Dear Mel,

"HILIC mechanisms are kinetically slower than the common mechanisms in RPLC, resulting in longer column equilibration times (typically two to four times longer than in RPLC).". Please see the attached file.

"The content of water in HILIC separations is very important. The percentage of water in the mobile phase can vary (5–40%). A minimum of 2% is essential for formation of the aqueous layer involved in the separation and provides for the complete solubilization of the hydrophilic analytes" (Mariana Roberto Gama, Raquel Gomes da Costa Silva, Carol H. Collins, Carla B.G. Bottoli. Hydrophilic interaction chromatography. Trends in Analytical Chemistry, Vol. 37, 2012).

Hi Mel, you wrote that equilibration is time consuming. That's the sticking point. You can't fool the physics behind the transport and diffusion processes. From my own personal experience I will give you an example: A hplc run using a HILIC column (run time: 3.5 min) needs an equilibration time of 10 min. I control the equilibration by using the system pressure of the hplc system. If the system pressure is constant again the equilibration process has been completed. Greetings Markus 

Dear Mel,

"HILIC mechanisms are kinetically slower than the common mechanisms in RPLC, resulting in longer column equilibration times (typically two to four times longer than in RPLC).". Please see the attached file.

"The content of water in HILIC separations is very important. The percentage of water in the mobile phase can vary (5–40%). A minimum of 2% is essential for formation of the aqueous layer involved in the separation and provides for the complete solubilization of the hydrophilic analytes" (Mariana Roberto Gama, Raquel Gomes da Costa Silva, Carol H. Collins, Carla B.G. Bottoli. Hydrophilic interaction chromatography. Trends in Analytical Chemistry, Vol. 37, 2012).

It's hard... In my experience anhydrous solvents may required 2-5% of water, but after days at RT conditions... the concentration of water in the organic solvent can reach even 3-8% so I might recommend to use water scavenger in the B reservoir. Deactivated Zeolite in pellets 4A and 5A are fine. Eventually you can replace the Organic phase more frequently. To stabilized the retention and the reconditioning process the water content in the B phase is the key point, if you are dealing with inconsistent chromatography.

A karl Fischer titration, or colorimetric kit, can help to measure the % of water in the B phase.

Finally ethyl and propyl linker coupled to amide end-cap on silica solid phases are less susceptible to water fluctuations and quickly equilibrate at 40-45C 

You need about 10-20 column volume to equilibrate. If my flow rate is 0.5 mL/min during the run, after the last peak is eluted and ready for equilibration, I would increase to 1 mL/min or as much as you can (limit by the pressure) and diver the LC outlet to waste, instead of to the detector (if you use LC/MS). This will reduce the time significantly. You need a good reproducible run-to-run.

I totally agree with Narong.

See additionally the attached tipp.

It is matter of fact that the samples most chromatographers have to face are rather complex for sample number and compound variety. In these situations, gradient elution is largely preferred for the separation of analytes with different retention factors. In this way it is possible to narrow the peak width of the later eluted compounds and shorten the run time. However, gradient separations have a principal drawback: after each run they require column equilibration with the starting concentration of the mobile phase before the next sample can be injected. In comparison to RPLC stationary phases, HILIC phases need gradients with lower slope and longer equilibration times. This is because in HILIC the separation is driven by the thickness of the water layer that originates from the water in the mobile phase. Changing the mobile phase composition will change the water layer. However, this is a slow process and requires several column volumes before the water in the water layer reaches equilibrium with the mobile phase. A HILIC mobile phase must always contain at least 2–3% of water in the final mobile phase in order to allow the formation of the water layer on the surface of stationary phase. Then a gradient elution should be performed increasing the water content up to a maximum of 40–50% in 20 minutes. Each HILIC column requires a different amount of column volume in order to reach a complete equilibration. Let’s consider a general column of dimensions 2.1 mm × 150 mm. The column volume is ~300 µL (for calculation of the column volume, see HPLC Solutions #101: Estimating Column Volume). HILIC columns need 20 column volumes, or more, for proper equilibration. This means that at a mobile phase flow-rate of 200–400 µL/min, the equilibration time will be between 30 and 15 minutes. Insufficient column equilibration will cause shifting of the retention times, resulting in poor reproducibility (Figure 1). In order to shorten the equilibration time it is possible to perform the equilibration step at a flow-rate higher, even two-fold, than the one used for the analysis. In HILIC this is possible owing to the low viscosity of the mobile phase at high organic content, which gives a low back pressure. Another possibility of increasing the analysis throughput shortening the equilibration step is to run the samples continually allowing a fast but constant equilibration time among the runs. In this case, the column reaches a dynamic equilibrium and even if there is not a complete equilibration, it is possible to obtain good reproducibility. In the forthcoming instalment, we will discuss the effect of sample diluent on HILIC retention and peak shape.

Giorgia Greco is currently a Post Doc researcher at the Analytical Research Group at the Technische Universität München, Germany. She received a PhD in Chemistry at the University of Naples, Italy. During her research, she specialized in the analysis and separation of metabolites from human and food matrices, as well as of organic contaminants in waste water samples, by hyphenated RPLC/MS and HILIC/MS techniques. She has also focused on the theoretical elucidation of the HILIC retention mechanism with the aim of providing scientific bases for the fast development of HILIC separations. Thomas Letzel, Associate Professor, is head of the Analytical Research Group at the Technische Universität München, Germany. He received his PhD in Chemistry with Aerosol Analysis, worked as Post-Doc performing pharmaceutical analysis, built up his research group in bioanalysis with Habilitation in 2009 and extended his analytical experience from then in food and water analysis. In all areas he developed novel analytical platforms based on LC–MS for the characterization of organic molecules in complex matrices. Thereby techniques (such as HILIC or RP-UHPLC) are applied for new analytical solutions often in direct flow-coupling with (bio)functional assays. He is the author of more than 50 publications and two books. Thomas Letzel wants to share his experience in liquid chromatography, especially in HILIC, with the community to accelerate the dissemination about HILIC theory and practical handling.

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Entedhar Sarhat continues to plagerise other people's work and often presents it as his own on this site.