FULVIC ACID SYNTHESIZED BY CONDENSATION OF CATECHOL AND GLYCINE

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Synthesis in the C-H-O system

Fulvic acids have been synthesized in the C-H-O system by two different polymerization reactions, including the autopolymerization of catechol and the polymerization through condensation of catechol and acetic acid. Those substances obtained from the first reaction will be designated below by SFA_1, while those obtained by the second reaction will be referred to as SFA_2.

Autopolymerization of catechol

The synthesis procedure to obtain SFA_1 was as follows. Catechol (44.04 g) was first dissolved in 4 liters of ultra pure water to obtain a 0.1 M solution. The pH of the reaction medium was adjusted to pH = 10 by using NaOH 1.0 M. The resulting solution was allowed to stand in the dark at 25°C (± 0.1°C), under constant stirring at 3000 rounds per minute (rpm) for homogenization. Successive samplings indicated that both the viscosity and color intensity of the solution were increasing as a function of reaction progress. The color first turned to light brown, then dark brown, and finally black after approximately three days. The polymerization was carried out for 63 days, and a constant value of pH was maintained by adding NaOH 0.1 M on a daily basis. The synthesis procedure described above differs from that originally proposed by Andreux et al. (1980), who performed the synthesis of their humic-like polymers at pH = 7.9 and under a constant current of pure oxygen while the synthesis was carried out in contact with the atmosphere in the present study. All samples collected over the period of the synthesis have been deep frozen, lyophilized (see below), ground to fine powder, and then stored at 4°C to await analysis.

Polymerization through condensation of catechol and acetic acid

A mixture of SFA_2 containing alcohol (―OH) and perhaps carboxylic acid (―COOH) groups has been obtained by dissolving catechol (44.04 g) and acetic acid (24.02 g) in equimolar (0.1 M) proportions in 4.0 L of ultra pure water. The pH of the reaction medium was again adjusted to pH = 10 by using NaOH 1.0 M, and the synthesis was carried out at 25°C, in the dark and under a constant stirring of 3000 rpm for 63 days. The same observations of increasing viscosity and color intensity were made, with the color of the reaction medium slowly turning into black as a function of the progress of the polymerization reaction. Samples of the solution were withdrawn just after the initiation of the reaction (zeroth day) and every five days for one month. These samples were again deep frozen, lyophilized, ground to powder and stored at 4°C to await analysis.

Synthesis in the C-H-O-N system

Fulvic acids synthesized in the C-H-O-N system will be designated in the following text by SFA_3. These substances which contain alcohol (―OH), amine (―NH2), and perhaps carboxylic acid (―COOH) groups have been synthesized using a procedure which is identical to that described for SFA_2, except that glycine was used instead of acetic acid. Note that catechol and glycine are the reactants used in the original synthesis of humic acids by Andreux et al. (1980). Catechol (44.04 g) was dissolved with glycine (30.03 g) to obtain an equimolar (0.1 M) mixture in 4.0 L of ultra pure water. NaOH 1.0 M was added daily to adjust the pH of the reaction medium to pH = 10. The synthesis was again carried out at 25°C, in the dark and under a constant stirring of 3000 rpm for 63 days. The same observations of increasing viscosity and color intensity were made, although in this case a pink color was initially observed, which has been attributed by Jung et al. (2005) to the formation of N-substituted quinones. Samples were withdrawn from the solution just after the initiation of the reaction (zeroth day,) and then every five days for one month. These samples were deep frozen, lyophilized, ground to powder and stored at 4°C to await analysis.

Synthesis in the C-H-O-N-S system

Fulvic acids synthesized in the C-H-O-N-S system will be designated in the following text by SFA_4. These substances obtained by reacting catechol with cysteine again contain alcohol (―OH), amine (―NH2), and perhaps carboxylic acid (―COOH). In addition, the presence of the thiol (―SH) group in cysteine may allow polymerization by cross-linking, resulting in the formation of sulfide or disulfide groups. Catechol (44.04 g) was dissolved with cysteine (48.06 g) to obtain an equimolar (0.1 M) mixture in 4.0 L of ultra pure water. The synthesis was carried out at 25°C, in the dark and under a constant stirring of 3000 rpm for 63 days while the pH of the reaction medium was adjusted to pH = 10 by using NaOH 1.0 M. The solution again showed an increase in darkening with increasing reaction progress. Samples were withdrawn from the solution just after the initiation of the reaction (zeroth day,) and then every five days for one month. These samples were deep frozen, lyophilized, ground to powder and stored at 4°C to await analysis.

