320 Reprinted from LA."iGi\n;IR. 1994. 10.Copyright @ 1994 by the .\merican Chemical Society and reprinted by pennission of the copyright owner.

Interaction of Pyrophosphate with Calcium Phosphates

T. V. V asudevan t and P. Somasundaran

Langmuir Center for Colloids and Interfaces, Columbia University, New York, New York 10027

C. L. Howie-Meyers, D. L. Elliott,! and K. P. Ananthapadmanabhan.

Unilever Research, Edgewater, New Jersey 07020

Received April 22. 1993. In Final Form: October 25. 19938

Interactions of pyrophosphate (Pp) with hydroxyapatite (HAP), brushite, and calcium pyrophosphate(CaPP) have been studied using electrokinetic and ESCA measurements. PP adsorbs on HAP and brushiteeven when their surfaces are highly negatively charged. Importantly, the HAP zeta potential curves asa function of PP concentration show a precipitous drop in the low concentration range. Also, curves atdifferent pH values show a progressive shift in the potential toward that of CaPP. These observationssuggest the formation of CaPP on HAP surface and this has been further conflflDed by ESCA studies.Thus, adsorption on HAP appears to involve a surface precipitation or chemisorption followed by surfaceprecipitation or surface reaction phenomenon. It is also suggested that in the case of brushite the dissolvedcalcium from the mineral competes for the PP and in turn reduces its adsorption.

and X -ray photoelectron spectroscopic (~CA) techniques.These techniques measure the changes in the interfacialproperties, namely of charge and surface chemical com-position of the substrate and, therefore, can provide insightinto the mechanism of adsorption of PP on HAP andbrushite.

Introduction

Regulation of calcium phosphate crystal growth isimportant in a wide variety of applications such as in theprevention of scale formation in industrial heat exchangers,in the removal of phosphates from polluted water, and,most importantly, in the biological mineralization of teethand bones.l-5 Pyrophosphates (PP) are known to inhibitformation and growth of calcium phosphate precipitates.Reported studies6-10 show that the inhibitory activity ofPP is due to its ability to adsorb on the growing nuclei andin turn block the crystal growth sites. In this regard, theadsorption of PP on hydroxyapatite (HAP) has beenconducted and correlated with the crystal growth inhibitionbehavior in the past;6-10 however, the mechanism ofadsorption itself has not been fully established. Also,interaction ofPP with brushite and other forms of calciumphosphate which exist as precursors to HAP during theformation of dental tartarl1 have not been examined.Significantly, while PP is an excellent inhibitor of HAPformation, it is only a moderate inhibitor of brushite crystalgrowth.12'. In the present investigation, the interaction of PP with

HAP and brushite has been studied using electrokinetic

Experimental Section

Materials. Calcium Phosphates. HAP and brushite powderswere obtained from Albright and Wilson. The surface areas ofthese samples have been reported to be 26 and 3 m2fg, respectively,as measured by the BET nitrogen adsorption technique. Calciumpyrophosphate (CaPP) was purchased from Alfa Products andused without further purification.

Pyrophosphate. Reagent grade tetrapotassium pyrophc.phate(TKPP) was purchased from Sigma Chemical Co.

Other Reagents. Sodium hydroxide and hydrochloric acid,used as pH modifiers, and sodium chloride, used for adjustingthe ionic strength, were supplied by Fisher Scientific Co. Triplydistilled water was used for preparing the solutions and thesuspensions.

Methods. Zeta Potential.\-Ieasurements. Samples of solids(0.05 g) were added to 50 mL of 2 x 10".3 kmol/m' NaClsolution,of preadjusted pH. in a 150.-mL beaker. The suspension wasstirred for 2 h using a magnetic bar and the pH was measuredat the end. Following this, 50 mL of 2 x 10".3 kmol/m' NaCIsolution containing the desired concentration ofTKPP was addedto the suspension. Prior to addi tion, the pH of the TKPP solutionwas adjusted to that of the suspension. The solids wereconditioned for 1 h with TKPP. At the end of the conditioningperiod, the pH of the suspension was measured and about 50 mLof the suspension was transferred to the cell of the zeta meter(Zeta Meter, Zeta Meter Inc.). The mobilitiesofl0to 15 particleswere measured and zeta potential was calculated. A chartprovided with the zeta meter manual was used for the conversionof mobility readings to zeta potential values.

