- Email: [email protected]
The effect of gamma irradiation on physical, thermal and antioxidant properties of kraft lignin
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 6 2 1 e6 2 9
Available online at www.sciencedirect.com
H O S T E D BY
ScienceDirect
Journal of Radiation Research and Applied Sciences journal homepage: http://www.elsevier.com/locate/jrras
The effect of gamma irradiation on physical, thermal and antioxidant properties of kraft lignin Nakka Rajeswara Rao a,*, Thummu Venkatappa Rao a, S.V.S. Ramana Reddy a, Buddhiraju Sanjeeva Rao b a b
Department of Physics, National Institute of Technology, Warangal, 506004, India Department of Physics, Govt. Degree College, Mulugu, Warangal, India
article info
abstract
Article history:
Physico-chemical, thermal and antioxidant properties of lignin irradiated with gamma
Received 2 February 2015
source of doses 30, 60 and 90 kGy have been investigated by Electron spin resonance (ESR),
Received in revised form
Fourier transform infrared, Differential scanning calorimetry, X-ray diffraction, Scanning
5 July 2015
electron microscope (SEM) and spectrophotometer techniques. ESR studies confirm the
Accepted 21 July 2015
presence of poly-conjugated radicals in unirradiated lignin, whereas irradiated lignin
Available online 2 August 2015
possess as both poly-conjugated and peroxy radicals. The peroxy radicals decay near the glass transition point on thermal heating while poly-conjugated radicals are stable even at
Keywords:
a high temperature of 450 K. The absorption band at 1615 cm1 reveals the presence of
Gamma irradiation
conjugated structures whose concentration increases with the dose of irradiation. Upon
Poly-conjugate radicals
irradiation, an increase in glass transition temperature as well as amorphous nature of
Glass transition temperature
lignin was observed. SEM micrographs depicts that particle size was considerably
Antioxidant activity
decreased on irradiation. Antioxidant studies show that the irradiated lignin with decreased particle size and high P-conjugation exhibit high antioxidant activity compared to the unirradiated lignin. Copyright © 2015, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1.
Introduction
Lignin is a tri-dimensional phenolic polymer built from phenyl-propane units linked together by different bonds and one of the most abundant biopolymers on Earth. Lignin is one of the major polymeric components found in the cell walls of plants along with the other two major components, cellulose and hemicellulose. Lignin forms a highly efficient composite that is synthesized entirely from carbon, oxygen, hydrogen and energy from the Sun.
There are several factors restricting the use of lignin, namely: a non-uniform structure and unique chemical reactivity. A few decades back, lignin was treated as an industrial waste or byproduct which was left unused. Later, it gained interest as a source of fuel due to its enormous amount available as a byproduct of the pulp and paper industry. In fact, due to its non-toxic nature, renewability and extreme versatility, lignin applications have been increasing in industry. As a neutralizer or inhibitor, lignin is an effective free radical scavenger, which can reduce oxygen radicals and
* Corresponding author. E-mail address: [email protected] (N. Rajeswara Rao). Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications. http://dx.doi.org/10.1016/j.jrras.2015.07.003 1687-8507/Copyright © 2015, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
622
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 6 2 1 e6 2 9
stabilize oxidation reactions (Gul, Farhan, Anand, Qureshi, & Irfan, 2013; Yang et al., 2010). The radiation induced modification of biopolymers is a feasible method either to produce materials or to tailor the properties according to usage. From the molecular point of view, irradiation was shown to generate free radicals on polymers and then induce various reactions like scission or crosslinking depending upon both the environmental conditions and the radiation dosage. Irradiation is a promising green tool in this respect and optimized/alternative methods to utilize irradiation to improve lignin as a precursor in a cost effective manner. The radioactive degradation of kraft lignin has been the subject of numerous investigations. Dry distillation is one of the important methods in industrial wood processing. In this method the wood is thermally heated or exposed to radiation under air free conditions. Dry distillation lead to the formation of liquid organic product (Metriveli et al., 2011). Radiation dry distillation (RDD) is more advantageous than thermal distillation as entire volume of material is exposed when compared to dry distillation. In thermal dry distillation the sample is heated from its periphery to center. RDD yields higher liquid products and many times less expensive. (Chulkov, Bludenko, & Ponomarev, 2007) have reported electron beam medicated dry distillation of lignin. During this process they have reported the formation of many organic products like methanol, ethanol, benzene and its derivatives, phenol and its derivatives etc. Usually aromatic polymers are more thermally/radiation resistant, as the energy is dissipated in aromatic groups (Alexander & Charlesby, 1955). One such material is phenyl formaldehyde. Since lignin also has characteristics of aromatic groups, blending of lignin with phenyl formaldehyde (PF) (Cazacu et al., 2004) is expected to improve much thermal stability. In this context, efforts have been made by researchers to blend lignin with natural fiber and synthetic thermoplastic materials (Rozman et al., 2000, Rozman, Tan, Kumar, & Abubakar, 2001) and PF (Khan, Ashraf, & Malhotra, 2004a, 2004b). (Tejado, Pena, Labidi, Echeverria, & Mondragon, 2007) have observed that the formulation developed PF-lignin composites have higher thermal decomposition temperature. Thermal mass loss was delayed due to incorporation of lignin resulting in resin with more thermal stability. Thermal properties of phenol formaldehyde adhesive and lignin substituted phenol formaldehyde adhesives have been investigated by (Khan et al., 2004a, 2004b) using Differential scanning calorimetry (DSC) and thermogravimetry. Incorporation of lignin has resulted improved adhesiveness and shear strength. The scope of the present study is as follows. Radicals produced on gamma irradiation of lignin are identified but their thermal stability has not been reported. Therefore the authors have recorded Electron spin resonance (ESR) spectra of gamma irradiated lignin from room temperature (RT) to higher temperature (473 K). Existence of poly conjugated structures in gamma irradiated lignin has not been proved. Fourier transform infrared (FTIR) spectra of irradiated and unirradiated lignin are compared to ascertain the formation of conjugated structures. The effect of thermal property, such as glass transition process, has not been explored. Therefore the authors have recorded DSC thermograms of unirradiated and
irradiated lignin. The effect of particle size on gamma irradiation was investigated by recording Scanning electron microscope (SEM) micrographs under different conditions. (Braca, Sortino, Politi, Morelli, & Mendez, 2002) have reported that phenolic model compounds recognized as indigenous to the lignin structure which possess significant antioxidant property. Structures with conjugated double bonds in propyl side chains have higher antioxidant activities compared to structures with saturated side chains or isolated double bonds. Lignin also contains ortho-distributed phenolic groups. In addition, ortho-methoxy groups might provide stabilization to the incipient phenoxy radical formed by resonance structures. (Qi et al., 2012) have investigated antioxidant properties of non-nano scale lignin and nano scale lignin. These authors have employed super critical anti-solvent (SAS) process to prepare nano scale lignin, which has high antioxidant property. These authors have characterized nano scale lignin by FTIR, X-ray diffraction (XRD) and SEM techniques. The nano scale lignin has high solubility when compared to non-nano scale lignin and possesses important aspects like high DPPH radical scavenging activity, superoxide radical scavenging activity (SRSA) and reducing power. These authors have proposed that due to decrease of particle size, rapid increase of dissolution rate was observed by nano scale lignin. With the increase of solubility, antioxidant properties of nano scale lignin were observed to increase. Therefore, nano scale lignin is superior to non-nano scale lignin in pharmaceutical and food industry applications. Lignin aromatic structures suggest that it is suited as a radical scavenger in existing commodity thermoplastics. Free radicals formed in plastic by irradiation with UV light which is the major causes of quick degradation of plastics, so kraft lignin can be substituted for more expensive UV stabilizers in polyethylene for minor effects on mechanical properties (Gosselink et al., 2004). There are limited numbers of papers dealing with gamma irradiation of lignin as a source for Antioxidant activity. This type of research is one of the first steps in modifying renewable lignin so that it is more consistent and reliable antioxidant for further applications. Antioxidant properties of lignin are reported to be particle size dependent. The nano scale lignin synthesized by SAS method is reported to possess more antioxidant properties than nonnano scale lignin. Solubility of nano scale lignin is increased many times than non-nano scale lignin which accounts for the improvement in the antioxidant properties. Radiation treatment of polymers results in decrease of particle size (Dizhbite et al., 2012). Therefore the authors have studied the antioxidant properties of irradiated lignin and found that there is an increase in antioxidant property.
