the behaviour of rhodanine-the type of the plateused and the development procedure employed can significantly influence the quality of the SERS spectrum. Presented at Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary September 1–3, 1999     

Identification of subnanomole amounts of PTH-amino acids by high performance thin-layer chromatography

Summary High performance thin-layer chromatography (HPTLC) of PTH-amino acids on 5×5 cm silica gel plates precoated with a fluorescence marker gives 10–20 fold increase in sensitivity compared to ordinary silica gel plates. Separation of PTH-Leu from PTH-Ile is easily achieved in contrast to chromatography on polyamide sheets. Only two solvent systems are required and as many as 12 samples can be chromatographed on each plate. However, if the sample is contaminated with N-phenylthiourea a third solvent system is necessary.     

Advances in the thin layer chromatographic analysis of counterfeit pharmaceutical products: 2008–2019

Publications reporting thin layer chromatography (TLC) screening and high performance TLC (HPTLC)-densitometry quantification analyses of counterfeit pharmaceutical products are reviewed for the 2008–2019 period. Screening using TLC methods published in the Global Pharma Health Fund (GPHF) Minilab Manual and U.S. Food and Drug Administration (FDA) Compendium, as well as in other sources, are covered. Also included are publications on TLC analysis hyphenated with Raman and mass spectrometry; analyses of counterfeit traditional herbal medicines; earlier published reviews; transfer of screening methods for counterfeit pharmaceutical products in the Minilab Manual and FDA Compendium to HPTLC-densitometry using a model process; development of HPTLC-densitometry methods for pharmaceutical products not included in the Minilab Manual or FDA Compendium using the model process followed by development of corresponding Supplemental FDA Compendium TLC screening methods; and modified Minilab methods with simplified detection based on heating of silica gel F layers to produce fluorescence quenching zones (thermochemical activation) rather than detection using spray, dip, or vapor phase derivatization reagents. Some thoughts on future prospects for the field are also offered.     

Thin-layer chromatography combined with MALDI-TOF-MS and 31P-NMR to study possible selective bindings of phospholipids to silica gel

High-performance thin-layer chromatography (HPTLC) is a highly established separation method in the field of lipid and (particularly) phospholipid (PL) research. HPTLC is not only used to identify certain lipids in a mixture but also to isolate lipids (preparative TLC). To do this, the lipids are separated and subsequently re-eluted from the silica gel. Unfortunately, it is not yet known whether all PLs are eluted to the same extent or whether some lipids bind selectively to the silica gel. It is also not known whether differences in the fatty acyl compositions affect the affinities to the stationary phase. We have tried to clarify these questions by using a readily available extract from hen egg yolk as a selected example of a lipid mixture. After separation, the complete lanes or selected spots were eluted from the silica gel and investigated by a combination of MALDI-TOF MS and ³¹P NMR spectroscopy. The data obtained were compared with the composition of the total extract (without HPTLC). Although there were significant, solvent-dependent losses in the amount of each lipid, the relative composition of the mixture remained constant; there were also only very slight changes in the fatty acyl compositions of the individual PL classes. Therefore, lipid isolation by TLC may be used without any risk of major sample alterations.

Utilization of a scientifically operated charge-coupled device detector for high-performance thin-layer chromatographic analysis of tetracyclines

A high-performance thin-layer chromatography (HPTLC) method using a scientifically operated charge-coupled device detector is described for the assay of tetracycline pharmaceutical products. Quantitative information can be obtained for all samples on a TLC plate within a few seconds. The separation efficiency and detection limits were determined on both normal phase and reverse phase TLC plates. Fluorescence detection mode offers higher sensitivity than fluorescence quenching mode. The dynamic range, sensitivity, accuracy and precision of the system were evaluated. Detection limits of the impurities are in the range of 0.1 to 0.5 ng or 0.3 to 1% of tetracycline, depending on the compound, with a recovery percentage over 85%. The existing impurities in tetracycline capsules were determined using both HPLC and HPTLC techniques. All of the impurities were below the regulation level.     

