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Evidence-based GLP-1 & peptide discussion since 2023
ForumsCOA & Analytical TestingHPLC vs UPLC for peptide purity — method comparison study Page 3

HPLC vs UPLC for peptide purity — method comparison study

kate.chem Mon, Jun 8, 2026 at 11:18 AM 18 replies 450 viewsPage 3 of 4
mona_PHX
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Jun 8, 2026 at 2:50 PM#11
Dr.CardioMD said:
They are not contradictory, they measure different things, and this is the single most common misreading of a certificate.

Genuinely useful, thank you. I had the facts and not the framework. Printing the relevant bit and taking it with me.

46 19RetaRick_CA, JenPlateau, SallyK_inj and 43 others
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PeptideChemSF
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Jun 8, 2026 at 3:45 PM#12

Adding the clinical framing, because it changes how the question reads.

HPLC results interpretation for certificates of analysis: when you get your Janoshik report back, here's what the numbers mean:

  • Purity %: Should be >97%. Below 95% is concerning.
  • Retention time: Should match reference standard for the peptide
  • Related substances: Degradation products should be <2% total
  • Single impurity: No individual impurity should exceed 1%

My last batch: 99.3% purity, 0.1% total related substances. Clean chromatogram with single sharp peak at expected retention time. Exactly what you want to see.

Last edited: Jun 8, 2026 at 7:45 PM
47 20dave_SLC, FDA_TrackerJim, ricardo_MIA and 44 others
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mike_nyc
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Jun 8, 2026 at 4:39 PM#13
LindaRN_retired said:
I tested my certificates of analysis batch through Janoshik — 98.7% purity.

Reconstituted peptide stability data for certificates of analysis: I had Janoshik test my reconstituted vial at day 1, day 14, and day 28 stored in the refrigerator.

Day 1: 99.4% purity
Day 14: 98.5% purity
Day 28: 99.2% purity

Minimal degradation over 28 days when properly refrigerated. This confirms the standard 28-day beyond-use date is appropriate. Beyond 28 days, I'd reconstitute a fresh vial to be safe.

48 21Dr.EM_Chicago, pete_RVA, CarlaRPh_TPA and 45 others
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Sigma-Aldrich — Research-Grade Standards

Certified reference materials, analytical reagents, and research-grade standards for peptide verification. Trusted by laboratories worldwide.

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labquiet_amy
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Jun 8, 2026 at 5:34 PM#14
kate.chem said:
The provenance questions are more useful than the numbers.

Mass spectrometry confirmation for certificates of analysis: beyond HPLC purity, mass spec tells you the molecule's exact molecular weight. This confirms identity — not just purity.

Why this matters: HPLC can show you a "pure" compound, but if it's pure of the WRONG compound, purity is meaningless. Mass spec at m/z 4113.58 Da confirms it's actually semaglutide. My Janoshik report includes both HPLC and mass spec.

If your testing provider only offers HPLC, consider upgrading to include mass spec for at least your first batch from a new vendor.

49 22bri_stats, pete_manc_UK, anna.melb_AU and 46 others
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Admin
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Jun 8, 2026 at 6:28 PM#15

Moderator note: leaving this open. It is being argued well and the disagreement is the useful part. Tagging this one for the weekly digest.

Last edited: Jun 8, 2026 at 10:28 PM
50 23PeptideSynthNJ, Dr.KarenChen, Dr.NateNeph and 47 others
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