Parameters of preparative centrifugal thin-layer chromatography

Summary The separation performance of preparative, centrifugal thin-layer chromatography was investigated for various flow rates and rotational speeds by means of a modified commerical instrument. High flow rates give rapid separations but require large amounts of solvent. Maximum separation (that is, greatest resolution R_s) is obtained at medium speeds and flow rates. The maximum sample size for a successful separation depends primarily on the type of substance. It varies from 10 to 1 000 mg per compound and per mm of layer thickness. Centrifugal TLC is superior to conventional layer chromatography. Possible improvements of the instrument are pointed out.     

Influence of solvent strength in possible optimization of adsorption thin-layer chromatography

Considerations of TLC process optimization have been based on the thermodynamic theory of adsorption from multicomponent solvents using experimental and theoretical R_{M1, 2} = f (Φ₁) relationships. It was found that a relationship exists between the A_z parameter (log k where k is the partition coefficient of the substance chromatographed) of the above theory and pK_a values of substances as well as the solubility parameter δ of the mobile phase components. Analysis of the A_z values of substances shows that a slight variation therein is associated with lower selectivity of chromatographic separation.     

Progress in forced-flow planar chromatography

This paper summarizes progress in forced-flow planar chromatography (FFPC) and demonstrates the importance of the different techniques like rotation planar chromatography (RPC), overpressured-layer chromatography (OPLC), and electro-planar chromatography (EPC). Special attention is paid to a novel analytical FFPC method in which continuous development and continuous evaporation of the mobile phase from the end of the chromatographic plate ensure forced-flow development. A simple, but powerful preparative forced-flow technique is also reported; in this technique hydrostatic pressure is used to increase mobile-phase velocity. Parallel- and serially coupled layers open up new vistas for the analysis of a large number of samples (up to 216) for high throughput screening and for the analysis of very complex matrices. The special features of fully off-line and fully on-line RPC, OPLC, and EPC are compared in a table. New detection methods—on-line coupling of OPLC with radiodetection and on-line OPLC–MS—are also discussed. The role of a new spraying device for post-chromatographic chemical detection and for biological detection is also discussed. Some applications, relating to different classes of substances, are given to demonstrate the versatility of the various FFPC techniques.     

Therapeutic drug monitoring by high performance thin-layer chromatography

Summary Methods for measuring concentrations of therapeutic drugs in blood serum have been devised which involve separation of the drugs by high-performance thin-layer chromatography (HPTLC) on silica gel followed by quantification by reflectance or fluorescence scanning. Aliquots of from 20 to 300 L serum are extracted with organic solvent. Portions of the extract containing approximately 1/4 of the drug present are deposited on a Contact Spotter (Clarke Analytical Systems), concentrated to a pin-point and applied to the HPTLC plate which is then developed and scanned.HPTLC permitted 12 samples and three standard solutions of drugs used to treat cardiac arrhythmias to be assyed for 6 drugs simultaneously, with scanning at a single wavelength. Results of these assays correlated well with those by EMIT^® immunoassays and by HPLC. No interference by other common drugs was observed. The total time required was two hours, making the work output esiily competitive with that of moderately rapid HPLC. Since sample preparation and quantification by scanning required only a small portion of this time, and the chromatography, which was time consuming, could be done on all the samples simultaneously, analysis of twice as many samples requires only an additional 1/2 hour. HPTLC thus offered the possibility of highly efficient mass production with relatively little capital equipment. Assay of chloramphenicol in serum, which in our laboratory requires fairly rapid turnaround of nnly a few samples at one time, required a similar length of time. Since standards and quality assurance samples could be assayed simultaneously, the analytical output was again comparable to that from a single HPLC instrument.Dedicated to Professor A. Zlatkis on the occasion of his 60th birthday.     

Gradient thin-layer chromatography of plant extracts

Summary A modified Niederwieser chamber for stepwise gradient elution, consisting of a PTFE capillary to store the series of eluents and a horizontal glass sandwich chamber with a glass distributor was used for the chromatographic separation of complex plant extracts (Seboren, Hemorigen and Pectosol) used in therapy. Densitograms demonstrate markedly improved separations of the extracts in comparison to isocratic elution.Produced by Polish Reagents, Melgiewska 18, 20-234 Lublin, Poland     

Gradient thin-layer chromatography of extracts fromDigitalis lanata andDigitalis purpurea

Summary Glycosides isolated fromDigitalis lanata andDigitalis purpurea (1985–1991 crops) were chromatographed on High-Performance precoated silica layers using stepwise gradient elution, the eluents containing increasing concentrations of methanol in ethyl acetate. The extracts were separated into about 20 components, presumably glycosides, some of which were identified using a mixture of standards. Densitograms at 365 nm were also obtained. The adsorbent-eluent system was very efficient and the separation was rapid owing to the use of a non-aqueous mobile phase. The method is suitable for the comparative analysis of lanatosides in plant extracts.     

