Langmuir 2000, 16, 9261-9266
9261
Factors Influencing Additive Interactions with Calcium Hydrogenphosphate Dihydrate Crystals M. Sikiric´,*,† V. Babic´-Ivancˇic´,† O. Milat,‡ S. Sarig,§ and H. Fu¨redi-Milhofer§ i er Bosˇ kovic´ ” Institute, Zagreb, Croatia, Institute of Physics, University of Zagreb, “Rud Zagreb, Croatia, and Casali Institute of Applied Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel Received May 22, 2000. In Final Form: August 30, 2000 The effect of aspartic acid, glutamic acid, hexaammonium tetrapolyphosphate, calcium phytate, and polyaspartic acid on calcium hydrogenphosphate dihydrate (CaHPO4‚2H2O, DCPD) crystal growth morphology was studied. The mechanism of additive-DCPD crystal interaction depends on the size and structure of the additive molecule and the structural fit between the organic molecule and the ionic structure of particular crystal face. Small molecules with low charge (aspartic and glutamic acid) do not have a significant effect, whereas small molecules with several functional groups (citrate and hexaammonium tetrapolyphosphate) adsorb on lateral faces primarily by electrostatic interactions. Molecules with hindered structures (phytate) and macromolecules (polyaspartic acid) specifically adsorb on the dominant (010) face, for which a certain degree of structural fit is necessary.
* To whom correspondense should be addressed at the Laboratory i er Bosˇkovic´” Institute, Bijenie`ka 54, of Radiochemistry, “Rud 10 000 Zagreb, Croatia. E-mail:
[email protected]. i er Bosˇkovic´” Institute. † “Rud ‡ Institute of Physics, University of Zagreb. § The Hebrew University of Jerusalem.
inhibitor adsorbs on some crystal faces, but not on others, it will retard crystal growth in the direction perpendicular to that face. The affected face will appear larger than in nonaffected crystals and, as a result, crystal morphology will change. Thus, by comparing the morphology of crystals grown in the presence and absence of an additive and observing changes in growth morphology, the mechanisms of crystal-additive interactions can be deduced. The aim of this study was to determine the factors underlying calcium hydrogenphosphate dihydrate (DCPD) interactions with several structurally different additives: glutamic and aspartic acid, sodium citrate, hexaammonium tetrapolyphosphate, calcium phytate, and polyaspartic acid. DCPD is one of the most important phosphate minerals. It can be found in small proportions in urinary and dental stones8,9 and has an important role as a precursor in their formation.10 It is also used for different applications in the cosmetics and pharmaceutical industries. Glutamic and aspartic acid, both dicarboxylic amino acids, are the most abundant amino acids in mucoproteinlike materials, which constitute the organic part of calcium stones.11 Citrate, a tricarboxylic acid, is a urinary constituent that is also used in the prevention of kidney stone formation.12 Hexaammonium tetrapolyphosphate is a known crystal growth inhibitor and is commercially used in the prevention of scale formation on heat exchanger surfaces in industrial water systems. Phytate, myoinositol hexaphosphate, is also a potent crystal growth inhibitor,13,14 found in nature in cereals and cells. It may be involved in calcium metabolism.15 Polyaspartic acid, an acidic macromolecule, has a partial β-sheet conformation
(1) Fu¨redi-Milhofer, H.; Sarig, S. Prog. Cryst. Growth Charact. Mater. 1996, 32, 45. (2) Addadi, L.; Berkovich-Yellin, Z.; Weissbuch, I.; van Mill, J.; Shimon, L. J. W.; Lahav, M.; Leiserowitz, L. Angew. Chem., Int. Ed. Engl. 1985, 24, 466. (3) Addadi, L.; Weiner, S. Angew. Chem., Int. Ed. Engl. 1992, 31, 153. (4) Addadi, L.; Weiner, S. Proc. Natl. Acad. Sci. (USA) 1985, 82, 4110. (5) Berman, A.; Addadi, L.; Weiner, S. Nature 1988, 331, 546. (6) Fu¨redi-Milhofer, H.; Moradian-Oldak, J.; Weiner, S.; Veis, A.; Mintz, K. P.; Addadi, L. Connec. Tissue Res. 1994, 30, 251. (7) Tunik, L.; Addadi, L.; Garti, N.; Fu¨redi-Milhofer, H. J. Cryst. Growth 1996, 167, 748.
