J. Phys. Chem. B 2000, 104, 3405-3406
3405
Cluster Formation in Aqueous Electrolyte Solutions Observed by Dynamic Light Scattering Yannis Georgalis,*,† Andrzej M. Kierzek,‡ and Wolfram Saenger† Institut fu¨ r Kristallographie Freie UniVersita¨ t Berlin, Takustrasse 6, 14195 Berlin, Germany, and Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland ReceiVed: January 10, 2000
Using dynamic light scattering we show that aqueous super- to undersaturated solutions of NaCl, (NH4)2SO4, and Na-citrate contain submicrometer size clusters at room temperature. The particle size distributions deduced by Laplace inversion of the spectra contain two predominant components. The smaller components with radii below 1 nm are attributed to mixtures of solvated ions and the larger with radii between 50-500 nm to ion clusters. Knowledge of the mesoscale structure of concentrated electrolyte solutions may be necessary to describe effective protein-protein interactions which are of importance in biochemical applications such as protein crystallization.
Introduction It is commonly accepted that undersaturated aqueous solutions of salts contain dissociated and hydrated ions. This view contrasts evidence gained from conductivity measurements in alkali halides1 and from studies of (NH4)H2PO4,2,3 citric acid,4 and sucrose solutions5 where cluster formation was assumed to explain experimental observations. Raman spectroscopy of aqueous salt solutions has suggested the presence of clusters in NaNO3,6 KH2PO4, and (NH4)H2PO4.7 Recent theoretical studies8 indicated that (NH4)2SO4 may form clusters in water, but as yet no experimental results corroborate the computational findings. Using dynamic light scattering we show here directly that NaCl, (NH4)2SO4, and Na-citrate (salts commonly used to induce protein crystallization9) form at least two, spatially welldistinguished components in both aqueous super- and undersaturated solutions. Materials and Methods Dynamic light scattering (hereafter abbreviated DLS, see ref 10 and references therein) is the best technique for directly detecting cluster populations in solution. Monochromatic laser light scattered by a solution at a given angle is analyzed in terms of the temporal field autocorrelation function g(1)(τ) sampled over a wide range of times τ. Assuming that the characteristic relaxation times typify diffusion processes, the obtained spectra can be Laplace inverted to provide the most probable distribution of hydrodynamic radii N(Rh). All three salts were employed in their “Suprapur” form (purity 99.5%, Merck Darmstadt). Supersaturated solutions were prepared in fresh, triply distilled water, equilibrated overnight, and centrifuged for 5 min at 13 000 rpm in a bench-top centrifuge prior to DLS experiment. The molarities quoted in Figures1 and 2 correspond to those reported in standard tables.11 Light scattering experiments were performed in 10 mm diameter round quartz precision light scattering cells at an angle * To whom correspondence should be addressed. Telephone: ++04930838-4588. Fax: ++04930-838-6702. E-mail:
[email protected]. † Institut fu ¨ r Kristallographie Freie Universita¨t Berlin. ‡ Institute of Biochemistry and Biophysics, Polish Academy of Sciences Warsaw Poland.
Figure 1. Field autocorrelation functions g(1)(τ) of aqueous solutions (A) NaCl, (B) (NH4)2SO4, and (C) Na-citrate, plotted as a function of sampling time τ. Open symbols indicate supersaturated solutions (O) and dilutions from the same stock, (0, 4, 3). Closed symbols (b) indicate filtered (Minisart, 200 nm pore size sterile filters) supersaturated solutions. All experiments were conducted at 293.2 K. The lines through the points are the ALV-CONTIN fits to the data, (see Figure 2). Concentrations are given individually for each salt in the inset.
of 45° and at 293.2 K. The apparatus employed was an ALV/ SP-86 spectrogoniometer (ALV, Langen, Germany) equipped with a Spectraphysics Stabilite 2017 Ar+ laser operating at a
10.1021/jp000132e CCC: $19.00 © 2000 American Chemical Society Published on Web 03/29/2000
3406 J. Phys. Chem. B, Vol. 104, No. 15, 2000
Figure 2. Mass-squared weighted radii distributions, M2 P(q Rh) N(Rh) normalized to unity, plotted as a function of the hydrodynamic radii, Rh, of (A) NaCl, (B) (NH4)2SO4, and (C) Na-citrate computed from the correlograms shown in Figure 1A to 1C as described in the Materials and Methods section.
