Download Characterization and Surface Properties of Amino-Acid

Langmuir 2007, 23, 12233-12242
12233
Characterization and Surface Properties of Amino-Acid-Modified
Carbonate-Containing Hydroxyapatite Particles
Kevin S. Jack,* Timothy G. Vizcarra, and Matt Trau
Centre for Nanotechnology and Biomaterials, LeVel 5 East, Australian Institute for Bioengineering and
Nanotechnology (AIBN), UniVersity of Queensland, St Lucia, QLD 4072, Australia
ReceiVed June 22, 2007. In Final Form: September 17, 2007
The surface properties (nature, strength, and stability of interaction of functional groups) and bulk morphologies
of a series of amino-acid-functionalized carbonate-containing hydroxyapatite (CHA) particles were investigated. It
was found that the amino acids were both occluded in and presented on the surface of the CHA particles. Furthermore,
their presence enhanced particle colloidal stability by retardation of Ostwald ripening and in some cases increasing
the magnitude of the ζ-potential. Measurements of adsorption isotherms and ζ-potential titrations have shown that
the amino-acid-surface interactions are weak and reversible at pH 9 and consistent with a model in which the carboxyl
terminus interacts with calcium ions in the CHA lattice. Complexities in adsorption behavior are discussed in terms
of different adsorption mechanisms that may be prevalent at different pHs.
Introduction
Hydroxyapatite (HA), [Ca10(PO4)6(OH)2], is one of the most
stable forms of the calcium orthophosphates and the major
inorganic component of bone and teeth in mammals.1,2 In nature,
stoichiometric HA is very rarely found and it is more often formed
with partial substitution by carbonate, Mg2+, Na+, etc. The
mechanical and biological properties of pure and substituted HA
have been well studied and are the subjects of many publications,
including reviews and texts.1-4 Of these properties its relatively
high stiffness and yield strength, osteoconductive nature, and
biocompatibility (with calcified tissues) has led to much interest
into the applications of HA and substituted HAs in the fields of
bone tissue engineering and orthopaedic therapies.2,5-9 In addition,
the surface adsorption properties of these materials has led to
applications in affinity chromatography,10-12 wastewater remediation,13,14 and drug delivery systems.9,15
There exist numerous synthetic strategies for producing HA
and substituted HAs including wet precipitation, hydrothermal
methods, sol-gel, and solid-state synthesis.1,3,16,17 Recently,
Gonzalez-McQuire et al.17 utilized a hydrothermal method in
which stable dispersions of ultrafine, rodlike, or prolate ellipsoidal
(1) Dorozhkin, S. V. J. Mater. Sci. 2007, 42, 1061-1095.
(2) Biomaterials Science: an Introduction to Materials in Medicine, 2nd ed.;
Ratner, B. D., Ed.; Elsevier Academic Press: Amsterdam, 2004.
(3) Hyroxyapatite and Related Material; Brown, P. W., Constantz, B., Eds.;
CRC Press: Ann Arbor 1994.
(4) Yaszemski, M. J.; Payne, R. G.; Hayes, W. C.; Langer, R.; Mikos, A. G.
Biomaterials 1996, 17, 175-185.
(5) Ben-Nissan, B.; Milev, A.; Vago, R. Biomaterials 2004, 25, 4971-4975.
(6) Gonzalez-McQuire, R.; Green, D.; Walsh, D.; Hall, S.; Chane-Ching, J.Y.; Oreffo, R. O. C.; Mann, S. Biomaterials 2005, 26, 6652-6656.
(7) Ma, P. X. Mater. Today 2004, 30-40.
(8) Wei, G. B.; Ma, P. X. Biomaterials 2004, 25, 4749-4757.
(9) Choi, A. H.; Ben-Nissan, B. Nanomedicine (London, United Kingdom)
2007, 2, 51-61.
(10) Kawasaki, T. J. Chromatogr. 1991, 544, 147-184.
(11) Bernardi, G.; Giro, M. G.; Gaillard, C. Biochim. Biophys. Acta 1972, 278,
409-420.
(12) Gorbunoff, M. J.; Timasheff, S. N. Anal. Biochem. 1984, 136, 440-445.
(13) Barba, F.; Callejas, P. J. Mater. Sci. 2006, 41, 5227-5230.
(14) de-Bashan, L. E.; Bashan, Y. Water Res. 2004, 38, 4222-4246.
(15) Perkin, K. K.; Turner, J. L.; Wooley, K. L.; Mann, S. Nano Lett. 2005,
5, 1457-1461.
(16) Milev, A. S.; Kannangara, G. S. K.; Wilson, M. A. Langmuir 2004, 20,
1888-1894.
(17) Gonzalez-McQuire, R.; Chane-Ching, J. Y.; Vignaud, E.; Lebugle, A.;
Mann, S. J. Mater. Chem. 2004, 14, 2277-2281.
Scheme 1: Scheme of Amino Acids
(cigar shaped) HA particles (ca. 4-15 nm wide by 30-150 nm
long) were prepared in aqueous solution. The synthetic scheme
reported employed addition of an aqueous ammonium phosphate
solution to an aqueous solution containing calcium nitrate plus
an amino acid at pH 9; glycine, alanine, valine, asparagine, serine,
lysine, arginine, and aspartic acid were all reported. The particles
produced were found to be smaller and of a higher aspect ratio
than those of the control sample (without amino acid present)
and generally more stable to aggregation. The presence of amino
acids at the particle surfaces were confirmed by both FTIR and
zeta (ζ) potential measurements. Furthermore, the authors showed
that it was possible to cross couple the amino-acid functionality
at the surface of the particles to produce aggregated clusters with
some degree of preferential elongation along the long axes of
the particles.
In this work we report a detailed investigation of the nature
and stability of the interaction of a series of amino acids with
the surface of carbonate-containing hydroxyapatite (CHA)
particles using a range of techniques including measurements of
the adsorption isotherms of the amino acids, ζ-potential titrations,
surface characterization by XPS, and bulk characterization by
XRD and TGA. The amino-acid-functionalized CHA particles
were prepared by the method of Gonzalez-McQuire et al.,17 and
further to their initial findings, it is demonstrated that the amino
acids are both adsorbed at the CHA surface and occluded within
the primary particles. Moreover, measurements of adsorption
10.1021/la701848c CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/27/2007
12234 Langmuir, Vol. 23, No. 24, 2007
isotherms and ζ-potential titrations have shown that the interaction
between amino acids and the surface of CHA is weak and
reversible and that the stability and functionality provided by the
amino acids can be readily lost during purification procedures.
