Comparison of silica-based cyanopropyl and octyl reversed-phase packings for the separation of peptides and proteins.
The performance of a silica-based C8 packing was compared with that of a less hydrophobic, silica-based cyanopropyl (CN) packing during their application to reversed-phase high-performance liquid chromatography (linear trifluoroacetic acid-water to trifluoroacetic acid-acetonitrile gradients) of peptides and proteins. It was found that: (1) the CN column showed excellent selectivity for peptides which varied widely in hydrophobicity and peptide chain length; (2) peptides which could not be resolved easily on the C8 column were widely separated on the CN column; (3) certain mixtures of peptides and small organic molecules which could not be resolved on the C8 column were completely separated on the CN column; (4) impurities arising from solid-phase peptide synthesis were resolved by a wide margin on the CN column, unlike on the C8 column, where these compounds were eluted very close to the peptide product of interest: and (5) specific protein mixtures exhibited superior resolution and peak shape on the CN column compared with the C8 column. The results clearly demonstrate the effectiveness of employing stationary phases of different selectivities (as opposed to the more common optimization protocol of manipulating the mobile phase) for specific peptide and protein applications, an approach underestimated in the past.
Zhou
N E
NE
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Mant
C T
CT
Kirkland
J J
JJ
Hodges
R S
RS
D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000302:isolation & purification*; D011506:Proteins / Q000302:isolation & purification*; D012822:Silicon Dioxide / Q000737:chemistry*
D010455:Peptides; D011506:Proteins; D012822:Silicon Dioxide