Calcium Binding Isotherms. One-milliliter samples of 10 wt% TKPP solution were added to 99 mL of solutions of varyingcalcium chloride concentration. The pH of the resulting sus-pension was adjusted to the desired value (pH 7.5 or pH 11.0)and the calcium activity measured using a pH meter equippedwith an Orion Model 93-20 calcium selective electrode.

XPS Analysis. X-ray photoelectron spectrO6COpic analysis ofthe HAP, brushite, and Capp samples was carried out at PhysicalElectronics Laboratories, Edison, NJ, using a Perkin-ElmerModel 5(XX)LS ~CA spectrophotometer. Samples for XPSanalysis were prepared by treatment of HAP and brushitepowders with 200, 500, and 1000 ppm of TKPP in aqueous

@ 1994 American Chemical Society

, Present address: Unilever Research.t Present address: Calgon Corp.8 Abstract published in Advance ACS Abstracts. December 15.

1993.(11 Zuhl. R; Amjad. z.; Muler. W. F.J. Cool. Tower Inst. 1987.8,41.(21 FergUlOn. J. F.; Jenkins. D.; Eastman. J. J. Water Pollut. Control

Fed. 1973.45,620.(3) NancoUu. G. H. Biological Mineralization and Demineralization;

Nancollas. G. H.. Ed.; Springer Verlag: Berlin. 1982; p 79.(4) P~ner.A. S.; BlumenthaL N. C.; Betta, F. Chemiatryand Structure

of Hydroxyapatites. Phosphate Minerals; Nriagu, J. 0.. Moore. P. B..Eds.; Springer Verlag: Berlin. 1984, Chapter 11. pp 330-350.

(5) Salimi. M. H.; Heughebaert, J. C.; NancoUu. G. H. Langmuir 1985.1. 119.

(6) Fleisch. H.; Russel. R. G. G.; Straumann. F. Nature 1*. 212.901-903.

(7) Krane,S. M.;Glimcher. M. J.J. Bioi. Chem.196Z.237. 2991-2998.(8) Burton. F. G.; Neuman. M. W.; Neuman. W. F. Curro Mod. Bioi.

1969.3.20-26.(9) Junc. A.; Bi88%. S.; Fleisch. H. Calcif. Ti.tsue ReI. 1973, 11. 269-

280.(10) Moreno. E. C.; Aoba, T.; Margolis. H. C. Compend. Contin. Educ.

Dent. Suppl. 1981, No.8. S25&-S266.(11) Schroeder. H. E.; et al. Formation and Inhibition of Dental

Calcuiul; Hana-Huber: Vienna, 1969; pp 109-121.(12) Elliott. et al.. 8ubmitted for publication in Arch. Oral Bioi.

0743.7463/94/2410-0320$04.50/0

Interaction of PP with HAP Lan,muir, Vol. 10, No. I, 1994 321

solution. followed by centriru,ation and dryinJ to yield tIDepowdered solidi.

Results and DiscussionInteraction of Pyrophosphate with HAP. Zeta

Potential. The zeta potential behavior of HAP wasdetermined as a function of PP concentration at twodifferent pH values and the results are shown in Figure1. For comparison, the zeta potential of CaPP is alsoplotted in the same rlgure. In the absence of PP, HAPunder both the tested pH conditions exhibits a negativecharge. Interestingly, the potential at low levels of PPexhibits a precipitous drop to values close to that ofCaPP.Beyond this concentration, the dependence of HAPpotential on PP concentration becomes similar to that ofCaPP. Note that the behavior is essentially the same atboth pH values (7 and 11). The sharp reduction in thepotential of HAP at low levels ofPP, even under conditionswhen the HAP surface is highly negatively charged as atpH II, clearly shows the strong afrmity of PP for thesurface. This behavior is indicative of an adsorptionprocess involving a phase change, possibly the formationof CaPP on HAP surface. The observation that thepotential of CaPP itself depends on PP concentration insolutIOn is not surprising since the latter is a potentialdetermining ion for the surface.

