J. Phys. Chem. 1995,99, 3743-3747
3743
Behavior of Native Xanthan in the Biphasic Region: a 23Na+Counterion NMR Study L. Bezemer and J. C. Leyte* Department of Physical and Macromolecular Chemistry, Gorlaeus Laboratory, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands Received: April 6, 1994; In Final Form: December 6, 1994@
The isotropic phase and the biphasic region of salt-free native xanthan were investigated using 23Na NMR techniques. Visual inspection of the samples between crossed polarizers demonstrates the formation of anisotropic domains in the biphasic region beyond 9 g/L, and it was found to coincide with the observation of quadrupolar splitting (QS) of the 23NaNMR signal. Before QS and birefringence are observable, the first indication of domain formation is a strong increase of the fast component of the apparent transverse relaxation rate at 5 g/L. Hence, the boundary concentration of the isotropic phase, a limit for further conformational analysis by NMR, can now be established with more accuracy. The QS of the 23NaNMR signal depends on magnetic field strength, orientation, and time. A sample which was rapidly concentrated from the isotropic phase to the onset of the biphasic region displayed an increased relaxation rate, which decayed toward equilibrium in ca. 50 days. For the first time, using NMR, we were able to monitor the development of this slow equilibration. In the biphasic region, the QS reaches equilibrium after 1.5 year. Many observations of apparent hysteresis and irreversibilities in the literature may be related to such a slow equilibration.
Introduction Xanthan is the high molecular weight polysaccharide produced from fermentation of glucose by Xanthomonas campestris bacteria.' The cellulosic backbone contains on every second glucose residue a well-defined trisaccharide side chain consisting of mannose and glucuronic acid residue^.^^^ The side chain possesses weakly acidic carboxylic groups which can be titrated with NaOH to the desired degree of dissociation, introducing charges which are compensated for by the Na+ counterion. Without added salt, two conformational transitions can be observed at high temperatures when the charge density of the polymer is gradually increased by titrati~n.~ Possibly, unfolding of the side chains from the backbone and, next, partial dissociation of a double-helix structure or rupture of side-byside aggregated chains may take place.4 Xanthan is an intrinsically stiff p~lyelectrolyte~~~ (intrinsic persistence length, Lp = 1000-1200 A) and if isotropic solutions are concentrated, a phase separation takes place toward an anisotropic, cholesteric, liquid-crystalline phase.7 In the intermediate biphasic region, the solution is thought to be made up of a dispersion of anisotropic domains in an isotropic phase.8 The occurrence of anisotropic domains may be assessed by optical observation9(crossed polarizers), and it may be associated with the occurrence of weak gel properties, in viscosity7 and light-scattering lo experiments. In the literature, various values for the boundary concentration of the isotropic phase, i.e., the concentration up to which no birefringence or weak gel behavior is observed, are reported, depending on the molecular weight and concentration of added salt. For our biochemically purified native xanthan sample without added salt, the precise boundary concentrationof the isotropic phase cannot be deduced from the literature. The results of Sato et al." (M, = 6.14 x 1@) can be extrapolated with some difficulty to obtain a boundary concentration of less than 10 g/L. Sat0 et al. induced the phase separation by centrifugation. From the relative column heights after centrifugation, they deduced the relative volume of the isotropic phase and the 'Abstract published in Advance ACS Abstracts, February 15, 1995.
anisotropic phase in the biphasic region. The dependence of these relative volumes on the overall polymer concentration, the concentration of added salt, and the molecular weight has recently been investigated for sonicated xanthan."J2 Fair agreement between theory and experiment was obtained for these fractionated, relatively low molecular weight (lo5 < M, < lo6) samples in aqueous solution with 5-1000 mM added A theoretical approach of our results is complicated by the polydispersity (Mw/Mn 2-3, according to the literathe high molecular weight, and the therefore wormlike properties of the macromolecules used here. In this paper, a 23NaNMR method is presented to determine the threshold concentration for the phase separation of xanthan, which enables a forthcoming 23Naf counterion NMR study of the above-mentioned conformational transition^,'^ without interference of phase separation effects. The N M R method is compared with observations using crossed polarizers. In the biphasic region, the NMR behavior of the counterions is further investigated. Since detailed analysis of the 23Na NMR relaxation rates is omitted here for the following reasons, only a qualitative analysis is presented. (1) Multiple quantum filtered NMR experiments necessary to detect hidden quadrupolar splittings are severely hampered by the poor signal-to-noise ratio of these samples. (2) A vast amount of spectrometer time would be required to determine the spectral densities of the pure isotropic, the pure anisotropic, and the biphasic regions. (3) The measurements were initially hampered by the discovery of very slow equilibration after sample preparation, which imposed repetitive measurements for monitoring the development toward equilibrium.
