Faraday Symp. Chem. Soc. 1983 18 115-129 Molecular Viscoelasticity of Xanthan Polysaccharide BY SIMON B. ROSS-MURPHY* Unilever Research Colworth House Sharnbrook Bedford MK44 1LQ AND VICTORJ. MORRIS ARC Food Research Institute Colney Lane Norwich NR4 7UA AND EDWIN R. MORRIS National Institute for Medical Research Mill Hill London NW7 1AA Received 14th July 1983 Previous studies of xanthan polysaccharide using molecular probes of local chain geometry such as optical rotation and n.m.r. have demonstrated a cooperative disorder-order transition in aqueous solution on cooling or on addition of salt. In the present work we have investigated chain geometry of the ordered species at a 'macromolecular level' using quasi-elastic light scattering transient electric birefringence and elongational flow birefringence and have used viscoelastic measurements to probe the 'supramolecular' organisation responsible for the 'weak-gel' properties of xanthan solutions.Solution viscoelasticity at fixed xanthan concentration was drastically modified by changing the counterion to the polyelectrolyte and by treatment with urea although in all cases the local ordered structure was unaffected. 'Macromolecular' studies under conditions which minimise intermolecular interactions (Na+ salt form in the presence of urea) and at substantially lower concentrations indicate a persistence length comparable to that of other highly persistent biopolymers such as double helical DNA and the triple helical polysaccharide schizophyllan. We conclude that in aqueous solution xanthan may be regarded as a highly extended worm-like chain interacting by non-covalent association to develop a weak-gel network which is readily reversible under shear.Xanthan is an anionic polysaccharide produced commercially by fermentation of the bacterium Xanthomonas campestris which in nature occurs as a plant pathogen. Aqueous solutions of the polymer show distinctive 'weak-gel ' properties' which form the basis of its technological utility. The primary structure2* is based on a linear chain of 1,4-linked/3-~-glucopyranosyl residues as in cellulose but with charged trisaccharide sidechains attached to alternate residues of the polymer backbone to give a pentasaccharide repeating sequence (fig. 1). Characterisation of the xanthan macromolecule is for a number of reasons much more complex than for most synthetic polymers.In particular being a polyelectrolyte its chain dimensions would be expected to change in response to changes in ionic strength. Under conditions of elevated temperature and/or comparatively low ionic strength this is indeed the case and the local chain flexibility (as monitored by for example the timescale of n.m.r. rela~ation)~ is very similar to that of other coil-like cellulose derivatives. On cooling or on addition of salt however the molecule undergoes a cooperative conformational transition to a rigid ordered struct~re.~-~ The disorder-order transition (fig. 2) may be monitored by a variety of physical techniques including optical r~tation,~-~ differential scanning circular dichr~ism,~? calorimetry,* solution viscosity6$ and loss of detectable high-resolution n.m.r.~ignal.~ 115 MOLECULAR VISCOELASTICITY OF XANTHAN POLYSACCHARIDE backbone y2-I OH I sidechain II 0 ) Fig. 1. Pentasaccharide repeating sequence of xanthan. The proportion of pyruvate and acetate substituents present may vary appreciably from sample to sample. 1 .o . 0.8-0.6 -f$ 0.4-0.2 -0.0 I -A The transition (which is fully reversible and shows no thermal hysteresis) obeys first-order kinetics** and seems to occur without an associated change in molecular weight.'