*2.3. Catalysts Stability*

As it is well-known, a key issue in the field of biodiesel production with heterogeneous catalysts is the chemical stability of the solids in the reaction medium, which is directly connected with both the possibility of reutilizing the catalyst and the quality of the produced biodiesel and glycerol, in particular, the fulfillment of the corresponding standards for their typical uses.

In this work, Mo (500) and s-Mo (8) catalysts were recovered after reaction, thoroughly washed with tetrahydrofuran (THF), which is an excellent solvent for the reaction mixture medium [40], calcined at their respective original temperatures (500 ◦C and 800 ◦C, respectively), and used again. Mo (500) yielded almost the same catalytic activity results than during its first use. XRD and N2-adsorption data evidenced that no substantial changes took place during reaction. To check the possible occurrence of molybdenum leaching into the reaction mixture, the Mo (500) catalyst was removed after reaction by centrifugation. The upper fraction was transferred to a rotary evaporator to remove the unreacted methanol, resulting in a liquid that acquired an intense blue color that evidenced the presence of molybdenum. The molybdenum concentration was measured by ICP atomic emission spectroscopy resulting in ca. 1000 ppm of Mo. Additionally, Mo (500) was mixed with methanol for 4 h under the typical conditions used in this work. The solid was removed afterwards by centrifugation and the methanol was used in a reaction run. An XTG value of 75% was obtained after 4 h of reaction, which is only slightly lower than the value of 82% achieved in the presence of the solid catalyst (see Figure 5). Clearly, leached molybdenum species were capable of homogeneously catalyzing the transesterification and esterification reactions. The fact that the recovered solid gives the same feedstock conversion than the fresh catalyst is not strange provided that the solid represents a sufficiently excess amount with respect to its solubility in the reaction medium, thus guaranteeing the activity during several re-utilization cycles. As a matter of fact, it is likely that the concentration of ca. 1000 ppm of Mo measured is close to the solubility limit of the molybdenum species in the polar phase. Ferreira Pinto et al. [23] reutilized unsupported MoO3 in eight consecutive cycles of acidified soybean oil methanolysis. The catalyst was filtered from run to run and was used without washing. A very low loss of activity was observed only during the two last cycles; however, information about Mo leaching was not provided.

Regarding s-Mo (8), catalyst recovery, washing with THF, calcination, and reuse was repeated four times. The XTG values recorded after 8 h of reaction are shown in Figure 8. A gradual decrease of the triglycerides conversion takes place during the first three reaction cycles. The conversion stabilizes at values about 40% after a fourth reaction cycle; a conversion 47% lower than the original value.

As described before for Mo (500), s-Mo (8) was mixed also with methanol for 4 h under the typical conditions used in this work. When used in a reaction run, the recovered methanol yielded XTG and XFFA values of 41% and 46%, respectively, after 4 h of reaction. These conversions were substantially lower than the 60% and 73% values obtained in the presence of fresh s-Mo (8). Mo leaching after the first reaction cycle was investigated following the procedure described above for the unsupported catalyst. Although the Mo concentration could not be measured, the color of the liquid phase resulting after methanol removal evidenced the presence of dissolved Mo. On the whole, the results point to a somewhat improved stability of the supported catalysts compared to the unsupported ones that could be attributed to the interaction established between MoO3 and Al2O3. This interaction has been evidenced by the XRD results that showed the formation of aluminum molybdate, which seems to contribute to an improved dispersion and anchorage of the molybdenum species. This leads to a comparatively high activity for low MoO3 loadings and reduced leaching in comparison with the unsupported catalysts.

**Figure 8.** Triglycerides conversion (XTG) obtained after 8 h of reaction during the number of reuse cycles indicated for the catalyst s-Mo (8). Reaction conditions: 100 ◦C, 30 atm, methanol/feedstock molar ratio of 12:1, 2 wt.% catalyst referred to the feedstock mass. Feedstock: refined sunflower oil containing 5 wt.% free oleic acid.

#### **3. Materials and Methods**

MoO3 was synthesized by thermal decomposition of ammonium heptamolybdate tetrahydrate (AHM, from Merck, Darmstadt, Germany) in air (6 h) using a muffle furnace at different temperatures (300, 500 and 700 ◦C). The resulting solids were labeled as Mo (300), Mo (500) and Mo (700), respectively. Supported molybdenum catalysts were prepared by incipient wetness impregnation of γ–Al2O3 (Spheralite 505, Procatalyse) in powder form (particle size between 100 and 200 μm) previously calcined at 800 ◦C (6 h) in order to remove adsorbed impurities. Different impregnating AHM aqueous solutions were prepared to adjust the Mo salt concentration in order to obtain several MoO3 contents in the final catalyst, namely 6, 8, 10, 13 and 16 wt.%. After impregnation, the solids were dried for 8 h at 80 ◦C and calcined for 6 h at 800 ◦C in a muffle furnace. The calcination temperature was selected in order to promote the interaction of molybdenum species with the alumina support with the aim of improving their resistance to leaching during the reaction. The supported catalysts were referred to as s-Mo followed by the corresponding nominal MoO3 content between parentheses.

