Analysis of Sugars and Sugar Alcohols in Energy Drink by Prominence-I with Differential Refreactive Index Detector

LAAN-A-LC-E258 Application High Performance Liquid Chromatography News Analysis of Sugars and Sugar Alcohols in Energy Drink by Prominence-i with Differential Refractive Index Detector No.L481 Sugars and sugar alcohols display almost no ultraviolet absorption, and are therefore typically detected using a differential refractive index detector or evaporative light uRI scattering detector. By using a ligand exchange column for 80 ■ Peaks sugar analysis, it is possible to distinguish among the different 1. maltose 70 2. glucose isomers based on the position of the hydroxyl group in the 1 4 3. fructose chair conformation of glucose and fructose for example. In 4. erythritol 60 5 other words, the hydroxyl group of the sugar and the metal 5. mannitol ion of the stationary phase form a complex, making it 2 3 6. sorbitol possible to achieve separation due to the difference in the 50 6 strength of the complex formation. Also, maintaining a 40 column temperature of 80 °C suppresses sugar anomer separation and peak dispersion, thereby achieving good 30 separation of adjacent peaks. The new Prominence-i integrated high-performance liquid 20 chromatograph can be connected to the RID-20A differential refractive index detector. The column oven, which can 10 accommodate a 30 cm column and maintain temperature 0 control up to 85 °C, therefore supports applications that require a long column. In Application News No. 467, we introduced an example of 0 5 10 15 20 25 min 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 Fig. 1 Chromatogram of a Standard Mixture of Six Sugars sugar alcohols in an energy drink using the Prominence-i and (10 g/L each, 10 µL Injected) RID-20A. n Analysis of a Standard Mixture of Six Sugars Sorbitol, xylitol, mannitol and erythritol are a type of sugar uRI alcohol that because of their relative sweetness, are used as 80 sweeteners. When conducting simultaneous analysis of sugars ■ Peaks and sugar alcohols, a hydrophilic compound analytical column, 70 1. maltose such as the SPR-Ca or SPR-Pb, is suitable along with the use of 2. glucose 2 3. fructose 60 1 a combination of the size exclusion and ligand exchange 4. mannitol modes of analysis. Fig. 1 shows the results of analysis of a 5. xylitol 3 standard solution of six sugar alcohol substances (10 g/L each 50 6. sorbitol of maltose, glucose, fructose, erythritol, mannitol and sorbitol) using the SPR-Ca column with a 10 µL injection. The analytical 40 4 conditions are shown in Table 1. 5 Fig. 2 shows the results of analysis of a standard solution of six 30 6 sugar substances including sugar alcohols (10 g/L each of maltose, glucose, fructose, mannitol, xylitol, sorbitol) using a 20 10 µL injection, and Table 2 shows the analytical conditions that were used. The SPR-Pb was used as the analytical column. 10 Table 1 Analytical Conditions 0 Column : Shim-pack SPR-Ca (250 mm L × 7.8 mm I.D., 8 µm) Mobile Phase : Water Flowrate : 0.6 mL/min 0 10 20 30 40 min Column Temp. : 80 °C Injection Volume : 10 µL Detection : RID-20A Polarity +, Cell temp. 40 °C, Response 1.5 sec Fig. 2 Chromatogram of a Standard Mixture of Six Sugars (10 g/L each, 10 µL Injected) Table 2 Analytical Conditions Column : Shim-pack SPR-Pb (250 mm L × 7.8 mm I.D., 8 µm) Mobile Phase : Water Flowrate : 0.6 mL/min Column Temp. : 80 °C Injection Volume : 10 µL Detection : RID-20A Polarity +, Cell temp. 40 °C, Response 1.5 sec Application No.L481 News n Linearity Table 3 Content of Respective Sugars in Energy Drinks Fig. 3 shows the calibration curves generated using the Content (g/L) analytical conditions of Table 2. When generating the Energy Drink A Energy Drink B curves for the six components over a concentration Glucose ND 59 range of 0.2 to 10 g/L (using the average of three area Fructose ND 101 values, respectively), excellent linearity with a coefficient Xylitol 25 ND 2 of determination greater than R =0.9999 was obtained Sorbitol 14 ND for each component. Area (×104) Area (×104) 150 150 maltose glucose uRI 10 100 100 ■ Peaks 5. xylitol 6. sorbitol 8 50 50 5 R² = 0.9999981 R² = 0.9999992 0 0 0 5 10 0 5 10 6 Concentration (mg/L) Concentration (mg/L) Area (×104) Area (×104) 150 150 4 6 fructose mannitol 100 100 2 50 50 0 R² = 0.9999976 R² = 0.9999975 0 0 0 5 10 0 5 10 Concentration (mg/L) Concentration (mg/L) 0 10 20 30 40 min Area (×104) Area (×104) 150 150 xylitol sorbitol Fig. 4 Chromatogram of Energy Drink A (10 µL Injected) 100 100 50 50 R² = 0.9999982 R² = 0.9999967 0 0 uRI 0 5 10 0 5 10 ■ Peaks Concentration (mg/L) Concentration (mg/L) 2. glucose 30 3. fructose Fig. 3 Calibration Curves of a Standard Mixture of Six Sugars (0.2 – 10 g/L, 10 µL Injected) 3 20 2 n Analysis of Energy Drink Figs. 4 and 5 show the chromatograms obtained from measurement of energy drinks A and B, respectively. 10 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 0 those of Table 2. Xylitol and sorbitol were detected in energy drink A, 0 10 20 30 40 min 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. Fig. 5 Chromatogram of Energy Drink B (10 µL Injected) First Edition: Jan. 2015 For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2015.

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