High Performance Liquid Chromatography (HPLC)
HPLC is a powerful separation technique for a wide range of dissolved samples. Analytes are pumped through a column bed and interact by specific physicochemical properties such as hydrophobicity, charge, size or immunoaffinity, depending on the stationary phase. Separated compounds elute and can be detected by UV absorption, fluorescence or mass spectrometry.
Chromatography is applied in our labs mainly as a preparative separation technique and complements gel-based techniques, especially for peptide mixtures. We have HPLC systems for microanalytical, micropreparative and preparative applications in flow ranges between 3µl/min up to 5 ml/min. For analyte detection UV and fluorescence can be monitored. Automated fraction collection is done in refrigerated devices to ensure sample integrity for following separation and detection techniques.
Separation modes include
- C18 phases for separation of peptides
- C8 phases for separation of large peptides and small proteins
- C4 phases for separation of proteins
- strong cation exchange (SCX)
- weak anion exchange (WAX)
- size exclusion chromatography (SEC)
- affinity chromatography
Selected applications of preparative HPLC separations include
- profiling of peptides and small proteins in complex protein mixtures for subsequent proteolytic digest and LCMS analysis
- SCX of complex proteolytic peptide pools as a second separation mode in iTRAQ and MuDPIT approaches
- separation of proteolytic peptides for subsequent internal Edman sequencing
- immunodepletion of high abundant serum/plasma proteins
Depending on the requirements of project we can choose the right separation mode and column size with regard to downstream analysis.
- nanoHPLC for online coupling to high resolution mass spectrometers (proteomics)
- microHPLC for combined detection with UV and high resolution MS (for characterisation of synthesis products and metabolomics)
- analytical HPLC for micro-preparative separation, e. g. of proteolytic peptides for Edman sequencing
- preparative HPLC for purification of synthesis products
Fast Protein Liquid Chromatography (FPLC)
For large scale protein separation in liquid phase FPLC is the method of choice. is a chromatographic technique used to separate the components, in a liquid mixture. The liquid sample (mobile phase) flows over a solid material packed into a column using a flow of liquid solvent under normal pressure. Preparation and purification of selected target proteins is the main application.
Different separation principles are available for FPLC
- IEX ion exchange (IEX)
- hydrophobic interaction (HILIC)
- size exclusion (SEC)
- affinity separation (e. g. sepharose A for antibodies)
Preparative continuous-elution electrophoresis (Prep Cell)
The preparative electrophoretic separation of proteins with collection of purified analytes in the liquid phase is especially useful to obtain pure antibody heavy and light chains for subsequent proteolysis and MS-based analyses.
Different HPLC/FPLC selectivities and other separation techniques are available upon request. Please contact our specialists to discuss your project.