The mechanism of stabilization of the CHA particles was also
investigated in further detail, and it is shown that in addition to
providing increased charge stabilization in the case of certain
amino acids, retardation of the rate of Ostwald ripening (and
hence a reduction in interparticle attractive forces and the rate
of sedimentation) also plays a significant role. This final point
is surprisingly not well stated in the literature, although it is well
recognized as the basis of methods to control the shape and
morphology of HA and CHA particles; see, e.g., refs 18-21.
Measurements of adsorption isotherms of various amino
acids,22-28 small organic molecules,29-33 as well as macromolecules (peptides, proteins, lysosomes, etc.)25,34-39 onto the surface
of HA, CHA, or fluorinated HA particles have been previously
reported. Additionally, the presence of different amino acids in
solution has been shown to significantly reduce the kinetics of
crystallization of HA from a supersaturated solution onto seed
particles.27,28,40-44 In general, the authors apply the Langmuir
model to the experimental isotherms or particle growth data to
quantify the relative affinities and capacities of the adsorbents
onto HA surfaces. This can be used to better understand the
efficiency of HA columns for separating peptides and related
compounds or provide insight into the functional groups that
may be interacting with the HA surface. Alternatively the
adsorption of serine onto HA at pH 7 has been modeled with a
Freundlich isotherm; it was suggested that this was indicative
of the weak nature of the interaction between the amino acid and
the surface.24
Variations in the nature of the particles used (e.g., the degree
of substitution by carbonate or other ions) or conditions at which
the isotherms were measured (e.g., pH) make it difficult, however,
(18) Sonoda, K.; Furuzono, T.; Walsh, D.; Sato, K.; Tanaka, J. Solid State
Ionics 2002, 151, 321-327.
(19) Oener, M.; Dogan, O. Prog. Cryst. Growth Charact. Mater. 2006, 50,
39-51.
(20) Zhang, H. G.; Zhu, Q. Chem. Lett. 2005, 34, 788-789.
(21) Zhang, H. G.; Zhu, Q.; Wang, Y. Chem. Mater. 2005, 17, 5824-5830.
(22) Aoba, T.; Moreno, E. C. J. Colloid Interface Sci. 1985, 106, 110-121.
(23) Kresak, M.; Moreno, E. C.; Zahradnik, R. T.; Hay, D. I. J. Colloid Interface
Sci. 1977, 59, 283-292.
(24) Benaziz, L.; Barroug, A.; Legrouri, A.; Rey, C.; Lebugle, A. J. Colloid
Interface Sci. 2001, 238, 48-53.
(25) Garcia-Ramos, J. V.; Carmona, P.; Hidalgo, A. J. Colloid Interface Sci.
1981, 83, 479-484.
(26) Misra, D. N. J. Colloid Interface Sci. 1997, 194, 249-255.
(27) Moreno, E. C.; Kresak, M.; Hay, D. I. Calcif. Tissue Int. 1984, 36, 48-59.
(28) Spanos, N.; Klepetsanis, P. G.; Koutsoukos, P. G. J. Colloid Interface Sci.
2001, 236, 260-265.
(29) Misra, D. N. J. Biomed. Mater. Res. 1999, 48, 848-855.
(30) Chirdon, W. M.; O’Brien, W. J.; Robertson, R. E. J. Biomed. Mater. Res.,
Part B 2003, 66B, 532-538.
(31) Mangood, A.; Malkaj, P.; Dalas, E. J. Cryst. Growth 2006, 290, 565570.
(32) Vega, E. D.; Narda, G. E.; Ferretti, F. H. J. Colloid Interface Sci. 2003,
268, 37-42.
(33) Misra, D. N. Langmuir 1988, 4, 953-958.
(34) Misra, D. N. J. Colloid Interface Sci. 1996, 181, 289-296.
(35) Barroug, A.; Lemaitre, J.; Rouxhet, P. G. Colloids Surf. 1989, 37, 339355.
(36) Barroug, A.; Fastrez, J.; Lemaitre, J.; Rouxhet, P. J. Colloid Interface Sci.
1997, 189, 37-42.
(37) Barroug, A.; Lernoux, E.; Lemaitre, J.; Rouxhet, P. G. J. Colloid Interface
Sci. 1998, 208, 147-152.
(38) Tsortos, A.; Nancollas, G. H. J. Colloid Interface Sci. 2002, 250, 159167.
(39) Capriotti, L. A.; Beebe, T. P., Jr.; Schneider, J. P. J. Am. Chem. Soc. 2007,
129, 5281-5287.
(40) Koutsopoulos, S.; Dalas, E. J. Colloid Interface Sci. 2000, 231, 207-212.
(41) Koutsopoulos, S.; Dalas, E. J. Cryst. Growth 2000, 217, 410-415.
(42) Koutsopoulos, S.; Dalas, E. Langmuir 2000, 16, 6739-6744.
(43) Koutsopoulos, S.; Dalas, E. J. Cryst. Growth 2000, 216, 443-449.
(44) Koutsopoulos, S.; Dalas, E. Langmuir 2001, 17, 1074-1079.
Jack et al.
Table 1. pKa Values and Isoelectric Points (pI) of the Amino
Acids Used in This Work; from Nelson and Cox45
amino acid
glycine
alanine
serine
aspartic acid
lysine
sample
code
GLY
ALA
SER
ASP
LYS
pKa
COOH
pKa
NH2
2.34
2.34
2.21
1.88
2.18
9.60
9.69
9.15
9.60
8.95
pKa
side chain
pI
3.65
10.53
5.97
6.01
5.68
2.77
9.74
to compare the affinities or maximum adsorbed amounts obtained
from the various authors and hence for a wide range of aminoacid structures. Moreover, while Koutsopoulos and Dalas40-44
studied the effects of a wide range of amino acids on the rate
of crystallization of HA, it is not clear if the parameters obtained
from Langmuir-type fits to these data are directly comparable
to those obtained from measurements of the adsorption isotherms.28 In addition Misra29 investigated the adsorption of a
range of molecules from both aqueous and nonaqueous solution
and also studied the reversibility or irreversibility of these
adsorption processes. It was concluded that adsorption of low
molecular weight solutes from an aqueous solution generally
involves an ion-exchange process and that additional processes
such as precipitation of salts of the solute can lead to the apparently
anomalous adsorption parameters.
The amino acids studied in this work are shown in Scheme
1, and some of the physical properties45 of these amino acids are
summarized in Table 1. The isoelectric point of the amino acids
(pI) shown in this table represents the pH at which the total
number of positive and negative charges on the amino acid are
balanced; i.e., at pH > pI there will be a net negative charge.
The amino acids were chosen to represent a series of simple
structures with increasing size (GLY < ALA < SER) and sidegroup charge (ASP negative, GLY neutral, LYS positive).
