Effect of Aspartic Acid and Glycine on Calcite Growth - Crystal Growth

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Effect of Aspartic Acid and Glycine on Calcite Growth G. Montanari,*,† L. Z. Lakshtanov,†,‡ D. J. Tobler,† K. Dideriksen,† K. N. Dalby,† N. Bovet,† and S. L. S. Stipp† †

Nano-Science Center, Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark Institute of Experimental Mineralogy RAS, 142432 Chernogolovka, Russia

‡

S Supporting Information *

ABSTRACT: Organic molecules control calcite growth and crystal morphology, influence biomineralization processes, and offer clues for optimizing antiscalants for industry. Here we quantified the effect of amino acid monomers, aspartic acid (Asp1), and glycine (Gly1), and their polymers (Aspn, Asp5, and Gly5), on calcite growth rate, in a constant composition setup. Asp1 and its polymers inhibit growth, with rate decreasing as amino acid chain length increases. For 2 mM Asp1, fractional inhibition (FI, where 1 represents complete inhibition) was 0.54; for 0.0012 mM Aspn, FI = 0.94. Gly1 and Gly5 only marginally affect growth (−0.1 < FI < 0.1); indeed, they slightly promote growth at most tested concentrations. Fitting of adsorption isotherms (Langmuir, Langmuir−Freundlich, Flory−Huggins) confirmed that Asp polymers adsorb strongly, explaining their strong control on calcite growth, but Gly1 and Asp1 adsorb less due to competition with carbonate ions. ΔGads (Aspn) = −39 kJ/mol; ΔGads (Asp5) = −50 kJ/mol; ΔGads (Asp1) = −21 kJ/mol; and ΔGads (Gly1) = −22 kJ/mol. The morphology was equally affected. Crystal edges became rougher, and corners, more rounded. Overall, the number of carboxyl groups and length of the carbon chain correlated with the lowest growth rate.

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focused on the effect of their smaller building blocks.11 Some studies have included simple to more complex sugars,12,13 amino acids and peptides,14,15 alcohols,16,17 and carboxylic acids.18 In particular, the effects of amino acids, such as aspartic acid (Asp) and glutamic acid (Glu), on calcite growth have been extensively studied both as monomers and as longer chain polymers.11,19−22 Both poly(Asp) and poly(Glu) act as strong calcite growth inhibitors, even at concentrations as low as 0.3 ppm.23 Elhadj and colleagues21 argued that the extent of growth inhibition is related to the number of aspartic acid units in the polymer21 but geometric matching of the molecule with surface crystal structure and electrostatic interactions between the organic compound and the calcite surface also influence adsorption and, thus, growth rate.22−24 Despite the extent of work on this topic, a systematic, quantitative study is needed to show how amino acids affect bulk calcite growth rate and morphology under conditions close to those of natural waters, which are often slightly supersaturated with respect to calcite and where saturation levels remain constant. The ratio of amino acid molecules to calcium and carbonate ions is often quite low in such environments, and this has not been considered in much of the previous work.

INTRODUCTION Organisms produce organic molecules, which can direct the nucleation, growth, and morphology of biominerals.1,2 This often leads to materials that have stronger and more resilient structures and textures than the same mineral formed in the absence of the organisms. For example, although made of the same mineral, teeth, bones, and shells are quite different than inorganic apatite, aragonite, and calcite. The study of biomolecules and their role in biomineral formation has engaged the scientific community for several decades and has opened doors for the synthesis of new biomaterials with predetermined structures, size, and function, specifically designed for a variety of environmental, industrial, and medical applications.3−5 Similarly, knowledge about how biomolecules control and regulate mineral growth has guided the design of organic antiscaling agents for use in fluid handling systems (e.g., pipelines, boreholes, tanks, and heat exchangers).6−8 However, there is still considerable uncertainty about how organic molecules actually control mineral growth rates. Biogenic calcite is produced by some mollusks and coccolithophorids, algae species that cover their one cell with calcite discs. In both cases, the biomineral is known to have an external organic matrix, composed primarily of lipids, polysaccharides, and proteins.9,10 To understand how these complex organic macromolecules interact with calcite and to determine the functional groups that control the growth process, several experimental and modeling studies have © XXXX American Chemical Society

Received: November 20, 2015 Revised: July 23, 2016

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DOI: 10.1021/acs.cgd.5b01635 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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In this study, we used the constant composition method25−29 to quantify the effect of aspartic acid (Asp1) and glycine (Gly1) and their polymers, Aspn, Asp5, and Gly5, on calcite growth. In the experiments, equimolar CaCl2 and Na2CO3 solutions were added in equal volumes to maintain a constant pH of 8.3 ± 0.05 and to buffer the pH decrease resulting from calcite precipitation: Ca 2 + + HCO3− ↔ CaCO3(s) + H+

At steady state, the added moles of CaCl2 and Na2CO3 correspond to the moles of calcite formed: nCaCl 2(added) + nNa 2CO3(added) = nCaCO3(s) + 2nNa + + 2nCl−

Figure 2. Typical plot from a constant composition experiment, showing the trends for pH (blue) and the added volume of titrants (red, i.e. precipitation rates). The amino acid inhibitor (Asp1 in this case) was added ∼30 min after the addition of the calcite seed, and this led to a slight decrease in precipitation rate (represented by the slope). For all experiments carried out in this study, similar growth curves were obtained.

