Isolation of KH2PO4 from crude glycerol purified using Acromonia aculleata charcoal as adsorbent and its application in the synthesis of methyl salicylate.

doi:10.21203/rs.3.rs-4682809/v1

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Isolation of KH2PO4 from crude glycerol purified using Acromonia aculleata charcoal as adsorbent and its application in the synthesis of methyl salicylate.

https://doi.org/10.21203/rs.3.rs-4682809/v1

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Activated charcoal was prepared from Acrocomia aculeata (macaúba) endocarp by ZnCl₂ activation. The charcoal was used for the adsorptive purification of pretreated crude glycerol (CG) and potassium dihydrogen phosphate (KH₂PO₄ or KDP) was formed by the addition of H₃PO₄ to the crude glycerol containing KOH, which was used as catalyst in the production of biodiesel and glycerol. The KH₂PO₄ was isolated and purified by recrystallization from aqueous solution. The KH₂PO₄ was characterized by X-Ray diffraction (XRD), thermal analysis (TGA/DTA) and spectrophotometry in the infrared region – FTIR. From 216.0 g of crude glycerol, 20.1038 g of purified KH₂PO₄ was obtained. The KH₂PO₄ was used in a simple, regioselective, green method as a selective base for the methylation of the salicylic acid (SA) carboxylate group using DMS as the methylating agent in the production of MS at reflux temperature (90 ^oC). The present method has a short reaction time, and it is simple with excellent yields of MS. In this process, DMS was used in excess because it has been shown to be an excellent eco-friendly, aprotic solvent for the S_N2 mechanism. The reuse of organic waste was accompanied by the transformation of DMS into the K₂SO₄ drying agent and H₃PO4 into KH₂PO₄, a source of potassium and phosphate in fertilizers. This reaction is the first example of the use of KH₂PO₄ as a useful selective base for methylation, and the present method obeys the reduce-reuse-recycle (RRR) principle for the development of a green protocol. Microbial susceptibility tests were accomplished by adding suspensions of microorganisms and spreading them on the surfaces of the Mueller Hinton and YPMD agar culture media, in which there were central wells containing 60 µL of pure MS, which inhibited the growth of Escherichia coli, Proteus mirabilis, Sporothrix sp, Rhodotorula mucilaginosa, Galactomyces geotrichum, Candida orthopsilosis and Candida akabanensis.

Environmentally friendly processes

potassium dihydrogen phosphate

waste management

crystallization process

wintergreen oil

cytotoxicity

Potassium dihydrogen phosphate (KH₂PO₄) is a salt commonly used in buffer solutions [1], as a food additive, and in fertilizer. Its crystal is also employed with lasers in fusion experiments [2]. Among the methods for KH₂PO₄ preparation found in the literature, the crystallization methods, such as the use of N-methyldiethanolamine (MDEA) as an extractant to produce KH₂PO₄ from KCl and H₃PO₄, where MDEA presented good water solubility and extraction selectivity, large extraction capacity, low toxicity and moderate price, was described by Tang et al. [3] and others [4–6]. Other methods for the production of KH₂PO₄ involve neutralization technology based on the thermal-process reaction between H₃PO₄ and KOH or K₂CO₃ [7], the direct chemical conversion method, the ion exchange method, and extraction technology [8–11].

Tan et al. [12] increased water resistance of magnesium oxychloride cement (MOC) without gel-like P5 in the hydration product by mixing phosphoric acid in the MgO-MgCl₂-H₂O system. According to Ying Li et al. [13], the incorporation of KH₂PO₄ increased the performance of MOC with respect to the long-term compressive strength, water resistance, thermal stability, qualitative and quantitative composition, micro morphology, and pore structure of MOC.

As the KH₂PO₄ is a material with several isomorphs, which are archetypal of hydrogen-bonded material. KH₂PO₄ possesses piezoelectric, ferro-electric, electro-optic, and nonlinear optical properties, to which many researchers from various fields are strongly attracted. Recently, Jayaprakash et al [14] described the effect of doping KH₂PO₄ with magnesium chloride crystal (MCKH₂PO₄) on structural, thermal, optical, nonlinear optical and electrical properties of KH₂PO₄. Jayaprakash et al. employed the slow evaporation technique with millipore water as a solvent to grow single crystals of pure KH₂PO₄ and MCKH₂PO₄ at room temperature. The investigations were performed to determine the optical, mechanical nonlinear optical (NLO) and thermal behaviors of KH₂PO₄ and MCKH₂PO₄. KH₂PO₄ is employed in various applications as a piezoelectric transducer in various sound reproduction systems, and it is the first material to possess the key property of nonlinearity, which has lead to a magnificent and trending era in the field of photonics [15].