Experimental Section
Materials. Carbonate-Containing Hydroxyapatite (CHA). The
syntheses of CHA and amino-acid-functionalized CHA particles
were performed in a manner similar to that described by GonzalesMcQuire et al.17 Typically, 35.29 g of Ca(NO3)2·4H2O (Ridedel-de
Haën, AR grade) and 2 mol equiv of the respective amino acid
(Sigma, AR grade) relative to the amount of Ca2+ was dissolved in
150 mL of milliQ water. The pH was adjusted to 9 via addition of
28% NH3(aq) solution (Ajax, AR grade), and the mixture was stirred
and heated to 80 °C in an oil bath. A 3.37 mL aliquot of 85%
ortho-phosphoric acid (Fluka, AR grade) was dissolved in 150 mL
of milliQ water, and the pH was adjusted to 9 with 28% NH3(aq).
This was added instantaneously to the Ca2+/amino-acid mixture,
after which a white solid was observed to precipitate immediately
from the solution. The particles were aged by stirring and refluxing
at 80 °C for 18 h. With the exception of CHA-ASP, this produced
CHA dispersions that did not sediment for several weeks.
CHA Washing Procedure. CHA particles were purified by
centrifugation at 2000 rcf for 1 min, removal of the supernatant, and
resuspension in fresh solvent of interest. Typical washing procedures
were repeated 5 times to ensure removal of excess amino acids.
Particle Dialysis. A 10 mL aliquot of freshly synthesized CHAGLY particles was purified by dialyzing against excess glycine
solution, prepared by dissolving 7 g of glycine into 1 L of distilled
water and adjusting the pH to 9 by the addition of 0.25 M KOH.
Solvent renewal was carried out once a day for 7 days and the
solutions were stirred during dialysis.
Measurement of Amino-Acid Adsorption Isotherms. CHA for these
experiments was synthesized as above without amino acid present.
Particles were collected by centrifugation and washed using the
(45) Nelson, D. L.; Cox, M. M. Lehninger principles of biochemistry, 3rd ed.;
Worth Publishers: New York, 2000.
Modified Carbonate-Containing Hydroxyapatite Particles
Langmuir, Vol. 23, No. 24, 2007 12235
procedure described previously, followed by drying in an oven. The
aggregated crystals were then pulverized using a mortar and pestle.
The CHA powder was heated in an oven at 500 °C for 24 h to
remove residual organic matter. BET analysis revealed the specific
surface area of the resultant particles to be 34.5 m2 g-1.
Isotherms of the adsorption of the amino acids onto CHA were
constructed using a depletion method. The concentration of amino
acid in solution before and after equilibration with CHA particles
was measured by reaction with a dye and subsequent detection by
spectrophotometry (see details below). The difference between these
concentrations was taken as the amount adsorbed onto the CHA
surface.
For each of the amino acids 5 mL of the respective amino-acid
solution (concentrations ranging between 1 and 40 mM) was added
to 90 mg of particles, and the pH was adjusted to 9 via addition of
0.25 M KOH. The suspensions were then agitated for 24 h, a time
determined experimentally to be sufficient for equilibrium to be
attained. The suspensions were centrifuged at 4000 rcf for 10 min,
after which 5 µL of supernatant was extracted and lyophilized at
0.25 Torr for 4 h to remove water.
The concentration of amino acid remaining in the extracted
supernatant was determined via reaction with ninhydrin and
subsequent measurement of the absorption at 570 nm.46 A 50 µL
amount of 80% phenol in ethanol (Fluka), 50 µL of KCN in water/
pyridine (Fluka), and 50 µL of 6% ninhydrin in ethanol (Fluka) were
added to each of the lyophilized amino acids, which were then heated
at 95 °C for 5 min. A 3.6 mL amount of 60% v/v ethanol (Ajax,
AR grade) in water was added to each solution, which was then
agitated to ensure complete mixing. The concentration of each
solution was determined by measuring the absorbance at 570 nm
and using the Beer-Lambert law. Calibration plots were constructed
to determine the molar extinction coefficients for the Ruheumann’s
Blue complexes and performed for each amino acid to account for
the different sensitivities of each amino acid to the ninhydrin reaction.
Two replicates of each isotherm were measured and subsequently
averaged. Blank experiments indicated that no background response
resulted from reaction of ninhydrin with CHA or from light scattering
from residual CHA particles.
Instrumental Methods. X-ray Diffraction. Samples were analyzed
in a Bruker D8 Advanced X-ray diffractometer equipped with a
graphite monochromator, copper target, and scintillation counter
(detector). Measurements were conducted from 2θ ) 20° to 70° in
0.050° steps using a step time of 12 s per step. The powder patterns
were compared with database fingerprints (Powder Diffraction File,
second Release 2003) using the DIFFRACplus Evaluation Package.
InductiVely Coupled Plasma-Optical Emission Spectroscopy.
Particle bulk compositions were determined using a Varian VistaPro CCD Simultaneous ICP-OES spectrometer fitted with a
concentric nebulizer and a forward power setting of 1200 W. Samples
were dissolved in 5% nitric acid prior to analysis.
ζ-Potential Titrations. Electrokinetic measurements were conducted on a Malvern Instruments Nano Series ZS Zetasizer (model
ZEN3600) fitted with a 633 nm laser and a MPT-2 autotitration unit.
Samples were diluted by a factor of 50:1 in Millipore water with
a background electrolyte concentration of 1 mM KCl so that a count
rate of 1800-2000 kilocounts per second was maintained. Thirty
runs were performed and averaged for each measurement. ζ-potentials
were calculated using the Henry equation
UE )
2zf(ka)
3η
(1)
where UE is the particle electrophoretic mobility, is the solvent
dielectric constant, η is the solvent viscosity, f(ka) is Henry’s function,
and z is the ζ-potential. Smoluchowski’s approximation was used,
and f(ka) was taken to be 1.5. Changes in pH were obtained via
addition of 0.25 M HCl and 0.25 M KOH. CHA particles were also
titrated with 0.25 M K3PO4 as well as 0.25 M CaCl2 in order to
understand the effects of these potential-determining ions.
(46) Friedman, M. J. Agric. Food Chem. 2004, 52, 385-406.
Infrared Spectroscopy. IR spectra were recorded from 4000 to
525 cm-1 on a Nicolet Nexus 8700 FTIR spectrometer with a singlereflection diamond Smart Omni-Sampler ATR sampling accessory.
Sixty-four scans were recorded at a resolution of 8.00 cm-1 and a
mirror velocity of 0.6329 cm-1.
BET Analysis. Particle specific surface areas were determined
using a NOVA 1200 Quantachrome apparatus. N2 partial pressures
were varied between 4.5 × 10-2 to 2.5 × 10-1, and a cross-sectional
area of 16.2 Å2 was assumed for nitrogen.
