Computer-aided optimization of stepwise gradient and multiple-development thin-layer chromatography

Summary Equations for the final R_F values for mobile phase gradient development and gradient multiple development are presented. Computer simulations of gradient development for both modes of thin-layer chromatography are discussed for the preliminary optimization of gradient programs.     

Improvement of separation in zonal preparative thin-layer chromatography by gradient elution

Summary Complex extracts of the plants Azulan and Hemorigen were separated by zonal micropreparative thin-layer chromatography in sandwich chambers of the ES and DS type which permitted zonal application of large volumes of sample, without auxiliary equipment. Application from the edge of the layer, in the frontal chromatography mode, markedly improved the separation efficiency and capacity owing to displacement effects which narrow the initially broad zones. Further improvement of separation efficiency and purity of fractions, revealed by densitometry, was observed using stepwise gradient elution. This was confirmed by extraction of some of the separated fractions from the layer and rechromatography; the composition of these fractions were generally simpler than for the corresponding isocratic chromatograms.     

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.     

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     

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.     

TLC as a pilot technique for the optimization of gradient HPLC II. Use of data obtained from RP-18 plates for the prediction of gradient programs in reversed-phase liquid chromatography

Summary A method is described and experimentally verified permitting the determination of the optimal gradient programs in HPLC on the basis of isocratic TLC data. The proposed method has many advantages and can also be used in reversed-phase systems. A satisfactory agreement between the theoretical and experimental of V_R/V_O values has been confirmed. Some discrepancies for more strongly retained solutes are, probably due to the respective equilibrium and nonequilibrium state of the column in isocratic and gradient elution.Part I: Ref. [1].     

Optimisation technique for stepwise gradient elution in reversed-phase liquid chromatography

An optimisation technique of reversed-phase liquid chromatographic separations based on gradient elution with a stepwise variation pattern of the volume fraction phi of the organic modifier in the water-organic mobile phase is presented. It uses a non-linear least-squares programme with a Monte-Carlo search for initial estimates in order to determine the best variation pattern that leads to the optimum separation of a mixture of solutes. The validity of the above methodology was tested by separating eight catechol-related solutes with mobile phases modified by methanol or acetonitrile and variation patterns of two, three or four steps in the psi values. It was found in all cases a very satisfactory accuracy of the predicted gradient elution times, which is of the same order with the accuracy of the retention times predicted under isocratic or linear gradient conditions. In addition, it was shown that the proposed optimisation technique is both effective and flexible but well-shaped chromatograms are obtained under electrochemical detection only if steps with increasing psi are used and the change in psi is programmed to occur at the intermediate of the predicted peaks.     

Advanced simplex optimization of two-factor selectivity by high performance thin-layer chromatography

Summary A computer-assisted mixture design simplex method is presented for optimization of the separation of a mixture of five benzoic acid derivatives in reversephase HPTLC and twelve PTH-amino acids in normal phase HPTLC. A two-factor selectivity rectangle concept was used. The method is based on a special polynomial estimated from nine experimental runs, using the R_F difference as the selection criterion, with connection to a general simplex method for optimization using a microcomputer. Excellent agreement is obtained between predicted data and experimental results, and more than half the number of experiments required in the general simplex method can be omitted.     

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.     

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.     

Purification of vandaterosides from Vanda teres (Orchidaceae) by stepwise gradient centrifugal partition chromatography

Vandaterosides are polar glucosyloxybenzyl eucomate derivatives found in Vanda teres (Orchidaceae), which display biological activities that slow the skin ageing process. In order to obtain larger quantities to allow us to go further in the bioassays, the hydroalcoholic extract of aerial parts (leaves and stems) of V. teres were fractionated by centrifugal partition chromatography, combining isocratic, gradient, and dual elution modes. The first fractionation was performed on the extract maintained in the stationary phase as water saturated in butanol, while increasing the polarity of the mobile phase by changing the proportions of ethyl acetate/1‐butanol/water, in order to obtain two enriched fractions. Vandateroside I was then purified by isocratic mode with ethyl acetate/ethanol/water (46:14:40), while vandateroside II was obtained by combining isocratic elution with ethyl acetate/isopropanol/water (30:20:50) followed by a multiple dual mode with ethyl acetate/ethanol/water (46:14:40). In this manner, hundreds of milligrams of vandateroside I and II were recovered from 10 g of V. teres extract.     

Computer simulation and optimization of preparative thin-layer chromatography under conditions of isocratic and stepwise gradient elution

Summary An equilibrium sandwich chamber for continuous thin-layer chromatography was used to study overloaded systems. Mixtures of two or three dyes were used as the model samples, and wide starting zones were formed (volume-overloaded systems). The movement of the zones was recorded during continuous elution. The effect of sample volume and the mode of development (isocratic or gradient) on the maximum separation yield was investigated. A computer program was developed for the calculation of the final R_F values for the front and rear edges of the bands.Satisfactory agreement was found between the experimental R_F values of zone boundaries and the values predicted by computer simulation.Presented at the 2nd International Symposium on Preparative and Up-Scale Liquid Chromatography, February 1–3, 1988, Baden-Baden (FRG).     

Separation of DABS derivatives of amino acids by multiple gradient development (MGD) in thin-layer chromatography

Summary A mixture of 13 DABS-amino acids has been chromatographed on high-performance silica gel layers developed with eluents containing increasing concentrations of ethyl acetate in heptane + chloroform, using a modification of stepwise multiple development MGD described in a earlier paper. Densitograms were obtained at 485 nm. The MGD method was very efficient, separating all 13 DABS-amino acids, and rapid, owing to the use of a non-aqueous mobile phase.     

Computer-aided target optimization for traditional Chinese medicine by ultra-performance liquid chromatography

A new strategy of target optimization was developed for resolving the separation problems of complex samples. Computer-aided target optimization for separation of complex samples by ultra-performance liquid chromatography (UPLC) was studied. Taking traditional Chinese medicine (TCM) (Rhizoma Coptidis) as an example, separation conditions of target compounds - overlapping peaks and all components - were optimized based on moved overlapping separation ranging map (OSRM) and artificially intervention. After calculating retention parameters and peak shape parameters of the target peaks, the optimization was operated in an emulational picture through the computer without further experiments. The emulational chromatogram was proved to be authentic by the estimated values, which were almost identical to experimental results. The method was very helpful for obtaining the satisfied separation conditions of target compounds rapidly and efficiently.     

Correlation in continuous development thin-layer chromatography

Summary Various relationships in continuous development TLC using a binary solvent system are discussed. These include the dependanc of analysis time and required plate length on the shape of a ZR_fvs. solvent composition curve and the dependendancy of analysis time on the spot separation required.Dedicated to Professor A. Zlatkis on the occasion of his 60th birthday.     

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.     

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.     

Thin-layer chromatography and densitometry of anthocyanins in the petals of red poppy during development of the flowers

Summary The variation in anthocyanins content of the petals of red poppy (Papaver, Rhoeas) during development of the flower from the closed bud stage have been investigated. Six-stage gradient thin-layer chromatography of the polyzonalstepwise type was used, starting from ethyl acetate, the last eluent fraction being isopropanol-water-acetic acid (65323). Silica Si 60 was used as adsorbent. The densitograms were recorded at 465 nm; they show gradually increasing peaks of red anthocyanins in the petals of closed buds, partly opened buds and fully developed flower and changes in the proportions of several anthocyanins, with various R_F values, during the growth of the flower.Editor's note: see also J. P. Goiffon, M. Brun and M.-J. Bourrier, J. Chromatogr.517, 101 (1991)