2.
Materials & methods
2.1.
Irradiation & chemicals
Kraft lignin in the form of powder was purchased from Sigma Aldrich, USA with an average molecular weight (Mw) of 10,000 and pH value of 10.5. Three lignin sample packets were prepared for gamma irradiation with optimum radiation doses
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 6 2 1 e6 2 9
i.e. 30, 60 and 90 kGy. Gamma irradiation was executed using Gamma Chamber 900 (GC-900) BRIT, India. The calibrated 60Co based GC-900 with a central dose of 0.15 kGy/hr with different doses has been used in present study. The uncertainty in doses delivered to samples is ±5%, so the actual doses are 30 ± 1.5 kGy, 60 ± 3 kGy and 90 ± 4.5 kGy. The distribution of radiation dose is almost uniform and is positioned at the center of the chamber using a stand. The total dose given to the sample is controlled by time of irradiation. Chemicals like 1, 1-diphenyl-2-picrylhydrazyl (DPPH), L-ascorbic acid, trichloroacetic acid (TCA) and other reagents were purchased from HiMedia Laboratories to study their radical scavenging activities.
2.2.
Electron spin resonance
ESR spectra were recorded on JES-FA200 ESR spectrometer, JOEL at an operating frequency of 9.4 GHz (X-band) and 100 kHz modulation frequency at RT. The samples were accommodated in quartz tubes with a weight of 3 mg in order to get quantitative comparison of the ESR line intensities under different conditions. The spectrometer was equipped with a variable temperature facility, so that irradiated samples at RT could be annealed to higher temperatures to study thermal stabilities.
2.3.
Fourier transform infrared spectrometer
FTIR spectra of the sample pellet prepared by adding KBr were recorded on Perkin Elmer spectrometer that is used to characterize the chemical structure of lignin before and after irradiation.
2.4.
Differential scanning calorimeter
DSC measurements were performed on a DSC-TA Q10 model calorimeter to study the thermal properties. Approximately 4 mg of samples were sealed into the aluminum crucibles and heated from 50 to 400 C at a heating rate of 20 C/min in flushing Nitrogen gas. The thermograms were recorded at second heating in order to remove the thermal history of the sample.
2.5.
X-ray diffraction
VEGA 3 LMU. The micrographs were captured with a magnification of 100 .
2.7.
Antioxidant activity assays
Lignin antioxidant capacity was determined by using DPPH scavenging activity. The method was followed from (Gul et al., 2013) with minor modifications. The scavenging activity of lignin was measured in terms of hydrogen donating or radical scavenging ability using the stable radical DPPH (Braca et al., 2002). The absorbance of the prepared solution was measured at 517 nm after 30 min of incubation. Methanol (95%), DPPH solution and L-ascorbic acid were used as blank, reference and control respectively. The reducing power (RP) of the lignin was determined by the method of Qi et al. (2012) with L-ascorbic acid as control. The UV absorbance of the prepared solution was measured at 700 nm. The sample with higher UV absorbance has stronger RP (Yang et al., 2010). Water and L-ascorbic acid was used as solvent and positive control respectively.
DPPH Scavenging activity ð%Þ ¼
ðA AÞ 100 A
Where Ao is the absorbance of the control reaction (containing all reagents except the test sample) and A is the absorbance of the test sample. IC50 value (the concentration of the extracts required to scavenge 50% of radicals) was calculated for different radiation doses of lignin.
3.
Results and discussion
3.1. study
Identification and behavior of free radicals: ESR
3.1.1.
Dose dependent studies
ESR spectrum of unirradiated lignin is shown in (Fig. 1). The spectrum is a singlet with g-value of 2.0035 and it is assigned to P poly-conjugated radicals (Dizhbite et al., 2012; Kuzina, Brezgunov, Dubinskii, & Mikhailov, 2004). ESR spectra of lignin irradiated to 30, 60 and 90 kGy dose of radiation are as shown in (Fig. 2). The g-values of the radicals responsible for the spectra are between 2.002 and 2.004. The ESR spectrum is assigned to two types of radicals.
The X-ray diffractograms were recorded on Bruker D8 Advance X-ray diffractometer (XRD). The X-rays were produced using a sealed tube and the wavelength of X-ray was 0.154 nm (Cu Ka). The XRD pattern was recorded in the 2q range from 5 to 60 , at 0.2 steps and at a fixed counting time of 10sec. The X-rays were detected using a fast counting detector based on Silicon strip technology.
2.6.
Scanning electron microscope
The samples were fixed on a cylindrical microscope stub covered with carbon strip and surfaces of the sample were gold coated using a sputter (Polaron E5200) set at 25 mA for 60 s. The morphology was then examined under TESCAN
623
Fig. 1 e ESR spectrum of unirradiated lignin.
624
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 6 2 1 e6 2 9
Fig. 2 e Dose dependent ESR spectra of lignin irradiated to 30, 60 and 90 kGy of radiation dose.
Fig. 3 e ESR dose response (-) and Temperature response (⊠) of gamma irradiated lignin.
i. Peroxy radicals ii. Poly-conjugated radicals
3.1.2. Formation of these radicals can be explained as follows. Lignin contain phenols, alcohol groups and glycosidic, acetal oxygen groups. When lignin is irradiated with gamma rays, dissociation of OH groups and degradation occurs through the cleavage of CeOeC bond producing primary radicals. The primary radicals are supposed to be trapped in the frozen lignin matrix even at RT, as glass transition temperature (Tg) of the lignin is around 130 C. Most of the free radicals are inaccessible to oxygen and only some of them are converted to peroxy radicals under oxygenated conditions. Some of the primary free radicals undergo secondary process like dehydration and water elimination leading to the formation of double bonds or poly-conjugated structures (Ershov & Isakova, 1984). The peroxy radical in lignin is characterized by its g-value (g ¼ 2.0033) while g-value of poly conjugated radical is reported to be 2.0030 (Kuzina et al., 2004). The ESR signal of peroxy radicals decays at normal temperatures i.e. around RT on thermal heating; while poly-conjugated radicals are thermally stable even up to elevated temperatures of 420 K. Temperature dependent spectra of irradiated lignin supported this view. Free radicals produced on gamma irradiation of lignin gives ESR singlet spectra with little width and g-value. € radicals localization of unpaired electron is on In case of RO 2pz orbital of oxygen resulting in little g-anisotropy. In poly conjugated radicals the unpaired electron is delocalized over the carbon atom of conjugated systems. (Kuzina et al., 2004) have used D-band ESR spectroscopy to resolve the singlets and detected the existence of poly-conjugated radicals. Therefore overall ESR spectrum of irradiated lignin is a superposition of singlets arising due to the poly-conjugated radicals and peroxy radicals. Intensity of ESR spectra gradually increased with radiation dose which suggests the formation of more number of free radicals upon irradiation of lignin. The plot of ESR intensity against radiation dose is as shown in (Fig. 3).