Scanning Electron Microscopy. Particle suspensions at concentrations of approximately 0.01% w/w were dropped onto aluminum
stubs coated with a carbon adhesive and covered with glass slips.
Platinum coating was performed using an EIKO IB-5 Sputter Coater
for 3 min, resulting in coat thicknesses of approximately 10 nm.
Images were obtained using a JEOL JSM 6300 at an aperture of 4
and an accelerating voltage of 6 kV.
UV Measurements. UV absorption measurements were conducted
using a Perkin-Elmer Lamda 2 UV/vis spectrometer. Scans were
conducted from 800 to 190 nm in data intervals of 1.0 nm and a
speed of 1 nm s-1.
TGA Measurements. TGA measurements were conducted using
a Shimadzu Thermogravimetric Analyzer. Approximately 25 mg of
sample was used in each measurement, which was conducted in a
nitrogen atmosphere (80 mL min-1). Samples were heated to
600 °C at a rate of 5 °C min-1.
Results and Discussion
Particle Synthesis, Characterization, and Morphology. All of
the amino-acid systems studied produced visibly cloudy suspensions during the reaction and subsequent aging and stirring
process. Suspensions prepared without any amino acid present
were observed to form dense white sediments within a few minutes
of being stood on the bench after 18 h of maturation. The
suspension of CHA-ASP was also observed to sediment when
left to stand for a period of hours. The remaining CHA-aminoacid dispersions, however, did not sediment over a period of
greater than 2 weeks. Moreover, SEM measurements of the CHA
particles precipitated without amino acids showed that the particles
were initially of the order of 150 nm in length after 2 h of
maturation and proceeded to grow to ∼1-2 µm over a period
of 4 weeks. In contrast to the untreated CHA particles, the particles
prepared with amino acids were found to not increase significantly
in size after the initial maturation process of a few hours.
SEM images of the particles produced by the precipitation
and maturation (18 h) process are shown in Figure 1. These
images were collected from a drop of the sample dispersion
which has been rapidly evaporated onto a carbon stub. Aggregation of the particles observed in the images is likely, in part
at least, to be a result of the drying process. The approximate
mean sizes (length and maximum width) of the particles were
determined by averaging ca. 100 primary particles in each of the
images and are reported in Table 2. The ratios of calcium to
phosphorus (Ca:P) in the particles were measured by both ICPOES and XPS. The ICP method measures the bulk Ca:P, while
XPS probes only the top ca. 1-5 nm of the surface of the crystals.
For both sets of measurements the particles were washed to
remove excess ions and amino acid. These ratios are shown in
Table 3. The bulk Ca:P ratio of 1.88 determined for the CHA
control sample is larger than the stoichiometric value of 1.67 for
pure HA but is in a range which is reported for carbonatesubstituted HA.47 Furthermore, it can be seen that the bulk Ca:P
ratio shows little change upon addition of the amino acids.
Powder XRD measurements for the particles are shown in
Figure 2. These XRD patterns are all consistent with poorly
(47) Milev, A. S.; Kannangara, G. S. K.; Wilson, M. A. J. Phys. Chem. B 2004,
108, 13015-13021.
12236 Langmuir, Vol. 23, No. 24, 2007
Jack et al.
Figure 1. SEM images of CHA particles prepared in the presence
of (a) no amino acid (CHA control sample), (b) 1 M GLY, (c) 1 M
ALA, (d) 1 M SER, (e) 1 M LYS, and (f) 1 M ASP.
Table 2. Sizes of CHA-AA Particles Determined by SEM
sample code
lengtha
(nm)
widtha
(nm)
aspect ratio
(L/W)
none
GLY
ALA
SER
ASP
LYS
258 ( 6
162 ( 1
96 ( 2
118 ( 3
57 ( 1
123 ( 2
28 ( 1
29 ( 1
30 ( 1
29 ( 1
20 ( 1
32 ( 1
9.2
5.6
3.2
4.1
2.9
3.8
a
Mean sizes determined by averaging ca. 100 randomly selected
primary particles.
Table 3. Ca:P Ratio and Organic Content of Washed CHA-AA
Particles
amino
acid
Ca:P
ICP-OES ( 0.05
Ca:P XPS ( 0.1
organica content
(%) ( 3%
none
GLY
ALA
SER
ASP
LYS
1.88
1.72
1.75
1.85
1.88
1.88
1.6
2.1
1.8
2.0
2.0
1.9
3
10
6
9
20
9
a
Samples washed to remove surface- bound molecules, and the organic
content is defined as the % mass loss from 100 to 600 °C (see text).
crystalline CHA.17,47 Moreover, FTIR measurements (see Supporting Information) of these particles were performed and found
to be consistent with HA containing CO3 substitutions.47-49 The
carbonate substitution in these particles is estimated to be ca. 4
wt % from the FTIR spectra and the empirical relationship of
Featherstone et al.49 and typical for apatite particles prepared at
ambient CO2 concentrations. It is not possible to assess the
carbonate content of the HA samples reported by Gonzalez(48) Kumar, R.; Prakash, K. H.; Cheang, P.; Khor, K. A. Langmuir 2004, 20,
5196-5200.
(49) Featherstone, J. D. B.; Pearson, S.; LeGeros, R. Z. Caries Res. 1984, 18,
63-66.
McQuire et al.;17 however, it seems likely that the level will be
similar given the similarity in the method of preparation. It can
be seen from Figure 2 that the peaks were broadened compared
to those in the XRD of the CHA control sample, indicating that
crystalline domains within the particles functionalized with amino
acids are smaller or significantly more disordered than those in
the CHA control. Incorporation of the amino acids also increased
the fraction of amorphous material in the particles, which can
be seen as a broad featureless peak (‘amorphous halo’) under the
crystalline reflections in Figure 2. Qualitatively, it can be seen
from the broadening of the 002 reflection at 25.9° and the
amorphous halo in Figure 2 that the more negatively charged
amino acids cause the most retardation or disorder along the
long axis, i.e., ASP < SER < GLY ≈ ALA < LYS.
The above results show that the additions of the amino acids
to the reaction solution affected the particle size, morphology,
composition, and stability of the dispersions. Such a result is in
line with previous reports in the literature.17 In contrast to the
work of Gonzalez-McQuire et al.,17 however, the particles
prepared in this work did not show such well-defined needlelike shapes and were found to be larger and of lower aspect ratio
(see Figure 1). These differences are presumably due to subtle
differences in the methods of preparation. Adsorption of the
amino acids onto or into the CHA particles is further confirmed
from the appearance of amino-acid-specific bands in the ∼15001700 cm-1 region of the FTIR spectra (see Supporting Information), which correspond to asymmetric stretching and bending
of COO- and NH3+ groups.50-54 The high degree of overlap of
amino-acid peaks in the 1500-1700 cm-1 region with those of
CHA limits the ability to observe shifts in vibrational energies
that may occur upon interaction of the functional groups of the
amino acids with CHA and its constituent ions. In the case of
the CHA prepared in the presence of ASP, a loss of the carboxylicacid stretching vibration (1685 cm-1) is observed. This observation implies that the protonated ‘free’ acid side chains present
in ASP are being converted to the carboxylate form and
presumably chelating with Ca2+ ions. It is not possible, however,
to determine if the interaction of the carboxyl group occurs with
calcium that is contained with the CHA crystal or to free calcium
ions in solution.
