STRUCTURE ANALYSIS OF POLYSACCHARIDE ESTERS

Water solubility of cellulose acetate: dependence on the pattern of functionalisation a Methods applied: 1 deacetylation of cellulose triacetate with aqueous acetic acid, 2 reaction of c

For modified polysaccharides, the analysis goes far beyond the structural cation The chemical structure of the ester function introduced, the DS, and thedistribution of the functional groups at both the level of the RU and along thepolymer backbone (Fig 8.1) can strongly influence the properties and need to bedetermined comprehensively.

verifi-Fig 8.1 Schematic plot of the possible patterns of functionalisation for the repeating units (A) and

for the distribution along the polymer chain (B) of polysaccharides with three reactive sites

The chemical functionalisation may be associated with side reactions ing the polymer backbone additionally, maybe to a rather low extent only These

modify-“structural impurities” introduced have to be revealed as well because they are notremovable from the polymer chain Consequently, an efficient and reliable analy-sis (type of functionalisation, DS, the pattern of substitution) is indispensable forthe establishment of structure–property relations of the modified polymers Thetailored modification of substitution patterns can be used to “fine tune” productproperties, e.g solubility behaviour, as shown for the water solubility of celluloseacetate in Table 8.1 [89]

Table 8.1 Water solubility of cellulose acetate: dependence on the pattern of functionalisation

a Methods applied: (1) deacetylation of cellulose triacetate with aqueous acetic acid, (2) reaction

of cellulose triacetate with hydrazine, and (3) acetylation of cellulose with acetic anhydride in

DMAc/LiCl

b Determined by 13 C NMR spectroscopy

Analytical data are also necessary to confirm the reproducibility of a synthesisand the resulting product purity Unconventional polysaccharide esters, e.g withsensitive heterocyclic moieties, can often not be analysed by “standard methods”and this has required the development of new analytical tools

Most of the structural features of the polymer backbone are accessible via cal spectroscopy, chromatography and NMR spectroscopy, as discussed in Chap 3.Specific techniques useful to determine the result of an esterification, the DS, andthe pattern of functionalisation are described herein The evaluation of the pattern

opti-of functionalisation is illustrated in detail for the most important polysaccharideester, cellulose acetate Detailed spectroscopic data for other polysaccharide estersare given in Chaps 3 and 5

From the synthesis chemist’s perspective, the most reliable, powerful and cient method for the detailed structure elucidation at the molecular level is NMRspectroscopy A number of interesting new chromatographic tools have been de-veloped over the last two decades, with a potential of gaining defined structuralinformation, but they are combined with a variety of complex functionalisationsteps making them susceptible to analytical errors and, therefore, should be usedonly by experienced analysts

effi-8.1 Chemical Characterisation – Standard Methods

Saponification of the ester function in the polysaccharide derivatives with aqueousNaOH or KOH and back-titration of the excess base is one of the oldest and easiestmethods for the determination of the DS In the case of short-chain aliphaticesters (C2–C4) of glucans, galactans and fructans, it is a fairly accurate method.For polysaccharide esters containing acidic (COOH) or basic (NH) functions, anabsolute deviation may occur

For longer aliphatic esters, strong hydrolysis conditions need to be applied(0.5 N NaOH, heating 48 h at 50◦C [372]) Saponification in solution, e.g in acet-one, has been used to determine the total acyl content in mixed polysaccharideesters, e.g cellulose acetate propionates and acetate butyrates This procedureovercomes some of the difficulties encountered in the commonly used heteroge-neous saponification, in that it is independent of the condition of the samples, can

be run in a shorter elapsed time, and is a little more accurate [373] Analysis ofpolysaccharide esters containing many of the C2–C4acid esters is carried out bycompletely hydrolysing the ester with aqueous alkali, treating with a high-boilingmineral acid in an amount equivalent to the alkali metal content, distilling the re-sulting mass to obtain all of the lower fatty acids in the form of a distillate, titrating

a given amount of the distillate with standard alkali, and determining the differentamounts of acids by titration after fractionated extraction [374]

