Before a research sample is analysed further — with HPLC, mass spectrometry or in a functional assay — one basic figure has to be established: the actual concentration of the peptide in solution. A label or certificate of analysis gives the quantity at the moment of packaging, but reconstitution, dilution and pipetting steps introduce uncertainty. UV-Vis spectrophotometry at 280 nm is one of the most widely used laboratory methods for checking that concentration quickly and non-destructively.
The principle: the Beer-Lambert law
UV-Vis concentration determination rests on the Beer-Lambert law, which links the absorbance of a solution to the concentration of the dissolved substance:
A = ε × c × 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 of the cuvette in centimetres (usually 1 cm). Once ε is known for a specific peptide, a single absorbance measurement is enough to calculate the concentration.
Why 280 nm in particular?
At 280 nm it is mainly the aromatic side chains of tryptophan (Trp) and tyrosine (Tyr) that absorb, with a smaller contribution from disulfide-bonded cysteine (cystine). Phenylalanine barely absorbs at this wavelength. That immediately implies the main limitation of the method: a peptide without Trp, Tyr or disulfide bridges generates little to no signal at 280 nm, regardless of the actual concentration. For such sequences A280 is simply not usable and an alternative method has to be found.
Calculating the extinction coefficient
For a peptide with a known amino acid sequence, ε can be estimated theoretically by adding up the individual contributions of the aromatic residues. The reference values commonly used at 280 nm are:
- Tryptophan: approximately 5500 M⁻¹cm⁻¹
- Tyrosine: approximately 1490 M⁻¹cm⁻¹
- Cystine (disulfide-bonded cysteine pair): approximately 125 M⁻¹cm⁻¹
The sum of these contributions, multiplied by the number of times each residue occurs in the sequence, yields a theoretical ε. This estimated value sometimes deviates from the actual, experimentally determined extinction coefficient, partly because of local conformational effects. For accurate quantitative research an experimentally validated ε is therefore preferable to a purely calculated value.
Practical considerations for the measurement
A number of factors affect the reliability of an A280 measurement in research practice:
- Turbidity and aggregation: light scattering by undissolved particles raises the apparent absorbance and distorts the calculated concentration.
- Buffer interference: some buffer components, stabilisers or solvent residues absorb at or near 280 nm themselves and have to be accounted for in a blank measurement.
- Cuvette quality and path length: quartz or UV-transparent plastic cuvettes with a controlled path length are necessary; ordinary glass or standard polystyrene cuvettes do not transmit UV light sufficiently.
- Working range of the instrument: at too high an absorbance (usually above A ≈ 1.5) the detector saturates, which requires a dilution step before measurement.
When an alternative method is needed
For peptides without aromatic residues, research practice often falls back on absorption at 205–214 nm, based on the peptide bond itself, or on colorimetric assays such as BCA or Bradford, which react with specific chemical groups in the peptide. Each method has its own sensitivity, interference profile and required starting volume, and the choice depends on the sequence, the quantity of sample available and the accuracy required. Mass spectrometric quantification, discussed earlier on this blog, also remains a point of reference for identity checking alongside concentration determination.
UV-Vis spectrophotometry remains, despite these limitations, valued in research laboratories for its speed, its non-destructive character and its low cost per measurement. Careful interpretation — with attention to sequence composition, buffer background and instrument calibration — remains essential for reliable results.
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