Practical Considerations in the Use of High Performance Liquid Chromatographic Columns

Abstract

Abstract The practical aspects of high performance liquid chromatographic (HPLC) column preparation and operation are reviewed. Recent advances in columns are briefly covered. Porous layer beads and the new microparticles are compared. The design of HPLC columns with respect to optimum dimensions in analytical and preparative techniques and the materials used for column construction are detailed. The effects on column performance of extra-column volumes (i.e., connecting tubing and fittings) and injection procedures and techniques are pointed out. Procedures for preparing the column, including slurry packing techniques for microparticles, are given. The influence of solvent purity in isocratic and gradient elution in column operation and practical techniques for removing impurities are presented. Solvents and buffers useful for operation in the far ultraviolet region (190—210 nm) are tabulated, and a practical example of the use of this wavelength region for the detection of carbohydrates is presented. The practical aspects of liquid-solid chromatography (LSC) including activity and retention time reproducibility are discussed. Difficulties encountered in LSC are listed and suggestions are made to solve them. The advantages and disadvantages of the new chemically bonded phase packings are covered. Details of a new technique, ion-pair partition chromatography, are presented along with suggested applications.