The separation of mixtures of complex sugars can be dramatically enhanced by the use of a stationary phase based on zwitterionic hydrophilic interaction chromatography (Zic-Hilic)
SeQuant has recently obtained baseline separation of a mixture of ribose, sucrose (Glc-Fru), raffinose (Gal-Glc-Fru) and stachyose (Gal-Gal-Glc-Fru) using a mobile phase consisting of acetonitrile:ammonium acetate (100mM, pH=5.6) [70:30].
Hydrophilic interaction chromatography (Hilic) with a zwitterionic stationary phase allows the analyst to use a polar mobile phase to readily separate polar samples such as sugars.
Typically, the order of elution is inverted relative to reverse phase separation and hydrophobic compounds are eluted before hydrophilic compounds.
The technique provides significant benefits compared to the use of normal phase or reverse phase chromatography.
When a normal phase is employed, the mobile phase is typically non-polar and the solubility of the compounds of interest is quite low.
This leads to a relatively low signal and difficulties in the detection process; this is a significant problem when mass spectrometry is employed.
When a reverse phase separation is employed, the polar mobile phase is normally capable of solvating a sufficient quantity of the sample, but the resolution of the components of the sample can be quite difficult.
In some cases, a complex gradient is required to separate the sample.
The Zic-Hilic column that was used in these studies has a very high capability for separation of complex sugars.
The separations are rapid, sensitive and provide superb resolution.
In addition, it should be noted that separations on Zic-Hilic stationary phases are readily scalable, and can be used for preparative work.