HPTLC Determination of Cefpodoxime Proxetil in Formulations

An HPTLC method has been developed that coelutes both the isomers of cefpodoxime proxetil (CFP). CFP was chromatographed on a silica gel 60 F₂₅₄ TLC plate using toluene:acetonitrile (6:4) as a mobile phase and was quantified at 234 nm. The method was validated with respect to linearity, accuracy, precision and specificity. The limit of detection and limit of quantification for CFP were found to be 0.150 and 0.4 μg spot^?1, respectively. The proposed method was successfully used to determine the amount of CFP present in the marketed tablets and self-nanoemulsifying systems.     

Improvement of chemical analysis of antibiotics. X. Determination of eight tetracyclines using thin-layer and high-performance liquid chromatography

Analytical methods for eight tetracyclines (TCs) were established using silica gel high-performance thin-layer chromatography (HPTLC), reversed-phase thin-layer chromatography (RP-TLC) and high-performance liquid chromatography (HPLC). Good separations of eight TCs were obtained using chloroform-methanol-5% disodium ethylenediaminetetraacetate solution (65:20:5) (lower layer) and methanol acetonitrile 0.5 M oxalic acid solution (1:1:4) (pH 3.0) on silica gel HPTLC and C8 TLC plates, respectively. A combination of HPTLC and RP-TLC made possible the identification of the eight TCs. Each calibration graph was linear between 0.1 and 1.0 microgram using UV densitometry except for rolitetracycline. For detection reagents, the diazonium salts including Fast Violet B gave variously coloured spots with the eight TCs and good sensitivities were obtained except with minocycline. In HPLC, the simultaneous analysis of the eight TCs on a C8 column was possible using methanol-acetonitrile-0.01 M oxalic acid solution (1:1.5:7) adjusted to pH 3.0 as the mobile phase. A linear relationship was obtained between 1.0 and 10 ng using the usual sample preparation except for rolitetracycline. The direct determination of rolitetracycline was possible using tetrahydrofuran, dimethyl sulphoxide and the mobile phase as solvents for preparation of the sample. For the determination of residual rolitetracycline, it was effective to measure the amount of rolitetracycline as tetracycline by HPLC, HPTLC and RP-TLC after conversion of rolitetracycline to tetracycline by incubating for 5 min in methanol at 50 degrees C.     

HPTLC Determination of Cefpodoxime Proxetil in Formulations

An HPTLC method has been developed that coelutes both the isomers of cefpodoxime proxetil (CFP). CFP was chromatographed on a silica gel 60 F₂₅₄ TLC plate using toluene:acetonitrile (6:4) as a mobile phase and was quantified at 234 nm. The method was validated with respect to linearity, accuracy, precision and specificity. The limit of detection and limit of quantification for CFP were found to be 0.150 and 0.4 μg spot⁻¹, respectively. The proposed method was successfully used to determine the amount of CFP present in the marketed tablets and self-nanoemulsifying systems.

     

HPTLC on inert pre-coated layers of cellulose and of silica 50000

Summary In a manner analogous to that for surface-active silica gel, HPTLC pre-coated plates for nano TLC have also been developed from two inactive sorbents. The two materials are microcrystalline cellulose and a synthetically produced, porous silica (Silica 50000) with a very low specific surface area. The chromatographic properties of these inert sorbents and of the new HPTLC pre-coated plates prepared therefrom are examined in relation to separations of amino acid mixtures and carbohydrate mixtures and are related to the chromatographic properties of the inactive sorbents and TLC precoated plates used hitherto. The figure 50000 characterizes the type of silica. The average pore diameter of this sorbent is about 5000 nm.     

Determination of chenodeoxycholic acid on 3-cyanopropyltrichlorosilane-treated high-performance thin-layer chromatographic plates

Summary Chenodeoxycholic acid in commerical drugs was analyzed by high-performance, thin-layer chromatography (HPTLC) using a cyanoalkyl chemically-bonded stationary phase, prepared by treating pre-coated silica with 3-cyanopropyltrichlorosilane (3CPTS). The 3CPTS-treated plates were used to evaluate commerical chenodeoxycholic acid in drug capsules. After development with methanol on the 3CPTS-treated plate, the chenodeoxycholic acid spot in the chromatogram was measured with a TLC densitometer equipped with a dual-wave length TLC scanner at λ=370nm. By this method, the fluorescence intensity of chenodeoxycholic acid was measured within 2.6% error over the range 30–240ng and the limit of detection was 30ng.     

