26
2.5 mM Ammonium acetate
10 mM Ammonium acetate
15 mM Ammonium acetate
20 mM Ammonium acetate
In summary, the recommendations we offered for mobile phase selection for HILIC applications are:
• Use acetonitrile or other polar, water-miscible organic modifiers. Remember that the elutropic strength
is inverse to what observed in RPLC (as outlined in table on page 20). Aprotic solvents give longer
retention than protic solvents.
• Have a high organic content, between 60 to 97%; a minimum of 3% water is necessary to ensure
sufficient hydration of the stationary phase.
• An increase in organic solvent will lead to an increase in retention.
• Use buffer salts such as ammonium acetate and ammonium formate to avoid peak tailing and to
control retention times of charged analytes.
• Buffer salts concentrations are 2−20 mM, although 20 mM is recommended for organic content of
below 90% only. Higher concentrations are not soluble in high levels of organic and could impair
MS or CAD signals.
• When using gradients, buffer both mobile phases, do not run buffer gradients.
• Do not run gradients from 100% organic to 100% aqueous.
• The charge state of the stationary phase can affect HILIC retention of ionizable compounds, depending
on the mobile phase pH.
Column:
Hypersil GOLD HILIC
, 5 µm
100 × 4.6 mm
Mobile Phase:
90/10 acetonitrile/ammonium acetate
Flow Rate:
1.0 mL/min
Inj.Volume:
5 µL
Temp.:
30 ˚C
Detection:
248 nm
Sample:
1. Uracil
2. Uridine
3. Cytosine
4. Cytidine
Time (min)
2.5
5
7.5
10
12.5
15
17.5
20
0
2.5
5
7.5
10
12.5
15
17.5
20
0
2.5
5
7.5
10
12.5
15
17.5
20
0
2.5
5
7.5
10
12.5
15
17.5
20
0
1
2
3
4
Separation of a mixture of bases on Hypersil GOLD HILIC (anion exchanger)