Experimental Section Commercially available xanthan, Keltrol T (a product of Kelco, Lot 370109V), was biochemically purified according to the method described el~ewhere.~ Characteristics of the native sample are M, = (3 f 1) x lo7 and degrees of substitution of pyruvic acetal and acetyl Dpyr= 0.5 f 0.1 and Dace= 0.8 f 0.1. With the glucuronic acid residue and the pyruvic acetal substitution, our samples contain 1.5 carboxylic acid group per
0022-3654/95/2099-3743$09.00/0 0 1995 American Chemical Society
Bezemer and Leyte
3744 J. Phys. Chem., Vol. 99, No. 11, 1995 monomer (defined as two glucose residues and a side chain). In the presence of excess salt, the polymer consists of roughly 50 f 30 Kuhn segments, given the reported intrinsic persistence length5 of ~1OOOA. But even without added salt, the native polysaccharide is wormlike rather than rodlike. Deionized stock solutions were prepared from the original stock solution in 0.16 M NaC1, by extensive dialysis with 0.01 M HC1 (p.a. grade) or by treatment with an Amberlite MB-3 mixed-bed ion-exchange resin to obtain the protonated form, followed by titration with NaOH to a degree of dissociation 8 = 0.73. The Na+ counterion and xanthan concentrations in the deionized stock solutions were determined using a Perkin-Elmer 460 atomic absorption spectrometer and an Ionics 1555B carbon analyzer, respectively. The concentrationsof the Na+ counterion and the monomeric xanthan concentration of a 1 glL deionized xanthan solution are [Na+] = 1.4 mM and Cp = 1.2 mM. The residual amount of NaCl was determined with argentometric titrations and with 35Cl NMR spectroscopy, using calibrated samples of known NaCl concentration. The ratio of the concentrations of residual salt and polymer is [Cl-]/[Cp] = hence, the residual salt can be neglected. The concentration series was prepared by concentrating the deionized stock solution in a carefully rinsed rotatory evaporator at 30 "C and 15 mmHg. Under these no excess salt conditions, xanthan is just below the midpoint of the first conformational tran~ition.'~ Glasswork and quartz tubes used for preparation of the NMR samples were thoroughly rinsed in boiling NaHCO3 and EDTA solutions and cleaned with water purified by a Milli-Q (Millipore) apparatus (conductance < 1.5 pS cm-l). Either carefully rinsed quartz or Teflon NMR tubes were used of 8- and 6-mm diameter, respectively. 23NaNMR relaxation measurements and spectra were obtained at 71.4 MHz, using a modified Bruker S X P spectrometer equipped with a 6.3-T superconducting magnet (Oxford Instruments). The temperature of the samples was controlled using a Bruker B-VT 1000 air thermostat. The dependence of the 23Na quadrupolar splitting on the magnetic field was investigated with use of Bruker spectrometers operating at 2.1, 4.7, and 9.3 T, respectively. The relaxation curves were obtained using inversion recovery and spin-echo methods of 1024 data points each for R1 and Rz, respectively, and they were fitted using a MarquardtLevenberg algorithm using the functions of expressions l a and lb. The biexponential transverse relaxation curves were fitted with the theoretical weighting factor of 312 held fixed in both the isotropic and the biphasic solutions. In the biphasic region, roughly 200 OOO scans were needed to obtain spectra with an acceptable SIN ratio. The width, height, and phase of the satellites and central lines which determine the peak areas were obtained by a Lorentzian fit.
