Maintaining fixed band spacing when changing column dimensions in gradient elution

Journal of Chromatography. a
J W Dolan, L R Snyder

Abstract

In gradient elution separations, it may be required to change either column length (to increase resolution or shorten run time) or column diameter (for an increase in sensitivity or for preparative separations). In either of these changes of column dimensions, it is usually desired to maintain the same relative band spacing (selectivity), so as to increase resolution in proportion to (column plate number)1/2 when increasing column length, or to maintain constant resolution when changing column diameter. A general rule for avoiding changes in band spacing in these situations is to maintain the quantity [(gradient time) x (flow-rate)/(column volume)] constant, while holding the initial and final gradient mobile phase compositions (%B) fixed. This rule is only valid as long as the equipment hold-up volume (dwell volume) is negligible, or if all sample components are strongly retained at the start of the gradient. When neither of the latter conditions apply, then significant changes in band spacing may result when changing column size. Rules are presented for recognizing this potential problem for a given sample/HPLC-equipment combination, and adjustments in separation conditions that can avoid this problem are discussed. Changes i...Continue Reading

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Citations

Oct 25, 2008·Analytical and Bioanalytical Chemistry·Vilém GurycaChristophe D Masselon
Nov 11, 2008·Journal of Pharmaceutical and Biomedical Analysis·G Hendriks
Sep 25, 2007·Journal of Chromatography. a·Dwight R StollSarah C Rutan
Aug 21, 2007·European Journal of Pharmaceutics and Biopharmaceutics : Official Journal of Arbeitsgemeinschaft Für Pharmazeutische Verfahrenstechnik E.V·Davy GuillarmeJean-Luc Veuthey
Jan 11, 2005·Journal of Separation Science·Pavel JanderaElsa Lundanes
Oct 26, 2005·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Sharon S Chen, Ruedi Aebersold
Nov 28, 2007·Electrophoresis·Peter HorvatovichRainer Bischoff
Dec 3, 2014·Journal of Chromatography. a·Ayse BeyazaAdam P Schellinger
Nov 18, 2008·Journal of Chromatography. a·Colin F Poole, Salwa K Poole

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