(8) Daudon, M.; Donsimoni, R.; Hennequin, C.; Fellahi, S.; Le Moel, G.; Paris, M.; Troupel, S.; Lacour, B. Urol. Res. 1995, 23, 319. (9) Werness, P. G.; Bergert, J. H.; Smith, L. H. J. Cryst. Growth 1981, 53, 166. (10) Pak, C. Y. C. J. Clin. Invest. 1969, 48, 1914. (11) Garcia-Ramos, J. V.; Carmona, P. J. Cryst. Growth 1982, 57, 336. (12) Tomson, C. R. V. Br. J. Urol. 1995, 76, 419. (13) Amjad, Z. Can. J. Chem. 1988, 66, 2181. (14) Freche, M.; Lacout, J.-L. Eur. J. Solid State Inorg. Chem. 1993, 30, 847. (15) Thomas, W. C.; Tilden, M. T. John Hopkins Med. J. 1972, 131, 133.
Introduction Interactions between inorganic crystals and organic molecules and macromolecules underlie crystallization processes in various fields (geology, biological and pathological mineralization, scale formation in heating systems, etc.1) and have therefore attracted the attention of scientists for a number of years. Only in recent years have attempts been made to define the general principles governing such interactions and to use this knowledge to propose additives with specific influences on the crystallization processes of interest.2 As part of such endeavors, scientists have been trying to understand biological mineralization processes, in which organic macromolecules and/or molecular assemblies are utilized to control the size, shape, and orientation of inorganic crystals.3 In 1985, Addadi and Weiner proposed4 a new method for the in vitro study of the effect of acidic macromolecules on the crystallization of biominerals, which has by now been implemented to investigate the interactions of a variety of additives with growing crystals.5-7 The principle of the method is based on the observation that the morphology of a growing crystal is determined by the relative growth rates of its faces. The faster the growth rate in the direction perpendicular to a particular face, the smaller that face appears. If an effective growth
10.1021/la000704m CCC: $19.00 © 2000 American Chemical Society Published on Web 11/03/2000
9262
Langmuir, Vol. 16, No. 24, 2000
Sikiric´ et al.
Figure 1. Scanning electron micrographs of calcium hydrogenphosphate dihydrate crystals obtained (a) in the control system c(CaCl2) ) c(Na2HPO4) ) 0.021 mol dm-3 and c(NaCl) ) 0.3 mol dm-3, pH 5.5, 37 °C (without additives), and in the presence of (c) citrate, ccit ) 3 × 10-5 mol dm-3; (d) and (e) hexammonium tetrapolyphosphate, cHATP ) 6 × 10-7 and 2 × 10-6 mol dm-3, respectively; (f) phytate, cPhy ) 1 × 10-7 mol dm-3; and (g) polyaspartate, cpAsp ) 1 × 10-5 mol AA dm-3. (b) Miller indices of lateral faces of crystal with the most dominant morphology.
