Particle size, pressure and analysis time in routine high-performance liquid chromatography

Abstract

The maximum number of theoretical plates that can be generated in a column can be calculated from the empirical equations u min d p = 10 and h min = 4 d p (where the minimum linear velocity of the eluent, u min, is given in mm/sec and the average size, d p , in μm), if the average particle size d p is based entirely on chromatographic particle measurements. It was demonstrated experimentally that 30-cm silica or reversed-phase columns having over 7000 plates could be packed with d p = 10 μm. Such columns can resolve, with baseline peak separation, mixtures that possess relative retentions greater than 1.12. In routine work, the relative retentions are usually greater and it is advantageous to use only 7.5-cm long columns. Analysis times of 10 min or less can be achieved even with velocities around u min. Consequently, most routine separations require column pressures of less than 50 atm. For research studies and for the optimization of conditions for routine work, columns packed with 5-μm particles are often necessary. The reproducibility of column packing with d p < 5 μm is poor. It was shown that up to 600 plates/cm and over 100 plates/sec (capacity ratio = 1) can be generated on silica columns with d p = 3.2 μm. Such small particle sizes are particularly advantageous for trace analysis because, as our experience indicates, these short and efficient columns can be loaded with samples larger than the amount calculated from the weight of the stationary phase present.