KH₂PO₄ has been employed in the NMR studies of disorder in condensed systems [16]. Phosphate is also important in the enzymatic phosphorolysis of amylose and other polysaccharides [17]. Phosphate participates in the partitioning of proteins during countercurrent chromatography [18].

The synthesis of p-enaminones and p-enamino esters has been achieved using KH₂PO₄ as a catalyst [19]. KH₂PO₄ has also been employed as an acidic catalyst in the production of hydroxymethylfurfural (HMF) from cellulose [20] and in the conversion of phenolic-oil methyl ethers [21]. Mahinuddin et al. [22] demonstrated that KH₂PO₄ could be employed as a catalyst for the monobromination of aralkyl ketones with N-bromosuccinimide.

Methyl salicylate (MS) is produced by several species of plants, where it acts as a signaling molecule, which is released when subjected to some type of biotic stress, thus being a defense signal in the early detection of possible infections [23]. MS can also be used to control pests and as a raw material for the production of insecticides and pesticides [24–26]. However, its importance goes beyond agriculture and extends to other sectors, such as the pharmaceutical and food industries. In the pharmaceutical industry, it is recognized as an analgesic and anti-inflammatory agent, being used to treat muscle pain [27, 28]. In the food industry, it is used as a flavoring in foods and beverages, as it has a floral and mentholated fragrance [29–31]. There are also reports of its antimicrobial activity against bacteria and fungi, however, these references are scarce in the literature.

Oloyede [32] reported that the essential oil from Laportea aestuans (Nettle), mostly made up of methyl salicylate, has an inhibitory potential, especially against microorganisms such as Klebsiella pneumoniae, Salmonella typhi, Candida albicans, Rhizopus stolon and Aspergillus niger.

Javani et al. [33] proposed the stepwise isolation of K₂HPO₄ and KH₂PO₄ as a means of compensating the cost of biodiesel production. Recently, we studied the “Preparation of activated charcoal from Acrocomia aculeata for purification of pretreated crude glycerol” [34]. We decided to study the isolation of KH₂PO₄ from the medium containing glycerol and KOH that results from the transesterification of waste cooking oil (WCO). This study sought to compensate for the cost of producing biodiesel and glycerol and decrease the environmental problems resulting from the storage of these residues (glycerol and KOH). The KH₂PO₄ was used as basis in the regioselective methylation of salicylic acid using DMS as agent in the production of MS. In this work we also, the antimicrobial activity of MS was evaluated against Escherichia coli, Proteus mirabilis, Salmonella enterica, Edwardsiella sp. and Shigella sp; and against the yeasts Candida akabanensis, Candida orthopsilosis, Sporothrix sp, Galactomyces geotrichum and Rhodotorula mucilaginosa.

Considering the bacteria and yeasts used as targets in these studies, they are briefly described. 1) Escherichia coli can cause diarrhea, urinary tract infections, bacteremia, meningitis, septicemia and pneumonia [35]. 2) Proteus mirabilis is considered to be an opportunistic microorganism in humans. It can reach the urinary, digestive and respiratory systems, in addition to causing infections in the bloodstream, ears and nose, resulting in wounds [36]; 3) Salmonella and 4) Shigella are enteric bacteria. The first (Salmonella) is considered to be one of the most prevalent pathogens in most foodborne diseases, resulting in several global outbreaks with high mortality [37, 38]; and the latter (Shigella) is a highly infectious intestinal pathogenic bacterium, which is present in polluted waters and causes symptoms such as fever, nausea, vomiting and watery diarrhea [39]. 5) Edwardisiella sp. is an intracellular bacterium that infects several species of commercially farmed fish and amphibians, as well as birds, reptiles and mammals, causing septicemia and mortality [40–42]. 6) Sporothrix causes the disease sporotrichosis; it is a yeast that mainly causes subcutaneous mycosis that affects the skin and mucous membranes [43]. 7) Rhodotorula mucilaginosa and 8) Galactomyces geotrichum are also yeasts that generally do not present pathogenicity [44–45]. However, R. mucilaginosa is considered to be an opportunistic yeast and causes infections in immunocompromised patients [46], whereas G. geotrichum has been associated with damage to machinery because they accumulate spores in food processing environments and cause their deterioration [47, 48].There are few reports regarding the yeasts 9) Candida orthopsilosis and 10) Candida akabanensis; however, there is a report regarding their association with cases of malignant otitis externa, leading to osteomyelitis of the skull base [49].

