**5. Determination of molar mass by HPSEC technique with MALLS detector and RI**

Size exclusion chromatography (also called gel permeation chromatography) has been widely used to analyze the molar mass distribution of polymers. The eluted fractions can be examined by different detection techniques such as: colorimetric methods, refractive index, ultraviolet, light scattering and viscosity measurements.

Size exclusion chromatography has been used to determine the molar mass of polysaccharides. In many instances, a calibration curve, previously obtained by using dextran molar mass standards, is used for this purpose. However, in size exclusion chromatography, molecules in solution are separated by their size rather than by their molar mass. Since the size is related to the conformation of the polysaccharide in a given solvent, the use of dextrans as molar mass standards is highly restricted. Experimentally, a problem in this approach is the absence of commercial molar mass standards for the different types of polysaccharides, including pectins.

The use of high performance size exclusion chromatography coupled to a differential refractometer (RI) and a multi-angle laser light scattering (MALLS) detector allows the study of the molar mass distribution of pectins as well as the determination of the absolute molar mass. While RI gives a signal proportional to the concentration, MALLS response increases with the concentration and molar mass. The MALLS instrument enables the determination of absolute molar masses of polymers from below 1000 g/mol to hundreds of millions without a calibration curve with reference standards. The molar mass is estimated directly from the angular dependence of scattered light intensity as a function of concentration, as formulated by light scattering theory. However, the molar mass calculation is only possible when the eluted fraction is detected by both RI and MALLS. An accurate determination of the refractive index increment with concentration, dn/dc, is required [37; 38]. Figure 1 displays a unimodal distribution of molar mass for a polysaccharide with *M*W 1.39 × 106 g/mol, determined by HPSEC-MALLS.

In addition to the average molar mass from HPSEC analyses coupled to light scattering detection it is also possible to obtain: a) the ratio *M*W/*M*N which is used as an index of polydispersity; b) the radius of gyration; c) the differential molar mass distribution plot which shows how much material (differential weight fraction) is; distribution for the same polysaccharide extracted using three different times of; extraction); d) the cumulative distribution of molar mass curve which gives for each molar mass, the weight fraction of material having molar mass less than the given molar mass (Figure 3 shows the cumulative distribution of molar mass for a same polysaccharide extracted using two different times of extraction); e) the log-log plot of root mean square radius of gyration as a function of molar 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 determined [40].

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

332 Chromatography – The Most Versatile Method of Chemical Analysis

ultraviolet, light scattering and viscosity measurements.

and molar mass of pectins [36].

of polysaccharides, including pectins.

**detector and RI** 

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

Size exclusion chromatography (also called gel permeation chromatography) has been widely used to analyze the molar mass distribution of polymers. The eluted fractions can be examined by different detection techniques such as: colorimetric methods, refractive index,

Size exclusion chromatography has been used to determine the molar mass of polysaccharides. In many instances, a calibration curve, previously obtained by using dextran molar mass standards, is used for this purpose. However, in size exclusion chromatography, molecules in solution are separated by their size rather than by their molar mass. Since the size is related to the conformation of the polysaccharide in a given solvent, the use of dextrans as molar mass standards is highly restricted. Experimentally, a problem in this approach is the absence of commercial molar mass standards for the different types

The use of high performance size exclusion chromatography coupled to a differential refractometer (RI) and a multi-angle laser light scattering (MALLS) detector allows the study of the molar mass distribution of pectins as well as the determination of the absolute molar mass. While RI gives a signal proportional to the concentration, MALLS response increases with the concentration and molar mass. The MALLS instrument enables the determination of absolute molar masses of polymers from below 1000 g/mol to hundreds of millions without a calibration curve with reference standards. The molar mass is estimated directly from the angular dependence of scattered light intensity as a function of concentration, as formulated by light scattering theory. However, the molar mass calculation is only possible when the eluted fraction is detected by both RI and MALLS. An accurate determination of the refractive index increment with concentration, dn/dc, is required [37; 38]. Figure 1 displays a unimodal distribution of molar mass for a

In addition to the average molar mass from HPSEC analyses coupled to light scattering detection it is also possible to obtain: a) the ratio *M*W/*M*N which is used as an index of polydispersity; b) the radius of gyration; c) the differential molar mass distribution plot which shows how much material (differential weight fraction) is; distribution for the same polysaccharide extracted using three different times of; extraction); d) the cumulative distribution of molar mass curve which gives for each molar mass, the weight fraction of material having molar mass less than the given molar mass (Figure 3 shows the cumulative distribution of molar mass for a same polysaccharide extracted using two different times of extraction); e) the log-log plot of root mean square radius of gyration as a function of molar

polysaccharide with *M*W 1.39 × 106 g/mol, determined by HPSEC-MALLS.

**5. Determination of molar mass by HPSEC technique with MALLS** 

A comparison between MALLS and LALLS detector in HPSEC has been shown to give results in very good agreement [40]

**Figure 2.** Differential molar mass distribution for a polysaccharide obtained after 15 min, 15 h, and 48 h of extraction [39].

HPSEC-MALLS/RI was used to compare the molar mass distribution of pectins obtained from the dried pomace of eleven apple cultivars [41]. Although the pectins were obtained under the same extraction conditions, some differences were identified among the fractions. According to the results, pectins from different variety of apple can display differences in their molar mass profiles as depicted in Figure 4 (for clarity only RI detector is shown).

Characterization of Apple Pectin – A Chromatographic Approach 335

Information on the composition of pectins can be obtained by different chromatographic methods. Regardless of the method used to determine the monosaccharide composition, the pectin under study needs to be previously hydrolyzed in order to release its monomeric units. It is well known that the glycosidic linkages with acidic monosaccharides are more resistant to hydrolysis. For pectins, the different sensitivities to acid hydrolysis are: GalA-GalA > GalA-Rha > Rha-GalA > neutral sugar-neutral sugar 42. The chosen conditions must

Although the more simple chromatographic methods, such as paper chromatography (PC) and thin layer chromatography (TLC), can give only qualitative information about the composition of pectins, they can be useful to check the adequacy of the conditions of

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

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,

be a compromise between maximum hydrolysis with minimal degradation.

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

pomace of eleven apple cultivars (Table 1) [41].

specificity, and sensitivity 43.

**6. Monosaccharide composition** 

hydrolysis.

**Figure 3.** Cumulative molar mass distribution for a polysaccharide obtained after 15 min and 48 h of extraction [39].

**Figure 4.** HPSEC elution profiles of pectins obtained from the dried pomace of eleven apple cultivars (columns: Waters Ultrahydrogel 2000/500/250/120 connected in series; eluent: 0.1M NaNO2 solution, containing NaN3 (0.5 g/l); flow: 1.5 mg/ml) [41].