and therefore represents an appropriate model molecule to study the correlation between the crystal lattice and an acidic polymer of defined structure. Materials and Methods Analytical grade chemicals and deionized water were used. Calcium chloride and sodium phosphate stock solutions were prepared from CaCl2‚2H2O and Na2HPO4‚2H2O (Merck), respectively, which were dried overnight in a desiccator over silica gel. Both stock solutions contained 0.3 mol dm-3 NaCl, and 0.01% sodium azide was added to the phosphate stock solution to avoid bacterial contamination. Anionic and cationic reactant solutions used for crystal preparation were diluted from the respective stock solutions. In the anionic reactant solution, the pH was adjusted to 5.5 with HCl. Commercially available sodium citrate (BDH Laboratory Supplies), aspartic acid, glutamic acid, hexaammonium tetrapolyphosphate, calcium phytate and polyaspartic acid (MW 5-15 kDa, Sigma) were used as additives. Stock solutions of the additives were prepared in the same way as the phosphate stock solution. Crystallization Experiments. Crystallization systems were prepared under controlled conditions by rapid mixing of equal volumes of the anionic and cationic reactant solutions. Crystals were grown without further stirring at 37 °C. The initial conditions were pH 5.5, c(CaCl2) ) c(NaHPO4) ) 0.021 mol dm-3, and c(NaCl) ) 0.3 mol dm-3. The respective additive was added to the anionic component prior to pH adjustment. In the control system, calcium and phosphate concentrations were the same, but no additive was added. After commencement of the reaction,
changes in pH were monitored, thus providing a qualitative estimate of the rate of crystallization.16 In addition, samples were taken at given time intervals for observation by light and polarized optical microscopy. The time that elapsed before detection of the first crystals was between 1 and 24 h, depending on the nature and concentration of the additive. When the crystals were grown, the reaction was discontinued and samples were taken for analysis (for specific times, see the Results section). Crystal Characterization. Crystals were characterized by i) optical microscopyssamples of suspensions were taken at given time intervals, observed, and photographed by light and polarized microscopy using a Nikon AFX IIA optiphot photomicroscope. ii) SEMssamples for SEM were prepared by placing a drop of the suspension on a stub covered with carbon glue and removing the supernatant with filter paper. The remaining crystals were washed by placing a drop of deionized water and removing the excess water with filter paper. After drying the sample in a desiccator over silica gel for several hours, the stubs were gold plated, and crystals were observed by a JEOL JXA-8600 “Superprobe” scanning electron microscope. iii) powder X-ray diffraction patterns (XRDP)scrystals were filtered through a 0.45-µm Millipore filter, washed with deionized water, dried in a desiccator over silica gel, gently crushed, and spread on a specimen holder using a spatula. Powder X-ray diffraction patterns were obtained using a Phillips PW diffractometer with graphite filtered CuKR radiation. (16) Despotovic´, R.; Filipovic´, N.; Fu¨redi-Milhofer, H. Calc. Tissue. Res. 1975, 18, 13.
Additive Interactions with Calcium Crystals
Langmuir, Vol. 16, No. 24, 2000 9263
Figure 2. X-ray powder diffraction patterns (graphite-filtered CuKR) of calcium hydrogenphosphate dihydrate crystals obtained (a) in the control system, c(CaCl2) ) c(Na2HPO4) ) 0.021 mol dm-3 and c(NaCl) ) 0.3 mol dm-3, pH 5.5, 37 °C (without additives), and in the presence of (b) citrate, ccit ) 1 × 10-4 mol dm-3; (c) hexammonium tetrpolyphosphate, cHATP ) 8 × 10-7 mol dm-3; (d) phytate, cPhy ) 2 × 10-7 mol dm-3; and (e) polyaspartate, cpAsp ) 8 × 10-6 mol AA dm-3. (f) An ideal DCPD powder pattern (according to ref 19). iv) FTIRssamples were prepared as for X-ray powder diffraction. FTIR spectra were obtained with an ATI Mattson Genesis Series FTIR spectrophotometer, using KBr micropellets. v) Weissenberg diffractogramssoscillation and Weissenberg diffraction patterns were obtained with a Siemens-Halske Weissenberg camera, φf ) 2rf ) 57 mm, using Ni-filtered CuKR radiation. XRDP were recorded from single crystals that were selected out of the batch on the basis of representative morphology and large enough size. Each crystal was aligned so that its largest dimension coincided with the oscillation axis, which was found to correspond to the [001] crystallographic axis. Such alignment provided unambiguous determination of the direction of the [010] axis using Weissenberg photographs of the (hk0)* plane. vi) determination of crystal morphologysDCPD crystals formed as (010) plates as verified by Weissenberg XRDP. The Miller indices of the side faces were determined from SEM micrographs of individual crystals oriented with their (010) faces approximately in the plane of the picture. The angles between side faces of such crystals were measured and compared to the angles between the corresponding planes on the reciprocal lattice (calculated according to data from Curry and Jones17).4 vii) supersaturation in the control system and distribution curves of glutamic and aspartic acid species were calculated with a previously described18 computer program.