The presence of amino acids during precipitation of the particles
was found to significantly reduce the dimensions of the CHA
particles along their long axis, although they were observed to
produce a negligible reduction in the shorter axis with the
exception of ASP (see Table 2). In general, the aspect ratio,
defined as the length divided by the width, was found to decrease
when the amino acids were present in the reaction mixture with
only minor variations observed between the different amino acids
used. This finding is in contrast to that reported previously;
however, the control sample (CHA matured for 18 h) studied in
this work was found to be significantly longer (260 nm, cf. 80
nm) and of greater aspect ratio (9.3, cf. 4) than that reported in
the work of Gonzalez-McQuire et al.17. It is also interesting to
note that the crystalline domain sizes determined by Scherrer
analysis in the work of Gonzalez-McQuire et al. are similar in
terms of overall sizes and trends with addition of amino acids
(50) Lopez Navarrete, J. T.; Hernandez, V.; Ramirez, F. J. Biopolymers 1994,
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(51) Rosado, M. T.; Duarte, M. L. T. S.; Fausto, R. Vib. Spectrosc. 1998, 16,
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6, 2441-2445.
(53) Selemenev, V. F.; Zagorodni, A. A. React. Funct. Polym. 1999, 39, 5362.
(54) Jarmelo, S.; Reva, I.; Carey, P. R.; Fausto, R. Vib. Spectrosc. 2007, 43,
395-404.
Modified Carbonate-Containing Hydroxyapatite Particles
Langmuir, Vol. 23, No. 24, 2007 12237
Figure 2. XRD spectra of CHA particles prepared in the presence of 1 M (a) GLY, (b) ALA, (c) SER, (d) LYS, (e) ASP and (f) no amino
acid (CHA control sample).
that would be determined if the same analysis were applied to
this work. It is possible that the larger overall primary particle
sizes measured by SEM in this work are a result of a greater
degree of agglomeration of these crystalline domains during the
reaction and maturation
To determine whether amino acids may be occluded within
these particles, the organic contents of the particles were
determined by thermogravimetric analysis (TGA). The particles
were washed prior to the TGA measurements to remove surfacebound amino acid. The residual organic content is, therefore,
assigned to material occluded within the particles. Representative
mass-loss curves are shown in Figure 3, and the percent organic
material determined from these curves is presented in Table 3.
The percent organic material is defined in this work as the
difference in mass between 100 and 600 °C divided by the mass
at 100 °C to allow for an initial mass loss due to adsorbed water.
Moreover, the relatively high uncertainty quoted for the percent
organic content is estimated to account for the loss of volatiles
observed in the CHA control sample, which are not due to amino
acid. It can be seen from this table that a significant amount of
organic material (up to approximately 10% of the mass of the
particle or 20% in the case of the sample containing ASP) may
be trapped within the primary particles. To put these weight
fractions into perspective, from the measurement of the adsorption
Figure 3. Representative mass-loss curves measured by TGA for
CHA (top-most curve) and CHA initially prepared in the presence
of 1 M ALA, SER, and ASP (in descending order from the CHA
curve). All of the samples were washed, as described in the text, to
remove surface-bound amino acid before the TGA measurements
were carried out.
isotherms presented below (see Table 4) it can be calculated that
the weight percent of amino acid that could be attributed to
12238 Langmuir, Vol. 23, No. 24, 2007
Jack et al.
Figure 4. ζ-potential titrations of CHA (×) and CHA prepared in
the presence of 1 M GLY (2), SER (b), ASP (9), ALA (+), and
LYS ([). Lines are shown as guides for the eye with CHA shown
as a solid line. Samples were measured as fresh reaction sols diluted
by a factor of 50 in milliQ water.
Table 4. Parameters Obtained from Langmuir Isotherm Fits of
Amino-Acid Adsorption onto Plain CHAa
amino acid
K (L mol-1)
GLY
ALA
SER
ASP
LYS
GLY (pH 7)
GLY (pH 11)
110
40
31
32
140
36
no adsorption
a
Γmax (×10-6 mol m-2)
5.1
9.4
10.1
6.9
4.6
12.0
no adsorption
Isotherms were measured at pH 9 unless otherwise stated.
surface adsorption to the primary particle is of the order of
1-5.5%; the maximum value is calculated for the SER sample
which has the highest adsorbed amount. As noted above, the
XRD patterns (Figure 2) for the CHA-amino-acid particles show
both an increase in the content of amorphous or noncrystalline
material and a significant broadening of the peaks compared
with the CHA control sample. Sarig55 hypothesized on the basis
of crystallographic data and geometric arguments that ASP could
be able to cocrystallize into carbonate-substituted HA. Moreover,
occlusion of the amino acid into the CHA particles may also be
the origin of the increased amorphous content that can be observed
in Figure 2 upon addition of amino acid (particularly in the ASP
and SER samples).
Particle Stability. In order to better understand the stability
of the CHA dispersions in the presence of amino acid, ζ-potentials
of the CHA particles were measured as a function of the pH of
the as-prepared particle dispersions. These are presented in Figure
4. As noted above, it was observed that the mean size of the
particles prepared in the presence of the amino acid remained
constant when stored, whereas the control sample was found to
increase in size with time due to Ostwald ripening. Additionally,
the CHA particles functionalized with GLY, ALA, SER, and
LYS were all found to be stable (i.e., they did not sediment) over
a period of weeks at pH ≈ 9, while the control sample and the
particles prepared with ASP were observed to sediment. From
Figure 4 it can be seen that the ζ-potentials at pH 9 are CHA
≈ -10 mV, CHA-ASP ≈ 0 mV, CHA-ALA ≈ CHA-SER
≈ +10 mV, CHA-GLY ≈ +21 mV, and CHA-LYS ≈ +27
mV. The reason for the instability of the CHA-ASP particles
at this pH can be seen to be due to the almost zero surface
(55) Sarig, S. Bone 2004, 35, 108-113.
Figure 5. ζ-potential titrations of CHA (×) and CHA initially
prepared in the presence of 1 M GLY (2), SER (b), ASP (9), ALA
(+), and LYS ([) and then dialyzed against milliQ water for 7 days.
