FUNCTIONALIZATION OF ALGINATE VIA PHOSPHORIZATION: PREPARATION, CHARACTERIZATION

W. M. Abou-Taleb 1 , G. D. Roston 2 , M. S Mohyeldin 3,4 , A. M Omer 3 , * T. M. Tamer 3 , E. F. Shehata 1 and A. M. Hafez 1 . 1. Physical Department, Faculty of Education, Alexandria University, Alexandria, Egypt. 2. Physics Department, Faculty of Science, Alexandria University, Alexandria, Egypt. 3. Polymer Materials Research Department, Advanced Technology and New Materials Research Institute, City of Scientific research and advanced technology. New Boarg El-Arab City,21934, Alexandria, Egypt. 4. Chemistry Department, Faculty of Science, University of Jeddah, Jeddah, 21589, Saudi Arabia. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

In this study, different degrees of phosphorated alginate were prepared via new method by utilizing epichlorohydrin as an activating agent and orthophosphoric acid as a phosphorus source.

Methods
Prepared 1 gm of Alginate powder (Alg) dissolved in 50 ml distilled water followed by the addition of ECH in different molar ratios; 1: 0.25, 1: 0.5, 1: 1 and 1: 2. The activation process carried at (60°C). After 6 hours, equivalent amounts of orthophosphoric acid were added to the reactor and continuously stirring for additional 6 hr. The obtained mixture of Alg-ph polymer was precipitated using ethanol. Alg-ph was rinsed ten times using absolute ethanol to remove any excess of free reactants. Four different substitutions Alg-ph 1, Alg-ph 2, Alg-ph 3 and Alg-ph 4 were prepared and characterized in comparison with neat Alginate Alg-ph 0.

Moisture content
Desired weight of dry samples was located in a humidity chamber with 80% humidity ratio, for 24 hr and then reweighed. Moisture content was calculated as follows equation; Moisture content % = [M -M 0 ] / M 0 X 100 Where M 0 is the initial weight of the dry sample and M is the final weight.

Infrared spectrophotometric analysis (FTIR)
The structures of Alginate and phosphonated Alginate derivatives were determined by FTIR analysis was carried out using Fourier transform infrared spectrophotometer (Shimadzu FTIR-8400 S), Japan.

Raman spectroscopy analysis
The micro-Raman scattering spectra were recorded with a Renishaw Raman RM1000 equipped with the 532 nm laser line, an electrically refrigerated CCD camera, and a notch filter to eliminate the elastic scattering. The spectra shown here were obtained by using a 25× microscope objective. The output laser power at the samples was about 0.2 mW. Spectral resolution was 4 cm −1 . The spectral scanning conditions were chosen to avoid sample degradation.

Scanning electron microscopic analysis (SEM)
Morphological changes of the samples surface were followed using SEM (Joel Jsm 6360LA), Japan.

UV-Vis Spectroscopic analysis
The electronic absorbance of Alginate and phosphonated Alginate derivatives (2.5 mg/ml) were done using spectrophotometer scanned from 200 -500 nm.

Results and discussion:-
The derivatization strategies for alginates depend on reactivity of alginate function groups. Alginate can be modified at the two secondary -OH positions (C-2 and C-3) or the one -COOH (C-6) position. In the current work, phosphorization of alginate was done through activation its hydroxyl groups (at C-2 and C-3) using epichlorohydrin then phosphorization using orthophosphoric acid as shown in Figure 1.

Electronic spectrum
Alginates have carboxylate as the intrinsic chromophore and hence display circular dichroism around 210 nm matching to the n-π* transition of the carboxyl group. The three types of block sequences present in alginates display very different circular dichroism behavior, the spectrum of poly-L-guluronate being entirely negative, whereas that of poly-D-mannuronate has a strong positive band and mixed sequences show intermediate behavior.
Spectra of intact alginates show a peak at ~200 nm, and a trough at ~215 nm ( Figure 3), with relative magnitudes varying with composition. The ratio of peak height to trough depth varies almost linearly with the ratio of mannuronate to guluronate residues. A shoulder was observed around 250-290 nm. Absorbance within the 250-290 nm range is commonly attributed to π − π* electron transitions in aromatic and poly-aromatic compounds [22], which could connect with the impurities such as humic acid, phenol, DNA or proteins [23]. The decrease of the

Infrared spectrophotometric analysis (FTIR)
The FT-IR spectra of alginate and its phosphonate derivatives were done and presented in figure 4. Alginate demonstrates typical characteristic bans related to its polysaccharide structure. A broadband at 3150 cm −1 was attributed to stretching vibration of hydroxyl groups. Bands at 1656 and 1417 cm −1 present in the IR spectrum are assigned to asymmetric and symmetric stretching peaks of carboxylate salt groups. Also, the bands around 1320 cm