? From this and other evidence it is proposed* that the fundamental structural unit is a single helix stabilised intramolecularly by ordered packing1* of sidechains along the polymer backbone and that the 'weak-gel' properties arise from higher levels of non-covalent interaction between helically stabilised species." Depending upon the nature of the growth medium the xanthan polyanion is associated with mixed proportions of the cations Na+ K+ and Ca2+.At the same time different growth conditions also alter the levels of pyruvate and acetate substituents S. B. ROSS-MURPHY V. J. MORRIS AND E. R. MORRIS 117 on the sidechains.l2? l3 Both factors are known to modify the viscoelastic behaviour in solution. Moreover the molecular-weight distribution is believed to be broad and fractionation of samples by sonication and/or size-exclusion chromat~graphy~~ l5 presents serious difficulties.Molecular characterisation of xanthan solutions is further frustrated by the presence of a microgel fraction the amount of which seems to be sensitive to for example the proportion of cations the ionic strength and the time after dissolution. In this paper we discuss the application of physicochemical techniques acting over longer distances than the ‘molecular probes’ such as optical rotation and n.m.r. which have been discussed previously. These we have arbitrarily divided into ‘macromolecular probes ’ acting over tens of nanometres in dilute solution including in particular quasi-elastic light scattering transient electric birefringence and extensional flow birefringence and ‘supramolecular probes’ such as oscillatory and steady-shear viscoelasticity for more concentrated solutions.In all cases reported here considerable care was taken to assess and apply a consistent regime of sample preparation in view of the complications outlined above. Results from ‘macromolecular’ and ‘ supra-molecular’ techniques are assessed in terms of the available information on local conformational rigidity from ‘molecular ’ probes. EXPERIMENTAL SAMPLE PREPARATION Commercial xanthan (Sigma) was freed of cellular debris by centrifugation of aqueous dispersions (ca. 76000 g; 1-3 h). Clarification was assessed by phase contrast and electron microscopy. The resulting clarified dispersions were ion-exchanged (Dowex SOX-W8) to single-salt forms (Na+ K+ Ca2+) and freeze dried. Solutions were prepared by adding the freeze-dried material to deionised water and stirring at room temperature until homogeneous (ca.12 h). Supramolecular aggregates (microgel particles) were removed by the techniques described in a subsequent section. Where heating was employed evaporation losses were corrected by addition of solvent after cooling. All samples contained 0.02 % sodium azide as preservative and were stored at 4 OC. RHEO-OPTICAL TECHNIQUES QUASI-ELASTICLIGHT SCATTERING (QELS) Measurements were made at 25 OC on a Malvern 4300 goniometer (Precision Devices Malvern) using homodyne detection. Single clipped autocorrelation was employed over the angular range 30-1 50°. The background was calculated from measurements of the clipped and unclipped counts and the number of samples.Results were expressed in terms of the normalised function C(t)= [g‘2’(t) -l]/Eg‘2’(0)-11 where g(2)(t)is the second-order correlation function.1s TRANSIENT ELECTRIC BIREFRINGENCE (TEB) The TEB apparatus was built to a standard design,17 which has been fully described elsewhere.1B Linear detection” was used to determine the sign of the birefringence whilst studies of the field dependence of the equilibrium birefringence and of the time decay were made using quadratic detection. ELONGATIONAL FLOW BIREFRINGENCE (EFB) Elongational flow fields were generated using two opposed jets operating under ~uction.~~~ 2o Strain rates (i) were calculated from the measured flow velocity and the geometry of the apparatus. The induced flow birefringence was monitored using quadratic detection.” The light transmitted by the analyser was detected by a photomultiplier and displayed on an oscilloscope.For all three rheo-optical techniques the incident wavelength was 633 nm. MOLECULAR VISCOELASTICITY OF XANTHAN POLYSACCHARIDE VISCOELASTIC MEASUREMENTS These were performed using a Rheometrics RMS-605M mechanical spectrometer (Rheo- metrics Inc. New Jersey U.S.A.).Both sensitive (ST-10) and mid-range (TC-2000) transducers were used and experiments were performed under both oscillatory and steady shear using cone- and-plate geometry. Sample temperature was maintained at 25 k0.5OC; in all experiments reported here a fixed polymer concentration of 0.5%w/w was used. RESULTS DETECTION AND