Characterisation and Traceability of Calcium Carbonate from the Seaweed Lithothamnium calcareum

Abstract

Calcium carbonate (CaCO₃) from the seaweed Lithothamnium calcareum is a suitable dietary supplement for the prevention of osteoporosis, due to its chemical composition. This study compared CaCO₃ from L. calcareum to CaCO₃ from oyster shell and inorganic minerals that are already used in the pharmaceutical industry. The Rietveld refinement of the XRD showed that the mineral fraction of L. calcareum is composed of aragonite (50.3 wt%), magnesian calcite (45.3 wt%), calcite (4.4 wt%), comin contrast to oyster shell and inorganic minerals, which contain only calcite. The morphology of L. calcareum carbonate particles is granular xenomorphic, which is distinct from the scalenohedral form of inorganic calcite and the fibrous and scale-like fragments of oyster shell. The crystal structures of aragonite and magnesian calcite, present in L. calcareum, have higher contents of oligoelements than the pure calcite in other materials. The isotopic composition (stable isotopes of carbon and oxygen) is heavy in the CaCO₃ from L. calcareum (δ¹³C = 1.1‰; δ¹⁸O = −0.1‰) and oyster shell (δ¹³C = −4‰; δ¹⁸O = −2.8‰) in marked contrast to the much lighter isotopic composition of inorganic mineral CaCO₃ (δ¹³C = −19.2‰; δ¹⁸O = −26.3‰). The differences indicated above were determined through principal component analysis, where the first and second principal components are sufficient for the clear distinction and traceability of CaCO₃ sources.

1. Introduction

Calcium carbonate (CaCO₃) is a mineral that is widely distributed in nature, originating from both biological sources [1,2,3,4] and inorganic mineralogical processes [5,6]. CaCO₃ is used in the production of civil construction materials [7], as well as in agriculture, food, cosmetics, and pharmaceuticals [8,9].

In biomedicine, CaCO₃ can be used in the form of nanoparticles to create drug delivery systems for cancer treatment [10]. In the pharmaceutical industry, CaCO₃ is commonly used as a low-cost excipient [11,12]. In addition, an interesting use of CaCO₃ is as an active ingredient in food supplements to prevent osteoporosis [13,14].

Osteoporosis is a bone metabolic disease characterised by low bone strength, leading to fragility and the risk of fracture, which is observed globally [15,16]. For optimal performance and processing capacity of a pharmaceutical formulation, it is essential to perform adequate physical and chemical characterisation of its active ingredient [17].

Biogenic CaCO₃ from seaweed has been increasingly used in dietary supplementation to prevent osteoporosis [18,19]. Lithothamnium calcareum (L. calcareum) is a seaweed from the Corallinacea family, with a typical reddish colour, which crystallises calcium carbonate in its cell walls [1,20,21] and, additionally, is rich in trace elements [20,22]. Products containing L. calcareum CaCO₃ are used in several countries [23] due to its bioavailability and beneficial anabolic effects on bone calcification in animals [19,24] compared to other CaCO₃ sources [25].

There is a paucity of the literature on the physical and chemical characterisation of L. calcareum CaCO₃. The published data focus on its chemical composition, especially with regard to trace elements [22,26,27]. However, aspects such as the flow properties, polymorphism, morphology and compositional variations on a microscopic scale, surface area and porosity are fundamental parameters for the production of CaCO₃ tablets [28].

Another aspect that is rarely explored in the literature is the composition of the rare earth elements (REE), trace elements and stable isotopes [29] of CaCO₃. The study of their composition allows for the distinction of carbonates from different origins and can be a useful tool for the traceability of inputs used in the pharmaceutical industry [30,31].

Thus, considering the gap in the scientific literature regarding the characterisation and traceability of L. calcareum CaCO₃, this study aims to accurately characterise the methods of powder flow, surface area and porosity, thermal analysis, scanning electron microscopy and energy-dispersive spectroscopy, X-ray diffraction, chemical analysis and stable isotopes of L. calcareum CaCO₃ in terms of its physical, chemical, mineralogical and isotopic properties (C, O) using, for comparison purposes, CaCO₃ from oyster shell and inorganic minerals.

