Peptide Concentration by A280: Extinction Coefficients in Practice
A balance reports how much solid went into the tube. It cannot say how much of that solid is peptide. Measuring peptide concentration by A280, the UV absorbance at 280 nm, answers the question most experiments actually depend on: how many molecules of the peptide are in this solution. The method is quick, uses little material and needs nothing more than a spectrophotometer, but it works only for certain sequences and only when the extinction coefficient is set up correctly. This guide covers the calculation, a worked example, the usual sources of error and how to use the method when checking many vials from one lot.
Why labs measure peptide concentration by A280
A lyophilised peptide is rarely just peptide. The weighed solid usually includes a counter-ion such as trifluoroacetate or acetate, some residual moisture, and occasionally an excipient. A concentration calculated from the balance reading therefore overstates the peptide, and by an amount that can differ between lots. For the background, read net peptide content explained.
Absorbance sidesteps that. Only molecules containing the absorbing residues contribute, so the salt and water in the vial are invisible to the reading. The governing relationship is the Beer-Lambert law:
A = ε × c × l, so c = A ÷ (ε × l)
Here A is the measured absorbance, ε the molar extinction coefficient in M-1cm-1, c the molar concentration and l the path length in centimetres. With a standard 1 cm cuvette, everything hinges on ε.
Building the extinction coefficient from the sequence
At 280 nm nearly all the absorbance in a peptide comes from three structures, and their contributions simply add up. Widely used values, derived from model compounds in a fully unfolded state, are:
| Chromophore | Contribution at 280 nm (M-1cm-1) |
|---|---|
| Tryptophan (each) | about 5,500 |
| Tyrosine (each) | about 1,490 |
| Disulfide bond (each cystine) | about 125 |
So ε280 = 5,500 nTrp + 1,490 nTyr + 125 nS-S. Free cysteine thiols contribute essentially nothing; only formed disulfides count. The value is calculated, not measured, which is why a confirmed sequence and a known oxidation state are prerequisites.
A worked example
Suppose a 2,200 Da peptide contains one tryptophan, one tyrosine and no disulfides.
- ε = 5,500 + 1,490 = 6,990 M-1cm-1
- A stock made up by weight at 0.6 mg/mL would be about 273 µM if the solid were pure peptide, reading roughly 1.9 undiluted. That is above the comfortable linear range of many instruments, so dilute it four-fold before reading.
- If the diluted sample reads 0.42, then c = 0.42 ÷ 6,990 = 60.1 µM in the cuvette.
- Multiply by the dilution factor: 240 µM in the stock.
- Convert with the molecular weight: 240 µM × 2,200 g/mol ≈ 0.53 mg/mL of peptide.
The absorbance result is about 88% of the weighed figure. The missing share is counter-ion and water, and it is precisely that gap the measurement exists to reveal. A heavier salt load would widen it further.
Getting a reliable reading
- Blank against the exact solvent. Zero the instrument on the same buffer, from the same bottle, in the same cuvette.
- Stay in the linear range. Aim for readings roughly between 0.1 and 1.0. Below that, noise dominates; above it, stray light bends the response.
- Use quartz or UV-transparent plastic. Ordinary polystyrene absorbs strongly near 280 nm.
- Know your path length. Micro-volume instruments use sub-millimetre paths and usually normalise to 1 cm. Check which figure you are copying down.
- Read in replicate. Three independent dilutions reveal pipetting error that a single reading hides.
- Consider a denaturant for careful work. The tabulated values assume exposed residues. Folded structure can shift a buried tyrosine’s contribution slightly, so guanidine hydrochloride is sometimes added to match the reference conditions.
Errors that push the number up or down
Scattering from aggregates. Particles scatter light, and the detector cannot tell scattered light from absorbed light. A well-dissolved peptide should read near zero between 320 and 350 nm. Anything above that indicates scatter inflating the 280 nm value. Subtracting the long-wavelength reading gives a rough correction; clarifying the solution is the better fix.
Oxidised tryptophan. Oxidation products of the indole ring absorb differently, so a partly oxidised sample no longer matches the calculated ε.
Loss to surfaces. Dilute solutions of cationic or hydrophobic peptides stick to tubes, tips and cuvettes. The reading falls for reasons unrelated to the stock.
Absorbing additives. Some buffer components, preservatives and nucleic acid contamination absorb near 280 nm and add to the signal.
Sequences with no chromophore at 280 nm
Many research peptides contain no tryptophan, no tyrosine and no disulfide. For them the 280 nm reading is baseline noise, and any concentration derived from it is meaningless.
The fallback is the far-UV region around 205 to 214 nm, where the peptide bond itself absorbs. Every peptide responds there, but the trade-offs are significant. The coefficient must be estimated from composition and is less certain. Buffers, solvents and dissolved oxygen absorb heavily, so the blank becomes critical. Signals are much stronger, forcing high dilutions that amplify pipetting error. For an absolute figure on such sequences, amino acid analysis remains the reference approach; see amino acid analysis.
Using A280 across a multi-vial lot
For a lab that has received many vials of one peptide, the method doubles as a consistency check. Dissolve several vials chosen from across the shipment to the same nominal concentration, read each one, and compare. Close agreement supports even filling and uniform content; an outlier deserves a second look before it enters an experiment.
Record the ε value and its derivation alongside every result, together with the dilution factor, path length and blank. A number without those details cannot be reproduced by a colleague or compared with the next lot.
Remember too what the reading does not show. A deletion impurity that kept its tryptophan absorbs just like the parent molecule, so A280 says nothing about purity. Pair it with a chromatographic method such as the one described in what an HPLC area percentage measures.
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