ELIMINATION OF MICROGEL ’ BY ULTRAFILTRATION Filtration of aqueous dispersions of Na+ xanthan (which had previously been freed of cellular debris by centrifugation) through 0.22 pm filters proved virtually impossible indicating extensive association of the polymer into supramolecular aggregates (‘microgel ’).On centrifugation a gel-like deposit was obtained which contained a substantial fraction of the total polymer mass (e.g. centrifugation of a 0.1 % disper-sion at 100000g for 1 h resulted in deposition of ca. 15% of the polysaccharide). The supernatant solution could then be filtered although with some difficulty. Dispersions which had undergone prolonged heating (90 OC 3 h) no longer gave gel-like deposits on centrifugation and could be filtered although again with difficulty (filtration of a 0.1 % w/v sample through a 0.22pm membrane resulted in a polymer loss of ca.5%). After a similar heating regime but in the presence of urea (2-8 mol dm-3) there was no detectable loss of polymer on centrifugation or filtration. Solutions of xanthan prepared directly from fermentation broth with no precipitation or drying stage show5 the filtration characteristics that we obtained only after removal of microgel. It therefore appears that the formation of stable aggregates is promoted by chain-packing in the solid state. RHEO-OPTICAL MEASUREMENTS QUASI-ELASTIC LIGHT SCATTERING The results obtained from QELS were very sensitive to sample history (fig. 3). For example ‘dispersions ’ (solutions from which polymer could be extracted by filtration or centrifugation) gave rise to extremely slowly decaying correlation functions [even when plotted as In C(t)];heat treatment of aqueous ‘dispersions’ produced a faster decay whilst ‘dispersions’ which had been centrifuged and/or filtered after heat treatment or which had been heat treated in the presence of urea produced C(t) functions which decayed to zero within ca.30 ms. Heat treatment was insensitive to urea concentration in the range 2-8 mol dm-3 (fig. 4). TRANSIENT ELECTRIC BIREFRINGENCE TEB studies of heat-treated xanthan solutions (4 mol dm-3 urea) have been de- scribed elsewhere,18 and data on heat-treated aqueous b dispersions ’ will be reported in detail at a later date. In both cases the sign of the birefringence was positive and at low-electric-field amplitude the Kerr law was obeyed,17 but at sufficiently large- electric-field amplitude the birefringence saturated the saturated birefringence then varying linearly with polymer concentration (c) for 0.02 < c (% w/w) < 0.08.Semi-logarithmic plots of the field-free decay were curved indicating a broad distribution of relaxation times. At given c this spectrum was dependent upon the magnitude IEI of the applied-electric-field amplitude but at given 1 El decay curves were relatively insensitive to c. Similar relaxation data were obtained for samples heat treated in water or 4 mol dm-3 urea. Fig. 5 shows normalised curves for cases in which [El was sufficient to induce saturation of the birefringence.S. B. ROSS-MURPHY V. J. MORRIS AND E. R. MORRIS I 1 1 1 I 0.0 1 2 3 4 5 tl10-2 s Fig. 3. Time dependence of the normalised autocorrelation function C(t) for an aqueous dispersionof xanthan (0.1 % w/v) initially (a),after heat treatment (m)and after centrifugation and filtration (A).Scattering angle = 40'; temperature = 25 OC. 0 0.01 1 1 1 1 0 1 2 3 4 5 tl10-2 s Fig. 4. NormaIised autocorrelation function C(t)for xanthan (0.1% w/w) after heat treatment in the presence of the following concentrations of urea/mol dm-3 2 (a) and 4 (a),6 (0) 8 (0).Conditions as in fig. 3. ELONGATIONAL FLOW BIREFRINGENCE These measurements proved difficult for samples heated in urea so only data for filtered heat-treated aqueous samples are reported here.These showed a progressive increase in birefringence (fig. 6) (An,) with increasing i rather than the sudden onset of An at a critical strain rate normally observed for flexible polymer c0i1s.l~ Turbulence at high velocity gradients prevented the measurement of the saturation birefringence so the rotatory diffusion coefficient could not be determined from the