High-Performance Liquid Chromatography (HPLC) and Certificates of Analysis (COAs) are fundamental tools used across scientific and analytical laboratories. They provide critical information about purity, identity, and composition of research compounds, helping researchers validate that a material meets required experimental standards.

This guide explains what HPLC is, how it works, why it matters, and how to understand a COA, using clear, compliance-safe language suitable for Australian research environments.
No part of this article refers to medical or therapeutic use.

Overview: What Is HPLC?

High-Performance Liquid Chromatography (HPLC) is an analytical technique used to separate, identify, and quantify components within a mixture. It is widely used in peptide analysis, small-molecule profiling, and general laboratory verification.

Why HPLC Matters in Research

• Confirms compound purity
• Detects impurities or degradation products
• Validates identity and stability
• Ensures consistency across research batches
• Supports reproducibility in experimental design

Researchers rely on HPLC data to evaluate whether a compound is suitable for in-vitro, biochemical, or analytical studies.

How HPLC Functions in Scientific Research

HPLC operates by passing a liquid sample through a packed column under high pressure. Different molecules interact with the column’s stationary phase differently, causing them to separate as they pass through.

Core Mechanisms

• Mobile Phase – solvent carrying the analytes
• Stationary Phase – column material interacting with compounds
• Retention Time (RT) – time it takes for a compound to exit the column
• Detector Response – produces peaks that represent components

What Researchers Examine

• Purity of synthesized peptides
• Stability of compounds under experimental conditions
• Presence of unwanted byproducts
• Batch-to-batch consistency
• Analytical fingerprints for identification

For peptides and small molecules, HPLC is one of the most trusted methods to verify material quality before use in laboratory workflows.

Understanding the Certificate of Analysis (COA)

A Certificate of Analysis (COA) accompanies most research compounds and provides verified test results from analytical laboratories.

Key Sections of a COA

Most COAs include:

1. Product Identification
   • Name of compound
   • CAS number (if applicable)
   • Batch or lot number
2. Appearance
   • Description of the lyophilised powder or solution
3. Purity (%)
   Usually determined by HPLC.
   • A single sharp peak suggests high purity
   • Peak area percentages show how much of the sample is the desired compound
4. HPLC Chromatogram
   • Graph showing retention time and peak height
   • Major peak represents the primary compound
   • Smaller peaks represent impurities or related substances
5. Mass Spectrometry (if included)
   • Confirms molecular weight
   • Ensures identity of the compound
6. Solubility / Stability Notes
   • Information relevant to laboratory preparation
7. Storage Guidelines
   • Helps maintain sample integrity during research use

How to Interpret HPLC Purity

Purity (%) displayed on the COA reflects the proportion of the main peak relative to total peaks.

Example:
If the main peak represents 99.12% of the total area, the compound is considered high purity.

Researchers examine:

• Retention Time – should match reference standards
• Peak Shape – symmetrical peaks indicate stability
• Impurity Peaks – small, isolated peaks are expected in raw analytical data

What Peaks Mean in Research Context

• Primary Peak – the target compound
• Secondary Peaks – minor impurities, byproducts, or degradation fragments
• Baseline Noise – normal signal fluctuations

HPLC data helps researchers make decisions about suitability for cell-based assays, protein-binding tests, or general biochemical analysis.

Key Research Areas Using HPLC & COA Analysis

HPLC and COA interpretation support a broad range of scientific fields, including:

• Peptide synthesis verification
• Metabolic pathway research
• Biochemical purity assessments
• Protein and enzyme kinetic studies
• Stability testing under various laboratory conditions
• Analytical method validation

Best Practices for Researchers Interpreting HPLC & COA Data

1. Confirm Batch Number & Identity

Always match the COA’s batch or lot number with the vial being used in the study.

2. Review Purity and Retention Time

Ensure results align with expected reference values for your experimental design.

3. Evaluate Peak Patterns

• Broad peaks may indicate instability
• Multiple large peaks may suggest impurities

4. Note Any Stability or Solubility Notes

COAs often include conditions that maintain molecular integrity.

5. Store Documents With the Material

Maintaining a COA logbook helps with reproducibility and compliance.

6. Perform Your Own Verification When Needed

Some labs run their own HPLC to cross-reference supplier reports.

Conclusion

HPLC and COAs are essential tools for verifying the purity, identity, and consistency of research compounds. Understanding how to read chromatograms, purity percentages, and batch data helps ensure accurate, reproducible results across laboratory environments.

PurePeptix provides a wide catalogue of research-grade peptides and compounds, each accompanied by clear analytical details to support informed scientific work.
Explore our product pages and Research Library for more information on laboratory handling, stability, and analytical interpretation.

For supporting documents, visit our Research Library, FAQ Hub, and Peptide Storage Guide.