23Na Relaxation and Line Splitting near the Phase Separation. 1. Isotropic Phase. For I = 312 spins such as in 23Na, the relaxation rates are driven by the interaction of the nuclear quadruple moment with the fluctuating electric field gradient tensor at the site of the nucleus. The field gradients originate from the water molecules solvating the Na+ ions and from the charges on the polyelectrolyte. The relaxation rates are determined by the spectral densities, which are the Fourier transforms, at multiples of the Larmor frequency, of the autocorrelationfunction of the randomly fluctuating interaction. For semidilute polyelectrolyte solutions of several millimolar. polymer concentration, the 23Na+counterion NMR relaxation is generally outside the extreme narrowing limit. In theory, both the longitudinal and the transverse relaxation rates are biexponential. However, due to weighting factors in the
equations for the time-dependent magnetization after the n pulse and because the slow and fast rates are similar, Rt, FZ Rlf, often only the transverse relaxation rate is observed to be biexponential, cf eqs l a and lb. In these expressions, 4 is the flip M,(T)
- Mo =
+ 4/5exp(-R,,?)) = Mo sin 4(3/5exp(-R2+) + 2/5exp(-R,,?))
M,(cos MJT)
4 - l)('I5 exp(-Rl+)
(la) (lb)
angle of the preparation pulse. In isotropic solutions, the 23Na NMR signal is a superposition of two lines of width nc-1R2s and n-lRzf, which is usually observed as an apparent singlet line. From the particular 23Na+counterion relaxation behavior, several characteristics of the polymer solution can be derived. For instance, conformational transitions of DNA16 and PMA" have been monitored this way. From the dependence of the relaxation rates on the degree of dissociation, 8, in isotropic xanthan solutions, the effects of the conformational transitions were examined.15 For 8 > 0.5, the dependence on 8 is very weak. At the present degree of dissociation (8 = 0.73), increasing the xanthan concentration will affect the conformational equilibrium slightly, but the changes in the experimental relaxation rates are expected to be small. The observed changes in the relaxation rates in the biphasic region are, however, much larger than the effects which are observed during conformational transitions in the isotropic phase.l5 2. Anisotropic Phase. In the liquid-crystalline phase, the anisotropy results in a non-zero average of the electric field gradients at the nucleus, and a QS of the NMR signal is observed. For I = 312 spins such as for 23Na in anisotropic solution, the quadrupolar splitting is much smaller than the Larmor frequency, so the NMR signal becomes a triplet with an intensity ratio1*of 3:4:3. For the central line and satellites, relaxation rates are different due to the different spectral densities. 3. Biphasic Region. In the biphasic region, a mixture of the signals of the isotropic and the anisotropic phases is obtained. If the QS is smaller than the line width of the central line, the broad component of the hidden satellite yields an extra contribution to the apparent relaxation rate R2f. As the anisotropy increases with increasing concentration, the QS shows up and it becomes possible to investigate the effects of the magnitude and relative orientation of the magnetic field on the observed quadrupolar splitting. Furthermore, assuming that the signal intensity of the central line and the satellites is given by the relative amounts of the isotropic (miso) and the anisotropic phases (@'aniso), the amount of anisotropy can be calculated from the ratio of surfaces of the central line and the satellites with the following equations:
(3) in which Fc and F, denote the relative values of the surfaces of the central line and the two satellites with respect to the total surface, respectively.
Native Xanthan in the Biphasic Region
J. Phys. Chem., Vol. 99, No. 11, 1995 3745
T
A
a
a'
" 0.1
1
10
0.1
xanthan concentration (g/L)
1
10
100
1000
time(days)
Figure 1. Dependence of the relaxation rates, Rzf (O), Rzs (V),and RI (0) on the xanthan concentration (g/L). Solutions were wellequilibrated.
Figure 2. Evolution of R2f after concentrating an isotropic solution of 2 g/Lto 5.5 g/L.The horizontal dotted line indicates the fial value which is in agreement with the equilibrium value: 115 f 5 s-l, c.f. Figure 1.