Temperature dependent studies
Annealing experiments were conducted by recording ESR spectra of irradiated lignin at different temperatures which are shown in (Fig. 4). A singlet spectrum is of up to 333 K was observed, while a doublet is visible at 363 K. Beyond 363 K a singlet was observed and it continued to appear even up to a temperature of 450 K. Irradiated lignin may possess different types of radicals, which have different thermal stabilities. The poly-conjugate radicals have higher thermal stability when compared to others. At room temperature both peroxy radicals and poly conjugated radicals are trapped in the rigid matrix of lignin. As the temperature is increased, the radicals gain thermal energy and begin to interact with themselves or with other radicals. When the temperature of observation is nearer to transition point, the radical reactions are optimum (Bartos & Klimova, 1996). In irradiated lignin the doublet like structures appeared around 363 K and disappeared at 413 K i.e. around glass transition temperature of lignin. The decayed component is assigned to peroxy radical. Since the poly conjugated radical have high thermal stability, the singlet at 450 K corresponds to poly-conjugated radicals (Kuzina et al., 1993). A variation of ESR intensity against temperature is as shown in (Fig. 3). Non-linearity of the curve is an indication that heterogeneous free radicals are present in irradiated lignin.
3.2.
Chemical structure investigations: FTIR study
(Fig. 5) shows the infrared spectra of unirradiated and irradiated lignin. FTIR spectra reflect the change in the chemical structure of the lignin after irradiation. The absorption bands appearing at 3440 cm1, 2928e2937 cm1, 1715e1712 cm1, 1603e1598 cm1, 1506 cm1 and 1328 cm1 are attributed to the stretching vibration of hydroxyl group of phenolic and aliphatic hydroxyl groups, CH stretching in aromatic methylene/methoxy/methyl groups, unconjugated carbonyl (C]O) groups, ring stretching of aromatic groups, aromatic OH and
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 6 2 1 e6 2 9
625
Fig. 4 e Temperature dependent ESR spectra of lignin (90 kGy) annealed to 333 K, 363 K, 413 K and 453 K.
bending vibrations of OH groups respectively (Faix, 1992). The weak absorption band observed for unirradiated lignin at 1615 cm1 is assigned to conjugated structures (Bellamy, 1954; Socrates, 2001). On irradiation, the intensity of the 1615 cm1 absorption band increased with dose as shown in subplot of (Fig. 5). As this band is assigned to conjugated structures, enhancement in conjugation of lignin after irradiation is proposed.
3.3.
Thermal studies: DSC study
DSC thermograms of unirradiated and irradiated lignin are as shown in (Fig. 6). The thermograms contain a first order transition around 130 C and 2 s order transitions (exothermic
Fig. 5 e FTIR spectra of lignin unirradiated and irradiated to radiation dose of 30, 60 and 90 kGy. The inset picture depicting 1615 cm¡1 absorption band which shows an increase in conjugation on irradiation.
peaks) around 280 C and 370 C. The first order transition at 130 C is reported to be associated with the glass transition temperature of lignin (Bouajila, Dole, Joly, & Limare, 2006; Nada, Yondef, & Gohray, 2002). The first exothermic peak (Ep1) is assigned to be due to thermal induced chemical reactions (Haz, Jaslonsky, Orsagova, & Surina, 2013) and the second exothermic peak (Ep2) is assigned to combustion of polysaccharides (Reh, Kraperlin, & Lamperckf, 1986). Usually the exothermic transitions are may be due to chemical reactions or crystallization process. Since lignin is an irregular amorphous polymer, assignment of exothermic peak to crystallization process is not improbable; instead they are attributed to chemical process occurring in lignin on thermal
Fig. 6 e DSC thermograms of unirradiated and irradiated lignin of radiation doses 30, 60 and 90 kGy.
626
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 6 2 1 e6 2 9
Table 1 e Glass transition temperature (Tg) and exothermic peaks of unirradiated and irradiated lignin. Radiation dose (kGy)
0 30 60 90
Glass transition temperature (Tg) C 133.2 141.5 145.6 142.4
Exothermic peaks ( C) Ep1
Ep2
271 280 285 290
362.4 368.1 366.5 364.8
treatment. Variation of Tg and exothermic peaks temperature (Ep1 & Ep2) against gamma radiation dose are as listed in (Table 1). Glass transition temperature of lignin is increased from 130 C (unirradiated) to 142 C (irradiated) with the increase of radiation dose (from 0 to 90 kGy). Variation of Tg against radiation dose is as depicted in (Fig. 7). The increase of Tg with radiation dose is assigned to plasticization effect. Lignin contains phenyl propane groups which are connected by different linkages which act as plasticizers (Lisperguer, Perez, & Silvio, 2009). Due to gamma irradiation, the intermolecular interactions are disassociated, reducing the plasticization effect causing an increase in Tg on irradiation. With regard to variation of exothermic peaks temperature against radiation, both the peaks have suffered a temperature shift with the increase of radiation dose. Radiation exposure causes chain cleavages and decrease in molecular weight. As a result, exothermic peaks suffered a change in the value of temperature.
3.4.
crystalline structure of lignin is assumed to unaffected by irradiation and retains amorphous nature after irradiation also.
3.5.
Morphological studies: SEM study
The SEM micrographs of unirradiated and irradiated lignin are shown in (Fig. 9). Considering the morphology of lignin, the size of the particles and surface has significant difference after irradiation. The average size of particles in unirradiated lignin is around 100 mm (‘a’ of Fig. 9) and also the particles are smooth on surface and spherical in shape. On irradiation, the size of the particles are gradually reduced and at 90 kGy dose of radiation (‘d’ of Fig. 9) the average size of the particle is 60 mm and particles bear rough patches on the surface.
3.6. Radical scavenging activities of lignin by DPPH and reducing power DPPH assay provides the scope of antiradical activity of many plant extracts and foods which indicates the presence of phenolic and flavonoid compounds in sample extracts (RiceEvans, Miller, & Paganga, 1996). DPPH is a stable free radical in a methanolic solution with maximum absorption at 517 nm (Lu & Foo, 2001). (Fig. 10A) depicts the change in DPPH inhibition values of unirradiated and irradiated lignin of
Crystalline studies: XRD study
Effect of gamma irradiation on crystalline structure of lignin is investigated by recording the X-ray diffractograms before and after irradiation as shown in (Fig. 8). Both unirradiated and irradiated lignin shows a broad diffraction peak centered around 2q ¼ 13.08 (Qi et al., 2012). On irradiation, neither the peak position nor the peak intensities are affected. Therefore
Fig. 7 e Variation of glass transition temperature (Tg) of lignin with radiation dose.
Fig. 8 e XRD diffractograms of unirradiated and irradiated lignin of radiation doses 30, 60 and 90 kGy.
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 6 2 1 e6 2 9
627
Fig. 9 e SEM micrographs of unirradiated (a) and irradiated lignin of doses 30 (b), 60 (c) and 90 kGy (d).
various concentrations. The antioxidant activities of unirradiated and irradiated lignin were found to be concentration dependent and there is a notable change in the % inhibition values with the increase of radiation dose. The IC50 values of 0(unirradiated), 30, 60 and 90 kGy lignin are 211 ± 0.63, 166 ± 0.45, 153 ± 1.12 and 139 ± 0.83 mg/mL respectively. In reducing power assay, the presence of reductants
(antioxidants) in samples would result in the reduction of Fe3þ to Fe2 by donating an electron. Reducing power is associated with antioxidant activity and may serve as a significant reflection of the antioxidant activity (Hsu, Coupar, & Ng, 2006). From the (Fig. 10B), we can observe that UV absorbance value of lignin solution is concentrated dependent and increases with the dose of irradiation. At highest
Fig. 10 e DPPH radical scavenging capacity of unirradiated and irradiated lignin (A) and reducing power of unirradiated and irradiated lignin (B).
628
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 6 2 1 e6 2 9
dose, the UV absorption value of lignin solution is thrice the value of the unirradiated lignin. (Tatiana, Galina, Jurkjane, & Uldis, 2004) have studied the radical scavenging activity of lignins and observed some structural property relations. According to their studies, nonesterified OH groups, phenolic groups, hydroxyl groups and double bonds between the outer most carbon atoms in side chain increase the radical scavenging activity (RSA). On the other hand high molecular weight, heterogeneity in terms of component composition i.e. carbohydrate admixture and polydispersity decrease RSA. In the present study, unirradiated lignin poses many intermolecular chain interactions and high molecular weight when compared to irradiated lignin. Due to irradiation, decrease in molecular weight chains is the most obvious effect. Therefore increase of RSA in irradiated lignin is expected. Consequent from XRD study to an increase in amorphousity of lignin, the solvent can easily diffuse into lignin matrix thus increasing its solubility. Decrease in particle size is also a cause for increasing solubility. Dissolution rate of poorly soluble ingredients can be increased by decrease of particle size (Choi et al., 2004). According to Prandf equation, small particle size results in shorter diffusional distance. From SEM micrographs, it is observed that the destruction of lignin granule after irradiation may lead to increase of solubility.
