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Biochemical Engineering Journal 67 (2012) 173– 185

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Biochemical Engineering Journal

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Regular Article

Lignin as natural radical scavenger. Effect of the obtaining and purificationprocesses on the antioxidant behaviour of lignin

Araceli Garcíaa, María González Alriolsa, Giorgia Spignob, Jalel Labidia,∗

a Department of Chemical and Environmental Engineering, University of the Basque Country UPV-EHU, Plaza Europa, 1, 20018 Donostia-San Sebastián, Spainb Institute of Oenology and Food Engineering, Universitá Cattolica del Sacro Cuore, Via Emilia Parmense, 84, 29122 Piacenza, Italy

a r t i c l e i n f o

Article history:Received 11 April 2012Received in revised form 23 May 2012Accepted 20 June 2012Available online xxx

Keywords:LigninBiorefineryAntioxidant capacityAgricultural wastesUltrafiltrationPurification

a b s t r a c t

In the present work, the antioxidant activity of 14 lignin samples obtained from apple tree pruningwas studied. Different solvents (water, soda–water 7.5% (w/w), ethanol–water 60:40 (v/v) and aceticacid–water 60:40 (v/v)) under different operation conditions were used in order to extract the ligninand different techniques (ultrafiltration, differential precipitation and lignin purification) were appliedwith the aim of improving the lignin properties. The obtained lignin samples were characterized bydifferent analytical techniques evaluating their chemical structure (ATR-IR), thermal behaviour (TGA andDSC) and molecular weight (GPC). Further analyses (13C NMR and Folin–Ciocalteu method) were carriedout in order to determine the purity and the total phenolics content present in the lignin samples. Theantioxidant activity of the analyzed lignins was evaluated by the radical ABTS assay. The results revealedthe high influence that the lignin obtaining process had on the lignin properties, determining its possiblecommercial application. Results also indicated that autohydrolysis, organosolv and some ultrafiltratedsoda lignins showed antioxidant efficiency comparable with a powerful natural antioxidant, as catechin,and a commercial one, Trolox®.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Nature provides plenty of compounds which can be transformedin a variety of products. In this way, vegetal kingdom is the bestnatural and renewable supplying source of products and chemicalprecursors for daily commodities used in food, medicine, buildingand many other industrial sectors. Cellulose, hemicelluloses andlignin (carbohydrates and aromatics) mostly form the structure ofvegetal cells, whereas waxes, lipids and other compounds foundin smaller amounts have a non-structural function. Among all thecompounds that could be found in vegetal cell wall in trees, plantsand herbs, those containing a polyphenolic structure are provingto be of great interest [1–3].

Polyphenols are biosynthesized compounds that contain one ormore hydroxyl groups in their aromatic structure. They are dis-tributed along the plant (in seeds, stems, leaves and roots) fulfillingthe main mission of protecting it against external aggressions dueto weather, fungi and insects attack. These aromatics compoundsrange from simple molecules to highly complex polymerized com-pounds, such as lignins, the most abundant polyphenols in nature.They can be categorized into several classes (flavonoids, quinones,

∗ Corresponding author. Tel.: +34 943 017178.E-mail address: [email protected] (J. Labidi).

lignans, tannins, etc.) and their proper extraction for medical anddietetic applications, due to their favourable health effects (suchanti-inflammatory, antibacterial and anticarcinogenic activity), hasbeen widely investigated [2–5]. One of the most studied propertiesof these compounds is their antioxidant activity, i.e. their capacityto act as radical scavenger, with the aim of using them as naturaladditives for functional food or in cosmetic and polymeric formu-lations [6–8]. An antioxidant agent is any molecule able to preventor retard oxidation processes induced by oxidizing species, suchas free radicals [7,9,10]. Many natural compounds, which have apolyphenolic structure, show this property (see Fig. 1). Antiox-idants are also being exploited as retarding of oxidation or bioor photo degradation of polymer blends, using generally artificialor semi-artificial compounds for these applications. For this rea-son, the extraction technologies of new antioxidant from naturalsources are being extensively investigated.

