Free amino acids are the ones already loose in a sample. Total amino acids also include everything bound in proteins and peptides, released by acid hydrolysis, which destroys or converts several amino acids along the way. We currently measure free amino acids only.
Free amino acids are the ones already unbound in a sample, while total amino acids include every residue locked in proteins and peptides, measured after hydrolysis breaks the peptide bonds. The two numbers answer different questions. We currently measure free amino acids only; total amino acids are not offered yet.
Two numbers, two preparations
A free amino acid analysis measures what is in solution as it stands, with no hydrolysis step. HPLC is widely used for this measurement 1. Protein is removed first, as described in sample preparation, and the free pool is then derivatized and measured.
A total analysis first hydrolyzes proteins and peptides back into their amino acids, then measures the combined pool 2,4. The result describes the composition of everything in the sample, bound or not.
The gap between the two is the bound fraction. For protein hydrolysates, one review calculates the share of amino acids still in peptides as (total amino acids − free amino acids) ÷ total amino acids × 100 1. That calculation needs both measurements.
What acid hydrolysis does
The standard method heats the sample in 6 M hydrochloric acid at 110 °C for 24 hours 1,5. Waters' protocols run liquid-phase hydrolysis at 110 °C and vapor-phase hydrolysis at 112–116 °C, both for 24 hours 3. Oxygen is excluded with nitrogen purging or vacuum, and phenol is added as an oxygen scavenger 3. An AOAC method adds phenol to prevent halogenation of tyrosine 5.
Hydrolysis is not gentle, and it is not uniform. Waters notes that the rate and extent of hydrolysis vary across amino acids, which is why time-course studies are used 3.
Known artifacts
| Amino acid | What standard acid hydrolysis does | Published workaround |
|---|---|---|
| Tryptophan | Destroyed 1,2,5 | Alkaline hydrolysis with NaOH or KOH, or methanesulfonic acid 2 |
| Asparagine, glutamine | Converted to aspartate and glutamate 1,2,5 | None within acid hydrolysis; only the combined totals can be reported |
| Cysteine, cystine | Partly destroyed 2 | Performic acid oxidation or alkylation before hydrolysis 2; conversion to a stable derivative in one AOAC method 5 |
| Methionine | Partial loss 1,2 | Performic acid oxidation to methionine sulfone 4 |
| Serine, threonine, tyrosine | Lower recoveries 2 | Time-course hydrolysis 3; for tyrosine, pure acid, oxygen exclusion and phenol 3,5 |
| Isoleucine, leucine, valine | Low yields if hydrolysis is incomplete 3 | Full hydrolysis temperature and time 3 |
The asparagine and glutamine row matters more than it looks. After hydrolysis, the aspartate result includes the asparagine that was there, and the glutamate result includes the glutamine. A total analysis cannot tell them apart.
Which one your question needs
| Your question | Measurement | Why |
|---|---|---|
| What are my cells consuming from the medium? | Free | Consumption shows up in the free pool |
| How is my fermentation progressing? | Free | Monitoring targets the free amino acids in broth |
| What is the amino acid composition of my protein? | Total | Most of it is bound |
| How far has my hydrolysate been broken down? | Both | The bound share is total minus free |
Media consumption and fermentation monitoring: free
In cell culture, amino acids are consumed as cells grow and must be replenished to reach target cell density and product concentration 7. Thermo Fisher notes that frequent monitoring of amino acid concentrations in bioreactors maximizes production efficiency 6. What the cells draw on is the free pool in the medium. Free amino acids in broth need only filtration and, in some cases, protein precipitation before analysis 6.
Glutamine makes the case on its own. It is consumed by cells and also decomposes over time at physiological pH 6. Acid hydrolysis would convert whatever glutamine is left into glutamate 1,5. Tracking glutamine therefore requires a free measurement. See cell culture media and our note on glutamine, glutamate and ammonia in fed-batch.
Protein composition and label protein: total
The composition of a protein is a total question, because nearly all of its amino acids are bound. Label protein is a related but different question. Under US regulations, the grams of protein on a Nutrition Facts label may be calculated as 6.25 times the food's nitrogen content 8. That is a nitrogen measurement, not an amino acid one. Where a percent Daily Value for protein is declared, the protein amount is corrected by an amino acid score adjusted for digestibility (PDCAAS) 8. That score describes the protein's amino acid make-up, which is a total measurement. Free amino acids alone do not support it. See label claims.
Hydrolysates: often both
Protein hydrolysates contain free amino acids, small peptides and large peptides 1. The proportions depend on the protein source, the proteases and the process 1. A free profile tells you what has been fully released. A total profile tells you the overall composition. Together, they give the bound share 1. Our note on free amino acids in hydrolysates and the protein hydrolysates page cover this in more depth.
What we measure today
We measure free amino acids. The AA-1 profile covers 19 amino acids plus ammonia, reported in µM and mg/L. The AA-2 extended panel adds GABA, ornithine, citrulline, taurine and hydroxyproline. Cysteine and cystine are available on request (AA-3) with a reduction–alkylation workup. Details are on our amino acid analysis page.
Because a free analysis skips hydrolysis, asparagine, glutamine and tryptophan are reported as themselves. Those are exactly the amino acids that standard acid hydrolysis converts or destroys 1,2.
Total amino acids after acid hydrolysis are not offered yet. If you are unsure which measurement your question needs, our free feasibility call is a good place to start.
References
- Protein hydrolysates in animal nutrition: Industrial production, bioactive peptides, and functional significance — Hou Y, Wu Z, Dai Z, Wang G, Wu G, Journal of Animal Science and Biotechnology, 2017.
- Introduction to Hydrolysis — Waters Corporation, Comprehensive Guide to Hydrolysis and Analysis of Amino Acids (primer), n.d.
- Hydrolysis of Purified Proteins and Peptides — Waters Corporation, Comprehensive Guide to Hydrolysis and Analysis of Amino Acids (primer), n.d.
- Hydrolysis of Food and Feed Samples — Waters Corporation, Comprehensive Guide to Hydrolysis and Analysis of Amino Acids (primer), n.d.
- Total Amino Acids by UHPLC-UV in Infant Formulas and Adult Nutritionals — Jaudzems G, Guthrie J, Lahrichi S, Fuerer C, Journal of AOAC International 102(4), 2019 (pre-print).
- Amino acid analysis of mammalian cell culture medium by liquid chromatography with UV and fluorescence detection and derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate — Thermo Fisher Scientific, Application Note 73057, 2019.
- Monitoring Cell Culture Media for Amino Acids Using an ACQUITY UPLC H-Class PLUS System — Maziarz M, Waters application note, 2018 (revised 2022).
- 21 CFR 101.9 — Nutrition labeling of food — U.S. Food and Drug Administration, Electronic Code of Federal Regulations, current as accessed 2026.
OpenChemWorks Laboratory · Reviewed by the laboratory director, PhD chemist · Published September 23, 2026