4.
Conclusions
Anti-radical activity of lignin is found to be structure dependent. Since structural properties are radiation dependent, gamma irradiation effect in lignin have been investigated with spectroscopic, thermal and morphological techniques. Radical scavenging efficiency has considerably increased after irradiation. DPPH radical scavenging activity and reducing power assays confirm these aspects.
references
Alexander, P., & Charlesby, A. (1955). Radiation protection in copolymers of isobutylene and styrene. Proceedings of Royal Society, A230, 136e143. Bartos, J., & Klimova, M. (1996). Relationship between macroradical decay kinetics and alpha-segmental dynamics in glassy amorphous polymers. Journal of Polymer Science A: Polymer Chemistry, 34, 1741e1746. Bellamy, L. J. (1954). The infrared spectra of complex molecules. New York: John Wiley & Sons, Inc. Bouajila, J., Dole, P., Joly, C., & Limare, A. (2006). Some laws of a lignin plasticization. Journal of Applied Polymer Science, 102, 1445e1451. Braca, A., Sortino, C., Politi, M., Morelli, I., & Mendez, J. (2002). Antioxidant activity of flavonoids from Licania licaniaeflora. Journal of Ethnopharmacol, 79, 379e381. Cazacu, G., Pascu, C., Profire, L., Kowarskik, A. L., Mihaies, M., & Vasile, C. (2004). Lignin role in a complex polyolefin blend. Industrial Crops and Products, 20, 261e273. Choi, W. S., Kim, H. I., Kwak, S. S., Hang, Y. C., Chung, H. Y., Keiji, Y., et al. (2004). Amorphous ultrafine particle preparation for improvement of bioavailability of insoluble drugs: grinding
characteristics of fine grinding mills. Journal of Mineral process, 74S, S165eS172. Chulkov, V. N., Bludenko, A. V., & Ponomarev, A. V. (2007). Electron-beam mediated dry distillation of lignin. Short Communications Radiation Chemistry, 41, 470e472. Dizhbite, T., Ponomarneko, J., Andersone, A., Dobele, G., Lauberts, M., Krasilnikova, J., et al. (2012). Role of paramagnetic polyconjugated clusters in lignin antioxidant activity (in vitro). International conference on functional material & nanotechnologies, 38, 1e4. Ershov, B. G., & Isakova, O. V. (1984). Formation and thermal transformations of free radicals in gamma radiation of cellulose. Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, 6, 1276e1280. Faix, O. (1992). Fourier transformed infrared spectroscopy. In S. Y. Lin, & C. W. Dence (Eds.), Methods in lignin chemistry (pp. 458e464). Berlin-Heidelberg: Springer-Verlag. Gosselink, R. J. A., Snijder, M. B. H., Kranenbarg, A., Keijsers, E. R. P., de Jong, E., & Stigsson, L. L. (2004). Characterization and application of NovaFiber lignin. Industrial Crops and Products, 20, 191e203. Gul, Z. M., Farhan, A., Anand, K., Qureshi, A., & Irfan, G. (2013). Antioxidant and antiproliferative activities of Abrus precatorius leaf extracts - an in vitro study. BMC Complementary and Alternative Medicine, 13, 1e12. Haz, A., Jaslonsky, M., Orsagova, A., & Surina, I. (2013). Determination of temperature regions in thermal degradation of lignin. In 4th International Conference on renewable energy source, High Tatras, Slovak Republic. Hsu, B., Coupar, I. M., & Ng, K. (2006). Antioxidant activity of hot water extract from the fruit of the Doum palm, Hyphaene thebaica. Food Chemistry, 98, 317e328. Khan, M. A., Ashraf, S. M., & Malhotra, V. P. (2004a). Eucalyptus bark lignin substituted phenol formaldehyde adhesives: a study on optimization of reaction parameters and characterization. Journal of Applied Polymer Science, 92, 3514e3523. Khan, M. A., Ashraf, S. M., & Malhotra, V. P. (2004b). Studies on thermal characterization of lignin substituted phenol formaldehyde resin as wood adhesives. International Journal of Adhesion & Adhesives, 24, 485e493. Kuzina, S. I., Brezgunov, A. Y., Dubinskii, A. A., & Mikhailov, A. I. (2004). Free radicals in the photolysis and radiolysis of polymers: IV. Radicals in g - and UV-irradiated wood and lignin. Radiation chemistry, 38, 298e305. Kuzina, S. I., Demidov, S. V., Brezgunov, A. Y., Poluektiv, O. G., Grinberg, O. Y., Dubinski, A. A., et al. (1993). Study of freeradical centers in lignin with 20 mm band ESR spectroscopy. A Polymer Science, 35, 798e802. Lisperguer, J., Perez, P., & Silvio, U. (2009). Structure and thermal properties of lignins: characterization by infrared spectroscopy and differential scanning calorimetry. Journal of Chilean Chemical Society, 54, 460e463. Lu, Y., & Foo, L. Y. (2001). Antioxidant activities of polyphenols from sage (Salvia officinalis). Food Chemistry, 75, 197e202. Metriveli, A. K., Metriveli, P. K., Bludenko, A. V., Chulkov, V. N., Ponomarev, A. V., & Ershov, B. G. (2011). Radiation and postradiation distillation of biopolymers: lignin and chitin. High Energy Chemistry, 45, 470e474. Nada, A. M. A., Yondef, H. A., & Gohray, S. E. (2002). Thermal degradation of hydrolyzed and oxidized lignins. Journal of Thermal Analysis and Calorimetry, 68, 265e273. Qi, L., Minghua, Z., Yuangang, Z., Wenjun, L., Yang, L., Ying, Z., et al. (2012). Comparative antioxidant activity of nanoscale lignin prepared by a supercritical antisolvent (SAS) process with non-nanoscale lignin. Food Chemistry, 135, 63e67. Reh, U., Kraperlin, G., & Lamperckf, I. (1986). Use of differential scanning calorimetry for structural analysis of fungally
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 6 2 1 e6 2 9
degraded wood. Applied Environmental Microbiology, 52, 1101e1106. Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1996). Structureantioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine, 20, 933e956. Rozman, H. D., Tan, K. W., Kumar, R. N., & Abubakar, A. (2001). Preliminary studies on the use of modified ALCELL lignin as a coupling agent in the biofiber composites. Journal of Applied Polymer Science, 81, 1333e1340. Rozman, H. D., Tan, K. W., Kumar, R. N., Abubakar, A., Ishak, Z., & Ismail, H. (2000). The effect of lignin as a compatibilizer on the physical properties of coconut fiberepolypropylene composites. European polymer Journal, 36, 1483e1494.
629
Socrates, G. (2001). Infrared and Raman characteristic group frequencies (p. 68). Wiley. Tatiana, D., Galina, T., Jurkjane, V., & Uldis, V. (2004). Characterization of the radical scavenging activity of lignins ee natural antioxidants. Bioresource Technology, 95, 309e317. Tejado, A., Pena, C., Labidi, J., Echeverria, J. M., & Mondragon, I. (2007). Physico-chemical characterization of lignins from different sources for use in phenol-formaldehyde resin synthesis. Bioresource Technology, 98, 1655e1663. Yang, Q., Pan, X., Kong, W., Yang, H., Zhang, L., & Zhang, Y. (2010). Antioxidant activities of malt extract from barley (Hordeum vulgare L.) toward various oxidative stress in vitro and in vivo. Food Chemistry, 118, 84e89.