Development and validation of a thin-layer chromatographic method for determination of chloramphenicol residues on pharmaceutical equipment surfaces

A thin-layer chromatographic (TLC) method with densitometric quantitation using the absorption reflectance mode at 280 nm was developed and validated for the determination of chloramphenicol residues in controlling pharmaceutical equipment cleanliness. Simulated samples at residue levels 0.5, 1, and 1.2 mg/m2 were prepared by spreading the calculated amount of chloramphenicol solution on a 10 dm2 stainless steel surface. After evaporation of the solvent, the residue was removed by 2 methanol-wetted cotton swabs, which were then extracted with methanol. The extract was applied on a high-performance TLC (HPTLC) silica gel F254 plate together with standards ranging from 10 to 60 ng. Plates were developed in a horizontal developing chamber from both sides (36 applications per plate) by using n-hexane-ethyl acetate (35 + 65, v/v) as developing solvent. The mean recovery (n=6) at 1 mg/m2 was 95.8%, and the coefficient of variation was 5.8%. The absolute detection limit was 3 ng, and the quantitation limit 10 ng. The method detection limit was 0.3 mg/m2 by swabbing 2.5 dm2 and 0.075 mg/m2 by swabbing 10 dm2. Chloramphenicol was stable on the plate 2 h before and 24 h after development. Additionally, it was stable during 7 days storage on the cotton swabs in the solvent at room temperature and in diluted standard solution stored in darkness at 4 degrees C. The method can be applied to routine control of pharmaceutical equipment cleanliness by sampling from the stainless steel surface areas of 2.5 to 10 dm2, and an acceptable residue limit of 1 mg/m2.     

Ultrathin-Layer Chromatography (UTLC)

Ultrathin-layer chromatography (UTLC) differs from high-performance thin-layer chromatography (HPTLC) and from thin-layer chromatography (TLC) in two basis things: the layer thickness, and the migration distances of the analytes. UTLC has a monolithic or a nanostructured stationary silica gel phase bound directly to the glass plates. Layer thickness in UTLC is 10 μm, instead of 100–250 μm in HPTLC. Migration distances are in the range of 1–3 cm for UTLC, instead of 8–10 cm for HPTLC. Therefore, the major advantages of UTLC over HPTLC and TLC are the shorter development times and higher separation efficiency and sensitivity. Moreover, separations on UTLC plates require smaller reagent and sample volumes. However, the UTLC plates are very difficult to manage with the TLC and HPTLC equipment currently available. Therefore, the next challenge in this area is the development of an inexpensive solution with appropriate instrumentation (sensitive optical scanners and sample application systems). UTLC had been used for separations of many compounds, e.g., pharmaceutically active ingredients, pesticides, plasticisers, natural products, and other chemical substances.     

Application of iodine-azide reaction for detection of amino acids in thin-layer chromatography

The iodine-azide reaction was employed to TLC detection of sulphur-containing derivatives of protein and some non-protein amino acids. The derivatization reaction with phenyl isothiocyanate (PITC) took place directly on the plate before the developing step. Subsequently, the plates were sprayed with a mixture of sodium azide and starch solution in NP-TLC and in the case of RP-TLC sodium azide solution with starch incorporated into mobile phase and then exposed to iodine vapour. The spots became visible as white spots on violet-grey background. The obtained detection limits of PTC-derivatives have been compared with other visualizing techniques commonly used in TLC practice (UV254 and iodine vapour). The iodine-azide system has been proved to be the most favourable and enabled to detect quantities per spot in the range of 1-60 pmol (HPTLC) and 3-100 pmol (TLC).     

Analysis of sucrose esters--insecticides from the surface of tobacco plant leaves

Sucrose esters from the surface of leaves of Nicotiana tabacum L. have been shown to possess interesting biological activities. We developed a simple and effective method for their analysis using HPTLC silica gel plates, n-hexane-ethyl acetate (1:3, v/v) as developing solvent and aniline-diphenylamine as a detection reagent. Off-line TLC-MS was also used for the detection and identification of the compounds. Solutions containing sucrose esters upon alkaline hydrolysis give sucrose, which is used for indirect estimation by TLC of the sucrose ester content. The method is applicable for the screening for sucrose esters in plant extracts. The extract obtained from the surface of green leaves of oriental tobacco type Prilep P-23 contains sucrose esters and is effective against Myzus persicae (Sulzer) in laboratory and field experiments.     

