How to Measure Peptide Concentration: UV Absorbance, Amino Acid Analysis and Dye Assays

Nominal versus measured concentration

When a powder is weighed and dissolved, the label concentration is the weighed mass divided by the volume. That number assumes the whole mass is peptide. In practice the powder also contains water and counter-ions, and some peptide can be lost to labware, so the amount in solution is lower than the nominal value. The difference is the net peptide content, explained in the guide on purity versus net peptide content. Chromatographic purity is a different quantity and is not changed by any of the methods below.

Measuring concentration matters whenever results are expressed per mole: binding constants, potency values, spectroscopic quantities such as mean residue ellipticity, and comparisons between laboratories.

UV absorbance at 280 nm

Absorbance follows the Beer-Lambert law: A = ε × c × l, where ε is the molar absorption coefficient (extinction coefficient), c the molar concentration and l the path length. Near 280 nm the absorbing groups are tryptophan, tyrosine and disulfide-bonded cystine. Coefficients can be calculated from the sequence, and an analysis of 116 measured values gave the widely used equation ε280 = 5,500 × (number of Trp) + 1,490 × (number of Tyr) + 125 × (number of cystines) M⁻¹ cm⁻¹. The same study found predictions reliable for sequences that contain tryptophan and less reliable for those that do not, and recommended measuring the coefficient where possible.^[1]

A worked example: Tirzepatide contains two tyrosines and one tryptophan and no disulfide, so ε280 ≈ 5,500 + 2 × 1,490 = 8,480 M⁻¹ cm⁻¹. A solution reading A280 = 0.44 in a 1 cm cell, blank-subtracted, contains 0.44 / 8,480 = 51.9 µM. At a molecular weight of 4813.5 g/mol that is 0.250 mg/mL. The peptide properties calculator gives the A280 coefficient for a typed sequence.

Far-UV absorbance at 205 or 214 nm

Many research peptides contain no tryptophan or tyrosine, and for them A280 is useless. The peptide bond itself absorbs strongly below 220 nm. Anthis and Clore proposed predicting ε205 from the sequence: 2,780 M⁻¹ cm⁻¹ for each backbone peptide bond, plus side-chain terms such as 20,400 for tryptophan, 8,600 for phenylalanine, 6,080 for tyrosine, 5,200 for histidine, 1,830 for methionine and 1,350 for arginine.^[2] Per-residue coefficients have also been published at 214 nm, a wavelength used for HPLC detection.

A worked example: BPC-157 (GEPPPGKPADDAGLV) has 15 residues, 14 peptide bonds and none of the listed side chains, so ε205 ≈ 14 × 2,780 = 38,920 M⁻¹ cm⁻¹. Because the coefficient is high, a stock is diluted first: a nominal 1 mg/mL solution diluted 100-fold should read 0.01 / 1419.5 × 1,000,000 = 7.04 µM, or A205 ≈ 0.274. If the reading is 0.230, the diluted solution is 5.91 µM and the stock is about 0.84 mg/mL, 84% of nominal. Selank (TKPRPGP) works the same way: 6 peptide bonds plus one arginine give ε205 ≈ 16,680 + 1,350 = 18,030 M⁻¹ cm⁻¹.

Far-UV readings need care. Many buffer components, including carboxylates, Tris and HEPES, can absorb strongly below 220 nm, so measurements are usually made in water or dilute phosphate against a matched blank, and absorbance should stay within the linear range of the instrument, roughly 0.1 to 1.

Amino acid analysis

Amino acid analysis (AAA) is the classical reference method for peptide quantity.^[3] A measured aliquot is hydrolyzed, typically in 6 M hydrochloric acid at about 110 °C for around 24 hours, and the released amino acids are separated by chromatography and quantified against standards. Hydrolysis has known limitations: tryptophan is largely destroyed, asparagine and glutamine are converted to aspartate and glutamate, cysteine and methionine can oxidize, serine and threonine are partly lost, and bonds between bulky residues such as valine and isoleucine are slow to break. Quantity is therefore calculated from the stable residues.

A worked example: a 50 µL aliquot of a BPC-157 solution yields 1.42 nmol alanine, 0.70 nmol leucine and 0.71 nmol lysine. The sequence contains two alanines and one each of leucine and lysine, so the three estimates are 0.71, 0.70 and 0.71 nmol of peptide, with a mean of 0.707 nmol. That is 0.707 nmol / 50 µL = 14.1 µM, or 0.0201 mg/mL at 1419.5 g/mol.

Reference laboratories use isotope-dilution LC-MS/MS for the amino acid step, and quantitative NMR (qNMR) can measure the intact peptide without hydrolysis. In a study on an angiotensin II reference material, both methods had to be corrected for related peptide impurities, and the trifluoroacetate counter-ion made up nearly a quarter of the mass.^[4]

Why BCA and Bradford assays mislead for peptides

Colorimetric protein assays are convenient, but they report concentration relative to a calibration curve built with a reference protein, usually bovine serum albumin.

In the BCA assay, protein reduces Cu²⁺ to Cu⁺ in alkaline solution and bicinchoninic acid forms a purple complex with the Cu⁺.^[5] Cysteine, tryptophan, tyrosine and the peptide bond all reduce copper, and studies with di-, tri- and tetrapeptides found the colour was not simply the sum of those groups.^[6] A peptide rich or poor in those residues gives a different colour per microgram than albumin. Reducing agents and copper chelators in the sample also interfere.

The Bradford assay uses Coomassie dye binding.^[7] The dye interacts chiefly with arginine, gives only slight responses with other basic and aromatic residues, and requires a macromolecular form to bind.^[8] Short peptides can therefore give little colour, and the response varies with sequence.

An illustrative example: if a peptide gives 40% of the BCA colour of albumin per microgram, a sample reading 0.20 mg/mL against albumin standards actually contains about 0.50 mg/mL. Unless the assay is calibrated with the same peptide at a known concentration, the result is an albumin-equivalent number, not a peptide concentration.

Choosing a method

A practical summary of the options:

• A280. Fast and non-destructive for sequences with tryptophan or tyrosine; uses a predicted or measured coefficient.
• A205 or A214. For sequences without aromatic residues; needs a far-UV-transparent solvent and careful blanking.
• HPLC peak area. Quantifies against a reference standard of the same peptide, or with 214 nm coefficients; also shows impurities. See reading an HPLC chromatogram.
• Amino acid analysis or qNMR. Reference methods for an absolute value, such as assigning net peptide content.
• BCA or Bradford. Suitable for comparing similar samples, not for absolute peptide concentration unless calibrated with the same peptide.

A record checklist

For a concentration value to be interpretable, record:

• Sample. Sequence, modifications, molecular weight used and the nominal concentration from weighing.
• Method. Wavelength, coefficient and its source, path length, blank, solvent and dilution factor; or hydrolysis conditions and residues used for AAA.
• Result. Measured concentration, replicates, and the ratio of measured to nominal concentration.

This guide describes general laboratory methods and published literature only. It is not a statement about the content or behaviour of any particular material. Terms used here are defined in the glossary, and questions can be sent to support.