29


Figure 1: α-ketoglutaric acid (blue)


each experiment refer to final total concentrations. Ammonium formate at pH 3 was prepared using ammonium formate and adjusted with formic acid. The ammonium formate pH 6.5 was used without pH adjustment. A SeQuant®


ZIC®


glutamic acid (white)


ketoglutaric acid, but not for glutamine, could be expected.


-HILIC column,


150 x 2.1mm (5µm, 200 Å) was operated at a flow rate of 0.4mL/min, with an eluent of 70%


ACN and 30% buffer (v/v) for all experiments. The chromatographic separations were carried out isocratically on a Shimadzu LC 10 system with column oven set at 30°C. The sample injected was 5µl with 150µg/ml of each compound dissolved in the eluent. Due to the analytes low UV absorbance detection was with a Sedere Sedex 85 ELSD equipped with a low flow nebuliser cell.


Three analytes were chosen for this study. Glutamine (Gln), which is a zwitterionic amino acid, with a hydrophilic side chain containing an amide functionality. Glutamine has two pKa values 2.2 and 9.1 thus exhibiting a stable state of ionisation in the studied pH range. Glutamic acid (Glu), which is an acidic amino acid with a carboxylic side chain has pKa values of 2.2, 4.2


and 9.7. α-Ketoglutaric acid, is a dicarboxylic acid with pKa values of 2.4 and 4.4.


Results Acidic pH & low buffer strength


At pH 3.0 and 3mM buffer, the sidechain carboxyls (pKa ~4.2) will be principally protonated, and thus act as neutral polar groups, whereas the alpha carboxyls of Glu


and Gln (pKa 2.2) and α-ketoglutarate (pKa 2.4) will be largely ionic and repelled by the negatively charged sulfonates of the column. In the amino acids Glu and Gln the negative charge is balanced by the positively charged amino group. Separation of the amino acids relies on the small polarity differences of the side chains: a neutral carboxyl v an amide functionality. The more repelled, negatively


charged α-ketoglutaric acid has the shortest retention time.


Neutral pH & low buffer strength


At pH 6.5 there is an overall increase in polarity from the, now, fully ionised carboxyls; an increased retention for both glutamic and α-


Figure 2: Separation at 3mM ammonium formate pH 3.0. Retention order is α ketoglutaric acid (blue), glutamic acid (white) and glutamine (green).


However, as the analytes carboxyls are now unprotonated and thus negatively charged, they are now repelled by the negatively charged distal sulphonates on the stationary phase. This repulsion limits the analytes ability to partition into the water layer on the stationary phase. Their retention therefore doesn’t increase as might be expected from the increase in hydrophilicity.


The only visible effect of changing the pH is that the glutamic acid is ‘moved’ to a shorter retention time due to the increased electrostatic repulsion. This resulted in all three components being baseline separated in three minutes.


glutamine (green) Neutral pH & medium buffer strength


Increasing the buffer strength to 30 mM effectively shields the charges on both the column stationary phase and analytes. Again the glutamine is unaffected by the change in eluent composition. None of its functional groups are affected by changing the pH, and due to the close proximity of the charges, their electrostatic interactions with the stationary phase are also unaffected when changing buffer strengths.


The effect on the two acids, especially the α- ketoglutaric acid, is however dramatic when


increasing the ionic strength. It is deceptive that it has approximately the same retention time in Figures 1 and 2, but here the high hydrophilicity of this doubly negatively charged


Figure 3: Separation at 3mM ammonium formate pH 6.5. Analytes as listed in Figure 1.


Figure 4: Separation at 30mM ammonium formate pH 6.5. Analytes as listed in Figure 1.


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