A systematic approach to HILIC method development is recommended. Method parameters should be considered and adjusted sequentially, one at a time. The following sequence is a good guide:

  1. Determine analyte properties
  2. Select the most appropriate type of column stationary phase
  3. Select the most appropriate mobile phase starting conditions
  4. Optimize mobile phase
  5. Optimize column geometry

HILIC method development workflow

1407788307028

Understanding the physico-chemical properties of an analyte is essential to the selection of a suitable HILIC column.  A combination of log P, log D and pKa data will help guide the stationary phase selection process.

HILIC is generally recommended for compounds with negative log P values. Another important analyte parameter is the pKa (-log Ka) value; Ka gives us an indication of the acidity of a molecule. The larger the pKavalue, the weaker the acid. log D provides a good indication of the ionization state of a compound in solution at a specific pH. Log D is more representative of hydrophobic character in buffered conditions than log P would.

Choose between these 8 different column types:

See more information about these columns on the Stationary Phase page.

In HILIC the mobile phase is highly organic (generally > 60-70% acetonitrile). Besides column selectivity, the organic modifier/aqueous ratio is the principal factor controlling retention. An increase in the percentage of organic solvent leads to an increase in retention when acetonitrile concentrations are between 60-90%. Little retention is achieved when the acetonitrile content is less than 60%.

The use of volatile buffers such as ammonium formate or ammonium acetate is recommended in order to:

  • Maintain good peak shape
  • Control retention of charged analytes
  • Ensure compatibility with MS detection
1407851275832

As a general recommendation for HILIC standard methods, select a 100 or 150mm long column, packed with 3 - 5µm particles and with a 4.6mm id, if using UV detection. If HILIC is coupled to MS or CAD detection, use a smaller column, e.g. 100 x 2.1mm or 50 x 2.1mm, packed with 3-5µm particles. Reduce column length only  after having considered sample complexity and detector selectivity. For higher separation efficiency and resolution we suggest using  either sub 2µm fully porous particles or solid core materials or longer columns.

While there will be differences due to analyte kinetics and stationary phase, for equivalent column dimensions and particle size, HILIC columns are typically run at flow rates similar to or slightly lower than reversed phase.

HILIC-Column-Care
Analyte Properties
1407788307028

Understanding the physico-chemical properties of an analyte is essential to the selection of a suitable HILIC column.  A combination of log P, log D and pKa data will help guide the stationary phase selection process.

HILIC is generally recommended for compounds with negative log P values. Another important analyte parameter is the pKa (-log Ka) value; Ka gives us an indication of the acidity of a molecule. The larger the pKavalue, the weaker the acid. log D provides a good indication of the ionization state of a compound in solution at a specific pH. Log D is more representative of hydrophobic character in buffered conditions than log P would.

Column Selection

Choose between these 8 different column types:

See more information about these columns on the Stationary Phase page.

Mobile Phase Consideration

In HILIC the mobile phase is highly organic (generally > 60-70% acetonitrile). Besides column selectivity, the organic modifier/aqueous ratio is the principal factor controlling retention. An increase in the percentage of organic solvent leads to an increase in retention when acetonitrile concentrations are between 60-90%. Little retention is achieved when the acetonitrile content is less than 60%.

The use of volatile buffers such as ammonium formate or ammonium acetate is recommended in order to:

  • Maintain good peak shape
  • Control retention of charged analytes
  • Ensure compatibility with MS detection
1407851275832
Column Geometry

As a general recommendation for HILIC standard methods, select a 100 or 150mm long column, packed with 3 - 5µm particles and with a 4.6mm id, if using UV detection. If HILIC is coupled to MS or CAD detection, use a smaller column, e.g. 100 x 2.1mm or 50 x 2.1mm, packed with 3-5µm particles. Reduce column length only  after having considered sample complexity and detector selectivity. For higher separation efficiency and resolution we suggest using  either sub 2µm fully porous particles or solid core materials or longer columns.

While there will be differences due to analyte kinetics and stationary phase, for equivalent column dimensions and particle size, HILIC columns are typically run at flow rates similar to or slightly lower than reversed phase.

HILIC-Column-Care
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