In this sense, lignocellulosic material, such as agriculturalresidues, forestry and industrial wastes, represents an interestingsource of antioxidants compounds because of its renewable andsustainable nature, as well as for its polyphenolic compoundscontent. Recent studies have investigated the isolation of antiox-idants from agricultural residues [1,2,8] in order to revalorizeby-products that usually have few or any use. One of the mostcommonly generated in the industry and poorly used naturalpolyphenolic compounds is the lignin. Several works have argued

1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.bej.2012.06.013


174 A. García et al. / Biochemical Engineering Journal 67 (2012) 173– 185

Fig. 1. Different phenolic structures corresponding to natural or commercial compounds that show antioxidant capacity.

about a better utilization of this lignocellulosic component [11–13]and have demonstrated that the physicochemical properties oflignin are strongly affected by the isolation process [6,14].

The antioxidant activity of lignin and the influence of the obtain-ing process on it has been previously evaluated [6,7,15]. However,the aim of the present study was to better understand this fac-tor, evaluating the impact that the lignin structure and purity (interms of hemicelluloses and other components content) had on theradical scavenging capacity. For this purpose, different fraction-ation processes (autohydrolysis, soda and organosolv processes)were used to fractionate an agricultural waste (apple tree prun-ing) in order to obtain a broad set of lignin samples. The sampleswere characterized for their chemical composition and structure,total phenolics content and antioxidant activity. Different sec-ondary treatments (differential precipitation, ultrafiltration andacid-purification) were also investigated as intensification pro-cesses to improve lignin purity and antioxidant efficiency.

2. Materials and methods

2.1. Lignin obtaining processes

Apple tree pruning wastes, kindly supplied by an independentfarmer from Gurutze (Oiartzun, Spain) were used as raw material.

After drying at room temperature until constant moisture, andgrounding (Retsch 2000 cutting mill provided with a 4 mm sieve),the chemical composition of the raw material was determined

according to TAPPI standards [11]. Moisture (T264 cm-97), ash(T211 om-93), hot water (T264 cm-97) and 1% NaOH solubilities(T212 om-98), ethanol–toluene extractives (T204 cm-97), lignin(T222 om-98), holocellulose [16], cellulose and hemicelluloses [17]were determined. The results of these analyses are shown in Table 1.

Fig. 2 summarizes the different fractionation processes andsubsequent treatments used for the obtaining of the 14 ligninsamples analyzed in the present study, and the yields of obtainedsamples referred to the treated raw material are indicated. Theautohydrolysis was carried out in a pressurized reactor of 4 L, dur-ing 30 min at 180 ◦C. A solid to liquid ratio of 1:10 was used. Afterreaction, liquid and solid fractions were separated by filtrationand to improve lignin precipitation, the recovered liquid fractionwas vacuum evaporated up to approximately 50% (w/w) in total

Table 1Chemical characterization results (%, w/w on dry basis) of the apple tree pruningwastes.

Assay Standard Composition (%, w/w)

Moisture TAPPI T264 cm-97 9.06 ± 0.05Ash TAPPI T211 om-93 9.28 ± 0.21Hot water solubility TAPPI T264 cm-97 17.98 ± 0.621% NaOH solubility TAPPI T212 om-98 49.24 ± 1.58Ethanol–toluene extractives TAPPI T204 cm-97 1.09 ± 0.13Lignin TAPPI T222 om-98 17.00 ± 3.41Holocellulose Wise et al. [16] 62.84 ± 1.81Cellulose Rowell [17] 30.60 ± 2.40Hemicelluloses Rowell method 32.24 ± 0.60


A. García et al. / Biochemical Engineering Journal 67 (2012) 173– 185 175

Fig. 2. Solvents, operation conditions and treatments applied for the obtaining of the lignin samples (S/L solid to liquid ratio). In brackets the yield g of obtained sample per100 g of raw material. For purified lignin the yield was calculated from the indicated percentage of dissolved sample after acidic treatment.

dissolved solids [6]. The resulting concentrate was then treatedwith two volumes of cold acidified water (with 72% (v/v) sulphuricacid to pH 2) were added and after vacuum filtration, the resultinglignin (HYD sample) was washed twice with acidified water forimpurities removal and then oven-dried at 60 ◦C.