2. Results and Discussion

L. calcareum CaCO₃ showed a lower Carr index (CI) and Hausner ratio (HR) than the other CaCO₃ samples used in the pharmaceutical industry, with all the samples having non-ideal flow characteristics (

Table 1. Flow properties of CaCO₃ samples: apparent density, compacted density, Carr index and Hausner ratio of Lithothamnium calcareum, inorganic mineral and oyster shell CaCO₃ and ±range (n = 3).

Samples	Apparent Density (g/mL)	Compacted Density (g/mL)	Carr Index (%)	Hausner Ratio
L. calcareum CaCO3	0.49 ± 0.01	0.80 ± 0.01	38.75 ± 0.01	1.63 ± 0.01
Inorganic mineral CaCO3	0.30 ± 0.01	0.53 ± 0.02	43.40 ± 0.02	1.77 ± 0.02
Oyster shell CaCO3	0.74 ± 0.03	1.32 ± 0.02	43.94 ± 0.02	1.78 ± 0.02

). Poor flow characteristics may be partly related to the distribution of particle size and porosity, among other factors [32]. In L. calcareum CaCO₃, there may have been a greater interaction between the particles as a consequence of their morphology. The flow of CaCO₃ particles is compromised by the cleavage of crystals, which form particles of an anisotropic (rhombohedral) shape, deviating from the ideal spherical shape [33]. The smaller the particle size, the lower the CI and HR, which are features that distinguish L. calcareum CaCO₃ from other carbonates [34,35]. As a result, during material processing, a deficient flow of powder in the compressing machine can significantly impact the final product, contributing to weight variability and influencing the quality of the tablets. In light of this, it is more appropriate to use the wet granulation process to obtain CaCO₃ tablets for food supplements, whatever their source [36,37].

The fluidity of CaCO₃ is also related to its surface area and porosity. A decrease in the pore size of L. calcareum and oyster shell CaCO₃ increases the compacted density of the material (

Table 2. Surface area, volume and pore size of Lithotthamnium calcareum, inorganic mineral and oyster shell CaCO₃ samples.

Sample	A(BET) (m2.g−1)	Vp (×10−3 cm3.g−1)	Pore Size (Å)
L. calcareum CaCO3	8.118	1.585	3.906
Inorganic mineral CaCO3	7.857	3.829	9.746
Oyster shell CaCO3	2.935	5.081	3.463

), making the tablets fragile, brittle and easily chipped during large-scale production [33].

In terms of porosity, L. calcareum CaCO₃ had the characteristics of an ultra-microporous material, with a pore size of < 7 Å; the form shown in the literature for this type of material is slit or curled [38,39]. These characteristics can represent a mechanical interlock between the particles and influence the fluidisation of the material [32]. Figure 1 shows that L. calcareum CaCO₃ had H3-type hysteresis [40]. The shape of a hysteresis loop depends on the sample’s pore size. In fact, like the pore sizes, the hysteresis loop was dissimilar between the CaCO₃ samples. A hysteresis loop means that the desorption and adsorption curves are not identical. However, type H3 indicates non-rigid particle aggregates and is related to capillary condensation of porous solids [41]. Inorganic mineral CaCO₃ also showed a tendency towards H3-type hysteresis, while for oyster shell CaCO₃, we did not identify a hysteresis characteristic.

Microporous materials are classified according to the International Union of Pure and Applied Chemistry (IUPAC), which considers materials with pore sizes between 7 and 20 Å as microporous and those with a pore size < 7 Å as ultra-microporous [42]. Oyster shell CaCO₃ was ultra-microporous like L. calcareum CaCO₃, while inorganic mineral CaCO₃ was microporous.