Raman Spectroscopy
The Raman spectroscopy analysis of Alginate and different phosphonate derivatives are shown in Figure 5. All the samples exhibit the characteristic asymmetric and symmetric bands of carboxylate ion (COO − ) at 1616-1611 cm −1 and near 1446 cm −1 , respectively. In the region 1400-1270 cm −1 , three bands assigned to the C-H deformation vibration, and a band around 1200 cm −1 assigned to the C-O stretching vibration are shown [25].
Alginate samples show several bands in the region between 1200-950 cm −1 ; this region deals with C-OH deformation, C-C-H bending, C-O, and C-C stretching vibrations; every band may be due to contributions of two or more kinds of motions. [26] The region between 950 and 750 cm −1 is called the 'fingerprint' or the anomeric region; the spectra of alginate samples present a band near 800 cm −1 assigned to skeletal stretching and deformation modes, and another around 770-730 cm −1 due to ring breathing. Below 700 cm −1 , four bands related to the deformation of pyranose rings and C-O-C vibration of glycosidic linkage is shown [25].

Thermal gravimetric analysis (TGA)
Thermal gravimetric analysis of alginate and its phosphonated derivatives were exanimate at temperature ranged from ambient to 600 ºC ( figure 6). Alginate demonstrates a general moisture loss band that starts from ambient to around 150 ºC. presence of highly hydrophilic bands such hydroxyl and carboxylic groups along backbone simplified trapping of moisture molecules inside chains. Hydrophilicity of polymers was increased significantly by phosphorization process. Moisture content was increased dramatically from 13.26 % on neat polymer alginate (Algph 0) to be 12.62, 20.03, 23.14 and 27.03% for (Alg-ph 1, Alg-ph 2, Alg-ph 3 and Alg-ph 4) respectively.
Alginate exhibits a second weight loss starting from 220 ºC that attributed to destructive decomposition of pyranose ring. In the other hand, Phosphonated alginate has a different decomposition behavior. New decomposition rate was observed consequently after elevation of moisture content (i.e., at 150 ºC) can be sign to evaporation of physically attached phosphoric acid.

Differential scanning calorimetry (DSC)
Differential scanning calorimetric analysis of the alginate and its phosphonate derivatives was measured and presented in figure 7. The first endothermic peak observed below 100 °C is attributed to elevation moisture which is attached on internal chains. Alginate exhibits a significant exothermic peak at 245 ºC as a result of cleavage of the glycoside ring. Phosphonate alginate demonstrates a nice endothermic peak with maximum at 140 ºC that can refer to evaporation of physically attached phosphoric acid (boiling point of phosphoric acid 158 ºC).

Morphological characterization (SEM)
Surface morphological study of alginate and different phosphonate derivatives were performed with Scanning electron microscope. Figure 8 show microstructure of powers surface. From figures, it's clear increase the surface roughness at low phosphorization degree (Alg-ph 1) that can attribute to effect of phosphorization on distortion of crystal structure of alginate. Identification of immobilized phosphornic groups on polymeric matrix after preparation step was studied by EDX in order to quantitative determine of P % via illustrate elemental analysis. EDX offers use of the X-ray spectrum emitted by a solid sample bombarded with a focused beam of electrons to obtain an elemental chemical analysis. Qualitative analysis involves the identification of the P line (i.e.; P K at 2.013 kev) in the spectrum. Figure 9, illustrate EDX spectrum of alginate and different phosphonate derivatives. It was clear increase P% from 0 in Algph 0 to 0.01, 0.02, 0.03, 0.08 % for Alg-ph 1, Alg-ph 2, Alg-ph 3 and Alg-ph 4 respectively.  Figure 9:-EDX analysis of alginate and different phosphonate derivatives.

X-Ray diffraction analysis (XRD)
The XRD patterns of sodium alginate and its phosphonated derivatives were shown in Figure 10. Sodium alginate is usually crystalline due to the strong interaction between the alginate chains through intermolecular hydrogen bonding of hydroxyl and carboxylic groups [27]. Three diffraction peaks at 2θ values 13.5º, 22º and 39º were observed for sodium alginate due to the reflection of their (110) plane from poly guluronate unit, (200) plane from poly Mannuronate and the other from amorphous halo [28].
functionalization of alginate with phosphonic groups via epichorhydrane exhibit distortion of its crystal form.