initial slope of Anf against 6. MOLECULAR VISCOELASTICITY OF XANTHAN POLYSACCHARIDE 0. OL 1 1 1 I 1 0 1 2 3 1 5 t/10-3 s Fig. 5. Normalised birefringence decay curves for xanthan (0.066% w/v) after heat treatment in water (A) and for the following concentrations of xanthan (% w/v) after heat treatment in 4 mol dm-3 urea 0.04 (@) 0.06 (0) The amplitude of the applied electric and 0.08 (u).field was sufficient to induce saturation of the equlibrium-induced birefringence An,(O) at elapsed time t= 0. The solid lines show the best theoretical fits based on a log-normal distribution of particle size. I 1 & I 1 2 3 strain rate e/i03 s-l Fig. 6. Dependence of the induced elongational flow birefringence (An,) on the applied strain rate (i)for a 0.02% w/w solution of xanthan in water following heat treatment at 90 OC for 3 h. S. B. ROSS-MURPHY V. J. MORRIS AND E. R. MORRIS VISCOELASTIC STUDIES ‘NATIVE ’ XANTHAN Viscoelastic measurements for native xanthan heated with and without urea have been published previously,113 21 but for completeness they are summarised briefly below. 1 OL 10’ m a -.u 1oo m a \ u lo-’ 10-2 10” 1oo 10’ lo2 o/rad s-’ Fig. 7. Frequency dependence of dynamic storage and loss moduli G’ and G for xanthan (0.5%w/w; 0.02 mol dm-3 KCl) in the presence of the following concentrations of urea/mol dm-3 0.02 (0, 0) and 4 (m m). (Reproduced with permission from J. Polyrn. Sci. Polym. Lett. Ed.). In the absence of urea the mechanical spectrum of 0.5% w/w solutions is such that over the frequency range 10-1-102 rad s-l G’ is greater than G and neither shows much frequency dependence over this range (‘gel’ spectrum) (fig. 7); the linear strain region extends to y x 0.05-0.20 for both native and single-cation samples. Essentially identical spectra are given for ‘native’ samples heat treated with 0.02 mol dm-3 urea.For the heat-treated 4 mol dm-3 urea samples the spectrum is quite different. At low frequency G” > G’ and both moduli approach the dependence G” oc w and G’ cc w2 as w becomes lower. At higher o (> 30 rad s-l) G’ > G consistent with physical 122 MOLECULAR VISCOELASTICITY OF XANTHAN POLYSACCHARIDE entanglement and G” is similar to that observed in 0.02 mol dm-3 urea. Intermediate urea concentrations produce intermediate effects. Fig. 8 shows the shear-rate dependence of steady-shear viscosity measured for solutions which had been treated with progressively increasing urea concentrations. In 4 rnol dm-3 urea typical pseudoplastic polymer solution behaviour is observed with a low shear rate Newtonian plateau (qox 0.4 Pa s) for i < 1 s-l.At this urea concentration q* values calculated from the data of fig. 7 are in close agreement with q at equivalent co and i (Cox-Merz superposable),22whereas with no added urea q* > q over the same range. This lack of superposition is at least partly an effect of the strain deformation applied to the system (low in oscillatory shear ‘high’ in steady shear). loi \ \ \ \ \ \ 10‘ \ \ \ \ ‘\ \ CA ‘\ 2 \ c-loc \ \ \ \ \ \ lo-’ \ \ \ \ \ \. \ 10-2 10” loo 10’ lo2 103 -+-’ Fig. 8. Shear rate (1;) dependence of viscosity (q) for ‘native’ xanthan (0.5% w/w; 0.02 mol dm-3 KC1) in the presence of the following concentrations of urea/mol dm-3 0.02 (O),0.2 (a), 2 (A)and 4 (a).The dashed lines indicate the approximate upper and lower shear-stress limits of the instrument for the test configuration used.(Reproduced with permission from J. Polym. Sci.,Polym. Lett. Ed.). The progressive disruption of structure by urea treatment is seen in fig. 8; with decreasing urea concentration q at low i shows a progressive increase until by 0.02 mol dmh3there is little sign of a Newtonian plateau and the j dependence is much greater than that normally observed for polymer solutions (viz.d log q/d log i 7 -0.9 compared with ca. -0.7),23 including disordered polysaccharides. At high y all the curves appear to converge to a common value of q(j).All the results for q against j and G’ G” against w are completely reversible i.e.independent of whether i is S. B. ROSS-MURPHY V. J. MORRIS AND E. R. MORRIS I23 increased or decreased. Xanthan solutions might therefore be regarded as ‘shear-reversible gels ’. Under the same conditions of low urea the proportionality constant between G’(o) and Nl(j),with Nl the primary normal stress at equivalent CL) and j (i.e. the normal stress analogue24 of the Cox-Merz rule) is significantly greater than ca. 2-3 usually observed for flexible polymers (and for disordered polysaccharides) and approaches ca. 6. Further discussion of this effect will be published later. 