Ultra-microporous and microporous materials give rise to a type III isotherm, i.e., point B in relative pressure (p/p⁰) is not determined. The three CaCO₃ samples had type III isotherms, and since gas molecules have a greater affinity for one another, the isotherms were not common [43]. We extracted data from the angular coefficient line of L. calcareum in which the constant (C) was 75.0 i.e., a characteristic of adsorbate molecules with significant interactions between them. The linear coefficient of the L. calcareum CaCO₃ sample was 0.999. The inorganic mineral and oyster shell CaCO₃ samples showed linear coefficients of 0.999 and 0.998, respectively, and each had a different value of C—inorganic mineral CaCO₃ had C = 52.28, while oyster shell CaCO₃ had C = 15.90. In general, a microporous material has a pore-filling mechanism with adsorption occurring instantaneously [40], but this behaviour occurred only in the L. calcareum and oyster shell CaCO₃ samples, which showed similar profiles, as seen in the graph of volume versus p/p⁰ (Figure 1).

L. calcareum CaCO₃ had the largest surface area compared with other samples and, consequently, a smaller pore volume, in addition to nitrogen adsorption and desorption isotherms similar to those observed by Xia et al. [44]. Inorganic mineral CaCO₃ had surface area, pore volume and adsorption and desorption isotherm values similar to those of L. calcareum CaCO₃, while the values of oyster shell CaCO₃ were lower than those of L. calcareum CaCO₃.

The thermogravimetric profile of L. calcareum CaCO₃ indicated decomposition by CO release at 670.9 °C (

Table 3. Thermo-analytical results for Lithothamnium. calcareum, inorganic mineral, and oyster shell CaCO₃.

Samples	Event	Tpico peak DTG (°C)	Tinitial (°C)	Tonset (°C)	Tfinal (°C)	Weight Loss (%)	Tpeak DTA (°C)
L. calcareum CaCO3	Decomposition	720.3	587.0	670.9	757.1	40.4	727.3
Inorganic mineral CaCO3	Decomposition	731.9	572.0	668.9	760.3	39.9	736.8
Oyster shell CaCO3	Decomposition	734.9	693.1	675.1	751.0	42.7	737.5

DTG, derived thermogravimetry; DTA, differential thermal analysis.

and Figure 2), a temperature consistent with data from the literature [45]. This decomposition event corresponds to the endothermic peak identified from differential thermal analysis (DTA) at the same temperature. The mass loss of L. calcareum CaCO₃ was similar to that reported by Li et al. [46].

The oyster shell CaCO₃ showed a slightly higher decomposition temperature than the L. calcareum CaCO₃, and the mass loss and exothermic peak increased in magnitude.

Scanning electron microscopy (SEM) images of L. calcareum CaCO₃ revealed xenomorphic crystals, where the external form was not intrinsically related to the crystal structure (Figure 3A). Crystals were granular, with a size range below 2 μm. Energy-dispersive spectroscopy (EDS) analysis (Figure 3B) revealed the presence of Ca and Mg, which is consistent with the mineralogical analysis.

Inorganic mineral CaCO₃ (Figure 4A) presented well-defined scalenohedral crystals, which are defined by crystallographic planes [47,48]. The crystal size range was below 1 μm, which was smaller than that of L. calcareum CaCO₃. The EDS plot (Figure 4B) revealed the presence of calcium, carbon and oxygen as the major components. Oyster shell CaCO₃ (Figure 5A) had a plate-like morphology, formed by fibrous particles on a 1 nm scale. EDS analysis (Figure 5B) indicates the presence of calcium.

X-ray diffractograms (Figure 6) of the three samples show that L. calcareum CaCO₃ contains aragonite (50.3 wt%), magnesian calcite (45.3 wt%) and calcite (4.4 wt%), as determined by the XRD–Rietveld method. Inorganic mineral and oyster shell CaCO₃ samples were composed solely of calcite.

The co-existence of calcite and magnesian calcite in L. calcareum CaCO₃ suggests that either different biominerals are precipitated in different tissues or, alternatively, the biomineral composition is re-equilibrated in secondary processes, such as the lixiviation of recrystallisation. Magnesian calcite is thermodynamically unstable under ambient conditions. It is currently formed by several marine organisms, including red coralline algae. Mg intake in calcite reflects the Mg/Ca ratio in seawater and is caused by the assemblage of amorphous crystallisation precursors [49]. In the XRD dataset presented (Figure 6), L. calcareum CaCO₃ has less intense and broader peaks compared with inorganic mineral and oyster shell CaCO₃, indicating a lower degree of crystallinity reducing the intensity and smaller crystallite size of L. calcareum magnesian calcite.