As for the catalysts characterization, XRD analyses were carried out in a D-Max Rigaku diffractometer (Akishima-shi, Tokyo, Japan). Specific surface areas were measured through N2 adsorption at −196 ◦C using a Micromeritics Gemini V 2380 apparatus (Norcross (Atlanta), GA, USA). Acidity was characterized by NH3-TPD in a Micromeritics Autochem 2920 equipment (Norcross (Atlanta), GA, USA). Pyridine adsorption was carried out using a purpose-made quartz IR cell connected to a vacuum adsorption device with a residual pressure lower than 10−<sup>4</sup> Pa. The samples, in the form of wafers, were activated and pyridine introduced into the cell at room temperature. Spectra were recorded in a Thermo Nicolet 380 spectrophotometer (Waltham, MA, USA) with a DTGS/KBr detector (Waltham, MA, USA) and accumulating 128 scans at a spectral resolution of 4 cm<sup>−</sup>1. X-ray photoelectron spectroscopy (XPS) analyses were carried out on a SPECS system equipped with an Al anode XR50 source operating at 150 W and a Phoibos 150 MCD-9 detector (Berlin, Germany). The pass energy of the hemispherical analyzer was set at 25 eV and the energy step was set at 0.1 eV. Charge stabilization was achieved by using a SPECS Flood Gun FG 15/40. The sample powders were pressed to self-consistent disks. Data processing was performed with the CasaXPS program (Casa Software Ltd., Teignmouth, UK).

Catalytic tests were carried out in batch mode in a Parr 4843 stainless steel autoclave reactor with mechanical stirring under controlled temperature and pressure. The feedstock consisted of refined sunflower oil (Urzante, Navarra, Spain; Acid Value of 0.07 mg KOH/g) to which pure oleic acid (Sigma Aldrich, San Luis, MO, USA) was added until it reached an FFAs content of 5 wt.% (Acid Value of 10.0 mg KOH/g). Catalyst concentration was set at a relatively low value of 2 wt.% referred to the feedstock (oil and FFAs mixture) mass. Simultaneous esterification of FFAs and transesterification of the triglycerides (TG) with methanol (Scharlau, HPLC grade, Barcelona, Spain) was carried out at 100 ◦C and methanol/feedstock molar ratio of 12:1. The amounts of the several substances used were: sunflower oil 60 g, FFAs 3 g, catalyst 1.26 g, and methanol 27.5 g. After reaching the reaction temperature, the reactor was pressurized with nitrogen until reaching an absolute pressure of 30 atm. Samples withdrawn from the reactor were analyzed by size exclusion chromatography as described elsewhere [40]. Conversion of triglycerides (XTG) and free fatty acids (XFFA), and yields of diglycerides (YDG), monoglycerides (YMG), and methyl esters (FAMEs) produced by transesterification (YME,trans.) were calculated through Equations (1)–(5), respectively:

$$X\_{\rm TG} = \frac{N\_{\rm TG,0} - N\_{\rm TG}}{N\_{\rm TG,0}} \tag{1}$$

$$X\_{FFA} = \frac{N\_{FFA,0} - N\_{FFA}}{N\_{FFA,0}} \tag{2}$$

$$\mathcal{Y}\_{\rm DG} = \frac{\mathcal{N}\_{\rm DG}}{\mathcal{N}\_{\rm TG,0}} \tag{3}$$

$$Y\_{\rm MG} = \frac{N\_{\rm MG}}{N\_{\rm TG,0}} \tag{4}$$

$$\mathbf{Y}\_{\text{ME,trans.}} = \frac{\mathbf{N}\_{\text{ME}} - \left(\mathbf{N}\_{\text{FFA},0} \mathbf{X}\_{\text{FFA}}\right)}{\mathbf{N}\_{\text{TG,0}}} \,, \tag{5}$$

where *NTG*,<sup>0</sup> and *NFFA*,<sup>0</sup> stand for the initial number of moles of triglycerides and FFAs, respectively, and *NTG*, *NDG*, *NMG*, *NME*, and *NFFA* stand for the number of moles of triglycerides, diglycerides, monoglycerides, FAMEs and FFAs, respectively, present in a sample taken at a given reaction time. Note that in Equation (5), in order to calculate the yield of methyl esters produced by transesterification, the number of moles of methyl esters produced by the esterification of FFAs (*NFFA*,0·*XFFA*) is subtracted from the total number of methyl esters present in the sample.