Available online at www.sciencedirect.com
H O S T E D BY
ScienceDirect
Journal of Radiation Research and Applied Sciences journal homepage: http://www.elsevier.com/locate/jrras
The effect of gamma irradiation on physical, thermal and antioxidant properties of kraft lignin Nakka Rajeswara Rao a,*, Thummu Venkatappa Rao a, S.V.S. Ramana Reddy a, Buddhiraju Sanjeeva Rao b a b
Department of Physics, National Institute of Technology, Warangal, 506004, India Department of Physics, Govt. Degree College, Mulugu, Warangal, India
article info
abstract
Article history:
Physico-chemical, thermal and antioxidant properties of lignin irradiated with gamma
Received 2 February 2015
source of doses 30, 60 and 90 kGy have been investigated by Electron spin resonance (ESR),
Received in revised form
Fourier transform infrared, Differential scanning calorimetry, X-ray diffraction, Scanning
5 July 2015
electron microscope (SEM) and spectrophotometer techniques. ESR studies confirm the
Accepted 21 July 2015
presence of poly-conjugated radicals in unirradiated lignin, whereas irradiated lignin
Available online 2 August 2015
possess as both poly-conjugated and peroxy radicals. The peroxy radicals decay near the glass transition point on thermal heating while poly-conjugated radicals are stable even at
Keywords:
a high temperature of 450 K. The absorption band at 1615 cm1 reveals the presence of
Gamma irradiation
conjugated structures whose concentration increases with the dose of irradiation. Upon
Poly-conjugate radicals
irradiation, an increase in glass transition temperature as well as amorphous nature of
Glass transition temperature
lignin was observed. SEM micrographs depicts that particle size was considerably
Antioxidant activity
decreased on irradiation. Antioxidant studies show that the irradiated lignin with decreased particle size and high P-conjugation exhibit high antioxidant activity compared to the unirradiated lignin. Copyright © 2015, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1.
Introduction
Lignin is a tri-dimensional phenolic polymer built from phenyl-propane units linked together by different bonds and one of the most abundant biopolymers on Earth. Lignin is one of the major polymeric components found in the cell walls of plants along with the other two major components, cellulose and hemicellulose. Lignin forms a highly efficient composite that is synthesized entirely from carbon, oxygen, hydrogen and energy from the Sun.
There are several factors restricting the use of lignin, namely: a non-uniform structure and unique chemical reactivity. A few decades back, lignin was treated as an industrial waste or byproduct which was left unused. Later, it gained interest as a source of fuel due to its enormous amount available as a byproduct of the pulp and paper industry. In fact, due to its non-toxic nature, renewability and extreme versatility, lignin applications have been increasing in industry. As a neutralizer or inhibitor, lignin is an effective free radical scavenger, which can reduce oxygen radicals and
* Corresponding author. E-mail address: [email protected] (N. Rajeswara Rao). Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications. http://dx.doi.org/10.1016/j.jrras.2015.07.003 1687-8507/Copyright © 2015, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
622
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 6 2 1 e6 2 9
stabilize oxidation reactions (Gul, Farhan, Anand, Qureshi, & Irfan, 2013; Yang et al., 2010). The radiation induced modification of biopolymers is a feasible method either to produce materials or to tailor the properties according to usage. From the molecular point of view, irradiation was shown to generate free radicals on polymers and then induce various reactions like scission or crosslinking depending upon both the environmental conditions and the radiation dosage. Irradiation is a promising green tool in this respect and optimized/alternative methods to utilize irradiation to improve lignin as a precursor in a cost effective manner. The radioactive degradation of kraft lignin has been the subject of numerous investigations. Dry distillation is one of the important methods in industrial wood processing. In this method the wood is thermally heated or exposed to radiation under air free conditions. Dry distillation lead to the formation of liquid organic product (Metriveli et al., 2011). Radiation dry distillation (RDD) is more advantageous than thermal distillation as entire volume of material is exposed when compared to dry distillation. In thermal dry distillation the sample is heated from its periphery to center. RDD yields higher liquid products and many times less expensive. (Chulkov, Bludenko, & Ponomarev, 2007) have reported electron beam medicated dry distillation of lignin. During this process they have reported the formation of many organic products like methanol, ethanol, benzene and its derivatives, phenol and its derivatives etc. Usually aromatic polymers are more thermally/radiation resistant, as the energy is dissipated in aromatic groups (Alexander & Charlesby, 1955). One such material is phenyl formaldehyde. Since lignin also has characteristics of aromatic groups, blending of lignin with phenyl formaldehyde (PF) (Cazacu et al., 2004) is expected to improve much thermal stability. In this context, efforts have been made by researchers to blend lignin with natural fiber and synthetic thermoplastic materials (Rozman et al., 2000, Rozman, Tan, Kumar, & Abubakar, 2001) and PF (Khan, Ashraf, & Malhotra, 2004a, 2004b). (Tejado, Pena, Labidi, Echeverria, & Mondragon, 2007) have observed that the formulation developed PF-lignin composites have higher thermal decomposition temperature. Thermal mass loss was delayed due to incorporation of lignin resulting in resin with more thermal stability. Thermal properties of phenol formaldehyde adhesive and lignin substituted phenol formaldehyde adhesives have been investigated by (Khan et al., 2004a, 2004b) using Differential scanning calorimetry (DSC) and thermogravimetry. Incorporation of lignin has resulted improved adhesiveness and shear strength. The scope of the present study is as follows. Radicals produced on gamma irradiation of lignin are identified but their thermal stability has not been reported. Therefore the authors have recorded Electron spin resonance (ESR) spectra of gamma irradiated lignin from room temperature (RT) to higher temperature (473 K). Existence of poly conjugated structures in gamma irradiated lignin has not been proved. Fourier transform infrared (FTIR) spectra of irradiated and unirradiated lignin are compared to ascertain the formation of conjugated structures. The effect of thermal property, such as glass transition process, has not been explored. Therefore the authors have recorded DSC thermograms of unirradiated and
irradiated lignin. The effect of particle size on gamma irradiation was investigated by recording Scanning electron microscope (SEM) micrographs under different conditions. (Braca, Sortino, Politi, Morelli, & Mendez, 2002) have reported that phenolic model compounds recognized as indigenous to the lignin structure which possess significant antioxidant property. Structures with conjugated double bonds in propyl side chains have higher antioxidant activities compared to structures with saturated side chains or isolated double bonds. Lignin also contains ortho-distributed phenolic groups. In addition, ortho-methoxy groups might provide stabilization to the incipient phenoxy radical formed by resonance structures. (Qi et al., 2012) have investigated antioxidant properties of non-nano scale lignin and nano scale lignin. These authors have employed super critical anti-solvent (SAS) process to prepare nano scale lignin, which has high antioxidant property. These authors have characterized nano scale lignin by FTIR, X-ray diffraction (XRD) and SEM techniques. The nano scale lignin has high solubility when compared to non-nano scale lignin and possesses important aspects like high DPPH radical scavenging activity, superoxide radical scavenging activity (SRSA) and reducing power. These authors have proposed that due to decrease of particle size, rapid increase of dissolution rate was observed by nano scale lignin. With the increase of solubility, antioxidant properties of nano scale lignin were observed to increase. Therefore, nano scale lignin is superior to non-nano scale lignin in pharmaceutical and food industry applications. Lignin aromatic structures suggest that it is suited as a radical scavenger in existing commodity thermoplastics. Free radicals formed in plastic by irradiation with UV light which is the major causes of quick degradation of plastics, so kraft lignin can be substituted for more expensive UV stabilizers in polyethylene for minor effects on mechanical properties (Gosselink et al., 2004). There are limited numbers of papers dealing with gamma irradiation of lignin as a source for Antioxidant activity. This type of research is one of the first steps in modifying renewable lignin so that it is more consistent and reliable antioxidant for further applications. Antioxidant properties of lignin are reported to be particle size dependent. The nano scale lignin synthesized by SAS method is reported to possess more antioxidant properties than nonnano scale lignin. Solubility of nano scale lignin is increased many times than non-nano scale lignin which accounts for the improvement in the antioxidant properties. Radiation treatment of polymers results in decrease of particle size (Dizhbite et al., 2012). Therefore the authors have studied the antioxidant properties of irradiated lignin and found that there is an increase in antioxidant property.
