Fourier transform Raman and infrared spectroscopy of pectins

Abstract

The FT-Raman and FT-IR spectra of polygalacturonic (pectic) acid, potassium pectate and its derivatives, as well as commercial citrus and sugar beet pectins were measured and interpreted. Methyl and acetyl esters of potassium pectate derivatives have several characteristic Raman and IR bands that allow both this groups to be distinguished. The very intense Raman band at 857 cm⁻¹ is sensitive to the state of uronic carboxyls and to O-acetylation. The wavenumber of this band decreases with methylation (min. 850 cm⁻¹) and increases with acetylation (max. 862 cm⁻¹). The acetylation of potassium pectate, as well as its acetylation together with methylation, causes drastic changes in the Raman spectra in the region below 700 cm⁻¹. Sugar beet pectin, but not citrus pectin, showed Raman bands at 1633 and 1602 cm⁻¹ and IR band at 1518 cm⁻¹. All these bands rise from feruloyl groups and can be used for identification of pectins containing feruloyl groups.

Introduction

Pectins are widespread plant polysaccharides that are commonly used in the food industry as gelling and stabilising agents (Thakur et al., 1997, BeMiller, 1986, Van Buren, 1991). Basically, pectins are polymers of (1→4) linked partially methyl esterified α-d-galacturonic acid. Pectins with more than 50% methyl ester groups are classified as high-methoxyl (HM) and those with less than 50% methyl ester groups as low-methoxyl (LM).

Natural pectins are not simple galacturonans. Particularly, l-rhamnose residues may interrupt the galacturonic acid backbone of pectins forming rhamnogalacturonan. Several pectins are also partially O-2 and O-3 acetylated and may contain neutral sugar side chains attached to rhamnose units (Thakur et al., 1997, Thibault, 1986). In some cases, feruloyl groups are linked to neutral sugar side chains by ester bonds (Ralet et al., 1994, Colquhoun et al., 1994, Rombouts and Thibault, 1986, Thibault, 1986).

The structure of pectins influences their physical–chemical properties significantly as well as their applications in food technology. Among various analytical methods, mid-infrared FT-IR spectroscopy is an excellent tool for structural and quantitative analysis of polysaccharides, including pectins (Kačuráková & Wilson, 2001).

FT-IR spectra of natural pectins and their derivatives have been recorded for KBr discs (Coimbra et al., 1998, Kamnev et al., 1998, Manrique and Lajolo, 2002), in emulsions with nujol (Kamnev et al., 1998), for films (Filippov, 1978, Filippov, 1992), and powders (Engelsen and Nørgaard, 1996a, Engelsen and Nørgaard, 1996b), D₂O-phosphate buffer solutions (Bociek & Welti, 1975), water solutions and gels (Wellner, Kačuráková, Malovı́ková, Wilson, & Belton, 1998).

FT-IR methods for determining the content of different groups, such as free carboxyls, carboxylate anions, methyl esters, acetyls and amides, in pectin solutions (Bociek & Welti, 1975) and films (Filippov, 1984, Filippov and Kohn, 1986, Filippov and Vaskan, 1987a, Filippov and Vaskan, 1987b) have been presented. Coimbra et al. (1998) has studied uronic acid and neutral sugars in pectic samples using FT-IR and multivariate analysis. The degree of methylesterification of pectins have been estimated by FT-IR using second derivative algorithm and peak decomposition (Chatjigakis et al., 1998) as well as using an outer product PLS1 regression (Barros et al., 2002).

Diffuse reflectance FT-IR has obvious advantages in the analysis of pectin derivatives compared with other techniques. This infrared technique has been successfully applied to record and analyse spectra of dried powder pectins for the quality control of commercial samples (Engelsen and Nørgaard, 1996a, Engelsen and Nørgaard, 1996b), and for the determination of the polygalacturonic acid content in pectin extracts and the degree of esterification of pectin (Chatjigakis et al., 1998, Gnanasambandam and Proctor, 2000, Monsoor et al., 2001a, Monsoor et al., 2001b). Diffuse reflectance FT-IR is especially attractive since commercial pectins and their derivatives are slightly soluble or insoluble in common solvents used in FT-IR. For example, hydrophobically modified amidated derivatives of HM citrus pectin (CP) have been prepared and analyzed by diffuse reflectance FT-IR spectroscopy (Sinitsya, Čopı́ková, Prutyanov, Skoblya, & Machovič, 2000).

In contrast, Raman spectroscopy is not a widespread method for the analysis of pectins, but it is widely applied in structural investigations of polysaccharides. Raman spectra of amylose (Cael et al., 1973, Cael et al., 1975a), cellulose (Cael, Koening, & Blackwell, 1975b), carrageenans (Malfait et al., 1987, Sekkal and Legrand, 1993), chitin (Gałat & Popowicz, 1978), and other polysaccharides (Cabassi et al., 1978, Kačuráková et al., 1999, Zhbankov et al., 2000, Gałat, 1980) have been interpreted and used in structural analysis of these polysaccharides. Engelsen et al. (1996a,b) compared FT-Raman spectra of amidated pectins with FT-NIR and FT-IR data for the determination of quality parameters. The assignment of Raman and infrared bands has been carried out in this paper.

It is evident that infrared and Raman spectroscopic methods are complementary for the structural analysis of carbohydrates (Zhbankov, Andrianov, Ratajchak, & Markhevka, 1995). Some vibration bands that are very weak or overlapped by stronger bands and cannot be detectable in IR can be indicated and studied by Raman spectroscopy and vice versa. Both these methods are rapid, non-destructive and do not need complex procedures for sample preparation.

In this work we describe the complementary use of IR and Raman spectroscopy for the analysis of pectins and their derivatives. FT-Raman and diffuse reflectance FT-IR spectra of citrus and sugar beet pectins as well as their acetylated, amidated and additionally methyl esterified derivatives were measured and the spectral changes were interpreted in connection with the state of pectic C-6 carboxyls and the degrees of methylation and acetylation.