Amino acid analysis versus HPLC purity
What AAA measures that HPLC cannot, what it cannot tell you, why it is not on most research peptide COAs, and when to commission it.
Amino acid analysis (AAA) and HPLC purity are both routinely quoted for peptides and answer different questions: HPLC asks how much of the peptide fraction is the target sequence, AAA asks how much peptide there is and whether its composition is right. Neither replaces the other. This note explains what AAA measures, when a laboratory needs it, why it is not on most research peptide COAs, and how it fits alongside HPLC and mass spectrometry. All Alphex products are for laboratory and in-vitro research only.
What amino acid analysis does
The peptide is hydrolysed completely (typically 6 M HCl at 110 C for 24 hours) into its constituent amino acids, which are then separated, derivatised and quantified by chromatography against calibrated standards. The output is two things: the molar ratio of each amino acid, which is compared to the ratio predicted from the sequence as a compositional identity check, and the absolute quantity of amino acids recovered, which gives the mass of peptide in the sample and hence the net peptide content of the solid.
What it tells you that HPLC does not
- Net peptide content. HPLC area percent says nothing about counter-ions or water. AAA gives the peptide mass directly, which is how the "70-90% net content" figures for lyophilised peptides are established. See net peptide content versus gross vial weight.
- Composition. A peptide with the right mass but the wrong residues (unlikely but possible for isobaric substitutions) would be caught by an unexpected ratio.
- Absolute concentration of a solution, for peptides without aromatic residues that cannot be quantified by UV at 280 nm. See 220 nm versus 280 nm.
What it does not tell you
- Purity in the HPLC sense. Deletion sequences, diastereomers and truncations hydrolyse to the same amino acids in nearly the same ratios; AAA cannot separate them from the target. This is why "purity by AAA" is not a meaningful phrase.
- Sequence order. Hydrolysis destroys it.
- Modifications. Acetylation, amidation and lipidation are lost or altered on hydrolysis; tryptophan is destroyed by acid hydrolysis unless special conditions are used, and asparagine and glutamine are converted to their acids.
Why it is not on most research peptide COAs
AAA is slower and more labour-intensive than HPLC-plus-MS, consumes material, and answers a question (net content) that most research applications handle by assumption or by in-house measurement. Manufacturers run it when qualifying reference standards; independent laboratories run it on request. The Alphex COA reports chromatographic purity, which establishes that the material is 99+% pure; net content is not on the standard certificate and the label mass should be treated as gross. See what HPLC purity actually tells you.
When to commission it
- Concentration-critical or molar work where an assumed net content is not acceptable.
- Qualifying your own in-house reference standard.
- Comparing batches or suppliers on a mass-of-peptide basis rather than a mass-of-solid basis.
Any contract analytical laboratory offering protein chemistry services will run it; ask for quantitative AAA with a stated hydrolysis method and internal standard. If you need it for an Alphex batch, ask via the COA request form and we will tell you whether the testing laboratory has the data.
Frequently asked questions
Can AAA confirm identity on its own?
Composition only. Identity is HPLC retention against a standard plus mass spectrometry; AAA is a supporting check. See mass spectrometry explained.
Is nitrogen (elemental) analysis a substitute?
For net content, roughly: total nitrogen relates to peptide mass if the sequence is known. It is cheaper and cruder than AAA.
Alphex research peptides are supplied for laboratory and in-vitro research by qualified researchers only. They are not authorised for human or veterinary use.
Research use only. Alphex products are supplied exclusively for laboratory and in-vitro research by qualified researchers. They are not authorised for human or veterinary use, and nothing in this note is advice on any such use.
Published 5 May 2026. Alphex reviews research notes when the testing method, the catalogue or UK guidance changes.