The organosolv treatments were carried out in the same reactorused for hydrothermal fractionation, testing different solvents, sol-vent to solid ratios, reaction times and temperatures. In the aceticacid–water (60%, v/v) treatment the raw material was fractionatedat 150 ◦C, with a solid to liquid ratio of 1:10 during 30, 45 and75 min, obtaining three lignin samples (AC1, AC2 and AC3, respec-tively) after precipitation with of two volumes of cold acidifiedwater, impurities removal, vacuum filtration and drying of the pre-cipitate. For the ethanol–water (60%, v/v) treatment the apple treepruning was fractionated during 90 min at 180 ◦C and with a solidto liquid ratio of 1:10. Afterwards reaction and solid fraction sepa-ration, the influence of decantation time was investigated, dividingthe obtained liquid fraction into two aliquots. Both of them weretreated with two volumes of cold acidified water. In one case ligninwas immediately filtered (ET1) whereas in the other case (ET2) thelignin was allowed to decant for 24 h before filtration, then washedand dried at 60 ◦C.

For alkaline lignin obtaining, the raw material was fractionatedwith an aqueous solution of soda (7.5%, w/w) in a glass atmo-spheric reactor of 25 L, at 90 ◦C during 90 min with two differentsolid to liquid ratio (1:18 and 1:10). After reaction and filtration,the liquid fractions were submitted to two different processes. Dif-ferential precipitation was applied to the liquid fraction obtainedwith a S/L of 1:18. This liquid fraction was acidified to pH 5 byadding sulphuric acid (72%, v/v). The precipitated lignin (S1.1) wasfiltrated, washed twice with acidified water and dried at 60 ◦C. Theresulting filtrate was further acidified to pH 2, obtaining a secondlignin sample (S1.2). Lignin samples from the alkaline liquid frac-tion obtained with an S/L of 1:10 were obtained after differential

precipitation and ultrafiltration processes. For this, a part of theliquid fraction was acidified with sulphuric acid (72%, v/v) obtain-ing the sample S2.1 as the resulting precipitate between pH 4 and2. The second aliquot of the alkaline liquor from S/L of 1:10 wassubmitted to an ultrafiltration process [18,19]. For this purposefour ceramic membranes (IBMEM – Industrial Biotech Membranes,Frankfurt, Germany) of different cut-offs (300 kDa, 150 kDa, 50 kDaand 15 kDa) were used in order to obtain lignin samples with nar-rower size distributions: greater than 300 kDa (S2.2), between 300and 150 kDa (S2.3), between 150 and 50 kDa (S2.4), between 50and 15 kDa (S2.5) and smaller than 15 kDa (S2.6). The lignin con-tained in the resulting permeates was isolated by acidification ofthe resulting liquid fractions with sulphuric acid to pH 2, washed,dried and grounded before chemical and instrumental analyses.

2.2. Physicochemical characterization of lignins

2.2.1. Determination of lignin samples compositionThe obtained lignins were chemically characterized, determin-

ing moisture, ash, acid insoluble lignin (AIL), acid soluble lignin(ASL) and hemicellulosic sugars (glucose, xylose and arabinose)contents. Moisture and ash contents were thermogravimetricallyquantified by TGA analysis in a TGA/SDTA RSI analyzer of MettlerToledo, heating the samples at a rate of 10 ◦C/min from 35 to 800 ◦Cin an air oxidizing atmosphere. Moisture H (%, w/w) was graphicallydetermined as the percentage of lost mass recorded after the firstmass loss step of the thermogram (between 120 and 145 ◦C), whilethe inorganic or ash A (%, w/w) content was determined as the finalresidue at 800 ◦C.