2.1. Raw materials and chemicals

Crude glycerol was obtained from triacylglycerides (waste cooking oil - WCO) by the transesterification reaction [34]. MeOH (analytical grade), H₃PO₄, salicylic acid, DMS and KOH were supplied by Vetec, São Paulo, Brazil. BaSO₄ (analytical grade) was supplied by Synth, São Paulo, Brazil. H₂SO₄, BaCl₂.2H₂O and C₆H₁₂O₆ (anhydrous dextrose) were obtained from Dinâmica Química Contemporânea, São Paulo, Brazil. Nutrient agar, nutrient broth, yeast extract, malt extract, soy peptone and agar agar were supplied by Kasvi, Paraná, Brazil. Ágar Mueller Hinton (HiMedia) was provided by Exodus Ciência, São Paulo, Brazil. The bacterial cultures were obtained from the teaching collection of the Microbiology Laboratory of the Institute of Science and Technology at UFVJM. The yeast strains were accessed in the culture bank of the Laboratory of Microbiology Applied to Biofuels at the UFVJM, registered in SISGEN under the code A18446E.

2.2. Instrumentation

MS content and conversion rate were determined with a GC/MS-QP 2010/AOC 5000 AUTO INJECTOR/Shimadzu Gas Chromatograph/Mass Spectrometer equipped with a 30 m Agilent J&W GC DB-5 MS column. Direct insertion spectra were measured at 70 eV. Quantitative analyses were performed on a Shimadzu GC-2010 gas chromatograph equipped with a flame ionization detector [50]. ¹H- and ¹³C-NMR spectra were recorded on Bruker Avance 400 Spectrometers as has been previously described [30]. All the reactions were monitored by TLC using Silica Gel 60 F 254 on aluminum. The chromatograms were visualized by UV light or by using the ethanolic vanillin developing agent [50]. The Purification of the products was achieved by flash column chromatography using a 9/1 mixture of hexane/ethyl acetate as the eluent [50].

2.3. Typical procedures

The crude mixture (216.0 mL) of glycerol, pH 9, and KOH resulting from the transesterification of WCO [34] was treated with phosphoric acid (20 mL) to pH 2. The precipitated KH₂PO₄ (20.1038 g) was filtered and purified by recrystallization. The procedure for the production and recrystallization of KH₂PO₄ was performed four times to produce four different samples.

2.4. Purification of KH₂PO₄ by recrystallization

The KH₂PO₄ (20.1038 g) resulting from the pretreatment of crude glycerol was washed with 6 x 25 mL of diethyl ether by stirring with a magnetic stirrer for 20 minutes to remove less polar impurities. The sample was filtered under reduced pressure, and the filtrate was analyzed by TLC to confirm the removal of non-polar impurities. The sample was dried in an oven at 120°C and weighed (13.6724 g). The KH₂PO₄ was dissolved in 130 mL of boiling distilled water containing 1% w/w (0.1367 g) of activated charcoal, filtered and allowed to crystallize. After recrystallization, the KH₂PO₄ was dried at 120°C to furnish 9.5081 g of purified salt.

2.4.1 Elemental analysis based on energy-dispersive X-Ray fluorescence (EDXRF) of KH ₂ PO ₄ (Chemical or elemental composition of KH₂PO₄)

The energy-dispersive X-Ray fluorescence (EDXRF) of KH₂PO₄ was performed on an X-ray fluorescence spectrometer, model EDX 720 (Shimadzu®; Kyoto; Japan) equipped with an x-ray tube, and liquid nitrogen was used as the cooling agent. The software used was PCEDX (version 1.11 Shimadzu®).

2.4.2. Experimental X-ray diffraction of KH₂PO₄. Characterization of the material by powder XRD

The measurements were accomplished using a Shimadzu model XRD-6000 diffractometer employing CuKα monochromatic radiation (λ = 0.15406 nm − 40 kV and 30 mA) at a scan rate of 2.0 degrees.s^− 1 covering the 2θ scale from 10–80°. KH₂PO₄ was dried at 120°C before analysis by X-ray diffraction.

2.4.3. Spectrophotometry in the infrared region – FTIR of KH₂PO₄.

Fourier transform infrared spectroscopic (FTIR) analyses were performed on an IRPrestige-21 spectrophotometer (Shimadzu) using the transmittance method. The samples were prepared with KBr oven dried at 120 ^oC for 3 h and crushed in a press. The resolution used was 2.0, and 20 scans were taken.

2.4.4. Thermal gravimetric (TG) analysis of KH₂PO₄.

KH₂PO₄ was dried at 120°C prior to thermogravimetric analysis (TG). Thermogravimetric analyses involving sample characterization were performed in the TA Instruments Q-600 – The KH₂PO₄ samples were placed in a platinum crucible and heat treated in a TA Instruments model Q-600 analyzer - simultaneous DTA/TGA/DSC thermal analysis module, operating under a synthetic air atmosphere, with a heating rate of 10°C min^− 1 from room temperature to 1000°C. The pure KH₂PO₄ salt was used as a reference for the acquisition of thermogravimetric (TG) curves.

2.5. Methylation of SA in DMS using KH₂PO₄ as a selective base.

All the experimental procedures were performed in a laboratory fume hood. The procedure utilized for the methylation process was based on various trials to determine the optimum conditions for this reaction.

