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Hydrophilic Interaction Liquid Chromatography (HILIC) is a subset of normal phase chromatography where some percentage of the mobile phase contains water, and the organic solvents used are water-miscible. The water molecules in the mobile phase forms a thin aqueous layer on the surface of the stationary phase, and thus, become a part of the semi-permanent stationary phase. Analytes are retained by this aqueous layer as well as the underlying solid stationary phase. So that, not all normal phase chromatographys are HILIC, but all HILICs are normal phase.**

Normal phase uses a relatively polar stationary phase and a relatively non-polar mobile phase. Normal phase separation is often considered to be less robust and reproducible to reversed-phase separations; HILIC is considered to be in between. HILIC chromatography can potentially be used for any types of polar compound separations, e.g., polar small molecules, simple sugars, glycans, and peptides. Traditionally, normal phase utilizes pure organic solvents (often non-water-miscible) as the mobile phase.

Practical tips on running HILIC

  • Sample solvent should be the same composition as the initial mobile phase
  • Equilibrate HILIC column with 10 to 60 column volumes depending on the sample. For quicker equilibration, run it at higher temperature, higher buffer concentration, or shallower gradient.
  • Acetonitrile and water combination is the most popular mobile phase composition. Other organic solvents can be substituted. Maintain at least some percentage of water.
  • Buffer selection and concentration has great effects on selectivity on some samples. Typically, you should try various composition between 5 to 50 mM.
  • Adjust pH and temperature to further tune the selectivity
  • Many types of HILIC stationary phases with complementary selectivity are available
  • basic analytes are retained strongly follow by phosphorylated ones
  • Bare Silica HILIC Columns

    The underivatized silica or bare silica has negatively-charged polar surfaces with siloxane and silanol functional groups. The ionized silanol interact with analytes in cation-exchange mode that causes peak tailing, especially for basic compounds. To suppress this, mobile phase around or less than pH 3 are used. Another major cause of peak tailing is the metal impurities in silica. Type-B silica, currently used by almost all column manufacturers, is recommended stationary media due to its lower level of metal impurities compare to the type-A silica. Pore size should be compatible with your analytes, i.e., small polar molecules do well around 100A, where as peptides may require 200A or larger.

    Derivatized Silica HILIC Columns - (neutral, ion exchange, and zwitterion / mixed-mode)

    • Neutral HILIC - Amide, Diol, Cyanopropyl
      Amide column has the retention mechanism of hydrogen bonding by being both hydrogen bond acceptor and donor. It is popular among sugar analyses sometimes at elevated temperature. Dihydroxypropyl (diol) derivatized silica is less polar than bare silica and thus more wettable with water, good for protein analysis. The cyano columns are hydrogen bond acceptor but not donor. It produces excellent separation at low pH 2.8 for amines. Gradient elution can easily be used on a cyanopropyl column because it can rapidly reach equilibrium. Both diol and cyanopropyl columns can be used either under HILIC or reversed-phase modes.
    • Anion Exchange HILIC - NH2, triazole
      The positively-charged stationary phases allow these columns to operate in weak anion-exchange or HILIC modes simply by adusting the pH. Amino HILIC columns are often used for glycan analysis, but it can easily be used for the separation of other polar hydrophilic compounds. However, silica-based amino columns are notoriously unstable. A more stable amino columns are composed of polymer or silica-polymer hybrid materials. Polymer-based columns are generally less efficient producing broad peaks. Silica-polymer hybrid amino HILIC column is the best choice (see below). Triazole column is an unique HILIC phase that has cyclic amino groups.
    • Cation Exchange HILIC - PolySULFOETHYL A
      Similar to anion exchange HILIC, the proper adjustment of pH determines the mode of operation.
    • Zwitterion HILIC - MN-HILIC
      NUCLEODUR HILIC is a special zwitterionic modified stationary phase based on ultra spherical NUCLEODUR particles. The betaine character of the ammonium-sulfonic acid ligands results in total charge equalization and in an overall neutrally charged but highly polar surface. PolyLC PolyHYDROXYETHYL A has covalently bonded poly(2-hydroxyethyl aspartamide) functional groups exhibiting neutral zwitterion at pH 4.4.
    • Mixed-Mode HILIC - Primesep N, Obelisc N
      Primesep N has embedded acidic groups (negatively charged, thus cation exchange) on a polar bonded phase. Obelisc N has both negatively and positively charged group on the same long chains of polar bonded phase. The ion exchange groups give these columns enhanced selectivity. In some cases, you may be able to use less acetonitrile to achieve the same separation. A good starting condition is ACN/water=75/25 with 10 mmol of ammonium formate pH-3 (or 0.1% of formic/acetic acid). For Obelisc N column you don't need high buffer concentration for strong retention.

    Silica-polymer HILIC Columns

    • Amino HILIC - Chromenta EP-NH2 - hybrid material has the best of both silica and polymer. As with other amino HILIC columns, EP-NH2 retains analytes through hydrogen bond donor, hydrogen bond acceptor, and ionic interations. Saccharide separations can be achieved with high efficiency of silica particles, while retaining the chemical strength of polymers. It is designed for HILIC mode operation, but it can be used in normal phase or weak anion-exchange modes. The amino columns at pH 6.0 generally produce better peak shape than amide columns for phosphorylated compounds, including triphosphates.

    Non-Silica - polymer and zirconia-based HILIC Columns

    • Amino HILIC - Jordigel polyamino
    • Zircornia HILIC - Zirchrom

    ** The exact mechanism of HILIC separation is still under debate.

    Normal Phase / HILIC Columns - Prep Capillary HPLC Columns

    Bare-Silica HILIC Columns
    BA Promosil Silica
    BA Venusil XBP Silica
    BA Venusil XBP-L Silica
    Chromenta BB-SiO2 (300A)
    Chromenta KB-SiO2
    Dikma Inspire Silica
    MN Nucleodur unmodified
    MN Nucleosil unmodified
    Nacalai COSMOSIL SL-II

    Derivatized-Silica HILIC Columns
    Neutral HILIC
    BA Promosil CN
    BA Unisol Amide
    BA Venusil XBP CN
    BA Venusil XBP Diol
    Chromenta KB-CN
    Chromenta KH-Diol
    Dikma Inspire Diol
    MN Nucleodur CN/CN-RP
    MN Nucleosil CN
    MN Nucleosil OH (diol)
    Nacalai COSMOSIL CN-MS

    Anion Exchange HILIC
    BA Durashell NH2
    BA Promosil NH2
    BA Venusil XBP NH2
    Chromenta EP-NH2
    Chromenta KB-NH2
    MN Nucleodur NH2/NH2-RP
    MN Nucleosil Carbohydrate (amino)
    MN Nucleosil N(CH3)2
    MN Nucleosil NH2
    Nacalai COSMOSIL HILIC (triazole)

    Cation Exchange HILIC
    MN Nucleosil NO2 (nitrophenyl)
    Nacalai COSMOSIL CM

    Zwitterion HILIC
    MN Nucleodur HILIC

    Mixed-Mode HILIC
    Sielc Obelisc N (Zwitterion & NP)
    Sielc Primesep AP (weak AEX & NP)
    Sielc Primesep N (weak CEX & NP)

    Non-Silica HILIC Columns
    Anion Exchange HILIC
    Chromenta EP-NH2 (aminopropyl)
    Polymer-based particles

    Jordi Amino

    BA = Bonna-Agela Technologies
    MN = Macherey-Nagel


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