R.P.W. Scott

Mode of operation and performance characteristics of microbore columns for use in liquid chromatography

Abstract The production and operating characteristics of microbore columns are described and details of the modification of the LDC UV monitor to give the necessary low dead volume detection cell for use with such columns are given. A method of connecting the Valco high-pressure sampling valve to microbore columns to provide the necessary small sample volume is described and a system given that permits the operation of the Waters Assoc. 6000 pump over the flow-rate range of 2 to I00 ,ul/min. The effect on column performance of column radius and particle diameter of the packing is examined in detail and height equivalent to a theoretical plate curves and reduced plate height curves are included; the effect of coiling the column on efficiency is also reported. Examples of the use of microbore columns for high-speed and high- efficiency separations are given and include results obtained from reversed-phase columns. The construction and performance of a column having 750,000 plates is described.

Examination of five commercially available liquid chromatographic reversed phases (including the nature of the solute-solvent-stationary phase interactions associated with them)

Abstract Five commercially available reversed phases are examined, and the effects of chain length, carbon content and extent of derivatization on wettability, retention and selectivity are investigated. The nature of the stationary phase surface, when in contact with the mobile phase under conditions of wetting, is examined and the mechanism of solute-stationary phase interaction identified. The location of the ion-pair reagent, when the reversed phase is used for ion-pair chromatography, is identified for both lyophilic and lyophobic conditions of chromatographic development.

Interface for on-line liquid chromatography—mass spectroscopy analysis

Abstract An interface is described that permits the on-line operation of a liquid chromatograph and quadrupole mass spectrometer combination. The interface consists of two differentially pumped chambers through which a wire train system continually takes a sample of the column eluent, evaporated the solvent, and subsequently vaporizes the residual solute in the ionizing chamber of the mass spectrometer. The feasibility of the system is confirmed and its use as an alternative method for probe injection is demonstrated. Examples of its used with a liquid chromatograph in the separation of a fermentation extract are given and the results are compared with those obtained using the instrument as a probe sampling system to provide spectra from fractions collected from the simple chromatographic separation of the same mixture. The sensitivity of the present system is ca. 10 6 −10 5 g/ml of solute, which is only just adequate for liquid chromatography separations if the full separation potential of the column is to be realized. The quadrupole mass spectrometer used in this work was aged and had poor resolution. It is estmated that a moderrn quadrupole with improved resolution fitted withe suitable interfaces would provide sensitivities of 10 7 −10 6 g/ml. which would cope with the majority of separation problems where 1 to 2 mg of a complex sample were available.

The exclusion properties of some commercially available silica gels

Abstract The exclusion properties of ten commercially available silica gels have been determined and their relative merits for use as stationary phases in exclusion chromatography discussed. Examples are given of the use of three selected silica gels in the separation of synthetic mixtures of high-molecular-weight standards. The effect of the exclusion properties of silica gel on retention data is considered and a modified retention volume equation that takes into account these exclusion properties is given. The validity of the equation is tested against retention data determined for three different solutes chromatographed on three silica gels of widely different porosities. The use of silica gel as an exclusion medium in conjunction with a column having 250,000 theoretical plates is also demonstrated.

Use of microbore columns for the separation of substances of biological origin

A gradient system for use with microbore columns is described and the repeatability of the system determined. A simple sampling device for use with aqueous biological samples is described, and the advantages of the high mass sensitivity of microbore columns for the analysis of such samples are discussed. The sensitivity of the system is demonstrated by the determination of acetophenone in water at a level of one part per billion. The use of the apparatus for the separation of low-molecular-weight, non-ionic compounds contained in blood is also demonstrated. It is shown that excellent separations of such components can be obtained from as little as 400 microliter of serum.

Solute-solvent interactions on the surface of silica gel. II

The interactions of solute and solvent on silica gel have been further examined and include adsorption isotherms for non-polar and polar solvents. The formation of a solvent bilayer on the surface of silica gel when using hydrogen-bonding polar solvents is further substantiated, and alternative explanations involving inhomogeneous site activity and steric hindrance are shown not to explain the bilayer formation. It is concluded that when polar-modifying solvents are employed in liquid chromatography at low concentrations, solutes interact with a primary layer of polar solvent wihtout displacing the solvent. At higher concentrations solutes interact with the primary layer, displacing solvents in the second layer, but do not interact directly with the silica gel surface itself. However, for solutes eluted at high K′ values or solutes that have a polarity similar to that of the modifying solvent, competition with the primary layer can take place. Under these circumstances, the solute interacts directly with the silica gel surface.

