Measuring Concentration by Absorbance at 280 Nanometres
Start with the disqualifying condition, because it rules out a large share of catalog sequences. Absent tryptophan, absent tyrosine and absent any disulfide, nothing in the molecule absorbs measurably at 280 nm. Plenty of peptides sit in that category, and for them this technique simply is not on the table: the instrument returns baseline noise, and a concentration computed from noise is meaningless. That same absence also rules out 280 nm as a detection wavelength during chromatography on those sequences, a limitation that keeps resurfacing for exactly this reason.
Where one of the three is present, though, the payoff is considerable.
Why the spectrophotometer beats the balance
Put a vial on a balance and you learn the mass of everything in it: peptide, counter-ion, whatever water the solid has taken up, any excipient present. Shine light through a solution instead and only molecules carrying the absorbing residues respond, which means the reading tracks the peptide itself and ignores however much salt and moisture travelled with it.
That difference is not marginal for material supplied as a trifluoroacetate salt holding several percent residual moisture. A figure derived from mass runs systematically high, and how high shifts between lots. The root of the problem is laid out in net peptide content explained.
The arithmetic underneath
Beer-Lambert states that absorbance is the molar extinction coefficient multiplied by concentration and by path length: A = εcl. Rearrange and c = A / (εl). The cuvette fixes path length, conventionally at 1 cm, and the instrument supplies absorbance, so everything rests on knowing ε for the particular molecule in the cuvette.
At 280 nm three contributors account for essentially all of it, and their contributions simply add.
| Tryptophan | roughly 5,500 M-1cm-1 |
| Tyrosine | roughly 1,490 M-1cm-1 |
| Cystine, that is, a disulfide already formed | roughly 125 M-1cm-1 |
Which gives ε280 ≈ (5,500 × Trp count) + (1,490 × Tyr count) + (125 × disulfide count). Nobody measures this; it is computed straight from the sequence, and that is why having the sequence is a precondition for using the method.
Running it on an actual solution
Suppose a 3,000 dalton peptide with a single tryptophan, two tyrosines and no disulfides. Then ε = 5,500 + (2 × 1,490) = 8,480 M-1cm-1.
At 1 mg/mL that solution is 1/3,000 molar, which is 0.333 mM, and in a 1 cm cuvette it would read 8,480 × 0.000333 = 2.82. Too high to trust, since instrument linearity typically gives out somewhere around 1.5 to 2, so the solution gets diluted and the dilution factor carried back through the calculation.
Reversing the process from a real reading: measure 0.85 on a five-fold dilution, and c = 0.85 / 8,480 gives 100 µM inside the cuvette. Multiply back and the stock is 500 µM, which at 3,000 Da works out to 1.5 mg/mL.
Dropping to the low wavelength
Between roughly 205 and 214 nm the peptide bond itself absorbs, so every sequence responds and the coverage problem disappears. Three new problems arrive in its place.
- Response scales with the number of peptide bonds, so a coefficient has to be estimated from chain length rather than from a few well-characterized residues, and such estimates are appreciably shakier.
- Nearly everything else absorbs down there as well, solvent, buffer components, dissolved oxygen, trace contaminants, which puts an enormous amount of weight on the blank.
- Absorbance is far stronger, forcing very dilute solutions, and dilution amplifies whatever volumetric error is present.
Use it where nothing else is available. It does not substitute cleanly for a 280 nm reading on a tryptophan-containing peptide.
Things that quietly shift the reading
Aggregates scatter light, and a spectrophotometer has no way to distinguish scattering from absorbance. Look for it at 320–350 nm, where a properly dissolved peptide ought to show nothing at all; any signal there means the 280 nm value is inflated. Convention is to subtract the scattering contribution, though repairing the solution is the better answer. Oxidized tryptophan absorbs unlike intact tryptophan, so a partially oxidized sample carries a coefficient slightly displaced from the calculated one. Environment matters too: published coefficients describe residues denatured and fully exposed to solvent, so a folded peptide tucking a tyrosine away shifts that residue’s contribution a little, which is the argument for measuring in a denaturing solvent when the work warrants it. Finally, dilute cationic peptides give material up to the cuvette and to whatever tube the dilution was made in, and read low for reasons having nothing to do with the spectroscopy.
What the number does and does not establish
A 280 nm reading establishes how concentrated the molecules bearing the expected aromatic residues were, in that solution, at that instant. Purity is untouched by it. A deletion sequence that kept its tryptophan absorbs precisely as the parent does and gets counted as parent.
Treat it as a concentration method rather than an identity or purity method, and pair it with the methods that do cover those, described in what “98% by HPLC” is a percentage of and amino acid analysis.
