Quantitative HPLC-UV/DAD Purity Assay
High-Resolution Reverse-Phase Liquid Chromatography for Absolute Purity Determination, Degradant Profiling, and Related Substance Quantitation in Synthetic Peptides and APIs.
The Gold Standard in Chemical Purity Quantitation
High-Performance Liquid Chromatography coupled with Diode Array UV Detection (HPLC-DAD) is the recognized global reference method for separating, identifying, and quantifying chemical compounds in a complex mixture. At Zenith BioChemical Laboratories HK, our chromatography protocols achieve baseline resolution of target active ingredients from structurally similar synthesis impurities, truncated peptides, and degradation products.
Samples are prepared in ultra-pure mobile phases (Optima LC/MS grade acetonitrile and water with 0.05% to 0.1% trifluoroacetic acid or formic acid modifier) and injected into temperature-controlled Agilent 1290 Infinity II UHPLC systems. Dual-wavelength monitoring at 214 nm (peptide backbone amide absorption) and 280 nm (aromatic side chains: Tyr, Trp, Phe) ensures accurate quantification across all molecular fractions.
What This Analysis Delivers
Comprehensive chromatographic reporting conforming to international pharmacopeial standards.
Peak Area Integration
Mathematical integration of all eluting peaks with baseline correction, tailing factor evaluation (T ≤ 1.5), and theoretical plate count calculation (N > 2000).
Impurity & Degradant Profiling
Quantification of diastereomers, deamidation products, oxidation species, and sequence truncation errors down to 0.05% relative abundance.
NIST-Traceable Standards
Retention time (tRT) alignment against certified reference materials (CRMs) run in identical batch sequences under calibrated column conditions.
Raw Chromatogram Curves
Complete multi-wavelength absorption curves, gradient elution tables, and peak integration data tables included on your official Certificate of Analysis.
Standard Operating Parameters (SOP-HPLC-01)
| Parameter | Specification / Condition | Notes |
|---|---|---|
| Instrument Platform | Agilent 1290 Infinity II UHPLC with 1260 DAD | Quaternary high-pressure gradient pump |
| Stationary Phase | ZORBAX Eclipse Plus C18 (2.1 × 100 mm, 1.8 µm) / C8 | Sub-2-micron core-shell technology |
| Mobile Phase A | 0.1% TFA in LC-MS grade ultrapure H₂O (18.2 MΩ·cm) | Degassed under continuous helium/vacuum |
| Mobile Phase B | 0.1% TFA in LC-MS grade Acetonitrile (ACN) | Linear gradient: 5% → 65% B in 15–25 min |
| Flow Rate & Temp | 0.30 – 0.50 mL/min @ 35.0°C ± 0.2°C | Thermostatted column compartment |
| Detection Wavelengths | Primary: 214 nm (Bandwidth 4 nm); Secondary: 280 nm | Full UV spectral scan 190–400 nm |
| Injection Volume | 2.0 – 5.0 µL (0.5 – 1.0 mg/mL sample concentration) | Cooled autosampler @ 4.0°C |
Sample Submission Requirements
- Minimum Quantity: 3 – 5 mg dry lyophilized powder, or 1 intact sealed vial.
- Liquid Samples: Minimum 0.5 mL solution in a secure, leak-proof screw-cap cryovial.
- Packaging: Wrap vials in bubble wrap or rigid vial tubes. Lyophilized peptides remain stable at ambient temperature during express transit.
- Blinded Option: You may submit coded samples (e.g. "Sample #A1") with zero disclosure of vendor or claimed purity.