Improved Analysis of Simple Sugars Using apHera™ NH2 HPLC Columns

Hugh M. Cramer, Ric Cone

Reporter US Volume 25.2

ric.cone@sial.com

Chromatographic analysis of simple sugars is important to the food and beverage, pharmaceutical and biotech industries. Effective separations of sugars can be challenging because these compounds are highly polar, uncharged, and lack a chromophore.

The preferred mode of HPLC separation for simple sugars is hydrophilic interaction chromatography (HILIC), an aqueous-organic variation of normal-phase chromatography where water is used as the strong mobile phase (ie. increasing water percentage decreases retention). HILIC provides the advantage of retaining highly polar analytes^1-3 that are difficult to separate on reversedphase columns such as C18. Simple sugars are well suited for this type of chromatography due to their polar nature.

Silica bonded with an aminopropyl silane is a popular phase for HILIC separation of sugars. Aminopropyl groups bonded to silica, however, tend to be susceptible to hydrolysis and are thus less stable than other silica-based phases. The propensity of the aminopropyl ligand toward ionization also limits suitability of the phase for mass spectrometric (MS) detection.

Polymeric columns bonded with aminopropyl groups offer improved stability and MS compatibility over silicabased amino phases. The apHera NH₂ column, now available from Supelco/Sigma-Aldrich, is based on covalently bonded polyamine to a copolymer that offers stability from pH 2-12, mechanical and chemical strength, and high column efficiency. Figures 1A and 1B provide a comparison of a separation of several simple sugars on a polymeric polyamine (apHera NH₂), versus a silica-based aminopropyl stationary phase. The apHera NH₂ column shows significantly greater efficiencies as exhibited by the relatively sharp peaks it produces. In addition, alternative selectivity is observed between the two phases indicating that the surface plays a significant role in the chromatographic separation mechanism. In this case it is assumed that the selectivity difference is due to strong secondary interactions of the sugar analytes with surface silanol groups.

Figure 1. Comparison of Five Simple Underivatized Sugars Separated on apHera™ NH2 vs Aminopropyl Silica (56401AST)

MS bleed analyses for both the aminopropyl silica and apHera NH₂ columns are shown in Figure 2. Depicted are accumulated background scans over a one-minute period with each of the respective columns in line. As is readily apparent, the aminopropyl silica-based phase exhibits strong background ions that may interfere with spectral analysis, make trace components difficult to find and ultimately cause source fouling/instrument down time. The background observed for the apHera NH₂ polymeric phase is very limited and thus suggests the phase is much more compatible with MS detection.

Figure 2. Analysis of Column Effluent by MS

In summary, the apHera NH₂ polymeric column is an excellent choice for the separation of closely-related polar analytes such as simple sugars using HILIC chromatography. These columns demonstrate higher efficiencies than their silica counterparts for this application, and the extended pH range and low bleed offer a much wider range of applications and improvements for use in LC-MS. Other potential applications include derivatized sugars, complex carbohydrates, oligonucleotides, glycopeptides, amino acids, peptides, and polar organic acids and bases.³ The apHera NH₂ column was recently used in an LC-MS assay for taurine and methionine in a carbohydraterich energy drink, with detection limits as low as 20 μg/L for taurine, and 50 μg/L for methionine.⁴

References

1. Alpert AJ. 1990. Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds. Journal of Chromatography A. 499177-196. https://doi.org/10.1016/s0021-9673(00)96972-3

2. Churms SC. 1996. Recent progress in carbohydrate separation by high-performance liquid chromatography based on hydrophilic interaction. Journal of Chromatography A. 720(1-2):75-91. https://doi.org/10.1016/0021-9673(95)00306-1

3. Neue ed U. 1997. HPLC Columns. New York: Wiley-VCH.

4. de Person M, Hazotte A, Elfakir C, Lafosse M. 2005. Development and validation of a hydrophilic interaction chromatography-mass spectrometry assay for taurine and methionine in matrices rich in carbohydrates. Journal of Chromatography A. 1081(2):174-181. https://doi.org/10.1016/j.chroma.2005.05.052