J.H.M. Van Den Berg

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.

Post-column reactor systems in liquid chromatography

Summary The sensitivity and selectivity of standard liquid chromatographic detectors such as photometers, fluorimeters and coulometric cells can be substantially improved by coupling the exit of the chromatographic column to a chemical reaction system. The additional band broadening in these reactors should be reduced to an acceptable value by an appropriate design of the system. Three types of reactors are used: tubular reactors, packed bed reactors and gas-segmented liquid flow systems. The performances and characteristics of these reactor systems are compared and some specific instrumental problems (mixing, noise level) are discussed. Rules for optimal design are given. The choice of the reactor system depends on the speed and the complexity of the reaction involved. This is illustrated by a number of practical applications of both simple, fast reactions and slower reactions that consist of several steps. Results on band broadening in the reactors and on detection limits are given. Some systems are suitable for the determination of sub-nanogram amounts.

The determination of cortisol in human plasma: Evaluation and comparison of seven assays

In a comparative study, seven different methods for the determination of cortisol in human plasma were evaluated, using routine patient samples. Four of these methods used radioactive steroids (125I- or 3H-labelled) and in three no radioactivity was needed. For the statistical evaluation a direct 3H-radioimmunoassay was arbitrarily taken as the independent variable. It was found that all other methods correlated well with this assay. However, the simplest method, the fluorimetric, cannot be recommended mainly because of its non-specific fluorescence and troublesome interference from some widely-used drugs. Of the methods evaluated a radioimmunoassay is recommended. For laboratories having no equipment for measurements of radioactivity, the more elaborate and time-consuming fluorometric method of Clark may represent a good alternative.

Determination of trace amounts of hydroperoxides by column liquid chromatography and colorimetric detection

Abstract The sensitive and selective determination of separated compounds in effluents from liquid chromatographic columns can be carried out by continuously adding a suitable colorimetric agent to the column effluent and continuously monitoring the absorbance of the reaction mixture. However, a considerable amount of additional broadening of the chromatographic peaks may occur in these systems, especially if slower reactions make it necessary for longer residence times to be used in the reactors. It is shown how this additional broadening can be reduced to an acceptable level by using packed reactors. Some general characteristics of these reactors are discussed and rules for the optimal design are given. A method is described for the determination of hydroperoxides in reaction mixtures from the oxidation of hydrocarbons. Separations are carried out by adsorption chromatography. In an on-line packed reactor, iodine is formed by hydroperoxides in an acidic solution of sodium iodide, and the absorbance of the reaction mixture is measured at 362 nm. A delay time of 1.5 min at a temperature of 70° in the reactor involved a standard deviation of the residence time distribution of less than 1 sec. Nanogram amounts of hydroperoxides can be determined by means of this colorimetric detector.

Reversed-phase liquid chromatography coupled on-line with capillary gas chromatography : I. Introduction of large volumes of aqueous mixtures through an on-column interface

Abstract The feasibility of on-line reversed-phase liquid chromatography (LC)-capillary gas chromatography (GC) using an on-column interface was investigated. Large volumes of acetonitrile—water mixtures were introduced into a retention gap which was coated with a very thin film of Carbowax. The retention gap was tested regulary after reapeted acetonitrile—water injections; its lifetime was several months. The maximum water content in the eluent must be close to taht in the azeotropic mixture, otherwise water which is left in the gap after evaporation of the azeotropic mixture will distort the analysis. An acetonitrile—water mixture allows the introduction of 20–40 μl with an introduction speed of up to 100 μl/min by using the retention gap technique. This means that 1 mm I.D. LC columns can be used in on-line reversed-phase LC—capillary GC work.

Study on the efficiency of support-coated open-tubular columns for steroid analysis

Glass support-coated open-tubular columns for the analysis of steroids in biological samples were prepared with varous support materials. The reproducibility of the procedure of preparing the column was investigated and some fundamental chromatographic properties were studied.

Column liquid chromatography of tricyclic antidepressants.

A column liquid chromatographic system for the analysis of tricyclic antidepressants in serum is described. A high separation efficiency can be obtained with a mixture of ethyl acetate, n-hexane and methylamine as eluent on a silica gel column. The retention is easily regulated by varying the concentration of n-hexane, the modifier methylamine and the water content of the ethyl acetate. Examples are given of separation of test mixtures of tricyclic antidepressants and of some of these drugs in serum. UV detection permits determinations down to the 10-ng level in serum.

A post-column extraction system for the determination of tertiary amines by liquid chromatography with chemiluminescence detection

SummaryThe combination of an ion-pair extraction detection system with peroxyoxalate chemiluminescence detection has been investigated. Ion-pairs of protonated tertiary amines with a chemiluminescent counter ion are on-line post-column extracted to 1,2-dichloroethane containing bis(2,4,6-trichlorophenyl)oxalate (TCPO). Hydrogen peroxide is added to the organic phase by means of a solid-state perhydrit reactor. The influence of the base catalyst (imidazole) on the chemiluminescence reaction in apolar solvents was studied. Some sulfonated chemiluminophores were compared with respect to their chemiluminescence, ion-pairing and extraction properties, and 5-dimethylaminonaphthalene-1-sulfonate was found to be the most suitable reagent. The detection limit of the potential drug secoverine is in the sub-nanogram range.

Separation of copolymers according to composiiton with special emphasis on the effect of block structure

Abstract Block copolymers are a separate but important branch of copolymers. Like all copolymers, they are made of two (or more) different monomers, usually in a sequential manner. The characterization of block copolymers requires, among other things, the measurement of the precursor and other by-products. Gradient high-performance liquid chromatography, which has been used for the separation of statistical copolymers according to composition, can, under suitable conditions, also separate block copolymers with different molecular structures. This method has proved to be efficient even in cases where size-exclusion chromatography has failed. Block copolymers have longer retention times than statistical copolymers of the same composition. This is result of the cooperative effect of adjucent repeat units.

Contribution of electronic effects to the lipophilicity determined by comparison of values of log P obtained by high-performance liquid chromatography and calculation

Abstract In a series of 49 aryl sulphoxides, the partition coefficients (log P ) estimated by high-performance liquid chromatography were compared with log P values calcualted according to the hydrophobic fragmental method of Rekker and a modification of the method of Moreau. Moderate correlations were found. The introduction of Hammett sigma values as a measure of electronic effects proved to be good correction factors for the calculated log P values. In the presence of strong electron-withdrawing substituents, the sulphoxide moiety is far less hydrophobic than in the absence of such groups.