A New HILIC Column for Saccharide Analysis

Due to the highly polar nature of saccharides, the analysis of sugars is typically achieved using hydrophilic interaction chromatography (HILIC). Using a high polarity packing material, separation is based on the hydrophilic interactions of the sugars on the stationary phase surface typically using an eluent with a majority of polar organic solvent.

HILIC columns with amino functional groups are often used to prevent the separation of anomers; however, one drawback of the amino functional group on the packing material is the low recovery rate of reducing sugars, such as glucose and mannose. Reducing sugars are able to adhere to the packing material through the formation of a Schiff base, and must be hydrolyzed with acid for removal.

Shodex introduces the VG-50 4D column packed with a durable polymer based packing material modified with chemically stable tertiary amino functional groups. Shodex VG-50 4D is suitable for saccharide analysis, has demonstrated the separation of reducing sugars, and has been compared to two competitor silica-base amino columns.

Experimental Conditions

The analysis of four saccharides is accomplished with Shodex VG-50 4D (4.6 mm I.D. × 150 mm, 5 µm), a polymer-based amino HILIC column. The separation is compared to the same analysis using two silica-based amino columns under the same conditions. Column temperature was 40 ºC and flow rate was 0.4 mL/min for the VG-50 4D analysis and 1.0 mL/min for the silica-based amino analyses. Eluent conditions are 80% acetonitrile in water. Injection volume of 5 µL of 5 mg/mL of each sugar was used for each experiment. The HPLC system was coupled with RI detector.

The analysis of four saccharides is accomplished with Shodex VG-50 4D (4.6 mm I.D. × 150 mm, 5 µm), a polymer-based amino HILIC column. The separation is compared to the same analysis using two silica-based amino columns under the same conditions. Column temperature was 40 ºC and flow rate was 0.4 mL/min for the VG-50 4D analysis and 1.0 mL/min for the silica-based amino analyses. Eluent conditions are 80% acetonitrile in water. Injection volume of 5 µL of 5 mg/mL of each sugar was used for each experiment. The HPLC system was coupled with RI detector.

Figure 1: The analysis of sugars using VG-50 4D and two silica-based amino columns. Column: Shodex VG-50 4D (top) and silica-based amino columns (middle and bottom); column temperature: 40 °C; injection volume: 5 µL; eluent: CH₃CN/H₂O = 80/20; flow rate: 0.4 mL/min for VG-50 4D and 1.0 mL/min for silica columns. Detector: Shodex RI. Sample: 1. fructose, 2. mannose, 3. glucose, and 4. sucrose.

Results

The saccharides, fructose, mannose, glucose, and sucrose, were analyzed successfully by HPLC and RI detection with VG-50 4D and two silica-based amino columns (Figure 1). A comparison of the recovery ratio of mannose, a reducing sugar, in relation to sucrose, a nonreducing sugar, is demonstrated in Figure 2. Shodex VG-50 4D demonstrates baseline separation of the four sugars with an elution volume of 20 min and allows up to 90% recovery rate of mannose. Silica-based amino column 1 shows baseline separation of the four compounds. However, the recovery ratio of reducing sugars is 40%.

Silica-based amino column 2 cannot separate the four sugars, and the recovery ratio is less than 20%.

Figure 2: Comparison of Shodex VG-50 4D and silica-based amino columns demonstrating recovery ratio of mannose. Recovery ratio = (peak area mannose)/(peak area sucrose).

Conclusions

Shodex VG-50 4D, a polymer-based hydrophilic interaction (HILIC) chromatography column suitable for saccharide analysis, has demonstrated the recovery ratio of reducing sugars compared with silica-based amino columns.

Shodex™/Showa Denko America, Inc.
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Website: www.shodex.net