The dependence of the amount of anisotropy in the biphasic region on the xanthan concentration and the molecular weight has been studied in the literature for Cs = 5 mM-1 M NaCl with the use of crossed p o l a r i ~ e r s . ~For ~ ' ~low ~ ~ ~molecular weight samples (-105), a linear growth of anisotropy with the concentration was found in all cases. In this paper, we will investigate the dependence of the amount of anisotropy on the polymer concentration in the salt-free case for the high molecular weight native sample by NMR.
TABLE 1: Results of Increasing the Xanthan
ReSUltS
Relaxation Rates and the Formation of Anisotropic
Domains. A first concentration series was prepared by partial lyophilization. After stirring for several weeks, the NMR samples were taken from these solutions. In Figure 1, the dependence of Rzf, Rzs, and R1 on the concentration of xanthan for these samples is shown. The relaxation rates of these solutions did not change over several months. From this figure, we deduce from the extreme growth of the apparent R z ~a threshold concentration of ca. 5 g/L,in agreement with extrapolated data of Sat0 et a l l 1(< 10 g/L). The drastic increase of the apparent Rzf above 5 g/L, in contrast with Rzs and R1, is consistent with the assumption of the onset of a QS smaller than the line width in anisotropic domains beyond 5 g/L. The increase of the apparent relaxation rates is much larger than the effects of conformational transitions only. The strong concentration depence of R2f beyond 5 g/L was used to investigate the development of a nonequilibrium xanthan solution obtained by rapidly concentrating a well-equilibrated isotropic sample (8 = 0.73; Cp = 2 g/L) to the biphasic region (5.5 g/L) in a rotatory evaporator at 30 "C and 15 mmHg. The result of these manipulations will be local differences in the concentration of the solution: the areas at the surface of the solution film are sheared by rotating, perhaps inducing some orientational order, and next they are concentrated by evaporation, whereas the bulk of the solution remains relatively dilute. From this concentrated solution, we immediately prepared a 23Na N M R sample, and the ensuing behavior of R2f is shown in Figure 2. At least 50 days are required to establish the equilibrium; the final value of 115 f 5 s-l, indicated by the dotted line, agrees well with the result of Figure 1 at 5.5 g/L. During the next 800 days, this value does not change, which demonstrates the long-term stability of the sample. For this sample, with 8 = 0.73 and Cp = a, the midpoint of the first conformational transition is found at 29 "C, according to the recently developed relation between Tm and It is therefore expected that
Concentration xanthan concn,
current obsvns with 23NaNMR and polarizers phase 0 < Cp < =5(6) normal relaxation isotropic biphasic =5 (6) < Cp < = 9 (10.8) enhanced app Rzf =9 (10.8) < Cp < QS (=OS-2 kHz) and biphasic Fz 45 (54) birefringence =45 (54) 4 c p not examined here anisotropic g L
increasing the concentration using a rotatory evaporator at 30 "C will not lead to dramatic conformational effects. If a disruption of (helical) structure takes place at this temperature, then increasing the concentration would lead to partial reconstitution of (helical) structure. Reconstitution of the polymeric structure is known to lead to an increase of relaxation rates for solutions of DNA.16 The observed decrease of the relaxation rate in Figure 2 is in contrast with these effects of conformational origin. The first and second conformational transitions of xanthan were recently found to be completely reversible in temperature-dependent circular dichroismZoand N M R ~tudies.'~ The most probable explanation for this slow relaxation process toward equilibrium would be that averaging out the concentration gradients after concentrating in the rotatory evaporator involves slow reorientation and translation processes in the lattice of intertwined polymeric chains. The areas of higher concentration experience some anisotropy, leading to the enhanced 23Na NMR relaxation rates. As the concentration differences diminish, the anisotropy also diminishes, and consequently, the relaxation rates decrease to the equilibrium value. The concentration of this sample is 5.5 g/L or 0.55%. In many studies of the conformational transition of xanthan, the concentration is of this order of magnitude. We expect that a similarly slow equilibrationprocess takes place if fresh xanthan solutions are prepared by dissolving the xanthan powder. Therefore, to our view, the interpretation of many observations of irreversibilities and apparent hysteresis in the literature may be complicated by this slow equilibration process. Evidence for a slow equilibration process near the mesophase formation was reported by Carnali in a viscosity experiment, although the time scale of the process could not be determined.8 Aging effects over several months for concentrations below 5 g/Lin solutions of ammonium acetate and urea, after lyophilization or precipitation of the native polysaccharide, were reported by Dintzis et Quadrupolar Splitting and Birefringence. The formation of liquid-crystalline domains was inferred from the observation
Bezemer and Leyte
3746 J. Phys. Chem., Vol. 99,No. 11, 1995 2ooo
1500h
T
T.
