HILIC: Limitations
One of the most common challenges in HILIC is distortion of peak shape that arises from mismatch of sample solvent
and mobile phase as shown below
Using an aqueous sample solvent (with high elution strength), which impairs the partitioning of the analytes into
the stationary phase is detrimental to the chromatographic peak shapes. Other negative ‘side effects’ that could be
experienced if using an aqueous sample solvent are easy column overload, reduced retention and resolution. To ensure
good chromatographic performance it is recommended that the sample solvent has an organic content greater than 50%
[17]. However, this can cause solubility issues with highly polar analytes. Johnson
et al
. addressed this problem by using
an injection loop partially filled with a central sample plug, surrounded by the focusing organic solvent [18].
Other challenges associated with HILIC are:
• HILIC mechanisms are kinetically slower than the common mechanisms in RPLC, resulting in longer column
equilibration times (typically two to four times longer than in RPLC).
• Accurate measurements of mobile phase pH cannot be made when using high proportions of organic solvent.
McCalley [19] showed that acetonitrile/ammonium formate 85/15 (v/v), where the aqueous buffer pH was 3,
presented in fact a pH of about 5.2 to the column and analytes.*
There is a common misconception that all HILIC columns will retain polar compounds in a similar manner. Indeed,
one of the most recurrent uncertainties in HILIC method development is the difficulty in identifying the most
suitable column to use.
*Although mentioned here, this issue will not be discussed further in this guide. When reference to mobile phase pH is made,
we always refer to the pH of the aqueous part.
1
2
3
4
Injection solvent: 10 mM NH
4
AcO
N = 7174
10
Column:
Hypersil GOLD HILIC
, 5 μm
150 × 4.6 mm
Mobile Phase:
10:90 (v/v) ammonium acetate
10 mM, pH 5.0/acetonitrile
Flow Rate:
0.6 mL/min
Inj.Volume:
1 μL
Temp.:
30 °C
Detection:
UV at 258 nm
Sample:
1. Benzophenone
2. Uracil
3. Cytosine
4.Acyclovir
1
2
3
4
Injection solvent: 10 mN NH
4
AcO/ACN (50:50)
N = 14705
1
2
3
4
Injection solvent: Mobile phase
N = 53975
1
2
3
4
Injection solvent: ACN
N = 53995
Effect of sample solvent on peak shape, under HILIC conditions
