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The rapid separation of mixtures of six peptides using porous polymer monolithic layers in electrophoresis and pressurized planar electrochromatography modes has been achieved. The separations in the former mode were performed on a generic hydrophobic poly(butyl methacrylate-co-ethylene dimethacrylate) layer with no ionizable functionalities and required 2 min. This layer also enabled the separation of three oligonucleotides. The separation in the pressurized planar electrochromatographic mode was carried out using a negatively charged layer prepared via cografting of 2-acrylamido-2-methyl-1-propanesulfonic acid and 2-hydroxyethyl methacrylate on top of the generic hydrophobic monolith and was completed in 1 min.

This report describes the first results from coupling of pressurized planar electrochromatography (PPEC) and desorption electrospray ionization (DESI) mass spectrometry (MS). Nine analytes were separated in 2.0 min by PPEC and detected by direct analysis of the TLC plate using DESI coupled to a tandem mass spectrometer. The total analysis time was less than 10 min and sample preparation was minimal. Combination of DESI–MS with PPEC enhances the specificity and sensitivity of detection of analytes in a mixture.

Recent developments in planar electrochromatography (PEC) in both the normal-phase and the reversed-phase modes, and at both atmospheric and elevated pressure, are reviewed. Other forced-flow techniques in planar chromatography are also briefly covered. Mobile phase migration in PEC is primarily due to electroosmotic flow, which is controlled by the applied electric field. Capillary mediated flow is an important secondary contributor to migration, and occurs because the layer is unsaturated as a consequence of liquid evaporating from the layer due to Joule heating. The magnitude of the electric field and the concentration of ions in solution are important variables that control both electroosmotic flow and Joule heating. Separations are faster and more efficient than those obtained by conventional planar chromatography, provided appropriate experimental conditions are selected. With inappropriate conditions, either mobile phase accumulates on the surface of the sorbent layer, or Joule heating causes excessive evaporation. The former results in poor spot shape, and the latter can cause the layer to dry. Good separations are obtained when there is a balance between these two effects. The problems associated with mobile phase accumulating on the surface of the sorbent layer, and with excessive evaporation of mobile phase, do not occur with pressurized planar electrochromatography. This technique is performed at high pressure, under conditions that allow heat to be removed form the sorbent layer. This allows the use of a substantially higher electric field than in PEC, and results in a high mobile phase flow rate.

Pressurized planar electrochromatography (PPEC) is a new planar chromatographic technique in which the mobile phase is driven by electroosmotic flow, while the sorbent layer is pressurized in a manner that allows heat to flow from the layer through an electrically insulating, thermally conducting, sheet of aluminum nitride ceramic. A prototype apparatus for performing PPEC is described. Separation by PPEC is faster than by conventional TLC, and an example is presented of a 24-fold enhancement in the speed of separation. PPEC was performed on both regular and high-performance C18 layers, and the latter yield substantially faster separation. The sorbent layer requires conditioning at elevated temperature before use, and solute migration velocity increases with this temperature. The flow rate increases in a linear manner with increasing voltage and diminishes in a nonlinear manner with increasing pressure. Both electrical current and Joule heating diminish with increasing pressure, and the diminution of flow at high pressure can be compensated by an increase in voltage. PPEC is more efficient than classical TLC. Theoretical plate heights diminish with increasing Rf and are in the range 29-21 and 55-27 microm for the high-performance and regular plates, respectively. PPEC retains the advantages of classical TLC but has the ability to separate a substantially higher number of samples simultaneously. An example is presented on the separation of nine samples in 1 min on a 2.5 cm x 10 cm sorbent layer.

Planar electrochromatography in the reversed-phase mode has been performed in a commercially available development chamber, which requires only small modification for this purpose. This report illustrates that good separation quality and good reproducibility can both be obtained by use of this apparatus. Only short migration distances - less than 4.0 cm - were obtained under the conditions used.