Thus, pH and saturation index, SI, remain constant in the experiments once a steady state is established, with calcite precipitation rates matching the CaCl2 and Na2CO3 addition rate. Based on determined calcite precipitation rates, SI is estimated to have varied somewhat between experiments from 0.3 to 0.6 (where SI = 0 represents equilibrium conditions). Asp1 has one more carboxyl (−COOH) than Gly1, but they both have one amino group (−NH2). In solution, the deprotonated carboxyl is electrostatically attracted to calcium surface sites, whereas the protonated amino group is known to interact with carbonate sites through hydrogen bonding.19,30 The organic molecules are different in terms of chain length, charge, and number of carboxyl and amino groups, which are both involved in the peptide bonds (Figure 1).

polypeptide (Gly5). Specific aliquots of these stock solutions were used in the experiments to produce the concentrations listed in Table 1. The concentration of the polymers was determined from the single unit of the corresponding amino acid residue with MWAsp unit = 115 g/ mol and MWGly unit = 57 g/mol. Merck calcite powder (99.95% Suprapur) was used as the seed material. Prior to the experiments, the powder was recrystallized to ensure uniform calcite surfaces by a method adapted from Stipp and Hochella.31 The fresh calcite powder was exposed to CO2 saturated Milli-Q water (with partial pressure of 1 atm). Carbon dioxide was bubbled into the solution with a constant gas flow. After 24 h, the solution was removed, the solid was rinsed with fresh deionized water, and the cycle was repeated; that is, the solid was put through two cycles of 24 h with the same procedure. At the end of the treatment, the remaining solution was removed, and the treated seed material was freeze-dried and stored dry, at room temperature, until further use. The recrystallized seeds had a surface area of 0.3 m2 g−1, determined by a 5 point Nitrogen BET measurement (0.1 ≤ p/p0 ≤ 0.3) using an Autosorb-1 M from Quantachrome. Experimental Setup. The constant composition method has been described in detail in previous work.25−29 In short, continuous precipitation of calcite at constant saturation is maintained by the constant addition of equal volumes of equimolar calcium and carbonate solutions into a mechanically stirred reactor with controlled pH (8.3), ionic strength (0.1 M), and temperature (25 °C). Figure 2 shows a plot from a typical constant composition experiment, where the total volumes of added calcium and carbonate solutions (red line) and pH (blue line) were recorded as a function of time. At the start of each experiment, 50 mL of 0.1 M NaCl solution containing 2 mM CaCl2 and 2 mM NaHCO3 was added to the reaction vessel. This solution was slightly supersaturated with respect to calcite. However, nucleation of calcite requires substantial supersaturation,32 and we observe no pH drop prior to addition of the seeds, showing that the saturation index, SI, was lower than that required for nucleation, where

Figure 1. Chemical structure of the amino acids, Asp1 and Gly1, and their polymers, Aspn, Asp5, and Gly5.

To compare differences in growth rate with the level of interaction between the calcite surface and the additives, we used X-ray photoelectron spectroscopy (XPS) to monitor the extent of adsorption. Scanning electron microscopy (SEM) was used to check for any changes in crystal morphology.

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MATERIALS AND METHODS

Solution and Calcite Seed Preparation. A set of reactant stock solutions (0.1 M CaCl2, 0.1 M NaHCO3, 0.1 M Na2CO3, and 0.1 M NaCl) were prepared using ultrapure deionized water (Milli-Q; specific resistivity 18.2 MΩ cm) and reagent grade chemicals purchased from Sigma-Aldrich. All solutions were filtered with acetate filters (0.45 μm) to remove dust particles. Three stock solutions were prepared using L-aspartic acid sodium salts: (Asp1), Asp-Asp-Asp-Asp-Asp polypeptide (Asp5), and poly(α,β)-DL-aspartic acid sodium salt (Aspn). Aspn is a polymer of undefined length with a molecular weight between 2000 and 11000 g/ mol and a degree of polymerization ranging from 15 to 73. Two stock solutions were prepared using glycine (Gly1) and Gly-Gly-Gly-Gly-Gly

SI = log Ω = log

IAP K sp

(1)

Ω = saturation ratio, IAP = ion activity product, and Ksp = calcite solubility product. After solution mixing, pH increased slightly, presumably as a result of slight CO2 degassing. Once the solution pH stabilized (usually within 5 min, Figure 2), 30 mg of recrystallized calcite seeds was added to initiate calcite precipitation. This resulted in a sudden pH drop below 8.3, triggering the addition of equal amounts of 0.1 M CaCl2 and 0.1 M Na2CO3 solutions from syringe pumps. The B

