11
HILIC Method Development
A systematic approach to method development should be taken when developing a method in HILIC.
Method parameters should be considered and adjusted sequentially, one at the time.
This section provides some guidelines for:
• which type of stationary phase is the most appropriate to use
• what effects the mobile phase composition and pH can have on the separation
• how the column temperature can influence HILIC analysis.
The HILIC method development process can be summarized in the flow chart below (and on page 30),
which outlines the necessary sequential method development steps in a logical manner.
For a review of Column Group 1
and Column Group 2 see page 17
Determine
analyte log D,
pKa, solubility
Neutral,
zwitterionic or
mixture of
acids + bases
Preliminary Method
Development Steps
Method
Optimization
Steps
Column Group 1:
zwitterion, amide, urea
or
Group 2: anion
exchanger, silica
Column Group 1:
zwitterion or amide
or
Group 2a:
anion exchanger
Column Group 1:
zwitterion, amide, urea
or
Group 2b:
silica
-ve charged
(acidic)
Increase ACN content
Change in 1-5% steps
Change pH
Use pK’s and the
molecular state required
to determine what pH
(charged or uncharged)
Change buffer
concentration/buffer type
Typical ranges: 5-20 mM for
mid-polar to polar compounds
100-200 mM for very polar
compounds
Change in 2.5 mM steps
Change column type but
within same group
+ve charged
(basic)
HILIC not
applicable
Initial mobile phase: Isocratic
ACN/100 mM NH40Ac 80/20
Buffer pH: 4.8
Initial mobile phase: Isocratic
ACN/100 mM NH40Ac 80/20
Buffer pH: 6.4
Initial mobile phase: Isocratic
ACN/100 mM NH40Ac 80/20
Buffer pH: 3.3
Increase ACN content
Change in 1-5% steps
Change pH
Use pK
3
’s and the
molecular state required
to determine what pH
(charged or uncharged)
Change buffer
concentration/buffer type
Typical ranges: 5-20 mM for
mid-polar to polar compounds
100-200 mM for very polar
compounds
Change in 2.5 mM steps
Change column type but
within same group
Change column
temperature
Working Assay
No
No
Yes
Yes
What is the
analyte overall
charge
Log D> 1
Does the
analyte have suitable
retention time
Are analytes
separated
