Absorbance at 280 nm measures three things in a peptide and nothing else: tryptophan, tyrosine, and cystine — the disulfide-bonded form of cysteine. If a sequence contains none of those three, its extinction coefficient at 280 nm is approximately zero and no concentration of it will give a reading. That is not a sensitivity limit a better spectrophotometer or a longer path length gets round; it is the absence of a chromophore, and most of this catalogue falls into that category. Below: how the coefficient is calculated, which catalogue compounds can and cannot be measured this way, how we determined that, and what to do for the ones that cannot.
How does absorbance at 280 nm give a concentration?
Through the Beer-Lambert relationship, A = ε c l: absorbance equals the molar extinction coefficient times the molar concentration times the path length in centimetres. Rearranged, c = A / (ε l). Measure the absorbance in a 1 cm cuvette, divide by the extinction coefficient, and you have the molarity — provided you know ε.
ε at 280 nm is not usually measured for a new molecule. It is predicted from the sequence, because the absorbance there is the sum of a small number of independent contributions. Pace and colleagues analysed measured coefficients across 80 proteins and gave the equation most laboratories still use:
ε(280) = (number of Trp × 5,500) + (number of Tyr × 1,490) + (number of cystines × 125) M⁻¹cm⁻¹
Gill and von Hippel's earlier paper gives a slightly different set — 5,690 for tryptophan, 1,280 for tyrosine, 120 per cystine — with a published erratum noting that the cysteine count must be halved to give cystines first. The two differ by a few percent and either is defensible; what matters is saying which you used. The ExPASy ProtParam tool implements this calculation, which is where most people get the number without thinking about where it came from.
Look at the size of the terms. One tryptophan contributes roughly forty times what one cystine does, and tyrosine about a tenth of a tryptophan. In practice A280 quantification is a tryptophan measurement with a tyrosine correction.
Which residues are actually invisible?
All the others. An aromatic ring is necessary but not sufficient.
| Residue | Absorbance around 280 nm | Note |
|---|---|---|
| Tryptophan | Strong | The dominant contributor |
| Tyrosine | Moderate | About a tenth of tryptophan |
| Cystine (disulfide) | Very weak | Only the oxidised, bonded form |
| Phenylalanine | Effectively none at 280 | Absorbs near 257 nm; its band has fallen away by 280 |
| Free cysteine (reduced thiol) | Effectively none | It has to be a disulfide to count |
| Histidine, methionine, arginine, everything else | None | Transparent at 280 |
Phenylalanine is the trap. It is aromatic, so a sequence containing it looks like it should absorb — and it does, near 257 nm, where its weak benzene band sits. By 280 nm that band has essentially gone, so a peptide can be aromatic on paper and flat at 280.
Free cysteine is the second trap. A reduced thiol contributes nothing meaningful; the 125 M⁻¹cm⁻¹ belongs to the disulfide, so two cysteines count only if they are bonded to each other. Our cysteine and disulfide post covers why the oxidation state is not a detail.
Which catalogue compounds can be measured this way?
Here is the honest table, and the section after it explains exactly how each row was determined.
| Compound | Trp | Tyr | Cystine | Predicted ε(280) | A280 quantification |
|---|---|---|---|---|---|
| Kisspeptin-10 | 1 | 1 | 0 | 5,500 + 1,490 = 6,990 | Works well |
| Melanotan-1 | 1 | 1 | 0 | 5,500 + 1,490 = 6,990 | Works well |
| DSIP | 1 | 0 | 0 | 5,500 | Works |
| Melanotan-2 and PT-141 | 1 | 0 | 0 | 5,500 | Works |
| BPC-157 | 0 | 0 | 0 | ≈ 0 | Not possible |
| KPV | 0 | 0 | 0 | ≈ 0 | Not possible |
| Epithalon | 0 | 0 | 0 | ≈ 0 | Not possible |
| Selank | 0 | 0 | 0 | ≈ 0 | Not possible |
| Semax | 0 | 0 | 0 | ≈ 0 | Not possible, despite a phenylalanine and a methionine |
| L-glutathione | 0 | 0 | 0 as supplied | ≈ 0 | Not possible in the reduced form |
Two rows deserve a sentence each. Semax demonstrates both traps at once: its published sequence is Met-Glu-His-Phe-Pro-Gly-Pro, so it has an aromatic residue and a sulfur atom, and neither is the right one — the phenylalanine absorbs at the wrong wavelength and the sulfur is methionine's. L-glutathione is a cysteine-containing tripeptide whose cysteine, in the reduced form the catalogue supplies, is a free thiol rather than half a disulfide. Its oxidised dimer does contain a cystine, worth about 125 M⁻¹cm⁻¹ — far too little to quantify with.