Lines are shown as guides for the eye. CHA is shown as a solid line.
potential which provides little charge repulsion and hence a low
energy barrier to aggregation. This has also been noted by previous
authors.17
The magnitude of the surface potential on the CHA control,
however, is similar to that of the ALA- and SER-prepared samples,
although the CHA sample was observed to readily sediment
while the ALA and SER samples were stable over time. This
difference in stability is, therefore, due to the overall reduction
and stabilization of particle size that is achieved by the presence
of amino acid, i.e., the retardation of the Ostwald ripening process
that is observed in the CHA sample. Moreover, incorporation of
amino acid into the particles would lead to a reduction in the
bulk density of the particles. Therefore, such an increase in the
observed stability is to be expected as the attractive forces between
the particles become weaker with decreasing particle size and
density, thereby increasing the overall barrier for particle
aggregation.56 In addition, the rate of sedimentation will also
decrease as particle size and density decreases. Any additional
increase in the magnitude of the surface potential from adsorption
of the amino acids would further increase the particle stability.
Presumably the reduction of crystal growth is also critical in the
reported stabilization of nanometer-sized CHA particles by
addition of polyelectrolyte and surfactants.
Figure 5 shows the ζ-potential measurements for the particle
dispersions following dialysis against Millipore water for 7 days.
In addition, it was observed that the samples contained a large
amount of sediment following dialysis. It can be seen from this
figure that ζ-potentials as a function of pH for all of the samples
show similar trends and have a common isoelectric point (iep).
Moreover, the magnitudes of the ζ-potentials for all of the samples
originally prepared with amino acid are reduced toward that of
the pure dialyzed CHA sample. These results suggest that the
surface-bound amino acids are in equilibrium with the surrounding
solution and are removed to a large extent by the dialysis process.
Such a finding is important as the ability to covalently react
other moieties to the amino-acid-functionalized CHA surface
may be limited by the necessity to remove residual ions and
amino acids following the initial preparation stage. Moreover,
redispersing of the functionalized particles even after reactive
coupling may ultimately lead to a loss of the functionalized layer.
The behavior of the ζ-potential titrations seen in Figure 5, i.e.,
a common iep with differing ζ-potentials, is consistent with
(56) Hunter, R. J. Foundations of colloid science; Oxford University Press:
Oxford, 2001.
Modified Carbonate-Containing Hydroxyapatite Particles
Langmuir, Vol. 23, No. 24, 2007 12239
Figure 6. ζ-potential titration of CHA initially prepared in the
presence of 1 M GLY and then dialyzed against 0.1 M GLY solution
for 7 days. The line is shown as a guide for the eye.
Figure 8. Adsorption isotherms of (a) ALA ([), GLY (+), and
ASP (9) and (b) SER (b) and LYS (2) adsorbing onto CHA (prepared
without amino acid) at pH 9. The solid lines show the results of
fitting the data to eq 3.
Figure 7. ζ-potential salt titrations of plain CHA with the potentialdetermining ions (a) Ca2+ and (b) PO43- at pH ) 7. Lines are shown
as guides for the eye.
dispersions containing particles with a common surface (in this
case CHA) but differing concentrations of indifferent ions. For
CHA, Ca2+, PO43-, and OH- are known to be potential
determining,57,58 and the effect of adding Ca2+ and PO43- to
dispersions of CHA at pH ) 7 is shown in Figure 7, while the
effect of adding OH- can be observed in Figure 5 (CHA control
sample). Note also that the decrease in ζ-potential observed in
the CHA upon dialysis (from -10 to -15 mV) is consistent with
removal of excess Ca2+ ions from this solution during dialysis.
Given that the samples were first purified by dialysis and prepared
with a fixed concentration of background electrolyte, it is not
(57) Bell, L. C.; Posner, A. M.; Quirk, J. P. J. Colloid Interface Sci. 1973, 42,
250-261.
(58) Garcia Rodenas, L.; Palacios, J. M.; Apella, M. C.; Morando, P. J.; Blesa,
M. A. J. Colloid Interface Sci. 2005, 290, 145-154.
clear what the exact source of the indifferent ions is. By
comparison of Figures 4 and 5, it is possible that the small increase
in ζ-potential with increasing pH observed at ca. pH ) 8.5 in
the case of the LYS and ALA samples in Figure 5 is due to
residual amino acid that has not been completely removed from
the CHA by dialysis for 7 days. Given the above finding that
significant amounts of the amino acids are likely to be occluded
into the crystal and, therefore, only removed by dissolution and
reprecipitation of the CHA particles, it may take a significant
amount of time to completely remove all of the amino acids. We
have not attempted to study the rate of dissolution or the rate of
CHA crystal growth in these dialyzed samples.
Finally it is noted that removal of ions from the reaction mixture
without removal of the amino-acid functional groups and the
particle stability can be achieved by dialysis of the reaction mixture
against a solution containing an excess of amino acid at pH )
9. ζ-potential measurements as a function of pH for a solution
of CHA particles prepared in the presence of GLY and dialyzed
against a 0.1 M solution of glycine at pH ) 9 for 7 days are
shown in Figure 6. The approximate trend of the ζ-potential is
similar to that of the GLY sample measured in the reaction solution
shown in Figure 4, although the magnitudes of the ζ-potentials
are reduced compared to those of the nondialyzed sample. This
is most likely due to the reduction of the excess ions in the
original reaction mixture.
Nature of the Adsorption of Amino Acids. In addition to
measurements of ζ-potential, adsorption isotherms of the amino
acids onto CHA particles which had been prepared without amino
acid present were measured to gain insight into the nature of the
interaction of the amino acids with the surface of CHA. In Figure
8 the adsorption isotherms for the five amino acids at pH 9 are
presented. Adsorption isotherms for GLY at pH 7 and 11 were
also measured, and it was found that the amounts of GLY adsorbed
12240 Langmuir, Vol. 23, No. 24, 2007
Jack et al.
Figure 9. Adsorption isotherms for GLY onto CHA (prepared
without amino acid) at pH 7 (b) and 9 (2). The solid lines show
the results of fitting the data to eq 3.
onto CHA at pH 11 were below the detection limit of the method
used here (Figure 9). It should be noted that the concentration
of amino acid used in the preparation of the particles shown in
Figure 1 (i.e., in the presence of 1 M amino acid) lies in the
plateau region (surface saturation) for all of the isotherms.
However, the solutions used in the measurement of the ζ-potential
titrations were necessarily diluted by a factor of 50, and
equilibrium concentrations of amino acids are, therefore, approximately 0.02 M.
The measured isotherms were all fitted to a Langmuir-type
isotherm shown in eq 2
Γads
Kc
)
Γmax 1 + Kc
(2)
where Γads is the number of moles of adsorbed amino acid per
unit surface area of CHA at a given equilibrium concentration
(c) of amino acid in solution, Γmax is the maximum number of
moles of amino acid that can be adsorbed (i.e., complete
monolayer adsorption), and K is the affinity constant.
