Acidic mobile phases have found widespread applications in the reversed-phase HPLC separation of many important pharmaceutical and environmental compounds.
Acidic mobile phases have found widespread applications in the reversed-phase HPLC separation of many important pharmaceutical and environmental compounds. Analytes such as pharmaceuticals and biomolecules often show peak shape, retention, and selectivity changes when the mobile phase pH is changed from neutral to acidic pH (pH=1.0). In fact, lowering the pH helps to suppress silanol interactions between basic solutes and the residual surface silanols, thus resulting in less tailing and better retention for acidic compounds (pKa lower than 2), in turn increasing retention and altering selectivity. For example, hydrophobic peptides and protein separations use TFA for solubility. Additionally, basic analytes are preferably positively charged in the HPLC eluent for positive-mode electrospray LC–MS applications thus requiring low pH mobile phases and high column robustness.
The surface of the silica gel packing of the Silia Chrom columns is treated with an organic form of silicon to increase the number of silanol groups on the surface. After this, the surface is bonded with two types of octadecyl silanes. One of them has a protecting group that shields the area under it and protects the surface from an acid attack from the mobile phase. (Figure 1)
The new SiliaChrom SB C18 3 μm phase was packed in-house. The column configuration was 4.6 mm × 150 mm. The low pH stability test was performed using a Thermo Surveyor HPLC with PDA detector monitoring absorbance at 260 nm, using a flow rate of 0.80 mL/min with the column heated at 35 °C. A sample containing uracil (void marker), phenol, and benzoic acid was injected to get k' between 0.5 and 1.0 in order to be very sensitive to minute variations of the stationary phase. We have monitored all injections every 6 h for a total of 48 h. The flow was kept constant during 48 h (0.80 mL/min, 35 °C) with an aggressive mobile phase of methanol/buffer: 10 mM potassium phosphate monobasic (1:1). The pH was adjusted to 1.0 using TFA (1.5% v/v) before adding methanol.
The chromatograms illustrate separations where the retention time for each analyte is constant. Figure 2 shows no modification of retention time from the beginning (0 h) and 2 days later (48 h) with the flow kept constant with the low pH mobile phase.
Novel SiliaChrom SB C18 is a powerful stationary phase for extremely acidic mobile phases. The phase itself is made directly from silica. The HPLC study clearly demonstrates the robustness for very low pH conditions. With this new HPLC column, any strongly acidic compounds can be analyzed without any limitation.
Table I: Retention time versus time
SiliaChrom SB C18 is a trademark of SiliCycle Inc.
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