How we determined this
Three steps, and we are spelling them out because a residue count asserted without a method is not verifiable.
- Residue composition came from sequences this site already publishes. BPC-157's fifteen residues, kisspeptin-10's ten, DSIP's nine, Semax's seven, Selank's seven, Epithalon's four and KPV's three are set out on their own product pages and posts, sourced to PubChem when those pages were written. Melanotan-1 and melanotan-2 are analogues of α-melanocyte-stimulating hormone, whose thirteen-residue sequence our melanotan certificates post gives in full: the tyrosine at position 2 and the tryptophan in the central His-Phe-Arg-Trp motif are both present in melanotan-1, while melanotan-2 and PT-141 retain the motif and its tryptophan without the tyrosine.
- Absence of cysteine was cross-checked against the molecular formula in
src/compound-data.json. A formula with no sulfur cannot contain cysteine, which settles the cystine term for BPC-157 (C62H98N16O22), KPV (C16H30N4O4), Epithalon (C14H22N4O9) and Selank (C33H57N11O9). Where sulfur is present the formula proves nothing — Semax is C37H51N9O10S and that sulfur is a methionine — so the sequence decides. The site's cysteine post reaches the same conclusion for most of the catalogue. - We did not extend the table to compounds whose sequences this site does not already publish. The coefficients above are predictions from a cited equation with the arithmetic shown, not measurements.
One caveat on the predictions: Pace and colleagues derived and validated their equation on folded proteins in water. Short unstructured peptides are if anything a simpler case, but the validation set was proteins, and a predicted ε is a prediction. If a concentration has to be right to better than a few percent, measure the coefficient rather than calculate it.
What the arithmetic looks like in practice
Take kisspeptin-10, molecular weight 1302.4 g/mol from compound-data.json, predicted ε(280) of 6,990 M⁻¹cm⁻¹.
A 1 mg/mL solution is 1 ÷ 1302.4 = 7.68 × 10⁻⁴ M, and in a 1 cm cuvette that reads A280 = 6,990 × 7.68 × 10⁻⁴ = 5.4 — well above the linear range of most spectrophotometers, so the sample needs diluting perhaps twenty-fold first. Equivalently, ε ÷ MW = 6,990 ÷ 1302.4 = 5.4 per mg/mL per cm, which is the form worth writing on the tube because it converts a reading straight into mg/mL. Run the same calculation for BPC-157 and every term is zero: a 10 mg/mL solution of it reads as water. The molarity post covers the mg-to-molar conversion, and the concentration table does the arithmetic for every vial size.
What to use instead
Four practical alternatives, in roughly descending order of how often they are the right answer for a short synthetic peptide.
Use the certificate and the arithmetic. For a lyophilised vial of known fill and known net peptide content, the concentration is net milligrams divided by millilitres of diluent, with no spectrophotometer involved. This is the normal route for this catalogue, and it is why the net peptide content post matters: gross fill weight and net peptide content are different numbers, and using the wrong one is a systematic error in every concentration downstream. What this route cannot see is material lost after reconstitution, to tube walls or to aggregates — the surface adsorption post covers that loss.
Absorbance at 205 nm. The peptide bond absorbs in the far UV, so every peptide has a chromophore there regardless of composition, and Anthis and Clore published a sequence-specific method for predicting molar absorptivity at 205 nm precisely so that peptides lacking tryptophan and tyrosine can be quantified. The catch is the diluent: at 205 nm many common buffer components, chloride included, absorb strongly, so the method needs a UV-transparent diluent and a scrupulous blank, and it will not work on a stock made in a preserved water.
Quantitative HPLC against a reference standard. Inject a known amount of the same peptide, build a calibration curve, read the sample off it. The most reliable route and the most demanding, because it needs an authentic reference standard of the same compound. Every certificate in the COA library reports purity by RP-HPLC at 214 nm, a wavelength chosen for exactly the reason this post exists: 214 nm sees the peptide bond, so it detects peptides that are invisible at 280.
