P. Kucera

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.

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.

Total amino acid analysis using pre-column fluorescence derivatization

Abstract An approach to total amino acid analysis utilizing off-line pre-column fluorescence derivatization is described. The full array of natural primary amino acids was treated with o -phthaldialdehyde (OPA) in the presence of mercaptans, and the highly fluorescent reaction products were separated on an ODS 5-μm reversed-phase column with gradient elution development. Due to their instability in solution and the relatively poor response of the OPA derivatives, cysteine and cystine were oxidized with performic acid and separated as the OPA cysteic acid reaction adduct, which is highly fluorescent. Secondary amino acids, such as proline and hydroxyproline, were reacted with 4-chloro-7-nitrobenzofurazan, and the separation was carried out on the same column. The stability of the reaction adducts was investigated and the derivatization reactions were optimized with respect to reaction time and temperature. It is shown that the detection limits for most amino acids are in the 0.1–1.0-pmole range. The reproducibility of the method was limited by the derivatization procedure and the gradient elution employed, but, by using an on-line computer data handling system, the retention time could be measured within ± 0.1% relative standard deviation and the relative peak areas, based on the internal standard calculation methodology, were within ± 3% or less. The sensitivity of lysine and hydroxylysine was improved by forming a sodium dodecylsulfate micellar solution around these amino acids to protect the fluorescent adducts from rapid decomposition. The sensitivity and separation appeared to be very much dependent on the pH of the mobile phase. Experimental details for the determination of total amino acid residues in pharmaceutically important proteins, such as thymosin α 1 and insulin, are given. Advantages and disadvantages of the system described are discussed.

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.

Design and use of short microbore columns in liquid chromatography

Abstract The various aspects of design and performance of short microbore columns for use in liquid chromatography are discussed. The basic hydrodynamic factors important in column design are considered. Consideration is given to instrumental time constant and solute band broadening due to the injection system, the column and the detector. The design and construction of a chromatographic system using commercially available components permitting the use of short microbore columns is described and experimental details for operation in isocratic and gradient elution modes are given. It is shown that a column 25 cm in length with 1 mm internal diameter can be packed with a 10-μm particle diameter silica gel to the limiting plate height of 20 μm to achieve 50,000 plates per meter. Examples of the use of the system for separations of compounds of pharmaceutical interest are given and advantages of the system are discussed.

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.

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.

use of microbore columns for rapid liquid chromatographic separations

Abstract The use of microbore columns for high-speed chromatographic separation is investigated, and apparatus is described that can be usd with such columns. Specific details of detector cell construction are given, and photosensor specifications are suggested to provide the necessary fast detector response time. Specifications are also provided for appropriate data acquisition equipment. The performance of microbore columns operated at high mobile phase velocities is examined, and the effect of mobile phase velocity on column efficiency investigated. The precisions of quantitative analysis by peak height and peak area measurements are compared for a microbore columns operated at high linear mobile phase velocity. An example of the separation of a seven-component mixture in less than 30 sec is included.