Acid–base titration is an approach to evaluate ester cross-linking of charides with polycarboxylic acids such as 1,2,3,4-butanetetracarboxylic acid, bymeasuring the concentrations of ester and free carboxylic acid using calcium acet-ate back-titration [375, 376]

polysac-In the case of polysaccharide dicarboxylates and esters with hydroxy boxylic acids, i.e citric acid, malic acid or tartaric acid, potentiometric titration

polycar-is used [377, 378] Unsaturated esters such as starch acrylic acid esters have beencharacterised via the bromide/bromate method or with permanganometric titra-tion [379]

For esters containing heteroatoms, a convenient method is elemental analysis.The DS is calculated by Eq (8.1)

DS= % Analyte· M r(RU) − 100· a · M r(RU)

100· b · M r(Analyte)· M r(Introduced mass) (8.1)

%Analyte (e.g heteroatom) obtained by elemental analysis

a=number of analyte in the repeating unit

b=number of analyte in the introduced group

M r(RU)=the molar mass of the repeating unit

8.2 Optical Spectroscopy

In addition to the characteristic signals for the polysaccharides given in Table 3.2,the strong C=O stretch band at 1740–1750 cm−1is characteristic of an ester moiety

For unsaturated esters, the signal is shifted to lower wave numbers (about 20 cm−1)and, in esters with strong electron withdrawing groups, e.g trifluoroacetates, is inthe region of 1760–1790 cm−1[188].

IR spectroscopy has been used for a quantitative evaluation of the amount ofbound carboxylic acid and the distribution of the primary and secondary hydroxylgroups It is a valuable tool for low-substituted derivatives and has been used toestimate the DS Consequently, it is applied for the analysis of low-substitutedstarch acetates, showing good reproducibility [380] Moreover, the FTIR bandsassigned to sodium acetate produced by saponification of starch acetate can bedetected by means of the ATR method, which can be used to determine the DS,showing a good correlation with values obtained from NMR [381]

In IR spectra of highly substituted cellulose acetates and mixed esters (0.15–3.0% solution in methylene chloride), two signals at 1752 and 1740 cm−1are ob-served, corresponding to acyl moieties at positions 2 and 3 and at position 6respectively (Fig 8.2) The signal areas can be used for the calculation of the ratio

of substitution at the different reactive sites

Fig 8.2 The C=O signal region of an IR spectrum

(0.15–3.0% solution in methylene chloride, ured in a KBr cell) of highly substituted cellulose acetates (1, C=O signal at positions 2 and 3, 2, C=O signal at position 6, adapted from [383])

meas-The wave number is not influenced by the length of the chain in aliphatic acidesters, as shown in Fig 8.3, and thus the technique can be extended to mixed esters.Comparable information concerning the distribution of substituents is acces-sible by evaluation of the signal region of the OH groups (Fig 8.4)

A signal at 3660 cm−1 corresponds to the primary OH unit Furthermore,signals at 3520 and 3460 cm−1 are caused by secondary hydroxyl moieties Theabsorption at 3580 cm−1can be attributed to hydrogen bonding of the primaryhydroxyl group [382, 383] Line shape analysis or deconvolution of the spectra ishelpful, and can be carried out with many modern FTIR instruments

In addition to IR spectroscopy, Raman and NIR spectroscopy are frequentlyutilised for the investigation of polysaccharide esters The potential of these twomethods is discussed in [384] DS determination for a number of cellulose deriva-tives, including tosyl cellulose and cellulose phthalate, has been carried out aftercalibration with standard samples of defined DS It has been shown that confo-

Fig 8.3 FTIR spectra of aliphatic acid esters of

cellulose with different numbers of carbons in the range C 5 –C 18 (reproduced with permission from [95], copyright Wiley VCH)

Fig 8.4 IR spectrum (0.15–3.0%

solu-tion in methylene chloride, measured in

a KBr cell) of cellulose acetates (OH group region, 1, primary OH, 2, hydrogen bond

of primary OH group, 3 and 4, secondary

OH functions, adapted from [382])

cal Raman spectroscopy is a very valuable method to study surface properties ofsuch derivatives Additionally, remote Raman sensing is a valuable tool for thedetermination of kinetic and chemical engineering data of esterification reactions,which are obtained in a direct and non-invasive inline manner by using remoteRaman sensors This is illustrated by the synthesis of cellulose acetate and cellulosephthalate In Fig 8.5, the development of Raman spectra versus time is shown for

a reaction mixture consisting of cellulose dissolved in DMAc/LiCl and phthalic hydride at 70◦C over 10 h The disappearance of signals for the anhydride at 1760,

an-1800 and 1840 cm−1is visible on the one hand On the other hand, the development

of a signal for the phthalate at 1720 cm−1is observed

Fig 8.5 The development of the Raman spectra versus time for a reaction mixture consisting of

cellulose dissolved in DMAc/LiCl and phthalic anhydride at 70 °C over 10 h (reproduced with permission from [384], copyright ZELLCHEMING)

