The stationary phases used in HILIC are quite diverse and users often have the misconception that HILIC columns are interchangeable. Understanding the possible HILIC retention mechanisms involved for a particular column-solvent-analyte system is crucial in the process of selecting the right HILIC stationary phase, leading to a successful application.

HILIC stationary phases should be characterized in terms of:

  • Degree of hydrophilicity
  • Selectivity for hydrophilic-hydrophobic groups
  • Selectivity for positional and conformational isomers
  • Degree of electrostatic interactions
  • Acid-base nature of the stationary phase

Column selection

In order to get the optimum retention of polar analytes, it is important to match the analyte log P or log D values to the degree of polarity of the HILIC phases. This  chart illustrates the relative hydrophilicity and ion-exchange properties for Thermo Scientific HILIC columns and can be used as a preliminary guide in stationary phase selection. 

HILIC selectivity

Stationary phase characteristics can be illustrated by 2-D radar graphs, which allow a simple visual assessment and comparison of column selectivity. Each axis of the radar plots below represents one of the following parameters:

  1. α (CH2) – degree of hydrophobicity
  2. α (OH) – degree of hydrophilicity
  3. α (V/A) – separation factor for configurational isomers
  4. α (2dG/3dG) – separation factor for positional isomers
  5. α (AX) – degree of anion exchange interactions
  6. α (CX) – degree of cation exchange interactions
  7. α (Tb/Tp) – acidic-basic nature of the stationary phase
  8. k U – absolute retention

Note: A selectivity value of 1 corresponds to no separation (since α= k2/k1, α= 1 when k1= k2).

Thermo Scientific™ Syncronis™ HILIC columns provide enhanced retention of charged and neutral polar compounds. Outstanding peak shape and sensitivity are demonstrated on this phase.

Syncronis HILIC stationary phase carries a zwitterionic functional group of the sulfobetaine type, monomerically bonded to highly pure, high surface area silica. The sulfobetaine zwitterion has both positive (quaternary ammonium) and negative (sulfonic acid) groups in a 1:1 ratio, so that the net surface charge is zero. Due to counterbalanced electrostatic forces, which lead to weaker secondary electrostatic interactions with the analyte, lower buffer concentrations are required to disrupt these interactions. The charge density of this phase is pH-independent and the optimization of the mobile phase pH is solely dictated by the analyte.

Analysis of edrophonium, neostigmine and pyridostigmine from plasma by LC-MS/MS


Column: 
Syncronis HILIC, 1.7 µm, 100 mm x 2.1 mm
Guard column: UHPLC filter/holder
Mobile phase A:acetonitrile / ammonium formate, 100 mM, pH 3.3 (90:10 v/v)
Flow rate: 500 µL/min
Column temperature: 25°C
Pressure: 210 bar
Injection volume: 2µL
Detection: MS, +ESI 275 °C, 3.0 kV

  • Amide phase bonded onto 150 Å pore diameter solid core particles
  • High retention of a broad range of hydrophilic analytes in HILIC mode
  • Recommended for hydrophilic biomolecules such as glycans


The amide bonded phases provide strong hydrogen bonding interaction between the stationary phase and the analytes, resulting in unique selectivity compared to other HILIC phases. Combined with larger pore size of the solid core particles, the Thermo Scientific™ Accucore™ 150-Amide-HILIC column is well suited for separating a variety of hydrophilic molecules, including carbohydrates and peptides. As a result the Accucore 150-Amide-HILIC is an excellent choice for glycan separations.

2-AB labeled dextran ladder

Column: Accucore 150-Amide-HILIC, 2.6µm, 100 x 2.1mm
Part Number: 16726-102130
Mobile Phases: A) Acetonitrile B) 50mM ammonium formate (pH 4.5)
Flow: 500µL/min
Backpressure: 110bar
Temperature: 60°C
Injection: 2µL, 5µL
Detection: FLEra 300nm, Era 420nm
Sample: 2-aminobenzamide labeled dextran ladder

Gradient

Time (min) B%  
0 20
40 50
45 50
45.5 20
50 20
  • (A) 2µL injection of sample, 11 glycans separated
  • (B) 5µL injection of sample, zoomed in to later part of gradient rise. 10 further glycans were detected.

