CRYSTAL GROWTH & DESIGN 2007 VOL. 7, NO. 1 25-29
Articles Precipitation of Calcium Phosphate from Simulated Milk Ultrafiltrate Solutions N. Spanos,‡ A. Patis,† D. Kanellopoulou,† N. Andritsos,§ and P. G. Koutsoukos*,† ICE/HT/FORTH and Department of Chemical Engineering, UniVersity of Patras, P.O. Box 1414, 265 00 Patras, Greece, School of Science and Technology, Hellenic Open UniVersity, 262 22 Patras, Greece, and CPERI/CERTH and Department of Mechanical and Industrial Engineering, UniVersity of Thessaly, Pedion Areos, 383 34 Volos, Greece ReceiVed July 25, 2005; ReVised Manuscript ReceiVed September 8, 2006
ABSTRACT: The present work deals with the influence of pH and temperature on the spontaneous precipitation of calcium phosphate from simulated milk ultrafiltrate (SMUF) solutions. The pH range investigated is 5.7-7.0, and the temperature varied from 55 to 75 °C. It was found that the precipitates were prisms of hydroxyapatite (HAP) with low crystallinity preceded by amorphous calcium phosphate (ACP). Crystallinity improved with solution aging. Microscopic particles in the range of 200 nm were obtained at relatively high supersaturation (pH ) 6.8), whereas at relatively low supersaturation (pH ) 6.0), aggregates in the range of 1 µm were formed. Moreover, it was found that although the SMUF solution is also supersaturated with respect to magnesium phosphate, no magnesium salt was identified in the precipitates, thus precluding the coprecipitation of magnesium phosphate. At the same temperature, the supersaturation was regulated by adjusting the solution pH. Kinetics study of precipitation showed a parabolic dependence of the (initial) rates on the solution supersaturation, suggesting a surface diffusion-controlled mechanism with activation energy equal to 96 kJ mol-1. The rates of precipitation were significantly reduced in the presence of citrate ions due to the decrease of the solution supersaturation caused by complexation of citrates with Ca2+ ions. Additionally, a decreasing effect of citrates on the precipitation rate resulted from the adsorption of these species on the formed crystals, blocking some active sites of crystal growth and diminishing the constant of precipitation rate. 1. Introduction Fouling of heat exchangers during milk processing is a major problem in the dairy industry, causing loss of processing time, cleaning costs and effluent problems due to intermittent cleaning.1 A large number of investigations to better understand the process of fouling have been reported in the past two decades.2-13 Nevertheless, the mechanism of fouling during heating of milk is still unclear. This is mainly due to the complexity of the dairy systems, which involve phenomena associated with protein aggregation and deposition as well as mineral deposition. A first approximation of the fouling mechanism suggests that whey proteins are adsorbed on the heated steel walls. The proteinmodified surface may be the basis for the further attachment of calcium phosphate particles, generated in the bulk of the solution upon heating. The particles that seem to be attached to the demetallized surface form the backbone of the fouling layer, into which the proteins are entrapped.5 The decreased solubility of calcium phosphate upon heating seems to be a major cause of fouling.8 Calcium phosphates constitute a significant part of * Corresponding author. † University of Patras. ‡ Hellenic Open University. § University of Thessaly.
milk deposits (up to 80% w/w) and their percentage tends to increase with increasing temperature. The calcium/phosphate system equilibrium is quite complex, and various calcium phosphate phases are encountered in the milk deposits, depending upon the bulk temperature, level of supersaturation, pH, ionic medium, etc. Amorphous calcium phosphate (ACP, stoichiometry corresponding to Ca3(PO4)2‚xH2O), dicalcium phosphate dihydrate (brushite, DCPD, CaHPO4‚2H2O), octacalcium phosphate (OCP, Ca8H2(PO4)6‚5H2O), hydroxyapatite (HAP, Ca5(PO4)3OH), β-whitlockite (β-TCP, β-Ca3(PO4)2) have been identified in milk deposits (e.g., refs 1 and 5). β-TCP is a high-temperature phosphate phase, and it has not been reported to form at temperatures below 100 °C. It is also generally agreed that the formation of HAP is usually preceded by a precursor phase, the spherullitic OCP at pH ) 6.7, the structureless ACP, or the thin platelet crystalline DCPD at pH 5-6.14 Recent studies with respect to mitigation of heat exchanger fouling devote only minor attention to the mechanism of precipitation of calcium phosphate in aqueous solutions. The scope of this work is to investigate the characteristics of calcium phosphate precipitates and the kinetics of their formation from simulated milk ultrafiltrate (SMUF) solutions in order to better
10.1021/cg050361w CCC: $37.00 © 2007 American Chemical Society Published on Web 11/29/2006
26 Crystal Growth & Design, Vol. 7, No. 1, 2007
Spanos et al.
