**4. The application of chromatographic techniques**

The properties and application of pectin are affected by several parameters related to pectin structure, including the composition, presence and distribution of side chains, degree of methyl-esterification (DE), degree of acetylation (DA), molar mass, and the charge distribution along their backbone. Differently from proteins, whose structure is defined in relation to a template, pectin are a mixture of heterogeneous polymers and preparations that contain pectins that have been isolated by chemical and enzymatic treatments likely to cleave covalent bonds. The conditions used for pectin extraction can also release other cell wall polymers. Preparative chromatography can be used to separate homogeneous fractions of pectic polysaccharides from a mixture.

Determining the fine structure of pectins is a challenging task which encompasses the determination of molar mass, monosaccharide composition, configuration and ring size of monosacchrides, identification of glycosidic linkages, sequence of glycosyl units in the polymer, determination of DA and DE, as well as the distribution of the acetyl and methyl esters groups along the chain. A combination of chemical, spectroscopic and chromatographic methods is commonly used to fully characterize pectic polysaccharides. In recent years, X-ray diffraction, circular dichroism, light scattering, electron and atomic force

microscopy and theoretical approaches has also been used in order to examine the fine structure and interactions of pectins. Although chromatography is primarily a separation technique, chromatography techniques are powerful tools to study the composition, DE, DA and molar mass of pectins [36].

Characterization of Apple Pectin – A Chromatographic Approach 333

mass which gives information about the molecular conformation. Theoretical slopes of 0.33, 0.50 and 1.0 occur for spheres, random coils in theta solvents, and rigid rods, respectively. Usually, most real random coils have slopes in the range 0.55-0.6 [37]; f) using a viscometer connected in series with MALLS and RI in the HPSEC system, the intrinsic viscosity can be

**Figure 1.** HPSEC elution profile of a polysaccharide isolated from seed [39].

results in very good agreement [40]

of extraction [39].

A comparison between MALLS and LALLS detector in HPSEC has been shown to give

**Figure 2.** Differential molar mass distribution for a polysaccharide obtained after 15 min, 15 h, and 48 h

determined [40].