The Langmuir isotherm assumes monolayer coverage, that
there are no lateral interactions between adsorbed solute
molecules, and that the surface is homogeneous with all adsorbing
sites on the surface equivalent in energy. In reality, most surfaces
are heterogeneous and molecules will tend to initially occupy
lower energy surface sites, progressively occupying higher energy
surface sites as the solute equilibrium concentration is increased.
Nonetheless, the assumption of a Langmuir-type isotherm is
often found to provide a useful method to determine equilibrium
coverage and allow for comparisons of the affinity of adsorption
between different but related systems.
It should also be noted that the adsorption isotherms reported
here were measured on CHA particles that were initially prepared
without amino acid (i.e., the control samples in Figures 1-4),
as described in the Experimental Section above, while the CHAamino-acid particles used in the previous analysis were precipitated in the presence of 1 M amino acid. Moreover, from the
TGA and XPS measurements it is most likely that there is amino
acid occluded within the particles shown in Figure 1, and it is
not suggested that the formation of CHA in the presence of the
amino acids is described by a Langmuir adsorption process.
Instead, the adsorption isotherm measurements are presented
here to determine if there is a correlation between the affinity
of the amino acids and their effects on particle formation in the
presence of amino acid and to better understand the ζ-potential
titrations, which are equilibrium surface processes. These points
are further discussed at the end of this section.
The values for K and Γmax determined from the measured
isotherms are shown in Table 4. It can be seen that the affinities
of the amino acid to the CHA surface at pH ) 9 are in the order
of LYS > GLY > ALA > SER ≈ ASP, although all of the K
values measured for the amino acids studied here span less than
1 order of magnitude (30-140 L mol-1). Moreover, this trend
is qualitatively similar to that observed for the increase in surface
potential at this pH (see Figure 4) and also for the degree of
positive nature of the side amino acid at pH ) 9, which scales
in the order of LYS (positive) > ALA, GLY (neutral) > SER
(partial negative) > ASP (negative); see also the isoelectric points
of the amino acids (pI) shown in Table 1.
From Table 3 it can be seen that the surface Ca:P ratio measured
by XPS (1.60) of the CHA control sample is lower than that of
the bulk ratio (1.88), implying that the surface is either depleted
in Ca2+ or rich in PO43-. It should be noted that the samples have
been ‘washed’, as described above, prior to the XPS measurements
to remove excess reactants. Similar changes in the surface Ca:P
ratios of washed HA samples have been observed by XPS.59
Brown and Martin60 proposed that the origin of this effect is due
to formation of thermodynamically stable calcium-deficient layers
at the HA surface via a dissolution and reprecipitation mechanism.
Such an effect is reported to be more pronounced for particles
of high surface area, such as those prepared in this work, and
in part responsible for the negative ζ-potential of the CHA particles
observed at pH > 5. The adsorption affinities (K) of the amino
acids measured here may, therefore, reflect an increased ease of
approach of the more positively charged amino acids toward the
negatively charged CHA interface. In addition, the relatively
narrow range of K values determined between LYS and ASP,
despite having opposite polarities in their side groups, suggests
that the principle interaction between the amino acid and the
CHA surface is not mediated through the side group.
It should also be stated that the affinity constants measured
for all of the amino acids investigated in this work are relatively
weak. For example the equilibrium concentration of amino acids
at one-half coverage on the isotherms, i.e., Γ/Γmax ) 0.5 (which
is equal to K-1; see eq 2), are in the range of 7-30 mM. Typical
values for high affinity adsorption are of the order of a few to
tens of micromolar, e.g., adsorption of a specific peptide39 as
well as catechol and related molecules30 onto HA and for
adsorption of a cationic surfactant onto silicon.61 In addition, the
affinity constants (K) determined in this work (30-140 L mol-1)
are of a similar order of magnitude to the stability constants (Ksp)
tabulated for the amino acid-Ca2+ complex;62 these range from
17-40 L mol-1. A number of previous authors have suggested
that this interaction involves some form of coordination between
the carboxyl groups and calcium ions at the surface of the HA
or within the crystal,17,23-26 and such a mechanism is consistent
with these observations at pH ) 9.
From the XPS measurements of the CHA particles formed in
the presence of the amino acids (Table 3) it can be seen that the
ratio of Ca:P at the surface is either the same or slightly elevated
with respect to the bulk. This finding suggests that the presence
of the amino acid either suppresses depletion of calcium ions
from the surface of the crystal during the washing process, as
discussed above, or leads to depletion of phosphate ions. Previous
studies on the adsorption of serine25 at pH 7 have suggested that
there is a concurrent loss of PO43- from CHA upon adsorption
of these molecules. However, in light of the previously discussed
(59) Amrah-Bouali, S.; Rey, C.; Lebugle, A.; Bernache, D. Biomaterials 1994,
15, 269-272.
(60) Brown, P. W.; Martin, R. I. J. Phys. Chem. B 1999, 103, 1671-1675.
(61) Pereira, E. M. A.; Petri, D. F. S.; Carmona-Ribeiro, A. M. J. Phys. Chem.
B 2006, 110, 10070-10074.
(62) Martell, A. E.; Smith, R. M. Critical stability constants; Plenum Press:
New York, 1974; Vol. I.
Modified Carbonate-Containing Hydroxyapatite Particles
finding that the amino acids stabilize particle growth by inhibiting
the Ostwald ripening process, suppression of the surface depletion
of Ca2+ from the amino-acid-functionalized surface is also
considered likely to occur. Moreover, if the loss of phosphate
alone was the main mechanism for the observed increase in the
surface Ca:P ratio (cf. the CHA) then it would be likely that there
would be a greater difference in the ratio for the differing amino
acids and that these differences would reflect the different amounts
of amino-acid adsorption seen in Figure 8 and the Γmax values
determined.
The increase in ζ-potential observed in Figure 4 upon addition
of the amino acids is, therefore, due to a complex balance of any
loss of PO43- and an increased retention of Ca2+ in addition to
the positive charge associated with the -NH3+ terminus and any
additional charge associated with the side group, which is
enhanced in the case of LYS (positive) and reduced in the case
of ASP (negative). The differences in the ζ-potentials of the
CHA functionalized with the neutral amino acids (GLY, ALA,
and SER) can then be further explained by considering the
adsorbed amounts at an equilibrium concentration of ca. 0.02 M
and pH ) 9, as shown in Figure 8. It can be seen that the SER
and ALA isotherms show similar adsorbed amounts but are higher
than that of the GLY. If adsorption is assumed to lead to loss
of PO43- from the CHA crystal and, therefore, an increase in the
solution concentration of PO43-, then from Figure 7 it can be
seen that such an increase in the concentration of PO43- would
lead to a reduction in the measured ζ-potential. Moreover, as the
excess amino acids and ions in the solutions are in equilibrium
with the surface, i.e. the surface amino acid is reversibly adsorbed
onto the CHA surface, the decrease in ζ-potential observed for
the GLY sample in decreasing pH from 9 to 7 is also consistent
with the higher adsorbed amount of GLY at pH 7 (see Figure
9) and the subsequent increase in the solution concentration of
PO43- upon this increased adsorption. Finally, the differences in
the values of Γmax observed at pH 9 are not simple to explain
in terms of molecular size or charge consideration.