We have used multiple linear regression to predict either initial rate, log initial rate or specificity for enzyme-catalyzed reactions performed in non-aqueous solvents. The Subtilisin Carlsberg catalyzed transesterification of N -acetyl- l -phenylalanine ethyl ester by methanol, 1-propanol, and 1-butanol was assayed in 30 non-aqueous solvents, and the lipase catalyzed transesterification of methyl methacrylate in 23 non-aqueous solvents. Both sets of reactions were performed at fixed thermodynamic water activity. The lipase catalyzed reactions were also performed in water saturated solvents and in dry solvents. The report illustrates that regression analysis may provide insight into how solvents can alter the activity and specificity of enzymes suspended therein. A regression model for the subtilisin catalyzed reaction suggests that solvents which have a flat hydrophobic region inhibit by competing with the substrate for an enzyme cleft. In the lipase catalyzed reaction, tetrachloroethylene is an outlier (i.e., behaves differently to other solvents) for all the regression models. This deviation, together with an element of structural similarity to the substrate, suggests that tetrachloroethylene acts as a competitive inhibitor. Log P is an important descriptor and it, or an expression containing log P , appears in all the regression equations. Log initial rate is predicted by a two-descriptor model for either enzyme system in solvents of high log P at fixed thermodynamic water activity. Regression models with the same two descriptors predict initial rate for the lipase system over the entire log P range for solvents maintained at fixed thermodynamic water activity and for dry solvents, but not for water saturated solvents.

Either a second order or a third polynomial equation accurately predicts R F in a mobile phase which is a binary mixture of a strong and weak solvent. The mole fraction of the strong solvent is used as the independent variable. The p -nitrobenzyl esters of fifteen dansyl amino acids were used as model solutes for planar chromatography, using each of five different mobile phases on silica gel layers. For either of the polynomial equations, a minimum of 98% of the predicted R F values, at mole fractions not used for establishing the equations, are within 0.05 or less of an R F unit (when compared to the experimental R F ), and 90% of the predictions are within 0.03 or less of an R F unit.

A method for selecting mobile phases for either one-dimensional (1-D) or two-dimensional (2-D) planar chromatography is described and is applied to the separation of steroids by overpressured layer chromatography [Formula: see text] a form of forced-flow thin-layer chromatography [Formula: see text] using both normal- and reversed-phase chromatography. Two metrics are used for evaluating the separation quality of simulated chromatograms for each of 100 (or more) subsets of a set of 30 steroids in each of 15 1-D, and 105 2-D systems. The subsets vary in size between five and 25 steroids. Butyl acetate/toluene on silica gel and aqueous 2,2,2-trifluoroethanol are, on average, the highest and second highest ranked 1-D systems, respectively, for separating all subset sizes. These two systems are the constituent members of the system that is, on average, the highest ranked 2-D system for all subset sizes. The probability of the above systems being highest ranked decreases with decreasing subset size. There is only a 17% probability of butyl acetate/toluene on silica being the best system for separating a subset of five steroids, while there is a 3% probability of this being the worst system for this subset size. The spot capacity of each system can be estimated by considering 100 subsets of each size and noting the largest subset size that yields an acceptable value of one of the metrics used for measuring separation quality. The mobile phase selectivity may be quantified using the actual values of either of the two separation metrics, or by a nonparametric approach. The latter is used in such a way that a difference of unity in the ranking (for a given subset size) of two systems corresponds to a 95% probability that the higher ranked system will yield the better separation.

We have investigated the effect of physical properties of 30 nonaqueous solvents on the specificity of Subtilisin Carlsberg for nucleophiles in the transesterification of N-acetyl-l-phenylalanine ethyl ester by methanol, 1-propanol, and 1-butanol at fixed thermodynamic water activity. In organic solvents, enzyme activity and nucleophile specificity are solvent-dependent, while in supercritical fluoroform, the activity and specificity are pressure-dependent. Losses in catalytic efficiency and substrate specificity are observed when subtilisin is exposed to hydrophilic organic solvents such as dioxane, tetrahydrofuran, and acetonitrile as compared to hydrophobic solvents (hexane and heptane). Log P is an important descriptor for correlating both the rate and the specificity of deacylation with solvent properties. A linear model of log initial rate against both log P and nonpolar unsaturated area provides the best two-variable fit to the data for solvents of high log P. A nonlinear model of specificity again...