From the results obtained above, we constructed two-dimensional graphs of the main components (Figure 7). The two new variables created in the same dimension—principal component analysis (PCA1 and PCA2)—retained 100% of the information contained in the original variables. We also verified that PCA1 explained more than 70%, while PCA2 explained 24.53% of the data variability, which was better defined only by pore size. For L. calcareum CaCO₃ (A), factor 1 interpreted the polymorphism, particle size and provided a well-represented surface area. In contrast, for inorganic CaCO₃ (B), the pore size stood out and, for oyster shell CaCO₃ (C), factor 1 mainly interpreted calcium content and mass loss. The oyster shell CaCO₃ sample was the most different from the other components, while the inorganic mineral CaCO₃ sample (B) registered a larger group of variables. Thus, we compared the three groups, found that the samples differed in all variables, while those that stood out the most were calcium content and pore size, with values above 0.999.

In terms of the chemical composition of the three CaCO₃ samples (

Table 4. Content of trace elements (ppm) of samples (in duplicate) of L. calcareum, inorganic mineral and oyster shell CaCO₃.

Chemical Elements	L. calcareum		Mineral CaCO3		Oyster CaCO3
Sc	0.50	0.49	0.07	0.07	0.03	0.03
Rb	0.87	0.91	0.33	0.33	0.11	0.11
Sr	291.0	307.8	158	161	868	842
Y	1.61	1.67	0.66	0.68	0.10	0.10
Zr	0.64	0.62	0.71	0.60	0.09	0.05
Nb	0.32	0.33	0.06	0.06	0.02	0.09
Cs	0.04	0.04	< 0.01	< 0.01	< 0.01	< 0.01
Ba	6.63	6.54	14.2	15.80	< 0.50	0.50
La	1.65	1.63	0.14	0.15	0.16	0.16
Ce	3.79	3.73	< 0.01	< 0.01	< 0.01	< 0.01
Pr	0.35	0.37	0.04	0.04	0.03	0.02
Nd	1.26	1.32	0.15	0.16	0.09	0.08
Sm	0.24	0.24	0.04	0.04	0.02	0.02
Eu	0.05	0.05	0.01	0.01	< 0.01	< 0.01
Gd	0.26	0.23	0.05	0.05	0.02	0.02
Tb	0.03	0.03	< 0.01	< 0.01	< 0.01	< 0.01
Dy	0.18	0.19	0.05	0.06	0.01	0.01
Ho	0.04	0.04	0.01	0.02	< 0.01	< 0.01
Er	0.10	0.10	0.04	0.04	< 0.01	< 0.01
Tm	0.01	0.02	< 0.01	< 0.01	< 0.01	< 0.01
Yb	0.09	0.09	0.04	0.04	< 0.01	< 0.01
Lu	0.01	0.01	< 0.01	< 0.01	< 0.01	< 0.01
Hf	0.02	0.02	0.01	0.01	< 0.01	0.01
Pb	0.88	0.90	0.13	0.16	0.03	0.03
Th	0.42	0.43	0.04	0.05	0.01	0.01
U	1.15	1.19	0.51	0.56	0.03	0.02

), the Sc, Rb, Y, Zr, Nb, Cs, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Hf, Pb, Th and U content was low (< 15 ppm), while the strontium (Sr) content was between 159.50 and 854.50 ppm. The presence of trace elements and the absence of toxicity represent advantages from a human health perspective [50,51]. The proportions of essential elements in food supplements are suitable for human consumption, since quantities of <0.01% of body weight are necessary in the daily diet, except for Al, Cr, Se, Cu, Mo and Pb, among others; high amounts of Pb, for example, are considered toxic [52]. The largest number of trace elements was identified in L. calcareum CaCO₃ compared with the other two samples, making them intrinsic characteristics related to the origin of the material. The presence of Sr in all three samples, but mainly in oyster shell CaCO₃, is not a restrictive factor, as this element is not on the WHO list of toxic elements [53]. In the same way that CaCO₃ plays a significant role in our body, trace elements also play an important role. Trace elements and their values are recognised by the European Food Safety Authority, which indicates how much the population should consume, in addition to calcium, regardless of their origin, to maintain a healthy diet [54].

