9
equation for both core-shell and UHPLC: this variable will allow optimum resolution to be found for the wide diversity of compounds types encountered in the pharmaceutical pipeline. Figure 4 shows the separation of a complex mixture of analytes using a C18, Diphenyl and PFP core-shell particle. The orthogonal selectivity offered by the 3 different stationary phases allows a suitable starting point to be made with scope for a highly qualitative method design. Peaks 2-5 not only show differing selectivity but also switches in elution order from phase to phase.
Figure 3. Selectivity (α) is a function of resolution, particle size (dp) and retention factor (k’).
Do UHPLC and core-shell particles offer enough selectivity? It is ultimately not down to the particle technology but to the stationary phases that are bonded to the particles. With both particles being contemporary offerings they are not currently as diverse in stationary phase choice as traditional particles, although this is something that should catch up fairly rapidly.
Analysis Requirements
What does the analyst require? In terms of LC columns the list includes, lifetime, loading, scaling reproducibility, selectivity, robustness, and peak shapes (effi ciency, been taken as a given here in context of the article). All of these variables have made silica popular over the years, even with the advent of carbon, zirconium, polymeric, hybrid and even diamond phases, nothing has replaced silica’s overall outstanding qualities. Core-shell and UHPLC particles both build on this and now increase substantially the effi ciency term.
Figure 4
stable well above the usual working limits of the commercial UHPLC systems on the lab bench.
Lifetime is also an interesting issue from a manufacturers and end-users point of view. In HPLC we would expect a lifetime of 1000- 2000 injections to be reasonable, sometimes much longer. With a 60minute run time and 5minute equilibration time, 2000 samples would take us approximately 90days working constantly 24hrs a day, 7 day a week. So approximately one column per assay per quarter.
However now if we have a UHPLC or core- shell column and our assay takes 10minutes with a 1 minute equilibration, then our 2000 samples take approximately 15days. Yet the number of times I have a customer say that the UHPLC column now doesn’t last 90days like their HPLC column did is strange. Why should it? Column lifetime is more related to sample throughput than to pure ‘calendar months’. If the UHPLC column did last 90 days that would mean by the same logic
that we would have analysed (90/15 x 2000) 12,000 samples instead on the same column.
So we have to compare apples with apples. If I have a core-shell particle column then I need to compare lifetime with another core- shell, not with my previous HPLC column and I need to use number of samples as the measure, not calendar days.
Selectivity
This leads me onto peak capacity (unit resolution, peaks per unit time), one of the variables that will change heavily in the move from HPLC to UHPLC or core-shell. As we improve N through reduced peak width we can go faster and faster with our analysis, however at a certain point we will start to run out of time for our peaks to elute (no matter how narrow they are). Therefore the one variable that does become most important to this high throughput chromatography is that of selectivity (Figure 3).
We need to incorporate selectivity into our
One of the main areas of interest for UHPLC when it emerged was a speeding-up of method development screening. Different columns and different mobile phases conditions could be quickly evaluated to assess the most suitable starting conditions. Once a suitable starting point was established, optimisation of the method could take place. This approach signifi cantly impacted on development time, potentially bringing down method development time to days instead of weeks. However the fi rst downside of this approach became evident when methods passed to other departments where scaling up to larger particles was required but not always possible.
Core-shell particles potentially have this same issue ahead. It will aid in the method development screening process, but at this time will have a scaling issue for those that wish to move from low quantitative methods to preparative scale chromatography. There are two factors that may limit the application of core-shell technology for method scale up. One is the cost of this new particle technology, with few manufacturers offering a preparative column format. The
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