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Start Up Guide
A systematic method development approach is provided in the main section of this guide and we suggest referring to it
for a comprehensive strategy.
As a general recommendation:
• Choose the column chemistry according to the nature of the interactions between analyte and stationary phase.
• For standard operations select a 100 or 150 mm long column, with a 4.6 mm id and packed with 3−5 µm
particles. The column length can be changed if resolution needs optimizing.
• Smaller column diameter is recommended if HILIC is coupled to MS detection.
Optimum flow rate varies from phase to phase and column id. Some investigations carried out into the kinetic
performance of 100 x 4.6 mm HILIC columns highlighted that the bare silica material has a wider range of optimal
flow rate conditions (0.9-1.7 mL/min) than the amide or the zwitterionic materials (0.7−1.0 and 0.6−0.8 mL/min,
respectively).
As outlined in the flow-chart reported on the next page, the starting mobile phase we suggest consists of 80/20
acetonitrile/aqueous buffer. Adjust the elution strength with small (<5%) changes in organic/aqueous ratio until
acceptable retention is achieved.
Alternatively, a generic gradient can be run (especially when separation of analytes with differing retention factors has to
be achieved), starting from 95% acetonitrile, holding for 2 minutes (to establish whether compounds are weakly retained
in HILIC mode) and gradually increasing the water percentage to about 40% over 15 minutes (for a 100 mm long
column). A 2 minute hold should follow, to establish whether compounds are strongly retained in HILIC. We discourage
to use fast gradients or gradients that run from 100% organic to 100% aqueous, as it would be very difficult to reach a
dynamic equilibrium with reproducible retention times and besides, the latter would fall outside the HILIC realm. We would
also suggest that the buffer concentration is maintained constant during the gradient (by adding the buffer to the organic
portion as well as the aqueous), unless using charged stationary phases - where increasing the buffer concentration
during the gradient would disrupt electrostatic interactions with the analyte.
If using gradients, it is vital to have a sufficiently long post gradient re-equilibration stage, to allow for the volume of the
water layer (as advocated by the HILIC partitioning model) to re-establish its initial conditions. It is recommended that
a post gradient re-equilibration of approximately 20 column volumes is used. The same level of equilibration is also
recommended for the initial column conditioning prior to the start of analysis. Failure to sufficiently equilibrate HILIC
columns will result in irreproducible chromatography, with drifting retention times.