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Table 1. Overview of Experimental Conditions, Including Concentrations of the Added Ligands, the N/CO3 Ratio from XPS Analyses, the PHREEQC Molar Ratio between Ca2+ Complexed to the Ligand and Free Ca2+, the Precipitation Rate before (R0) and after Ligand Addition (R1), the Calculated SI (eq 5), FI, and the Maximum % Dilution of the Inhibitor, due to the Continuous Addition of CaCl2 and Na2CO3 Solutions Ligand (L)

[L] (μM)

Asp1

20 20 100 100 200 200 400 400 1000 1000 2000 2000 0.25 0.25 0.8 0.8 2.1 2.1 3.4 4.2 4.2 8.4 8.4 12.6 12.6 0.06 0.07 0.3 0.3 0.6 0.6 0.7 0.7 1.2 1.2 1.4 1.4 1.4 1.4 2.1 2.1 4.8 4.8 20 20 20 20 100 100 100 100 200 200 200 400 400 400 400

Asp5

Aspn

Gly1

N/CO3

∑[Ca2+ − L]/[Ca2+(aq)] 10−4

0.01

0.01 0.02

10−2 -a

0.07 0.04

3 × 10−7

0.03

0.05 0.07

2 × 10−5 10−5

0.01 0.02

R0 (μmol m−2 s−1)

SIb

R1 (μmol m−2 s−1)

FI

Maximum inhibitor dilution (%)c

0.67 0.68 0.68 0.58 0.67 0.66 0.94 0.82 0.34 0.37 0.43 0.36 0.81 0.78 0.48 0.43 0.59 0.51

0.51 0.51 0.51 0.48 0.51 0.51 0.59 0.56 0.38 0.40 0.42 0.39 0.55 0.54 0.44 0.42 0.48 0.46

0.68 0.65 0.61 0.59 0.58 0.58 0.64 0.55 0.22 0.21 0.20 0.16 0.64 0.68 0.32 0.30 0.19 0.26

0.01 0.04 0.09 0.02 0.13 0.13 0.31 0.33 0.34 0.44 0.53 0.56 0.21 0.12 0.34 0.31 0.69 0.48

0.5 0.8 0.5 0.4 0.6 0.4 0.6 0.6 0.4 0.3 0.3 0.3 0.6 0.6 0.2 0.3 0.3 0.2

0.44 0.56 0.54 0.46 0.66 0.60 0.38 0.49 0.43 0.55 0.35 0.56 0.59 0.50 0.38 0.71 0.66 0.60 0.40 0.55 0.64 0.62 0.64 0.77 0.66 0.72 0.62 0.71 0.69 0.66 0.70 0.66 0.77 0.68 0.65 0.70 0.80 0.42 0.41

0.42 0.47 0.46 0.44 0.51 0.49 0.40 0.45 0.42 0.47 0.39 0.47 0.48 0.45 0.40 0.52 0.51 0.49 0.41 0.47 0.50 0.49 0.50 0.54 0.51 0.52 0.49 0.52 0.52 0.51 0.52 0.51 0.54 0.51 0.50 0.52 0.55 0.42 0.41

0.12 0.17 0.09 0.10 0.10 0.13 0.39 0.49 0.27 0.35 0.06 0.21 0.12 0.11 0.00 0.08 0.06 0.07 0.03 0.04 0.03 0.07 0.06 0.03 0.69 0.67 0.60 0.72 0.63 0.64 0.67 0.66 0.72 0.60 0.61 0.62 0.69 0.39 0.38

0.72 0.70 0.84 0.79 0.84 0.78 0.03 0.01 0.37 0.37 0.84 0.63 0.79 0.79 1.00 0.88 0.90 0.88 0.93 0.93 0.96 0.89 0.91 0.96 0.05 0.06 0.03 0.02 0.09 0.03 0.04 0.00 0.07 0.13 0.06 0.12 0.13 0.06 0.07

0.2 0.3 0.2 0.1 0.3 0.3 0.3 0.4 0.3 0.3 0.3 0.3 0.3 0.3 0.1 0.3 0.4 0.2 0.2 0.2 0.3 0.3 0.2 0.4 0.5 0.5 0.4 0.4 0.5 0.6 0.5 0.5 0.7 0.6 0.5 0.7 0.5 0.4 0.4

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Table 1. continued Ligand (L)

Gly5

[L] (μM) 540 540 540 700 700 1000 1000 0.2 4 5 20 100 480

N/CO3

0.01

∑[Ca2+ − L]/[Ca2+(aq)]

5 × 10−4 -a

0.02 0.01

R0 (μmol m−2 s−1)

SIb

R1 (μmol m−2 s−1)