To a 150-mL round bottom flask coupled to a reflux condenser, 2.7624 g (20.00 mmol) of SA and 1.6801 g (20.0 mmol) of KH₂PO₄ were added. The reaction mixture was heated on the heating mantle at 90°C for 30 min, and DMS (2.5226 g, 1.92 mL, 40.00 mmol) was added with the help of a glass syringe. The mixture was magnetically stirred for another 90 min at 90 ^oC. The reaction process was monitored by thin layer chromatography (TLC) throughout this period. The mixture was cooled (Quenching) to room temperature and transferred to a separatory funnel, and 20.0 mL of deionized water and 10.0 mL CH₂Cl₂ were added. The aqueous phase containing H₃PO₄, MeOH and H₂SO₄ [34] was transferred to a beaker, and KOH was added to pH 7. The resulting mixture (0.1790 g) containing KH₂PO₄ and K₂SO₄ was recrystallized from water and dried in a muffle furnace (600 ^oC for 2 h). The organic solution obtained was removed, transfered to another extraction funnel, and partitioned between 10 mL of CH₂Cl₂ and 20 mL of saturated NaHCO₃. The organic phase was dried with Na₂SO₄, filtered, and evaporated under reduced pressure. The resulting residue was subjected to GC/MS analysis, which demonstrated the absence of unreacted SA. The residue containing 96.23% of MS and 3.77% methyl 2-metoxibenzoate (benzoic acid 2 metoxy methyl ester), was then purified by flash column chromatography on silica using hexane:ethyl acetate (9:1) as the mobile phase to yield MS (2,9203 g, 96%) as a colorless oil.

2.6. Preparation of microbial culture inoculum

Considering that the inoculum is a critical step in susceptibility testing, the concentrations of bacteria used were standardized according to the McFarland scale, represented by a series of barium sulfate solutions (99.5% sulfuric acid solution 0. 18 M and 0.5% of 0.048 M barium chloride solution) [51].

The bacterial cultures Escherichia coli, Proteus mirabilis, Salmonella enterica, Edwardsiella sp. and Shigella sp. were grown in Nutrient Agar (na) medium (38 g L^− 1) for 48 h at 35 ºC, and the yeast cultures Candida akabanensis UFVJM R131, Candida orthopsilosis UFVJM 4G, Sporothrix sp, UFVJM 5G, Galactomyces geotrichum UFVJM R10 and Rhodotorula mucilaginosa UFVJM 2G were grown in YPMD Agar medium (3 g L^− 1 yeast extract; 3 g L^− 1 malt extract; 5 gL^− 1 peptone; 10 gL^− 1 glucose and 18 gL^− 1 agar) for 48 h at 28 ºC.

Bacterial suspensions for sensitivity tests were prepared by the transfer of a batch of cultures, previously grown in solid medium, to liquid medium containing Nutrient Broth (10 g L^− 1). The cultures were incubated at 35°C until the turbidity of the medium reached the McFarland standard (0.1 OD), measured on a spectrophotometer at 610 nm (BIOSPECTRO SP-22). Yeast suspensions were also obtained by transferring a portion of cultures previously grown in solid medium to YPMD liquid medium and incubation at 28 ºC and 150 rpm until the turbidity of the medium reached the McFarland standard, measured in a spectrophotometer at 610 nm.

2.7. Antimicrobial sensitivity test

The antimicrobial susceptibility test was performed by evaluating the direct action of the synthesized methyl salicylate on the microbial cells using the agar perforation methods (cup-plate) according to Ostrosky et al [52]. The bacterial suspension was inoculated onto the surface of Mueller-Hinton Agar solid medium contained in 90x15 mm Petri dishes by spreading it on the surface with the aid of a swab. After this step, the well technique was applied, which consisted of removing part of the solid culture medium with the aid of a previously sterilized 6-mm-diameter glass cylinder to form wells. Pure methyl salicylate (60 µL) was added to each well, followed by incubation at 35 ºC for bacterial cultures and 28 ºC for yeast for a period of up to 96 h.

The inhibitory action of methyl salicylate on the tested cultures was evaluated by the formation of a halo of inhibition of microbial growth around each well containing methyl salicylate. Antimicrobial activity was determined by measuring the growth inhibition zone in centimeters.

The KH₂PO₄ was obtained from pretreated crude glycerol (CG). Glycerol is a by-product of transesterification of triacylglycerides contained in WCO. FAME (Fatty Acid Methyl Esters; biodiesel) is the principal product. In the transesterification reaction, two phases were formed: the biodiesel phase and the glycerol phase (CG). CG contained glycerol along with many other materials such as soaps, remaining catalyst (in this case KOH used in excess), MeOH and esters (glycerides) formed during the process. The glycerol content was approximately 10 wt.%. In this study, the glycerol phase was treated with 85% H₃PO₄ to produce CG, a small organic phase containing a mixture of FAME, fatty acids and glycerides, and unpurified solid KH₂PO₄.

