**8. Determination of monosaccharide composition by HPLC and HPAEC-PAD**

High performance liquid chromatography (HPLC) is characterized by using high pressure to force chemical compounds to pass the column containing a stationary phase. Since monosaccharides released after total hydrolysis do not present enough volatility to be analyzed by GC, HPLC should be the most useful technique for the determination of monosaccharide composition. Monosaccharides cannot be detected by absorption due to the lack of chromophore in the molecular structure and are usually detected by refraction index detector. However, refraction index detector is sensitive to eluent composition and sample matrix, making the application of the gradient elution method limited and not allowing the complete separation of all monosaccharides [43].

Acid-catalysed or enzymatic hydrolysis followed by HPLC analysis to determine the GalA content in pectins has been proposed by different authors. When enzymes that release and specifically degrade pectins are used, the method can be useful for analysis of GalA in pectin samples as well as in the raw material that can be used for pectin extraction [45].

336 Chromatography – The Most Versatile Method of Chemical Analysis

**Table 1.** Monosaccharide composition of pectins from the dried pomace of eleven apple cultivars. Neutral sugars were determined as alditol acetates by GC using a DB-210 capillary column (0.25 mm internal diameter x 30 m), film thickness 0.25 μm and flame ionization detector. Contents of uronic acids

When carboxyl-reduction of glycosyluronic acid is performed the use of sodium borodeuteride (NaBD4) and GC-MS is preferred. The use of NaBD4 provides an easily identified tag by MS analysis that allows the quantitative determination of the content of

GC-MS has also been used to identify and quantify unusual sugars present in RG-II. This approach is very useful, since commercial standards are not available for all unusual monosaccharides present in RG-II. The contents of DHA, KDO, aceric acid, 2-methyl-xylose and 2-methyl-fucose have been determined by GC-MS of their trimethylsilyl-esters *O*-

**8. Determination of monosaccharide composition by HPLC and HPAEC-**

High performance liquid chromatography (HPLC) is characterized by using high pressure to force chemical compounds to pass the column containing a stationary phase. Since monosaccharides released after total hydrolysis do not present enough volatility to be analyzed by GC, HPLC should be the most useful technique for the determination of monosaccharide composition. Monosaccharides cannot be detected by absorption due to the lack of chromophore in the molecular structure and are usually detected by refraction index detector. However, refraction index detector is sensitive to eluent composition and sample matrix, making the application of the gradient elution method limited and not allowing the

Source: Sato et al., (2011)[41].

**PAD** 

(AUA) were determined by titration.

uronic acid as its corresponding neutral sugar [43].

complete separation of all monosaccharides [43].

methyl glycosides after acidic methanolysis and derivatization [43; 44].

In recent years, high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) had been used to directly analyze the contents of carbohydrates. The development of this new technology had readily solved the abovementioned problems. HPAEC-PAD can be used for simultaneous determination of monosaccharides and uronic acids. The method had been applied for the separation of GalA, GlcA, Rha, Fuc, Ara, Xyl, Man, Gal and Glc. Monosaccharides are weak acids with p*Ka* above 11. HPAEC uses NaOH as an eluent making sugars in their anionic form. In general, the elution sequence of monosaccharide is related to their p*Ka* value, and in the same conditions, the time of elution increases with the value of p*Ka* [46].