2
1 1000-
1 1
-2000
0
-1000
1000
2
0
2000
8
6
4
10
magnetic field (Tesla)
frequency offset (Hz)
Figure 3. 23NaN M R spectrum of a 22 g/L solution at 71.4 MHz and 25 "C: 208 OOO scans were recorded.
Figure 5. Dependence of the quadrupolar splitting on both the strength and the orientation of the magnetic field. (0)parallel orientation; (0) perpendicular orientation. Cp = 10 g L (12 mM); [Naf] = 14 mM.
imn
.-""1
loo
I
i
1500
0
200
400
600
800
time (days)
I
o
m 0
Figure 4. Evolution of the observed quadrupolar splitting. Open
circles, initial evolution; closed circles, after shaking the sample vigorously. Cp = 18 g/L (21.6 mM); [Naf] = 25.2 mM. The lines are drawn as an aid to the eye. of quadrupole splitting and macroscopic patterns observed between crossed polarizers beyond 9 g/L. Below this concentration, there is no evidence for anisotropy. A schematic representation of the observations related to the phase separation is given in Table 1. The birefringence patterns vary from sample to sample, yielding large amounts of small patches and a smaller amount of larger patches. Figure 3 shows the 23Na spectrum of a 22 g/L, xanthan solution at equilibrium, 1.5 years after sample preparation (rotatory evaporation at 30 O C ) : ma+] = 31 mM, 208 0oO scans. Figure 4 shows the development of the QS after preparation of 18 g/Lfrom 3 gjL (rotatory evaporation at 30 "C),similar to the experiment of Figure 2. The initial value is 300 Hz, and it develops in time. The FJFc ratio is very small and also develops in time. The QS at 18 g/L does not reach equilibrium in 1 year, but in both Figures 2 and 4,the quantities develop in time. Possibly, the size of the anisotropic domains increases in time. After 1 year, the sample was vigorously shaken on a vortex machine after which the QS disappeared completely. The QS returned quickly, and as if nothing had happened, the apparent equilibrium value was approached, as if large domains had been randomized but not destroyed. In Figure 5 , the dependence of the quadrupolar splitting on the applied magnetic field is shown. There are three data points at parallel orientation of tube and field, and there is one point of perpendicular orientation. The quadrupolar splitting of the latter point is much higher than expected from the other points,
0 10
20
30
40
50
xanthan concentration (g/L)
Figure 6. Dependence of the amount of anisotropy (0)in percent (left y axis) and the quadrupolar splitting (0)in Hz (right y axis) on the
initial xanthan concentration. The lines are drawn as an aid to the eye. Measurements were made more than 1 year after sample preparation in view of the results of Figure 4. which shows that the anisotropic domains possess some preferential orientation in the tube. Quartz and Teflon NMR tubes produced the same results. The strong dependence of the QS on the magnetic field strength for the parallel geometry is noteworthy: increasing the field strength leads to increased alignment of xanthan in the field. From the dependence at parallel orientation, a lower bound of -0.5 kHz is estimated for the QS at zero field strength. The dependence of the amount of anisotropy and the quadrupolar splitting on the xanthan concentration are shown in Figure 6 . Both quantities show a similar development: between roughly 5 and 10 glL,they increase sharply, and beyond roughly 10 g/L, the increase is almost linear in the concentration. The steep initial increase of the anisotropy above the threshold concentration is in contrast with other observation^.^**^
Conclusions With the use of the 23Na relaxation rates, a threshold concentration of 5 g/Lis established for the isotropic phase of our biochemically purified native xanthan. Near the phase separation, at least 50 days are required to obtain solutions at equilibrium. At higher concentration where QS is observed, the equilibration may take 1.5 years. It is expected that at slightly lower concentrations a similarly slow behavior exists.
