Decoding Amino Acids Using Ion Chromatography

Sometimes, you need the little guys to see the bigger picture. Amino acids are the little guys, the peptides are the bigger ones, and then the mega protein structural picture. To understand their structures, we must identify their sub-components. Like in troubleshooting, the problem arises, and then we put all the pieces together to solve it (or to see the story)!

There are many ways to analyze amino acids. The most recommended way is using post-column derivatization with the Pickering Pinnacle PCX amino acid analyzer solution, suggested by the European Pharmacopeia (EP) Monograph. It uses post-column ninhydrin derivatization. It requires some colorful staining and corrosive chemicals and can stain and corrode the instrument if it has a metal flow path. Some laboratories use inert flow path ion chromatography (IC) systems to avoid any corrosion. See the case study from Reading Scientific Services Limited (RSSL) about their amino acids application and setup. We know precolumn derivatization is highly susceptible to interference from the sample matrix, and complex sample matrices can reduce derivatization efficiency, causing high variability in amino acid recovery.

Although there are other amino acid methods, we often go with what is familiar to us and what we have around the laboratory. Sometimes it works, and sometimes not. It can divert and create other problems and challenges. I am a big fan of the ‘KISS’ method (Keep it stupidly simple). For many years we have demonstrated high-performance anion exchange (HPAE) chromatography for carbohydrate analysis with no derivatization as a simple and easy-to-use technique. Like carbohydrates, amino acids can easily be reduced and oxidized (Redox reaction). Hence, they can be analyzed by HPAE chromatography, just like carbohydrates, as demonstrated in our manual for Amino Acid Analysis (AAA) – Direct, MAN031481). With HPAE, with integrated pulsed amperometry detection (IPAD), the system can separate the amino acids and carbohydrates in complex matrices even in fermentation broth (See application update 152 and application note 150). The capability of AAA-Direct to analyze amino acids can be demonstrated in peptide samples, as types of hydrolysates, in Technical Note 50.

The use of AAA for total protein determinations provides advantages over colorimetric methods. Complete degradation of proteins to amino acids enables for the accounting of the composition completely, without bias from the specificity of dye-binding typical for colorimetric assays (BCA, Bradford, Lowry, etc.). The compositional analysis also provides information about the identity and purity of the protein not achieved using dye-binding assays. Direct detection of amino acids using AAA-Direct results in a lower cost in materials and labor. It also eliminates the risk of exposure to the hazardous chemicals used for pre- or post-column derivatization and helps to eliminate hazardous waste stream created by pre- or post-column derivatization techniques. Accurate total protein measurements can be achieved using AAA-Direct, with results equivalent to other AAA techniques (Application Note 163).

If you are studying proteins and peptides, you may want to know how to decode the amino acids. Why not KISS your analysis with AAA-Direct using HPAE-PAD? There is no messy derivatization, and it is much safer for you, your instrument and your laboratory.