Table 1. Synthesis of SMUF Solutionsa final solution conc (mM)
reagents Solution 1 KH2PO4 K3cit‚H2O Na3cit‚2H2O K2SO4
11.61 3.70 6.09 1.03 Solution 2
K2CO3 KCl
2.17 8.05
CaCl2‚2H2O MgCl2‚6H2O a
Solution 3 8.98 3.21
cit ) C6H8O73-
understand the crystallization processes taking place during the heating of dairy fluids. 2. Experimental Procedure Two series of experiments were performed. All solutions were prepared with triply distilled water using reagent grade chemicals. SMUF stock solutions, shown in Table 1, were prepared according to Jenness and Koops.15 In the first series of experiments, 20 mL of stock SMUF solutions 1 and 2 (Table 1) were added to 940 mL of distilled water and left in the experimental setup to reach the desired temperature. The pH of the solution was adjusted using HCl or KOH solutions. Then, 20 mL of stock solution 3 were added dropwise in about 3 min under magnetic stirring. The temperature of the solution was maintained at the desired temperature ( 0.5 °C. The course of precipitation was followed by pH monitoring. However, since in this system the pH change was about 0.1 pH units (maximum change 0.25 pH units in 1 h), additional monitoring was carried out by periodic measurements of light absorbance of the solution at 436 nm. Under certain conditions (high pH and high temperature), the solution became turbid immediately after or during the addition of solution 3. Another series of experiments was done at constant pH in a batchtype, stirred, double walled glass reactor thermostatted by circulating water from a thermostat. The SMUF supersaturated solutions, volume totaling 200 mL, were prepared in the reactor by mixing 100 mL of the solutions 1 and 2, preheated at the desired temperature (55-71 °C), with 100 mL of the solution 3 (Table 1). Supersatuartion was varied by changing the pH in the range 6.20-6.84. In both series of experiments, the same order of addition of solutions was observed. It should be noted that interchange of solution 2 with 1 and vice versa did not affect the results. Solutions 1 and 2 contain ions, which have a buffering capacity, and solution pH may be relatively easily adjusted. Solution 3 containing calcium and magnesium ions was always left for last because of the very low buffering capacity. Upon the initiation of the precipitation process, sodium hydroxide was added from a pH-stat controlled by a computer, so that the pH was kept constant throughout the precipitation process. The rates of precipitation were determined from the desupersaturation curves from the profiles of variation of calcium as a function of time. Typical curves illustrating the variation of calcium in the working solution with time are illustrated in Figure 1. During the precipitation process, small aliquots of sample were temporarily removed from the work solution, filtered (Millipore 0.2 µm), and diluted (1:100) for analysis of the filtrate for residual calcium ions by inductively coupled plasma spectroscopy (ICP, Perkin-Elmer Plasma 40). The morphology of precipitates, dried at 60 °C for 15 h, was observed by scanning electron microscopy (SEM, JEOL 6300) coupled with an energy dispersive X-ray microprobe (EDXS, OXFORD ISIS 300). The precipitates were characterized by powder X-ray diffraction (XRD, Siemens D500). Their chemical composition was assayed by ICP after dissolution of the precipitate in hydrochloric acid.
