Total and free amino acid analysis
AltaBioscience amino acid analysis service offers two main types of analysis, ‘total’ and ‘free’, for a diverse range of samples.
Types of analysis: for amino acids bound in proteins or amino acids existing as free entities.
Total amino acids analysis determines the amino acids which are bound in proteins as well as those amino acids that may be present as individual unbound units.
Free amino acid analysis determines the amount of each unbound individual amino acid i.e. not bound in a protein.
Some applications may require both analyses to determine the difference in bound and unbound amino acids.
Total amino acid analysis
Where the amino acid composition of a protein or peptide is of interest, or if the total protein concentration of a sample is required, then total amino acid analysis is the method of choice. The proteins and/or peptides within the sample are broken down by acid hydrolysis into their individual amino acids, the extract is therefore a sum of amino acids which were free in solution and those which were previously incorporated into proteins before hydrolysis. The amino acids within the hydrolysate are separated using a sodium citrate buffer system prior to detection.
Total amino acids analysis measures 18 of the 20 standard amino acids and can be used to determine:
- Protein content of food, feed and drinks products
- Protein content of specialist low protein dietary and nutritional products, e.g. those for people with phenylketonuria
- Amino acid ratios and percentage peptide content in purified peptides or proteins
- The extinction coefficient of recombinant proteins
Figure 1. Example chromatogram of an amino acid standard separated on the protein hydrolysate (sodium) system
Tryptophan and cysteine are usually lost during acid hydrolysis, however, estimates can be given for these amino acids when the sequence is known. For specific quantification of tryptophan or cysteine, separate additional testing methods are available. Read more about the analysis of tryptophan and cysteine.
Free amino acid analysis
For the analysis of samples such as physiological fluids including serum or cell growth media, the amino acids incorporated into proteins or peptides are not of interest, and it is the concentration of the individual unbound amino acids which is required. Therefore the proteins and other compounds that could interfere with the analysis are precipitated out, leaving only the free amino acids in solution. The free amino acids are then separated using a lithium citrate buffer system prior to detection. This high resolution method quantifies up to 40 components, including many non-standard amino acids.
It can be used for analysis of:
- Spent cell culture media to determine the changes in the amino acid content of the media as used by the cells
- Blood serum and other physiological fluids
- Taurine in pet foods (for example, cats must acquire enough additional taurine through diet to meet their needs) and sports nutritional supplements
- Ninhydrin positive substance and ammonium to European Pharmacopoeia 2.2.56 monographs
Although not included in our standard free amino acid analysis, we have also worked closely with customers to quantify compounds such as theanine, glucosamine and galactosamine. Please contact us for further information.
Figure 2. Example chromatogram of an amino acid standard separated on the physiological (lithium) system
For more information about the amino acid analysis service we provide , please contact us.
Author: Sarah Hawker, Senior Analyst