For the DS determination of maleinated starch by means of Raman

spec-troscopy, the calibration sets have very high linearity (r > 0.99) Combined with

simple sample preparation, Raman spectroscopy is a convenient and safe methodfor the DS determination of polysaccharide esters [385]

UV/Vis spectroscopy is usually applied after ester hydrolysis For calibration,standard mixtures of the polysaccharide and the acid are prepared and measure-ments at the absorption maximum of the acid are performed DS determination

is easily achieved by UV/Vis spectroscopy on mixtures of the saponified charide esters, e.g for bile acid esters of dextran The ester is dissolved in 60%aqueous acetic acid and a mixture of water/sulphuric acid (13/10, v/v) is added.The mixture is treated at 70◦C for 30 min and measured (after cooling) at 378 nm

polysac-to obtain the amount of covalently bound bile acid [216, 217] A similar technique

is applied for the analysis of the phthaloyl content of cellulose phthalate aftersaponification [386] and for polycarboxylic acid esters, e.g starch citrate [387]

An interesting and simple approach to determine the DS of starch esters withUV/Vis spectroscopy is the investigation of the iodine-starch ester complex Theeffect of acetylation on the formation of the complex has been studied by mon-itoring the decrease in absorbance at 680 nm (blue value), which decreases byincreasing the DS [381]

8.3 NMR Measurements

The application of NMR techniques was among the first attempts for the structureanalysis of polysaccharide esters that exceeds the simple DS determination Thepioneering work of both Goodlett et al in 1971 [388] using1H NMR spectroscopy,and of Kamide and Okajima in 1981 [389] applying13C NMR measurements oncellulose acetates opened major routes for further studies in this field, includingcomplete signal assignment, the determination of the functionalisation pattern

of polysaccharide esters depending on reaction conditions, and the establishment

of structure–property relationships Description of the sample preparation andrepresentative signals of the polymer backbones are given in Sect 3.2 NMR exper-iments on polysaccharide derivatives are most commonly performed in solutionstate The solubility of the derivatives strongly depends on the DS and the type ofpolymer Thus, a selection of NMR solvents used for the spectroscopy of polysac-charide esters is listed in Table 8.2 The preferred solvent for the investigation

Table 8.2 Typical NMR solvents used for the solution state13 C- and 1 H NMR spectroscopy of saccharide esters

of partially substituted esters is DMSO-d6, which dissolves the polymers within

a wide range of DS values and is comparably inexpensive

The application of13C NMR spectroscopy with focus on cellulose esters hasbeen reviewed [63] For the majority of (1→4) linked polysaccharides, e.g starchand cellulose, esterification of the primary OH group results in a downfield shift(higher ppm values, between 2–8 ppm) of the signal for the adjacent carbon

In contrast, the signal of a glycosidic C-atom neighbouring carbon adjacent to

an esterified OH moiety shows a high-field shift in the range of 1–4 ppm Thesignal splitting and the corresponding shifts of the other carbon atoms of thepolysaccharides strongly depend on the electronic structure of the ester moietybound This is illustrated for cellulose sulphuric acid half ester and cellulose acetate

in Fig 8.6 A complete assignment requires two-dimensional NMR techniques

In the spectra of partially functionalised derivatives, a mix of spectra is served (Fig 8.6) In combination with the line broadening caused by differentpatterns of substitution, the spectra obtained are very complex Nevertheless, thesignal splitting and the intensities of the carbon atoms of the glycosidic linkagegive an insight into the degree of functionalisation at the neighbouring positions

ob-Fig 8.6 Schematic13C NMR spectra of cellulose (spectrum in the middle) and completely sulphated (lower picture) as well as fully acetylated cellulose (upper picture) and the characteristic shifts caused

by the esterification

The structure elucidation of organic esters of polysaccharides can be wellillustrated with cellulose acetates, and the techniques can be similarly applied forother polysaccharide esters Table 8.3 shows representative13C NMR spectroscopicdata of cellulose triacetate.

