Anomalies in evaporative light scattering detection.

A two-dimensional (2-D) "heart-cutting" HPLC system was used to fractionate oligostyrenes into the respective diastereoisomers. For samples of known composition, the response of an ultraviolet (UV) absorbance detector followed the anticipated pattern. The response of an evaporative light-scattering (ELSD) detector on the other hand indicated quite different concentrations for the two diastereoisomers, relative to what was anticipated and what was indicated by the UV detector. Whereas approximately the same concentration was indicated by UV, ELSD in some cases indicated no detection of the later eluting isomer. The magnitude of the errors depended on both the molecular weight and the tacticity of the diastereomers. These anomalies appear to be an artifact of power transform functions imbedded within the firmware processor of the ELSD, invisible to the user.

Application of power functions to chromatographic data for the enhancement of signal to noise ratios and separation resolution.

Chromatographic detection responses are recorded digitally. A peak is represented ideally by a Guassian distribution. Raising a Guassian distribution to the power 'n' increases the height of the peak to that power, but decreases the standard deviation by radicaln. Hence there is an increasing disparity in detection responses as the signal moves from low level noise, with a corresponding decrease in peak width. This increases the S/N ratio and increases peak to peak resolution. The ramifications of these factors are that poor resolution in complex chromatographic data can be improved, and low signal responses embedded at near noise levels can be enhanced. The application of this data treatment process is potentially very useful in 2D-HPLC where sample dilution occurs between dimension, reducing signal response, and in the application of post-reaction detection methods, where band broadening is increased by virtue of reaction coils. In this work power functions applied to chromatographic data are discussed in the context of (a) complex separation problems, (b) 2D-HPLC separations, and (c) post-column reaction detectors.

Reproducibility of the finger pattern in viscous fingering.

The complex pattern of viscous fingering (VF) appears to be chaotic. Its early evolution seems predictable but, on a long time-scale, it is not so and the transition is complex. Detailed experimental observations of fingering systems have been hindered by various limitations. We present a new method for visualising VF in particulate beds. Our results show that the onset of VF and its initial evolution are reasonably reproducible at very low Reynolds numbers (Re < 0.005). The transition to irreproducibility of the fingering pattern develops progressively over long migration distances. When the flow velocity increases, changes in the finger distribution take place over shorter migration distances, become more important, and the fingers evolve faster. Understanding this new aspect might allow improvements in the efficiency of processes governed by VF, e.g. injection of large, concentrated samples or transfer of large fractions between two streams having different viscosities.

Adsorption isotherms and retention behavior of 1,1'-bis(2-naphthol) on CHIRIS AD1 and CHIRIS AD2 columns.

The separation of the atropoisomers of 1,1'-bis(2-naphthol) was studied on CHIRIS AD1 and CHIRIS AD2, two Pirkle-type chiral stationary phases. Satisfactory selectivity was found only on CHIRIS AD2. The ternary mobile phases comprised hexane, dichloromethane and methanol. The effects of their composition and of the temperature on the retention under analytical conditions and on the single-component and competitive isotherms were investigated. The retention of the R- and S-isomers on CHIRIS AD1 and CHIRIS AD2 is controlled by the enthalpic contribution to adsorption, but the effect of the mobile phase on the retention should be attributed mainly to the entropic contribution. The adsorption of the less retained R-isomer is controlled by the achiral interactions, which are the same as for the S-isomer. The single-component and competitive isotherms of the R- and S-isomers are adequately described by the sum of a Langmuir term for the achiral contribution to adsorption and a linear-term characterising the selective or chiral adsorption of the S-isomer in the concentration range experimentally available, i.e. within the solubility limit of 1,1'-bis(2-naphthol).

Study of the mass transfer kinetics in a monolithic column.

