Column and Mobile Phase Considerations for Complete Separation of Nutritional Sugars

Record: 588

Category: Column Selection
Product: NH2P-50 4E
Sample: Nutritional Sugars

Question

A customer needs to quantify sugars listed on a nutritional label, including:

• Maltose
• Sucrose
• Fructose
• Lactose
• Glucose

They are currently using a SugarPak-type column with 100% water (with EDTA) but has difficulty separating sucrose and maltose.

Shodex NH2P-50 4E appears capable of separating these sugars, but the catalog method uses a high acetonitrile (ACN) content. The samples are aqueous.

Can NH2P be used with 100% water, or must the ACN-based method be followed?

Answer

For accurate quantification of multiple sugars such as glucose, fructose, sucrose, maltose, and lactose, both chromatographic selectivity and sample preparation must be considered.

Columns such as SUGAR SP0810, KS-801, and SC1011 can operate under 100% aqueous conditions and may be considered when maintaining a fully aqueous method is required. However, these methods typically do not provide sufficient selectivity to completely resolve all five sugars simultaneously, particularly sucrose and maltose.

High-resolution separation of these sugars generally requires HILIC-based columns operated with acetonitrile/water mobile phases. Shodex HILICpak NH2P-50 4E is a commonly used option and provides effective separation under high ACN conditions.

Sample preparation plays an important role in HILIC separations. Even if the original samples are aqueous, compatibility can be improved by first dissolving the sample in water and then diluting with acetonitrile to closely match the mobile phase composition. This helps maintain retention, peak shape, and reproducibility.

Using 100% water with NH2P is not recommended, as retention in HILIC mode depends strongly on organic solvent content. Fully aqueous conditions will result in reduced retention and poor resolution.

It should also be noted that aminopropyl (NH₂) columns such as NH2P may show gradual changes in recovery over time for certain sugars due to Schiff base formation between reducing sugars and the stationary phase. This can lead to decreased response or variability in quantitative analysis.

As an alternative, polymer-based HILIC columns such as Shodex HILICpak VG-50 4E may be considered. These columns provide similar sugar separations under HILIC conditions while minimizing secondary interactions associated with aminopropyl phases.

In summary, aqueous methods may be used when simplicity is required, but HILIC conditions are generally necessary for full resolution of these sugars. Sample dilution with acetonitrile provides a practical way to adapt aqueous samples to HILIC methods.

Recommended Applications:

• Elution Volume of Saccharides (Selectable Analytes)
• Elution Volume of Saccharides (VG-50 4E)

Additional Comments

Shodex Technical Support can assist with mobile phase optimization if needed. Please contact us at support@shodexhplc.com

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