Influence of chamber saturation in thin-layer chromatography on theoretical and experimental parameters of mixture separations

The influence of chamber saturation in thin-layer chromatography on theoretical and experimental parameters of mixture separations were examined. Five different types of chambers for development of chromatograms were taken into consideration. The relation found experimentally differed widely from that calculated using O?cik's equation for different method of carrying out of the chromatographic process. The investigations presented here show the possibility of choosing chromatographic chambers which are optimum for theoretical studies on the process of thin-layer chromatography.     

Computer-aided optimization of stepwise gradient profiles in thin-layer chromatography

Summary Two mixtures, one containing model coloured solutes and the other glycosides from the Digitalis species were separated by stepwise gradient thin-layer chromatography, the first in the system silica-toluene + ethyl acetate, the second in the system silica-ethyl acetate + methanol. The optimal gradient programs (i.e., for which maximal spread of spots along the plate is observed) were determined using a computer program reported in an earlier paper. Good separation and satisfactory agreement of predicted and experimental R_F values were obtained; the non-aqueous system for the separation of lanatosides permits considerable shortening of elution time owing to the lower viscosity of ethyl acetate-methanol mixtures.Dedicated to Professor Leslie S. Ettre on the occasion of his 70th birthday.     

Forced flow,and physical field enhanced thin-layer chromatography

Planar chromatography was firstly introduced by Izmailow and Schreiber in 1938. Since then it has been used as a one of the basic techniques in liquid chromatography, but after introducing High Performance Liquid Chromatography in the 70’s of the past century by many investigators has been marginalized due to the worse separation capabilities and efficiency. The attempt of solving this problem was introducing forced flow planar techniques, and coupling it with other method of separation. In this article the ideas and history of Rotational Planar chromatography, Overpressured Layer Chromatography Planar Electrochromatography, and Thin Layer chromatography in Magnetic field have been presented. The short discussion on strong and weak sides of every presented technique and their further development possibilities was also included.     

Influence of development distance on resolution in thin-layer chromatography

Theoretical equations are given to relate resolution with development distance. Comparative experiments undertaken with quaternary ammonium compounds as test substances and using a selection of basic drugs are presented. As expected, resolution decreases with shorter development distance. However, this effect is less pronounced for compounds with small R_f values and when the spot size at the starting point is kept small. It was also shown that, for correctly assessing resolution in relation to development distance, the amounts of material spotted have to be decreased for shorter development distances.     

Improved separation of amino acids by thin-layer chromatography

Summary The separation of 35 amino acids on Avicel F layers was investigated and 6 solvent systems are recommended for use either singly or in combination in 2-dimensional chromatography. The mechanisms and limitations of these methods are discussed.     

Computer-assisted optimization of pesticides separation by two-dimensional high-performance thin-layer chromatography

Summary A computer-assisted method is presented for optimization of two mobile phase selection for separation of a mixture of eight pesticides in two-dimensional TLC. Optimization of the separation over the experimental region is based on two special polynomial estimations from preliminary runs of two groups. Using D (distance of two spots) as the selection criterion with a two factor statistical scanning technique excellent agreement is obtained between predicted and experimental results.     

Optimization of reversed-phase ion-pair chromatography by over-pressurized thin-layer chromatography III. Over-pressurized thin-layer chromatography using 10-camphor sulfonic acid as ion-pair reagent

