220 nm or 280 nm: peptide detection wavelengths
Why purity is measured at 220 nm, what 280 nm is actually good for, and how to read a COA reported at an unusual wavelength.
The wavelength printed on a chromatogram is not a technicality. It determines which impurities the detector can see, and therefore what a purity percentage means. A peptide that is 99.5% pure at 280 nm may be 96% pure at 220 nm, and both figures are correct measurements of different things. This note explains why 220 nm is the standard for peptide purity, what 280 nm and 214 nm show instead, and how to read a COA that reports at an unusual wavelength. All Alphex products are for laboratory and in-vitro research only.
What absorbs where
- 210-220 nm: the peptide bond itself. Every amide linkage in every peptide absorbs here, so the signal is roughly proportional to the number of residues, regardless of sequence. This is what makes it the universal peptide detection wavelength.
- 254-260 nm: phenylalanine weakly, and many aromatic contaminants (including benzyl alcohol from bacteriostatic water and residual scavengers from synthesis).
- 275-280 nm: tryptophan strongly, tyrosine moderately, cysteine disulfides weakly. Phenylalanine barely. Peptides with none of these residues are almost invisible at 280 nm.
Why purity is measured at 220 nm
Because the impurities in a synthetic peptide are themselves peptides (deletions, truncations, oxidised and deamidated forms) and the only thing guaranteed to make them visible is the peptide bond. At 220 nm a deletion sequence responds almost like the target and its peak area is a fair estimate of its abundance. At 280 nm, if the deleted residue happened to be the peptide's only tryptophan, the impurity vanishes from the trace entirely and purity appears higher than it is. Reporting purity at 220 nm (or 214 nm, which is similar but noisier) is the accepted standard for exactly this reason. See how HPLC purity is calculated.
What 280 nm is good for
Quantification, not purity. If a peptide contains tryptophan or tyrosine, its concentration in solution can be estimated from absorbance at 280 nm using the residues' extinction coefficients, which is a fast way to check net peptide content or a working solution. Of the Alphex catalogue, GLP-3 (RT) has tyrosine and tryptophan in the glucagon-family backbone and can be quantified this way; Ipamorelin's naphthylalanine absorbs in the 220-230 nm and 280 nm regions; BPC-157, TB-500 and GHK-Cu have no Trp or Tyr and cannot be quantified at 280 nm.
Reading a COA reported at 280 nm
Treat it with caution. Ask whether a 220 nm trace exists; a laboratory running peptide purity will almost always have collected both. If the supplier can only offer 280 nm, the purity figure is an upper bound and may be substantially optimistic for any peptide with few aromatic residues. Alphex reports purity at 220 nm on every COA. See how to read a research peptide COA.
Practical notes for the bench
- Benzyl alcohol absorbs at 220 nm and around 257 nm. Analytical samples of stocks made in bacteriostatic water will show its peak; prepare analytical samples in water or mobile phase, or exclude the peak. See benzyl alcohol explained.
- TFA in the mobile phase absorbs below about 215 nm; 220 nm is a compromise between sensitivity and baseline drift.
- Buffers such as HEPES and Tris absorb in the low UV; do not run purity in them.
Frequently asked questions
Is 214 nm better than 220 nm?
Slightly more sensitive, more baseline noise from mobile-phase additives. Either is standard; the point is that it is a peptide-bond wavelength.
Can I quantify BPC-157 by UV?
Only at 205-220 nm against a standard curve, and buffer absorbance makes that awkward. Amino acid analysis or a peptide assay is more reliable. See net peptide content.
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 13 August 2026. Alphex reviews research notes when the testing method, the catalogue or UK guidance changes.