The purpose of this work is to investigate the mass transfer kinetics of butylbenzoate on a monolithic RPLC column, with methanol-water (65:35, v/v) as the mobile phase. We used the perturbation method, measuring the height equivalent to a theoretical plate (HETP) of the peaks obtained as the response to small pulses of solute injected on a concentration plateau. The equilibrium isotherm of butylbenzoate was previously determined by frontal analysis. It is well accounted for by a liquid-solid extended multilayer BET isotherm model. The equilibrium data derived from the pulse method are in excellent agreement with those of frontal analysis in the accessible concentration range of 0 to 8 g/dm3. Plots of the HETP of small pulses. injected on eight different plateau concentrations, were acquired in a wide range of mobile phase flow velocities. The axial dispersion and the mass transfer kinetic coefficients were derived from these data. The validity of these measurements is discussed. The mass kinetics of butylbenzoate depends strongly on the plateau concentration. Processes involving adsorptive interactions between the solute and the stationary phase, e.g. surface diffusion and adsorption-desorption kinetics, combine in series to the external mass transfer kinetics and to effective pore diffusivity.

Comparison of the adsorption equilibrium of a few low-molecular mass compounds on a monolithic and a packed column in reversed-phase liquid chromatography.

Adsorption isotherm data were acquired by frontal analysis for several low-molecular mass compounds (3-phenyl 1-propanol, 4-tert.-butylphenol, butylbenzene, and butyl benzoate) on a classical packed column and a monolithic column using methanol-water RP-HPLC conditions. These columns have similar characteristics (C18-bonded silica, close specific surface areas and bonding densities). In each case, the isotherm model best accounting for the data was the same on both columns. The solute polarity determines the class of this model. For the two -OH compounds it was a Langmuirian adsorption isotherm. The hydrocarbon data were best modeled by an anti-Langmuir convex-downward isotherm model. The adsorption data for the aromatic ester exhibited a nearly linear behavior, depending on the methanol concentration of the mobile phase. A slightly convex downward isotherm was obtained at high methanol concentrations while the best fitting was obtained with a liquid-solid extended multilayer B.E.T. isotherm model at low concentrations. The validation of these models is discussed in detail. In all cases, similar values of the adsorption-desorption constants were found, underlining the closeness of the adsorption energies on both columns. By contrast, the adsorption capacity of the monolithic column was found to be approximately 1.4 greater than that of the packed column in spite of the close values of the surface areas of the silica in both columns.

Measurement and modeling of the equilibrium behavior of the Tröger's base enantiomers on an amylose-based chiral stationary phase.

The binary isotherms of the two enantiomers of Tröger's base were measured on a system made of pure 2-propanol as the mobile phase and of Chiralpak AD, a chiral stationary phase (CSP) based on amylose tri-(3,5-dimethylphenyl carbamate). The experimental data were acquired using both frontal analysis and the perturbation method. The results obtained are most unusual. The adsorption of the more-retained (-)-enantiomer is not competitive: the amount adsorbed onto the CSP at equilibrium with a constant concentration of the (-)-enantiomer is independent of the concentration of the (+) enantiomer. On the other hand, the adsorption of the less-retained enantiomer is cooperative: the amount of this (+)-enantiomer adsorbed by the CSP at equilibrium with a constant concentration of this enantiomer increases with increasing concentration of the (-)-enantiomer. Such a phenomenon has hardly ever been reported. A model equation is proposed that accounts well for all these isotherm data.

Experimental studies of pressure/temperature dependence of protein adsorption equilibrium in reversed-phase high-performance liquid chromatography.