Summary The use of over-pressurized thin-layer chromatography in ion pair system using acidic type pairing reagent has been studied. The most important aspect when reversed phase ion-pair TLC system is used to apply a suitable procedure for pre-treatment of the sorbents. Because of the acidic type of ion pair reagents cannot be bounded to the surface of the layer by immersion or pre-development with the reagent solution, double coating technique was used for the pre-treatment, the plate was firstly immersed in an ethanolic solution of cetrimide, then the immersion has been repeated by an ethanolic solution of acidic ion pair reagent. The necessary coating of the sorbents can be achieved by this technique. To find the optimal conditions for reversed phase ion pair TLC separation of organic amines, 10-camphor sulfonic acid as reagent, different aliphatic, aromatic amines and diamines and heterocyclic nitrogen compounds, respectively as model compounds were selected. The dependence of the selectivity and efficiency of the separation on the sorbents, on the concentrations of reagents (cetrimide and camphor sulfonic acid) applied for both immersion and in the eluent were investigated in detail. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Anticircular high performance thin-layer chromatography

The principal analytical details for the third of three possible modes in high performance thin-layer chromatography are given, namely the anticircular mode. Separation is achieved by allowing the mobile phase to enter the plate layer on a precise outer circle line, from where it flows towards the centre with nearly constant speed. This technique is theoretically and practically the fastest of all three possible in HPTLC. It permits maximum sample capacity with a minimum of time, layer and mobile phase consumption. It is therefore the most economical HPTLC technique. A new carrier-free mobile phase transfer principle is used. The conditions for qualitative and quantitative analysis are good: repeatability, reproducibility and accuracy of routine TLC analyses are superior to those achieved by the classical trough technique. The specially narrow spot-path in anticircular HPTLC facilitates automated quantitation. Compared with the linear and circular modes, the anticircular mode shows better separation and significantly increased sensitivity at higher Rf-values. The drawback, however, is that the separation power (expressed by the separation number) is lower compared with the other two modes.     

Analysis of gardenia blue in foods by thin-layer chromatography

In the present study, a thin-layer chromatography (TLC) method for the analysis of gardenia blue was described. Gardenia blue is obtained from the fruits of Gardenia augusta Merrill or Gardenia jasminoides Ellis. Recently, gardenia blue has frequently been used in Japan as a natural coloring in various foods. However, the structural characterization of gardenia blue components has not been yet clarified and even chromatographic separation of the components has not been reported. Synthetic colors in foods are generally analyzed by TLC; so, we therefore investigated the analysis of gardenia blue in foods with TLC. We established two TLC conditions; reversed phase C-18 TLC using a solvent system of aqueous 0.2% TFA–acetonitrile–ethanol (1:2:3) and TLC on cellulose plate using a solvent system consisting of acetone–3-methyl-1-butanol–water (6:5:5). Both conditions yielded three well-delineated spots with good separation. We applied these separations to the analyses of gardenia blue in coloring matter preparations from different manufacturers and in foods on the market. After the gardenia blue was extracted from the samples with water, the extract was evaporated and the residue was dissolved in water–methanol (1:1). Aliquots of the dissolved solutions were then applied to TLC and their chromatographic behaviors were observed. Each preparation showed characteristic spot patterns depending on the manufacturers. The R_f values of the separated spots when extracted from foods are slightly different from the R_f values of the spots observed for standards, and we were thus able to identify the manufacturers using the spot pattern of gardenia blue. The present study is considered to be useful for the establishment of a method of analysis for gardenia blue in coloring preparations and foods.     

A new technique for the determination of polyamines: Overpressured ion-exchange thin-layer chromatography

Summary An overpressured, thin-layer ion-exchange, chromatographic method was developed for the separation of basic amino acids and natural polyamines. This technique, combined with video-densitometric evaluation, appeared to be suitable for the quantitative determination of polyamines in tissue extracts prepared without any previous purification.     

Determination of methyl nicotinate in pharmaceutical creams by high-performance thin-layer chromatography

Summary A rapid, simple and specific high-performance, thin-layer chromatographic, photodensitometric method is described for the quantitative determination of methyl nicotinate. The HPTLC plates, coated with silica gel, are developed with a mobile phase which allows the separation of several active components in pharmaceutical creams. After quantitation of methyl nicotinate, a second solvent can be used for the identification of the cream base excipients.     

Separation of aliphatic and aromatic amines by thin-layer chromatography using silica gel plates

Summary 28 different aromatic and aliphatic amines have been separated by TLC; 20 solvent systems were examined.     

Determination of bismuth by forced-flow liquid chromatography

Bismuth(III) is retained on a cation-exchange column from dilute acid and is then separated from most other metal ions by elution with 0.5M hydrobromic acid. The elution curve can be measured spectrophotometrically and automatically recorded. This separation method is rapid and is selective for bismuth.