Results Control System. In the control system, DCPD crystals appeared after a 1 h reaction time in the form of relatively large platelets (approximately 200 × 100 µm and 5-µm thick), as shown in Figure 1a. The crystal morphology (17) Curry, N. A.; Jones D. W. J. Chem. Soc. A 1971, 3725. (18) Tonkovic´, M.; Sikiric´, M.; Babic´-Ivancˇic´, V. Colloids Surf., A 2000, 170, 107.
was typical for DCPD, which has been reported to grow in the form of thin platelets with prominent (010) and lateral (h0l) faces.19,20 The Miller indices of the prominent faces, as determined by the Weissenberg camera, were (010). The Miller indices of the lateral faces of crystals with the most dominant morphology were determined by combining this result with SEM micrographs (Figure 1b). The initial supersaturation in the control system was 10. A typical X-ray diffraction powder pattern of DCPD crystals obtained in the control system is given in Figure 2a. The positions of the peaks perfectly fit the corresponding d-parameters of the DCPD lattice, but the line intensities depend on the sample and the methods of its preparation. In particular, the peak intensity of the (020) line versus the (021) line intensity in Figure 2a is much higher than the intensity ratio of these two lines for the ideal DCPD powder (Figure 2f), with an isotropic distribution of grain orientations.19,20 A more or less enhanced peak intensity is present for the whole sequence of (0k0), k even, lines in all patterns of Figure 2. Generally, higher intensities of groups of lines in powder XRDP is a feature typical for deviation from an isotropic orientation of crystallite grains. In the case of our DCPD powder samples, which were prepared by gently crushing the precipitated crystals, this feature is interpreted in terms of crystallite morphology; namely, the higher the number of platelike grains in the sample, the higher the fraction of the (010) (19) Elliott, J. C. Structure and Chemistry of the Apatites and Other Calcium Orthophosphates; Elsevier: Amsterdam, 1994. (20) Powder Diffraction File Sets 16-18, Inorganic Volume; Joint Committee on Powder Diffraction Standards: Swarthorne, PA, 1974.
9264
Langmuir, Vol. 16, No. 24, 2000
Sikiric´ et al.
Figure 3. Packing arrangement of calcium hydrogenphosphate dihydrate crystals viewed on the (100) plane drawn according to data in ref 17.
planes preferentially aligned along the specimen surface, and consequently, the more prominent the intensity of the corresponding series of (0k0), k even, lines in the powder XRDP. According to their influence on DCPD crystal morphology, the investigated additives can be divided into three groups: i) glutamic and aspartic acid: Glutamic and aspartic acid showed no significant effect on the rate of growth and growth morphology of DCPD crystals. In the investigated concentration range (3 × 10-5 to 5 × 10-4 mol dm-3), crystals such as those obtained in the control system appeared after 1 h. ii) citrate ions and hexaammonium tetrapolyphosphate (HATP): The influence of citrate ions was investigated in the concentration range 1 × 10-5 to 5 × 10-4 mol dm-3. Up to 1 × 10-4 mol dm-3, citrate ions affected the morphology but not the DCPD growth rate, that is, pH curves were similar as in the control system but crystals that appeared after 1 h were typically rodlike (Figure 1 c). When the concentration of citrate ions exceeded 1 × 10-4 mol dm-3, both the morphology and the crystal growth rate were affected, that is, rodlike crystals could be observed only 24 h after sample preparation. The influence of HATP was investigated in the concentration range 1 × 10-8 to 5 × 10-6 mol dm-3. Up to 3 × 10-7 mol dm-3, the additive did not affect crystal morphology or growth rate, whereas at HATP concentrations above 3 × 10-7 mol dm-3, gradual changes of the growth morphology, from platelets to rodlike crystals, were observed (Figure 1d and 1e). Crystal growth was also inhibited, proportional to the additive concentration. The prominent face of the crystals obtained in the presence of both additives was (010), as determined repeatedly by Weissenberg photographs. The rodlike appearance of the crystals indicates that these two additives adsorbed preferentially on the lateral faces and retarded crystal growth in directions perpendicular to these faces. In the case of citrate, the XRDP (Figure 2b) fits very well the ideal powder pattern (Figure 2f19,20), revealing no preferential orientations of the rodlike crystallites. However, for lower HATP concentrations, the retention of a platelike crystallite morphology is strongly indicated by the pronounced peak intensities of the characteristic series of (0k0), k even, lines in the XRDP of Figure 2c.
