Amino Acid Analysis:
During Amino Acid Analysis, proteins and peptides are hydrolyzed to their component amino acids which are then separated by HPLC, detected by UV or fluorometry and quantitated.
When Amino Acid Analysis is used to measure the free amino acids that are not bound up in a protein or peptide but exist freely in a biological tissue like serum, spinal fluid and cells, hydrolysis is not used. Usually, higher molecular weight proteins and peptides are removed from the matrix by a filter. However, the assay is “blind” to the peptides and proteins and only the free amino acids are derivatized and seen in the working part of the chromatogram.
Amino Acid Analysis can be used to answer many biological questions. New technologies have permitted the assay to be scaled down and allow greater sensitivity and the analysis of less abundant samples which increases the usefulness of this assay greatly.
The assay is primarily used as the gold standard in protein and peptide quantification. The Bradford and other colorimetric assays are easy to perform and do not require much instrumentation. But these assays are not accurate and vary from sample to sample. The colorimetric assay can be calibrated, however, to an amino acid analyzed standard if one is working with the same protein(s) repeatedly.
Amino Acid Analysis is a useful tool in synthetic peptide qualification. The mole percent of each residue can be compared to the expected percents from the known sequence.
Some data-based searching can be done from the percent composition of a protein determined by amino acid analysis. But Edman chemistry and Mass Spectrometry are much more selective, have higher confidence limits and therefore are the analyses of choice for protein identification.
Amino Acid Analysis can be used in many creative ways. Just a few of the many past and present projects that utilize this service in our lab are:
- Qualification of MAPS peptides that cannot be easily purified and are not compatible with Mass Spectrometry
- Determination of resin loading
- Determination of drug loading on nanoparticles
- Measuring the rate of enzymatic efficacy in the hydrolysis of di-peptide libraries
- Measuring the changes in culture media amino acids over time
- Measuring the accumulation of marker amino acids (ex. DAPA in bacterial cell walls) over time
- Monitoring organic matter in drinking water related to the production of trihalmethanes from chlorination
- Quantitation and qualification of recombinantly produced proteins
- Measuring changes in Glutamic Acid in CNS during brain insult
Measuring changes in percent lysine in grains at different stages of growth
Amino Acid analysis is a service provided by the protein chemistry lab staff only. We can be reached by pcl@ag.tamu.edu
During Amino Acid Analysis, proteins and peptides are hydrolyzed to their component amino acids which are then separated by HPLC, detected by UV or fluorometry and quantitated.

Sample Submission
We recommend sending samples in screw cap (with gasket) Eppendorf tubes packed in such a way that are cushioned from the effects of FedEx. If a gasket equipped tube is not available, use Parafilm to assure that the cap does not pop off in shipping. Keeping the sample cool is at the discretion of the investigator. It is not necessary to preserve biological activity or structural integrity of the sample unless solubility will become an issue. Dried samples and samples on PVDF do not require being kept cool. Before sending a sample, many of our clients alert us by pcl@ag.tamu.edu that it is coming so we can look for its arrival.
The Amino Acid Analysis Sample Submittal Form should accompany the sample. This form is available on this website and can be filled out on-line. It cannot be sent electronically except as an attachment and we prefer that it is printed out and accompanies the sample. Click HERE for the form.
Sample Preparation
Proteins and Peptides
Sample buffer
The assay is fairly tolerant of salts and buffers in the millimolar range. Samples that are submitted in very dilute amounts might need to be concentrated which will also concentrate those buffers until they possibly begin to interfere with the derivatization chemistry which tags the amino acids with a chromophore immediately before injection. For this reason, we prefer that samples be submitted in the 0.5 to 2 mg/ml range. Only a few things are problematic in the assay. They are:
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- Non-volatile amines (TRIS is not acceptable in sample matrices)
- Glycerol (in any concentration)
- Large amounts of Lipids
- reducing sugars and sucrose interfere if the sample needs hydrolysis
If possible, water is the best matrix for sample submission. The sample can also be sent dried down if it is known that it does not have solubility or sticking problems.
Sample amounts
For the quantitation, three replicates of 5-10 µg each (15-30 µg total) is the best scenario. And if the sample is in a concentration of approximately 0.5-2 µg/µl this allows accurate pipetting while not adding a lot of buffer to the sample. We know that it is not always possible to submit this much sample and good results can be obtained with less. Below 5 µg of sample, call first so that a plan can be made to assure useful data. With our fluorometric detector, we can see picomoles of amino acids. However, background contamination becomes more and more problematic the lower the amount and makes interpretation of the data difficult.
