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.     

Computer-assisted transposition of conditions in high-performance thin-layer chromatography to high-performance liquid chromatography for the separation of pesticides

Summary A computer-assisted method is presented for mobile phase selection for the optimal separation of pesticides by HPTLC and HPLC. The system is based on a plot of solute retention value and separation criterion vs. binary mobile phase composition for graphic optimization. The result of HPTLC can be transposed to HPLC for optimal separation. The transposition equation is given.     

Determination of metabolites relating to the tryptophan-NAD pathway in rat urine by cellulose high performance thin-layer chromatography

High performance thin-layer chromatography (HPTLC) was used to determine 12 metabolites of the tryptophan-NAD pathway in rat urine. The method of separation described in the previous report was improved. The metabolites were separated on the cellulose plate by using five multiple developments with four solvent systems. The plate was scanned by the TLC scanner at 254 nm and the amounts of the metabolites were calculated by comparing peak areas of the scanning curves obtained for the urine sample and for the authentic standards. This procedure can be used as a semiquantitative determination method for the metabolites in biological fluids.     

Electrostatic interactions in normal and reversed-phase high-performance thin-layer chromatography. Preliminary experiments

Summary The retention behaviour of five polycyclic aromatic hydrocarbons having nearly equal ionization potentials but different molecular polarizability values was investigated using normal and reversed-phase high-performance TLC systems. Homologous series of hydrocarbons and n-alcohols were applied as the mobile phases. The electrostatic interactions operating in these chromatographic systems were analysed by studying the relationship between the log 1/R_F values and the molecular polarizabilities of solutes and solvents.     

Separation of eight selected flavan-3-ols on cellulose thin-layer chromatographic plates

The potential of microcristaline cellulose as sorbent in the separation of eight compounds: (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), (-)-epigallocatechin gallate (EGCg), procyanidin B1 and procyanidin B2 was studied. Cellulose HPTLC plates prewashed in water (not necessary, when water was used as developing solvent) and dried with a hair dryer, bandwise application and development in horizontal developing chamber (sandwich configuration) gave the best results. Detection was performed using vanillin-H3PO4 reagent. Four new developing solvent systems were proposed: water, 1-propanol-water (20:80, v/v), 1-propanol-water-acetic acid (4:2:1, v/v) and 1-propanol-water-acetic acid (20:80:1, v/v), and at least two of them were needed for the differentiation between all eight compounds. Surprisingly, water enabled the separation of epimers C from EC and GC from EGC, as well as the dimers procianidin B1 and B2. Additionally, C, EGC, B1 and B2 were separated from all the other compounds. The best choice for developing solvent is given for each of the studied compounds. The best separation of the five main catechins (EC, GC, EGC, ECg, EGCg) present in green tea extract was achieved using 1-propanol-water-acetic acid (20:80:1, v/v). The chromatograms of oak bark extract developed in solvents with higher water content (1-propanol-water (1:4, v/v) and 1-propanol-water-acetic acid (20:80:1, v/v)) showed less bands than chromatograms developed in solvents with higher organic modifier content (e.g. 1-propanol-water-acetic acid (4:2:1, v/v)). It was proved that such behavior was due to the presence of procyanidins beside the main component catechin.     

The use of chemically bonded stationary phases in thin-layer chromatography-a survey

An overview is given of the literature publisned in the field of thin-layer chromatography on chemically bonded phases. Aspects which merit further attention are: quantitative analysis, organic solvent selection, stationary phase characteristics, surface modification of precoated silica plates, ion-pair chromatography and correlation of thin-layer and column chromatographic data.     

Retention parameters of some isomeric 2-benzoylbenzoic acids in reversed-phase ion-pair high performance thin-layer and column chromatography

Summary The 2-benzoylbenzoic acid series was investigated by reversed-phase, high-performance, thin-layer and column chromatography using various alkylammonium salts and di(2-ethylhexyl)orthophosphoric acid as polar associating reagents. The effects of the individual substituents on retention were quantified by log k and R_M values. The compounds investigated differing in molecular structure (hydrophilic and hydrophobic substituents) commonly occurring groups in drugs and biologically active substances provide information on molecular interaction in these ion-pair systems. The combined effects on retention of organic modifier and ion-pair reagent concentration were investigated.     

Comparison of optical in-situ scanning of conventional and high-performance thin-layer chromatograms

Conventional TLC, linear and circular HPTLC are compared by using In-situ reflectance measurements. Chromatographic resolution, time requirement and reproducibility of quantitative scanning of the different TLC methods are investigated by means of three separation problems: benzodiazepines, corticosteroids, polycyclic aromatic hydrocarbons. In all three application examples, which were chosen without prejudice, the circular developing mode with subsequent quantitative scanning showed certain advantages over the corresponding linear HPTLC techniques. These were in the case of

• Benzodiazepines: Better reproducibility of quantitative assessment.
• Corticosteroids: Better resolution and slightly better quantitative reproducibility.
• Polycyclic aromatic hydrocarbons: Chromatography considerably faster.