FI

Maximum inhibitor dilution (%)c

0.33 0.36 0.39 0.40 0.35 0.41 0.38 0.31 0.44 0.53 0.50 0.48 0.54

0.38 0.39 0.40 0.41 0.38 0.41 0.40 0.36 0.42 0.46 0.45 0.44 0.47

0.33 0.40 0.41 0.42 0.36 0.44 0.42 0.34 0.46 0.57 0.54 0.52 0.56

0.02 0.12 0.06 0.06 0.04 0.07 0.11 0.11 0.05 0.08 0.07 0.09 0.03

0.5 0.5 0.5 0.6 0.5 0.6 0.5 0.3 0.4 0.5 0.5 0.4 0.6

Could not be determined due to the absence of equilibrium constants for Asp5 and Gly5. bEstimated based on the correlation between R0 and Ω given ref 28. cCalculated by dividing the final volume of titrants added at the end of the experiment by the initial volume of the solution, multiplied by 100. a

injection raises pH back to 8.3 (Figure 2). The addition of carbonate and calcium ions promoted further calcite growth, which decreased pH. This is constantly compensated by the addition of CaCl2 and Na2CO3 solutions. Calcite growth rate is proportional to the injected volume of CaCl2 and Na2CO3 titrants, which is continuously recorded during the experiments (Figure 2). Once a constant titrant injection rate, i.e, calcite growth rate, was observed (generally ∼30 min after the addition of the calcite seeds), one of the amino acid solutions was injected, at once, into the vessel. Pipetted aliquots of amino acid stock solutions ranged from 0.1 to 0.5 mL for Asp1, from 0.003 to 0.15 mL for Asp5, from 0.01 to 0.7 mL for Aspn, from 0.1 to 2 mL for Gly1, and from 0.009 to 1.5 mL for Gly5. The change in calcite growth rate as a result of the addition is expressed as fractional inhibition, FI:

FI =

R 0 − R1 R0

However, due to slightly variable and unknown equilibration with atmospheric CO2, the saturation indices for the different experiments will have varied slightly. This variation in atmospheric CO 2 equilibration between experiments complicates the PHREEQC calculations of the SI, which at constant pH depend on the amount of dissolved CO2 in addition to the Ca2+ activity. Instead, SI was estimated from the measured R0 based on its reported correlation with Ω in our previous study,28 where an identical experimental setup and calcite powder were used. Adsorption Isotherm Fitting. The Langmuir adsorption isotherm can be fitted to fractional growth inhibition data to interpret the adsorption behavior of the inhibitor,28 i.e., the interaction between the calcite surface and the amino acid or poly(amino acid). We tested three adsorption isotherm models: (1) Langmuir, which describes simple adsorption of an ideal gas onto distinct (empty) surface sites, (2) Langmuir−Freundlich, which takes into account multisite adsorption, and (3) Flory−Huggins, which is most suitable for predicting the adsorption behavior of polymers. These models are described by the following equations:

(2)

where R0 and R1 represent the injection rates prior to and following addition of the organic compound. The time required for stabilization of R1 varied between monomers and polymers and also depended on their concentration (between 1 and 6 h). If FI = 0, calcite growth rate is not affected by the addition of the additive. FI = 1 indicates that growth stops, i.e. complete inhibition. FI can also be negative, which means that growth is enhanced by the organic compound. R0 and R1 were determined from the slope of the titrant injection profile before and after the additive was injected, using linear regression with an in house Matlab code (details are included in the Supporting Information). A mean fractional inhibition was determined from the results of 2 to 4 replicate experiments. The constant concentration of Ca ions in solution was verified by analyzing solution aliquots at regular time intervals using atomic absorption spectroscopy (AAS). Except for a single sample, determined Ca concentrations were within analytical uncertainty (±5%) of the initial value, i.e., before the addition of seeds. The solution speciation for each experiment was determined using PHREEQC,33 using the minteq.dat database.34 Equilibrium constants exist in the database for Gly1 ligand complexation and protonation. For Asp1, values were taken from the NIST Standard Reference Database,35 and for Aspn, they were taken from the study by Wu and Grant.36 Wu and Grant36 reported the constants for Aspn with a molecular weight of 10000 g/mol, consisting of 72 aspartyl residues, which is similar to the Aspn polymer used in this work. For Asp5 and Gly5, constants have not been reported. Note that the continuous addition of titrants (CaCl2 and Na2CO3) hardly affected the concentration of the additive (maximum dilution was less than 1%, Table 1) and, thus, could be neglected. For a given experiment, pH, Ca concentration, and CaCl2 and Na2CO3 addition rates were kept constant; thus, calcite precipitation rate and saturation index were constant prior to inhibitor addition.

Langmuir:

θ=

KC 1 + KC

(3) n

Langmuir−Freundlich: Flory−Huggins:

θ=

KC 1 + KC n

(4)

θ = K (1 − θ ) j C

(5)

where θ represents the fractional coverage, K the affinity constant, C the concentration of the additive, n the Langmuir−Freundlich constant, and j the Flory−Huggins exponent. In this work, equilibrium concentrations were assumed to be equal to the initial concentration. This assumption was tested by changing the amount of calcite seed. The change in the extent of inhibition was insignificant, meaning that the change in concentration resulting from adsorption was negligible relative to the total amount added. The affinity constant, K, was obtained from fitting the fractional inhibition data with the three models and was then used to calculate the adsorption free energy, ΔGads:

ΔGads = − RT ln K

(6) −1

−1

where R is the gas constant (8.314 J mol K ) and T represents temperature (298 K). Solid characterization. Solid samples were removed from the reaction vessel periodically during the experiments for characterization with SEM and XPS. They were separated from solutions using vacuum filtration, quickly rinsed with calcite saturated solution (Milli-Q water equilibrated with calcite), and then dried at room temperature. D