2.
Materials & methods
2.1.
Irradiation & chemicals
Kraft lignin in the form of powder was purchased from Sigma Aldrich, USA with an average molecular weight (Mw) of 10,000 and pH value of 10.5. Three lignin sample packets were prepared for gamma irradiation with optimum radiation doses
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 6 2 1 e6 2 9
i.e. 30, 60 and 90 kGy. Gamma irradiation was executed using Gamma Chamber 900 (GC-900) BRIT, India. The calibrated 60Co based GC-900 with a central dose of 0.15 kGy/hr with different doses has been used in present study. The uncertainty in doses delivered to samples is ±5%, so the actual doses are 30 ± 1.5 kGy, 60 ± 3 kGy and 90 ± 4.5 kGy. The distribution of radiation dose is almost uniform and is positioned at the center of the chamber using a stand. The total dose given to the sample is controlled by time of irradiation. Chemicals like 1, 1-diphenyl-2-picrylhydrazyl (DPPH), L-ascorbic acid, trichloroacetic acid (TCA) and other reagents were purchased from HiMedia Laboratories to study their radical scavenging activities.
2.2.
Electron spin resonance
ESR spectra were recorded on JES-FA200 ESR spectrometer, JOEL at an operating frequency of 9.4 GHz (X-band) and 100 kHz modulation frequency at RT. The samples were accommodated in quartz tubes with a weight of 3 mg in order to get quantitative comparison of the ESR line intensities under different conditions. The spectrometer was equipped with a variable temperature facility, so that irradiated samples at RT could be annealed to higher temperatures to study thermal stabilities.
2.3.
Fourier transform infrared spectrometer
FTIR spectra of the sample pellet prepared by adding KBr were recorded on Perkin Elmer spectrometer that is used to characterize the chemical structure of lignin before and after irradiation.
2.4.
Differential scanning calorimeter
DSC measurements were performed on a DSC-TA Q10 model calorimeter to study the thermal properties. Approximately 4 mg of samples were sealed into the aluminum crucibles and heated from 50 to 400 C at a heating rate of 20 C/min in flushing Nitrogen gas. The thermograms were recorded at second heating in order to remove the thermal history of the sample.
2.5.
X-ray diffraction
VEGA 3 LMU. The micrographs were captured with a magnification of 100 .
2.7.
Antioxidant activity assays
Lignin antioxidant capacity was determined by using DPPH scavenging activity. The method was followed from (Gul et al., 2013) with minor modifications. The scavenging activity of lignin was measured in terms of hydrogen donating or radical scavenging ability using the stable radical DPPH (Braca et al., 2002). The absorbance of the prepared solution was measured at 517 nm after 30 min of incubation. Methanol (95%), DPPH solution and L-ascorbic acid were used as blank, reference and control respectively. The reducing power (RP) of the lignin was determined by the method of Qi et al. (2012) with L-ascorbic acid as control. The UV absorbance of the prepared solution was measured at 700 nm. The sample with higher UV absorbance has stronger RP (Yang et al., 2010). Water and L-ascorbic acid was used as solvent and positive control respectively.
DPPH Scavenging activity ð%Þ ¼
ðA AÞ 100 A
Where Ao is the absorbance of the control reaction (containing all reagents except the test sample) and A is the absorbance of the test sample. IC50 value (the concentration of the extracts required to scavenge 50% of radicals) was calculated for different radiation doses of lignin.
3.
Results and discussion
3.1. study
Identification and behavior of free radicals: ESR
3.1.1.
Dose dependent studies
ESR spectrum of unirradiated lignin is shown in (Fig. 1). The spectrum is a singlet with g-value of 2.0035 and it is assigned to P poly-conjugated radicals (Dizhbite et al., 2012; Kuzina, Brezgunov, Dubinskii, & Mikhailov, 2004). ESR spectra of lignin irradiated to 30, 60 and 90 kGy dose of radiation are as shown in (Fig. 2). The g-values of the radicals responsible for the spectra are between 2.002 and 2.004. The ESR spectrum is assigned to two types of radicals.
The X-ray diffractograms were recorded on Bruker D8 Advance X-ray diffractometer (XRD). The X-rays were produced using a sealed tube and the wavelength of X-ray was 0.154 nm (Cu Ka). The XRD pattern was recorded in the 2q range from 5 to 60 , at 0.2 steps and at a fixed counting time of 10sec. The X-rays were detected using a fast counting detector based on Silicon strip technology.
2.6.
Scanning electron microscope
The samples were fixed on a cylindrical microscope stub covered with carbon strip and surfaces of the sample were gold coated using a sputter (Polaron E5200) set at 25 mA for 60 s. The morphology was then examined under TESCAN
623
Fig. 1 e ESR spectrum of unirradiated lignin.
624
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 6 2 1 e6 2 9
Fig. 2 e Dose dependent ESR spectra of lignin irradiated to 30, 60 and 90 kGy of radiation dose.
Fig. 3 e ESR dose response (-) and Temperature response (⊠) of gamma irradiated lignin.
i. Peroxy radicals ii. Poly-conjugated radicals
3.1.2. Formation of these radicals can be explained as follows. Lignin contain phenols, alcohol groups and glycosidic, acetal oxygen groups. When lignin is irradiated with gamma rays, dissociation of OH groups and degradation occurs through the cleavage of CeOeC bond producing primary radicals. The primary radicals are supposed to be trapped in the frozen lignin matrix even at RT, as glass transition temperature (Tg) of the lignin is around 130 C. Most of the free radicals are inaccessible to oxygen and only some of them are converted to peroxy radicals under oxygenated conditions. Some of the primary free radicals undergo secondary process like dehydration and water elimination leading to the formation of double bonds or poly-conjugated structures (Ershov & Isakova, 1984). The peroxy radical in lignin is characterized by its g-value (g ¼ 2.0033) while g-value of poly conjugated radical is reported to be 2.0030 (Kuzina et al., 2004). The ESR signal of peroxy radicals decays at normal temperatures i.e. around RT on thermal heating; while poly-conjugated radicals are thermally stable even up to elevated temperatures of 420 K. Temperature dependent spectra of irradiated lignin supported this view. Free radicals produced on gamma irradiation of lignin gives ESR singlet spectra with little width and g-value. € radicals localization of unpaired electron is on In case of RO 2pz orbital of oxygen resulting in little g-anisotropy. In poly conjugated radicals the unpaired electron is delocalized over the carbon atom of conjugated systems. (Kuzina et al., 2004) have used D-band ESR spectroscopy to resolve the singlets and detected the existence of poly-conjugated radicals. Therefore overall ESR spectrum of irradiated lignin is a superposition of singlets arising due to the poly-conjugated radicals and peroxy radicals. Intensity of ESR spectra gradually increased with radiation dose which suggests the formation of more number of free radicals upon irradiation of lignin. The plot of ESR intensity against radiation dose is as shown in (Fig. 3).
Temperature dependent studies
Annealing experiments were conducted by recording ESR spectra of irradiated lignin at different temperatures which are shown in (Fig. 4). A singlet spectrum is of up to 333 K was observed, while a doublet is visible at 363 K. Beyond 363 K a singlet was observed and it continued to appear even up to a temperature of 450 K. Irradiated lignin may possess different types of radicals, which have different thermal stabilities. The poly-conjugate radicals have higher thermal stability when compared to others. At room temperature both peroxy radicals and poly conjugated radicals are trapped in the rigid matrix of lignin. As the temperature is increased, the radicals gain thermal energy and begin to interact with themselves or with other radicals. When the temperature of observation is nearer to transition point, the radical reactions are optimum (Bartos & Klimova, 1996). In irradiated lignin the doublet like structures appeared around 363 K and disappeared at 413 K i.e. around glass transition temperature of lignin. The decayed component is assigned to peroxy radical. Since the poly conjugated radical have high thermal stability, the singlet at 450 K corresponds to poly-conjugated radicals (Kuzina et al., 1993). A variation of ESR intensity against temperature is as shown in (Fig. 3). Non-linearity of the curve is an indication that heterogeneous free radicals are present in irradiated lignin.