Trace elements help us to distinguish between carbonates from different sources, as analysed in this study. L. calcareum CaCO₃ showed levels of trace elements around one order of magnitude higher than those of oyster shell and inorganic mineral CaCO₃. Among the trace elements analysed, several were below the detection limits for inorganic mineral and oyster shell CaCO₃ (Table 4).

Chemical differences between the three samples were related to the mineralogical composition, since L. calcareum CaCO₃ was composed of magnesian calcite, calcite and aragonite, while the other two samples of calcium carbonate were composed exclusively of calcite. There are differences in the partition of trace elements between aragonite, low-Mg calcite and high-Mg calcite due to differences between their crystal structures [55]. Figure 8 and Figure 9 show the content of trace elements (ppm) divided by the average composition of the upper terrestrial crust [56], a commonly used form of data representation in geochemistry. Regarding the REE (Figure 8), the L. calcareum CaCO₃ sample showed a practically horizontal pattern, indicating that there is no preferential selective incorporation of individual REE. The rare earth patterns of L. calcareum CaCO₃, with stable, differ from the composition of seawater, which has depleted cerium levels (negative Ce anomaly) and a trend of increasing amounts of heavy REE [57]. Inorganic elements in seaweeds have two likely sources: elements dissolved in seawater or solid particles in suspension [58]. The similarity of L. calcareum CaCO₃ to the average of the upper continental crust, in terms of the REE distribution, may suggest that the uptake of suspended solid particles played an important role in the mineralisation of this seaweed.

Inorganic mineral and oyster shell CaCO₃ samples had a lower concentration of REE, with several gaps in rare earth patterns due to elements that were below the detection limit, making it difficult to observe trends. Nevertheless, the inorganic mineral CaCO₃ sample showed a tendency for the enrichment of medium and heavy rare earth elements (Eu–Lu) compared to light rare earth elements (La–Sm), which is common in marine sediments. The multi-element diagram (Figure 9), where the elements are ordered by their ionic potential (ionic charge/radius), shows that the contents of practically all elements were higher in L. calcareum CaCO₃. Exceptions included barium, which was more abundant in inorganic mineral CaCO₃, and strontium, which was more abundant in oyster shell CaCO₃. Strontium, in particular, was an abundant trace element in all three samples, with levels in the range of hundreds of ppm (Table 4). The reasons are due both to the abundance of this element in natural systems and the similarity of its ionic radius and charge with calcium (Sr²⁺ ≈ Ca²⁺), which allows its uptake into natural calcium carbonates, whether biogenic or otherwise.

The data on the stable carbon and oxygen isotopes (Figure 10) of L. calcareum and oyster shell CaCO₃ fall within the category of shallow-water biogenic marine carbonates, with a small fraction in relation to the V-PDB standard (Vienna Pee Dee Belemnite international standard) [59]. Published data on stable isotopes of L. calcareum CaCO₃ are rare. Data on carbonate algae (Lithothamnion sp. and Halimeda sp., both composed of calcite and aragonite) were obtained by Rocha [60] in samples collected from the northeast Brazilian coast. The L. calcareum CaCO₃ sample analysed in this study is within the compositional group of 66 samples analysed by Rocha [60], who described variations in isotopic ratios in samples of the same species collected at different depths as a consequence of water temperature. Our data on the stable isotopic composition of the oyster shell of Crassostrea virginica is within the isotopic range of oysters found in the James River estuary, Virginia (USA), as published by Grimm et al. [61].

Inorganic mineral CaCO₃ showed negative carbon δ ¹³C and oxygen δ ¹⁸O values. Inorganic mineral CaCO₃ is an extra-light carbonate that is used in the pharmaceutical industry. It is chemically precipitated in a controlled process, and its isotopic composition reflects the composition of the raw materials, influenced by the temperature and kinetics of the crystallisation process.