It can be seen from Figure 4 that the trends in the ζ-potential
as a function of pH for the amino-acid-coated samples all follow
the same general pattern. That is, they show a decrease in
ζ-potential from pH 9.5, a plateau region at pH < ∼8 and then
an additional decrease to a second plateau in the case of ASP,
SER, and GLY, the onsets of which are dependent on the specific
amino acid (pH ≈ 7, 6.3, and 6, respectively). Moreover, from
Figure 6 it appears that there is a further decrease in ζ-potential
for the GLY sample for pH > 10. The complex behavior observed
in the ζ-potential titrations, i.e., multiple inflections and decreasing
surface charge with increasing H+ concentration (decreasing OHconcentration), suggests that there may be a complex multisite
adsorption process occurring, perhaps associated with different
faces of the CHA crystal. The changing of pH may effectively
‘switch’ on or off certain sites for adsorption, and it is further
likely that the nature of the interaction between the amino acid
and the CHA (e.g., a surface complex between COO- and Ca2+)
surface may be different at different sites. It is also interesting
to note that the inflections occur at similar pH values to inflections
observed in the CHA control, albeit with an opposite sign, and
that the point of inflection at lower pH values (<7) is modified
by the presence and type of the amino acid, although there is no
clear correlation with their pKa values shown in Table 1. The
complex nature of the CHA surface has been discussed in a
recent review of its possible dissolution mechanisms that have
been proposed for different pH ranges.63
(63) Dorozhkin, S. V. Prog. Cryst. Growth Charact. Mater. 2002, 44, 45-61.
Langmuir, Vol. 23, No. 24, 2007 12241
No correlation was found between the CHA-amino-acid
particle sizes and both the trends in the affinity (K) and the
maximum surface coverage (Γmax) measured at pH 9. Such a
result should not be unexpected as the former observations are
associated with the interaction of the amino acids during
precipitation and formation of the CHA particles while the latter
involve equilibrium between the acids and the maturated CHA
surfaces. Finally, the former observations have significant
importance in the control of the shape and morphology of the
CHA particles, while the latter are of great importance to the
nature, stability, and reactivity of the functionalized surfaces
produced.
Conclusions
Amino-acid-functionalized CHA particles were prepared with
GLY, ALA, SER, ASP, and LYS following the method of
Gonzalez-McQuire et al.17 TGA measurements of samples which
were washed to remove surface-bound amino acid have shown
that there are significant amounts of amino acid occluded within
the particles. These occluded amino acids are most likely trapped
within the amorphous phases of the particles, exhibited by the
XRD spectra.
Measurements of ζ-potential as a function of pH were collected
to gain a better understanding of the stabilities of the dispersions
and the amino-acid functionality. The colloidal stability and
reduced rates of sedimentation observed for the majority of aminoacid-functionalized CHA dispersions were assigned primarily to
retardation of particle growth due to adsorption of the amino
acid onto and into the CHA crystals. In addition, the GLY- and
LYS-functionalized surfaces lead to an increase in the magnitude
of the ζ-potential when compared to the CHA. The ζ-potential
of the ASP-functionalized surface was found to be almost zero
at pH 9, and the dispersions were observed to aggregate due to
the negligible charge repulsion between particles.
Dialysis of the amino-acid-functionalized CHA particles
against water with added OH- (at pH 9) lead to the removal of
the amino-acid functionality at the particle surface and ultimately
destabilization of the colloidal dispersions. That is, the surfacebound amino acids were in equilibrium with the surrounding
solution and occluded material was exposed to the surface by
Ostwald ripening. It was further demonstrated that the functionality and stability of the particles could be retained after
purification of the dispersions by dialysis against a solution
containing excess amino acid, although an excess of amino acid
was necessary to maintain the surface adsorption. We are currently
investigating methods for reacting chemical moieties to the
functionalized surfaces.
Measurements of the adsorption isotherms for the amino acids
have shown that the affinities of the acids are relatively weak
and of a similar order of magnitude, suggesting a common
mechanism of association with the surface. However, the small
differences observed show that the more positively charged amino
acids have a greater affinity toward the CHA surface, most likely
reflecting a decrease in repulsion between the net negative charge
of the surface and any residual negative charge on the aminoacid side chains. This does not preclude that the primary
association between amino acids and the CHA surface is via
interactions between -COO- and surface-bound Ca2+, only that
the additional charge interactions lead to changes in the affinities.
The decrease observed in the ζ-potential of the GLY sample
in lowering the pH from 9 to 7 may be explained by the increase
in the adsorbed amount of GLY at the equilibrium concentration
of the amino acid relevant to the measurements and the release
of PO43- upon adsorption. However the ζ-potential titrations
12242 Langmuir, Vol. 23, No. 24, 2007
suggest that the CHA surface may contain multiple adsorption
sites which become accessible for adsorption in different pH
ranges.
Acknowledgment. The authors thank Dr. Barry Wood, Ms.
Anya Yago (Brisbane Surface Analysis Facility), and Ms.
Wadcharawadee Noohom (Centre for Nanotechnology and
Biomaterials) for their assistance with XPS, XRD, and SEM
measurements, respectively. K.J. would like to thank Prof. Hans
Griesser (Ian Wark Research Institute, University of South
Australia) and Dr. Lisbeth Grondahl (School for Microbial and
Molecular Science) for helpful discussions during the preparation
of this manuscript. The authors also acknowledge the valuable
suggestions of the referees during the reviewing of the manuscript.
Jack et al.
The SEM images were collected in the Centre for Microscopy
and Microanalysis, University of Queensland, and with assistance
from centre staff. This work was produced as part of the activities
of the ARC Centre for Functional Nanomaterials funded by the
Australian Research Council under the ARC Centres of Excellence
Program. M.T. acknowledges the support of the Australian
Research Council Federation Fellowship (FF0455861).
Supporting Information Available: FTIR spectra of the CHA
particles prepared in the presence of 1 M amino acids, and the spectra
of the amino acids. This material is available free of charge via the
Internet at http://pubs.acs.org.
LA701848C