food & beverage

Separation of selected amino acids

Amino acids are the fundamental building blocks of all proteins and therefore essential constituents of many nutritional supplements and can also be present in pharmaceutical formulations. Due to the highly hydrophilic nature and zwitterionic charge at neutral pH of many amino acids and their structurally related degradation products, hydrophilic interaction chromatography is an attractive mode of separation that require no derivatization, while evaporative light scattering enables rather sensitive detection of these poorly light-absorbing compounds.

This application displays separation of a variety of selected amino acids ranging from rather hydrophobic to more hydrophilic, including several with acidic or basic side chains. The separation employed a shallow HILIC gradient elution on an amide stationary phase, using acetonitrile and water buffered to pH 3.2 with formic acid and ammonium formate. Included amino acids were tryptophan, phenylalanine, leucine, isoleucine, glutamine, valine, cysteine, glycine, serine, glutamic acid, aspartic acid, histidine and arginine.

Gradient elution of thirteen different amino acids on a TSKgel Amide-80 column (100×4.6 mm, 5µm) using a linear gradient pumped at 1 mL/min, at 25 °C. Solvent A was acetonitrile and B was water buffered to pH 3.2 using 100 mM formic acid and 30.7 mM ammonium formate. The gradient program was 0-2 min 87%A, 2-30 min 87-80%A, 30-35 min 80%A, after which the column was equilibrated for at least 15 min. The instrument setup comprised Shimadzu SCL-10Avp controller, DGU-14A degasser, two LC-10ADvp pumps, JetStream Plus column oven, Spark Holland Triathlon 900 autosampler, and Sedex 85 LT ELSD (3.6 bar air, 40 °C) detector, all controlled by Clarity 8.4 software. Injection of 20 µL standard diluted in eluent to concentrations of 100 mg/L of each amino acid, except histidine which was 200 mg/L.

Separation of selected amino acids

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