10-2 ,;-I 100 10’ lo2 103 1 7h-l Fig. 9. Shear-rate dependence of viscosity for Na+ xanthan before urea treatment (A)and after treatment with 0.02 mol dm-3 urea (0)and 2 mol dm-3 urea (m).The solid line is the data for ‘native’ xanthan in 0.02 rnol dmP3 urea (fig. 8). ‘SINGLE-CATION ’ XANTHAN The data reported above show that increasing urea concentration gradually reduces the ‘gel-like’properties of xanthan solutions at low deformation rates. This tendency is substantially affected by the nature of the cation for ion-exchanged samples. Fig. 9 gives the ~(j) dependence for Na+ xanthan without urea treatment and after treatment with 0.02 and 2 mol dm-3 urea. The behaviour of Na+ xanthan is quite different from that of the mixed-cation ‘native’ form with some evidence that a Newtonian plateau would be reached at lower shear rates than were experimentally accessible. Even this behaviour is so modified by treatment with 0.02 mol dm-3 urea that there is little further change when 2 mol dm-3 urea is used.By contrast K+ xanthan (fig. 10) behaves in the absence of urea very similarly to the ‘native’ form but there is a considerable decrease in low-shear viscosity on treatment with 0.02 mol dm-3 urea which has no detectable effect on ‘native’ xanthan. At higher concentrations of urea (2 mol dm-3) where the ‘gel-like’ properties of ‘native’ xanthan are diminished little further change is observed for the K+-salt form and the ~(9) profiles for ‘native’ and K+ xanthan in 2 mol dm-3 urea are again quite similar. Finally fig. 11 shows that the Ca2+ form of xanthan has at the same concentration a much greater increase in low-shear viscosity (i.e.ca. 8 x as great at 3= 0.1 s-l) compared with the native form and that this is scarcely altered by urea treatment. I24 MOLECULAR VISCOELASTICITY OF XANTHAN POLYSACCHARIDE lo2-10' -v) ." 0 a \ c loo -lo-' -If-2 16-1 160 161 102 1d3 ?Is-' Fig. 10. As fig. 9 but for K+ form in KC1. I t. lo2 ' 10' P .-0 a \ c loo. \ lo-'/ I 10-,;-2 10-1 100 101 102 103 ?is-' Fig. 11. As fig. 9 but for Ca2+ form; urea effect is negligible. Qualitatively the slow but progressive disruption in structure for 'native ' xanthan may be rationalised in terms of the relative contributions of the pure Ca2+ Na+ and K+ forms. DISCUSSION 'MACROMOLECULAR ' STRUCTURE Evidence from the rheo-optical techniques employed in this paper re-emphasised the importance of a consistent regime of sample preparations when studying xanthan (and other biopolymers).Furthermore the importance of using a range of physico- chemical techniques (preferably probing over different 'length ' scales) is reasserted S. B. ROSS-MURPHY V. J. MORRIS AND E. R. MORRIS 125 particularly when attempting to interpret rheological data on the basis of ‘molecular’ models. For example QELS studies on dispersions of xanthan (sodium-salt form) in water or urea are characterised by both ‘fast ’(macromolecular) and ‘slow’(‘ supramolecular’ or ‘gel’) modes of 25 However at the same polymer concentration (c) the slow mode was extremely sensitive to the method of sample preparation and could be eliminated by heat treatment in the presence of urea or heat treatment followed by filtration.Without the filtration step intermediate results were obtained (cf. fig. 3). Loss of the slow mode in QELS occurred (at lower c) under the same preparative conditions as those which drastically modify the viscoelastic mechanical spectrum from that typical of a polymer gel to that of a viscoelastic polymer solution (see fig. 7). Nevertheless the optical rotation (and n.m.r.) data for both native xanthan and the Na+ form demonstrate that the ordered helical structure is retained after heat treatment for both ‘aqueous’ and