chromatography industry, I have seen tremendous improvements in daily laboratory workflow through the years, in many cases, due to revolutionary products developed to save time and money for the researcher and provide more accurate and reproducible results day after day. I know how difficult it is to stay current and sort through all the new developments in the industry especially while you’re trying to execute daily activities. There is little time to explore all the new tools and techniques presented, and it’s hard to know which ones will benefit you. My goal, in this and future blog posts, is to discuss and provide resources on the latest tools and techniques that will help you improve and speed up your day-to-day work in the lab.
The first topic I have chosen today is Hydrophobic Interaction Liquid Chromatography or HILIC as it is frequently referred to.
What is HILIC?
Typically, HILIC separation is used for the retention and separation of polar compounds and has been around since 1951 when described by Gregor et al in the following article, titled, Studies on ion-exchange resins. IV. Selectivity coefficients of various cation exchangers towards univalent cations, published in the Journal of Colloid Science 6-323 (link to article).
So, in actuality, HILIC is not a new mode of separation but it is just a variation on normal phase and reversed phase separation. Polar sample components distribute between a polar stationary phase and a less polar mobile phase and compounds elute in order of decreasing hydrophobicity (increasing polarity which is opposite that of reversed phase) using an high organic solvent concentration with a small aqueous salt buffer concentration mobile phase.
Why would you want to use HILIC?
Better MS Signal to Noise, Lower limits of detection
You want to use a HILIC separation when your hydrophobic sample components are not well retained in your reversed phase method without the use of an ion pair agent. A very significant advantage to HILIC is for the use with ESI MS detection. A HILIC separation is performed using a buffered organic mobile phase which ensures efficient desolvation in the MS and in turn leads to higher signal to noise ratios and lower limits of detection.
Compatible with Solid-Phase Extraction extracts
A sample extract from Solid-Phase Extraction (SPE) can be directly injected in HILIC separations without the need to dry down and reconstitute in a RP compatible mobile phase. This is true because the organic solvent is the weak eluent under HILIC conditions. Polar analytes will focus near the column inlet upon injection.
The low viscosity of the organic rich mobile phase for HILIC separations results in lower column backpressure. This in turn allow you to run at higher flow rates than RP making separation times shorter. This is especially significant if your column particle size is less than 2 µm.
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What are the limitations of analysis using HILIC?
One common challenge in HILIC is peak shape distortion from mismatched sample solvent and mobile phase. Aqueous sample solvents can impair the partitioning of the analytes into the stationary phase. Aqueous sample solvents can also cause column overload and reduced retention and resolution. Typical sample solvent recommendations are at least 50% organic. If your sample is not soluble at that organic level, there may be injection loop options to help focus the sample as described by Jandera in Stationary and mobile phases in hydrophilic interaction chromatography: a review (link to article).
Who is using HILIC and for what type of analysis?
Every area of study is finding benefit in using HILIC including pharmaceutical, biopharmaceutical, environmental, and food and beverage for important analyses. Here are some examples of application in different arenas using HILIC separations. There are many more but I thought I’d provide a few in different areas of study to provide an idea of its flexibility.
- In the pharmaceutical arena, the HILIC technique was used for the analysis of pharma drugs, edrophonium, neostigmine and pyridostigmine in Plasma in the method described in Application Note 20787, LC-MS/MS Analysis of Edrophonium, Neostigmine, and Pyridostigmine in Plasma Using HILIC Chromatography and Weak Cation-Exchange SPE, (downloadable PDF). Fast analysis was achieved with a cycle time of less than 3 minutes.
- In the environmental test arena, the technique was used for the analysis of diquat and paraquat and is described in Application Note, HILIC Method for the Separation of Diquat and Paraquat, (downloadable PDF).
- In the biopharmaceutical arena, the HILIC technique was used for the analysis of human IgG glycans and is described in Application Note 20703, Analysis of Human IgG Glycans on solid Core Amide HILIC Stationary Phase, (downloadable PDF).
- In the Food and Beverage arena, the HILIC technique was used for the analysis of melamine and cyanuric acid and is described in Application Note, Analysis of Melamine and Cyanuric Acid Using a Core Enhanced Technology Accucore HILIC HPLC Column (downloadable PDF).
For more application examples, visit the Thermo Fisher Scientific web site, type HILIC and your specific interest in the search bar.
For more general information about the technique and more examples, you can download the HILIC Separation Technical Guide, (downloadable PDF after filling out a short form).
My next post will sort through aspects of sample preparation techniques.
Is HILIC analysis of interest to your laboratory? If so, I would like to hear your thoughts and experiences.