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SEM was used to monitor the change in morphology during the experiments. SEM samples were prepared by placing a small amount of the dry solids on double sided carbon tape which was fixed to aluminum sample holders. We collected images under high vacuum conditions using an FEI Quanta 3D instrument, with an accelerating voltage of 10 kV. XPS probes the chemical composition of the top 10 nm of a solid surface. We used XPS to quantify the extent of additive sorption. Solid samples were analyzed using a Kratos Axis UltraDLD system operated with a monochromated Al Kα X-ray source (power = 150 W, hν = 1486.6 eV). Pass energies of 20 and 160 eV were used for high resolution and wide scans. Data were analyzed using the CasaXPS software. Binding energy calibration was performed with the C 1s peak from carbonate, at 290.1 eV.31

calcite saturation of our experiments, no significant correlation was observed between FI and the calculated SI values. To explore the effect of amino acid chain length, structure, and geometry on calcite growth inhibition, we tested the 5 unit polymers of aspartic acid and glycine (Asp5 and Gly5) and a larger Asp polymer with undefined chain length, Aspn. Similar to the single unit aspartic acid, FI steadily increased as Asp5 and Aspn concentrations increased (Figure 3b). The polymers were more effective growth inhibitors than the single unit Asp. Much lower concentrations were needed to obtain the same FI. For example, 0.0084 mM Asp5 decreased growth rate by FI = 0.81, whereas a 7 times lower concentration of Aspn (0.0012 mM) decreased growth by FI = 0.94 (Figure 3b). Very different behavior was observed for Gly5. FI was slightly negative for a range of concentrations, meaning that Gly5 consistently enhanced precipitation rate slightly (Figure 3b), even at concentrations as high as where the monomer, Gly1, behaves as an inhibitor. Overall, the aspartic acid and its polymers are more effective at inhibiting calcite growth than Gly1 and Gly5. High resolution XPS of the calcite, taken periodically after the addition of the amino acids and poly(amino acid)s, showed a nitrogen peak at ∼400 eV, confirming their presence on the calcite surfaces. The extent of adsorption was quantified from the atomic ratio of nitrogen and the CO3 peak. At the highest Asp1, Gly1, and Gly5 concentrations (1−2 mM), the N/CO3 ratio was 0.02, whereas, for Asp5 and Aspn, the ratio was about 3 times higher, i.e. 0.06 (Table 1). Thus, Asp polymers adsorbed to a greater extent than the Gly polymer, probably because of a stronger interaction with the surface. The nitrogen peak was quite low for Asp1, Gly1, and Gly5, so further relationships between inhibitor type and concentration could not be resolved. Samples of the calcite were further examined by SEM to explore possible changes in crystal morphology. Samples from the inorganic system, i.e. the control, where no inhibitor was added, showed euhedral rhombohedra (Figure 4a and b) that are typical of pure calcite. The {1014̅ } crystal faces dominated. Some crystals had small, corner {101̅0} faces. After exposure to the slightly supersaturated solution without additive, particles had grown (Figure 4c). Terraces were flat and edges were sharp, indicating layer-by-layer growth (Figure 4c). Amino acid addition notably changed the morphology. Terraces and edges were rougher, and the {101̅0} crystal faces become more dominant (Figure 4d). This was true for crystals grown in the presence of all of the additives, but rounded corners were particularly apparent for calcite grown with the Asp polymers (Figure 4e and f). In summary, SEM demonstrated that amino acid addition changed calcite morphology in a similar way, but change was most pronounced with the polymers of aspartic acid. This is consistent with the rate of growth data.

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RESULTS Table 1 shows the measured growth rates before and after the addition of the organic ligands, along with values for the N/ CO3 molar ratio at the surface, the SI, the fractional growth inhibition (FI), and the degree of Ca2+ complexation by the ligands. Notably, the degree of complexation was very small and the PHREEQC calculations show that the addition of the organic ligands affected SI values by no more than 1%. The growth rate of calcite is decreased by most of the organic compounds tested, but the extent, i.e. FI, varied, depending on its composition and concentration (Figure 3). For the single

Figure 3. Fractional inhibition, FI, of calcite growth as a function of inhibitor type and concentration for (a) single unit amino acids (Asp1, Gly1) and for (b) all of the additives. Concentration is expressed as Asp units (MW = 115 g/mol) and Gly units (MW = 57 g/mol). Each point represents the mean value of the replicate experiments (error bars = standard deviation), except for Gly5.