3.2.
Chemical structure investigations: FTIR study
(Fig. 5) shows the infrared spectra of unirradiated and irradiated lignin. FTIR spectra reflect the change in the chemical structure of the lignin after irradiation. The absorption bands appearing at 3440 cm1, 2928e2937 cm1, 1715e1712 cm1, 1603e1598 cm1, 1506 cm1 and 1328 cm1 are attributed to the stretching vibration of hydroxyl group of phenolic and aliphatic hydroxyl groups, CH stretching in aromatic methylene/methoxy/methyl groups, unconjugated carbonyl (C]O) groups, ring stretching of aromatic groups, aromatic OH and
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 6 2 1 e6 2 9
625
Fig. 4 e Temperature dependent ESR spectra of lignin (90 kGy) annealed to 333 K, 363 K, 413 K and 453 K.
bending vibrations of OH groups respectively (Faix, 1992). The weak absorption band observed for unirradiated lignin at 1615 cm1 is assigned to conjugated structures (Bellamy, 1954; Socrates, 2001). On irradiation, the intensity of the 1615 cm1 absorption band increased with dose as shown in subplot of (Fig. 5). As this band is assigned to conjugated structures, enhancement in conjugation of lignin after irradiation is proposed.
3.3.
Thermal studies: DSC study
DSC thermograms of unirradiated and irradiated lignin are as shown in (Fig. 6). The thermograms contain a first order transition around 130 C and 2 s order transitions (exothermic
Fig. 5 e FTIR spectra of lignin unirradiated and irradiated to radiation dose of 30, 60 and 90 kGy. The inset picture depicting 1615 cm¡1 absorption band which shows an increase in conjugation on irradiation.
peaks) around 280 C and 370 C. The first order transition at 130 C is reported to be associated with the glass transition temperature of lignin (Bouajila, Dole, Joly, & Limare, 2006; Nada, Yondef, & Gohray, 2002). The first exothermic peak (Ep1) is assigned to be due to thermal induced chemical reactions (Haz, Jaslonsky, Orsagova, & Surina, 2013) and the second exothermic peak (Ep2) is assigned to combustion of polysaccharides (Reh, Kraperlin, & Lamperckf, 1986). Usually the exothermic transitions are may be due to chemical reactions or crystallization process. Since lignin is an irregular amorphous polymer, assignment of exothermic peak to crystallization process is not improbable; instead they are attributed to chemical process occurring in lignin on thermal
Fig. 6 e DSC thermograms of unirradiated and irradiated lignin of radiation doses 30, 60 and 90 kGy.
626
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 6 2 1 e6 2 9
Table 1 e Glass transition temperature (Tg) and exothermic peaks of unirradiated and irradiated lignin. Radiation dose (kGy)
0 30 60 90
Glass transition temperature (Tg) C 133.2 141.5 145.6 142.4
Exothermic peaks ( C) Ep1
Ep2
271 280 285 290
362.4 368.1 366.5 364.8
treatment. Variation of Tg and exothermic peaks temperature (Ep1 & Ep2) against gamma radiation dose are as listed in (Table 1). Glass transition temperature of lignin is increased from 130 C (unirradiated) to 142 C (irradiated) with the increase of radiation dose (from 0 to 90 kGy). Variation of Tg against radiation dose is as depicted in (Fig. 7). The increase of Tg with radiation dose is assigned to plasticization effect. Lignin contains phenyl propane groups which are connected by different linkages which act as plasticizers (Lisperguer, Perez, & Silvio, 2009). Due to gamma irradiation, the intermolecular interactions are disassociated, reducing the plasticization effect causing an increase in Tg on irradiation. With regard to variation of exothermic peaks temperature against radiation, both the peaks have suffered a temperature shift with the increase of radiation dose. Radiation exposure causes chain cleavages and decrease in molecular weight. As a result, exothermic peaks suffered a change in the value of temperature.
3.4.
crystalline structure of lignin is assumed to unaffected by irradiation and retains amorphous nature after irradiation also.
3.5.
Morphological studies: SEM study
The SEM micrographs of unirradiated and irradiated lignin are shown in (Fig. 9). Considering the morphology of lignin, the size of the particles and surface has significant difference after irradiation. The average size of particles in unirradiated lignin is around 100 mm (‘a’ of Fig. 9) and also the particles are smooth on surface and spherical in shape. On irradiation, the size of the particles are gradually reduced and at 90 kGy dose of radiation (‘d’ of Fig. 9) the average size of the particle is 60 mm and particles bear rough patches on the surface.
3.6. Radical scavenging activities of lignin by DPPH and reducing power DPPH assay provides the scope of antiradical activity of many plant extracts and foods which indicates the presence of phenolic and flavonoid compounds in sample extracts (RiceEvans, Miller, & Paganga, 1996). DPPH is a stable free radical in a methanolic solution with maximum absorption at 517 nm (Lu & Foo, 2001). (Fig. 10A) depicts the change in DPPH inhibition values of unirradiated and irradiated lignin of
Crystalline studies: XRD study
Effect of gamma irradiation on crystalline structure of lignin is investigated by recording the X-ray diffractograms before and after irradiation as shown in (Fig. 8). Both unirradiated and irradiated lignin shows a broad diffraction peak centered around 2q ¼ 13.08 (Qi et al., 2012). On irradiation, neither the peak position nor the peak intensities are affected. Therefore
Fig. 7 e Variation of glass transition temperature (Tg) of lignin with radiation dose.
Fig. 8 e XRD diffractograms of unirradiated and irradiated lignin of radiation doses 30, 60 and 90 kGy.
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 6 2 1 e6 2 9
627
Fig. 9 e SEM micrographs of unirradiated (a) and irradiated lignin of doses 30 (b), 60 (c) and 90 kGy (d).
various concentrations. The antioxidant activities of unirradiated and irradiated lignin were found to be concentration dependent and there is a notable change in the % inhibition values with the increase of radiation dose. The IC50 values of 0(unirradiated), 30, 60 and 90 kGy lignin are 211 ± 0.63, 166 ± 0.45, 153 ± 1.12 and 139 ± 0.83 mg/mL respectively. In reducing power assay, the presence of reductants
(antioxidants) in samples would result in the reduction of Fe3þ to Fe2 by donating an electron. Reducing power is associated with antioxidant activity and may serve as a significant reflection of the antioxidant activity (Hsu, Coupar, & Ng, 2006). From the (Fig. 10B), we can observe that UV absorbance value of lignin solution is concentrated dependent and increases with the dose of irradiation. At highest
Fig. 10 e DPPH radical scavenging capacity of unirradiated and irradiated lignin (A) and reducing power of unirradiated and irradiated lignin (B).
628
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 6 2 1 e6 2 9
dose, the UV absorption value of lignin solution is thrice the value of the unirradiated lignin. (Tatiana, Galina, Jurkjane, & Uldis, 2004) have studied the radical scavenging activity of lignins and observed some structural property relations. According to their studies, nonesterified OH groups, phenolic groups, hydroxyl groups and double bonds between the outer most carbon atoms in side chain increase the radical scavenging activity (RSA). On the other hand high molecular weight, heterogeneity in terms of component composition i.e. carbohydrate admixture and polydispersity decrease RSA. In the present study, unirradiated lignin poses many intermolecular chain interactions and high molecular weight when compared to irradiated lignin. Due to irradiation, decrease in molecular weight chains is the most obvious effect. Therefore increase of RSA in irradiated lignin is expected. Consequent from XRD study to an increase in amorphousity of lignin, the solvent can easily diffuse into lignin matrix thus increasing its solubility. Decrease in particle size is also a cause for increasing solubility. Dissolution rate of poorly soluble ingredients can be increased by decrease of particle size (Choi et al., 2004). According to Prandf equation, small particle size results in shorter diffusional distance. From SEM micrographs, it is observed that the destruction of lignin granule after irradiation may lead to increase of solubility.
