11


The resulting 3D chromatogram, obtained after processing data with Matlab, is given in Figure 5. The optimisation of gradient conditions in both dimensions was achieved thanks to thermodynamic (retention models), kinetic (Van Deemter plots) and column permeability data. The use of HT-UHPLC in the second dimension made it possible to obtain an ultra fast separation (<0.6 min).


Table 3. Optimisation of the instrumental design. The steps presented consist of a) conventional conditions in both dimensions, b) reduction of the first column diameter, c) UHPLC conditions in the second dimension, d) HT-UHPLC conditions in the second dimension.


stationary phase column geometry mobile phase


flow-rate (µL/min)


composition range (%) normalized gradient slope temperature (°C)


dimension 1 Hypersil Gold


100 x 1mm; 1.9µm


ammonium acetate 10mM / acetonitrile


20


1 to 50% 0.2 30


Table 4. Selected conditions for on-line 2D-LC separation of the BSA digest.


Figure 5. Zoom shot on a fraction of the 3D reconstructed chromatogram of a protein digest.


pressure in the second dimension (e.g. 1000 bar for flow-rates up to 1mL/min); (3) the maximum temperature in the second dimension (depending on the oven and the column stability); (4) the dwell volume in the second dimension. A calculation tool based on an Excel sheet was developed in our laboratory to help with dimensioning 2D separations.


Four examples of calculation are displayed in Table 3. The calculations were carried out so that the sampling rate was suitable (>3) and the injection volume was below 15% of the column dead volume. In conditions (a), the same internal diameter (2.1 mm i.d.) and the same temperature (30 °C) is used in both dimensions. This configuration leads to a very low practical peak capacity (600). In contrast, when the first column internal diameter is decreased to 1 mm, the practical peak


capacity is significantly higher (conditions (b)). Changing to a sub-2 µm column and increasing the pressure up to 1000 bar in the second dimension leads to another large improvement of the practical peak capacity (conditions (c)). Finally, when the temperature is raised in the second dimension, the peak capacity is further increased (conditions (d)). To sum up, a small internal-diameter column in the first dimension associated with an ultra- fast second dimension (HT-UHPLC) is a good combination to maximise the practical peak capacity.


It must be highlighted that the


columns in each dimension should be operated at their maximum pressure in order to maximise the peak capacities. Consequently, the maximal allowable pressure and/or the maximuml delivered flow-rate are limiting factors in the pursuit of very high peak capacities. Recent instruments that can withstand pressures up to 1200 bar and/or over a range of flow-rates up to 5 mL/min are hence very attractive with a view to reaching larger peak capacities. High temperatures increase the peak capacity since higher flow-rates can then be reached due to the decrease in mobile phase viscosity. In addition, increasing temperature is very attractive in case of charges compounds as it improves the peak shape as highlighted above.


Example of a 2D-LC separation The separation of a BSA digest was performed according to the conditions given in Table 4.


dimension 2


Acquity BEH Shield 50 x 2.1mm; 1.7µm TFA 0.05% /


acetontitrile 1300


1 to 30% 5.5 80


Conclusions The different steps necessary for the development of an online RPLC x RPLC separation have been discussed. The orthogonality between the two dimensions is needed. It was shown that combinations involving two silica-based columns operated at different pH provide the highest degree of orthogonality and the largest practical peak capacity for a sample of 17 ionisable compounds. When dimensioning the instrumentation, many parameters have to be taken into account. In particular, the injection volume in the second dimension is a critical parameter. It was shown that a volume as high as 15% of the column dead volume can be injected in the second dimension without causing a detrimental effect on the peak shape and hence on the peak capacity.


Specific instrumental constraints are also limiting parameters. A home-made calculation tool was very helpful to deal with all these constraints and hence to optimize 2D- separations. Using HT-UHPLC in the second dimension allowed accessing impressive peak capacities as illustrated by a separation of a complex mixture (protein digest).


Acknowledgements


Perkin Elmer is acknowledged for the loan of the micro pumps and Waters for providing the ten- port high pressure valve.


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