Measuring Concentration by Absorbance at 280 Nanometres

Weighing a peptide tells you the mass of a mixture. Measuring its absorbance tells you the number of molecules of one specific thing — but only if the molecule contains one of three residues, and only if you know its extinction coefficient.

The relationship

The Beer-Lambert law states that absorbance equals the molar extinction coefficient multiplied by concentration multiplied by path length: A = εcl. Rearranged, c = A / (εl).

Path length is fixed by the cuvette, conventionally 1 cm. Absorbance is what the instrument reads. So the whole measurement turns on knowing ε for that specific molecule.

Where the coefficient comes from

At 280 nm, absorbance in a peptide comes almost entirely from three sources, and their contributions are additive:

• Tryptophan — approximately 5,500 M^-1cm^-1
• Tyrosine — approximately 1,490 M^-1cm^-1
• Cystine, meaning a formed disulfide bond — approximately 125 M^-1cm^-1

So ε₂₈₀ ≈ (5,500 × number of Trp) + (1,490 × number of Tyr) + (125 × number of disulfides). This is calculated from the sequence, not measured, and it is why a sequence is a prerequisite for the method.

A worked example

Take a peptide of 3,000 daltons containing one tryptophan and two tyrosines, with no disulfides.

ε = 5,500 + (2 × 1,490) = 8,480 M^-1cm^-1.

A solution at 1 mg/mL is 1/3,000 molar, or 0.333 mM. Its absorbance in a 1 cm cuvette is 8,480 × 0.000333 = 2.82.

That is a high reading — most instruments are linear to about 1.5 to 2 — so the solution would be diluted before measuring, and the dilution factor carried back through the arithmetic. Working backwards from a measured absorbance of 0.85 on a five-fold dilution: c = 0.85 / 8,480 = 100 µM in the cuvette, so 500 µM in the stock, which at 3,000 Da is 1.5 mg/mL.

Why this is better than weighing

Weighing measures the vial’s contents: peptide plus counter-ion plus residual water plus any excipient. Absorbance measures only molecules containing the absorbing residues, which means it measures **the peptide itself**, independent of how much salt and water came with it.

For a peptide supplied as a trifluoroacetate salt with several percent residual moisture, the difference between the two numbers is not small. The mass-based figure is systematically high, and by an amount that varies lot to lot. The underlying problem is set out in net peptide content explained.

When it does not work at all

**No tryptophan, no tyrosine, no disulfide means no measurable absorbance at 280 nm.** A great many peptides fall into this category, and for them the method is simply unavailable — the reading is baseline noise and any concentration calculated from it is meaningless.

This is the same limitation that makes 280 nm useless for chromatographic detection on those sequences, and it comes up throughout this material for the same underlying reason.

The alternative wavelength, and why it is worse here

Absorbance near 205–214 nm comes from the peptide bond itself, so every peptide responds. That solves the coverage problem and introduces three others.

The response depends on the number of peptide bonds, so a coefficient has to be estimated from length rather than from a small number of well-characterised residues, and the estimates are less reliable. Almost everything else absorbs there too — the solvent, buffer components, dissolved oxygen, trace contaminants — so the blank matters enormously. And absorbance is much stronger, so solutions must be very dilute, which magnifies pipetting error.

It is used where there is no alternative. It is not a drop-in replacement for a 280 nm measurement on a tryptophan-containing peptide.

What else moves the number

• Light scattering from aggregates. Aggregated material scatters, and a spectrophotometer records scattering as absorbance. The tell is absorbance at 320–350 nm, where a properly dissolved peptide should absorb nothing. A reading there means the 280 nm figure is inflated, and the usual correction is to subtract the scattering contribution — or better, to fix the solution.
• Oxidised tryptophan absorbs differently from intact tryptophan, so a partly oxidised sample has a coefficient slightly different from the calculated one.
• Environment. The published coefficients are for residues in a denatured, fully solvent-exposed state. A folded peptide that buries a tyrosine shifts its contribution slightly, which is why the convention for careful work is to measure in a denaturing solvent.
• Adsorption losses. A dilute solution of a cationic peptide loses material to the cuvette and to the tube it was diluted in, and reads low for reasons that have nothing to do with the method.

What it establishes and what it does not

A 280 nm measurement establishes the concentration of molecules carrying the expected aromatic residues, in that solution, at that moment. It says nothing about purity: a deletion sequence retaining the tryptophan absorbs exactly as the parent does and is counted as parent.

It is a concentration method, not an identity or purity method, and it pairs naturally with the ones that are — described in what “98% by HPLC” is a percentage of and amino acid analysis.