Separation of 20 Amino Acids by Polymer-Based Amino HILIC Column

Determination and quantification of amino acids are important in many fields. For the HPLC analysis of amino acids, cation exchange mode method has been used with post-column derivatization. Recently, use of reversed-phase mode method is also increasing. However, because of amino acids' hydrophilic nature, they are usually not well retained and have less selectivity by reversed-phase mode without pre-column derivatization steps. In contrast, HILIC mode is an ideal separation mode for hydrophilic compound analysis. In addition, by employing an ESI-MS detector, target compounds that have similar retention time may also be separated based on their m/z. Cation exchange mode generally provides good separation for multiple amino acids, however the eluent used is not desirable for ESI-MS use. A volatile mobile phase used for HILIC mode is advantageous for the ESI-MS analysis. Typically used mobile phase is acetonitrile and buffer, containing ammonium acetate or ammonium formate owing to their high volatilities and sample solubility. In this application, 20 standard amino acids were analyzed simultaneously without additional derivatization steps.

Experimental Conditions

Separation was carried by Shodex NH2P40-2D (2.0 × 150 mm, 4 µm), a polymer-based amino HILIC column. Column temperature was set at 30 °C and flow rate used was 0.2 mL/min. Linear gradient was applied as following: (A) 100 mM ammonium formate, (B) acetonitrile. B% 75% (0 min) → 75% (10 min) → 50 % (11 min) → 50% (35 min). Sample contained 10 µg/mL each of 20 amino acids, diluted with water/acetonitrile = 25/75. Injection volume of 5 µL was used for the experiment. HPLC system was coupled with an ESI-MS and SIM mode was used for detection. Ala and Gly were detected as acetonitrile adducts and Cys was detected as cystine (as a product of ESI).

Results

All 20 standard amino acids were analyzed successfully by the HPLC (HILIC)-ESI-MS method (Figure 1). The method achieved baseline separations of isobaric amino acids such as Leu and Ile, and Lys and Gln providing accurate identification of each.

Figure 1: Separation of 20 amino acids by NH2P40-2D. Eluent: Linear gradient (A) 100 mM ammonium formate, (B) acetonitrile. B% 75% (0 min) → 75% (10 min) → 50 % (11 min) → 50% (35 min). Flow rate; 0.2 mL/min, Temp.; 30 °C, Detector; ESP-MS SIM mode.

Effects of ammonium formate concentration were tested during the solvent optimization step. By increasing ammonium formate concentration, tailing which was observed for few amino acids was reduced. The higher ammonium formate concentration also benefits in shortening the analysis time; 53 min (Solvent (A) as 50 mM ammonium formate) down to 33 min (Solvent (A) as 100 mM ammonium formate).

Conclusions

Shodex polymer-based amino HILIC column, NH2P40-2D, coupled with ESI-MS is feasible for the analysis of 20 standard amino acids within 35 min: NH2P40-2D provides baseline separations of isobaric amino acids such as Leu and Ile, and Lys and Gln. The eluent condition used for HILIC mode is desirable for ESI-MS measurements.

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