HILIC with Increased Sensitivity for the Analysis of Sugars

Hydrophilic interaction chromatography (HILIC) provides an alternative approach for the separation of highly polar substances. Using a high polarity packing material, separation is based on the hydrophilic interactions of the analyte on the stationary phase surface. Shodex Asahipak NH2P series is packed with a durable polymer based packing material modified with chemically stable amino functional groups. Shodex Asahipak NH2P-40 3E column is suitable for saccharide analysis, has demonstrated the separation of sugars at low concentration, and has achieved 2–3 times improved detection sensitivity even with RI detection.

Experimental Conditions

The separation was carried out by Shodex Asahipak NH2P-40 3E (3.0 mm i.d. × 250 mm, 4 µm), a polymer-based amino HILIC column. The separation is compared to the same analysis using Shodex Asahipak NH2P-50 4E (4.6 mm i.d. × 250 mm, 5 µm). Column temperature was set at 30 °C and flow rate was 0.35 mL/min for the NH2P-40 3E analysis and 1.0 mL/min for the NH2P-50 4E analysis. Eluent conditions are 75% acetonitrile in water. Injection volume of 10 µL of 0.2% each sugar used for each experiment. HPLC system was coupled with RI detector.

Results

The saccharides, arabinose, mannose, glucose, sucrose, and lactose, were analyzed successfully by HPLC with RI detection with NH2P-40 3E and NH2P-50 4E (Figure 1). Shodex NH2P-40 3E column has smaller dimensions of 3.0 mm i.d. × 250 mm length with a smaller particle size of 4 µm. The decrease in column dimensions requires a decrease flow rate of 0.35 mL/min. The decrease in particle size allows for an increase in the number of theoretical plates allowing for increased sensitivity of analytes. Of note, the NH2P-40 3E analysis uses one third the amount of eluent and provides up to three times improved detection sensitivity.

Figure 1: Increased sensitivity using NH2P-40 3E for the analysis of sugars. Column: Shodex NH2P-40 3E (left) and NH2P-50 4E (right); column temperature: 30 °C; injection volume: 10 µL; eluent: CH3CN/H2O = 75/25; flow rate: 0.35 mL/min and 1.0 mL/min, respectively; detector: Shodex RI.

Conclusions

Shodex Asahipak NH2P-40 3E, a polymer-based hydrophilic interaction (HILIC) chromatography column suitable for saccharide analysis, has demonstrated the separation of these sugars at low concentration and achieved 2–3 times improved detection sensitivity even with RI detection. The presented method using NH2P-40 3E provides an efficient analysis of saccharides that can be detected in small quantities while saving the amount of eluent used per analysis.

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