Electroblotted Samples
Alternatively, we can analyze amino acid composition from proteins that are electroblotted onto a hydrophobic support membrane. Tryptophan and Cysteine cannot be quantitated from PVDF. If these amino acid quantities are required, the sample should be submitted in a liquid form. Samples (> 5µg per lane) that are prepared by electrophoretic procedures (SDS-PAGE, non-denaturing PAGE, 2-D PAGE) should be blotted to polyvinylidene fluoride (PVDF). Nitrocellulose and nylon membranes are not compatible with hydrolysis. Electroblotting must be performed in non-Tris, non-glycine-containing buffers. The electroblotted proteins can be stained with Coomassie Brilliant Blue R-250 or Ponceau S. After destaining, the sample can be excised from the membrane using a razor blade and washed extensively by vortexing with HPLC grade water in a clean Eppendorf-style test tube. As PVDF membranes contain some number of contaminants themselves it is required that the client supply a blank piece of stained/destained PVDF membrane to act as a subtractive background control. Usually, several lanes of the target sample are needed for successful amino acid analysis. We estimate that ~50% of the sample loaded on the gel will finally be blotted to the PVDF. If you need blotting or staining protocols, recipes or advice you may press HERE or call us at the PCL.
Feed and Solid Samples
Many different types of solid samples can be acid hydrolyzed directly in the liquid HCl. Samples should be ground sufficiently finely to assure a homogeneous sampling. About 100 mg is a good sample size for this assay however, the assay can be scaled down to ~5 mg samples. Tryptophan and cysteine are not determined in this liquid hydrolysis assay.
Physiologicals
We are willing to try to develop assays for unusual amino acids and have some standard protocols on hand. Please drop in or call us and discuss your particular needs.
Free Amino Acids
Free amino acids can be quantitated. The matrix in which they are submitted should be free of large amounts of proteins, lipids and carbohydrates. The assay works well with serum which has been spun through a molecular weight 5000-6000 cutoff filter. Some free amino acids that are routinely assayed are glutamine, asparagine, citrulline, B-alanine, taurine, tryptophan and ornithine.
Results
Amino Acid Analysis Assays
We offer a range of assays for amino acid analysis, tailored to your sample type and specific requirements. Here’s a breakdown of our processes and the amino acids we report:
Purified Proteins (Acid Vapor-Phase Hydrolysis)
For purified proteins undergoing acid vapor-phase hydrolysis (6 N HCl), we report the analysis of 16 naturally occurring amino acids.
–Asparagine (Asn) and Glutamine (Gln) are deamidated to their respective acids during hydrolysis and are reported as ASX (Asparagine/Asparagic Acid) and GLX (Glutamine/Glutamic Acid).
–Cysteine is destroyed during hydrolysis but can be preserved if alkylated before the process. Please ensure you request Cysteine analysis on your sample submittal form if it is essential. Note that this requires a separate assay and incurs an additional charge due to the use of a different detector.
–Tryptophan is destroyed during HCl hydrolysis, so it requires a separate assay using Methanesulfonic Acid hydrolysis. This assay demands a larger sample size and may have some variability.
–Isoleucine (ILE) can sometimes be challenging to hydrolyze, and it may report lower than expected, especially if it is adjacent to other branched amino acids in the sequence (such as Isoleucine or Valine). This is a known issue that can affect the results.

Solid Samples (e.g., Feed or Meal)
Solid samples are processed via acid liquid-phase hydrolysis (6 N HCl), and we will report the 16 naturally occurring amino acids. Please note that Cysteine and Tryptophan are not preserved in this process and will not be reported.
Physiological Samples (e.g., Serum)
For physiological samples like serum, we report the 19 naturally occurring amino acids. In addition, we can include the following amino acids upon request:
Citrulline (Cit)
Beta-Alanine (B-Ala)
Taurine (Taur)
Ornithine (Orn)
Please specify these additional amino acids on your request form if needed.
The Excel Workbook – Results Summary
After analysis, you will receive a detailed Excel Workbook summarizing the results, which includes:
–Replicates Averaged: The data from multiple runs is averaged for accuracy.
–Percent Composition: Calculated for each amino acid in your sample.
–Sample Amount: The amount of sample in the aliquot is calculated in several ways, allowing for flexibility in how you view your results.
If you provide the molecular weight and sequence of the protein, we can calculate the amount of protein in nanomoles and compare the known molar percent composition to the results obtained from the assay.

For quantitative accuracy, we recommend running your samples in triplicate. Please note there is an additional charge for triplicates or duplicates, and it’s up to the investigator to specify the preferred number of replicates.

Data Retention
-The raw data is kept electronically for 1 year.
-The summary data in the Workbook is retained for 3 years.
This ensures that your data is securely stored and easily accessible for future reference or analysis.
For any specific requests or to clarify the details of your sample analysis, please don’t hesitate to contact us!
Instrumentation
Hydrolysis of samples is performed in a PicoTag Workstation. Amino acids are derivatized and separated on an Agilent 1260 liquid chromatograph with ”Chemstation” software that controls the LC and collects, analyzes and reports the data. The G1367E autosampler performs pre-column derivatization and multiple sample handling.
Derivatized amino acids are eluted from a narrow bore, (2.1 x 200 mm), (Hypersil AA-ODS), 5 um reverse phase column purchased from Thermo Fisher (part # 30105-202130). Solvent A consists of a 20mM Na acetate buffer with 0.018% v/v triethylamine (Fluka 90338), 0.05mM EDTA, (Sigma E4884) and 0.3% tetrahydrofuran (Fluka 87363) adjusted to pH 7.2 with weak acetic acid. Solvent B is a 20% 100 mM Na acetate buffer (pH 7.2) with 40% acetonitrile and 40% methanol. The working gradient begins at 0 minutes at 100% A at 0.45 ml/min and goes to 60% B over 17 minutes.