3. Results and Discussion Experiments were conducted at temperatures ranging from 55 to 75 °C. In the pH range of the experiments (5.7-7.0), the
Figure 1. Calcium concentration in the working solution as a function of time at conditions of constant pH: (9) T ) 70 °C, pH ) 6.8; ([) T ) 60 °C, pH ) 6.8; (b) T ) 55 °C, pH ) 6.8; (2) T ) 60 °C, pH ) 6.6; (1) T ) 60 °C, pH ) 6.4.
Figure 2. Correlation between the light absorbance change (a) and the pH drop (b). T ) 60 °C; initial pH ) 6.46.
SMUF solution is supersaturated with respect to all possible calcium and magnesium phosphate crystalline phases. The relative supersaturation, σ, is given by
σ ) Ω1/ν - 1
(1)
where ν is the number of the ionic species constituting a specific phase and Ω represents the supersaturation ratio defined as
Ω ) (IAP)/Ksp
(2)
where IAP is the ion activity product of the phase consider and Ksp is its thermodynamic solubility product. The solution speciation and the supersaturation ratios with respect to several calcium and magnesium phosphate phases were computed by the HYDRAQL computer code.16 In the case of variable pH, the onset of precipitation was detected by a small drop in pH or by the appearance of solution turbidity, as shown in Figure 2. The pH reported in these experiments corresponds to the initial pH prior to precipitation, pHin. Upon cooling, the precipitates were redissolved as it was evidenced from the loss of turbidity. The pH drop in the SMUF system is not large. At pHin ) 6.46, it does not exceed 0.25 pH units in 1 h after the start of precipitation (Figure 2, curve b), whereas at pHin ) 6.20, the pH drop in 1 h is lower than 0.1
Precipitation of Calcium Phosphate from SMUF Solutions
Crystal Growth & Design, Vol. 7, No. 1, 2007 27 Table 2. Values of the Conditions (Temperature, pH, and Initial Supersaturation) and the Determined Initial Rates for the Precipitation of Calcium Phosphate in SMUF Solutions at Conditions of Constant pH temp (°C)
pH
σHAP
R, 10-5 mol min-1 L-1
55
6.20 6.40 6.64 6.84 6.27 6.58 6.60 6.80 6.40 6.55 6.60 6.80 6.09 6.40 6.60 6.84 6.20 6.40 6.60 6.80
14.5 19.8 27.9 36.2 17.7 21.6 27.9 37.3 23.4 29.0 31.0 40.3 16.0 25.7 34.0 46.2 21.4 28.7 37.7 48.5
2.5 4.4 10.9 24.6 3.6 6.6 10.9 22.6 17.8 27.4 39.0 63.8 8.0 31.8 37.6 108.3 40.0 46.6 117.0 245.0
60
65
Figure 3. Variation with time of light absorbance from the working solutions at various initial pH values at conditions of variable pH. T ) 72 °C.
pH units. The lower pH drop in the case of lower pHin is due to the lower initial supersaturation corresponding to the lower pHin, which in turn results in a smaller extent of precipitation thus exhibiting lower pH drop. On the contrary, a sharp pH increase has been reported in the literature upon precipitation in the calcium phosphate system.17 Concerning aging of the precipitate, it was found that the pH drop kept on during aging, attaining a value of 0.4 units after a 24 h period. In the runs where the residual calcium was analyzed, light absorbance and pH changes were correlated well with the residual calcium concentrations in the filtrates. Typical light absorbance variations with time at various pHin values are depicted in Figure 3 for a constant temperature. It may be observed that precipitation commenced either immediately after solution preparation, at the highest pHin, or after an increasing delay period (i.e., induction time), as pHin decreases. This is anticipated due to the diminishing initial supersaturation as pHin decreases. Finally, Figure 3 shows that at the highest pHin value, that is, at the highest initial supersaturation, the precipitation process finished earlier than that at the pHin values corresponding to lower initial supersaturations. As regards the experiments carried out at constant pH, the conditions of temperature, pH, and initial supersaturation with respect to HAP and the determined initial rates of precipitation, R, are compiled in Table 2. The initial rates expressed in moles of HAP formed per minute and per liter of the working solution were computed from the initial slope of the curves illustrated in Figure 1. As may be seen in Table 1, at the same temperature, pH affects strongly the solution supersaturation. The dependence of the rate of precipitation upon the supersaturation may be described according to the phenomenological eq 318,19
R ) kσn or ln R ) ln k + n ln σ
(3)
where k is the precipitation rate constant, and the exponent n is the apparent growth order. The value of the exponent is considered to be indicative of the mechanism of the crystallization process.19-21 The kinetics data for the crystal growth of calcium phosphates were fitted according to the logarithmic form of eq 3, in which the relative supersaturation was taken with respect to hydroxyapatite. The curves obtained, shown in Figure 4, revealed very good linear fittings, from the slope and intercept of which the values of n and k were determined, respectively. The fact that the attained values of n corresponding to the various temperatures studied (Table 2) ranged between 2.31 and 2.50 suggested a surface diffusion-controlled mechanism typical for a number of sparingly soluble salts.22,23 The
71
60a
a
In the absence of citrates.
