HPLC Analysis
HPLC Analysis of Peptides
High-Performance Liquid Chromatography (HPLC) is the primary analytical technique for peptide purity assessment and purification.
Reversed-Phase HPLC Principles
Separation is based on hydrophobicity. Peptides interact with a non-polar stationary phase and are eluted with an increasing gradient of organic solvent.
Standard Method Parameters
| Parameter | Typical Setting |
|---|---|
| <strong>Column</strong> | C18, 4.6 x 250 mm, 5 um particle size |
| <strong>Flow Rate</strong> | 1.0 mL/min (analytical) |
| <strong>Mobile Phase A</strong> | 0.1% TFA in water |
| <strong>Mobile Phase B</strong> | 0.1% TFA in acetonitrile |
| <strong>Gradient</strong> | 5-65% B over 30 min |
| <strong>Detection</strong> | UV at 214 nm |
| <strong>Temperature</strong> | Room temperature or 40C |
Purity Assessment
- Crude purity: >50% (acceptable for initial synthesis)
- Moderate purity: >70% (suitable for preliminary studies)
- High purity: >95% (required for analytical standards)
- Ultra-pure: >98% (for crystallography, calibrants)
See [[Mass Spectrometry]], [[Solid Phase Peptide Synthesis]], [[Peptide Science Overview]]