Equilibration under HILIC mode can be very time consuming. It will take as long as it takes. That is the true scientific answer.

HILIC methods are known for their poor reliability and difficulty in maintaining a clean column between runs. Often an alcohol wash after several runs is needed to remove built-up material and re-equil the surface layer before equilibrating back in the mobile phase. *Not a good type of mode (HILIC is a mode of chromatography, not a "column" type) to start out with or learn on.

Mel, based on the wide range of questions you have been asking, you really would benefit from some solid introductory HPLC training courses and also reading up on some of the many great classic books on liquid chromatography too. "Introduction to Modern Liquid Chromatography" (ISBN: 978-0-470-16754-0) is a great one as is "Practical HPLC Method Development" (ISBN: 978-0-471-00703-6).  The books and web are a great help, but not a substitute for learning from an experienced practitioner directly. Under the right conditions it takes many years of practical experience to learn the basics of this technique.

https://hplctips.blogspot.com/2012/03/hydrophilic-interaction-chromatography.html

Equilibration under HILIC mode can be very time consuming. It will take as long as it takes. That is the true scientific answer.

HILIC methods are known for their poor reliability and difficulty in maintaining a clean column between runs. Often an alcohol wash after several runs is needed to remove built-up material and re-equil the surface layer before equilibrating back in the mobile phase. *Not a good type of mode (HILIC is a mode of chromatography, not a "column" type) to start out with or learn on.

Mel, based on the wide range of questions you have been asking, you really would benefit from some solid introductory HPLC training courses and also reading up on some of the many great classic books on liquid chromatography too. "Introduction to Modern Liquid Chromatography" (ISBN: 978-0-470-16754-0) is a great one as is "Practical HPLC Method Development" (ISBN: 978-0-471-00703-6).  The books and web are a great help, but not a substitute for learning from an experienced practitioner directly. Under the right conditions it takes many years of practical experience to learn the basics of this technique.

https://hplctips.blogspot.com/2012/03/hydrophilic-interaction-chromatography.html

Which operative temperature are you using ? 

Which is the spacer between the direct phase and the hilic?

A commercial column. My project was on separation not testing new material. Making new material has it's own problem. I agree the reason for asking is that i need to know today what I must do to complete the workload. Supervisor did not ask me to do work. Only expects me to finish some necessary tasks. This question arose yesterday evening. The internet does not give experience and it does seem rather difficult to learn hilic in academia without the experience. I like the concept of an alcohol wash in between. It is polar vs ACN so washes off polar contaminants post runs.

I search the internet but not always the correct way. Had I known that a course would benefit me I would have signed up. Since a method developer needs 1.1 I am highly unlikely to get a job in method development compared to QC. All academics should ideally get an industry intro course or be pushed to getting one early on in the project. I tried to find them but they are abroad or not within my time limits to travel. Not enough of those courses.

Hi Mel, when I use HILIC I control the equilibration by using the system pressure of the HPLC. When system pressure is constant you can assume that the equilibrium has been reached. If not you will see it by varying retention times.  Greetings Markus

I have never carried a HILIC separation myself but from what I read here it seems that to achieve reasonable reproducibility of the method is... not easy. Therefore, it is like asking for luck, and therefore would discourage me. I would rather look for a suitable column, these days various hybrid columns are commercial.

Hi Mel

Using HILIC chromatography has many pros and many cons but is basically a chromatography method that mimics the same concept of a dynamic stationary phase as used for ion pairing chromatography.

It is also difficult to reproduce when going from standards analysis to real samples where components in the sample change the dynamic phase and can even bind to the underlying stationary phase. In HILIC the idea is to use bare silica or a more polar bonded phase (amine or hydroxyl) and form a thin dynamic layer of aqueous phase that acts as the interacting phase for the analytes. The more water added the thicker and less stable the aqueous phase becomes and this layer then moves down the stationary phase without providing much analyte retention and loosing the selectivity. The aqueous phase can be buffered to enhance selectivity but these buffers need to be compatible with high organic phase concentrations and downline detection systems. As opposed to reverse phase separation increasing water concentrations reduce the retention time of the polar analytes and are typically only 3-10% water.

The retention is generally greatest for the most polar analyte which is the opposite retention order compared to reverse phase separations. The amount of water determines the dynamic layer "thickness" which in turn affects the retention time. Being a dynamic process it does take quite a large volume to achieve a good reproducible layer and if you are using water gradients it becomes a very difficult process to reproducibly achieve. The layer is also temperature, pH and organic phase type dependant, all of which can change the selectivity.

Another factor you must consider is the sample matrix. If the sample is dissolved in water it could elute the analyte due to passing through the column with the slug of the injected solvent and not show much retention at all. Any pH shift due to the sample matrix also has an effect on many polar analytes and the buffer concentration of the mobile phase.

When scouting for optimal elution conditions you must test all the parameters that can affect the separation and be careful to watch the sample solvent and potential matrix effects of other compounds in the sample. Test all the parameters one at a time using standards then spike a typical matrix with the standard and test that the separation still works after several injections.

Test different solvents, pH, buffer concentrations and try keep the gradient change in water concentration as small as possible to reduce between run re-equilibration times.

The pros are that there are many polar compounds that are well separated under HILIC conditions but not other techniques

Just curious, what types of compounds can be separated well only with the help of the HILIC technique, and cannot be separated using other HPLC options? What sample size does the technique is best used for?

HILIC is a hybrid technique used as an alternative means to retain samples with Log P < 1.0 using RP style organic mobile phases plus some water (on silica). IOW: For samples which are not retained on a C18 column (which would normally shoot through and elute at the void).

For more info, please refer to the linked article, "Hydrophilic Interaction Chromatography (HILIC)" for an overview.

https://hplctips.blogspot.com/2012/03/hydrophilic-interaction-chromatography.html

Thank you, Bill.

I, of course, read your reference as soon as you posted it. Without a priori excluding an easier approach for a compound, I just wonder if perhaps something like the Waters C18 CSH columns or some of the YMC Triart columns (they also have Triart Diol-HILIC packing) would allow to more easily run the separation. A Triart C18 can readily separate cytosine from uracil (at pH 6.9), for example. This is why I kind of was asking what kind of compounds we were talking about.

Thank you again for your suggestions - always very valuable...

Hi Jacek

You are in the right ball park with the polar compounds like cytosine, uracil, adenine etc but also some sugar separations work pretty well and the rather notorious melamine.

HILIC has its place, but is in my opinion over-rated as was ion-pairing about 20 years ago. Both rely on dynamic layer formation and that is sample and mobile phase dependent. Isocratic separations work fairly well but then there really is a limitation in the selectivity. However, peak shape is sometimes really good with HILIC, so I dont want to shoot it down completely.