The acid insoluble lignin fraction (AIL) was gravimetricallydetermined after acid hydrolysis. For each lignin sample 0.375 gwere treated with 3.75 mL of 72% (v/v) sulphuric acid at 30 ◦C during1 h, then diluted up to a final volume of 40 mL and heated at 100 ◦Cfor 4 h. Afterwards the insoluble fraction was separated by filtration



(202 Double Ring filter, Nahita Auxilab, Spain), washed with waterand dried at 105 ◦C for 24 h. The filtrate was spectrophotometri-cally analyzed [20] in a Jasco V-630 spectrophotometer measuringits absorbance at 205 nm for acid soluble lignin (ASL in % (w/w) drybasis) determination according to Eq. (1)

ASL = A205 · Vh · dfa · b · msample

× 100 (1)

where A205 is the absorbance at 205 nm, Vh is the final hydrolysatevolume, df is the dilution factor used for adjusting the hydrolysateabsorbance below 0.7, a is the absorption coefficient (110 L/g/cm), bis the width of the cuvette and msample is the analyzed lignin sampleweight.

Monosaccharides concentration in the obtained hydrolysateswas measured by high performance liquid chromatography (HPLC)in a Jasco LC-Net III-ADC chromatograph, equipped with a ROAOrganic Acid column (00H-0138-K0, Phenomenex). The usedmobile phase consisted in 0.005 N H2SO4 prepared with 100%deionized and degassed water (0.35 mL/min flow, 40 ◦C and injec-tion volume of 20 �L). High purity glucose, xylose and arabinose(provided by Fluka, with ≥99% of purity) were used for the calibra-tion procedure.

2.2.2. Instrumental analysesThe chemical structure of the lignin samples was evaluated

by attenuated-total-reflectance infrared spectroscopy (ATR-IR) bydirect transmittance in a single-reflection ATR System (ATR topplate fixed to an optical beam condensing unit with ZnSe lens) withan MKII Golden Gate SPECAC instrument. Spectra were recordedover 32 scans with a resolution of 4 cm−1 in a wavelength rangebetween 4000 and 700 cm−1.

Variations in lignin structure due the different obtaining pro-cess were evaluated by 13C NMR analyses. The lignin samples weredissolved in DMSO-d6 [21,22] and the spectra were recorded on aBruker Avance 500 MHz at 25 ◦C and 125 MHz. The spectral widthand the acquisition time were 30,000 Hz and 1.042 s, respectively.

Size exclusion chromatography (SEC) analysis was used toevaluate the average molecular weight (Mw) and polydispersity(Mw/Mn) of the obtained samples. They were examined through theN,N-dimethylformamide (DMF) eluted GPC technique [23], con-taining 0.1% LiBr in order to avoid sample aggregation, in a Jasco Inc.chromatograph provided with an interface LC-NetII/ACD interface,a column oven CO-2065Plus and a RI-2031Plus Intelligent Refrac-tive Index Detector. A guard column and two columns PolarGel-M(Varian Inc.) were employed at a flow rate of 0.7 mL/min and 35 ◦C.Calibration was made using polystyrene standards provided byFluka, ranging from 250 to 70,000.

Lignin thermal behaviour was analyzed by differential scan-ning calorimetry (DSC) in a Mettler Toledo DSC 821 analyzer [6].Dynamic temperature scans from 50 to 250 ◦C were run undernitrogen atmosphere with a constant heating rate of 10 ◦C/min. Inorder to release the moisture content, a previous scan from 25 to100 ◦C followed by an isothermal period of 5 minutes at 100 ◦C wasdone. For each analyzed sample the glass transition temperature Tg

was determined as the temperature at the inflection point in theendothermic step of the thermogram.

The thermal stability of the lignin samples was studied by ther-mogravimetric analysis (TGA), which was carried out in a MettlerToledo TGA/SDTA RSI analyzer. The samples (between 5 and 10 mg)were heated from 25 ◦C up to 600 ◦C at a heating rate of 10 ◦C/min,using a constant nitrogen flow as inert atmosphere during theexperiments. The temperature corresponding to the maximumdegradation of the sample (DTmax) was determined as the temper-ature of the minimum in the derived thermogram. The final residue

of the lignin corresponded to the percentage of remained sampleafter thermal analysis.

