Sugars and sugar alcohols display almost no ultraviolet absorption, and are therefore typically detected using a differential refractive index detector or evaporative light scattering detector. By using a ligand exchange column for sugar analysis, it is possible to distinguish among the different isomers based on the position of the hydroxyl group in the chair conformation of glucose and fructose for example.
In other words, the hydroxyl group of the sugar and the metal ion of the stationary phase form a complex, making it possible to achieve separation due to the difference in the strength of the complex formation. Also, maintaining a column temperature of 80 °C suppresses sugar anomer separation and peak dispersion, thereby achieving good separation of adjacent peaks.
The new Prominence-i integrated high-performance liquid chromatograph can be connected to the RID-20A differential refractive index detector. The column oven, which can accommodate a 30 cm column and maintain temperature control up to 85 °C, therefore supports applications that require a long column.
In Application News No. 467, we introduced an example of analysis of sugars in juice, in which the Prominence-i was connected to a differential refractive index detector. Here, we introduce an example of simultaneous analysis of sugars and sugar alcohols in an energy drink using the Prominence-i and RID-20A.
Analysis of a Standard Mixture of Six Sugars
Sorbitol, xylitol, mannitol and erythritol are a type of sugar alcohol that because of their relative sweetness, are used as sweeteners. When conducting simultaneous analysis of sugars and sugar alcohols, a hydrophilic compound analytical column, such as the SPR-Ca or SPR-Pb, is suitable along with the use of a combination of the size exclusion and ligand exchange modes of analysis. Fig. 1 shows the results of analysis of a standard solution of six sugar alcohol substances (10 g/L each of maltose, glucose, fructose, erythritol, mannitol and sorbitol) using the SPR-Ca column with a 10 µL injection. The analytical conditions are shown in Table 1.
Fig. 2 shows the results of analysis of a standard solution of six sugar substances including sugar alcohols (10 g/L each of maltose, glucose, fructose, mannitol, xylitol, sorbitol) using a 10 µL injection, and Table 2 shows the analytical conditions that were used. The SPR-Pb was used as the analytical column.
Fig. 3 shows the calibration curves generated using the analytical conditions of Table 2. When generating the curves for the six components over a concentration range of 0.2 to 10 g/L (using the average of three area values, respectively), excellent linearity with a coefficient of determination greater than R2= 0.9999 was obtained for each component.
Analysis of Energy Drink
Figs. 4 and 5 show the chromatograms obtained from measurement of energy drinks A and B, respectively. Energy drink A was diluted 10 : 1 with water, and energy B, 20 : 1 with water, and after each was filtered through a 0.2 µm membrane filter, 10 µL of each sample was injected. The analytical conditions were the same as those of Table 2.
Xylitol and sorbitol were detected in energy drink A, and glucose and fructose were detected in energy drink B. Table 3 shows the quantities of each of these sugars in the respective energy drinks.
Reprint of Shimadzu Application News No. L481, January 2015