suitable for USP L3
60 °C temp. range
600 bar max. pressure (9000 psi)
7.5 cm × 3 mm
<5 ppm metals
Fused-Core particle platform
90 Å pore size
2 - 9
Food and Beverages
hydrophilic interaction (HILIC)
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Polar peptides are quite amenable to HILIC separations; however, our experience with larger peptides has been only minimally successful - mainly due to solubility issues. Proteins are even more difficult due to the same issue. An additional problem with proteins is that they are often multiply charged. When IEX is performed on multiply charged analytes, you often get what is referred to as a rolling effect where the analyte interacts with ionic sites on the surface in many different ways as it 'rolls' down the column; this produces broad and misshapen peaks.
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.
We are not aware of any systematic studies with respect to the impact of flow rate on HILIC separations. Our concern would be that as you move to higher flow rates, you might observe peak shape issues due to the slow kinetics of IEX and adsorption mechanisms. If the retention mechanisms for a given system are partition dominated, this should be of less concern. It will be a case by case cause and effect.
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.
If you change organic, try to keep the overall buffer concentration (and all other parameters, for that matter) constant. There are times when you will want to change both and perhaps pH/temp/etc. simultaneously, but that drastically complicates the system and thus should be avoided, if possible.
Typically when we refer to pH in HILIC, we use the effective pH or the pH as measured after the addition of organic. The point is that we should always define what pH we are stating. The common way to distinguish is using notation of w/w pH or s/w pH (usually superscript/subscript). The notations mean superscript = solvent the pH is measured in (s would indicate some mixture of aqueous:organic) and the subscript = the solvent the pH meter is calibrated in (typically water (or w) as we readily have calibration standards).
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