Ascentis^® Express 90 Å HILIC (2.7 μm) HPLC Columns

Ascentis Express HPLC columns are capable of use on standard HPLC systems as well as UHPLC systems.  Columns are packed in high pressure hardware capable of withstanding the pressures used in UHPLC systems.

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Express Fused-Core™ particles were designed with LC-MS in mind. Even extremely short column lengths exhibit sufficient plate counts to show high resolving power. The flat van Deemter plots permit resolution to be maintained at very high flow rates to maximize sample throughput. All Ascentis stationary phases have been evaluated for MS compatibility during their development, and the Express phases are no exception. You can expect extremely low column bleed and background while maintaining longest possible column lifetime. A bonus of Ascentis Express columns for high throughput UHPLC and LC-MS is that they are extremely rugged and highly resistant to plugging, a very common failure mode for competitor columns.

If the acids are hydrophilic or you can adjust the pH to make them hydrophilic enough, any of the phases that exhibit HILIC partitioning are possible (bare silica, OH5, diol, Zwitterionic, amide). We typically go with the OH5 first to try and avoid any negative impacts on the like charge.

While operating pressures may not exceed the 400 bar (6,000 psi) capability of your traditional instruments, sustained pressures of about 200 bar (3,000 psi) will exceed the recommended pressure for conventional PEEK tubing and fittings at the column inlet. We recommend changing to stainless steel fittings in all high pressure locations and have designed special High Performance HPLC Fittings/Interconnects that will stay tight at pressures of 1,000 bar (15,000 psi) or greater, even when elevated column temperatures are employed.

Yes, it would be highly preferential (especially in this case where you want partitioning to dominate) to inject in high organic. That said, you can 'get away' with it if the injection volume can be kept small - much like we can inject low volumes of stronger solvents in RP mode, if needed. What you will want to do to minimize impact is to get as much retention on the analytes of interest as you can, this helps give the sample solvent some time to dissipate and negate the effects.

Based on the minimum in the van Deemter curves, higher flows than 5um particle columns are required in order to maximize Ascentis Express column efficiency. The suggested starting point for flow rate for Ascentis Express columns: 1.6 mL/min for 4.6 mm ID;  0.8 mL/min for 3.0 mm ID; and 0.4mL/min for 2.1 mm ID.

When running in HILIC mode, both isocratic and gradient practices result in instability. If you keep the re-equilibration times constant, gradient should not be a problem, but changing this parameter can have a significant impact. It is not so much that it is bad as it is different than we are used to in reversed phase. Usually, we assume that once equilibrated (5, 10, 15 min, etc.), we can leave the system for any time period and come back to the same results. This does not appear to be the case in our studies of HILIC. Knowing that the re-equilibration time has an impact, you should get in the habit of making several injections with known re-equilibration times prior to making any development decisions. To get around this, isocratic runs are recommended. Attached are two posters; the first was presented at HPLC 2013 (Amsterdam) and the second was presented at Balaton Symposium on High Performance Separation Methods 2013 (Hungary). Both show 'reproducibility' at any set re-equilibration time is good but both show that if you change the re-equilibration time; then retention, peak shape and selectivity can change especially where ionic interactions are prevalent.

Nothing special is required to use Ascentis Express HPLC columns. To obtain the full benefits of Ascentis Express, one should minimize dispersion or instrument bandwidth in the HPLC system (tubing, detector flow cell) as well as confirm the detector response system is set at a fast level. For more information, request Guidelines for Optimizing Systems for Ascentis Express Columns (T407102)

Yes. if possible you should always have at least a small amount of buffer in a HILIC system to help mediate/control IEX and other polar interactions that are bound to be present (even if at a low level). It is not so much the "buffering capacity" that is important, but the presence of the competing ions. We have found that their presence helps with day to day and column to column reproducibility. There are times when you need to eliminate the buffer, but aside from special circumstances, our recommendation is to include them.