wavelength of 488 nm. Scattering was monitored using an ALV/ SO-SIPD double photomultiplier detection unit. After amplification and discrimination, signals were directed to an ALV-5000/ E-FAST digital correlator and spectra are recorded on 255 channels, quasi logarithmically spaced in time. Usually 100 correlograms were acquired for 30 s each and averaged after discarding few outliers which were due to random transients of big clusters through the scattering volume. Mass-squared and form factor weighted radii distributions were computed using the regularized inverse Laplace transform, ALV-CONTIN12 package. Due to the unknown cluster morphology, the form factors P(q Rh) were approximated by those of hard spheres. Results and Discussion Figure 1 shows typical correlograms for aqueous NaCl, (NH4)2SO4, and Na-citrate solutions and dilutions from samples mildly centrifuged to remove residual dust contamination (open symbols). They indicate the presence of two predominant componets well separated in time. The smaller component corresponds to the mixture of solvated ions. whereas the larger one corresponds to molecular clusters and can be removed by a simple filtration (filled circles). Laplace inversion of the spectra in Figure 1 shows particle distributions with hydrodynamic radii below 1 nm and 300 to 500 nm for NaCl, 150 to 300 nm for (NH4)2SO4, and 50 to 150 nm for Na-citrate, Figure 2. From
Letters the filtered supersaturated solutions we were able to deduce radii distributions only for Na-citrate because the clusters are small enough to pass through the filter. As roughly estimated from the peak areas in Figure 2, the numbers of the large clusters are between one part per 10 thousands to one part per million of dissociated species, resembling a considerable population since concentrations are in the M range. More exact estimates will require knowledge of the cluster morphology. Our findings imply that in aqueous solutions simple electrolytes such as NaCl aggregate even at moderate concentrations. The observed clusters are too large to be detected within the spatial scales explored by X-rays and neutrons, but they are immediately detectable with visible laser-light, under the prerequisite that autocorrelation functions can be sampled over several decades in time. The exact nature of the interactions involved in cluster formation is not clear. It may be attributed to (1) direct electrostatic interactions among ions of opposite charge, (2) hydrophobic interactions among ions caused by exclusion of ions by water, and (3) “cement” water molecules that can be responsible for interaction between ions bearing identical charges. In the case of anions, “cement” water molecules direct two hydrogen atoms toward anions thus forming a kind of bridge. Cations can interact in similar fashion, dividing appropriately the two free electron pairs of oxygen atoms.13 If electrolytes are used to enhance protein crystallization, minute quantities of such clusters will modify drastically the solution properties since the clusters will act as excluded volume agents influencing both the diffusion of the protein molecules and the chemical potential of any species involved in the crystallization process. Excess pressure, free energy and chemical potential of these solutions are expected to undergo drastic changes, and protein molecules will nucleate in an unpredictable manner, as is the case in real laboratory life. We have examined a variety of other electrolytes using DLS and found them to form similarly sized clusters. A detailed account on these experiments is under preparation. Acknowledgment. Financial support to Y.G. by the Deutsche Forschungsgemeinschaft, the European Community Biocrystallogenesis network, and the Fonds der Chemischen Industrie is acknowledged. We also thank Mrs. H. Evers for expert technical assistance. References and Notes (1) Chatterji, A. C.; Singh, R. N. J. Phys. Chem. 1958, 62, 14081411. (2) Mullin, J. W.; Raven, K. D. Nature 1961, 4772, 251. (3) Mullin, J. W.; Raven, K. D. Nature 1962, 195, 35-38. (4) Mullin, J. W.; Leci, C. Philos. Mag. 1969, 19, 1075-1077. (5) Allen, A. T.; McDonald, M. P.; Nicol, W. M.; Wood, R. M. Natural Phys. Sci. 1972, 235, 36-39. (6) Rusli, I. T.; Schrader, G. L.; Larson, M. A. J. Cryst. Growth 1989, 97, 345-351. (7) Ceretta, M. K.; Berglund, K. A. J. Cryst. Growth 1987, 84, 577588. (8) Smith, P. E. J. Phys. Chem. B 1999, 103, 525-534. (9) Jancarik, J.; Kim, S.-H. J. Appl. Crystallogr. 1991, 24, 409-411. (10) Brown, W., Ed. Dynamic Light Scattering, the Method and Some Applications; Oxford University Press, Oxford, U.K., 1993. (11) Handbook of Chemistry and Physics, 65th ed.; CRC Press: Boca Raton, FL, 1984-1985. (12) Provencher, S. W. Comput. Phys. Comm. 1982, 27, 213-227. Provencher, S. W. Comput. Phys. Comm. 1982, 27, 229-242. (13) Degre´ve, L.; da Silva, F. L. J. Chem Phys. 1999, 110(6), 30703078.
