Mechanisms of polymer ca2+ interaction and their effects on the characteristics of alginate microspheres and films 3

Pectin and ι-carrageenan showed good miscibility with sodium alginate as shown by the high light transmittance values of uncross-linked AP and AI films Table 19.. This was reflected by d

Part III Influence of incorporating other polymers on the properties of the

alginate matrix

A Light transmittance, thickness and percent weight loss of films and FTIR spectroscopy

The copolymers chosen in this study are natural gums that have a long history

of applications in the food industry These gums are safe, biocompatible and biodegradable Their influence on the properties of the alginate matrix was investigated

Light transmittance measurements are often used as an empirical method for the determination of phase mixing in polymer composite materials (Krause, 1972) Composite materials which are not homogeneous i.e the individual polymers are not completely miscible, will reduce light transmittance because of the quality of scattered and reflected light This will result in lower transmittance values of the composite materials than those obtained from individual polymers (Zhou and Zhang, 2001)

Light transmittance through a film may also be affected by its thickness Preliminary studies using SA films showed insignificant difference in the percent transmittance values obtained if the film thickness varied within 0.0389 to 0.0578 mm (Table 18) Hence, this film thickness range was used for all test films in the subsequent measurement of light transmittance in all studies

Pectin and ι-carrageenan showed good miscibility with sodium alginate as shown by the high light transmittance values of uncross-linked AP and AI films

(Table 19) Yoshida et al (2001) found that the miscibility of polymers depended

strongly on polymer-polymer interactions, such that a slight difference in the conformation of the polymer chain might affect the miscibility of the polymers with

Table 18 The influence of thickness of SA films on light transmittance

0.0380 87.0 0.0396 86.7 0.0488 85.5 0.0526 84.8 0.0578 84.0

SA films were composed of sodium alginate (Manucol ® DH, uncross-linked)

*Only 1 determination of light transmittance was carried out for the films of different thickness

one another This was especially evident with the addition of κ-carrageenan to alginate κ-carrageenan is structurally similar to ι-carrageenan with the exception of having only 1 sulphate group (Figure 3) but the transmittance of uncross-linked AK was significantly lower than that of uncross-linked AI The addition of κ-carrageenan and gellan gum to sodium alginate gave rise to lower light transmittance values indicating that the polymers were not completely miscible (Table 19)

Thickness is an important film property as it affects the tensile strength and permeability of films The degree of cross-linking might also be reflected to a certain extent by the percent change in film thickness after cross-linking The changes in film thickness after cross-linking are shown in Figure 22a Although the amount of film former (sodium alginate with or without copolymer) used was constant, significant differences in film thickness were observed (p<0.05) This could be due to the different extent of interaction between the polymer chains (Table 20)

The thickness of SA film was greatly reduced after cross-linking with Ca2+ but less change in film weight was observed (Figure 22) Stochiometrically, one mole of calcium ion (atomic weight ≈ 40 g) displaces two moles of sodium ions (total atomic weight ≈ 46 g) As there is little difference between the corresponding weights of the cations exchanged, the weights of the film before and after cross-linking were expected to be comparable Any significant change in weight would imply a loss in polymer content through dissolution during the cross-linking process Hence, the minimal change in weight of SA film after cross-linking showed insignificant polymer loss during the cross-linking of sodium alginate Decrease in the film thickness could be due to the divalent calcium ions interacting with two binding sites

on different polymer chains, bringing the polymer chains closer together

Table 19 Light transmittance of films

Transmittance (%) Film code

Uncross-linked films Cross-linked films

(a)

(b)

Copolymer

Copolymer

Table 20 Thickness of films before and after cross-linking

Film thickness (mm) Film code

Before cross-linking After cross-linking

FTIR spectroscopy revealed interactions between Ca2+ and the free carboxyl groups in alginate The strong band due to a C=O stretch of the carboxylic acid group

at 1737cm-1 in alginic acid disappeared after cross-linking (Figure 23) This band was also absent in both uncross-linked and cross-linked SA films Furthermore, bands between 1600-1615 cm-1 and 1429-1417 cm-1 became more prominent These bands were attributed to the COO- asymmetric and symmetric stretches in the cross-linked alginates A slight shift to lower wavenumber for asymmetric COO- stretch and to higher wavenumber for symmetric COO- stretch after cross-linking indicated an exchange of Na+ for Ca2+