Solute interactions with the mobile and stationary phases in liquid—solid chromatography

Abstract A dynamic equation for the distribution coefficient is proposed that accounts for the different solute—phase interactions in liquid chromatography. The validity of the basic form of the equation is experimentally verified and the effect of solvent composition on retention is shown to be dependent on the probability of solute—solvent interaction and thus the concentration of polar solvent in the mobile phase. Using the equation, the change in retention volume with polar solvent concentrations can be accurately predicted. Where polar forces between solute and solvent are weak, it is shown that dispersive interactions are proportional to the density of the dispersing solvent. The magnitude of the polar interactive effects of both solute and solvent are shown to be proportional to the exponent of the polarizability per milliliter of the respective solute or solvent for a limited number of solutes and solvents.

Solute-solvent interactions on the surface of reversed phases : I. Stationary phase interactions and their dependence on bonding characteristics

Abstract The retention characteristics of a number of “brush”- and “bulk”-type reversed-phase column packing materials that are commercially available for high-performance liquid chromatography are examined in the situation where an abrupt change in mobile phase from pure methanol to pure water occurs. It is shown that the brush-type reversed phases are slow to come into equilibrium with pure water, and in contact with water it would appear that the hydrocarbon chains dispersively interact with themselves. This dispersive intramolecular interaction of the hydrocarbon chains results in a reduction in effective chromatographic surface area and consequently anomalously low retentive characteristics. In contact with solvent containing 10% (w/v) or more of organic solvent such as methanol, the brush-type materials exhibit normal retention characteristics. The bulk or polymeric bonded phases, however, equilibrate rapidly with water, and due to their more rigid structure do not appear to exhibit dispersive intramolecular interactions and thus exhibit normal retention characteristics. The bulk or polymeric type reversed phases, therefore, are to be preferred for examining solute-solvent/stationary phase interactions due to their more consistent chromatographic properties.

Some aspects of ion-exchange chromatography employing adsobed ion exchangers on reversed-phase columns

Abstract The nature of the retention of ionic solutes in the presence of alkyl sulphonates and quaternary alkylammonium halides is examined, and it is shown that the ion-exchange reagents adsorbed on the hydrocarbon stationary phase and act as adsorbed ion-exchange agents. Thus, in the system examined the major mechanism of retention is normal ion-exchange chromatography. This paper demonstrates that a layer of solvent exists over the surface of the bonded phase which can be shown by the nature of the adsorption isotherm of methanol on the bonded phase; this is also confirmed chromatographically. In a similar manner, the adsorption isotherm of sodium octane sulphonate is also determined, and the presence of the adsorbed layer of the octane sulphonate confirmed by chromatographic measurements. By the use of conductivity and nuclear magnetic resonance chemical shift measurements, it is shown that in the system examined and under the conditions where solutes are retained by an ion-exchange mechanism on the surface of the stationary phase, both solute and ion-exchange reagent are ionized.

Solute-solvent interactions on the surface of silica gel : III. Multilayer adsorption of water on the surface of silica gel

Abstract Employing techniques of thermal treatment, infrared examination and chemical reaction, at least three layers of adsorbed water have been identified on the surface of silica gel equilibrated at 23°C with an atmosphere containing 50% humidity. Each layer contains about 8.5-10 20 molecules of water per gram of silica, which agrees well with previous reported data for polar and non-polar solute layers on the surface of the same silica gel. The second and third layers are weakly held and can be removed completely with dry solvents or by heating to 120°C. The first layer is very strongly held, removal commencing at about 200°C and is not complete until 650°C. This latter water loss is completely reversible. The silanol groups do not commence condensing to siloxyl groups until a temperature of 450°C is reached, and at 750°C, 75% of the silanol groups still remain and are not completely removed until temperatures in excess of 1000°C are reached. Conversion of silanol to siloxyl groups does not appear to be reversible.