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Effect of buffer on peak shape of peptides in reversed-phase high performance liquid chromatography

McCalley, David V.



The effect of changing the buffer at constant low pH in the mobile phase is investigated with respect to the separation of a mixture of basic peptides. Considerably worse peak shapes, leading to poorer resolution of complex peptide mixtures, were obtained when using formic acid favoured in LC-MS applications compared with non volatile phosphate buffers or with trifluoroacetic acid (TFA). Poorer peak shapes were largely attributable to reduced column capacity for the peptides when using mobile phases of low ionic strength, due to the increased mutual repulsion of ions held on the hydrophobic column surface which is facilitated in these buffers. However, ion-pairing between the peptides and additives such as TFA or even phosphate may also lessen mutual repulsion effects, leading to greater column capacity. Overloading effects could be observed when sample masses around only 0.1 μg were injected on to standard size analytical columns in formic acid containing mobile phases; sample masses around only 1.5 μg may cause loss of half the system peak capacity in such mobile phases. Results were broadly comparable (after scaling sample size according to column diameter) on columns of both conventional (4.6 mm i.d.) and capillary (0.075 mm i.d.) dimensions. Ammonium formate may be a useful alternative buffer for some applications due to its higher ionic strength. © 2004 Elsevier B.V. All rights reserved.


McCalley, D. V. (2004). Effect of buffer on peak shape of peptides in reversed-phase high performance liquid chromatography. Journal of Chromatography A, 1038(1-2), 77-84.

Journal Article Type Article
Publication Date Jun 4, 2004
Journal Journal of Chromatography A
Print ISSN 0021-9673
Publisher Elsevier
Peer Reviewed Peer Reviewed
Volume 1038
Issue 1-2
Pages 77-84
Keywords peak shape, mobile phase composition, buffer composition, peptides
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