With the exception of AG 70/30 films, significant decrease in thickness of composite films containing at least 30 % of copolymer was observed after cross-linking with Ca2+ (p<0.05) Significant decrease in weight of composite films was

also observed It has been shown that sodium alginate did not undergo polymer

dissolution during cross-linking Hence, the decrease in the film weight was most likely due to the dissolution of the copolymer Greater % decrease in thickness of composite films and film weight were generally observed with increased proportion

of copolymer incorporated (Figure 22) The percent weight loss after cross-linking generally increased in the order of GG < KC < IC< PT A linear relationship (r2 = 0.984) between % decrease in thickness and % change in film weight was observed for KC, IC and PT and GG films This implied that the loss of copolymer during the cross-linking process had a considerable effect on film thickness Pectin dissolved readily in water at room temperature while carrageenans and gellan gum were less soluble This might contribute to the apparent greater % weight loss observed in PT and AP films after cross-linking as opposed to the other films The presence of an additional sulphate group in ι-carrageenan (Figure 3) makes it more hydrophilic and

soluble in water than κ-carrageenan (Imeson, 2000) This contributed to the greater loss of ι-carrageenan in the cross-linking process, compared to κ-carrageenan

Interestingly, the SA film did not exhibit significant weight change after linking (p>0.05) even though sodium alginate dissolved easily in water It could be inferred that the rate of interaction between sodium alginate and the Ca2+ was very rapid and the affinity of alginate for Ca2+ was much greater than the other copolymers, thus forming insoluble calcium alginate immediately upon contact with Ca2+ This was supported by a higher Ca2+ content obtained in cross-linked alginate films as compared to the other cross-linked films (Figure 24)

cross-The percent reduction in thickness of the composite films after cross-linking was generally markedly higher than the percent reduction in weight (Figure 22) Therefore, the loss in polymer contents could not account for the reduction in film thickness totally Interaction between Ca2+ and the remaining polymer also contributed to the reduction in film thickness The different Ca2+ contents of the films composed of a single polymer revealed that there might be varying affinity of the polymers for Ca2+ thus introducing the potential for varying degrees of cross-linking

in the film matrix

The interaction between pectin and Ca2+ occurs mainly through the carboxyl groups in pectin The strong band at 1743.3 cm-1 in pectinic acid is due to the C=O vibration of the methyl ester and free carboxyl groups (Figure 25) The decreased intensity of this strong band with increased prominence of bands between 1680-1670

cm-1 and 1417-1426 cm-1 were associated with asymmetric and symmetric COO- stretching vibrations of the carboxyl groups and they provided strong evidence of cation-carboxyl interaction Cross-linking of sodium pectinate with Ca2+ resulted in a slight decrease in wavenumber of the asymmetric COO- stretching band and an

Figure 24 Calcium content of polymeric films after cross-linking

increase in wavenumber of the symmetric COO- stretching band (Figure 25) Pectin creates junction zones by ordered side-by-side associations of the galacturonans, whereby specific sequences of galacturonic acid monomers in parallel or adjacent chains are linked intermolecularly through electrostatic and ionic bonding of carboxyl groups forming an ‘egg box’ structure similar to that proposed for the alginates (Grant

et al.,1973)

The formation of the ‘egg box’ structure between polymer chains of the same type enables close packing of the dimers formed A film with closely packed polymer chains would be expected to exhibit a lower light transmittance because of its greater ability to block light This was reflected by decreased film thickness and lower light transmittance of SA and PT films after cross-linking (Table 19 and Figure 22)

The % decrease in SA film thickness after cross-linking was >16 % and that of

PT films is >30 % However, the decrease in film thickness of AP 90/10 and AP 70/30 after cross-linking was less than 15 % despite having a high proportion of alginate It was also observed that for AP 50/50, the decrease in film thickness after cross-linking is only 18 % although PT films showed significant film thickness change (Figure 22a) The methoxy and amide groups scattered along the pectin chains

do not interact with Ca2+ Their presence could hinder the formation of the ‘egg box’ structure with alginate Therefore interactions between alginate and pectin are likely

to occur along some segments of the polymer chains, resulting in the composite film having a more loosely packed structure than the SA and PT films (Figure 26) The interaction between the polymers and Ca2+ were evident from FTIR studies (Figure 25) In uncross-linked AP films, bands at 1147.4 cm-1 and 1101.2 cm-1, which were due to C-O-C vibration of the gylcosidic linkage/ring and ring associated vibrations

Figure 26 Schematic representation of possible interactions between pectin and alginate with calcium ions ({)

Pectin

Sodium alginate

Region with methoxy groups

CO, CC, CCH, OCH) (Wellner et al., 1998) of pectin, were clearly observed (Figure

25)

However, these bands were either absent (band 1147.4 cm-1) or shifted to a lower wavenumber (band 1102.2 cm-1) in the cross-linked AP films In addition, 2 bands at 1126.2 cm-1(C-C stretch and C-O stretch) and 1097.3 cm-1 (C-O stretch and C-O-C stretch) in uncross-linked SA films were not present in uncross-linked AP films but became prominent in cross-linked AP films These 2 bands were associated

with G blocks of the alginate (Sartori et al., 1997) which mainly involved interactions

with Ca2+ Thus, the above observations from the FTIR spectra indicated a change in polymer ring and linkage conformation before and after cross-linking in both polymers, suggesting interactions between the two types of polymer chains in the cross-linking process The higher light transmittance of cross-linked AP films as compared to cross-linked SA and PT films further supported the presence of interactions between the polymers, mediated by Ca2+, with greater miscibility between the polymers