The urea phase bonded onto solid core particles provides a unique selectivity for polar analytes. Thermo Scientific™ Accucore™ Urea-HILIC columns provide good retention for charged and neutral polar compounds.

Analgesic compounds

  • Column: Accucore Urea-HILIC, 2.6µm, 100 x 2.1mm
  • Mobile Phase: A) Water B) Acetonitrile C) 100mM
    Ammonium Acetate adjusted to pH 4.9.
    Composition 10:80:10, A:B:C
  • Flow: 300µL/min
  • Run Time: 2 minutes Temperature: 35°C
  • Injection: 2µL into 10µL partial loop mode
  • Injection Wash Solvent: Water:Acetonitrile 20:80
  • Detection: UV at 230nm
  • Backpressure: 71bar

 

Thermo Scientific™ Acclaim™ HILIC-10 columns are based on high purity, fully porous silica covalently modified with a proprietary hydrophilic layer. The cation exchange properties identified on this material are due to the presence of residual deprotonated silanol groups (under the mobile phase pH conditions used for the characterization testing). Acclaim HILIC-10 provides good retention for charged and neutral polar compounds.

This phase shows dual mode retention behavior, with a ‘’U-shaped’’ retention profile as a function of acetonitrile content in the mobile phase. Increased retention is demonstrated at both low and high levels of acetonitrile (RPLC and HILIC modes, respectively).

Pharmaceuticals

Column: Acclaim HILIC-10, 3µm, 150mm x 4.6mm
Mobile phase: 90/10 v/v CH3CN/10mM (total) NH4OAc (pH 5)
Flow rate: 1mL/min
Injection volume: 2µL
Temperature: 30ºC
Detection: UV at 230nm
Sample: 1) Acetaminophen (0.1mg/mL) 2) Salicylic acid (0.1) 3) Aspirin (0.2) 4) Penicillin G (0.1) 5) Metformin (0.1)

Thermo Scientific™ Hypersil GOLD™ HILIC columns are recommended for the analysis of acidic or neutral polar compounds.

Column: Hypersil GOLD HILIC, 5µm, 150 x 4.6mm
Mobile Phase: 50nM ammonium acetate buffer (pH 5) / Acetonitrile (10:90)
Flow Rate: 0.4mL/min
Temperature: 30°C
Detection: 205nm
Injection Volume: 10µL
Sample: 1) Cyanuric acid 2) Melamine

 

  • Highly pure base deactivated silica media
  • Outstanding peak shape and sensitivity

Besides being the underlying support for all Hypersil GOLD phases, Thermo Scientific™ Hypersil GOLD™ Silica columns have wide applications under both HILIC and normal phase conditions. The cation exchange properties identified on this material are due to the presence of deprotonated silanol groups (under the mobile phase pH conditions used for the characterization testing). 

Hypersil GOLD Silica is recommended for the analysis of basic or neutral polar compounds.

Thermo Scientific™ Syncronis™ Silica columns offer highly pure, high surface area silica.

Serves as a powerful and efficient tool for the chromatography of moderately polar acids or neutral compounds in both HILIC and normal phase mode. The cation exchange properties identified on this material are due to the presence of deprotonated silanol groups (under the mobile phase pH conditions used for the characterization testing).

The cation exchange properties identified on this material are due to the presence of deprotonated silanol groups (under the mobile phase pH conditions used for the characterization testing).

Thermo Scientific™ Accucore™ HILIC columns are recommended for the analysis of basic or neutral polar compounds.

Catecholamines

Column: Accucore HILIC, 2.6µm, 50 x 2.1mm
Mobile Phase: 85:15 (v/v) acetonitrile / 100mm ammonium formate (pH 3.2)
Flow Rate: 2mL/min
Temperature: 40°C
Detection: UV at 280nm
Injection Volume: 5µL
Backpressure: 157bar
Analytes: 1) Catechol 2) 5-HIAA 3) DOPAC 4) Serotonin 5) L-tyrosine 6) Dopamine 7) L-DOPA

HILIC Resource Library

See this collection of resources to help you learn more about HILIC columns, their use and application. 

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