Sampling and Extra-Column Effects in High-Performance Liquid Chromatography; Influence of Peak Skew on Plate Count Calculations

Abstract

Chromatographic resolution and analysis accuracy can be seriously affected by broadening of very sharp bands associated with both high-performance size-exclusion chromatography and other liquid chromatography methods using less than 5 micron particles. The effect on column plate count by extra-column band broadening from injection systems, detectors, connectors and "guard" columns is discussed and exemplified with experimental data. The special cases of tailing and doublet peaks for a single species are investigated. These effects are eliminated by a special sample injection technique. Computer simulation studies demonstrated that serious errors can result from calculating column plate count with non-Gaussian peaks. The maximum allowable peak tailing is suggested and a new method for quantitating peak skew is proposed.

Development of a gas chromatography/mass spectrometry based metabolomics protocol by means of statistical experimental design

“…The asymmetry factor (Kirkland, 1977) (A s ) for valine 2TBDMS was included as a response as this peak was observed to suffer from asymmetric band broadening, whereas this type of peak distortion was weaker or absent for later eluting metabolites. A s was determined according to Eq.…”

Estimation of the band broadening contribution of HPLC equipment to column elution profiles

“…By plotting the total variance versus the length of the capillary tubes in a graph, and by extrapolating this line to length zero, the instrument variance is obtained [4]. In either method, however, the column has to be removed from the chromatographic system.…”

“…As a result of a sudden change in inner diameter and deviating flow behavior [4], these are probably important contributions to the peak dispersion.…”

Cromatografia líquida capilar: 2. Alargamento das bandas por efeitos extracoluna

“…[1] para os processos de dispersão intracoluna, um grande número de pesquisadores vem propondo e desenvolvendo vários métodos e equações específicas para estimar as contribuições das diversas fontes de dispersão extracoluna [11][12][13][14][15][16][17][18][19][20][21][22] . A seguir discutiremos as suas cinco principais fontes, a citar:…”