In composite films containing carrageenan, the reduction in film thickness was considerably greater than the change in film weight after cross-linking indicating that interaction between Ca2+ and polymer chains greatly influenced the film thickness (Figure 22a).The Ca2+ enhanced gelation of κ-carrageenan by stabilization of the helical conformation of the polymer, through shielding of the charge of sulphate groups by Ca2+ (Watase and Nishinari, 1986) This phenomenon enabled tight binding and enhanced aggregation of the helices, which accounted for the reduction in thickness of KC and AK films after cross-linking The increased aggregation of the helices was reflected by the lower transmittance of KC and AK films as compared to

SA films (Table 19) The gels formed by interaction of κ-carrageenan with Ca2+ had

been described as ‘turbid’ by Michel et al (1997) FTIR studies revealed possible

interactions of sodium alginate and κ-carrageenan with Ca2+ and with each other A series of bands in the region 1150-1000cm-1 of SAhad been reported to shift to lower wavenumbers as the calcium content increases and the authors attributed the shift to

C-C and C-O bond-sharing with calcium ion (Sartoris et al., 1997) The shift to lower

wavenumber for these bands in SA indicated binding with Ca2+ (Figure 27) Bands around 1260 cm-1 and 1230 cm-1 for carrageenans were due to O=S=O antisymmetric stretching vibration The shoulder around 1230 cm-1 in KC became visible only after cross-linking with Ca2+ (Figure 27) The possible interaction between anionic sulphate groups and Ca2+ was further supported by the presence of the band at 1236.2 cm-1 in cross-linked AK, as this was not observed in uncross-linked AK

The band at 1616.1 cm-1 in uncross-linked AK films shifted to a higher wavenumber after cross-linking This band is attributed to the asymmetric COO-stretch in SA However, in uncross-linked SA, this band was shifted to a lower wavenumber after cross-linking Thus, the band shift observed in AK films after cross-linking could not be attributed only to bonds between Ca2+ and alginate but also

to the possible interaction between the carboxyl groups of SA and other hydrophilic groups on KC This band in the range of 1640-1645 cm-1 in KC was due to polymer

bound water (Abad et al., 2003) Thus, the hydrophilic groups in KC could be

involved in interaction with SA in the composite films The band at 1419.4 cm-1 in uncross-linked AK shifted to 1429.0 cm-1 after cross-linking In uncross-linked SA, the band around 1419 cm-1 was due to the symmetric COO- stretch and this band shifted to a higher wavenumber in cross-linked SA Bands between 1500-1270 cm-1 in carrageenans were a result of coupling of CCH bending motions in the molecule (Sekkal and Legrand, 1993) Thus, it is highly likely that the significant band shift

Figure 27: FTIR spectra of uncross-linked and cross-linked SA, KC and AK films

from 1419.4 cm-1 to 1429.0 cm-1 observed in AK films after cross-linking was due to the band shift in cross-linking The shoulder at 1430.9 cm-1 in uncross-linked KC was

no longer observed in the FTIR spectrum of cross-linked KC The observations further supported alginate-KC interaction Hence, interactions between alginate and carrageenan in the presence of Ca2+ were responsible for the increased % reduction in film thickness in AK composite films with increasing proportion of KC (Figure 22a)

For ι-carrageenan, each cross-linking cation is coordinated to two sulphate groups, each from a different double helix, in such a way as to produce a Ca2+-sulphate-Ca2+-sulphate… chain running through the lattice parallel to the fibre axis Thus, each double helix is linked to its neighbours by cations forming a complete three dimensional system (Arnott and Scott, 1974) Therefore, Ca2+ not only connects and balances the charge on two sulphate groups from the different helices but it also brings the helices closer (Janaswamy and Chandrasekaran, 2002), producing thinner

IC and AI cross-linked films (Figure 22a) The prominent band around 1260 cm-1 in the FTIR spectra, attributed to antisymmetric stretch of O=S=O in IC, became less prominent and shifted to a higher wavenumber after cross-linking in cross-linked IC films (Figure 28) The observation suggests that there is interaction of Ca2+ with IC FTIR evidence also highlighted probable interactions between the sulphate groups in

IC and alginate Bands in uncross-linked AI at 848.5 cm-1 and 806.1 cm-1, associated with galactose-4-sulphate and 3, 6-anhydrogalactose-2-sulphate of iota carrageenan, were shifted to 894.8 cm-1 and 819.6 cm-1 respectively after cross-linking with Ca2+ These bands in IC were also shifted to a higher wavenumber after cross-linking, indicating interactions between Ca2+ and the macromolecules of AI However, the extent of the band shift was considerably larger with cross-linked AI than IC In