Choosing a Stationary Phase Surface for HILIC

The chemical composition of the stationary phase in HILIC is often more complex than traditional high-performance liquid chromatography (HPLC) and uses a combination of different molecules that interact with analytes to achieve maximum separation across several parameters.

Molecules for separation in HILICAris Suwanmalee | Shutterstock

Almost any polar chromatographic surface can be used in HILIC, which often consists of bare silica. Silica can be modified by adding surface functional groups to increase or lower the interaction strength between the stationary phase and analyte or solvent may be used to fine-tune retention time and improve separation between compounds.

Modes of separation

Many physicochemical interactions can be exploited in HILIC to separate molecules in a sample. The choice of stationary phase depends upon the molecules to be separated and the solvent (mobile phase), as separation occurs through differing retention strengths between molecules with the stationary phase.  

Separation based on chemical and physical interactions

Chemical interactions that contribute to retention time within the HILIC column include hydrogen bonding between a proton donor and hydrogen acceptor and donor-acceptor interactions between Lewis base electron acceptor and donor pair.

Physical interactions between sample molecules and the mobile phase with the stationary phase consist of electronic interactions between a dipole and a second dipole or ion, and Van der Waals forces. This type of interaction is used when separating charged and polar species.

Hydrophobic and hydrophilic forces, fundamental to HILIC, are also considered to be physical interactions. Analytes are separated in order of polarity by hydrophilic (polar) stationary phases.

Silica stationary phases

Silica gel is the most common substrate from which stationary phases are made in all types of column chromatography, due to its porous structure. Bare silica may be used as a stationary phase in HILIC due to the formation of silanol groups on exposed surfaces, making it highly polar. The three common types of silica gel, differing in their preparation, are termed type A, B, and C.

Type A silica gel

Type A silica gels contain metal impurities as they are precipitated from silicate solution without removing pollutants. The presence of metal ions activates silanol groups, and they may form complexes with certain chelating solutes. This may be exploited when working with some peptides, as they are often highly labile and break apart easily without chelation.

Type B silica gels

Type B silica gels are prepared through the aggregation of colloidal silica solutions in air and so contain far fewer impurities. Polar compounds are well separated by type B gels. The pH of the mobile phase may also be adjusted to encourage the ionization silanol groups on the surface of the silica, making cation exchange an important factor in retention time.

Type C silica gels

Type C silica gels are modified to possess a hydrosilated surface made up of non-polar silicon hydride groups. This is useful when a mobile phase with high organic solvent composition must be used, and more polar analytes elute more quickly from this type of column.

Coated silica stationary phases

Silica gel stationary phases may be modified with a variety of functional groups to finely adjust the retention strength depending on factors, such as hydrophobicity or pH. A common modification to bare silica gels is the incorporation of a hydroxyl group to the exposed silanol surface to create a diol group.

Diol groups are highly polar, protected from ionisation, and have strong hydrogen bonding properties. Thus, polarity and hydrogen bonding are the primary factors in column retention.

Anionic functionalized silica gels, obtained by the addition of amino groups, are also frequently used in HILIC. This creates a negatively charged surface that causes positively charged species to be more strongly retained than basic compounds.

Zwitterionic stationary phases have become increasingly used in HILIC and consist of silica or polymer substrate with exposed zwitterionic molecules such as, sulfoalkylbetaine. Zwitterionic stationary phases are highly polar and ion-exchange interactions are common. They demonstrate greater column efficiency and excellent separation compared to other types of column.

Further Reading

Last Updated: Feb 1, 2019

Michael Greenwood

Written by

Michael Greenwood

Michael graduated from the University of Salford with a Ph.D. in Biochemistry in 2023, and has keen research interests towards nanotechnology and its application to biological systems. Michael has written on a wide range of science communication and news topics within the life sciences and related fields since 2019, and engages extensively with current developments in journal publications.  

Citations

Please use one of the following formats to cite this article in your essay, paper or report:

  • APA

    Greenwood, Michael. (2019, February 01). Choosing a Stationary Phase Surface for HILIC. News-Medical. Retrieved on October 14, 2024 from https://www.news-medical.net/life-sciences/Choosing-a-Stationary-Phase-Surface-for-HILIC.aspx.

  • MLA

    Greenwood, Michael. "Choosing a Stationary Phase Surface for HILIC". News-Medical. 14 October 2024. <https://www.news-medical.net/life-sciences/Choosing-a-Stationary-Phase-Surface-for-HILIC.aspx>.

  • Chicago

    Greenwood, Michael. "Choosing a Stationary Phase Surface for HILIC". News-Medical. https://www.news-medical.net/life-sciences/Choosing-a-Stationary-Phase-Surface-for-HILIC.aspx. (accessed October 14, 2024).

  • Harvard

    Greenwood, Michael. 2019. Choosing a Stationary Phase Surface for HILIC. News-Medical, viewed 14 October 2024, https://www.news-medical.net/life-sciences/Choosing-a-Stationary-Phase-Surface-for-HILIC.aspx.

Comments

The opinions expressed here are the views of the writer and do not necessarily reflect the views and opinions of News Medical.
Post a new comment
Post

While we only use edited and approved content for Azthena answers, it may on occasions provide incorrect responses. Please confirm any data provided with the related suppliers or authors. We do not provide medical advice, if you search for medical information you must always consult a medical professional before acting on any information provided.

Your questions, but not your email details will be shared with OpenAI and retained for 30 days in accordance with their privacy principles.

Please do not ask questions that use sensitive or confidential information.

Read the full Terms & Conditions.

You might also like...
Intermittent fasting with protein pacing trumps calorie restriction in gut health and weight loss