A comparative study of the performance of acetonitrile and methanol in the multi-linear gradient separation of proteic primary amino acids

The performance of the separation of proteic primary amino acids using multi-linear gradients of acetonitrile and methanol was studied under an experimental-design basis, using an Inertsil ODS-3 column and pre-column derivatization with o-phthaldialdehyde (OPA) and N-acetyl-l-cysteine (NAC). Elution strength, peak properties, resolution, and analysis time, were examined. The optimal separation was established through modeling, using information obtained from isocratic data. By optimizing the separation with gradients of increasing complexity, acceptable resolution was possible, being glycine/threonine the critical pair. Multi-criteria decision-making (Derringer desirabilities) was applied to balance resolution and analysis time. The more favorable peak distribution for methanol gradients allowed a larger reduction of analysis times, keeping satisfactory resolution, but its smaller elution strength forces the use of concentrations significantly larger. Methanol is, however, less toxic, and the final cost is similar for both solvents.