Note that beyond the precipitous drop in zeta potentialof HAP in PP solutions, values of the potential are notidentical to those of CaPP but vary similarly to that ofCaPP. Even if PP forms a CaPP layer on HAP surface.differences between the potentials ofCaPP and PP-treatedHAP are not unreasonable. For example, PP, being apotential determining ion for CaPP, can lower the CaPPpotential. Therefore, identical values of zeta potential ofCaPP and PP-treated HAP can be expected if and onlyif the residual levels of all ionic species are the same inboth the systems. Inhomogeneties in the newly formedCaPP layer on HAP can also cause differences betweenthe potentials of CaPP and PP-treated HAP.

The pH dependence of the zeta potential of HAP treatedwith solutions containing different levels of PP is shownin Figure 2. Again for comparison, the zeta potential ofCaPP is included. It is evident from the zeta potentialcurves that the surface of HAP is becoming more negativewith increase in PP concentration. AJao, the curves appearto progressively shift toward that of CaPP. While this initself cannot be taken as evidence of CaPP formation, this

in combination with the data in Figure 1 suggests theformation of CaPP on HAP surface.

Formation ofCaPP on HAP can occur by either a surfaceprecipitation phenomenon or a surface reaction process.Surface reaction involves chemical interaction betweenthe adsorbate species and the adsorbent, in this casepyrophosphate and the HAP surface. The reaction beginsat the interface and proceeds inward. Surface precipi-tation, on the other hand, is the nucleation and growth ofa stoichiometric precipitate of the adsorbate species by acomplexing ion at the interface. In the present case, itwill be the precipitation of CaPP on HAP surface. Notethat often prior to surface precipitation, chemisorption ofthe adsorbate species can occur at the interface. Chemi-sorption can be distinguished from surface precipitationin the following manner. Chemisorption usually refers toadsorption resulting from the formation of a high-energybond, such as a covalent bond, between the adsorbatespecies and site/sites on the solid surface and this will notresult in the formation of a stoichiometric compound. Notethat chemisorption can occur even if electrostatic factorsare unfavorable for adsorption.

Adsorption has been shown to involve surface reactionor surface precipitation in a number of other mineraI;inorganic systems in the past.l3.l4 For eumple, formationof calcium carbonate and calcium fluorite on a HAP surfacein the presence of dissolved carbonatel3 and fluoride,respectively,l4 and formation of calcium phosphate oncalcite in the presence ofphosphatel3 have been shown toinvolve a surface reaction or surface precipitation phe-nomenon.

The results from the current study suggest that in thepresence of PP formation of CaPP can occur on the HAPsurface. In order to test whether this observation isconsistent with the expected thermodynamic behavior ofthe system, the following analysis was conducted. Es-sentially the feasibility of the reaction:

6H+ + Calo(PO4)s(OH)2(s) + 5P2O74- - 5C82P207(S) +

6HPO4~ + 20H- (1)

was examined using the literature values of free energy offormation of various species. The standard free energy

(13) So_undaran. P.; AmankOIIah.J. 0.; ADaDthaP8d_~.bhan, K.P. Colloid. Surf. 1985,15,309-333.

(14) ChaDder, S.; Fuersteoau. D. W. Colloid. Surf. 1985,13,137-144.LiD. J.; R.,havan. S.; Fuenteoau. D. W. Colloid. Surf. 1t81.3, 357-370

322 Langmuir, Vol. 10, No.1, 1994

Free Energy ot Formation tor Reaction ot H.-\Pand PP

Table I

free energy of fonnanonfrom elements. Gor. kcal/mol refspecies

0.3030-260.3-458.7-37.6

-132.3-748.6

-H+Ca1o<PO.)e<OH>2(slHPO.2-Pzo,'"OH-Caz+CaPP(s)

151515131515

!.f;.,C::c,i.,

-741.95 17

Table 2. Calculation of PP Levell Required To Form CaPP

~

;tAIa:QJ),

""'~---;{A#~~--"'"-748.6-743.6-741.95

.0 .. .. .. ..,.c- .