3.1. Pretreatment of glycerol

Several methods are currently used for purification of glycerol. In this study, the crude glycerol was initially treated with H₃PO₄ until the glycerol solution reached pH 2. The KH₂PO₄ precipitate obtained in the acidification step was filtered through a Buchner funnel and recrystallized. The glycerol was purified and clarified using charcoal produced from Acromonia aculleata as the adsorbent, according to the method previously described by our research group [34].

3.2. Production of KH₂PO₄

In a previous work, Javani et al. [33] described the production of K₂HPO₄ by the stepwise acidification of glycerol, obtained from saponification of WCO, with H₃PO₄ to pH 9.67. The pH of the glycerol filtrate was lowered to 4.67 by the addition of H₃PO₄, and the precipitated KH₂PO₄ was recovered by filtration. The salts were washed with isopropyl alcohol to eliminate traces of glycerol, fatty acids, and glycerides that remained and then recrystallized. In the present study, the crude glycerol was acidified with H₃PO₄ to pH 2, producing only KH₂PO₄.

H₃PO₄(aq) + KOH(aq) ◊ KH₂PO₄(aq) + H₂O(l)

3.3. Recrystallization of KH₂PO₄

To produce pure KH₂PO₄, a highly accurate knowledge of the solubility is necessary for the crystallization process [53–55].

3.4. EDXRF of the KH₂PO₄

The EDXRF is an apparatus used for the quantitative and quali-quantitative determination of chemical elements in a wide range of samples. The analyses performed involved qualitative and quantitative determinations, with only the pre-calibration of the equipment (Al ≥ 80% and the detection of Sn and Cu), using only atmospheric air, and restricting the detection of metals between ₁₃Al and ₉₂U. The samples were placed in a sample holder made of polypropylene film, and the analysis conditions were as follows: 10 mm collimator, scans with voltages of 0–40 KeV (Ti-U) and 0–20 KeV (Na-Sc) with a time of 100 seconds for each sample.

In this method, the material to be analyzed is struck with an X-ray beam that interacts with the atoms of the sample, causing the ionization of the innermost layers of the atoms. The filling of the resulting vacancies by more peripheral electrons induces the emission of X-rays characteristic of the constituent elements of the sample.

Energy scattering X-ray fluorescence is a non-destructive multi-element analytical technique capable of identifying elements with atomic number Z equal to or higher than 12. When the electrons of the innermost layer of the atom (for example, K and L) interact with photons in the X-ray region, these electrons are ejected, creating a vacancy. To recover the stability, the electronic vacancies are immediately filled by electrons from the closest layers (Kα, Kβ or Lα, Lβ), resulting in an excess of energy in the process, which is manifested in the form of the emission of X-rays characteristic of each atom present in the sample. EDXRF was used as a method for semi-quantitative elemental analysis, and the titanium derivatives were not quantified. Their concentrations were lower than the quantification limit. However, their presence was clearly observed in the XRD powder spectra. The XRD patterns of the KH₂PO₄ are shown in Fig. 1.

3.5 XRD patterns of the KH₂PO₄

On the basis of the XRD of KH₂PO₄ obtained from crude glycerol, the extraction/purification method was observed to be successful. In the highlighted XRD regions of 2θ ~ 55^o and 70-75^o, it can be seen that the principal impurities that were found in sample 1 had been removed in sample 4. The XRD pattern of sample 4 was very similar to that of the commercial sample, as well as that of a sample reported in the literature [56]. Small differences in intensity and position can occur because of the preferential orientation and the possible variations in the crystallization of KH₂PO₄ [57].

3.5. Spectrophotometry in the infrared region – FTIR of KH₂PO₄.

The vibrational spectra in the infrared region of commercial KH₂PO₄ and the KH₂PO₄ samples (four) are shown in Fig. 2. The same transmission bands can be seen in the FTIR spectra of the KH₂PO₄ standard and the four samples. The band corresponding to the O-H stretching vibration of water can be observed in the 3468-cm^− 1 region, as well as the band for the POH stretching vibration at 1631 cm^− 1, the band for the HO-P-OH bending vibration at 635 cm^− 1, the band corresponding to P-OH deformation or KO stretching at 540 cm^− 1, and a band for PO₄ stretching at 416 cm^− 1 (Table 1).

