Analytical Techniques

High-Performance Liquid Chromatography

HPLC serves as the primary analytical technique for peptide purity assessment and purification. Reversed-phase HPLC (RP-HPLC) separates peptides based on hydrophobic interactions with a nonpolar stationary phase, typically C18 or C8 bonded silica.

Analytical HPLC employs UV detection at 214-220 nm, where peptide bonds absorb strongly. Peak integration provides quantitative purity data expressed as percentage of total peak area.

Mass Spectrometry

Mass spectrometry confirms peptide identity by measuring molecular mass. Electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are the primary ionization methods for peptide analysis.

ESI-MS generates multiply charged ions from peptides in solution, useful for LC-MS applications. MALDI-TOF produces singly charged ions and provides rapid analysis of purified samples. Mass accuracy within 0.1% of theoretical confirms peptide identity.

Amino Acid Analysis

Amino acid analysis (AAA) determines peptide composition through hydrolysis and chromatographic quantitation of individual amino acids. The technique confirms the presence and ratio of expected amino acids in the peptide sequence.

Acid hydrolysis (6N HCl, 110°C, 24 hours) cleaves peptide bonds, releasing free amino acids for derivatization and chromatographic separation. Some amino acids (tryptophan, cysteine) are destroyed or modified during hydrolysis, requiring alternative methods for quantitation.

Peptide Content Determination

Peptide content represents the mass fraction of actual peptide in a lyophilized sample. The remainder consists of counterions (acetate, trifluoroacetate), water, and residual solvents. Typical peptide content ranges from 70-90%.

Peptide content is determined through amino acid analysis or UV spectroscopy using calculated extinction coefficients. This value enables accurate mass-based preparation of peptide solutions for research applications.

Water Content Analysis

Karl Fischer titration quantifies water content in lyophilized peptides. Water absorption occurs during handling and storage, affecting both weight-based calculations and long-term stability.

Typical specifications limit water content to less than 8-10% of total mass. Proper storage conditions (desiccated, frozen) minimize water uptake between analysis and use.

Endotoxin Testing

Limulus amebocyte lysate (LAL) assays detect bacterial endotoxin contamination. Endotoxin limits are specified when peptides are used in applications where bacterial contamination could affect experimental results.

Standard specifications for research peptides typically require less than 1 EU/mg (endotoxin units per milligram). Lower limits may be specified for specialized applications.

How Peptide Hackers COA Documentation Works

Every certificate we publish in our public COA library is lot-level — not a representative certificate for the product line, but the certificate for the specific lot in the vial. Where a certificate is not yet published, the product's COA page says so plainly rather than showing a substitute.

Each certificate reports what the independent testing lab measured: purity by HPLC (the share of the main compound after chromatographic separation, reported per vial when multiple vials were tested), identity confirmation by mass spectrometry, and measured content — the quantity of compound the lab found in the vial.

Figures on our COA pages are copied verbatim from the lab's own report, and every certificate links to the report on the lab's own site, so results can be verified without trusting ours.

For verified compounds we also publish reference identity data alongside the certificate — CAS registry number, molecular formula, and exact molecular weight — checked against PubChem's own records, never copied from secondary sources. Reference data is labeled separately from lab results: the lab certifies what it measured in the lot; the reference section describes the compound itself.

Purity, Measured

Our purity bar is 99% by HPLC, and the number on the product page is not the claim that matters — the number on the lot's certificate is. HPLC purity is the fraction of the chromatogram's total peak area attributable to the main compound; our COA pages show the lab's reported figure per vial, and for supported labs, the actual chromatogram trace from the lab's report.

Purity and identity are reported separately because they answer different questions: purity says how much of the sample is one compound; identity (by mass spectrometry) says that compound is the one on the label.