Figure 3. XPS analysis of hydroxyapatite treated with pyro-phosphate.

change accompanying the reaction can be estimated fromthe free energies of formation of the reactants and products.The standard free energy change (~GO) can be related tothe equilibrium constant for the reaction, K. as

K = [HPO.2-)6[OH-)2/[H+)8[P20.4-)5 (2)

Thus, if HAP is in equilibrium with the solution at a givenpH (i.e. H+,OH-.andHPO.~arerlXed), then the minimumamount of P2O74- required to force the above reaction tothe right can be estimated.

The values of free energy of formation of various speciesand the source of the data are given in Table 1. As canbe seen from the information in Table 1. there appears tobe some uncertainty with regard to the value of free energyof formation of CaPPo The first value (-748.6 kcal/mol)was obtained from ref 15. The latter two values wereobtained from unpublished work at Unilever Research.16.17The values -743.6 and -741.95 kcal/mol correspond toCaPP solubility product values of 1.2 x 10-15 and 1.9 x10-1', respectively. Since it was difficult to assess theaccuracy of these estimates, each value was used todetermine the corresponding three free energy and equi-librium constants for the above reaction. The results ofthis analysis are given in Table 2. The last two columnsin the table show the concentrations of PP and TKPP forthe present system at a pH of 6.9.

Note that because of pH-dependent hydrolysis reactions,Only about 5()% of the total PP will be P~74- at pH 6.9.This has been accounted for in the calculation of theconcentration of TKPP in equilibrium with both of thesolid phases. It is clear from Table 2 that from the threepossible estimates of PP levels, the worst case situation,i.e., the highest concentration of TKPP needed to causethe formation of CaPP on HAP. is just above 16.5 ppm.This simple analysis indicates that the formation ofCaPPon HAP in the presence of soluble PP at relatively lowlevels is consistent with the expected behavior, i.e., PPcan replace orthophosphate on the mineral surface.

X-ray Photoelectron Spectroscopic Analysis (XPS/ESCA). In order to further confmn the formation ofCaPPon the HAP surface in the presence of dissolved PP, anFSCA study was conducted. The XPS spectra of HAP .CaPP, and PP-treated HAP are shown in Figure 3. Figure

(15) LGnIe', Handbook 01 Chemistry; Dean, J. A., Ed.; Formerlycompiled by Lange, N. A.; McGraw-Hill: New York, 1985.

(16) Welch, G. J.; WOCMeY, S. Unpublished results, Unilever Research,P:>rt Sun\icht, 1975.

(17) Clark.. D. E.; Lee, R. S.; Lunt, T. J. Unpublished result., UnileverReHazch, Port Sunlight, 1974.

3 is divided into parts a, b. and c which represent thebinding energies corresponding to P2p (132-134 e V). Ca2p(347-348 eV), and 01s (531-532 eV), respectively. TheXPS spectra for- each element shows the spectra ofuntreated HAP. CaPP. and PP.treated HAP at PPconcentrations of 200, 500. and 1000 ppm. For eachelement, the PP-treated HAP samples are between thoseof HAP and CaPP. The binding energy moves closer toCaPP as the concentration of TKPP increases. Thisindicates that the HAP surface. upon PP treatment, isbecoming progressively similar to that of CaPP. Resultsof the XPS analysis thus support the inference, deducedfrom electrokinetic studies and thermodynamic analysis.of CaPP formation at the HAP surface.

Interaction of Pyrophosphate with Brushite. Asin the case of HAP. the interaction of PP with brushitewas also studied using zeta potential and ESCA techniques.The zeta potentials of brushite as a function of PPconcentration are given in Figure 4. Here again. brushiteis negatively charged under the test conditions and it showsa sharp reduction in potential after exposure to low levelsof PP. Interestingly, the absolute values of the potentialare lower than those observed for HAP. Also. beyond thisinitial drop there appears to be less dependence on PPconcentration. in contrast to the behavior of the HAP/PPsystem described above. Thus. while zeta potentialsuggests some significant adsorption of PP on brushite.whether it involves surface conversion to a CaPP-typesurface is not clear.