Liquid-liquid extraction

In the liquid-liquid extraction procedure, the solute is removed from one liquid phase by adding to that phase an immiscible solvent in which the solute is more soluble. The procedure, which was applied only to SFA_1, is depicted in Figure 1.5 and was the following. A sample of 4.06 g of solid SFA_1 was dissolved in ultra pure water and the pH of the resulting solution was adjusted to 2.052 using 1.0 M HCl. The acidified solution was extracted with ethyl acetate (heated up to ~ 60°C) using a glass liquid-liquid extractor. The extraction was carried out continuously for 63 hours. After separation of the organic phase and aqueous phase, the organic phase was evaporated to dryness and an orange solid was obtained with a 15% yield with respect to the initial mass of SFA_1 (4.06 g). The aqueous phase was deep-frozen and then lyophilized. A black solid was obtained with a 85% yield.

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X-ray diffraction

X-Ray diffraction is a versatile, non destructive analytical technique for the identification and quantitative determination of the various crystalline materials of compounds present in powdered and solid samples. Identification is achieved by comparing the X-ray diffraction pattern obtained from unknown sample with an internationally recognized database (ICDD) containing reference patterns. Solid samples of the synthesized products were investigated by comparing them with their starting materials.
For XRD analysis, the solid samples were collected using a D8 Bruker diffractometer with Co-Kα radiation (λ = 1.7902Å). General operating conditions were 35 kV accelerating voltage, 45 mA intensity, step-scanning at 0.035°(2θ) intervals, 3-sec counting time, and 3-75°(2θ) for disoriented powder.

Infrared spectroscopy

Infrared (IR) spectroscopy is an extremely reliable and well recognized fingerprinting method. It has long been used for the characterization of humic substances (Schnitzer et al., 1959; Ishiwatari, 1970; Stevenson and Goh, 1971). The technique of Attenuated Total Reflectance (ATR) has in recent years revolutionized solid and liquid sample analyses because it combats the most challenging aspects of infrared analyses, namely sample preparation and spectral reproducibility. ATR is an IR sampling technique that provides excellent quality data in conjunction with the best possible reproducibility of any IR sampling technique. It has revolutionized IR solid and liquid sampling through:
• Faster sampling.
• Improving sample-to-sample reproducibility and.
• Minimizing user to user spectral variation.
An attenuated total reflection accessory operates by measuring the changes that occur in a totally internally reflected infrared beam when the beam comes into contact with a sample (see Figure 1.7). An infrared beam is directed onto an optically dense crystal with a high refractive index at a certain angle. This internal reflectance creates an evanescent wave that extends beyond the surface of the crystal into the sample held in contact with the crystal. This evanescent wave protrudes only a few microns (0.5 μ – 5 μ) beyond the crystal surface and into the sample. Consequently, there must be good contact between the sample and the crystal surface. In regions of the infrared spectrum where the sample absorbs energy, the evanescent wave will be attenuated or altered. The attenuated energy from each evanescent wave is passed back to the IR beam, which then exits the opposite end of the crystal and is passed to the detector in the IR spectrometer. The system then generates an infrared spectrum.

Table of contents :