Native Xanthan in the Biphasic Region Several cases of irreversibilities and apparent hysteresis reported in the literature may be connected with this effect. The enhancement of the apparent relaxation rate (&f) above 5 g/L can be related to the formation of anisotropic domains, and evantually beyond 9 giL, the latter gives rise to quadrupolar splitting and birefringent patterns between crossed polarizers. The orientational ordering in the biphasic region depends on both magnetic field strength and orientation, and it depends on time. Just beyond the threshold concentration, the formation of anisotropic domains shows a steep initial increase, in contrast with earlier observations in the literature on sonicated xanthan with added salt. Extrapolation of the dependence of anisotropy on the concentration yields % 45 giL for the 100% anisotropic phase, which completes the list of observations related to the phase separation given in Table 1.
Acknowledgment. We thank Mr. J. B. Ubbink and Mr. J. A. de Kooker for performing some of the tedious NMR measurements. Dr.J. de Bleijser is thanked for his technical assistance and continuous interest in this work. Dr. J. R. C. van der Maarel is thanked for his useful comments on the manuscript. This work was supported by The Netherlands Foundation for Chemical Research (SON) with financial aid from The Netherlands Foundation for the Advancement of Pure Research (NWO).
J. Phys. Chem., Vol. 99, No. 11, 1995 3741
References and Notes (1) Jeanes, A.; Pittsley, J. E.; Senti, F. R. J. Appl. Polym. Sci. 1961,5, 519. (2) Jansson, P. E.; Kenne, L.; Lindberg, B. Curbohydr. Res. 1975.45, 275. (3) Melton, L. D.; Mindt, L.; Rws, D. A.; Sandersen, G. R. Curbohydr. Res. 1976, 46. 245. (4) Bezemer, L.; Ubbink, J. B.; de Kooker, J. A.; Kuil, M. E.; Leyte, J. C. Mucromolecules 1993, 26, 6436-6446. (5) Sato, T.; Norisuye, T.; Fujita, H. Polym. J. 1984, 16, 341. (6) Sho, T.; Sato, T.; Norisuye, T. Biophys. Chem. 1986, 25, 307. (7) Maret, G.; Milas, M.; Rinaudo, M. Polym. Bull. 1981,4 , 291. (8) Carnali, J. 0. J. Appl. Polym. Sci. 1991,43,929. (9) Milas, M.; Rinaudo, M. ACS Symp. Ser. 1981, 150, 25. (10) Southwick, J. G.; Jamieson, A. M.; Blackwell, J. Mucromolecules 1981,14, 1732. (11) Sato, T.; Kakihara, T.; Teramoto, A. Polymer 1990, 31, 824. (12) Inatomi, S. I.; Jinbo, Y.; Sato, T.; Teramoto, A. Macromolecules 1992, 25, 5013. (13) Dintzis, F. R.; Babcock, G. E.; Tobin, R. Carbohydr. Res. 1970, 13, 257. (14) Holzwarth, G. Carbohydr. Res. 1978, 66, 173. (15) Bezemer, L.; et al. To be published. (16) van Dijk, L.; Gruwel, M. L. H.; Jesse, W.; de Bleijser, J.; Leyte, J. C . Biopolymers 1987, 26, 261. (17j v k der Klink, J. J.; Zuiderweg, L. H.; Leyte, J. C. J . Chem.Phys. 1974, 60, 2391. (18) Abragam, A. The Principles of Nuclear Magnetism, 1st ed.; Oxford at Clarendon: Oxford, 1973; Chapter 7. (19) Bezemer, L.; Kuil, M. E.; Leyte, J. C. Curbohydr. Res. 1994,263, 197. (20) Bezemer, L.; van Haeringen, B.; Kuil, M. E.; Leyte, J. C.; van Grondelle, R. Submitted for publication. Jp940865X
