A FAQ List About SeQuant® Products

Which column should I start with?

Columns with 4.6 mm ID geometry are suitable in the analytical flow rate range (about 0.5 to about 3 mL/min) whereas 2.1 mm ID columns are more appropriate for HILIC-MS. It depends on the application which column length is the most suitable.

A 150 mm long column with 5 µm particles (P/N 2712-155) is a good choice if you are unsure on your needs. A fast and simple separation would be better off on a shorter column (e.g., 50 mm, P/N 2712-055), whereas a more complex separation is better off on a longer column (e.g., 250 mm, P/N 2712-255).

What is the recommended flow rates for 4.6, 2.1 and 1.0 mm ID ZIC®-HILIC columns?

Optimum flow rate, where maximum efficiency is obtained:
0.5 mL/min for a 4.6 mm column
0.1 mL/min for a 2.1 mm column
0.02 mL/min for a 1.0 mm column

All geometries can be used at both lower and much higher flow rates. For instance, maximum efficiency for a 2.1 mm id column is obtained at 0.1 mL/min. Increasing the flowrate to 0.5 mL/min lowers the separation efficiency to 20% of maximum, but if resolution is sufficient it saves time. The same rule is applicable to the other dimensions.

What is the name of ZIC®-HILIC functional group?

It is called "sulfobetaine-group".

We want to couple the ZIC®-HILIC to ICP-MS, but we are concerned about the load of organics in the mobile phase. Is there any alternative to Acetonitrile for this purpose?

The carbon depletion problem may be diminished by using other, more high-boiling solvents like dioxane, but that will ultimately depend on your analytes and the nature of the application.

What is the difference between ZIC®-HILIC and Silica HILIC Columns?

The ZIC®-HILIC column is based on a bonded phase having zwitterionic functional groups, while a silica HILIC column is just plain silica.

The ZIC®-HILIC phase allows creation of a stable aqueous layer on the stationary phase, and holds a lot of water at the surface. This makes the column very robust in both isocratic and gradient mode, and it allows aqueous samples to be injected (unless too large volumes are used). The column also has a very good lifetime.

A silica HILIC column has all the problems associated with straight phase chromatography; adsorption effects, peak band-spreading (for some compounds), and slow equilibration. It is furthermore sensitive to changes in water ratio in mobile phase, making it difficult to run gradients due to slow kinetics. Plain silica columns are also reported to have short life. It can be used for some HILIC applications.

What is useful pH range for a ZIC®-HILIC column compared with Silica HILIC Columns?

The ZIC®-HILIC zwitterionic groups maintain their charged but overall neutral hydrophilic form, independent of pH-value. This opens for a unique selectivity since the pH of the mobile phase can be used to moderate the dissociation and retention of the analyte(s). The silica based ZIC®-HILIC column can be used from pH 2-8 whereas the ZIC®-pHILIC polymeric column can be used in the pH range 2-10.

Oppositely, a plain silica column will act as a cation exchanger when pH is raised above 4, as the -OH groups forms negative silanol groups on the surface. Basic compounds then bind more strongly and more buffer salts are needed for elution, which is not favorable for MS detection.

I want to know the following specification of ZIC®-HILIC and ZIC®-pHILIC, pH-range, max-pressure, max-temp, pore size, surface area and carbon load.

Carbon load is relevant for RP columns, not for HILIC columns. The remaining parameters are summarized below:

pH-range; 3-8 pH-range; 3-8 pH-range; 2-10
max-pressure; 400 bar max-pressure; 400 bar max-pressure; 200 bar
max-temp; 70 °C max-temp; 70 °C max-temp; 50 °C
pore size; 100 Å pore size; 200 Å
surface area; 180 m2/g surface area; 135 m2/g

What sort of analyte lipophilicity range is the ZIC®-HILIC columns appropriate for (I was initially under the impression that they were only suitable for very highly polar and water soluble compounds)?

HILIC is a versatile separation technique for compounds with little or no retention under RPLC, or for such solutes that requires ion-pair reagents. This means that not only just the very highly polar and water soluble compounds are candidates.

One simple rule of thumb is based on the logP value. If it is less than about +0.5 for the solute, it can be regarded as a HILIC compound. Observe that manipulation of eluent pH can change the ionisation state of the compound, which then changes the logP value. A charged compound normally has a lower logP compared to its neutral form.

Some users have developed pragmatic polarity indexes in-house and use caffeine as a model compound to decide if the analyte is polar or not polar. Compounds with more retention under HILIC or less under RPLC, compared to caffeine, is considered polar.

Are acids and bases a problem the same way as they are in RPLC?

There are plenty of application notes available for acids and bases.

Normally one uses acetonitrile/water (buffer) mixtures for isocratic or gradients since acetonitrile is among the weakest solvent in HILIC, but methanol, ethanol, isopropanol, acetone, dioxane, etc. may very well also be used. It is preferred to use organic buffers (e.g., ammonium acetate) due to their higher solubility in organic solvents. Inorganic buffer salts should be avoided, as they easily precipitate. When the organic solvent content is about or less than 70 vol% of the mobile phase, low ionic strengths of inorganic salts could be tolerated though.

Is it possible to use TFA as mobile phase additive in HILIC while performing bioanalysis?

In general, TFA is unsuitable for HILIC as it acts as an ion-pairing agent and makes solutes less hydrophilic (as ion-pair). Commonly TFA is used for acidic precipitation of proteins, but it is better to use a high acetonitrile: sample dilution ratio instead. We would suggest that the protein (plasma) sample is added to acidic (formic acid) acetonitrile in 1:4 ratio. After mixing (orbital shaker) most proteins precipitate and may be removed by intensive centrifugation (>5000 rpm).

Secondly, the ZIC®-HILIC column (and other HILIC columns) may retain all kind of ions. A very special feature of ZIC®-HILIC is that even salts like NaCl can be separated, and Na+ and Cl- ions elute at different retention times. Sometimes this means that Na+ co-elute with the analyte which can give suppression of the MS signal. If there are salts in the sample matrix only, we suggest that the mobile phase is changed slightly, about 1-2% more or less acetonitrile. Typically the Na+ ion is then eluting on another retention time and the problem is solved (it may require more optimisation).

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