Elena Katz

Retenion characteristics of several bonded-phase liquid chromatography columns for some polycyclic aromatic hydrocarbons

Abstract We have compared the retention of twelve polycyclic aromatic hydrocarbons (PAHs) on columns packed with octadecyl bonded phases from eight different manufacturers. The capacity ratios and selectivity factors were calculated for each compound on every column, under conditions of isocratic elution with identical mobile phase strength and similar linear flow velocities. We observed a wide range of capacity ratios and significantly different selectivity on different material. These data did not correlate with the carbon content or the surface concentration of alkyl groups for the various stationary phases. We also report the retention and selectivity characteristics for these PAHs on different manufacturing lots of the octadecyl silica used for one of the commercial columns.

Distribution of a solute between two phases: the basic theory and its application to the prediction of chromatographic retention

Abstract A simple theory that explains the distribution of a solute between two phases is put forward and experimentally validated employing both previously published data and results from unambiguous liquid—liquid distribution experiments. The theory is tehn extended to liquid—liquidand liquid—solid distribution systems where association takes place between the components of the liquid phases. It is shown that under such circumstances a binary mixture becomes. in fact, a ternary system where the third component comprises the associated solvent. The properties of the ternary system are shown to be accounted for by the basic theory and, further, the theory can be employed to predict distribution behaviour in liquid—liquid systems where association occurs. It is shown that the theory can also be used to predict solute retention in reversed-phase liquid chromatography.

Peak dispersion and mobile phase velocity in liquid chromatography: the pertinent relationship for porous silica

The equations of Van Deemter, Giddings, Huber, Knox and Horvath for the height equivalent to a theoretical plate (H) are tested against over 25 data sets of experimental values of H and the mobile phase linear velocity (u) obtained for columns packed with silica gel. Each data set contains at least 10 complementary pairs of H and u values and furthermore, each H and u value was taken as the mean of at least three replicate measurements, thus, involving a total of over 750 individual and precise measurements of H and u. The maximum standard error for any set of replicate measurements was 2%. The data were obtained for silica gels having four different particle diameters, for six solvent mixtures and nine different solutes. It is shown that over the velocity range of 0.02–1 cm/sec, the Van Deemter equation accurately predicts the experimentally determined relationship between H and u. Consequently, under normal operating conditions in liquid chromatography, employing silica gel as the stationary phase Van Deemter equation can be employed with confidence in column design.

Liquid chromatography system for fast, accurate analysis

Abstract The design characteristics of a liquid chromatograph that are essential for very fast separations are examined theoretically. Equations are derived that permit the calculation of the column length, column diameter, maximum instrument time constant and flow-rate to effect a given separation in the minimum time. A gradient elution system, that can provide gradients covering a wide range of solvent concentrations and gradient profiles that can be delivered in a few seconds, is disclosed. Examples are given of the rapid separation of multi-component mixtures in less than 30 sec by both isocratic and gradient elution. The separation times obtained experimentally are compared with those predicted theoretically. The quantitative precision that was obtained from fast liquid chromatography separations is also reported.

Rapid separation, quantitation and purification of products of polymerase chain reaction by liquid chromatography.

The polymerase chain reaction (PCR), a new, powerful method for rapid enzymatic amplification of specific DNA fragments, has gained tremendous popularity in molecular biology. This paper describes the successful application of liquid chromatography to the analysis of products of the PCR. Efficient separation of both DNA restriction fragments and amplified PCR products were achieved in 10-12 min on a new ion-exchange column, DEAE-NPR, packed with 2.5-microns non-porous particles. The PCR products were quantitated with a reproducibility within 10%. Use of liquid chromatography was demonstrated for separation and quantitation of PCR products in amounts below those required for direct analysis by ethidium bromide gel electrophoresis or a Hoechst 33258 dye-based fluorescence assay. Liquid chromatography was also demonstrated to be effective for quick optimization of PCR procedures.