Table 1

FTIR assignments for KH₂PO₄.
KH₂PO₄ (cm^− 1)	Assignments
3468	O-H stretching, H-bonded
2900	O-H stretching
2810	Intermolecular H-bonded –OH stretching
2443	O-H stretching
1631	O-P -OH symmetric stretching
1463	O-H stretching
1290	CH₂ bending, P = O symmetric stretching
1091	P -O-H symmetric stretching
920	O = P-OH bending
635	HO-P-OH bending
540	P -OH deformation/K-O stretching
416	P-O₄ stretching

3.5. Thermal gravimetric (TG) analysis of KH₂PO₄.

The same percentage of mass loss, depending on temperature, was observed in the TG curve of the pure salt sample (Fig. 3) and those of the four samples of KH₂PO₄ salts (Fig. 4). The formation of potassium polyphosphate was also indicated.

A loss of 12% in mass in the region of 200 ^oC and 400 ^oC, which corresponds to the loss of a water molecule in the salt polymerization process, can be observed because, when KH₂PO₄ is heated continuously to higher temperatures, constitutional water is released to form long-chain potassium polyphosphate, known as Kurrol's salt. Kurrol Potassium Polyphosphate is easily obtained by further heating of KH₂P0₄ [58].

nKH₂PO₄(s) ⇄ K_nH₂P_nO_3n+1(s) + (n-1) H₂O(aq)

3.6. Synthesis of MS using KH₂PO₄ as proton acceptor

Development of the simple, efficient, and environmentally friendly synthetic procedure for the commonly used SA was one of the major challenges in the MS synthesis. KH₂PO₄ participated as a base in the simple, regioselective, green method for the preparation of MS from SA acid using DMS in excess at reflux temperature (90 ^oC). The present method has a short reaction time (2 h), and 96.23% conversions to MS are obtained and 3.77% to methyl 2-metoxibenzoate. The method became greener and more productive because the DMS and H₃PO₄ can be separated from the reaction mixture during work-up, and subsequently transformed in other, greener compounds.

KH₂PO₄ (pKa is approximately 12) is known to be easily available, inexpensive, ecologically favorable and safe to handle [59]. In this work we report an environmentally benign procedure. KH₂PO₄ was used in stoichiometric quantities in the regioselective acid-base reaction for the capture of the carboxyl hydrogen (Fig. 5).

Only 3.77% of SA was converted into the intermediate 2-benzoate oxide (carboxylate and phenoxide ions), which demonstrates the regioselectivity of KH₂PO₄ in capturing the carboxylic proton. In this reaction, KH₂PO₄ was used in the same proportion as SA. It was transformed into H₃PO_4, and the carboxylate ion generated in situ was demonstrated to be an excellent nucleophile in the presence of DMS (electrophilic reagent), which was used in excess because it was an excellent aprotic solvent for this S_N2 mechanism (Fig. 6).

The DMS in excess was transformed into K₂SO₄ and MeOH after the reaction by the addiction of water [60] during the work-up of the reaction mixture. Previously formed H₃PO₄ was removed by simple addiction of KOH and transformed into KH₂PO₄. The mixture containing K₂SO₄ and KH₂PO₄ can be used as fertilizer. Both salts are an excellent source of nutrition for plants [61, 62].

3.7. Infrared Spectrophotometry– FTIR of MS

The vibrational spectra in the infrared region of MS samples are shown in Fig. 7. The spectra of the two samples contain the same bands for MS. A broad band characteristic of OH stretching vibrations occur in the region of 3800 to 2600 with a peak at 3.2350 cm^− 1 due to hydrogen bonding. The peak at ~ 2955 cm^− 1 corresponds to the CH stretching vibrations of the CH₃ group and that at 2029 to the C-H stretching vibration of the arene. The absorption at 1680 cm^− 1 corresponds to the C = O carbonyl stretching vibration. The absorptions corresponding to the stretching vibrations of the benzene aromatic ring are in the 1600 − 1450 cm^− 1 region. The absorption at about 1200 cm^− 1 corresponds to the CO stretching vibration of an ester. Also, there are several characteristic peaks resulting from CO stretching vibrations around 1342 to 1250 and 1177 to 1079 cm^− 1 (where the O is directly bonded to the benzene ring as in a phenol). These data are in accordance with those found in the literature [63].

3.7. Infrared Spectrophotometry– FTIR of the mixture containing KH₂PO₄ and K₂SO₄

Some bands can be observed in the high frequency region of the spectrum, one close to 3444 cm^− 1 referring to OH stretching (ν OH), confirming the presence of H₂O in its constitution and another band around 1639 cm^− 1 referring to deformation angle of water molecules (δ OHO) present on the sample surface. The band at 1116 cm^− 1 represents an asymmetric stretching of SO₄²⁻ and the band at 615 cm^− 1 shows the deformation of the O-P-O stretching in PO₄ ^− 3, thus proving the presence of sulfate and phosphate in the sample (Fig. 8).