ESCA studies of brushite samples treated with PP werealso conducted. Unlike the case of HAP. brushite andCaPP have very similar spectra and therefore it is notpossible to ~e this technique to distinguish between thetwo phases.

One of the differences between HAP and brushite istheir water solubility, 'with HAP having a relatively lowsolubility compared to brushite. For example, at a pH of

Interaction of PP with HAP La"lmuir, Vol. 10, No. I, 1994 .'l23

-;:z~ .. -:'Y -;;;c;- l0 OS... -.

-sa

-60

Figure 5. Specific conductance oCHAP slurry in pyrophoephatesolutions.

-~

. ~. . . ---~- --I0 0.5 , LI a a.1 I 1.1Potas8Arn PynJpha8phaM. ~

Ficureo&. Zeta potential o(bruahite treated with pyrophc»phate,effect or pH.

i:-2..-0.-.os - - -

'-.8

/ ,_.--_.--_.

!rf_.-! . , , . . ,.

0 0.. I LS 1 1.5 J J.S

~ ~8Phat8 Conc8nIratian, ~

Ficun 6. Specific coDductaoce ofbruabite l1urry in PP IOlutioDl.

o.,= 2 . 10 -. M IIeCI0.05 wt~ .81'_.

-28-

ii '11' ~~ ~ ~~ --

~- ~ -~= ~~~.:::::::::::::~ ~:'" ~-"

.1c. -M,:-!N -80 ~

~-* ~

~ ~~ ,"11.0-\JO-

G I 2 J 4

Potassium Pyrophosphat., ~

Ficw'e 7. Zeta pountial VB pyrophc»pbau concentration, zetavalues corrected for ionic strength in PP solutions.

to different forms of empirical equations. The equationthat showed best fit (highest correlation coefficient) ofthe experimental data W88 obtained and is shown in Table3. This empirical equation shows that the negative zetapotential of all three surfaces increases 88 a function ofthe square root of the PP concentration. In the equationsshown in Table 3, the constant A repreaenta the zetapotential of the untreated calcium phosphate, while B canbe taken 88 a measure of the afrmity of TKPP to thecalcium ph~phate surface. Eumination of Table 3 shows

6.9, the concentration of calcium ions in equilibrium withHAP is about 2 ppm and that with brushite is about 28ppm. When a reagent such as PP is introduced into asuspension of HAP or brushite, there exists a competitionbetween complexation of bulk calcium va adsorption.Becauae of the higher solution levels of calcium in thebrushite suspensions, it is reasonable to expect that therewill be more bulk complexation than in the case of HAP.This is supported by the observation that addition of 10ppm ofPP into supernatant solutions of HAP and brushiteresulted in precipitation in the case of the latter and notin the case of the former. Thus, at a particular level ofpp, there will be more PP available for surface adsorption!conversion on the HAP surface than on brushite. Thismay account for the differences in the dependence of zetapotential of HAP and brushite on PP concentration. Thissolubility difference appears to provide favorable condi-tions for bulk precipitation of CaPP in the brushite-PPsystem. In contrast to this, because of low calcium levelsin solution, the interactions with HAP may be more likesurface conversion or surface precipitation and this is inline with the ~CA results.

Comparison of Affinities of PP to HAP, Brushite,and CaPP. Zeta potential results presented in Figures 1and 4 showed that addition of PP renders the surface ofHAP and brushite negative. Therefore, variation of thecharge density at the solid/liquid interface can be used asa meuure of adsorption. Zeta potential values can beused as a measure of charge density at the solid/liquidinterface and can be used for comparison provided theionic strength of the medium is maintained constant sinceonly the zeta potential. and not the charge density, dependson the ionic strength. As shown in Figures 5 and 6, additionofTKPP resulted in a significant increase of ionic strength,measured in terms of specific conductance. Therefore,the zeta potential results shown in Figures 1 and 2 werecorrected for ionic strength variation using the proceduredescribed by Hunter.ll

Figures 7 and 8 show the zeta potential curves ofTKPP-treated HAP and brushite corrected for a base ionicstrength of 2 x 10'"-1 kmollm3 NaCL Zeta potential CUrvesobtained for the CaPP-TKPP system are presented inFigure 7 for the purpose of comparison. These conectedpotentials can now be used to examine the dependence ofzeta potential on pyrophosphate concentration. Theapproach involved the use of CUrve fitting software (TableCurve, Jandel Scientific, CA) to fit the experimental data

--0

(18) Hunter, R. J. Zeta Pot~ntiGl in ColWid Sci~nce: Princip". andApplicatioM; Academic p~ New York, 1981; Chapter 2, pp 11-58.

