HPLC purity testing explained

When a peptide is synthesized, the final product is never 100% the target sequence. The synthesis process produces some quantity of truncations (shorter sequences where coupling failed), deletion products, diastereomers, residual solvents, and other byproducts. Purity is the measurement of how much of the final product is the actual target peptide versus everything else.

High-Performance Liquid Chromatography (HPLC) is how purity gets quantified. It's the analytical technique that produces the chromatogram on every Merit COA.

How HPLC works

A small sample of the peptide is dissolved in solvent and injected into a column packed with a stationary phase (typically C18 silica beads for reversed-phase HPLC). The sample is then pushed through the column under high pressure using a moving solvent gradient. Different molecules in the sample interact with the stationary phase differently — some elute (come out) faster, some slower. As each molecule exits the column, it passes through a UV detector (peptides are commonly detected at 220 nm, where the peptide bond absorbs strongly), and the detector records an absorbance trace over time.

The result is a chromatogram: a graph of detector signal versus time, with each peak representing a distinct molecule. The largest peak is the target peptide. Smaller peaks are impurities.

How purity is calculated

Purity is reported as the area under the curve (AUC) percentage of the main peak relative to all peaks above the noise threshold. If the main peak represents 99% of the total integrated peak area, the sample is 99% pure by HPLC.

A few subtleties worth knowing:

• UV-active impurities only. HPLC purity captures impurities that absorb UV at the detection wavelength. Things that don't absorb (residual salts, certain solvents) are invisible to this measurement — that's why we also test for residual solvents via separate methods.
• Method-dependent. Different HPLC methods (gradient profiles, columns, run times) can produce slightly different purity numbers for the same sample. A reputable lab uses a peptide-specific validated method.
• Reversed-phase is standard. For peptides, reversed-phase HPLC (RP-HPLC) with a C18 column is the standard. Other modes exist (ion-exchange, size-exclusion) but are used for specific purposes.

What "99% AUC purity" actually means

When a Merit COA reports 99.2% AUC purity, it means: of all UV-absorbing material in the sample, 99.2% of the integrated peak area came from the target peptide peak. The remaining 0.8% is distributed across smaller peaks representing synthesis-related impurities.

For research use, peptide-grade lots commonly target ≥95% purity. Many suppliers report 95–98%. Merit's release floor is 99% — lots that don't clear it are rejected before shipping. We document those rejections in our internal lot history but the customer never sees them.

What HPLC doesn't tell you

HPLC measures how much target peptide is in the sample. It does not tell you whether the peptide present is actually the labeled one. A misidentified peptide can have a perfectly clean HPLC chromatogram. That's why HPLC is always paired with mass spectrometry, which identifies the molecule by mass. The combination — HPLC for quantity, MS for identity — is what produces a complete purity-and-identity COA.

Reading a chromatogram

The Merit COA includes the full HPLC trace for the lot. The most important elements:

1. Main peak (target). The largest peak; its retention time should match the published reference for that peptide.
2. Integration window. Marked on the trace, showing the time range over which peaks are integrated.
3. AUC table. Shows each peak's retention time, height, and percentage of total area.
4. Method block. Records the column, gradient profile, flow rate, and detection wavelength — so the test is reproducible.

Why Merit publishes every chromatogram

Reproducibility matters. By publishing the actual chromatogram (not just the headline number), researchers can:

• Verify the integration was correctly done (no peaks excluded)
• See the impurity distribution, not just the total — small early-eluting peaks may matter for specific applications
• Compare across lots over time as Merit's process matures

You'll find the chromatogram for the current shipping lot linked on each product page. After purchase, the chromatogram for your specific lot is permanently linked to your order.