4.
Conclusions
Anti-radical activity of lignin is found to be structure dependent. Since structural properties are radiation dependent, gamma irradiation effect in lignin have been investigated with spectroscopic, thermal and morphological techniques. Radical scavenging efficiency has considerably increased after irradiation. DPPH radical scavenging activity and reducing power assays confirm these aspects.
references
Alexander, P., & Charlesby, A. (1955). Radiation protection in copolymers of isobutylene and styrene. Proceedings of Royal Society, A230, 136e143. Bartos, J., & Klimova, M. (1996). Relationship between macroradical decay kinetics and alpha-segmental dynamics in glassy amorphous polymers. Journal of Polymer Science A: Polymer Chemistry, 34, 1741e1746. Bellamy, L. J. (1954). The infrared spectra of complex molecules. New York: John Wiley & Sons, Inc. Bouajila, J., Dole, P., Joly, C., & Limare, A. (2006). Some laws of a lignin plasticization. Journal of Applied Polymer Science, 102, 1445e1451. Braca, A., Sortino, C., Politi, M., Morelli, I., & Mendez, J. (2002). Antioxidant activity of flavonoids from Licania licaniaeflora. Journal of Ethnopharmacol, 79, 379e381. Cazacu, G., Pascu, C., Profire, L., Kowarskik, A. L., Mihaies, M., & Vasile, C. (2004). Lignin role in a complex polyolefin blend. Industrial Crops and Products, 20, 261e273. Choi, W. S., Kim, H. I., Kwak, S. S., Hang, Y. C., Chung, H. Y., Keiji, Y., et al. (2004). Amorphous ultrafine particle preparation for improvement of bioavailability of insoluble drugs: grinding
characteristics of fine grinding mills. Journal of Mineral process, 74S, S165eS172. Chulkov, V. N., Bludenko, A. V., & Ponomarev, A. V. (2007). Electron-beam mediated dry distillation of lignin. Short Communications Radiation Chemistry, 41, 470e472. Dizhbite, T., Ponomarneko, J., Andersone, A., Dobele, G., Lauberts, M., Krasilnikova, J., et al. (2012). Role of paramagnetic polyconjugated clusters in lignin antioxidant activity (in vitro). International conference on functional material & nanotechnologies, 38, 1e4. Ershov, B. G., & Isakova, O. V. (1984). Formation and thermal transformations of free radicals in gamma radiation of cellulose. Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, 6, 1276e1280. Faix, O. (1992). Fourier transformed infrared spectroscopy. In S. Y. Lin, & C. W. Dence (Eds.), Methods in lignin chemistry (pp. 458e464). Berlin-Heidelberg: Springer-Verlag. Gosselink, R. J. A., Snijder, M. B. H., Kranenbarg, A., Keijsers, E. R. P., de Jong, E., & Stigsson, L. L. (2004). Characterization and application of NovaFiber lignin. Industrial Crops and Products, 20, 191e203. Gul, Z. M., Farhan, A., Anand, K., Qureshi, A., & Irfan, G. (2013). Antioxidant and antiproliferative activities of Abrus precatorius leaf extracts - an in vitro study. BMC Complementary and Alternative Medicine, 13, 1e12. Haz, A., Jaslonsky, M., Orsagova, A., & Surina, I. (2013). Determination of temperature regions in thermal degradation of lignin. In 4th International Conference on renewable energy source, High Tatras, Slovak Republic. Hsu, B., Coupar, I. M., & Ng, K. (2006). Antioxidant activity of hot water extract from the fruit of the Doum palm, Hyphaene thebaica. Food Chemistry, 98, 317e328. Khan, M. A., Ashraf, S. M., & Malhotra, V. P. (2004a). Eucalyptus bark lignin substituted phenol formaldehyde adhesives: a study on optimization of reaction parameters and characterization. Journal of Applied Polymer Science, 92, 3514e3523. Khan, M. A., Ashraf, S. M., & Malhotra, V. P. (2004b). Studies on thermal characterization of lignin substituted phenol formaldehyde resin as wood adhesives. International Journal of Adhesion & Adhesives, 24, 485e493. Kuzina, S. I., Brezgunov, A. Y., Dubinskii, A. A., & Mikhailov, A. I. (2004). Free radicals in the photolysis and radiolysis of polymers: IV. Radicals in g - and UV-irradiated wood and lignin. Radiation chemistry, 38, 298e305. Kuzina, S. I., Demidov, S. V., Brezgunov, A. Y., Poluektiv, O. G., Grinberg, O. Y., Dubinski, A. A., et al. (1993). Study of freeradical centers in lignin with 20 mm band ESR spectroscopy. A Polymer Science, 35, 798e802. Lisperguer, J., Perez, P., & Silvio, U. (2009). Structure and thermal properties of lignins: characterization by infrared spectroscopy and differential scanning calorimetry. Journal of Chilean Chemical Society, 54, 460e463. Lu, Y., & Foo, L. Y. (2001). Antioxidant activities of polyphenols from sage (Salvia officinalis). Food Chemistry, 75, 197e202. Metriveli, A. K., Metriveli, P. K., Bludenko, A. V., Chulkov, V. N., Ponomarev, A. V., & Ershov, B. G. (2011). Radiation and postradiation distillation of biopolymers: lignin and chitin. High Energy Chemistry, 45, 470e474. Nada, A. M. A., Yondef, H. A., & Gohray, S. E. (2002). Thermal degradation of hydrolyzed and oxidized lignins. Journal of Thermal Analysis and Calorimetry, 68, 265e273. Qi, L., Minghua, Z., Yuangang, Z., Wenjun, L., Yang, L., Ying, Z., et al. (2012). Comparative antioxidant activity of nanoscale lignin prepared by a supercritical antisolvent (SAS) process with non-nanoscale lignin. Food Chemistry, 135, 63e67. Reh, U., Kraperlin, G., & Lamperckf, I. (1986). Use of differential scanning calorimetry for structural analysis of fungally
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 6 2 1 e6 2 9
degraded wood. Applied Environmental Microbiology, 52, 1101e1106. Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1996). Structureantioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine, 20, 933e956. Rozman, H. D., Tan, K. W., Kumar, R. N., & Abubakar, A. (2001). Preliminary studies on the use of modified ALCELL lignin as a coupling agent in the biofiber composites. Journal of Applied Polymer Science, 81, 1333e1340. Rozman, H. D., Tan, K. W., Kumar, R. N., Abubakar, A., Ishak, Z., & Ismail, H. (2000). The effect of lignin as a compatibilizer on the physical properties of coconut fiberepolypropylene composites. European polymer Journal, 36, 1483e1494.
629
Socrates, G. (2001). Infrared and Raman characteristic group frequencies (p. 68). Wiley. Tatiana, D., Galina, T., Jurkjane, V., & Uldis, V. (2004). Characterization of the radical scavenging activity of lignins ee natural antioxidants. Bioresource Technology, 95, 309e317. Tejado, A., Pena, C., Labidi, J., Echeverria, J. M., & Mondragon, I. (2007). Physico-chemical characterization of lignins from different sources for use in phenol-formaldehyde resin synthesis. Bioresource Technology, 98, 1655e1663. Yang, Q., Pan, X., Kong, W., Yang, H., Zhang, L., & Zhang, Y. (2010). Antioxidant activities of malt extract from barley (Hordeum vulgare L.) toward various oxidative stress in vitro and in vivo. Food Chemistry, 118, 84e89.