How to read an HPLC chromatogram on a Janoshik COA

Every Lumera Labs lot ships with a Janoshik Analytical certificate that includes the full HPLC chromatogram, not just the headline purity number. Researchers who've never read a chromatogram before sometimes stop at "purity: 99.18%" and trust it. The chromatogram is where the actual evidence lives — peak shape, baseline drift, impurity placement, integration choices. Reading it takes about 60 seconds once you know what each element means.

The axes

A reverse-phase HPLC chromatogram has two axes:

• X-axis: retention time (minutes). Time between sample injection and when the compound reaches the detector. A peptide's retention time is a chemical fingerprint — same peptide on the same column with the same gradient should retention-elute at the same minute every run. Major impurities elute earlier (more polar) or later (less polar) than the target.
• Y-axis: absorbance (mAU at 214 nm or 220 nm). Peptide bonds absorb UV at 214/220 nm strongly. Peak height tracks concentration. Y-axis units are milli-absorbance units (mAU) — the absolute number depends on injection volume and concentration, so don't read it as "amount of peptide" without context.

The main peak

The target peptide is usually the dominant peak — the tallest, widest peak in the chromatogram. On a well-resolved Janoshik run, the main peak should:

• Be sharp and symmetric (Gaussian shape, not skewed)
• Return cleanly to baseline before the next peak begins
• Account for ≥99% of total integrated peak area (the "purity" number)

If the main peak shows shouldering (a hump on one side) or splits into a doublet, that signals chromatographic co-elution — two compounds eluting at very similar times that the column couldn't fully separate. The reported purity for that lot is approximate, not precise.

Impurity peaks

Smaller peaks before and after the main one are impurities. Each peak gets integrated (its area under the curve calculated) and the impurity is reported as a percentage of total area. Common impurity classes for synthetic peptides:

• Truncated sequences — peptide missing one or more residues from the C- or N-terminus. Usually elute EARLIER (more polar). Common in long peptides where coupling efficiency drops past 30+ residues.
• Deletion sequences — peptide missing a residue from the middle. Hard to separate from the target because mass differs by only one amino acid.
• Oxidized variants — methionine sulfoxide, tryptophan oxidation. Elute slightly earlier than the target. Sensitive to storage conditions.
• Deamidation products — asparagine → aspartate or isoaspartate. Common in stored peptides with N residues at hot spots.
• Residual solvents / reagents — TFA, scavengers, cleavage byproducts. Usually elute very early (well-resolved peaks near solvent front).

What ≥99% purity actually means

A reported purity of 99.18% means: of the total integrated peak area in the chromatogram, 99.18% is the main peak. That figure has several caveats:

• It's relative purity, not absolute concentration. A 99.2% purity vial could be 99.2% peptide and 0.8% impurities, OR it could be 99.2% peptide-relative-to-other-peak-things but only 70% of vial contents (the rest being water/excipient/salts). Lumera lots ship lyophilized with low residual moisture, so the practical difference is small — but it's why peptide content by mass (e.g. from amino acid analysis) is a stronger number than HPLC purity alone.
• It assumes all impurities absorb at 214 nm at similar molar absorptivity to the target peptide. UV-invisible impurities (some salts) won't show. This is why Janoshik also runs LC-MS for identity — to confirm the main peak is actually your target, not a UV-equivalent contaminant.
• It depends on integration choices — where the integrator places baseline and peak boundaries. Reputable labs use consistent automated integration. Suspect a COA if the integration parameters aren't disclosed or if the chromatogram has visible "manual" baseline corrections.

Comparing two COAs of the same peptide

If you have lots from two batches — or two suppliers — and want to compare:

1. Same x-axis scale. Make sure both chromatograms run on the same gradient (linear B 5-95% over 20 min is common). Different gradients shift retention times — direct comparison is meaningless.
2. Same column. Reverse-phase C18 5 μm, 150×4.6mm is the typical Janoshik configuration. Different column chemistries shift selectivity.
3. Compare peak shape, not just purity %. A 99.5% lot with broad/asymmetric peaks is lower quality than a 99.1% lot with sharp peaks.
4. Look at impurity placement. A "fresh" lot has the impurity profile from synthesis. A "degraded" lot accumulates new peaks adjacent to the main peak (deamidation, oxidation) that weren't in the original COA.

Where the Janoshik chromatogram lives

Every Lumera Labs lot's chromatogram is published at lumerapeptide.com/lab-results/, indexed by lot number. The lot number is printed on the vial label. Pull up the PDF, look at the main peak, scan for impurity placement, confirm the LC-MS identity panel matches the expected mass. ~60 seconds per lot.

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