The small number of isotopic data collected in the present study did not allow us to describe trends, but indicated that the isotopic composition can be used with additional data to distinguish biogenic carbonates. The carbon and oxygen stable isotope ratios of the raw materials are a potential tool for tracing the sources of calcium carbonates that are used in the pharmaceutical industry.

3. Materials and Methods

3.1. Samples

Calcium carbonate extraction from the seaweed Lithothamnium calcareum occurred mechanically from the harvest at sea, followed by drying. Subsequently, the seaweed was cut so that the crushing was homogeneous and, finally, a raw material was generated in powder form. The powder was micronised for commercial use as L. calcareum CaCO₃ [62,63]. L. calcareum calcium carbonate was kindly provided by Lithocálcio Indústria, Comércio, Importação, Exportação e Representação Ltd.a. (São Paulo, Brazil), calcium carbonate of inorganic mineral origin was provided by Valdequímica Produtos Químicos Ltd.a. (São Paulo, Brazil) and oyster shell calcium carbonate (Crassostrea virginica) was already processed as a raw material in powder form by Option Fênix Distribuidora de Insumos Ltd.a. (São Paulo, Brazil).

3.2. Assessment of Powder Flow

The flow of the powder samples was analysed by determining the apparent density, compacted density, Carr index (CI) and Hausner ratio (HR).

To measure the apparent density (d_ap) of the samples, each sample was transferred to a 100 mL beaker until it reached a volume of 50 mL. Subsequently, the weight was recorded on a semi-analytical scale, and the material was transferred to a standardised cylinder in a Tap Density densimeter (Ethik, São Paulo, Brazil). The compacted density (d_cp) was determined as described in the American Pharmacopoeia [64]. From the data obtained, the apparent and compacted density values were calculated as follows:

$${\mathrm{d}}_{\mathrm{ap}}={\mathrm{M}}_{\mathrm{i}}/{\mathrm{V}}_{\mathrm{i}}$$

(1)

$${\mathrm{d}}_{\mathrm{cp}}={\mathrm{M}}_{\mathrm{i}}/{\mathrm{V}}_{\mathrm{f}}$$

(2)

where

• d_ap = apparent density (g/cm³);
• M_i = initial mass (g);
• V_i = initial volume (cm³);
• d_cp = compacted density (g/cm³);
• V_f = final volume (cm³).

The CI and HR were calculated using the values obtained for the apparent density (d_ap and compacted density (d_cp), according to the following equations:

$$\mathrm{CI}\left(\%\right)=\frac{{\mathrm{d}}_{\mathrm{cp}}{-\mathrm{d}}_{\mathrm{ap}}}{{\mathrm{d}}_{\mathrm{cp}}}\times 100$$

(3)

$$\mathrm{HR}=\frac{{\mathrm{d}}_{\mathrm{cp}}}{{\mathrm{d}}_{\mathrm{ap}}}$$

(4)

where

• CI = Carr index;
• HR = Hausner ratio;
• d_cp = compacted density;
• d_ap = apparent density.

3.3. Surface Area and Porosity

To analyse the surface area and porosity of the samples, a NOVA 2200e gas adsorption analyser (Quantachrome Corporation, Boynton Beach, FL, USA) was used. The samples were desiccated at 90 °C and then subjected to the test at an equipment temperature between 0 and −196 °C. The Brunauer–Emmelt–Teller (BET) (Supplementary Materials) method described by Lowell et al. [43] and Klobes et al. [65] was used according to the equations described below. The parameters were programmed in isotherms from 52 points (31 points of adsorption and 21 points of desorption of ultrapure nitrogen gas). Relative pressures (p/p⁰) between 0.05 and 0.950 were established, and the analysis time varied from 3 to 8 h.

BET method:

$$\frac{p/p^0}{n \left(1-p/p^0\right)}= \frac{1}{n_{m}C}+ \frac{C-1 }{n_{m}C} \left(p/p^0\right)$$

(5)

where

• n = amount adsorbed at relative pressure;
• n_m = specific monolayer capacity;
• C = constant relative to the adsorption of the first monolayer;
• p/p⁰ = relative pressure.