Both first- and second-order regression models are presented that relate retention, as the R f of individual solutes, the log k' of individual solutes, or the average R f of a mixture of solutes, to the properties of the weak solvent in each of a series of 25 binary mobile phases consisting of a specified concentration of ethyl acetate as a common strong solvent. The stepwise procedure is used for constructing these models, which are for either simulated or experimental separations on silica gel. Similar regression models are used to predict separation quality, defined by a suitable metric. A comparison of the forward and backward stepwise procedures finds that the former is the more reliable method for constructing these models. The solutes are either steroids or the p-nitrobenzyl esters of dansyl amino acids, and the solvent descriptors are density, dipole moment, molar volume, polarizability, saturated surface area, and unsaturated surface area. The quality of regression fits obtained with models using computed dipole moment is comparable to that obtained with models using experimental (literature) dipole moment. Both nonstandardized and standardized regression models are presented. The relative contribution of each descriptor to the variability in retention may be estimated from the latter models. A set of three descriptors-dipole moment, polarizability, and saturated surface area-predicts R f for each of the amino acid derivatives at an ethyl acetate mole fraction of 0.30. A set of two descriptors-dipole moment and saturated surface area-predicts log k' for each of these compounds at an ethyl acetate mole fraction of 0.20. Such concordance in descriptors is not found in models predicting retention of individual steroids.

An evaluation of an empirical method for the determination of log P values for 24 phenols by thin-layer chromatography is described. Separation is on bonded C8 plates and aqueous methanol is used as the mobile phase. The method is based on the Soczewinski equation and uses standards to define a solvent composition at which RM is equal to log P. In addition, a new approach, based on a paired t-test is described. The t-statistic is used to compare the differences between the literature log P values and the computed RM values over a range of solvent compositions. The p-value corresponding to the t-score can then be used as the measure of agreement between the computed and chromatographic values of log P. The mean difference between the estimated and the literature log P values varies between 0.1 and 0.2 of a log P unit, depending on the method used.

Pressurized planar electrochromatography (PPEC) is a fast and efficient planar chromatographic technique. The mobile phase is driven by electroosmotic flow, while the system is pressurized in a manner that allows heat to flow between the sorbent layer and the pressurizing medium. The reproducibility of solute retention was not satisfactory in the initial report describing PPEC. In the current report, this reproducibility is improved by better control of several experimental variables. The pressure at which PPEC is performed is now free of drift, and the temperature at which the layer is preconditioned is maintained to within +/-1 degrees C. The best reproducibility of retention is obtained when the plate is soaked in the mobile phase for a defined time before each run. In the original prototype, the temperature of the sorbent layer was not controlled. In the present apparatus, water, at a constant temperature between 3 and 60 degrees C, is circulated through channels in the two die blocks that pressurize the layer. The highest efficiency is obtained at an intermediate temperature. This behavior is ascribed to high resistance to mass transfer at the lower temperatures and increased diffusion at higher temperatures. Efficiency, as measured by the number of theoretical plates, increases with increasing migration distance. The height equivalent of a theoretical plate diminishes with increasing migration distance, and values as low as 0.0106 mm are obtained under appropriate conditions. This extrapolates to 94 000 plates/m. Manual spotting was used in this report. Evidence is presented that substantially better efficiency would be obtained if the initial spot size were smaller. The efficiency of PPEC in its current form is illustrated by a chromatogram showing the separation of nine solutes in 2 min. PPEC was also performed with TLC plates in a back-to-back configuration, and this doubles the number of samples that can be simultaneously separated.