The effect of the average pressure and temperature of the column on the adsorption equilibrium of insulin variants on a C8 bonded silica was studied in isocratic reversed-phase HPLC. Analytical injections of samples of four different insulins (bovine, porcine. Lys-Pro and human recombinant) were carried out at constant flow-rate but under increased average pressure. The temperature dependence of the retention parameters over the range 25-50 degrees C was studied under two different average column pressures (47 and 147 bar). Substantial increases of the retention time (up to 300%) were observed when the pressure and/or the temperature were increased. Similar adsorption-induced changes in the partial molar volume at constant temperature (deltaVm approximately 102 ml/mol) were found for all the variants studied. Furthermore, deltaVm was revealed to be practically independent of the temperature, which suggests that the temperature has no or very little influence on the mechanism of the pressure induced perturbations in the molecular structure of the solute. This conclusion was also derived from the observed temperature dependence of the logarithm of the retention factor (k) measured under different pressures. The relation between the temperature and In k was nonlinear with a parabolic shape. Moreover, the shapes of the plots corresponding to the low and high pressures were found to be exactly the same, except that the curves were vertically shifted, due to the difference between the two average column pressures. These results indicate that pressure and temperature affect the retention behavior of insulins in a different and separate way.

Influence of the pH on the behavior of an imprinted polymeric stationary phase--supporting evidence for a binding site model.

The equilibrium isotherms of the two enantiomers of phenylalanine anilide (PA) were measured by conventional frontal analysis at three different pH on a thermally-treated imprinted stationary phase selective for the L enantiomer. The first of these pH (buffer pH=3.0, pH(app)=4.0) is well below the apparent pKa (6.4) of the two solutes, the second (buffer pH=5.8, pH(app)=7.0) slightly below this pKa, and the third (buffer pH=7.0, pH(app)=8.3) well above it. The experimental data were fitted to several isotherm models. The best estimates of the parameters of these models are reported and discussed. The corresponding isotherms are compared with the experimental ones. The contributions of the enantioselective and nonselective interactions could be separated. The results obtained show that the saturation capacity is always smaller for D-PA than for L-PA, the template. The analytical separation is best at pH=3.0 because of a good separation factor (2.82) and short retention times. A good compromise between the resolution and the saturation capacity is obtained at pH=5.8, for which the best preparative separation is found. Both analytical and preparative results are poor at pH=7.0 because the separation factor is low (1.32). At this pH, the isotherm remains nearly linear in the whole concentration range accessible to measurements. The number of nonselective sites increases with increasing mobile phase pH slightly faster than the number of selective sites, suggesting different pKa ranges for the two type of sites. Moreover, the binding energy and the homogeneity of the selective sites decreases with increasing pH. These results agree with a binding site model involving more than one carboxylic acid group, providing charge complementarity and hydrogen bond donors for binding of L-PA.

Comparative modeling of breakthrough curves of bovine serum albumin in anion-exchange chromatography.

The experimental results of a previous study of the mass transfer kinetics of bovine serum albumin (BSA) in ion-exchange chromatography, under nonlinear conditions, were reevaluated using the general rate model of chromatography. Solutions of this model were obtained numerically. The influences of axial dispersion, the resistance to mass transfer from the bulk mobile phase to the surface of the packing particles, and the intraparticle mass transfer resistances on the profiles of the breakthrough curves of BSA were investigated. The results obtained are compared to those of a previous investigation of the same data, using the simple transport-dispersive model and the lumped pore diffusion model. The results obtained show that the use of an oversimplified model for the analysis of chromatographic data can lead to erroneous interpretations of the experimental data and to misunderstandings of the fundamentals of the processes involved. Finally, a theoretical comparison between the properties and the range of application of the three models is provided.

Fitting adsorption isotherms to the distribution data determined using packed micro-columns for high-performance liquid chromatography.

