Peptide Purification
From crude synthesis to research-grade compound through advanced separation techniques.
Peptide Purification Methods
After solid-phase synthesis, crude peptide preparations typically contain the target sequence along with various impurities including deletion sequences, truncated fragments, and chemically modified byproducts. Purification is the process of isolating the desired peptide from this complex mixture to achieve the required purity grade.
Reversed-Phase HPLC
Reversed-phase high-performance liquid chromatography (RP-HPLC) is the most widely used method for peptide purification. The technique exploits differences in hydrophobicity between the target peptide and its impurities.
In RP-HPLC purification:
- The crude peptide mixture is loaded onto a column packed with silica particles coated with C18 (octadecyl) or C8 (octyl) chains
- An aqueous mobile phase (typically water with 0.1% TFA) initially holds all components on the column
- A gradient of increasing organic solvent (acetonitrile with 0.1% TFA) progressively elutes compounds in order of increasing hydrophobicity
- The eluent is monitored by UV detection at 214 nm to identify peptide-containing fractions
- Fractions containing the target peptide at acceptable purity are pooled and lyophilized
Ion-Exchange Chromatography
Ion-exchange chromatography separates peptides based on net charge at a given pH. Cation-exchange columns bind positively charged peptides and elute them with increasing salt concentration or pH. This method is particularly useful for separating peptides that co-elute on reversed-phase columns but differ in charge state. It is often used as an orthogonal purification step following initial RP-HPLC.
Size-Exclusion Chromatography
Size-exclusion chromatography (SEC), also called gel filtration, separates molecules by hydrodynamic radius. Larger molecules elute first because they cannot enter the pores of the column packing material. SEC is primarily used for desalting peptide solutions and for separating peptide monomers from aggregated species, rather than as a high-resolution purification technique.
Preparative vs. Analytical Scale
Analytical HPLC uses narrow-bore columns (4.6 mm diameter) to assess purity, while preparative HPLC uses wider columns (10-50 mm diameter) to purify gram quantities of material. The chromatographic conditions developed on analytical columns can typically be scaled to preparative columns, though flow rates and gradient times must be adjusted to account for the larger column dimensions.
Post-Purification Processing
After purification, peptide-containing fractions are typically processed through:
- Pooling: Combining fractions that meet purity criteria
- Lyophilization: Freeze-drying to remove water and organic solvent, yielding a stable powder
- Quality control: Final HPLC analysis to confirm purity, plus mass spectrometry for identity verification
- Packaging: Dispensing into amber glass vials under inert atmosphere (nitrogen or argon)
Related Topics
- Peptide Purity – Understanding purity measurements
- Peptide Synthesis – The manufacturing step before purification
For research use only. Not for human consumption. All products sold by Epiq Aminos are intended for laboratory research purposes only.