HPLC or UHPLC for peptide analysis?
Same technique, smaller particles: what changes on the chromatogram, what does not, and how to compare purity figures from each.
Some COAs say HPLC, some say UPLC or UHPLC, and buyers occasionally ask whether one is "better" or whether a purity figure from one is comparable to the other. They are the same technique at different pressures, and a purity figure from either is comparable if the method is sound. This note explains the difference, what it changes about the chromatogram, and what to look for regardless of which acronym is on the certificate. All Alphex products are for laboratory and in-vitro research only.
Same principle, smaller particles
High-performance liquid chromatography separates components on a packed column under pressure. Ultra-high-performance LC (UHPLC; UPLC is one manufacturer's trademark) uses columns packed with sub-2 µm particles instead of the 3-5 µm particles of conventional HPLC. Smaller particles give sharper peaks and better resolution, but they resist flow, so the instrument must run at several times the pressure (up to 1,000-1,500 bar against 200-400). The chemistry (reverse-phase C18, water-acetonitrile gradient, 0.1% TFA, UV detection at 220 nm) is identical.
What changes on the chromatogram
- Run time. UHPLC methods are typically three to five times faster; a 30-minute HPLC gradient becomes a 6-10 minute one.
- Peak width. Narrower peaks, so impurities that co-eluted as a shoulder on HPLC may resolve as separate peaks on UHPLC.
- Sensitivity. Sharper peaks are taller for the same area, so small impurities are easier to see above the baseline.
- Retention times. Different absolute values, so retention time is only comparable within one method, which is why a reference standard is run alongside. See reference standards.
The consequence for purity is that a well-run UHPLC method may report a slightly lower purity for the same batch than a coarse HPLC method, because it sees impurities the other merged into the main peak. That is a more accurate number, not a worse batch.
What does not change
The calculation (area percent at 220 nm), the assumptions behind it, the need for a chromatogram with axes and an integration table, the need for method conditions, and the need for a batch number on the trace. A poor UHPLC method with a steep gradient can hide impurities exactly as a poor HPLC method can. See how HPLC purity is calculated.
Reading a COA that says one or the other
- Either is fine. Judge the method, not the acronym: gradient length relative to peak count, resolution of minor peaks, wavelength, integration threshold.
- Be more sceptical of a very short run (two or three minutes) with a single peak claimed at 99.9%; speed without resolution is where impurities hide.
- Compare like with like when comparing suppliers: purity from a slow HPLC gradient and purity from a fast UHPLC gradient are both valid but not identical measurements of the same batch.
What Alphex's certificates use
Reverse-phase LC at 220 nm against a reference standard, with the gradient, column and conditions stated on the certificate. The laboratory selects HPLC or UHPLC by compound; the acceptance threshold (99+% area purity) and the reporting standard (trace, integration table, batch number, laboratory name) are the same either way. See how Alphex tests every batch.
Frequently asked questions
Is UHPLC purity "more accurate"?
Potentially, because of better resolution, provided the method is designed for it. It is not automatically so.
Can I compare our lab's HPLC re-test to the COA's UHPLC result?
Yes, with awareness that different resolution may give slightly different purity. A large discrepancy is meaningful; half a percent usually is not.
Which is more common for research peptides?
Both are in routine use. Conventional HPLC remains widespread; UHPLC is increasingly standard in contract laboratories.
Alphex research peptides are supplied for laboratory and in-vitro research by qualified researchers only. They are not authorised for human or veterinary use.
Research use only. Alphex products are supplied exclusively for laboratory and in-vitro research by qualified researchers. They are not authorised for human or veterinary use, and nothing in this note is advice on any such use.
Published 10 July 2026. Alphex reviews research notes when the testing method, the catalogue or UK guidance changes.