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DISCUSSION The inhibitory effect on growth rate and the changes in calcite morphology clearly show that aspartate, glycine, and their polymers interact with calcite surfaces and affect crystal growth. XPS indicates Asp and Gly monomers and polymers on the surface, and this after rinsing with calcite saturated solution, confirming that adsorption occurred and attachment was strong. Adsorption Isotherm Model Fitting. To fit the adsorption isotherm models to our data, we needed to determine the degree of surface coverage, θ, by the amino acids and poly(amino acid)s, as a function of their

unit Asp1, FI increased as Asp1 concentration increased from 0.02 to 2 mM (Figure 3a). The highest FI (0.54) was observed for Asp1 = 2 mM, whereas, at concentrations ≤0.02 mM Asp1, there was little to no effect. This behavior was similar for the experiments with Gly1. Concentrations ranging from 0.02 to 0.4 mM led to increased FI (Figure 3a), but for concentrations >0.4 mM, FI was negative, meaning that these high Gly1 concentrations enhanced calcite growth. For the relatively low E

DOI: 10.1021/acs.cgd.5b01635 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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3−5). The Langmuir model is used to estimate the adsorption of solutes on various types of substrates.44 It precludes assumptions such as limited adsorption capacity of the substrate, monolayer adsorption, identical and equal surface sites, and no interaction and steric hindrance between the adsorbed species.45 The Langmuir−Freundlich model also applies for multilayer, nonideal, and reversible adsorption.45 When the coefficient, n, is 1, it becomes the Langmuir isotherm. Positive cooperativity, when n > 1, indicates multilayer adsorption, and negative cooperativity, when n < 1, indicates weak interaction with the surface. The Flory−Huggins model is used to describe the behavior of polymeric solutions46 and can provide information about the tendency of adsorption to proceed.45 The fractional inhibition data fitted with these adsorption models are shown in Figure 5. We did not fit the data for Gly5 because its effect on calcite growth rate was at or below the detection limit for the rate of growth. The Langmuir and Langmuir−Freundlich isotherms both fit the Asp1 data (Figure 5a), especially at low concentrations, while the fit with the Flory−Huggins model is better at high concentrations. Fitting of the Asp1 data with the Langmuir−Freundlich isotherm resulted in n values equal to 1, which means it is identical to a Langmuir isotherm (eqs 3 and 4). None of the adsorption models produced a good fit for the Gly1 data (Figure 5b), probably because the FI values are very small and the uncertainty is high. Asp5 (Figure 5c) data are well represented by all of the models, in particular the Langmuir and Langmuir− Freundlich, which produce an identical isotherm. As for Asp1, fitting of the Asp5 data with the Langmuir−Freundlich isotherm resulted in a Langmuir isotherm (n = 1, eq 3 and 4). Figure 5d shows the fit to the Aspn data, where the main difference is between Langmuir and Flory−Huggins models. The Langmuir model fits better at higher fractional inhibition values, while the Flory−Huggins model fits better for lower FI. A good compromise is found with the Langmuir−Freundlich isotherm, which lies between the two others and fits the data particularly well. Fitting the fractional growth inhibition data with these 3 adsorption models provided values for the affinity constant, K. These were then used to determine the adsorption free energies, ΔGads (eq 6), for all fitted data sets (Table 2), to define and compare the driving force for adsorption with respect to calcite precipitation. The ΔGads estimates were quite similar from all three models for all of the additives except Aspn. The fit with the Langmuir− Freundlich isotherm gave a substantially lower ΔGads, −66 kJ/ mol compared with ∼ −42 kJ/mol from the other two models. To understand whether adsorption of amino acids and poly(amino acid)s is favored, we compared our calculated ΔGads with those for adsorption of calcium and carbonate ions on calcite. Calcite is an ionic solid, so the adsorption free energy is dominated by electrostatic interactions. Therefore, we expect ΔGads for the carbonate ion to be very similar to that of calcium. Amino acids and poly(amino acid)s with ΔGads more negative than ΔGads for calcium and carbonate ions would win in a competition for the kink sites. Therefore, assuming that the adsorption free energy of calcium is similar to that of carbonate and by comparing ΔGads for Asp1 and Gly1 from all of the adsorption models (Table 2), we can conclude that both the amino acids compete with carbonate ions (ΔGads Ca = −28.3 kJ/mol47), quite in the range of the values we calculated. This would explain the low fractional growth inhibition for Asp1 and

Figure 4. Typical SEM images of calcite samples collected from the experiments: (a) calcite crystal faces; (b) in the inorganic system (control; no amino acid added); (c) after 50 min of precipitation in the inorganic system, new calcite growth is visible; (d−f) calcite morphologies grown in the presence of (d) Asp1, (e) Asp5, and (f) Aspn. They all roughen terraces, round steps, and favor {1010̅ } face formation.

concentration. It is generally assumed that impurities inhibit crystal growth by blocking active growth sites, impeding Ca2+ and CO32− binding.28,37−39 When an impurity adsorbs to a kink site, it decreases the kink site density and blocks kink propagation.40−43 Dynamic equilibrium ensures that no species blocks a site forever but the rate of adsorption/desorption controls the extent of blocking. Even at the highest Asp polymer concentrations, we do not observe a complete halt of calcite growth, which is typical of the step pinning mechanism. Therefore, we interpret that the leading mechanism of inhibition in these experiments is kink blocking. We assume here that the number of growth units that were adsorbed is directly proportional to the fraction of the free surface.39,42 Then, we further assume that fractional inhibition, FI, is equal to the fractional surface coverage, θ, and we can test how the results match the adsorption isotherm models. Note that the assumption that FI is a function of theta only is a deliberate oversimplification. First of all, it depends on supersaturation as well as on the surface free energy (specific edge free energy). Moreover, different substances (inhibitors) can yield different ratios of crystal faces (as observed here). Nevertheless, we assume that, for a given type of inhibitor, this ratio is nearly constant. So the total coverage is an average over all the faces. The three models that are most commonly used to describe surface adsorption, especially of complex molecules, are Langmuir, Langmuir−Freundlich, and Flory−Huggins (eqs F

