HPLC Analysis¶
TL;DR¶
Reversed-phase HPLC with UV detection at 214 nm is the primary method for peptide purity determination. A C18 column with 0.1% TFA in water/acetonitrile gradient is the standard configuration.
Principle¶
Reversed-phase HPLC separates peptide components based on hydrophobicity. The peptide elutes as a peak, and purity is calculated as the area percentage of the main peak relative to all detected peaks.
What Are the Standard HPLC Method Parameters?¶
| Parameter | Typical Value |
|---|---|
| Column | C18, 4.6 × 250 mm, 5 μm |
| Mobile phase A | 0.05–0.1% TFA in water |
| Mobile phase B | 0.05–0.1% TFA in acetonitrile |
| Gradient | 5–60% B over 20–40 min |
| Flow rate | 1.0 mL/min |
| Detection | UV 214 nm |
| Temperature | 25 °C |
| Injection | 10–50 μL |
Purity Calculation¶
Acceptance Criteria¶
| Grade | Minimum Purity |
|---|---|
| Crude | — (baseline) |
| Research | ≥95% |
| Purified | ≥98% |
| GMP | ≥98% (per COA spec) |
Interpreting a Chromatogram¶
- Main peak: Target peptide
- Earlier eluting peaks: Typically deletion sequences (shorter)
- Later eluting peaks: Truncated peptides or hydrophobic impurities
- Baseline separation: Indicates good resolution
Related Tests¶
- Purity analysis
- Impurity profiling
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Method validation
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Peptide Purity Determination — Review the section above for key parameters, methods, and quality criteria.
- What Are the Standard HPLC Method Parameters? — See the section for detailed parameters and specifications.
- Purity Calculation — Review the section above for key parameters, methods, and quality criteria.
- Interpreting a Chromatogram — Review the section above for key parameters, methods, and quality criteria.
🔗 Related: Analytical HPLC System | Purity Analysis | LC-MS Testing | Peptide Quality Control Guide
Key Takeaways¶
- What Are the Standard HPLC Method Parameters? — See the section above for detailed parameters and specifications.
- Purity Calculation — Review the section above for key parameters, methods, and quality criteria.
- Interpreting a Chromatogram — Review the section above for key parameters, methods, and quality criteria.