Possibilities of new generation columns packed with 1.3µm core-shell particles in gradient elution mode.

The aim of this work was to evaluate the practical possibilities in gradient elution mode of a column packed with 1.3µm core-shell particles recently released on the market. For this purpose, two types of analytes possessing different diffusion coefficients were selected (small molecule and peptide). It appears that the new 1.3µm material was particularly well suited for fast separations, compared to other existing core-shell particle dimensions in gradient mode. The new material systematically outperforms the other existing ones for peak capacity up to 300 for small molecules and 700 (corresponding to t0=15min) for peptides. Based on these cut-off values, the advantage of column packed with 1.3µm was much more obvious for peptides vs. small molecules analysis. Further improvements in terms of column mechanical stability and system upper pressure capability could expand the limits of separation speed and efficiency to a different level. Again, because of the current pressure limitation and low permeability, a column length of more than 5-8cm is never desired for small molecules analysis in gradient elution. On the contrary, longer columns were useful for peptide analysis. As example, a column of 28cm packed with 1.3µm particles provides a peak capacity of 1000 in the case of peptides analysis. All the predicted values were experimentally confirmed using a standardized extract of Ginkgo biloba and a tryptic digest of a monoclonal antibody (Panitumumab). For the plant extract, the better performance was always achieved with a 5cm long column (P=267 and 268 for the 5 and 15cm, respectively, using a gradient time of 10 and 40min, respectively). Finally, in the case of peptide mapping, a 15cm long column packed with 1.3µm particles was the best choice (P=176 and 311 for the 5 and 15cm, respectively, using a gradient time of 10 and 40min, respectively).

Kinetic evaluation of new generation of column packed with 1.3 µm core-shell particles.

The goal of this study was to critically evaluate a new generation of columns packed with 1.3 µm core-shell particles. The practical possibilities and limitations of this column technology were assessed and performance was compared with other reference columns packed with 1.7, 2.6 and 5 µm core-shell particles. The column efficiency achieved with 1.3 µm core-shell particles was indeed impressive, Hmin value of only 1.95 µm was achieved, this would correspond to an efficiency of more than 500,000 plates/m. The separation impedance of this column was particularly low, Emin=2000, mostly due to a reduced plate height, h of 1.50. Comparing the kinetic performance of 1.3 µm core-shell particles to that of other particle dimensions tested in this study revealed that the 1.3 µm material could provide systematically the shortest analysis time in a range of below 30,000 theoretical plates (N<30,000).Despite its excellent chromatographic performance, it was evident that this column suffers from the limitations of current instrumentation in terms of upper pressure limit and extra-column band broadening: (1) even at 1,200 bar, it was not possible to reach an optimal linear velocity showing minimal plate height value, due to the low permeability of this column (Kv=1.7×10(-11)cm(2)), and (2) for these short narrow bore columns packed with 1.3 µm core shell particles, which is mandatory for performing fast-analysis and preventing the influence of frictional heat on column performance in UHPLC, it was observed that the extra-column band broadening could have a major impact on the apparent kinetic performance. In the present work, significant plate count loss was noticed for retention factors of less than 5, even with the best system on the market (σ(2)ec=2 µL(2)).