Method considerations for the analysis of amino acids
Analysis of amino acids is important in determining the quantity and quality of proteins in a sample. We use the most robust amino acid analysis method available: separation of a mixture of amino acids using ion exchange chromatography followed by post-column photometric detection. This makes us one of few laboratories in the world operating to such exacting standards with ISO 17025:2017 accreditation.
We can analyse free amino acids and total amino acids in a wide range of samples.
Benefits of post-column detection method
Combining ion exchange chromatography with post-column detection with ninhydrin derivatisation is the gold standard method. It is the most robust and accurate method and has the following advantages:
- Compared to other pre-column derivatisation methods, the preparation of the samples is faster. The reaction of ninhydrin with amino acids is specific and does not yield multiple derivatives, which can render the quantification more difficult and less accurate in the case of pre-column detection methods.
- This method is also superior to the Kjeldahl and Dumas methods, which are unable to detect non-protein nitrogen from proteinogenic nitrogen. As a result, this method does not give falsely high readings.
- This method doesn’t require pure protein and is suitable for a wide array of sample types, including but not limited to food and feed samples, cell culture media and serum. Check out our technical document or read about amino acid sample requirements.
- Natural and non-proteinogenic amino acids can be quantified as free amino acids or bound as proteins. Amino acid analysis is particularly interesting to the food and feed, drug and supplement industries to accelerate innovation in these fields.
- This method can also be performed as part of a quality control step to determine the net weight of a peptide obtained by custom peptide synthesis since synthesised peptides purified by reversed-phase HPLC contain a certain amount of residual water and salts.
- It is fast, and results can be obtained with a short turnaround time.
- It is also applicable to other biological nitrogen-containing compounds such as:
- Amino sugars: glucosamine and galactosamine: Although they can be readily detected under standard conditions, they require special hydrolysis conditions for accurate determination.
- Nitro amino acids.
- Taurine: An amino sulfonic acid -SO3CH2CH2NH3+ essential for cardiovascular function and often used as a dietary supplement in sports beverages and foodstuffs. It can be detected with high sensitivity and high specificity in the free (soluble) state using a lithium buffer system. Being a strong acid, taurine elutes early in the chromatogram under these standard pH conditions.
Specific considerations for amino acids sensitive to acid
The initial step to analyse the amino acid composition of proteins using the described method is the hydrolysis of proteins to break them down into their individual amino acid building blocks. This is done prior to the separation of amino acids by ion exchange chromatography and is achieved by treating proteins with hydrochloric acid for 24 hours at 110°C and in vacuo in order to reduce oxidative destruction. Although suitable for the majority of amino acids, under these conditions, certain amino acids that are sensitive to acid can be destroyed (tryptophan) or partially degraded (asparagine, glutamine and cysteine). It is still possible to accurately quantify them. However, separate analyses or considerations must be taken.
Asparagine and Glutamine
Asparagine (Asp) and glutamine (Glu) bear amides in their side chains and are, therefore, completely converted to their corresponding carboxylic acids during acid hydrolysis, forming aspartic acid (Asn) and glutamic acid (Gln), respectively. Both amino acids and their corresponding acids can be observed as separate compounds when analysed as free amino acids, as no prior hydrolysis is required in that case. The results are reported as the sums of Asp + Asn and Glu + Gln, often referred to as Asx and Glx.
Cysteine and Cystine
Cysteine is a sulphur-containing amino acid. Its thiol group (-SH) readily oxidised, forming either the dimer cystine (Cys-Cys) or disulphide bridges (-S-S-) between two cysteine residues in proteins. Cystine is unstable to the conditions used in normal acid hydrolysis, and recoveries are usually low. It is therefore oxidised with performic acid to the very stable cysteic acid before carrying out protein hydrolysis. However, this oxidation reaction interferes with other amino acids, so a separate analysis is required for cysteine. Read more about the analysis of cysteine and cystine.
Tryptophan is an essential amino acid. Unstable during acid hydrolysis due to the indole functional group on the side chain, tryptophane can be recovered reliably under alkaline conditions using barium hydroxide. It is, therefore, possible to quantify tryptophane accurately, but this requires separate analysis. Read more about the analysis of tryptophan.
We can detect and quantify hydroxyproline in samples. As it is usually impossible to fully resolve hydroxyproline from aspartic acid on the standard protein hydrolysate separation system, we use a special temperature gradient for the separation, requiring separate analysis.
Other points to consider
Peptide bonds linking the amino acids isoleucine and valine
Peptide bonds between two amino acids of valine and/or isoleucine (Ile- Ile, Val-Val, Ile-Val and Val-Ile) are slightly resistant to acid hydrolysis. The cleavage of these bonds is usually slower due to the steric hindrance caused by the alkyl side chains, but this can be overcome by increasing the reaction time.
D-Amino acids are amino acids where the stereogenic carbon bearing the side chain has a D-configuration. These cannot be resolved from the naturally occurring L-amino acids using this method.
Although readily detected, glucosamine and galactosamine show considerable losses under standard hydrolysis conditions. For optimum results, the samples must be hydrolysed at lower temperatures and shorter times, then analysed according to a protocol optimised for the analysis of amino sugars, which gives complete separation from the amino acids.
Compounds that are not detectable
Although this method is suitable for a wide range of compounds, it cannot detect carnitine, a quaternary ammonium carboxylic acid that plays a critical role in energy production and that can be used as biomarkers for many metabolic disorders, as well as creatine and creatinine.
Ion exchange chromatography (IEX) allows high-resolution separation of amino acids
Ion exchange chromatography (IEX) is a technique that allows the separation and purification of ionic molecules based on their affinity with an exchanger resin. The exchanger resin displays charged functional groups covalently bound to a stationary phase, attracting the corresponding oppositely charged analytes. The electrostatic interaction strength between the resin and the analytes depends on the analytes’ net charge. A weak attraction will result in analytes eluting first, whereas analytes interacting strongly with the stationary phase will be retained. Usually, after loading the column, it is first washed with an eluent to remove impurities. Then, buffers with increasing pH or different salt gradients are passed through the column to allow the exchange of ions and the displacements of the analytes of interest.
Ion exchange chromatography is particularly well suited for the separation of amino acids, peptides or proteins. Amino acids are zwitterionic compounds bearing amino and carboxylic acid functional groups, which depending on the buffer pH, can carry a net positive charge, no charge or a net negative charge.
When separating amino acids by IEX for amino acid analysis, the choice of the buffer pH of the eluent is crucial. The amino acids or proteins need to be stable under the chosen pH and in a form that is able to bind to the exchanger resin but sufficiently close to their point of elution.
Typically, a cation exchange resin is combined with either a lithium citrate buffer system for physiological samples or a faster sodium citrate buffer system for protein hydrolysates. Because each amino acid is unique and has distinct properties due to its side chains bearing varied functional groups, their retention time will vary. It is, therefore, possible to separate them with high resolution.
Ninhydrin derivatisation for post-column detection
The next step in this amino acid analysis method is coupling ninhydrin with the amino acids previously separated by IEX. The reaction produces a purple-coloured chromogen called Ruhemann’s purple (or yellow for secondary amines such as proline and hydroxyproline), which can be detected by photometry using 570 nm (Lithium) or 660 nm (Sodium). Under suitable conditions, the produced colour intensity is proportional to the concentration of amino acid, allowing the accurate quantification of amino acids in a sample.