urea-treated samples.l19 21 The evidence thus suggests that the ‘slow’ mode in the QELS spectrum and the predominantly elastic mechanical response (to o < 0.1 rad s-l) result from inter- molecular association of the polymer chains.The irreproducible and irreversible results obtained for example on xanthan aqueous ‘dispersions’ without heat treatment in both QELS and mechanical measurements strongly imply that such ‘dispersions’ involve supramolecular aggregates which only slowly ‘dissolve’. Measurement for example of MWz1 by integrated light scattering suggests that heat-treated aqueous samples still contain some very high Mwaggregates (microgel) which may be removed by prolonged centrifugation or filtration whilst measurements of Mw for samples heated in the presence of urea suggest that these are true solutions. The presence of microgel would account for the slight mass loss observed on filtration of aqueous solutions and fluctuations in the concentration of such aggregates in the sample volume would explain the tail in the correlation function and its removal on filtration.Thus heat treatment in the presence of urea or heat treatment followed by filtration may be used to prepare ‘molecular solutions’ of the Na+-salt form of xanthan. The choice of associated cation ionic strength and level of pyruvate and acetate substituents would of course also modify these effects. However the above evidence confirms that suitable pretreated solutions of xanthan in the sodium-salt form may be used to investigate the size shape and flexibility of the isolated macromolecule. For example preliminary information may be deduced from the course of the strain-rate dependence of birefringence under elongational flow.As mentioned earlier there was no evidence of the sudden increase in Anf above a critical strain rate which is typical of random coil polymers;lg rather the data suggest that xanthan behaves in the manner characteristic of a highly extended rod or persistent worm-like chain. TEB studies provide further information on the hydrodynamic properties of the xanthan macromolecule. The field-free decay curves (fig. 5) suggest that the relaxation process for heat-treated ‘aqueous’ and 4 mol dm-3 urea-treated solutions were quite similar and the magnitudes of the relaxation times furnish a guide to the local rigidity of the polymer.2s For flexible chains the electric dipole moments associated with individual monomeric units may orientate or disorientate virtually independentl~.~’? 28 For such coils electrical polarisation processes are insensitive to Mwand are virtually identical to those for solutions containing equivalent concentrations of the monomers.29-31 The relaxation behaviour ofmore persistent chains is morecomplicated.For example for cellulose derivatives (e.g. sodium carboxymethylcellulose and hydroxyethylcellulose) where the Kratky-Porod persistent length q is ca. 10-30 nm (depending upon conditions) the relaxation time z = 1-2 ps.32933 For rod polymers I26 MOLECULAR VISCOELASTICITY OF XANTHAN POLYSACCHARIDE z is proportional to L3,where L is the rod length (contour length); thus for worm-like chains z cc L3 for L xq (rod limit) but becomes independent of L for L % q (coil limit).34 In the present measurements the initial slopes in fig.5 yield z = 1600ps (4 mol dm-3 urea) and z = 580 ps (aqueous solution). Since the xanthan backbone (1,4-B-~-glucan) is the same as in the above cellulose derivatives we can estimate q for xanthan to be ca. 10 times as great as that for these derivatives i.e. ca. 100-300 nm. More precise estimates cannot be made from the present measurements but the results are comparable with other systems which are known to be extremely persistent chains. For example DNA (double helical) has q x 150 nm at low ionic strength decreasing to ca. 50 nm at high ionic strength,35- 36 and the triple helical polysaccharide from the bacterium Schizophyllum commune has q x 150-200 nm.379 38 Evidence from X-ray fibre diffraction suggests that xanthan exists as a five-fold helix with a pitch of ca.4.7 nm,l07397 40 which yields a mass per unit length (ML)of ca. 1000 dalton