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Figure 5. Langmuir, Langmuir−Freundlich, and Flory−Huggins adsorption models fit to fractional inhibition, FI, data for (a) Asp1, (b) Gly1, (c) Asp5, and (d) Aspn.

account, the true equilibrium concentration would be smaller. Because of this uncertainty and because of the similarities in the quality of the fit and the closeness of the ΔGads values derived from the three models, we do not need to judge which model is the correct one. Amino Acid and Poly(amino acid) Interaction with Calcite. The constant composition method is particularly accurate for determining growth rates because precipitation occurs at constant supersaturation, uncomplicated by homogeneous nucleation, so it allows systematic study of how growth kinetics is affected by additives, such as the effect of structure and composition, under conditions close to those at equilibrium in a natural system. In the case of calcite, single unit amino acids, Asp1 and Gly1, slightly affect calcite growth rate. Both molecules interact with the surface through their functional groups.19,30 At the pH of the experiments (8.3), carboxyl (−COOH) functional groups are deprotonated while the amino groups (NH3+) are protonated. Aspartic acid thus has an overall negative charge, with two −COO− and one −NH3+ functional groups, while glycine (in the zwitterionic form) has an overall neutral charge, having only one −COO− and one −NH3+ group.15 Thus, carboxyl (−COO−) is attracted to calcium surface sites and amino groups (−NH3+) interact with carbonate sites through hydrogen bonding. Computational simulations have shown that Gly1 binds to calcite through water molecules in the second hydration layer, as an outer sphere complex,47 and Nada49 showed that Asp1 can form outer sphere complexes and adsorb directly in an inner sphere configuration. Nada saw no difference energetically for Asp1 and Gly1, consistent with our ΔGads. It is also consistent with our XPS results, which show similar uptake of Asp1 and Gly1, and with the SEM images, which demonstrate equivalent changes in morphology. The difference in growth inhibition, visible from higher FI for Asp1, is probably caused by more extensive

Table 2. Adsorption free energies (kJ/mol) determined from the models ΔGads Langmuir (kJ/mol) Asp1 Gly1 Asp5 Aspn

−21 −21 −38 −43

ΔGads Langmuir− Freundlich (kJ/mol) −21 −20 −38 −66

(n (n (n (n

= = = =

1) 0.7) 1) 1.2)

ΔGads Flory− Huggins (kJ/mol) −22 −25 −40 −42

Gly1, even though the adsorption energies calculated for these two molecules do not differ by much. In contrast, the adsorption free energies obtained for the Asp polymers are higher (Table 2), suggesting they compete with carbonate ions, thereby affecting calcite growth considerably more than the monomers. From the Langmuir−Freundlich parameter n (Table 2, third column), we can gain more information about the additives and their interaction with the surface. For Aspn, n > 1, meaning the polymer occupies more than the available surface sites, indicating interaction with other Aspn molecules already adsorbed (positive cooperativity). For Asp1 and Asp5, n = 1, indicating Langmuir behavior, where adsorption is limited at a monolayer. For Gly1, n < 1, meaning its interaction with calcite is poor (negative cooperativity). This is consistent with the results of Magdans and colleagues,48 who proposed that glycine does not attach directly to the calcite surface but rather it substitutes for water molecules in the second hydration layer, indicating moderate electrostatic interaction, i.e. outer sphere complexation. It has to be considered that these adsorption models describe equilibrium conditions, whereas, in our experiments, we designed the amount of addition to ensure that the amount taken up was a negligible amount of the total. We discuss the results in terms of total concentrations, so taking this into G

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phobic surfaces, is not bound as strongly at step edges, where water adsorption is favored. Therefore, we interpret that the absence of growth inhibition, and even promotion of growth by Gly5 in our experiments, can be explained by hydrophobic interactions. The monomer, Gly1, has also only one carboxyl group, but because of its smaller size, it experiences higher attractive forces, meaning that Gly1 can inhibit calcite growth to a greater extent than Gly5. Above 0.4 mM, the inhibiting effect decreases to an extent similar to Gly5. A possible explanation is that calcite acts as a polymerization promoting agent for Gly1, leading to a longer molecule that has a similar structure and behavior in solution as Gly5, as proposed by Lambert.15 He claimed that amino acid polymerization favored by a solid surface is possible. For this explanation to be considered, a study to demonstrate polymerization would be required.