Effect of pressure on solute diffusivity, solvent viscosity and column temperature in liquid chromatography

Abstract The interrelationship between column pressure, solvent viscosity, solute diffusivity and column temperature is complex. Any increase in inlet pressure to provide a higher flow-rate and consequently a faster analysis increases solvent viscosity and column temperature and decreases solute diffusivity. However, a higher column temperature resulting from increased operating pressure reduces the solvent viscosity and increases the solute diffusivity, thereby masking the direct effect of pressure on these variables. Ipso facto, the net effect of pressure and temperature on solute diffusivity for an unthermostated column can be relatively small; consequently, the effect of pressure on column efficiency and column resolution can be minimal for unthermostated columns. However, the effect of this temperature increase on solute retention is very significant for unthermostated columns and leads to a 5% change in the value of k′ for a solute by merely changing the flow-rate from 0.5 to 5 ml/min. Hence, as the heat generated in the column is directly related to the flow-rate and further as the heat transfer through the packed bed of the column is very poor, the use of well thermostated small-bore columns could be mandatory for the precise measurement of solute retention.

Use of chromatographic data to determine the molecular weight of a solute eluted from a liquid chromatographic column

The diffusivities of 69 different compounds in a n-hexane—ethyl acetate solvent mixture have been determined and a precise relationship between solute diffusivity, molecular weight and molar volume established. The band dispersion for each of the same solutes has also been measured employing a liquid chromatographic column designed to emphasize the resistance-to-mass transfer factor and minimize thermal effects resulting from the use of high pressures and high mobile phase velocities. The effect of the capacity ratio of a solute on the resistance-to-mass transfer factor is determined and the relationship between the bandwidth of a solute, its diffusivity and its molecular weight established. A procedure is outlined for the determination of the molecular weight of a solute from the measurement of its bandwidth, when eluted from a liquid chromatographic column, within an error of 13% for 90% of the solutes examined providing the density of the solute lies between 0.85 and 1.25 g/ml.

Selectivity Factors for Several Pah Pairs on C18 Bonded Phase Columns

Abstract The selectivity factors for four pairs of polycyclic aromatic hydrocarbons (PAHs) have been found to be very different on the HC-ODS column in comparison to most other C18 bonded-phase columns. The selectivity factors for these PAH pairs varied slightly with different manufacturing lots of the HC-ODS material, the selectivity variations for each PAH pair being linearly correlated with those for the other pairs.

Low-dispersion connecting tubes for liquid chromatography systems

Abstract The dispersion that takes place in coiled and serpentine tubes of different dimensions is reported. The advantage of using serpentine tubes suitably encased as connecting tubes in liquid chromatography is demonstrated. Such connecting tubes have been shown to have a variance contribution of less than 0.05 μl 2 /cm of linear length and a pressure drop of less than 0.05 MPa/cm at a flow-rate of 3 ml/min.

Liquid chromatography column design

Abstract A liquid chromatography column design protocol is described utilizing three data bases which are defined as performance criteria, instrument constraints and elective variables. The optimum column length, column radius and particle size of the packing to provide minimum analyses time can be calculated from the information contained in these three data bases. An explicit equation is also derived to permit the optimum particle diameter to be calculated. It is shown that if the inlet pressure is limited, small particles are only suitable for use in short columns for simple separations. Conversely, difficult separations can only be achieved with larger particles packed in long columns. In liquid chromatographic analyses, operating at 6000 p.s.i. as opposed to 4000 p.s.i. results in a proportional reduction in analysis time (about 30%). It follows that a maximum inlet pressure of 4000 p.s.i. appears to be quite adequate and is to be recommended for general liquid chromatographic analysis. The optimum k′ value of the first solute of the critical pair of a complex mixture can range between 2 and 6; furthermore the optimization procedure compensates for changes in diffusivity by corresponding changes in optimum particle diameter and optimum column length. Consequently the numerical value of solute diffusivity is not critical. The quality of the packing remains important even for fully optimized columns and consequently, the best packing procedures should always be employed.