Related Experiment Video
Updated: Aug 15, 2026

Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid
Published on: December 4, 2017
Two-dimensional separation of peptides using RP-RP-HPLC system with different pH in first and second separation
Martin Gilar1, Petra Olivova, Amy E Daly
1Life Sciences Chemistry R&D, Waters Corporation, 34 Maple St., Milford, MA 01757, USA. Martin_Gilar@waters.com
Abstract:
Two-dimensional high performance liquid chromatography is a useful tool for proteome analysis, providing a greater peak capacity than single-dimensional LC. The most popular 2D-HPLC approach used today for proteomic research combines strong cation exchange and reversed-phase HPLC. We have evaluated an alternative mode for 2D-HPLC of peptides, employing reversed-phase columns in both separation dimensions. The orthogonality of 2D separation was investigated for selected types of RP stationary phases, ion-pairing agents and mobile phase pH. The pH appears to have the most significant impact on the RP-LC separation selectivity; the greatest orthogonality was achieved for the system with C18 columns using pH 10 in the first and pH 2.6 in the second LC dimension. Separation was performed in off-line mode with partial fraction evaporation. The achievable peak capacity in RP-RP-HPLC and overall performance compares favorably to SCX-RP-HPLC and holds promise for proteomic analysis.
Related Concept Videos
Two-dimensional Gel Electrophoresis
The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
Biological samples, such as cells...
High-Performance Liquid Chromatography: Introduction
In HPLC, two phases play a critical role in the separation process:
Ion-Exchange Chromatography
High-Performance Liquid Chromatography: Elution Process
Peptide Identification Using Tandem Mass Spectrometry
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...
Capillary Electrophoresis: Applications
Capillary zone electrophoresis (CZE) separates ionic components based on their electrophoretic mobility. It has been used to separate proteins, amino acids,...