Fluorescence detection by intensity changes for high-performance thin-layer chromatography separation of lipids using automated multiple development

Changes in emission of berberine cation, induced by non-covalent interactions with lipids on silica gel plates, can be used for detecting and quantifying lipids using fluorescence scanning densitometry in HPTLC analysis. This procedure, referred to as fluorescence detection by intensity changes (FDIC) has been used here in combination with automated multiple development (HPTLC/AMD), a gradient-based separation HPTLC technique, for separating, detecting and quantifying lipids from different families. Three different HPTLC/AMD gradient schemes have been developed for separating: neutral lipid families and steryl glycosides; different sphingolipids; and sphingosine-sphinganine mixtures. Fluorescent molar responses of studied lipids, and differences in response among different lipid families have been rationalized in the light of a previously proposed model of FDIC response, which is based on ion-induced dipole interactions between the fluorophore and the analyte. Likewise, computational calculations using molecular mechanics have also been a complementary useful tool to explain high FDIC responses of cholesteryl and steryl-derivatives, and moderate responses of sphingolipids. An explanation for the high FDIC response of cholesterol, whose limit of detection (LOD) is 5 ng, has been proposed. Advantages and limitations of FDIC application have also been discussed.     

Development of quantitative HPTLC-densitometry methods following a model approach for transfer of TLC screening methods for pharmaceutical products of atenolol,chloramphenicol, furosemide,glibenclamide, penicillin V potassium,and praziquantel

Transfer of six thin-layer chromatography (TLC) Global Pharma Health Fund E.V. Minilab manual protocols for detecting fake drugs in pharmaceutical products to quantitative high-performance TLC (HPTLC)-densitometry methods was performed following a previously published model process. The developed and validated methods for tablets or capsules containing atenolol, chloramphenicol, furosemide, glibenclamide, penicillin V potassium, and praziquantel involved use of a limited list of inexpensive, relatively nontoxic, readily available solvents and other reagents; silica gel 60?F₂₅₄ plates; automated bandwise sample and standard solution application; ascending mobile phase development of plates in a chamber; and automated slit scanning densitometry for detection, identification, and quantification. Validation data for methods developed in an early version of the transfer model process that did not include standard addition validation are reported for pharmaceutical products containing amitriptyline HCl, amodiaquine, diphenhydramine HCl, and mebendazole.     

Simultaneous Densitometric Determination of Rifampicin and Isoniazid by High-Performance Thin-Layer Chromatography

JPC – Journal of Planar Chromatography – Modern TLC - High-performance thin-layer chromatography (HPTLC) on silica gel 60 F254 TLC plates, with ethyl acetate-methanol-acetone-acetic...     

Comparison of high performance TLC and HPLC for separation and quantification of chlorogenic acid in green coffee bean extracts

Two chromatographic methods, high-performance TLC (HPTLC) and HPLC, were developed and used for separation and quantitative determination of chlorogenic acid in green coffee bean extracts. For HPTLC silica gel Kieselgel 60 F 254 plates with ethyl acetate/dichlormethane/formic acid/acetic acid/water (100:25:10:10:11, v/v/v/v/v) as mobile phase were used. Densitometric determination of chlorogenic acid by HPTLC was performed at 330 nm. A gradient RP HPLC method was carried out at 330 nm. All necessary validation tests for both methods were developed for their comparison. There were no statistically significant differences between HPLC and HPTLC for quantitative determination of chlorogenic acid according to the test of equality of the means.     

苦荞叶片中芸香甙的薄层色谱定量研究

根据芸香甙的理化性质，建立了一种测定植物样品中芸香甙含量的薄层紫外扫描法，采用硅胶ＧＦ２５４薄层板，样品经甲醇提取浓缩后点样，展开剂为乙酸乙酯－甲酸－水（８＋１＋１）。用薄层色谱扫描仪在波长３５５ｎｍ处测定芸香甙的吸收强度并直接定量。该法操作简便，灵敏度高，定量准确，结果稳定，应用于苦荞叶片中芸香甙含量测定，得到良好结果，线性范围为０～２．０μｇ／μＬ，斑点的最低检出限为５．０ｎｇ，回收率１０２％