Amino acid analysis. Hydrolyse and quantify the released amino acids against standards. The reference method for peptide content, destructive, and usually sent out rather than run in-house. If a number has to be defensible in print and there is no reference standard, this is the answer.
Colorimetric assays — Bradford, BCA and their relatives — get a caution rather than a recommendation: their response depends on residue composition too, differently for each assay, and their standard curves are built with proteins whose behaviour a short peptide does not reproduce. General laboratory practice treats them as unreliable for short peptides, and we have no peptide-specific validation to cite either way.
Four things that corrupt an A280 reading
Even for a peptide that does have a tryptophan, the reading can be wrong in predictable ways.
Light scattering from aggregates. Suspended aggregate scatters across the UV and inflates the apparent absorbance at 280. Read at 320 or 340 nm, where a clean peptide solution should be flat; anything there means scattering and a 280 figure that is too high. It is a useful early warning for the problem in the aggregation post.
Diluent background. Blank against the identical diluent, never against water. Benzyl alcohol, the preservative in a bacteriostatic water, is itself an aromatic molecule present at a concentration far above the peptide's, so a preserved stock is not a sensible sample for UV quantification at all — see the sterile water post.
Path length, and a coefficient from the wrong molecule. A microvolume instrument reports a normalised 1 cm equivalent from a much shorter path, which amplifies both signal and error, so check the reading sits inside the linear range rather than at the top of it. And recalculate ε from the actual sequence of the actual compound: acetylation or amidation changes almost nothing at 280, but a value copied from a neighbouring analogue can change a great deal.
Frequently asked questions
Why can I not just measure BPC-157 at 280 nm with a more sensitive instrument?
Because sensitivity is not the limitation. Its sequence contains no tryptophan, no tyrosine and no cystine, so there is nothing in the molecule that absorbs at that wavelength. The absorbance is not small, it is absent, and multiplying zero by a larger concentration or a longer path length still gives zero.
Does phenylalanine really contribute nothing?
Essentially nothing at 280 nm. Its absorbance band peaks near 257 nm and is weak even there, and by 280 it has fallen away to the point that the standard extinction-coefficient equations do not include a phenylalanine term at all. A peptide whose only aromatic residue is phenylalanine is not measurable at 280.
What about peptides with non-standard aromatic residues?
The standard equations cover only tryptophan, tyrosine and cystine, so a peptide containing a modified or non-canonical aromatic residue — a naphthylalanine, a dimethylated tyrosine — is outside their scope. Such a residue may well absorb near 280, but there is no published term to plug in, and a predicted coefficient would be a guess. Those compounds need a measured coefficient or a different method.
Can I use A280 to check whether a stock has degraded?
Not reliably. Oxidation, deamidation and backbone hydrolysis do not remove tryptophan or tyrosine, so the absorbance holds while the molecule changes — and tryptophan oxidation, covered in the oxidation post, moves it in a direction that is hard to interpret. The one useful signal runs the other way: rising absorbance at 320 or 340 nm means scattering, and therefore aggregation. For real degradation work the methods in the stability study post answer the question.
Does the certificate give me an extinction coefficient?
No. Our reference table post says why extinction coefficients are still absent from the site's own specification tables: they would have to be verified per compound rather than calculated and published as though measured. Certificates report identity, content and purity. Everything here about 280 nm is calculated from the sequence, not handed to you by the laboratory.
References
- Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. How to measure and predict the molar absorption coefficient of a protein. Protein Science 1995;4(11):2411-2423. doi.org/10.1002/pro.5560041120
- Gill SC, von Hippel PH. Calculation of protein extinction coefficients from amino acid sequence data. Analytical Biochemistry 1989;182(2):319-326. doi.org/10.1016/0003-2697(89)90602-7
- Anthis NJ, Clore GM. Sequence-specific determination of protein and peptide concentrations by absorbance at 205 nm. Protein Science 2013;22(6):851-858. doi.org/10.1002/pro.2253
- ExPASy ProtParam documentation, Swiss Institute of Bioinformatics. Extinction coefficient calculation. Read 29 September 2026. web.expasy.org/protparam/protparam-doc.html
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