Knowing the adsorption isotherms of the components of a mixture on the chromatographic system used to separate them is necessary for a better understanding of the separation process and for the optimization of the production rate and costs in preparative high-performance liquid chromatography (HPLC). Currently, adsorption isotherms are usually measured by frontal analysis, using conventional analytical columns. Unfortunately, this approach requires relatively large quantities of pure compounds, and hence is expensive, especially in the case of pure enantiomers. In this work, we investigated the possible use of packed micro-bore and capillary HPLC columns for the determination of adsorption isotherms of benzophenone, o-cresol and phenol in reversed-phase systems and of the enantiomers of mandelic acid on a Teicoplanin chiral stationary phase. We found a reasonable agreement between the isotherm coefficients of the model compounds determined on micro-columns and on conventional analytical columns packed with the same material. Both frontal analysis and perturbation techniques could be used for this determination. The consumption of pure compounds needed to determine the isotherms decreases proportionally to the second power of the decrease in the column inner diameter, i.e. 10 times for a micro-bore column (1 mm I.D.) and 100 times for capillary columns (0.32 mm I.D.) with respect to 3.3 mm I.D. conventional columns.

Retention and mass transfer characteristics in reversed-phase liquid chromatography using a tetrahydrofuran-water solution as the mobile phase.

The characteristics of the retention and the mass transfer kinetics in reversed-phase liquid chromatography (RPLC) were measured on a system consisting of a C18-silica gel and a tetrahydrofuran-water (50:50, v/v) solution. These parameters were derived from the first and the second moments of the elution peaks, respectively. Further information on the thermodynamic properties of this system was derived from the temperature dependence of these moments. Some correlations previously established were confirmed for this system, namely, an enthalpy-entropy compensation for both retention and surface diffusion and a linear free-energy relationship. These results are compared with those observed in other similar systems using methanol-water (70:30, v/v) and acetonitnile-water (70:30, v/v) solutions. The contribution of surface diffusion to intraparticle diffusion in C18-silica gel particles was shown to be important. The analysis of the thermodynamic properties of surface diffusion suggests that, in these three RPLC systems, its activation energy is lower than the isosteric heat of adsorption. The nature and the extent of the influence of the mobile phase composition on the parameters describing the retention and the mass transfer kinetics are different but the chromatographic mechanisms involved in RPLC systems appear similar, irrespective of the nature of the organic modifier in the mobile phase.

Analysis of the surface diffusion of alkylbenzenes and p-alkylphenols in reversed-phase liquid chromatography using the surface-restricted molecular diffusion model.

Surface diffusion of alkylbenzene and p-alkylphenol derivatives was measured in reversed-phase liquid chromatography on a C18-silica column with a methanol/water mixture (70/30, v/v) as the mobile phase. They were analyzed on the basis of the restricted molecular diffusion model for surface diffusion. The temperature dependence of the surface diffusion coefficient (Ds) arises probably from that of molecular diffusivity, suggesting a correlation between surface and molecular diffusion. Other correlations were also observed: (1) an enthalpy-entropy compensation of the retention equilibrium constant, (2) a linear free energy relationship between the retention and surface diffusion, and (3) a linear correlation between the restriction energy of surface diffusion and the isosteric heat of adsorption. The physical meaning of parameters involved in the surface-restricted molecular diffusion model is discussed on the basis of these results. A practical and convenient procedure for the estimation of Ds is also suggested. This study demonstrates how surface diffusion phenomena can be accounted for on the basis of the correlation between surface and molecular diffusion.

Visualization of solute migration in chromatographic columns influence of the frit porosity.

The dual-perspective, on-column detection method previously described was used to observe the effects of the inlet frit on the profiles of chromatographic bands. Visualization of bands of iodine was achieved by injecting its dilute solutions in carbon tetrachloride into a glass column packed with a C18-bonded silica and eluted with carbon tetrachloride, which has the same refractive index as the packing material. The bands were photographed on-column with two standard 35-mm SLR cameras oriented at right angle. The photographs were scanned and the digitized images of the sample bands analyzed with proper software. A number of columns, as similar as possible, were fitted with different 2- and 10-microm porosity stainless steel frits. Subsequent analysis of the digitized band images revealed irregularities in the band shape resulting from frit contributions to band dispersion. The 2-microm frits produced more dramatic effects overall than the coarser frits. Local axial dispersion coefficient values, expressed as local reduced plate height, were calculated. The results demonstrate the possibly damaging effects of the frit on the band shape.