     

High performance thin-layer chromatography of some sixteen-membered ring macrolide antibiotics

The use of HPTLC in the analysis of some sixteen-membered ring macrolide antibiotices was examined. In the case of Spiramycins, instrumentalized HPTLC proved to be very efficient for the separation and determination of these antibiotics. With the use of an internal standard together with the datapair technique in sampling and evaluation of the HPTLC plates, a coefficient of variation less than 1.5% could be achieved when determining the different Spiramycins. Other sixteen-membered macrolides, such as Tylosins, Turimycins and 9-Propionylmaridomycins can be separated with sufficient resolution for quantitative work, in spite of their extremely simular structures and large molecular weights. Detection is always at wavelengths which agree with the intrinsic absorption maximum of the chromophors of the components (e. g. 282 nm for Tylosins, 232 nm for Spiramycins and Turimycins, 195 nm for 9-Propionylmaridomycins).     

The identification of tricyclic neuroleptics and sulphonamides by high-performance thin-layer chromatography

Circular high-performance thin-layer chromatography (HPTLC) has been used to differentiate a series of twelve tricyclic neuroleptics, using both normal phase and reverse phase procedures. The use of normal phase systems also allows the resolution of geometric isomers of chlorprothixene, clopenthixol and flupenthixol. Thirteen sulphonamides and Trirnethoprim may also be distinguished using HPTLC.     

Synthesis and performance of HPTLC plates modified with cyanopropyl- and octadecyl-trichlorosilane

Chemical modification of commercial high performance thin-layer chromatography plates with various mixtures of cyano-propyltrichlorosilane and octadecyltrichlorosilane is described. Surface coverage by different treatments is demonstrated as well as the variations in chromatographic performance. With regard to the development in aqueous media the utility of CN/ ODS plates compared to ODS plates is also shown.     

Thin-layer chromatographic and high-performance thin-layer chromatographic ready-for-use layers with lipophilic modifications

Stable silica gel sorbents with aliphatic or aromatic groups are formed by chemical modifications of the silanol groups with special reactive silanes. Various lipophilic surface modifications on silica gels with varying pore structures are tested with regard to their chemical and physico-chemical characteristics, their wettability and their chromatographic retention data. The main problem in TLC is the preparation of abrasion-resistant layers on glass or on foils which meet the usual high standard of quality and are also suited for quantitative determinations. Thin-layer chromatography on reversed-phase layers can only be performed if the complete wettability of the lipophilic stationary phase in dry form is guaranteed by the mobile phase. Adsorption-chromatographic separations with lipophilic eluents and reversed-phase partition-chromatographic separations with hydrophilic eluents are performed, for example, with dyes, with polycyclic aromatic hydrocarbons and with lipids. The great differences in selectivity caused by the various modifications of the sorbent and the varying eluent composition are remarkable. Ready-for-use layers with lipophilic surface modifications complement the existing range of pre-coated layers and thus widen the application of TLC and HPTLC considerably.     

Quantitative determination of phospholipids in mitochondria using HPTLC and fluorimetric assay in situ

A method is described for quantitative determination of phospholipids of mitochondria following one-dimensional thin-layer chromatography without previous elution. Using high performance thin-layer chromatography (HPTLC) and an in situ fluorescence technique, the time needed for quantitative determination is relatively short. As the method is rather sensitive, small amounts of the extracts can be applied (about 200 ng of total phospholipids per spot). The reproducibility for the phospholipid fractions determined was in the range of 8–17%. The procedure was tested with lipid extracts of rat liver mitochondria. The method allows the determination of cardiolipin, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl choline, and sphingomyelin.     

Study of dispersive and inductive electrostatic interactions in reversed-phase thin-layer chromatography

Summary The retention behaviour of a group of polycyclic aromatic hydrocarbons having nearly equal ionization potentials but different molecular polarizability values was investigated using reversed-phase HPTLC. Normal alcohols were applied as the mobile phase. The relationships between the retention parameters and molecular polarizabilities of the solutes and solvents are discussed.     

Demixing effects in thin-layer chromatography with NH2-modified silica gel precoated plates

Summary Demixing effects in thin-layer chromatography have been investigated with NH₂-modified silica gel precoated plates and for frequently used hydroorganic binary solvents of various compositions with salt addition.The position of the solvent demixing front depends on: (1) solvent composition, (2) nature of the organic modifier and (3) salt concentration. Whatever the organic modifier, for a given water percentage in the developing solvent, solvent demixing is found to occur at the same concentration of NaCl.