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A study of column equilibration time in hydrophilic interaction chromatography

McCalley, David V.



© 2018 Elsevier B.V. The time taken to achieve full column equilibration for isocratic analysis of acidic, basic, and neutral solutes in hydrophilic interaction liquid chromatography (HILIC) was compared using the mobile phase disturbance method, column efficiency measurements and retention time stability. Full equilibration, which could take up to an hour, was best measured by the last procedure and was found to depend on the nature of the stationary phase, the pre-equilibrium (e.g. storage) solvent and the flow rate. While longer equilibration times are a relatively minor inconvenience in isocratic analysis, they are surprisingly not a barrier to the use of gradient elution in HILIC. A repeatable partial equilibration giving retention time stability equivalent to that in isocratic analysis was demonstrated for an equilibration time of only ∼5 min, using as few as 2 preliminary conditioning runs on a column that had taken the longest time to achieve full equilibration. Due to selectivity changes that occur dependent on the equilibration time, it is necessary to use identical gradient conditions in a series of analyses, which however appears to be facile on a modern HPLC instrument.


McCalley, D. V. (2018). A study of column equilibration time in hydrophilic interaction chromatography. Journal of Chromatography A, 1554, 61-70.

Journal Article Type Article
Acceptance Date Apr 6, 2018
Online Publication Date Apr 7, 2018
Publication Date Jun 15, 2018
Journal Journal of Chromatography A
Print ISSN 0021-9673
Electronic ISSN 1873-3778
Publisher Elsevier
Peer Reviewed Peer Reviewed
Volume 1554
Pages 61-70
Keywords hydrophilic interaction chromatography, HILIC, Equilibration
Public URL
Publisher URL
Additional Information Additional Information : This is the author's accepted manuscript. The final published version is available here:


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