Methods for Separating Sugars
Detection Methods (2)
Sugar separation methods were discussed in LCtalk 49 and detection methods were discussed in LCtalk 50. In response to a request from a reader, this page describes the post-column fluorescence detection method in more detail. Therefore, we will continue our discussion of detection methods by specifically focusing on the post-column fluorescence detection method, using arginine as the reagent.
When Is the Post-Column Fluorescence Method Necessary?
As mentioned in LCtalk 50, there are several choices for detecting sugars. In very approximate terms, if the sample concentration is sufficient to taste sweet, consider a refractive index detector as the first candidate. Refractive index detectors are used widely in the food industry.
However, even though they are foods, what about brewed foods like soy sauce?
The typical sugar in soy sauce is glucose, which can be detected using a refractive index detector, but presumably those analyzing soy sauce are more interested in the other non-glucose sugars (such as ribose, mannose, arbinose, galactose, and xylose. If so, then it can no longer be handled with a refractive index detector. In terms of sensitivity, an electrochemical detector could be considered, but soy sauce contains large amounts of amino acids, which could interfere with analysis. In such cases, the post-column fluorescence method is required.
What Is the Post-Column Method Using Arginine Reagent?
Shimadzu discovered that reducing sugar generates strongly fluorescent derivatives (excitation wavelength 340 nm and fluorescence wavelength 430 nm) when reacted with arginine, a basic amino acid, in the presence of boric acid in a thermal reaction (150 °C)1). Consequently, Shimadzu built an HPLC system with post-column fluorescence detection based on application of this reaction process. Figure 1 shows a flow line diagram of the system.
This method can be used with each method for separating sugars described in LCtalk 49, but it is most effective when combined with the borate complex anion exchange method. The borate complex anion exchange method offers superior separation of disaccharides from monosaccharides. To improve separation efficiency, however, gradient elution is required, which precludes using a refractive index detector. This post-column fluorescence detection using arginine reagent combined with the borate complex anion exchange method is currently still widely used.
What Are the Advantages of the Arginine Reagent?
There are several possible ways to perform post-column fluorescence detection of reduced sugar, but the biggest advantages of using arginine reagent are no doubt its easy availability, reasonable price, and superior safety.
Furthermore, the arginine method enables detection of sucrose, which is a non-reducing disaccharide. Though the detection sensitivity is only one-tenth compared to the reducing disaccharide, maltose, the fact that it can detect sucrose is a significant advantage.
More About Arginine Reagent
Later, Shimadzu studied how to add the arginine reagent to the mobile phase in advance. This method enabled increasing the reaction efficiency because it did not require a mixer or reaction reagent delivery pump,.. We also thought it would allow simplifying the instrument.
Based on our study results, we determined that arginine in the mobile phase had no effect on sugar separation using the borate complex anion exchange method and reacted efficiently when heated to 150 °C in the reaction coil.
What About Analyzing Soy Sauce?
Finally, returning to our previous question about analyzing sugars in soy sauce, let's look at the analytical results using this system. Figure 2 shows the analytical results for a commercially-marketed soy sauce product. It shows that trace quantities of non-glucose sugars are detected.
1) H.Mikami and Y.Ishida: Bunseki Kagaku, 32, E207 (1983).
2) H.Mikami and Y.Ishida: 10th International Symposium on Column Liquid Chromatography (1986).