HILIC: Advantages
The main benefits of HILIC can be summarized as follows:
• Retention of hydrophilic compounds that are difficult to retain in RPLC without the use of an ion
pair agent
•
Elution order is based primarily on analyte hydrophilicity, as opposed to
hydrophobicity, so HILIC selectivity is complementary to RPLC selectivity.
• Mobile phases have high organic content which generally allows:
•
Improved signal/noise ratio, (S/N) with MS detection This is illustrated below.
The use of HILIC is often highlighted as advantageous for use with ESI MS detection by virtue of the fact that the highly
organic mobile phases ensure efficient desolvation, which in turn leads to lower detection limits. In the example shown
below, in order to retain nicotine and cotinine using reversed phase chromatography, a highly aqueous mobile phase is
required (in this case 98%). This results in low S/N ratio, even after adjustment of the electrospray conditions.
When the same two compounds are analyzed using HILIC, the S/N ratio for nicotine is 15 times higher than in RPLC;
the S/N ratio for cotinine is 5 times higher in HILIC than in RPLC, as illustrated in the following chromatograms.
Time (min)
1.0
0.0
2.0
3.0
4.0
5.0
Time (min)
1.0
0.0
2.0
3.0
4.0
5.0
S/N: 551
S/N: 80
Nicotine
8
Column:
Hypersil GOLD HILIC
, 5μm
150 × 2.1 mm
Mobile phase:
10:90 (v/v) ammonium formate 50 mM
pH 3.5 / acetonitrile
Detection:
+ESI
Extracted Mass Chromatograms for nicotine and cotinine in HILIC
Time (min)
1.0
0.0
2.0
3.0
4.0
5.0
Time (min)
1.0
0.0
2.0
3.0
4.0
5.0
S/N: 551
S/N: 80
Cotinine
Time (min)
1.0
0.0
2.0
3.0
4.0
5.0
S/N: 35
S/N: 15
Nicotine
Column:
Hypersil GOLD
, 5μm
150 × 2.1 mm
Mobile Phase:
98:2 (v/v) water / acetonitrile + 0.1%
formic acid
Detection:
+ESI (spray conditions adjusted for higher
aqueous content of mobile phase)
Cotinine
Extracted Mass Chromatograms for nicotine and cotinine in RPLC
Time (min)
1.0
0.0
2.0
3.0
4.0
5.0
Time (min)
1.0
0.0
2.0
3.0
4.0
5.0
S/N: 35
S/N: 15
