Fast-liquid chromatography using columns of different internal diameters packed with sub-2 μm silica particles.

Research paper by Giovanni G D'Orazio, Anna A Rocco, Salvatore S Fanali

Indexed on: 15 Jun '11Published on: 15 Jun '11Published in: Journal of Chromatography A


Capillary columns (50, 75 and 100 μm I.D.) were packed with silica C₁₈ sub-2 μm particles for 50mm and were employed in nano-liquid chromatography (nano-LC) for fast chromatographic separations. Mixtures of nonsteroidal anti-inflammatory drugs (NSAIDs) and steroids were used as models. Separations no longer than 3 min were obtained with the three capillary columns employing a mobile phase of 35/65 (v/v) and 45/55 (v/v) ACN/H(2)O in 0.1% formic acid (HFo) for steroids and NSAIDs, respectively. Among the capillary columns used, best results, in terms of retention factor and selectivity, were achieved with the 50 μm I.D. column. The same sample mixtures were analyzed by micro-liquid chromatography (μ-LC), employing a commercial microbore column Hypersil GOLD™ (50 mm × 1.0 mm I.D.) packed with C₁₈ 1.9 μm particles, and working at the same linear velocity. The results were compared with those obtained with the 50 μm I.D. column in terms of analysis time, efficiency and selectivity. An evaluation of Van Deemter curves was also done for both columns. Finally both nano-LC and μ-LC methods, developed for the separation of the steroid mixture, were validated and applied to the determination of dexamethasone in commercial tablets. Relative standard deviation (RSD%), intra and inter days, of retention time were in the 0.1-1.0% and 2.3-3.5% ranges respectively, for nano-LC, while for μ-LC they were in the 0.6-1.4% and 0.9-1.6% ranges. Peak areas RSD% were also satisfactory (not higher than 6.3% for inter days values). LOD were between 0.010 and 0.040 μg/mL for nano-LC and between 0.1 and 0.5 μg/mL for μ-LC. To assess the linearity of both methods, six concentration levels were injected for three times in two different concentration ranges. Correlation coefficients (r²) of 0.998 and 0.997 were obtained for μ-LC and nano-LC respectively. Good recovery data were also found from analysis of real samples, utilizing 11α-hydroxyprogesterone as internal standard.