Table 8.3 Chemical shifts of the13 C NMR signals for cellulose triacetate (adapted from [390])

b The coupled resonance overlaps with the solvent resonance

The assignment of the signals is based on the comparison of chemical shifts

of model compounds such as peracetylated cellobiose, cellotetraose, cellopentaoseand cellulose [391–393] Data are available for the peracetylated homoglucanespullulan [102] and dextran [58], i.e the pullulan nonaacetate with its maltotriosestructure of the polymer backbone (Table 8.4)

As described above,13C NMR spectra of cellulose acetates provide structuralcharacterisation and determination of both total DS and the distribution of acetylfunctions within the RU concerning positions 2, 3 and 6 in partially functionalisedpolymers [394, 395] The investigation of cellulose acetate samples with DS values

of 1.7, 2.4 and 2.9 reveals the same NOE for C-1–C-6, which confirms quantitativeassessment of partial DS values at positions 2, 3 and 6 from the13C NMR spectrum.The signals at 59.0 ppm (C-6 unsubstituted), 62.0 ppm (C-6 substituted), 79.6 ppm(C-4, no substitution at C-3), 75.4 ppm (C-4 adjacent to substituted position 3),101.9 ppm (C-1, no substitution at C-2) and 98.9 ppm (C-1 adjacent to substitutedposition 2) are used for the calculation

The exact distribution of substituents in polysaccharide esters over a widerange of DS is not readily estimated by simple comparison of the relevant peak

intensities A major problem is the overlapping of signals at around 70–85 ppm,resulting from the unmodified C-2–C-5 and the corresponding acylated positions

2 and 3 as well as the influence of an acylated position 3 on the chemical shift of C-4

In addition, line broadening of the signals due to the ring carbons is frequentlyobserved in the quantitative mode of13C NMR measurements The fairly long

pulse repetition time applied causes T2relaxation of the relevant signals

Table 8.4 Assignment of the carbon signals in pullulan peracetate in ppm (position, see Fig 2.4,

RU as well as the hydrogen bond system of cellulose acetate with DS < 3 A

prelim-inary assignment of the C=O signals of cellulose acetate is achieved by applying

a low-power selective decoupling method to the methyl carbon atoms of the acetylgroups [397], and can be carried out via C-H COSY spectra of cellulose triacetate(Table 8.5, [398])

An elegant analysis of the structure elucidation of cellulose [1-13C] acetatesprepared in different ways with a wide range of DS values is described by Buchanan

et al [399] A total of 16 carbonyl carbon resonances can be identified using

a variety of NMR techniques, including INAPT spectroscopy The assignmentdiffers somewhat from that given in Table 8.5 In the case of C-2 and C-3 carbonyl

carbon resonance, it is possible to assign these resonances to repeating units with

a specific pattern of functionalisation

Table 8.5 Peak assignment of the carbonyl region in the13 C NMR spectrum of cellulose acetate (adapted from [397])

δ(ppm) Carbon at position Functionalised glucopyranose unit

Fig 8.7. 1H NMR spectrum of

a cellulose acetate (DS 2.37)

Nevertheless, cellulose acetate with a DS of 2.46 has been studied with sensitive COSY and relayed COSY NMR spectroscopy Comparison both of thespectra with simulated ones (nine different spectra) and model compounds, e.g.cellotetrose peracetate, shows that nine different types of spin systems are found.They are four types of 2,3,6-triacetyl glucose residues flanked by different acetylglucose units, two different types of 2,3-diacetyl glucose residues, a 2,6-diacetylglucose residue, and a 6-monoacetyl glucose residue [400].

phase-In contrast to1H NMR spectra of partially acylated polysaccharides, completelysubstituted polymers yield assignable spectra, as shown for cellulose triacetate inFig 8.8 and Table 8.6

Fig 8.8. 1 H NMR spectrum

of a cellulose triacetate produced with permission from [151], copyright Wiley VCH)

(re-Table 8.6 Chemical shifts of1 H NMR signals for cellulose triacetate

Signal δ(ppm) of cellulose triacetate