3.9. MS as microbiological agent

The results for the activity of methyl salicylate as an antimicrobial compound in cultures of Escherichia coli, Proteus mirabilis, Salmonella enterica, Edwardsiella sp., Shigella sp., Candida akabanensis UFVJM R131, Candida orthopsilosis UFVJM 4G, Sporothrix sp, UFVJM 5G, Galactomyces geotrichum UFVJM R10 and Rhodotorula mucilaginosa UFVJM 2G are found in Table 2.

Tabela 2. Inhibition by methyl salicylate using the cup-plate method for Escherichia coli, Proteus mirabilis, Salmonella enterica, Edwardsiella sp., Shigella sp., Candida akabanensis UFVJM R131, Candida orthopsilosis UFVJM 4G, Sporothrix sp, UFVJM 5G, Galactomyces geotrichum UFVJM R10 and Rhodotorula mucilaginosa UFVJM 2G, with 48 and 96 h of monitoring.

Microorganisms		Inhibitory action (Diameter of inhibition halo)
		Hours
		48	72	96
Bacteria	Escherichia coli	1,1	1,1	1,1
	Proteus mirabilis	1,4	1,4	1,4
	Salmonella entérica	negative	negative	negative
	Edwardsiella sp.	negative	negative	negative
	Shigella sp	negative	negative	negative
Yeast	Sporothrix sp UFVJM 5G	2,0	2,0	2,0
	Rhodotorula mucilaginosa UFVJM 2G	negative	negative	6,5
	Galactomyces geotrichum UFVJM R10	3,9	3,9	3,9
	Candida orthopsilosis UFVJM 4G	2,9	2,8	2,8
	Candida akabanensis UFVJM R131	2,9	2,9	2,9

The inhibition of the bacteria cultures by MS was positive for Escherichia coli and Proteus mirabilis. This finhibiton was demonstrated by the formation of translucent halos, measuring 1.1 and 1.4 cm in diameter, after 48 h (Fig. 1a and 1b). The inhibition results for yeast suggest a positive action of MS on all the cultures tested, as inhibition halos were observed with values ranging from 2.0 to 6.5 cm in diameter. The largest inhibition zone observed was for the culture R. mucilaginosa UFVJM 2G, which was 6.5 cm in diameter, and the smallest was for the yeast Sporothrix sp UFVJM 5G with 2.0 cm in diameter (Fig. 1c and 1d). The first indication of a halo for Rhodotorula mucilaginosa UFVJM 2G appeared only after 72 h. Before this period, there was no growth or an obvious halo of inhibition. Over a period of 96 h, a rapid growth of the culture at the edge of the culture medium could be seen, with a halo measuring 6.5 cm in diameter (Fig. 1d). Verdi et al. [63] reported that methyl salicylate has a broad antimicrobial activity, mainly affecting proteins located in the cell membrane, which interferes with the cell signaling process. Because of its lipophilic character, a strong interaction with the phospholipid layer of the cell membrane occurs, destroying and causing changes in the cytoplasm of bacteria and mycobacteria. Bitla et al. [67] synthesized triazole compounds from methyl salicylate and reported antifungal activity for the products, which were effective against microbial growth.

The production and isolation of KH₂PO₄ are simple and environmentally friendly processes because no special chemicals or equipment are required. Overall, the isolation process reported in this work can be scaled up to commercial levels without any significant changes in its parameters. Acidification of the soap in the crude glycerol to free fatty acids and KH₂PO₄ was accomplished with the addition of H₃PO₄ to a pH of 2. The KH₂PO₄ FTIR data helped us to analyze the vibrational functional groups. The thermal analysis by TG/DTA techniques reveals the thermal stability of the KH₂PO₄. The product KH₂PO₄ can be used as potash-phosphate fertilizer [68], in addition to more refined applications. KH₂PO₄ is a source of two nutrients, potassium and phosphorus. The KH₂PO₄ obtained satisfies the industrial grade standard. This process of production and isolation furnished a product of high purity in high yield and contributed to the removal of industrial waste from the environment. The KH₂PO₄ salt is usually applied in medical, food and industrial fields [7]. The synthesis of MS resulted in excellent yields without disadvantages such as long reaction times, precarious operational procedures or generation of organic waste in large quantities. Hence, the user-friendly and eco-friendly method for regioselective methylation of SA was achieved in this new synthetic organic protocol. The MS was shown to be capable of inhibiting the growth of Escherichia coli, Proteus mirabilis, Sporothrix sp, Galactomyces geotrichum UFVJM R10, Candida orthopsilosis UFVJM 4G and Candida akabanensis UFVJM R13, as demonstrated by the formation of translucent halos measuring up to 6.5 cm in diameter. The results suggest that the yeasts studied are more sensitive to the methyl salicylate. Further studies are necessary to comprehend the action of this compound on the biochemical mechanisms of bacterial and yeast cells.

Conflict of interest

This paper does not have any competing interests.

Ethical approval

This paper does not have any ethical statement, and this work does not involve any conflict of interest.

