HILIC column

 

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 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 the hydroxyl groups on analytes and the amides on the stationary media. 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. 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
  Properly adjusted pH can make weak ion exchange columns operating in HILIC modes. Amino columns are often used for glycan analysis, but it can easily be used for the separation of other polar hydrophilic compounds. 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 and Bonna-Agela Durashell NH2 - hybrid material has the best of both silica and polymer. Saccharide separations can be achieved with high efficiency of silica particles, while retaining the chemical strength of polymers.

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.

 

HILIC is a useful and complimentary method to reversed-phase chromatography (RPC) and is especially attractive in situations where compound retention is poor in RPC and very high levels
of water are required in the mobile phase for adequate retention.

Retention in HILIC is not well-understood but appears to be a combination of hydrophilic interaction, ionexchange and some reversed-phase retention. The aqueous layer which forms on the surface of HILIC particles promotes interaction with polar solutes. Retention in HILIC as a function of the mobile phase is just opposite from that in RPC.

The strongest mobile phase has a high concentration of water and the weakest has a high concentration of organic solvent. Therefore, for gradient separations, the initial mobile phase has a high concentration of organic solvent and the gradient is formed by increasing the aqueous concentration.

Greatest retention for basic and acidic analytes is found when using more than about 70% organic (e.g., acetonitrile) in acidic mobile phases. High organic concentrations are used in the mobile phases, therefore, HILIC is especially favorable for separations using mass spectrometry (MS) detection.

Acetonitrile is typically used as the weak organic solvent in the mobile phase. With this solvent, 95% is typically the upper limit and 60 - 65% the lower limit for adequate retention. At least 5% of the mobile phase should be the highly polar solvent such as water or methanol. Water should be the polar solvent if
a buffer is included because of solubility limitations. The organic solvent type can be varied to change retention and separation selectivity, much as in RPC. Solvent strength (from weakest to strongest) for HILIC generally is tetrahydrofuran <acetone < acetonitrile < isopropanol < ethanol < methanol <water, where water is the strongest elution solvent. To further increase retention in HILIC, replacing some of the water in the
mobile phase with another polar solvent such as methanol or isopropanol sometimes is effective.
For optimum column efficiency and reproducibility, buffers in the range of 10 - 20 mM concentration or additives in the 0.5% range are used the mobile phase. Phosphate buffers are not recommended because of their poor solubility in high organic mobile phases and incompatibility with MS detection.

Additives such as formic acid, trifluoroacetic acid and phosphoric acid at concentrations up to about 1% can be a part of the mobile phase. Volatile ammonium formate/formic acid buffers up to a final concentration of about 20 mM and pH 3 are especially effective for separating both basic and acidic compounds when using MS detection. (Acetonitrile/formate mobile phases seem to be a good starting point for many separations of both basic and acidic compounds.)
Ammonium acetate at pH ~5 also have been used at concentrations of 5 - 20mM, but are generally less effective for separating stronger basic and acidic compounds. Buffers or additives above pH 6 usually are not recommended because of slow dissolution of the silica support.

Reversed Elution Order Compared to RPLC

The elution order in HILIC is roughly the reverse of that in RPLC (Reversed-Phase Liquid Chromatography). A compound that elutes in the void volume on a RPLC column typically has high retention in HILIC, and vice versa, see Figure 1.

Compounds such as, acids, bases, ions, sugars, and other charged and neutral hydrophilic compounds that are troublesome to separate in RPLC, are much easier to separate in HILIC due to the different separation selectivity. Some compounds are possible to separate by more than one chromatographic technique, and HILIC does in that respect partly overlap both with RPLC, NPLC (Normal Phase Liquid Chromatography) and IC (Ion Chromatography). Figure 2 illustrates the overlap between these different chromatographic techniques

Fig. 1

HILIC 분리의 장점

대사산물과 같이 극성이 큰 물질의 보유능 우수

특별한 선택성

MS 감도 증가

샘플전처리 마지막 단계에서 빠른 전환
HILIC 컬럼은 극성화합물의 보유능 증가로 인지도를 확보하였으며, 모든 극성화합물(극성중성자, 극성산, 극성/비극성 염기) 분석이 가능하지만 극성과 이온반응 모두 보유능과 선택성에 영향을 미치는 특성이 있다.

HILIC 분석은 이렇게 하십시오.
HILIC은 역상과 반대로 극성도가 높은 순으로 용출되기 때문에 대부분 유기용매를 사용하여 분리합니다.
HILIC의 보유능에 영향을 주는 인자에는 친수성 상호반응, 이온교환, 몇몇의 역상 보유능의 결합 작용이 있습니다.
이동상의 60~95%는ACN과 수용액 버퍼이며, 휘발성 및 용해성으로 인해 10~20 mM 암모늄아세테이트 또는 암모늄포메이트가 필요합니다.
시료의 용매는 이동상의 세기 및 형태가 유사해야 하며, 이동상보다 유기물을 많이 포함해야 하지만 물이 많아서는 안됩니다.

1.     사용가능한 pH 범위 온도 범위 숙지

2.     평형 : 50:50=DW:A CN으로 컬럼 부피의 50배로 flushing, 20배의 양을 흘려 평형화 한다. 기울기 용리의 경우에는 컬럼 부피의 8-10부피의 용매로 평형화 한다.

3.     이동상 선택

가.   이동상에는 최소 5%이상의 극성용매(DW, MeOH, EtOH)있어야 한다.

나.   최소 40% 이상의 유기용매(A CN)이 포함되어 있어야 한다.

다.   Phosphate buffer보다는 phosphoric acid 권장

라.   Formic acid, acetic acid보다는 최소 10mM의 ammonium formate나 ammonium acetate가 좋은 재현성을 나타내며 formic acid나 acetic acid사용할 경우 0.2%권장

4.     Injection solvent

가.   가능한 100% 유기용매 사용 권장. 75:25= A CN : MeOH는 시료의 용해도와 피크 모양에 대하여 좋은 결과 기대, 용해도가 낮다면 0.2% formic acid 첨가

나.   시료 용매로 물과 DMSO는 peak 나쁜 피크 모양을 유발 할 수 있다.

5.     BEH column의 경우 silica보다 입자가 작기 때문에 머무름 시간이 적은 경향이 있다.

6.     머무름 시간의 증가는 MeOH aceton isopropanol을 물 대신 사용

Reference

http://www.hichrom.co.uk/assets/Products/AMT/HALO/Hilic-general-info.pdf
http://www.sequant.com/default.asp?ml=11625

http://labplus.co.kr/catalog/500000.asp?cat_no=505038&pro_sect1=100001&pro_sect1_name=%ED%81%AC%EB%A1%9C%EB%A7%88%ED%86%A0%EA%B7%B8%EB%9E%98%ED%94%BC&pro_sect2_name=HPLC%20%EC%BB%AC%EB%9F%BC&pro_sect3_name=Normal%20Phase%20(%EC%88%9C%EC%83%81)&pro_sect4_name=

http://www.columnex.com/hilic.php

https://phenomenex.blob.core.windows.net/documents/kinetex_verify_gu71811109_l.pdf