Figure 4. Kinetic curves corresponding to different temperatures.
rate of stirring did not have any effect on the measured rates of crystallization, suggesting that the contribution of bulk diffusion to the precipitation mechanism was negligible. Moreover, the precipitation rate constants obtained at the temperatures shown in Table 2 were tested according to the logarithmic form of the Arrhenius equation. The curve obtained, illustrated in Figure 5, showed a satisfactory linear fitting, the slope of which yielded the value of 96.04 kJ for the activation energy. This value is typical for sparingly soluble salts.24 Inspection of Table 2 shows that the precipitation rates obtained in the absence of citrates are much higher. This should be attributed to the fact that in the presence of citrates the concentration of Ca2+ ions decreases because of their complexation with citrate anions thus diminishing the supersaturation. An additional decreasing effect of citrates on the precipitation rate should be the fact that these species may be adsorbed on the formed crystals,25-34 thus blocking some active sites of crystal growth and resulting in a reduced value of the rate constant. Comparison of the intercepts of curves c and e of Figure 4 demonstrates that this is really the case. According to the XRD patterns (Figure 6), at the first stages amorphous calcium phosphate may be formed, hydrolyzing rapidly to a poorly crystallized HAP. The relatively low value
28 Crystal Growth & Design, Vol. 7, No. 1, 2007
Spanos et al.
Figure 5. Variation of the logarithm of rate constant of precipitation with the reciprocal of absolute temperature.
Figure 6. X-ray patterns of calcium phosphate precipitated at conditions of variable pH.
of the molar ratio Ca/P in these precipitates, estimated to be 1.50 ( 0.05, is indicative of the poor crystallinity of the formed HAP. HAP crystallinity seems to be independent of temperature, but it improves with solution aging. SEM results showed that the precipitates were prisms with size dependent upon the supersaturation. Specifically, at relatively high supersaturation (pH ) 6.8), microscopic particles in the range of 200 nm or smaller are observed, while at relatively low supersaturation (pH ) 6.0), aggregates in the range of 1 µm are formed, as illustrated in Figure 7. Finally, EDXS of the precipitates did not reveal the presence of any other element than Ca and P; that is, although the SMUF solution is also supersaturated with respect to magnesium phosphate, no magnesium salt was identified in the precipitates, thus precluding the coprecipitation of magnesium phosphate. 4. Conclusions Calcium phosphate, precipitated spontaneously from SMUF solutions, resulted in prisms of HAP with low crystallinity preceded by ACP. Crystallinity improved with solution aging. At relatively high supersaturation (pH ) 6.8), microscopic particles in the range of 200 nm are observed, while at relatively low supersaturation (pH ) 6.0), aggregates in the range of 1 µm are formed. Although the SMUF solution is also supersaturated with respect to magnesium phosphate, no magnesium salt was identified in the precipitates, thus precluding the coprecipitation of magnesium phosphate. The effect of pH on the solution supersaturation was found to be strong. Initial rates of
Figure 7. SEM micrographs of calcium phosphate precipitated at conditions of constant pH: (a) pH ) 6.8; (b) pH ) 6.0. T ) 71 °C.