Figure 4. (a) Schematic representation of phytate interaction with the (010) face of DCPD crystals. Distances between phosphate groups in the phytate molecule (6.19 Å) correspond to the distances between calcium ions in one layer of the DCPD structure (6.24 Å). (b) Schematic representation of polyaspartate interaction with the (010) face of DCPD crystals. Distances between carboxylic groups in the polyaspartic β-sheet (6.9 Å) correspond to the distances between neighboring calcium ions from two adjacent layers that constitute one Ca-HPO4 layer (6.95 Å). The DCPD packing arrangement is drawn according to data in ref 17.
iii) phytate and polyaspartic acid: DCPD crystal growth was investigated in the presence of phytate in the concentration range 1 × 10-8 to 1 × 10-5 mol dm-3. Up to a concentration of 5 × 10-8 mol dm-3, there was no significant influence of the additive on the morphology or growth rate of DCPD crystals. In concentration range 8 × 10-8 to 1 × 10-5 mol dm-3, phytate inhibited the crystal growth rate preferentially in the direction perpendicular to (010), so that large crystals with the same basic orientation as in the control system were obtained (Figure 1f). At phytate concentrations 5 × 10-7 to 1 × 10-6 mol dm-3, such crystals were obtained after 2 days and at concentrations 3 × 10-6 to 1 × 10-5 mol dm-3 after 6 days in contact with the mother liquid. The corresponding XRDP in Figure 2d reveals especially high intensities of the characteristic series of (0k0), k even, lines, indicating that, despite sample preparation by
Additive Interactions with Calcium Crystals
Langmuir, Vol. 16, No. 24, 2000 9265
Figure 5. Schematic representation of the interaction between calcium hydrogenphosphate dihydrate crystals and the additives investigated.
crushing, a very large number of DCPD crystallites, although smaller, remained in a platelike shape and therefore spread preferentially parallel to the specimen surface. The effect of polyaspartate was investigated in the concentration range 0.2 to 20 × 10-6 mol AA dm-3 (calculated assuming an average molecular weight of 10 kDa). At concentrations 5 × 10-6 < c < 7.5 × 10-6 mol AA dm-3, very large crystals with the same basic orientation as in the control system were obtained within 24 h (Figure 1g). At concentrations above 1.5 × 10-5 mol AA dm-3, crystallization of DCPD was completely inhibited, that is, even after several days no crystallization was apparent. Note that, in contrast to the crystals with a very smooth (010) facet, obtained in the presence of phytate, the crystals obtained in the presence of polyaspartate were striated, having a leaf-like appearance with fragmented stripes (Figure 1g). This difference in morphology is reflected by the XRDP (Figure 2e), in which the line intensities fit the intensities of an ideal isotropic powder XRDP (Figure 2f19,20). It seems that the absence of a preferred orientation of grains in these samples is due to easy fragmentation of the larger striated crystals. Discussion To fully understand interactions of such a variety of additives with DCPD crystals, the ionic structure of the affected crystal faces and the molecular structure of the additives has to be taken into account. DCPD crystallizes in the monoclinic system (a ) 5.812 Å, b ) 15.810 Å, c ) 6.239 Å, β ) 116°), space group Ia.17 The unit cell consists of alternating bilayers oriented parallel to the (010) plane, one of them consisting of calcium and hydrogenphosphate ions and the other one of water molecules (Figure 3). DCPD growth morphology reflects the crystal structure so that (010) is the prominent crystal face.21,22 It is a fair assumption that in aqueous solutions, bilayers of water molecules are, for most of the real time, exposed at the surface of the (010) face, whereas lateral faces have a mixed ionic character with intercalated water molecules.22 Because of this kind of ionic structure, (21) Abbona, F.; Christensson, F.; Franchini-Angela, M.; Lundager Madsen, H. E. J. Cryst. Growth 1993, 131, 331. (22) Haninen, D.; Geiger, B.; Addadi, L. Langmuir 1993, 9, 1058.