nm-l. Since optical rotation results suggest that this helical conformation is retained in solution we can calculated the contour length for a rod of known M,. The distribution of relaxation times observed in fig. 5 may be attributed either to a limited amount of flexibility or to a distribution of contour lengths. Of course in practice both effects would contribute simultaneously but by considering each in turn we can examine their relative contributions. For example if we assume that the relaxation-time spectrum is solely due to the distribution of rod lengths (molecular- weight distribution) and select a model for this distribution we can invert the relaxation data to obtain the length distribution.21 In this case we assume that the molecular-weight distribution follows a log-normal distribution (compared to the more general Schulz-Flory distribution); inversion of the relaxation data yields a length distribution (fig.12) with Mw = 1.06 x lo6. This is in very good agreement with the value from integrated-intensity light scattering uiz. (1.1 &-0.1) x lo6. Alternatively we can use the measured root-mean-square radius of gyration from integrated light scattering21 (250 & 10 nm) to calculate the persistence length assuming a monodisperse molecular-weight distribution.In more detail since (P)x250 nm and M z 1.1 x lo6,L (= (S2)i/d12) x870 nm; from the value of M and M we can also calculate the contour length as M/M = (1.1 x 106/103) nm = 1100 nm; the lower value from (S2)would imply some degree of flexibility. Using the Benoit-Doty expansion for (9)for the Kratky-Porod chain we can also estimate q from M/M, as suggested recently by Norisuye and F~jita.~l Using M = 1000 nm-l the optimum number of statistical segments per chain nk [= M/(2q M,)] was ca. 1.45 giving q x380 nm. This is probably too high when compared with DNA and schizophyllan but interestingly using a larger M, as was suggested by Paradossi and Brant,14 would produce a still larger value of q. In practice a number of possibilities exist for gaining more reliable estimates for q although most require the preparation of a number of samples of very narrow molecular-weight distribution.For example one could perform combined light- scattering/intrinsic-viscosity measurements as was done for the schizophyllan 38 or polysa~charide,~~~ using narrow-molecular-weight samples perform the Hagerman-Zimm analysis34 of TEB data as a function of (L/q)for the worm-like model. Perhaps the most reliable measurements are those for ‘infinitely dilute’ viscoelastic properties for example those by Hvidt and coworkers for the persistent rod-like protein myosin.42 The use of the worm-like chain model for xanthan has been discussed by H01zwarth.~~ S. B. ROSS-MURPHY V. J. MORRIS AND E. R. MORRIS /-\ effective rod length 1/10” m Fig.12. Calculated length distributions based on a log-normal distribution function fir) = (27r)-4 (al>-lexp { -0.5[In(l/rn)/~]~}>. The results shown provide the best fits to the experimental data in fig. 5 for xanthan after heat treatment in water (-) and in 4 mol dm-3 urea (--); the fitted parameters are Q = 0.6 rn = 0.58 pm and Q = 0.7 rn = 0.60 pm respectively. ‘SUPRAMOLECULAR’ STRUCTURE Results from viscoelastic measurements cast further light on the nature of the non-covalent intermolecular interactions which are believed to confer ‘weak-gel’ properties to xathan solutions. The earlier sections have dealt with probes of local chain flexibility and order (optical rotation and n.m.r.) or studies of macromolecular species over longer distances (TEB and QELS); the viscoelastic measurements are primarily concerned with supramolecular structure at higher concentration (0.5% w/w).In fact [q]for ‘native’ xanthan was found to be ca. 3 dm3 g-l so that the overlap parameter c [q]z 15. Nevertheless previous QELS studies of xanthan placed great emphasis on the presence of ‘slow ’ modes of relaxation.44* 45 As the concentration is increased these have been attributed to either hindered rotational effects or diffusional processes in a weak-gel network. The TEB data shown in fig. 5 do not show the marked concentration dependence