precipitation showed a parabolic dependence on the solution supersaturation, suggesting a surface diffusion-controlled mechanism typical for a number of sparingly soluble salts. In addition, an activation energy indicative for sparingly soluble salts equal to 96.04 kJ mol-1 was found. Citrate ions were found to reduce efficiently the rates of precipitation due to both their complexation with Ca2+ ions thus decreasing the solution supersaturation and their adsorption on the surface of the formed particles thus blocking some growth active centers and eventually diminishing the constant of the precipitation rate. Acknowledgment. N.A. wishes to thank the Commission of European Communities for the financial support of part of this work under Contract G5RD-CT-1999-00066. References (1) Burton, H. Ultra-high Temperature Processing of Milk and Milk Products; Elsevier: London, 1988. (2) Delplace, F.; Leuliet, J. C.; Tissier; J. P. 1994, Fouling Experiments of a Plate Heat Exchanger by Whey Protein Solutions. Fouling and Cleaning in Milk Processing; Department of Chemical Engineering, University of Cambridge: Cambridge, U.K., 1994; pp 1-8. (3) Fryer, P.; Belmar-Beiny M. T.; Schreier P. J. R. Fouling and Cleaning in Milk Processing. In Heat Induced Changes in Milk; Fox, P. F., Ed.; International Diary Federation: Brussels, 1995; pp 364-395. (4) de Jong, P.; Bouman, S.; van der Linden, H. J. L. J. J. Soc. Dairy Technol. 1992, 45, 3-10. (5) Visser, J.; Jeurnink, Th. J. M. Exp. Therm. Fluid Sci. 1997, 14, 407412.
Precipitation of Calcium Phosphate from SMUF Solutions (6) Morison, K. R.; Tie, S. H. Food Bioprod. Process. 2002, 80, 326332. (7) Andritsos, N.; Yiantsios, S. G.; Karabelas, A. J. Food Bioprod. Process. 2002, 80, 223-230. (8) Jeunrink, T. J. M.; Walstra, P.; deKruif, C. G. Neth. Milk Dairy J. 1996, 50, 407-412. (9) Schmidt, D. G; Both, P. Neth. Milk Dairy J. 1987, 41, 105-111. (10) Yoon, J.; Lund, D. B. J. Food Sci. 1994, 59, 964-969. (11) Barton, K. P.; Chapman, T. W; Lund, D. Biotechnol. Prog. 1985, 1, 39-48. (12) Dupeyrat, M.; Labbe, J. P.; Michel, F.; Billoudet, F; Daufin, G.; Michel, F. Lait 1987, 67, 465-472. (13) Changani, S. D.;Belmar-Beiny, M. T.; Fryer P. J. Exp. Therm. Fluid Sci. 1997, 14, 392-398. (14) Brule´, G.; Real del Sol, E.; Fauquant, J.; Fiaud, C. J. Dairy Sci. 1978, 61, 1225-1232. (15) Jenness, R.; Koops, J. Neth. Milk Dairy J. 1962, 16, 153-158. (16) Papelis, G.; Hayes, K. F.; Leckie, J. O. HYDRAQL: A program for the computation of chemical equilibrium composition of aqueous batch systems including surface compexation modelling of ion association at the oxide / solution interface; Technical Report No. 306, Stanford University: Stanford, CA, 1988. (17) Brecˇevic´, L.; Fu¨redi-Milhofer, H. Calcif. Tissue Int. 1979, 28, 131136. (18) Koutsoukos, P. G. Growth of calcium phosphates on different substrates: epitaxial considerations. In Calcium Phosphates in Biological and Industrial Systems; Amjad, Z., Ed.; Kluwer Academic Publishers: Boston, MA, 1989; pp 41-66. (19) Kapolos, J.; Koutsoukos, P. G. Langmuir 1999, 15, 6557-6562.
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CG050361W