CHAPTER 1. MATERIALS, EXPERIMENTAL METHODS, AND ANALYTICAL TECHNIQUES
1.MATERIALS
1.1. Reactants used in the synthesis of fulvic acids
1.2. Reactants used in the complexation studies
1.3. Dialysis membranes
2. EXPERIMENTAL METHODS
2.1. Synthesis of fulvic acids
2.1.1. Synthesis in the C-H-O system
2.1.1.1. Autopolymerization of catechol
2.1.1.2. Polymerization through condensation of catechol and acetic acid
2.1.2. Synthesis in the C-H-O-N system
2.1.3. Synthesis in the C-H-O-N-S system
2.2. Lyophilisation
2.3. Extraction
2.3.1. Solid-liquid extraction
2.3.2. Liquid-liquid extraction
3. ANALYTICAL TECHNIQUES
3.1. Elemental analysis
3.2. X-ray diffraction
3.3. Infrared spectroscopy
3.4. UV-visible spectroscopy
3.5. Electrospray ionization – mass spectroscopy (ESI-MS)
3.6. Atmospheric pressure chemical ionization – mass spectroscopy (APCI-MS)
CHAPTER 2. CHARACTERIZATION OF THE SYNTHESIZED FULVIC ACIDS
1. FULVIC ACID SYNTHESIZED BY POLYMERIZATION OF CATECHOL
1.1. Elemental analysis
1.2. Scanning electron microscopy
1.3. X-ray diffraction
1.4. ATR-FTIR spectroscopy
1.4.1. Catechol
1.4.2. Synthetic fulvic acid SFA_1
1.5. UV-Visible absorption spectrophotometry
1.6. Electrospray ionization – mass spectrometry (ESI-MS)
1.7. Atmospheric pressure chemical ionisation mass spectrometry (APCI-MS)
1.7.1. Analysis of raw SFA_1 by LC-APCI-MS-MS
1.7.2. Analysis of purified SFA_1 by APCI-MS
1.8. Discussion
1.8.1. Oxidative coupling
1.8.2. Quinone formation and its reactions
2. FULVIC ACID SYNTHESIZED BY CONDENSATION OF CATECHOL AND ACETIC ACID
2.1. Elemental analysis
2.2. Scanning electron microscopy
2.3. X-ray diffraction
2.4. ATR-FTIR spectroscopy
2.4.1. Acetic acid
2.4.2. Sodium acetate, hydrated
2.4.3. Synthetic fulvic acid SFA_2
2.5. UV-Visible absorption spectrophotometry
2.6. Electrospray ionization – mass spectrometry (ESI-MS)
2.7. Atmospheric pressure chemical ionisation mass spectrometry (APCI-MS)
2.7.1. Analysis of raw SFA_2 by LC-APCI-MS-MS
2.7.2. Analysis of purified SFA_2 by APCI-MS
2.7.3. Analysis by APCI-MS of the methanol extract obtained by solid-liquid extraction of raw SFA_2
2.8. Discussion
3. FULVIC ACID SYNTHESIZED BY CONDENSATION OF CATECHOL AND GLYCINE
3.1. Elemental analysis
3.2. Scanning electron microscopy
3.3. X-ray diffraction
3.4. ATR-FTIR spectroscopy
3.4.1. Glycine
3.4.2. Synthetic fulvic acid SFA_3
3.5. UV-Visible absorption spectrophotometry
3.6. Electrospray ionization – mass spectrometry (ESI-MS)
3.7. Atmospheric pressure chemical ionization mass spectrometry (APCI-MS)
3.7.1. Analysis of raw SFA_3 by LC-APCI-MS-MS
3.7.2. Analysis of purified SFA_3 by APCI-MS
3.8. Discussion
4. FULVIC ACID SYNTHESIZED BY CONDENSATION OF CATECHOL AND CYSTEINE
4.1. Elemental analysis
4.2. Scanning electron microscopy
4.3. X-ray diffraction
4.4. ATR-FTIR spectroscopy
4.4.1. Cysteine
4.4.2. Synthetic fulvic acid SFA_4
4.5. UV-Visible absorption spectrophotometry
4.6. Electrospray ionization – mass spectrometry (ESI-MS)
4.7. Atmospheric pressure chemical ionization mass spectrometry
4.7.1. Analysis of raw SFA_4 by LC-APCI-MS-MS
4.7.2. Analysis of purified SFA_4 by APCI-MS
4.8. Discussion
CHAPTER 3. URANYL(VI)-FULVATE COMPLEXATION
3.1. Objectives of the experiment and scientific background
3.2. Experimental section
3.2.1 Uranyl(VI)-SFA_1 systems at pH 4
3.2.2 Uranyl(VI)-SFA_1 systems at neutral pH (pH 7)
3.2.3 Uranyl(VI)-SFA_1 systems at basic pH (pH 10)
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