Surface area:

$$A_{\left(BET\right)={ }^{}}{n}_{m^{}}. L . a_{m^{}}$$

(6)

where

• A_(BET) = BET area;
• L = Avogadro constant.

Porosity:

$$InP/P_{0^{}=}\frac{-2\gamma \nabla }{rRT}$$

(7)

where

• r = surface tension of the liquid;
• ∇ = molar volume of the condensed liquid contained in a narrow pore of radius r;
• R = gas constant;
• T = temperature.

3.4. Thermal Analysis

The samples were analysed by thermogravimetry (TG), derived thermogravimetry (DTG) and differential thermal analysis (DTA). Extar TG 7200 equipment (Seiko Instruments, Tokyo, Japan) was used. The selected parameters were an inert nitrogen atmosphere with gas flow (100 mL min⁻¹), a heating rate of 10 °C min⁻¹ and a temperature from 30 to 1000 °C in a platinum crucible. In addition, the equipment was previously calibrated with a calcium oxalate standard, with the same heating rate and temperature range as those used for the samples.

3.5. Scanning Electron Microscopy (SEM) and Energy-Dispersive Spectroscopy (EDS)

The morphology of the samples was analysed at the Technological Characterization Laboratory (Polytechnic School of the University of São Paulo) using a Quanta 650 FEG environmental scanning electron microscope (FEI), equipped with EDS Quantax microanalysis systems (Bruker, MA, USA), image analysis (MLA-Mineral) and a secondary electron detector x Flash 4030 with silicon drift detector (SDD) technology operating under high vacuum conditions. The following instrumental parameters were selected: a 30 mm² window, and resolutions of <130 eV (Mn Kα) at 60 kcps, 138 eV at 130 kcps and 158 eV at 280 kcps.

3.6. X-ray Diffraction

X-ray diffraction analysis (powder method) of the samples was performed at the Multi-User Geoanalytical Center (Institute of Geosciences, University of São Paulo) using a Bruker D8 Advance diffractometer with a LYNXEYE detector (Bruker, MA, USA). The instrumental parameters selected were Cu Kα_1/2 radiation, a 40 mA current, a 40 kV voltage, an angular range of 2° to 65° (2θ), an angular step of 0.02° (2θ) and a scan speed of 38.4 s per step.

Polymorphs were identified using the High Score Plus 3.0 software (PANalytical B.V., Lelyweg, The Netherlands) and the Crystallography Open Database (COD) [66]. The quantitative analysis of phases in polyphasic samples was performed using the method of Rietveld [67] using the High Score Plus 3.0 program (PANalytical B.V., Lelyweg, The Netherlands).

3.7. Principal Component Analysis

The results of the sample characterisation were compared via multivariate analysis and principal component analysis (PCA) with Statistica^TM Inc. software 13.5.0.17 (TIBCO^® Software Inc., Tulsa, OK, USA). Powder flow (CI%), information obtained in the analysis of the surface area by the BET method (specific surface area, size and pore volume), information obtained from the thermal analysis (melting point (°C) and mass loss (%)), particle size obtained by SEM and polymorphism were adopted as original variables. After standardising the data, two main components were created, PCA1 and PCA2, which were used in the construction of two-dimensional graphics.

3.8. Chemical Analysis

The chemical analysis of trace elements was carried out at the Geoanalytic Multiuser Plant (Instituto de Geosciences, University of São Paulo) using ICP-MS ELAN 6100 DRCTM equipment (PerkinElmer^®, Waltham, MA, USA), according to the procedures described by Navarro et al. [68].

