GlossaryVendor & SourcingHPLC (High-Performance Liquid Chromatography)
Vendor & Sourcing

HPLC (High-Performance Liquid Chromatography)

Also known as: HPLC · liquid chromatography

HPLC (high-performance liquid chromatography) is a lab method that separates a sample into its components so the main compound's purity can be measured.

High-performance liquid chromatography (HPLC) pumps a dissolved sample through a packed column. Different molecules interact with the column material to different degrees, so they come out at different times. A detector, often an ultraviolet (UV) detector, records each one as a peak on a chart called a chromatogram. Reversed-phase HPLC is widely used for peptides; more polar peptides may need a different column chemistry, such as hydrophilic interaction chromatography (HILIC).

On a peptide COA, HPLC purity is normally reported as the main peak's share of the total detected peak area. A result of 98% means about 2% of the detected signal came from other substances, such as truncated sequences, oxidized forms or synthesis by-products. HPLC with a UV detector is good at measuring how much of the signal is the main peak; on its own it does not prove what that main peak is.

Why it matters

Synthetic peptides carry predictable impurities: sequences missing an amino acid or carrying an extra one, altered forms of individual residues, oxidation products and leftover reagents. Liquid chromatography is the established way to separate and measure them, which is why a COA's purity figure normally comes from HPLC.

The limits matter as much as the number. Analytical labs that test illegal peptide products pair liquid chromatography with mass spectrometry for identification and use UV signal for quantification. A purity figure also does not tell you how much peptide is in the vial: in one test-purchase study of online semaglutide, measured content was above the label while measured purity was 7.7% to 14.37%.

How to read an HPLC result

  1. Find the purity percentage and the method line (column type, detection wavelength). A purity number with no method stated is hard to judge.
  2. Look at the chromatogram if one is provided: one tall main peak with small side peaks is what a high-purity result should look like.
  3. Check that the report names the batch and the date of testing.
  4. Look for a separate mass spectrometry result. HPLC purity without an identity test leaves open what the main peak is.

HPLC purity (%) = main peak area ÷ total area of all detected peaks × 100

Worked example

A chromatogram shows a main peak with area 9,860 and side peaks totaling 140. Total area = 10,000. Purity = 9,860 ÷ 10,000 × 100 = 98.6%. Check: 140 ÷ 10,000 = 1.4%, and 98.6% + 1.4% = 100%.

Common mistakes

  • Reading a high HPLC purity as confirmation that the vial holds the right peptide.
  • Reading purity as the amount of peptide in the vial; it is a share of detected signal, not a weight.
  • Comparing purity numbers from different labs or methods as if they were directly equivalent.
  • Accepting a purity figure with no batch number, date or method attached.
  • Assuming HPLC purity says anything about sterility or endotoxin, which need separate tests.

Sources

  1. ReviewRelated impurities in peptide medicines. J Pharm Biomed Anal, 2014. PMID 25044089.
  2. ReferenceAnalysis of illegal peptide drugs via HILIC-DAD-MS. Talanta, 2017. PMID 28738623.
  3. ReferenceMultifactor Quality and Safety Analysis of Semaglutide Products Sold by Online Sellers Without a Prescription: Market Surveillance, Content Analysis, and Product Purchase Evaluation Study. J Med Internet Res, 2024. PMID 39509151.

Page updated . How we grade evidence: methodology.

Related Terms

COA (Certificate of Analysis)

A certificate of analysis (COA) is a lab report showing test results, such as purity and identity, for one specific production batch of a product.

Mass Spectrometry

Mass spectrometry (MS) is a lab method that measures the mass of molecules, used to confirm a peptide is the molecule it is supposed to be.

Vendor Trust Score

DoseCraft's vendor trust score is an editorial rating of a peptide seller's documentation, testing claims, reputation and business details, not a lab result.

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