5. Acknowledgements

The authors acknowledge the support by the PRPPG/UFVJM in response to Resolução 15/2019 and the Fundação de Apoio à Pesquisa do Estado de Minas Gerais - FAPEMIG - APQ-03286-21.

Data availability

The paper entitled ‘‘Isolation of KH₂PO₄ from crude glycerol purified using Acromonia aculleata charcoal as adsorbent and its application in the synthesis of methyl salicylate’’ is submitted for your kind considerations. The research data policy and data availability are not shared with anyone before publication.

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You are reading this latest preprint version

Isolation of KH2PO4 from crude glycerol purified using Acromonia aculleata charcoal as adsorbent and its application in the synthesis of methyl salicylate.

Status:

Version 1

Abstract

Figures

1. Introduction

2. Experimental

2.1. Raw materials and chemicals

2.2. Instrumentation

2.3. Typical procedures

2.4. Purification of KH₂PO₄ by recrystallization

2.4.2. Experimental X-ray diffraction of KH₂PO₄. Characterization of the material by powder XRD

2.4.3. Spectrophotometry in the infrared region – FTIR of KH₂PO₄.

2.4.4. Thermal gravimetric (TG) analysis of KH₂PO₄.

2.5. Methylation of SA in DMS using KH₂PO₄ as a selective base.

2.6. Preparation of microbial culture inoculum

2.7. Antimicrobial sensitivity test

3. Results and Discussion

3.1. Pretreatment of glycerol

3.2. Production of KH₂PO₄

3.3. Recrystallization of KH₂PO₄

3.4. EDXRF of the KH₂PO₄

3.5 XRD patterns of the KH₂PO₄

3.5. Spectrophotometry in the infrared region – FTIR of KH₂PO₄.

3.5. Thermal gravimetric (TG) analysis of KH₂PO₄.

3.6. Synthesis of MS using KH₂PO₄ as proton acceptor

3.7. Infrared Spectrophotometry– FTIR of MS

3.7. Infrared Spectrophotometry– FTIR of the mixture containing KH₂PO₄ and K₂SO₄

3.9. MS as microbiological agent

4. Conclusions

Declarations

5. Acknowledgements

Data availability

References

Supplementary Files

Status:

Version 1

Isolation of KH2PO4 from crude glycerol purified using Acromonia aculleata charcoal as adsorbent and its application in the synthesis of methyl salicylate.

Status:

Version 1

Abstract

Figures

1. Introduction

2. Experimental

2.1. Raw materials and chemicals

2.2. Instrumentation

2.3. Typical procedures

2.4. Purification of KH2PO4 by recrystallization

2.4.2. Experimental X-ray diffraction of KH2PO4. Characterization of the material by powder XRD

2.4.3. Spectrophotometry in the infrared region – FTIR of KH2PO4.

2.4.4. Thermal gravimetric (TG) analysis of KH2PO4.

2.5. Methylation of SA in DMS using KH2PO4 as a selective base.

2.6. Preparation of microbial culture inoculum

2.7. Antimicrobial sensitivity test

3. Results and Discussion

3.1. Pretreatment of glycerol

3.2. Production of KH2PO4

3.3. Recrystallization of KH2PO4

3.4. EDXRF of the KH2PO4

3.5 XRD patterns of the KH2PO4

3.5. Spectrophotometry in the infrared region – FTIR of KH2PO4.

3.5. Thermal gravimetric (TG) analysis of KH2PO4.

3.6. Synthesis of MS using KH2PO4 as proton acceptor

3.7. Infrared Spectrophotometry– FTIR of MS

3.7. Infrared Spectrophotometry– FTIR of the mixture containing KH2PO4 and K2SO4

3.9. MS as microbiological agent

4. Conclusions

Declarations

5. Acknowledgements

Data availability

References

Supplementary Files

Status:

Version 1

2.4. Purification of KH₂PO₄ by recrystallization

2.4.2. Experimental X-ray diffraction of KH₂PO₄. Characterization of the material by powder XRD

2.4.3. Spectrophotometry in the infrared region – FTIR of KH₂PO₄.

2.4.4. Thermal gravimetric (TG) analysis of KH₂PO₄.

2.5. Methylation of SA in DMS using KH₂PO₄ as a selective base.

3.2. Production of KH₂PO₄

3.3. Recrystallization of KH₂PO₄

3.4. EDXRF of the KH₂PO₄

3.5 XRD patterns of the KH₂PO₄

3.5. Spectrophotometry in the infrared region – FTIR of KH₂PO₄.

3.5. Thermal gravimetric (TG) analysis of KH₂PO₄.

3.6. Synthesis of MS using KH₂PO₄ as proton acceptor

3.7. Infrared Spectrophotometry– FTIR of the mixture containing KH₂PO₄ and K₂SO₄