Reversed phase Liquid Chromatography (RPLC)

Reversed phase Liquid Chromatography (RPLC)

The application of reverse-phase liquid chromatography (RPLC) in glycoproteomic analysis has attracted more and more attention. CD BioGlyco has a complete experimental system and powerful data analysis platform to meet the research needs of customers in glycan separation and purification. We are confident to provide customers with scientific help in the field of glycoproteomic analysis through a variety of strategies including RPLC.


RPLC uses hydrophobic interactions between biomolecules and ligands on chromatographic support to achieve separation. In the past decades, RPLC with C18 stationary phase has been widely used for peptide separations due to the relatively high hydrophobicity of peptides. In addition, C18-RPLC has also been used in large-scale glycopeptide separations. Since RPLC is a standard separation system, its compatibility with other proteomic research lines allows RPLC to be widely used in glycopeptide separation.

RPLC provides poor differentiation of glycan isomers, which limits the application of RPLC in glycoproteomic analysis. Therefore, RPLC is usually combined with various detection techniques for different research problems. Recent studies have reported that RPLC is coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS) for site-specific glycosylation analysis of highly heterogeneous glycoproteins in limited samples. RPLC has a great advantage of broad distribution because it is used in almost every bioanalytical research laboratory, making it an attractive platform for glycan analysis.

Fig 1. Workflow for the site-specific N-glycan characterization of AGP with a reversed-phase UHPLC-CZE-ESI-MS/MS approach.Fig.1 Workflow for the site-specific N-glycan characterization of AGP with a reversed-phase UHPLC-CZE-ESI-MS/MS approach. (Qu, 2017)


RPLC is a technique commonly used for the separation of glycans. At CD BioGlyco, we commonly combine RPLC with other detection techniques to achieve superior glycopeptide analysis. Services we offer include but are not limited to:

  • Use different chromatographic columns and eluent mixtures to explore the variation in separation efficiency.
  • Use various labeling reagents to increase hydrophobicity and detectability of glycans to develop the most effective separation method.
  • MS/MS analysis of N-glycopeptides from alpha-1-acid glycoprotein (AGP) with reversed-phase UHPLC prefractionation.


  • Purification and separation of glycans
  • Analysis of drugs at every stage of the drug development process
  • Development of biological therapeutics

Advantages of Us

  • Increased column efficiency and higher resolution
  • Enhanced MS sensitivity
  • Offer reduced analysis times
  • Multiple methods with high flexibility

CD BioGlyco is experienced in selecting different chromatographic columns for high-efficiency reversed-phase chromatographic analysis. We are pleased to develop tailor-made RPLC methods for our clients to accelerate their research projects in the field of glycomics. If you need scientific assistance in your research, please contact us for detailed information and technical support.


  1. Qu, Y.; et al. Site-specific glycan heterogeneity characterization by HILIC solid-phase extraction, RPLC fractionation, and capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry. Analytical Chemistry. 2018, 90(2): 1223.
  2. Vreeker, G.; Wuhrer, M. Reversed-phase separation methods for glycan analysis. Analytical & Bioanalytical Chemistry. 2017, 409(2):359-378.
This service is for Research Use Only, not intended for any clinical use.

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