Planar electrochromatography is performed by applying an electric field across a thin layer chromatography (TLC) plate. In addition to electroosmotic flow in the axial direction, there is also flow to the surface of the TLC layer, and this can substantially degrade the quality of separation. This effect is offset by Joule heating which causes evaporation of liquid from the layer surface, and which under some conditions causes degradation of separation quality by excessive drying of the layer. It is shown that pH, buffer concentration, and applied voltage control the balance between liquid being driven to the surface and liquid evaporating from the surface due to Joule heating. Conditions are discussed which result in good separation quality, or in separations degraded by either excessive wetting or drying of the layer. The above separations were performed at constant voltage. A chromatogram is presented that shows that a good separation is also obtained at constant power, i.e. under conditions where there is a constant amount of Joule heating.

Planar chromatography with electroosmotic flow is used to separate either a mixture of dyes using 80% aqueous ethanol as the mobile phase or a mixture of miscellaneous compounds using 45% aqueous acetonitrile as the mobile phase. Both mobile phases are 1.0 mM in N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS) buffer. Separations using this technique are faster and more efficient than the same separations by conventional TLC. The respective relationships between migration velocity and applied potential, and between analysis time and distance migrated, are presented.

Several methods for evaluating separation quality in two-dimensional thin layer chromatography are discussed. These include using either the distance between spot centers for the pair of solutes that is the most difficult to separate in a mixture, or the value of a suitable, separation function, as criteria of overall separation quality. Contour diagrams may be constructed in which solvent compositions for each of the two-dimensional developments are the independent variables and spot separation or the value of a suitable function is the dependent variable. Such diagrams indicate which are the best and the worst regions of the two-dimensional solvent domain. These diagrams should be of value for comparing different solvent systems for two-dimensional thin layer chromatography.

A method for the computer simulation of continuous-development thin-layer chromatography (TLC) for the separation of a mixture of 30 steroids id described. Both normal-phase—normal-phase and normal-phase—reversed-phase two-dimensional TLC are discussed. Examples are presented illustrating the agreement between predicted and experimental chromatograms.

A method is described whereby a transevaporatoer is used for sampling 60–100 μl of aqueous sample. Volatiles are stripped from the sample either by a stream of helium and collected on a porous polymer, Tenax, or by 0.8 ml of 2-chloropropane and collected on glass beads. The volatiles are thermally desorbed into a precolumn which is connected to a capillary gas chromatographic column for analysis. The technique is shown to be reproducible and suitable for determining chromatographic profiles for a wide variety of sample types.

A method is described for constructing overlapping resolution maps for thin-layer chromatography. The axes of the maps are time, length and binary solvent composition. These maps can be used to identify sets of compounds within a mixture such that each set is fully separated under appropriate conditions. The separation of a mixture of fifteen steroids is described, using this technique.

A procedure for the thin-layer chromatography of malto-oligosaccharides over a temperature range of 22 to 70° is described. Various mixtures of acetone, ethanol, 2-propanol and water were used as solvent. A method is proposed for estimating the temperature at which oligomers of a given degree of polymerization will be resolved.

Equations for predicting analysis time in thin-layer chromatography as a function of spot separation, solvent velocity constant, ΔRf and Rf, are presented and discussed.

Description d'une methode de separation de melanges complexes par developpements paralleles simultanes, resultant en un temps relativement court de separation. Les methodes de calcul utilisees pour la determination des conditions optimales pour des developpements paralleles peuvent etre etendues a la prevision des conditions optimales pour la chromatographie en couche mince bidimensionnelle

A method is described for the determination of antiarrhythmia drugs in serum by high-performance thin-layer chromatography. Baseline separations are achieved for all the drugs and clozapine, an internal standard, in two developments with solvents of different polarity. Lidocaine and diphenylhydantoin are scanned at 220 nm after the first development. Procainamide, propranolol and quinidine are scanned at 290 nm after the second development. The relative standard deviation of the determination varies between 3 and 14% depending on the nature of the drugs and its concentration.

A method is described for the determination of sucrose in molasses by high-performance thin-layer chromatography. A baseline separation of sucrose is achieved in about 40 min using acetone--water (90:10) as developing solvent. The relative standard deviation of determinations by this method is about 2.2%. Results agree with those obtained by isotope dilution analysis of the same molasses samples.