interaction through the extra carboxyl on step edges, as was suggested by Aschauer and colleagues.50 The changes in crystal morphology observed with SEM can be related to the molecule structure and its affinity for the calcite surface. Orme et al.,20 who studied the effect of Asp1 on {101̅4} cleaved calcite, showed preferential adsorption to step edges rather than terraces. They proposed that steps provide multiple attachment sites for carboxyl so growth then leads to a modification of step edge structure, promoting development of near-{hk.0} minor faces. Also, Tobler et al.51 observed rounded edges of calcite crystallized in the presence of higher concentrations of Asp1, morphology similar to the one obtained in our work. This fits with our SEM observations, where terraces roughen, step edges round, and {1010̅ } faces are favored (Figure 4). The aspartic acid polymers, Aspn and Asp5, decreased calcite growth rates considerably more than the monomers, which agrees well with previous studies.21,23 Elhadj et al.21 used atomic force microscopy (AFM) and suggested that Aspn (n = 1−6) at slightly higher concentrations replaces water molecules at step edges. They also observed that Asp with a higher chain length considerably decreases calcite growth rate at much lower concentrations than short chain molecules. Our growth rate data also showed that Asp1 and Asp5 are weaker inhibitors than Aspn (Figure 2A), and from adsorption energy, binding is stronger for higher molar mass (Table 2). Heterogeneity in chain length probably also enhances surface site occupation, which explains both the slower rate of growth (Figure 3b) and stronger effect on morphology (Figures 4e and f) but we cannot state this with certainty because we do not know the details of our polymers’ structure. We can also compare our results with those of a previous study that examined alcohol adsorption on calcite52 and derive information about surface coverage. Methanol, ethanol, pentanol, and tert-butanol all adsorb through their hydroxyl group to Ca atoms. A full alcohol layer gives a C−OH/CO3 ratio of 0.15 ± 0.01.52 For Asp5 and Aspn, the N/CO3 ratio was 0.06. If the poly(amino acid)s lie on the surface, the footprint for the 1 Asp unit would certainly be broader than 1 alcohol molecule standing up, so the N/CO3 ratio for a complete Asp monolayer would lie between 0.07 and 0.15. Thus, the amino acid coverage in our experiments is between half a monolayer and a monolayer, if we assume that no multilayers are formed. Gly5 has an interesting behavior, not inhibiting calcite growth and even promoting it slightly. This can be explained by considering its molecular structure. It has only one carboxyl group, and although it is attracted to the calcite surface, it is short, so bonding is not strong enough to keep it tightly attached to the surface, as was also suggested by Aschauer et al.50 Their study of a range of carboxylic acids demonstrated that the number of functional groups determines the extent of adsorption, meaning that Gly5 continuously adsorbs and desorbs as a result of a weak and nonselective interaction with the surface. Chen et al.53 showed that moderate electrostatic interactions of functional groups permit amino acids and poly(amino acid)s to weakly adsorb while moderate hydrophobic interactions for the rest of the molecule disrupt water adsorbed on the surface. This allows calcium and carbonate ions to approach the surface, accelerating growth. The computational study by Tran et al. showed that polypeptide with a backbone of Gly units behaves hydrophobically.54 Work in our own group16,55 also shows that alcohol, which covers calcite in preference to water, creating hydro-

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CONCLUSIONS The results of this study improve the kinetic and mechanistic understanding of calcite growth in the presence of amino acid molecules, such as are ubiquitous in biomineralising systems. The constant composition experiments show that calcite growth rates drastically decrease in the presence of small concentrations of aspartic acid. Chain length controls the extent of inhibition. Less than 0.002 mM of Asp5 and Aspn leads to growth reduction by 58% and 94%, whereas at such concentrations of the monomer, no growth reduction is observable. Slightly higher amounts of Asp5 could inhibit growth up to 81%, whereas 2 mM of Asp1 causes inhibition up to 54%. Gly1 and Gly5 modified the morphology of the calcite surface but did not lead to any considerable changes in growth rate (i.e., >10%) at all studied concentrations. In some cases, the Gly molecules even promoted growth. Fitted adsorption energies were obtained for Asp1 (−21 kJ/mol), Asp5 (−50 kJ/ mol), Aspn (−39 kJ/mol), and Gly1 (−22 kJ/mol). This indicates that Asp polymers adsorb readily on calcite, whereas the monomers compete with carbonate ions. The results offer better insight into the interaction of monomeric and polymeric molecules with calcite surface sites and clarify which parameters are most influential in growth inhibition. Insight for improving design of calcite scale inhibitors comes from the change in crystal morphology and the influences of chain length and the number of carboxylic groups, which affect the degree of their interaction with the mineral surface.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.5b01635. Detailed description of the constant composition experimental setup (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +45 21181104. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Keld West and Maria B. Bjørn for laboratory support, to Diwaker Jha for creating a Matlab code for analysis of growth rates from constant composition experiments, and to H

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the NanoGeoScience group members for very helpful discussions. The work was supported by funding from the European Commission for the MINSC project, a Marie Curie Initial Training Network, Grant Agreement No: FP7-290040, and the UK Engineering and Physical Sciences Research Council (EPSRC grant number EP/I001514/1), which funds the Materials Interface with Biology (MIB) Consortium.

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