predicted for hindered rotational effects.46 In addition the saturation birefringence for heat-treated solutions in water and 4 mol dm-3 urea varies linearly with polymer concentration.21 Both factors are consistent with independent non-interacting particles for the more dilute solutions.Hence the slow modes observed in QELS result from association of the polymers. The extent of this interaction is here shown to depend on the method of sample preparation the nature of the cations and the ionic strength of the medium. The present datal1g2l do not support previous ~uggestions~~-~~ that heat treatment in the presence of urea results in a disordered ‘random-coil’ xanthan at low temperatures. As far as the supramolecular (rheological) measurements are concerned previous results have shown how differing proportions of acetate and pyruvate groups on the sidechains (again introduced by subtle changes in the bacterial growth medium) can modify the shear-rate-viscosity pr0fi1e.l~ The present data show that quite drastic MOLECULAR VISCOELASTICITY OF XANTHAN POLYSACCHARIDE changes in the viscoelastic response may be induced by modifying the balance of associated cations.Unfortunately we cannot unequivocally characterise the nature of the intermolecular interactions; what is clear is that the effect is much greater than would be expected for non-specific entanglement coupling. The sensitivity to urea might suggest a contribution from hydrogen bonding or modification of dispersive forces due to the change in dielectric constant of the medium while ionic interactions are modified by counterion concentration and type and by pyruvate content. Again these effects are much more pronounced than would be expected purely from the polyelectrolyte character of the sidechains.Another possibility is that the structuring is associated with the separation of an ordered anisotropic phase (isotropic-nematic phase transition). According to the argument of Fl~ry,~O this would occur at a volume fraction CD given by where yn is the (number average) length/diameter ratio of a rigid rod in an assembly of such rods. Using the molecular data presented earlier yn x 400 and thus CP x 0.02. Of course both the curvature associated with large q and the effect of aggregation would tend to increase CDc (by reducing yn) but for xanthan the partial specific volume51 is ca. 0.62 so that the weight fraction of polymer (w*)at CDc is given by w* x 1.6 CD, i.e.w* x 0.032. Two report an apparent 0,transition at ca. 2.9%w/w and ca. 3.5% w/w respectively. In neither case are sufficient details of sample electrolyte or solution preparation given so that no rigorous comparison with results predicted above may be made but qualitatively the agreement with the above calculation is reasonable and the quantitative prediction is in keeping with results for synthetic rod-like Nevertheless w* is greater than the concentrations reported here uiz. 0.005 and weak-gel properties of xanthan have been reported1 down to 0.1% i.e. w = 0.001 so that the evidence seems to rule out liquid-crystalline structuring in this regime. We conclude therefore that overall properties of xanthan in dilute and semi-dilute solution are governed by non-covalent association of worm-like chains (which may be modified by solvent quality and ionic environment) to produce at concentrations down to ca.0.1% a weak-gel network but one which is readily reversible under shear. We thank C. Turner and D. Franklin (Physics Department Brunel University) K. I’Anson G. R. Chilvers and S. R. Ring (FRI) and R. K. Richardson S. A. Frangou and L. A. Linger (URL Colworth) for their help and M. J. Miles (FRI) for providing unpublished flow birefringence data (fig. 6). P. J. Whitcomb and C. W. Macosko J. Rheol. 1978 22,493. P. E. Jansson L. Kenne and B. Lindberg Carbohydr. Res. 1975 45 275. L. D. Melton L. Mindt D. A. Rees and G. R. Sanderson Carbohydr. Res. 1976,46 245. E. R. Morris D.A. Rees G. Young M. D. Walkinshaw and A. Darke J. Mol. Biol. 1977 110 1. D. A. Rees Biochem. J. 1972 126 257. G. Holzwarth Biochemistry 1976 15 4333. M. 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