J.F.K. Huber

Solvent-generated ion-exchange systems with anionic surfactants for rapid separations of amino acids.

The retention behaviour of amino acids in phase systems consisting of a hydrophobic solid support as the stationary phase and water-organic solvent mixtures containing a small amount of an anionic detergent as the mobile phase was investigated. Such phase systems are found to behave like conventional ion-exchange systems. The degree and order of retention of amino acids can be influenced by changing the temperature, the nature of the hydrophobic support, the pH and the nature and concentration of the anionic detergent, organic constituent and counter ion in the eluent. In many instances this solvent-generated (dynamic) ion-exchange chromatography shows a greater selectivity than conventional ion-exchange systems towards amino acids. The results obtained so far indicate that a complete separation of the 19 protein amino acids by applying solvent gradients or/and multi-column system is possible within 30 min.

Evaluation of dynamic gas chromatographic methods for the determination of adsorption and solution isotherms

Abstract Various dynamic gas chromatographic methods for the determination of distribution isotherms in gas-solid and gas-liquid-solid systems are compared. The influence of the column length, the carrier gas velocity and the nature of the carrier gas is discussed. Isotherms of different shapes are studied. An equation for the calculation of distribution isotherms from gas chromatographic data is derived which applies not only at low mole fractions but also at higher mole fractions.

Temperature gradients in HPLC columns due to viscous heat dissipation

SummaryTemperature effects in HPLC columns due to viscous heat dissipation are examined. For the case when the thermostatted column wall and mobile phase at the column inlet are at the same temperature an explicit solution of the heat transport equation is given. The predicted temperature profile is parabolic at large distances from the column entrance; the magnitude of the effect is proportional to the square of the mobile phase velocity, and is of the order of a few degrees centigrade. At the upper end of the column a relaxation occurs over a length of a few centimers. Experimental results confirm the validity of the predictions made and indicate that the various assumptions and approximations are justified. Plate height curves obtained with two mobile phases with differing viscosities show a much smaller efficiency for the less viscous mobile phase. The curves show an upward curvature at high reduced velocities. Both phenomena can be related to thermal effects. It is concluded that viscous heat dissipation constitutes an obstacle to obtaining higher speed and efficiency in HPLC by the use of smaller particles. Possible remedies, such as the use of smaller bore columns or special thermostatting devices, look troublesome from the experimental point of view.

Column swithiching in high-pressure liquid chromatography

Abstract A high-pressure micro multiport switching valve, its performance and application are described. Its is shown that the contribution of column switching to the peak width is negligible with this valve. Different modes of operation are demonstrated: (1) Control of the total elution of the sample, by-passing the column. (2) Stripping of a part of the sample, using a sectional column system. (3) Separation of a complex mixture on a given phase system using columns of different phase ratio.

Quantitative analysis of trace amounts of estrogenic steroids in pregnancy urine by column liquid-liquid chromatography with ultraviolet detection

Abstract The influence of the sample volume on peak height and resolution in elution column chromatography was studied. Graphs which enable the choice of the optimum sample volume are given. The quantitative analysis of estrogens by column liquid–liquid chromatography with ultraviolet detection was investigated. The method has been applied to the rapid quantitative determination of estriol in urine. After preparing the urine sample for chromatography, the quantitative information can be obtained in 20 min with a precision of 5″6 for a concentration of 3 mg l. The noise of the sample-free detector corresponds to 60 ng of estriol.

Separation of acidic compounds by high-pressure liquid-liquid chromatography involving ion-pair formation

Abstract The distribution of acidic compounds in the phase system tri- n -octylamine-aqueous perchloric acid was investigated. The distribution equilibrium can be expressed by a simple formula which describes the dependence of the distribution coefficient on the single equilibrium constants, pH and ion concentration in the aqueous phase. The theoretical expression was shown to agree well with the experimental data. The distribution of strong acids was caused mainly by ion-pair formation and for weaker acids at low pH by the liquid-liquid distribution of the acid, while at larger pH ion-pair formation can also be involved in the distribution equilibrium. Special selectivity effects can be obtained by changing the temperature, pH or type and concentration of the anion in the aqueous phase. It is shown that the tri- n -octylamine-aqueous perchloric acid system is very suitable for separation of acidic compounds like sulphonic and carboxylic acids by high-pressure liquid-liquid chromatography.

Reduction of theoretical plate height in gas chromatography by increase of pressure and decrease of particle size

Abstract The influence of particle size and pressure on the column efficiency was investigated. The experimental results are explained by means of the theory of the theoretical plate height in chromatography. Columns of small diameter (0.75–1.5 mm) were prepared from porous solid supports of small particle diameters (down to 30 μm) which were coated with low liquid loadings (3–4% w/w) and operated at elevated pressure (inlet pressure up to 50 bar). As a result, a maximum of more than 10,000 theoretical plates per meter of column length were obtained for 30–35-μm particles at an inlet pressure of 25 bar at a mobile phase velocity of 2.5 cm/sec.

Application of high-pressure gas chromatography with columns packed with small particles

Abstract The application of high-pressure gas chromatography using columns with an I.D. of 1.0 mm, packed with small porous particles (below 60 μm), coated with a non-polar or polar stationary liquid, is described. Plots of the theoretical plate height as a function of the mobile phase velocity demonstrate the low mixing characteristics of this type of column. A linear relationship was found between the number of theoretical plates and column length at a high ratio of the inlet and outlet pressures; 55,000 theoretical plates could be achieved at an inlet pressure of 44 bar with a 6-m long column. For use in solving separation problems by gas chromatography, a nomogram has been derived that enables the proper conditions to be chosen. Some chromatograms that demonstrate the potential of these columns are presented. Finally, an example of the dependence of the retention behaviour on the average column pressur is shown.

Influence of static and dynamic effects on the reproducibility of retention data in gas-liquid chromatography

Abstract The influence of static and dynamic effects on the reproducibility of retention data in gas—liquid chromatography was investigated. The static effects considered are the effects due to adsorption, non-ideality of the gas—liquid partition equilibrium and the temperature. Equations are given for estimating the error caused by the non-ideality and the temperature. Adsorption is found to occur in practice mainly at the solid surface and to a smaller extent at the gas—liquid interface. The dynamic effects considered are the effects of the plate number, the resolution, the mole fraction, the retardation of the inert tracer and the time constant of the detector. Equations are given for the quantitative estimation of the errors caused by these effects.

Adaption of the ninhydrin reactor to highly efficient columns for amino acid analysis by chromatography

SummaryThe choice of optimal conditions for the ninhydrin reaction detection of amino acids was studied. By increasing the temperature to 140°C it was possible to reduce the necessary reaction time to about one minute. By using a glass-bead packed bed reactor, peak broadening could be lowered to a level corresponding to a standard deviation of 18 mm3. This allows fast and sensitive amino acid analysis without significant loss in resolution due to the flow reactor. The characteristics of the ninhydrin amino acid detection system such as sensitivity, detection limit and reproducibility are described. Detection limits in the picomole range are obtained.