324 Langmuir, Vol. 10, No. I, 1994 Vasudevan et al.

hydroxyapatite 7.1AJ 0.9310.74 <0.10

brushite 7.60 24.0010.85 1.50

calcium p)Tophoephate 7.55 0.8210.~ <0.10

supernatant of calcium phosphate suspensions wouldcontain calcium ions which would compete with the surfacefor PP molecules. The affinity ofPP to calcium phosphatesurface would thus depend on both the concentration ofdissolved calcium and its relative affinity to the surface.

Table 4 shows the dissolved calcium concentration inthe supernatants of HAP. bnlShite. and CaPP at pH valuesof 7.5 and 11. It can be seen that the dissolved calciumconcentration is much higher at the lower pH for all threeminerals. The dissolved calcium is much higher in bnlShitesupernatant. Presence of larger levels of dissolved calciumcan account for the lower afimity of PP to calciumphosphate surface at lower pH and also its lower affinityto bnlShite surface compared to that for HAP and CaPP.The dissolved calcium concentration in CaPP supernatantis similar to that of HAP supernatant However. theaffinity ofTKPP is higher for HAP than for CaPP (Table3). This is explained by comparing the electrostatic

that in all cases, except for the brushite-TKPP system atpH 11, the equation that best fitted the experimental datawas of the same type in which the zeta potential showeda square root dependence on TKPP concentration. Thequality of fit can be seen from Figures 9,10, and 11 to begood for HAP and CaPP. Also, it can be seen from theM B" value that the affmity of TKPP is highest for HAP

followed by CaPP and brushite. Further, the affinity ofTKPP to the calcium phosphate swface is greater at higherpH. This is posaibly due to differences in reactivity ofPZO74- present at high pH values vs HP2O73- and otherforms at lower pH.

Dissolved Calcium in the Supernatant of CalciumPho8phate Su8pension8. Review of past resultslO andthe results obtained in the present study suggest that theadsorption of PP on calcium phosphates involves chemicalinteractions and ultimately results from the formation ofCaPP on HAP surface. Because of dissolution, the

Langmuir, Vol. 10, No. I, 1994 325Interaction of PP with HAP

repulsion at the two surfaces, where the repulsion betweenthe negatively charged phosphate species and the surfacewould be higher for CaPP than for HAP, as evidenced bythe much higher negative charge density of the CaPPsurface (ZP = -41 mY) compared to that of HAP (ZP =-13.5 mY).

, .potential of CaPP at pH 6.9 and 10.95, indicating that itis a potential-determining species for the surface.

(3) Formation of CaPP on the HAP surface wassupported by the results from XPS and electrokineticstudies.

(4) Affinities ofPP for HAP are found to be higher thanthat for brushite. Lower affinity for brushite was attrib-uted to the presence of dissolved calcium in the super-natant which competes with surface calcium for PP.

(5) An empirical fit of the zeta potential data showsthat the llt:gaLlve potellLiai:; vii rit"l.1, CdLJ P, and u,u.,J.J.;tt:increase as a function of the square root of the PPconcentration. Greater affinity between PP and thesurface was observed for both HAP and CaPP at the higherpH (~11) than at the lower pH (~7).

Conclusions

(1) Electrokinetic studies showed the negative zetaDotentiAI of HAP and brushite to increase sharply in thepresence of very low concentrations ol 1> P indicating strongaffinity of the solute to the surface.

(2) Adsorption of negatively-charged PP species onhighly negatively charged surfaces suggested chemisorp-tion of solute on the surface. PP increased the negative