The Malaysian Journal of Analytical Sciences Vol 15 No
2 (2011): 273 – 287
PEPTIDE
SEPARATION BY CAPILLARY ELECTROPHORESIS WITH ULTRAVIOLET DETECTION: SOME SIMPLE
APPROACHES TO ENHANCE DETECTION SENSITIVITY AND RESOLUTION
(Pemisahan Peptida Oleh Elektroforesis Rerambut Dengan Pengesan
Ultra-Lembayung: Pendekatan Mudah Untuk Meningkatkan Isyarat Pengesanan Dan
Resolusi)
L.
Noumie Surugau
Industrial
Chemistry Program,
School of
Science and Technology,
Universiti
Malaysia Sabah,
Locked Bag 2073,
88999 Kota Kinabalu, Sabah, Malaysia.
*Corresponding author: lnoumie@ums.edu.my
Abstract
Capillary electrophoresis
(CE) is one of the leading separation technologies for analysis of
water-soluble analytes. CE has many advantages over the more established
methods such as liquid chromatography and gel electrophoresis particularly in
rapid analysis, require very little sample, use less or no toxic organic
solvent, high peak efficiency and ease of automation. Despite the many
attractive advantages of CE, CE users continue to seek improvements
particularly on detection sensitivity, resolution and selectivity. This paper presented several simple
approaches to improve detection sensitivity using simple sample
pre-concentration called field-enhanced sample injection (FESI) and
chromatographic-based ZipTip C18 pre-concentrator. Also, some improvements in the resolution of
complex peptides mixture when using two strategies namely, capillary coating
and manipulation of the hydrophobicity of peptides using perfluorinated
acids as background electrolyte (BGE), which have anionic conjugate base forms with
hydrophobic character. As test
compounds, standard peptide mixture and proteins digests were used for these
studies. The results showed that FESI has significantly enhanced the detection
signal of peptide standards and bovine serum albumin (BSA) tryptic digests. As
for the use of ZipTip C18 pre-concentrator, selective enhancement in
detection signal was particularly notable on the late migrating peptides.
Coating the capillary proved to have little changes on the CE of peptides when
used in conjunction with acidic BGE. Electropherograms of BSA tryptic peptides
in pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA) showed
interesting profile, with notable resolution improvement for peptides with
close similarity in electrophoretic mobilities.
Keywords: Capillary electrophoresis,
peptide, protein digests, pre-concentration, detection sensitivity, resolution.
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