3.9. Analysis of Stable Isotopes

The stable isotopes of carbon (δ¹³C) and oxygen (δ¹⁸C) were analysed at the Research Centre for Geochronology and Isotopic Geochemistry (Institute of Geosciences, University of São Paulo) using a isotopic ratio mass spectrometer (IRMS) Thermo Fisher Scientific^TM -Delta V, Waltham, MA, USA). The isotopic data are presented in the delta (δ) notation, expressed in per thousand (‰), which relates each sample’s isotopic ratios to those of international reference standards, as shown in the following equations. The accuracy of the δ¹³C results was 0.05‰ and the accuracy of the δ¹⁸O results was 0.07‰.

$$\mathsf{\delta }{}_{}{}^{18}\mathrm{O}{}_{\mathrm{V}-\mathrm{PDB}}=\frac{{}_{}{}^{18}\mathrm{O}/{}_{}{}^{16}\mathrm{O}{}_{\mathrm{amostra}} -{}_{}{}^{18}\mathrm{O}/{}_{}{}^{16}\mathrm{O}{}_{\mathrm{V}-\mathrm{PDB}}}{{}_{}{}^{18}\mathrm{O}/{}_{}{}^{16}\mathrm{O}{}_{\mathrm{V}-\mathrm{PDB}}} \times 1000$$

(8)

$$\mathsf{\delta }{}_{}{}^{13}\mathrm{C}{}_{\mathrm{V}-\mathrm{PDB}}=\frac{{}_{}{}^{13}\mathrm{C}/{}_{}{}^{12}\mathrm{C}{}_{\mathrm{amostra}} -{}_{}{}^{13}\mathrm{C}/{}_{}{}^{12}\mathrm{C}{}_{\mathrm{V}-\mathrm{PDB}}}{{}_{}{}^{13}\mathrm{C}/{}_{}{}^{12}\mathrm{C}{}_{\mathrm{V}-\mathrm{PDB}}} \times 1000$$

(9)

where

• ¹⁸O/¹⁶O_sample and ¹³C/¹²C_sample = isotopic ratios of the samples;
• ¹⁸O/¹⁶°v_–PDB and ¹³C/¹²C_V–PDB = isotopic standards of the Vienna Pee Dee Belemnite international standard.

4. Conclusions

In view of the results presented in this study, it is possible to state that seaweed L. calcareum CaCO₃ is significantly different in terms of flow properties, surface area, volume and pore size, thermogravimetric profile, morphology, crystalline phases, number of trace elements, rare earth elements and isotopic composition from inorganic mineral and oyster shell CaCO₃. All samples analysed in this study have a compromised flow due to their anisotropic morphology, which may have an influence on the particle size distribution, hindering tablet production.

L. calcareum CaCO₃ is an ultra-microporous material, with a pore size of <7 Å, and has the highest specific surface area (8.1 m².g⁻¹) among the studied materials. Thermal events cause similar decomposition and mass loss in the three types of CaCO₃, although L. calcareum CaCO₃ exhibits DTG (720.3 °C) and DTA (727.3 °C) peaks at a slightly lower temperature compared with other samples. However, the mineralogical composition of L. calcareum CaCO₃ includes three phases—aragonite, magnesian calcite and calcite—in contrast to the monomineral composition (calcite) of inorganic mineral and oyster shell CaCO₃. The more complex mineralogical composition of the L. calcareum sample leads to a chemical composition that is richer in Mg and trace elements compared with other samples.

When evaluating the multivariate analysis results, it is possible to clearly distinguish the three sources of CaCO₃. All variables (flow properties, surface area, porosity, thermogravimetric profile, morphology and crystalline structures) have an effect, and those that have the greatest effect are calcium content and pore size.

In terms of the chemical composition of L. calcareum CaCO₃, trace elements are more evident than in other samples. However, Al, Cr, Se, Cu, Mo, Pb and others are not present in high quantities in any of the samples. In addition, it is possible to distinguish between the trace elements in the studied carbonates. Analysis of stable isotopes of carbon and oxygen clearly distinguishes biogenic (CaCO₃ from L. calcareum and oyster shell) from inorganic mineral calcite, which is enriched in lighter isotopes of carbon and oxygen.

Detailed analysis of L. calcareum CaCO₃ indicates that this material provides not only Ca, but also Mg and a wide range of trace elements. By using a combination of analytical techniques, L. calcareum CaCO₃ can be clearly distinguished from CaCO₃ from other sources used in the pharmaceutical industry, providing a basis for its traceability.

Further studies, such as wet powder rheometry, are needed to determine the best wet granulation conditions for the production of tablets using the Lithothamnium calcareum CaCO₃. These studies are ongoing in our laboratory.