DCPD represents a good model crystal for studying several aspects of interactions between additives and crystals. It should be possible to assess: i) the importance of molecular size and structure of the additive, that is, small molecules or macromolecules, number of functional groups in the molecule and the overall charge; ii) the importance of a structural fit between the organic molecule and the ionic structure of a particular crystal face; and iii) the influence of the hydration layer exposed on the surface of the crystal. Glutamic and aspartic acid had no detectable effect under our experimental conditions. The distribution curves of these two amino acids show that at the investigated pH they exist as Glu- and Asp- species, that is, both behave as a monocarboxylic acid. It seems that one negative charge of these species is not sufficient for significant interaction with any of the DCPD crystal faces. Indeed, in previous studies it has been shown23,24 that for effective interaction between additive and crystal, the molecule of the additive has to have several functional groups. Furthermore, even dicarboxylic acids have no significant effect on DCPD crystallization unless additional polar groups, such as the -OH group, are present in the molecule.24 In contrast to glutamic and aspartic acids, citrate and HATP retarded DCPD crystal growth and induced similar morphological changes, HATP being by far more effective than citrate ions. In the presence of both additives, rodlike crystals with a well developed (010) face were obtained, indicating their adsorption on the DCPD lateral faces. We can assume that interaction is primarily electrostatic and that additives recognize calcium ions exposed on the lateral crystal faces. The difference in effectiveness of citrate and HATP could be explained by their molecular structure. Under the experimental conditions investigated, citrate exists in the form of an HL2- species (according to the ion distribution curves24), and adding to the polarity of the molecule is the hydroxyl group. On the other hand, (23) Sarig, S.; Glasner, A.; Epstein, J. A. J. Cryst. Growth 1975, 28, 295. (24) Brecˇevic´, Lj.; Sendijarevic´, A.; Fu¨redi-Milhofer, H. Colloids Surf. 1984, 11, 55.
9266
Langmuir, Vol. 16, No. 24, 2000
HATP has four functional phosphate groups and therefore acts as stronger inhibitor. This is in accordance with the results of related studies on the effectiveness of polyphosphates as crystallization inhibitors.13,25,26 In both cases, however, the interactions were not strong enough to enable the additives to penetrate the water bilayer exposed on the (010) face. Phytate and polyaspartate generally acted as stronger inhibitors than citrate and HATP and specifically interacted with the dominant (010) face, rather than with the side faces of the crystals. This would indicate that, in addition to strong electrostatic interactions, a structural and stereochemical fit probably exists between the periodical spacings of the inhibitor’s polar groups and the interionic distances on the affected crystal face.5,6,27 Also, based on the different morphologies of (010) faces (Figure 1f and 1g) and X-ray diffraction patterns (Figure 2d and 2e) of crystals grown in the presence of phytate and/or polyaspartate, it seems that their interactions with DCPD crystals are not quite the same. Indeed, in the case of phytate, the distances between phosphate groups (6.19 (25) Koutsoukos, P. G.; Amjad, Z.; Nancollas, G. H. J. Colloid Interface Sci. 1981, 83, 599. (26) Amjad, Z. Langmuir 1987, 3, 1063. (27) Moradian-Oldak, J.; Frolow, F.; Addadi, L.; Weiner, S. Proc. R. Soc. London B 1992, 247, 47.
Sikiric´ et al.
Å) correspond to the distances between calcium ions within one layer of the (010) crystal plane (6.24 Å, Figure 4a). In the case of polyaspartate, however, such a structural fit exists between the distances of carboxylic groups in the polyaspartic β-sheet (6.90 Å) and the distances of neighboring calcium ions from two adjacent layers within one Ca-HPO4 bilayer (6.95 Å), which is positioned parallel to the (010) plane (Figure 4b). It thus seems that molecules of polyaspartate are intercalated, which could explain the leaf-like appearance of the affected crystal plane (Figure 1g). We can conclude that the mechanism of additive-DCPD crystal interaction depends on the size and structure of the additive molecule and the structural fit between the organic molecule and the ionic structure of the particular crystal face. Small molecules with low charge (aspartic and glutamic acids) do not have a significant effect, whereas small molecules with several functional groups (citrate and hexaammonium tetrapolyphosphate) adsorb on lateral faces primarily by electrostatic interactions. Molecules with hindered structures (phytate) and macromolecules (polyaspartic acid) specifically adsorb on the dominant (010) face, for which a certain degree of structural fit is necessary (Figure 5). LA000704M
