**7. Determination of monosaccharide composition by GC**

Gas-liquid chromatography (GC) is widely used for the analysis of carbohydrate. GC is used in the separation and analysis of complex mixtures of many components that can be vaporized without decomposition. For monosaccharides released after total hydrolysis, derivatization is necessary due to polar groups of carbohydrates which make them nonvolatile. Derivatization methods consist of the substitution of the polar groups of monosaccharides in order to increase their volatility. Many types of derivatives can be employed in the analysis of monosaccharide composition by GC, acetylated and silylated derivatives being the most popular. The advantages of acetylated derivatives include the presence of a single peak for each derivatized monosaccharide and their high stability. The derivatization reaction involves reduction with sodium borohydride followed by the acetylation itself. However, in the acetylation only the hydroxyl groups are derivatized. Complete quantification and identification of individual neutral and acidic sugars using acetylated derivatives can be accomplished by an additional step, which includes the carbodiimide-activated reduction of the carboxyl groups of uronic acids in order to give the corresponding neutral sugars [43]. Alternatively, GC can be used just to quantify the neutral monosaccharides, while titration or colorimetric method can be used to evaluate the amount of acidic units. This approach was chosen to study the composition of pectins from the dried pomace of eleven apple cultivars (Table 1) [41].

In GC analysis, compounds that have similar properties often have the same retention times. Sometimes extraneous background peaks can be a problem for the identification and quantification of monosaccharides present in minor amounts in complex mixtures. Gas chromatography associated to mass spectroscopy (GC-MS) can be used to overcome this difficulty. GC-MS combines two techniques to form a single method of analyzing mixtures of chemicals. For a pectic polymer, gas chromatography separates the monosaccharides derivatives present in the mixture and mass spectroscopy characterizes each of the derivatives individually by their mass fragments. MS has the advantages of high selectivity, specificity, and sensitivity 43.


Characterization of Apple Pectin – A Chromatographic Approach 337

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].

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

**9. The use of chromatographic techniques for determining DA and DE** 

DE is usually determined, together with galacturonic acid content, by titration. However, the presence of acetyl can contribute to an overestimation of DE, since the acetic acid released during the saponification will be titrated. Chromatographic methods can be used for determination of DE and DA. An HPLC method for simultaneous determination of DE and DA was proposed by Voragen et al (1986) [47] and modified by Levigne et al (2002) [31]. In the former, an Aminex HPX-87H column is used while the second method uses a C18 column. The pectin is saponified and precipitated followed by methanol and acetic acid separation from the supernatant by HPLC and quantification by refractometry using an internal standard. The method allows accurate determination in a single run and within a short time, the DE and DA using low amounts of pectins (~ 5mg). This method presents some advantages when compared to that using FT-IR for DE calculation. FT-IR method requires pectins standards with different DEs in order to construct a calibration curve and

Huisman et al. (2004) [48] proposed a method for DE determination using head-space GC. The approach is similar to that used in the HPLC method. Head-space GC is used for the quantification of methanol released from pectin by saponification. A lower amount of pectin is needed (~2mg) and the chromatogram shows a symmetrically shaped methanol peak which is very easy to integrate. However, the method is only applicable for the

Chemical and enzymatic hydrolysis has been used to produce fragments of pectic polysaccharides in order to study the fine structure of pectins. The differences in the lability of glycosidic linkages to acid hydrolysis (see section 3.2) allowed the homogalacturonan region to

determination of DE and another approach has to be used in order to estimate DA.

**10. Chromatographic techniques as an aid in the study of the fine** 

same conditions, the time of elution increases with the value of p*Ka* [46].

the presence of acetyl can contribute to an overestimation of the DE.

**structure of pectic polysaccharides** 

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

**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 (AUA) were determined by titration.

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 uronic acid as its corresponding neutral sugar [43].

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