Effect of the mobile phase on the retention behaviour of optical isomers of carboxylic acids and amino acids in liquid chromatography on bonded Teicoplanin columns.

Conditions for separation of enantiomers of underivatized amino acids phenyl glycine and tryptophan and of mandelic acid as test compounds were studied on a Chirobiotic T column packed with amphoteric glycopeptide Teicoplanin covalently bonded to the surface of silica gel. The effects of the mobile phase composition on the retention and selectivity under analytical conditions, on the profile of the adsorption isotherms of the enantiomers and on the overloaded separation were investigated. The concentration of ethanol or of methanol in aqueous-organic mobile phases and the pH of the mobile phase affect not only the retention and selectivity, the saturation capacity and the isotherm profile, but also the solubility of the acids, which should be taken into account in development of preparative separations. A compromise between the separation selectivity and the solubility should be made in selecting the mobile phase suitable to accomplish preparative separations at acceptable production rate and throughput of the operation.

Validation of a chromatography data analysis software.

The performance of chromatography data analysis software packages is of cardinal importance when the precision and the accuracy of a chromatographic system are evaluated. Users cannot rely on a procedure generating chromatographic data of known accuracy. Holistic approaches cannot always be entirely trusted. We propose a new method consisting in validating a data analysis package against computer generated chromatograms of exactly known characteristics by feeding these chromatograms into the vendor supplied software and comparing the results supplied by the software and the exact answers. We simulated symmetrical and tailing chromatograms and processed these signals with the Agilent Technologies (formerly Hewlett-Packard) ChemStation software. The noise profile (i.e. the power spectrum of the baseline) was determined for a HPLC UV detector prior to the calculations, and chromatograms of different signal-to-noise ratios were used for the analysis. For every chromatogram, we simulated 25 replicates with identical signal-to-noise ratios but different noise sequences. In this manner, both the random and the systematic errors of the retention data and peak shape characteristics can be evaluated. When analyzing tailing peaks, we simulated the effects of extra-column band broadening and those of column overload. Our calculations show that the general performance of the data analysis system studied is excellent. The contribution of the random error originating from the data analysis procedure is in most cases negligible compared to the repeatability of the chromatographic measurement itself.

Identification of the factors that influence the reproducibility of chromatographic retention data.

Principal component analysis was used to identify the parameters that influence the column-to-column and batch-to-batch reproducibility of retention times and retention factors measured on Symmetry C18, Kromasil C18, Luna C18 (2) and Vydac RP C18, all reversed-phase silica columns. We devised a procedure that allows the determination of the differences in column volume and packing density between two columns, provided that these columns are packed with identical stationary phases (i.e., phases that originate from the same batch). Principal component analysis of the retention times confirmed that the column-to-column variations of the column volume and the total porosity of the bed are the factors that influence the reproducibility of the retention times, the column volume being the major factor. For the fluctuations of the retention factors, the column phase ratios (or the bed porosities) and some specific, secondary retention mechanisms are responsible. All the C18 columns investigated proved to behave in a very similar fashion. Two principal components were always sufficient to characterize the variations of either the retention times or the retention factors.

Repeatability and reproducibility of retention data and band profiles on reversed-phase liquid chromatography columns. V. Results obtained with Vydac 218TP C18 columns.

The general goal of this work is to investigate the precision of chromatographic data and to determine which properties of chromatographic columns influence this factor. Chromatographic data were acquired under five different sets of experimental conditions for 30 neutral, acidic and basic test compounds on columns packed with Vydac 218TP C18, a polymeric, wide-pore silica-based stationary phase. Five columns packed with samples from the same batch of this packing material were used to measure the column-to-column reproducibility and six columns packed with material from six different batches to measure the batch-to-batch reproducibility. The parameters studied were the retention time, the retention and separation factors, the hydrophobic and the steric selectivities, the column efficiency, and the tailing factor.