3. then, the crystallinity, morphology and particle size
3. Results and discussion3.1 Seed characterizations Prior to investigating the effect of organic acids on the crystal growth of aragonite, an experiment for obtaining suitable aragonite seed for finding the effect of organic acids was conducted. It is important to use small seeds to determine the effect of organic acids during the growth of crystals. This is because the organic acids incorporated into the structure affect the crystallinity and morphology during crystal growth. Therefore, aragonite seeds were synthesized under the same experimental conditions with various reaction times (1, 2, 6 hr). And then, the crystallinity, morphology and particle size analysis were conducted.Fig. 1 shows that the XRD peak positions of the sample with all diffraction peaks were well consistent with aragonite reference data (JCPDS card No. 01-078-4339). Therefore, it was confirmed that the aragonite crystals grew regardless of the reaction time. However, the crystallinity increased with the reaction time. SEM analysis showed that as the reaction time increases, the crystal grows and the size increases. The sample reacted 2 hr shows that average particle size is approximately 2 ?m. When they reacted for 6 hr, the size was increased as 5-6 ?m. Considering that the final size of coprecipitated aragonite in the presence of organic acid is about 40 ?m, both of conditions, reaction times, are sufficient to be used as a seed in seeded constant-addition experiments. Even less than 0.1 weight percent of incorporated organic acids in the minerals is sufficient to affect the properties of minerals (Weiner et al., 2000). Therefore, we decided to use the smallest particle size of aragonite as seed. Using the smaller size of seeds will increase the amount of organic acids that can be incorporated into the crystals during mineral growth, and thus the changes in the physicochemical properties of the minerals can be investigated. The average particle size of samples from 2 hr reaction results was confirmed to be approximately 2 ?m on average through size distribution analysis (Fig. 3). 3.2 Crystallinity 3.2.1 X-ray Diffraction (XRD) analysis XRD peak positions of the aragonite samples are consistent with those of an aragonite reference data (JCPDS card No. 36-1451). Therefore, it is confirmed that aragonite seeds in the presence of organic acids grow well without producing other minerals in the supersaturated solution (Fig. 4). Higher intensity of the peak generally means the material has high crystallinity (Geffory et al., 1999, Katoyannis and Vagenas, 1999). The XRD results reveal that aragonite has relatively high intensities at 26°, 46° within the range of 20 to 60 degrees. In the case of the XRD results of aragonite in the presence of citric and malic acid, the peak height was decreased compared with other samples (Fig. 4). These results may demonstrate that some specific organic acids could adsorb onto the growing crystal face and suppress further growth. Tricarboxylic acid had a greater inhibitory capacity on crystallinity than the dicarboxylic acids and monocarboxylic acid. Also, organic acids having amino groups has been confirmed that even it has two carboxyl groups, it has no effect on the crystallinity, and the same results have been found in the presence of phthalic acid. The aragonite with citric acid prepared at different concentration is given in Fig. 5. It shows that the degree of crystallinity was affected by the concentration of organic acid. However, The crystal sizes of the samples are no marked differences regardless of concentration and type of organic acids. These results mean that within the given experimental condition, the especially concentration range of organic acids, it is possible to control crystallinity and the growth rate but it can not be completely inhibited the crystal growth. The reason for this tendency is explained in section 3.4 with PHREEQC species calculations. 3.2.2 Transmission Electron Microscopic (TEM) analysis with EDX SAED analysis is a method that can confirm the crystal structure or crystallinity with the electrons diffracted by injecting the electron into the sample. The crystals having a constant lattice structure are incident and diffracted so that electrons appear as a series of points perpendicular to the plane of the crystal. This allows obtaining a diffraction image in a localized region of a micro-size and identifying the molecular arrangement and symmetry structure of the polymer crystal from the diffraction information to confirm the crystal structure and crystallinity. It means that precipitates with some organic acid which exerted as an inhibitor of crystal growth have low crystallinity. From XRD results above mentioned that the crystallinity of samples with some organic acids (citric acid, malic acid) was declined due to the disorder of crystal lattice. TEM Selected Area Electron Diffraction (SAED) pattern also shows that precipitated in the presence of organic acids, especially citric and malic acid, have been shown more disorder than aragonite samples synthesized without organic acids (Fig. 5-7). These irregular arrangements were more prominent in aragonite synthesized with citric acid than with malic acid. Therefore, these results show that the organic acid, which having more carboxyl groups affects the crystallinity during the growth of aragonite crystals. Electron Diffraction X-ray Spectroscopy (EDX) analysis was performed to confirm the ion composition ratio of these samples. When an incident beam from a target of an electron microscope collides with the surface of a material, various kinds of electrons, ions, and characteristic X-rays having such characteristics are emitted from the surface of the material. In this case, the EDX device detects only the emitted characteristic X-rays separately and displays it on the screen according to the energy level of the beam to analyze the composition ratio of the ions. As a result of analyzing this principle, it was confirmed that there was no significant differences of composition ratio between pure aragonite and aragonite samples in the presence of organic acids. (Table 1). Considering the error ranges of the EDX analysis, it was found that the organic acid may not much affect the composition ratio of the minerals. Studies by Cal et al, (2012) have reported that less than 1% of organic acids incorporated into the structure can interfere with the stability of the structure. Because the amount of organic acid incorporated in the mineral is very small, it seems to be difficult to detect by chemical analysis through EDX. Therefore, we conducted another chemical analysis for the determination of the amount of incorporated organic acid, which is covered in section 3.4 through HPLC analysis. 3.3 Scanning electron microscopic (SEM) analysis As studied by Wang et al., (2013) the reason why prismatic or needle shape crystal morphology are dominantly shown in aragonite crystal is that the continuous epitaxial oriented overgrowth is expedited by preferential precipitation from solution and attachment of crystallites on both edges of the end of the crystal with rod shape. In this study, synthesized aragonite without organic acid also revealed the flower-shaped structure that is composed of nanorods (Fig. 13). Magnified image shows that flower-shaped structures are constituted by the accumulation of several hundreds of sharp-tipped aragonite nanorods. the typical diameters of these crystals are approximately 40 ?m (Fig. 13). However, citric and malic acid, which affected the crystallinity of minerals, also affected the morphology (Fig. 14-15). The nanorods aggregated on the mineral surface changed from sharp tipped to bumpy shape. These tendencies were in the order of citric and malic acid, which had a greater effect on the decrease in the crystallinity of the minerals. In the case of citric acid, no sharp-tipped shape could be found anywhere on the crystal surface of the mineral, but in the case of malic acid, the surface was less interrupted or had some sharp-tipped shape. Among the organic acids applied in the experiment, citric acid, which had the greatest influence on the crystallinity of the mineral, contained the most carboxyl groups in the structure. And citric acid has the greatest effect on mineral morphology either. Therefore, it was found that the more the carboxyl groups in the structure of the organic acid decrease the crystallinity and effect on the morphology of the minerals. 3.4 High Performance Liquid Chromatography (HPLC) analysis with PHREEQC species calculations. HPLC analysis of samples dissolved in acid was performed to determine the amount of organic acid incorporated into the aragonite structure. The citric and malic acids which affect the crystallinity and morphology of aragonite minerals were partially (~1 wt.%) incorporated into the structure (Table 1). Wang et al. (2016) studied that less than 0.1% of organic acids which incorporated into CaCO3 minerals are sufficient to exhibit other physicochemical properties. This is because the carboxyl group of the organic acid coexists with the surface while the mineral grows, causing the lattice structure to be mutated.In this study, The aragonite coprecipitation experiment with some organic acids also show the same results. Citric acid, malic acid, and acetic acid which have the same structure except for the number of carboxyl groups were used to determine the degree of influence on the crystal growth of the mineral according to the number of carboxyl groups in organic acids. Citric acid, malic acid, and acetic acid have 3, 2, and 1 carboxyl groups in order. There are a plenty of studies have reported that carboxyl groups interfere with the growth of CaCO3 (Reddy and Hoch 2000; Wada et al., 2001; Westin and Rasmuson 2003; Aschauer et al., 2010; Kim et al., 2011; Karar et al., 