title: Separation of therapeutic peptides with cyclofructan and glycopeptide based columns in hydrophilic interaction liquid chromatography.
abstract: Three cyclofructan-based, two glycopeptide-based, and one zwitterionic column used in the HILIC mode were assessed within a graphical framework based on different functional characteristics contributing to selectivity. The characteristics of these six HILIC columns are put in the perspective of 33 columns evaluated previously. The isopropyl carbamate modified cyclofructan 6 (CF6) stationary phase, Larihc P, showed reduced component contributions for hydrophilicity and hydrogen bonding relative to the native cyclofructan 6 column (Frulic N). Both Frulic N and Larihc P exhibited cation exchange attributed primarily to deprotonation of residual unsubstituted silica with the greater exchange ascribed to the reduced loading of CF6 observed for Larihc P. The cyclofructan 6 column with a polymeric styrene divinylbenzene support (MCI GEL™ CRS100) showed distinct selectivities consistent with its decreased cation exchange attributable to its nonionic core. The Chirobiotic T, Chirobiotic V, and ZI-DPPS columns displayed hydrophilicity and ion exchange selectivities similar to other zwitterionic stationary phases. All of the more hydrophilic columns showed excellent separation for the four classes of therapeutic peptides investigated: microbial secondary metabolites used as immune suppressants, synthetic gonadotropin hormones, synthetic cyclic disulfide-linked hormone-regulating hormones, and non-ribosomally derived polycyclic antibiotics. Resolution provided by these columns and ZIC-HILIC is compared for each class of peptide. Frulic N is primarily suitable for use in the HILIC mode whereas Chirobiotic T, because of its increased efficiency and selectivity, can be useful in both HILIC and reverse phase modes. In some Chirobiotic T applications, addition of low levels of a strong additive (trifluoroacetic acid, formic acid, etc.) to the mobile phase can be beneficial. In these peptide analyses, a relative weakening of the often-dominant ionic interaction between analyte and residual charge on the stationary phase improved resolution and selectivity.
authors.affiliation: Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX 76019, USA; College of Life and Health Sciences, Northeastern University, Shenyang 110189, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Shu
authors.affiliation: Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX 76019, USA; AZYP LLC, Arlington, TX 76019, USA.
authors.forename: John C
authors.initials: JC
authors.lastname: Lang
authors.affiliation: Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX 76019, USA.
authors.forename: Zachary S
authors.initials: ZS
authors.lastname: Breitbach
authors.affiliation: Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX 76019, USA.
authors.forename: Haixiao
authors.initials: H
authors.lastname: Qiu
authors.affiliation: Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX 76019, USA; AZYP LLC, Arlington, TX 76019, USA.
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Smuts
authors.affiliation: Mitsubishi Chemical Corporation, 1-1-1, Marunouchi, Chiyoda-ku, Tokyo 100-8251, Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Kiyono-Shimobe
authors.affiliation: Mitsubishi Chemical Corporation, 1-1-1, Marunouchi, Chiyoda-ku, Tokyo 100-8251, Japan.
authors.forename: Mari
authors.initials: M
authors.lastname: Yasuda
authors.affiliation: Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX 76019, USA; AZYP LLC, Arlington, TX 76019, USA. Electronic address: sec4dwa@uta.edu.
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Armstrong
mesh_terms: D002853:Chromatography, Liquid; D005630:Fructans / Q000737:chemistry*; D006020:Glycopeptides / Q000737:chemistry*; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D010455:Peptides / Q000302:isolation & purification*
chemical_list: D005630:Fructans; D006020:Glycopeptides; D010455:Peptides; C557358:cyclofructan 6
keywords: Cyclofructan stationary phase; Glycopeptide stationary phase; HILIC; Peptide drugs; Selectivity