Primary amino acids (tagged with OPA, Agilent Item # 5061-3335) are detected at 338/390 nm by the Variable Wavelength (UV) detector (G1365D) and secondary amino acids (tagged with FMOC, Agilent Item #5061-3337 ) at 266/324 nm.
Our flourometric detector (G1321B) monitors the primaries at excitation/emission 340/450 and the secondaries at 266/305.
Assay Description
Protein and peptide samples are aliquoted and mixed with Internal Standards (see Quantitation), dried in glass tubes (6 x 50 mm, Fisher PN 14-957AA) in a vacuum concentrator and subjected to vapor phase hydrolysis by 6N HCl (Thermo Sci # 24308) at 150ºC for 1.5 hours under argon atmosphere in the presence of phenol (2%, Sigma #P5566) which limits the halogenation of Tyrosine residues. The samples are subsequently reconstituted in 0.4 N Borate Buffer (Agilent # 5061-3339) to bring the eventual pH to 10 for optimum derivatization and transferred to the Agilent G1367E autosampler for automated derivatization and loading. In cases where Cysteine data is requested, Dithiodiproprionic Acid (Acros Organics # 14800500) is added at the beginning of the assay to convert Cysteine to S-2-carboxyethylthio-L-cysteine (Cys-MPA) which is preserved during hydrolysis.
Insoluble samples (feeds, waste, cloth etc.) are weighed, Internal Standards added, and liquid-phase hydrolysis is used. Enough 6N HCl is added to cover the sample and samples are exposed to 100ºC for 22 hours. Since the alkylation reagent for Cysteine is not soluble in acid, cysteine conversion and quantitation is not available for this assay. Liquid HCl samples are filtered after hydrolysis and a portion of the filtrate is dried down, the HCl is pulled off, and reconstituted in 0.4 M Borate buffer (pH 10) prior to derivatization.
Note: In cases where a proteinaceous sample undergoes hydrolysis, any free amino acids that are present in the sample matrix and survive the hydrolysis will be included in the quantitation. While this is not a consideration for purified proteins and peptides, it is a consideration in complex matrices like serum or culture media.
Note: In all cases where hydrolysis is used, Asparagine and Glutamine are deamidated to their respective acids. Results for these residues are reported as ASX and GLX to denote that these data contain the combined amounts from both the amide and the acid. Acid hydrolysis also destroys Tryptophan. If it is necessary to quantitate this residue, a separate assay is performed using 4 N Methansulfonic Acid, Sigma # M4141(with 0.2%w/v tryptamine) for hydrolysis. The Tryptophan analysis has a very high %RSD (~25%) and requires ~50-100 µg of sample. It requires two assays and there is an additional charge.
Physiological samples or samples where only the free amino acids are of concern might be filtered to remove proteins or dried down and then reconstituted in 0.4 N Borate Buffer to bring the eventual pH to 10 for optimum derivatization. No hydrolysis is involved for free amino acid determination and Tryptophan can be measured. Cystine can be measured using a different detector but Cysteine cannot be measured.
The Agilent 1260 HPLC analyzes all samples by pre-column derivatization of the amino acids with o-phthalaldehyde (OPA), Agilent #5061-3335 and 9-fluoromethyl-chloroformate (FMOC) Agilent #5061-3337. OPA reacts with primary amino acids and FMOC with secondary amino acids (Proline). Both reagents react rapidly and quantitatively and give highly fluorescent and UV-absorbing isoindole derivatives. The derivatized amino acids are separated by reverse phase HPLC and detected by UV absorbance (primaries at 338/390 nm and secondaries at 266/324 nm) with a variable wavelength detector (G1365D) or by fluorescence (primaries at excitation/emission 340/450 and secondaries at 266/305) using an in-line fluorescence detector (G1321B).
Amino Acid Codes
Amino Acid | Three letter Code | One letter Code |
Alanine | Ala | A |
Cysteine | Cys | C |
Aspartic Acid | Asp | D |
Glutamic Acid | Glu | E |
Phenylalanine | Phe | F |
Glycine | Gly | G |
Histidine | His | H |
Isoleucine | Ile | I |
Lysine | Lys | K |
Leucine | Leu | L |
Methionine | Met | M |
Asparagine | Asn | N |
Proline | Pro | P |
Glutamine | Gln | Q |
Arginine | Arg | R |
Serine | Ser | S |
Threonine | Thr | T |
Valine | Val | V |
Tryptophan | Trp | W |
Tyrosonine | Tyr | Y |
Citrulline | Cit | |
Hydroxyproline | Hyp | |
Ornithine | Orn | |
B-Alanine | B-Ala | |
Asparagine/Aspartic Acid | Asx | B |
Glutamine/Glutamic Acid | Glx | Z |
cannot be differentiated. Therefore, the resulting mixtures are designated as Asx and Glx.