2014;). In this study, organic acids with the same structure except for carboxyl groups were incorporated into the lattice during crystallization and the morphology was affected by the increase of carboxyl groups. The incorporation of these organic acids into the Ca-Complex form can be expected through the PHREEQC species calculation. In this experiment, the reaction solution was adjusted to pH 8.3, the carboxyl groups of organic acids are fully deprotonated (pKa3 = 6.4 for citric acid; pKa2 = 5.2 for malic acid; pKa1 = 4.8 for acetic acid; pKa2 = 4.1 for glutamic acid; pKa2 = 3.7 for aspartic acid; pKa2 = 5.5 for phthalic acid). most of the citric acid species existed in the form of Ca-Citrate (-1), while malic acid was Ca-Malate (0) 80%, Malate (-2) 20% And Acetic acid was composed of Acetate (-1) 70% and Ca-Acetate (+1) 30%. Gefforoy et al. (1999) suggested that during the growth of CaCO3 crystals, the minerals are hydrated and have partially positive charged surface. We inferred that the negative charged Ca-Complex has more strong affinity for mineral surface and is readily incorporated during mineral growths. Ca-Citrate complex with -1 charge was partially incorporated as 0.63 wt.%. it is expected that the epitaxial growth cannot be performed because there is no change, so that the amount of incorporated organic acids are less than that of citric acid. Acetic acid has a Ca-Complex, but it is positively charged and is not expected to be incorporated into the lattice structure due to ionic repulsion. In the case of organic acids having a amino groups in this experiment, non-deporotonated amino structure (pKa3 = 9.5 for glutamic acid; pKa3 = 9.6 aspartic acid) may interfere with the binding due to the electron repulsion (Table 2). Also, It is expected that because of glutamic acid and aspartic acid are more complex and bigger than other organic acids. it was demonstrated that the lower the chelate structural complexity, the more stable the crystal is bound to the mineral crystals (Stumm, 1998). The structural complexity of these chelates is expected to be the reason that minerals cannot be incorporated during growth. Likewise, phthalic acid also expected to have not been incorporated into mineral because of its structural complexity because it has more chelate structures than others. Geffory et al. (1999) noted that during the growth of CaCO3 crystals, the carboxyl group of the Ca-Complex binds to the crystal surface along with the hydroxylic group and that it can grow epitaxially. we also inferred that organic acids are incorporated through the bidentate linkage with Ca ion on the mineral surface (Fig. 22). However, we also find a possibility that the Ca-complex present in the solution can be incorporated into the mineral during crystal growth through the modeling results (Fig. 21).These arguments for preferential incorporation of organic acids remain speculative, but suggest a model for further testing. In this study, it was confirmed that less than 1 wt.% of organic acids were incorporated into the minerals but could affect the crystallinity and morphology of the aragonite minerals, which ultimately controlled the growth rate of the minerals and the aragonite minerals used in various industries. It can be used as a material to help synthesize with desired properties. 4. Conclusions In this study, we investigated the effect of organic acid on the growth of aragonite mineral crystals by structural characteristics of organic acids. Some organic acids with carboxyl groups structure were found to be incorporated into aragonite minerals and it was confirmed that even if the amount of organic acids incorporated is less than 1 wt.%, it is sufficient to affects the crystallinity and morphology of minerals. In particular, citric acid and malic acid, which exist as Ca-Complex in the experimental conditions and have uncomplicated chelate structure compared with other organic acids applied in the experiment, are incorporated into the mineral to lower the crystallinity and the morphology shape changed to bumpy shape from sharp-tipped shape. It is expected that the organic acids were incorporated into the structure together during the growth of minerals in the Ca-Complex form, and It affects the physicochemical properties of the minerals.Aragonite minerals are used in many fields to improve the pharmacy series, cosmetics, and other industrial fillers. It is important to make these aragonite minerals with the desired properties for each use. This study investigated the role of organic acids as a factor influencing the physiochemical properties of aragonite and confirmed that the specific structure of organic acids can affect the crystal growth, crystallinity, and morphology of aragonite. This study can be used as a factor to control the growth rate of minerals in the future and it can be used as a basic material to help research on nano-biomaterial synthesis which is being actively studied recently.