2.3. Antioxidant power determination

Two different procedures were used in order to evaluate theantiradical scavenging activity of the samples: the Folin–Ciocalteu(F–C) and the ABTS assay.

2.3.1. Folin–Ciocalteau (F–C) assayThis method has for many years been used and is still com-

monly used as a measure of total phenolics in natural products,but the basic mechanism is an oxidation/reduction and, as such,can be considered an antioxidant method [24].

The F–C analysis was carried out in lignin samples (2 g/L inDMSO) and results expressed as total phenolic content (TPC) usinggallic acid as a reference [1,6]. The calibration curve of gallicacid (>98% HPLC, Fluka) in dimethyl sulphoxide (DMSO > 99.5%GC, Sigma) was constructed with six different concentrations (inthe range 100–1000 mg/L). For the analysis 0.5 mL of sample weremixed with 2.5 mL of Folin–Ciocalteu reagent (Merck) and 5 mL ofNa2CO3 and the volume brought with distillate water to 50 mL. Thesamples were kept in a thermostatic bath at 40 ◦C for 30 min beforespectrophotometric measure of the absorbance at 750 nm (UV-1601 Shimadzu spectrophotometer) against the blank (the samereagents but with 0.5 mL of DMSO instead of the sample). The TPCin lignin samples cGAE was expressed as mg of gallic acid equivalentsper litre of dissolution

cGAE = A750

kcal(2)

where A750 is the absorbance at 750 nm and kcal is the slope of thecalibration curve. TPC was also expressed as the percentage of gallicacid equivalents (% GAE on dry basis) in the dried lignin samplerelating it with the tested sample concentration csample (expressedas mg/L).

GAE = cGAE

csample(1 − (Hsample/100))× 100 (3)

The total phenolic content was also determined for Trolox® (97%,Aldrich) and (+)-catechin (98% HPLC, Fluka) samples for comparisonpurposes.

2.3.2. ABTS testWith this assay the antioxidant activity was determined as the

capacity of the lignin to reduce the ABTS radical [1], on the samesamples (2 g/L in DMSO) used for the TPC determination describedin Section 2.3.1. Briefly a radical solution (7 mM ABTS and 2.45 mMpotassium persulphate) was prepared and left to stand in the darkat room-temperature for 12–16 h before using. This solution wasthen diluted with 50% (v/v) ethanol–water to an absorbance of0.70 ± 0.02 at 734 nm and equilibrated at 30 ◦C. For the analysis,2 mL of the diluted radical solution were mixed with 20 �L of thesample and the absorbance at 734 nm was read against 50% (v/v)ethanol–water (in a Perkin Elmer UV/VIS Lambda Bio 40). A blankwith 20 �L of DMSO into 2 mL of ABTS solution and a sample with2 mL of the diluted ABTS solution were included for each test.The absorbance at 734 nm of all the samples was registered after6 minutes and the reduction of the ABTS, or antioxidant power AOP,was determined by the following equation:

AOP = % reduction of ABTS734 nm = A734,blank − A734,sample

A734,ABTS× 100

(4)



The analysis was also carried for each lignin sample at differentconcentrations attempting to cover the complete range of 0–100%inhibition.

In order to elucidate the effect on the antiradical activity of thesample composition and the lignin structure, and subsequently ofthe extraction process, some specific parameters were defined [1].The specific antioxidant power per �g of lignin sample (AOPsample)was determined relating the AOP to the tested lignin sampleconcentration clignin (expressed as �g/mL), using the followingequation:

AOPsample = AOP0.020 · clignin

(5)

The specific antioxidant power related to the TPC of the samplesand expressed as AOP per �g of gallic acid equivalents (AOPGAE),and the AOP related to the �g total content of lignin (AOPlignin), i.e.the sum of acid soluble and soluble lignin determined in Section2.2.1, were also considered for results interpretation and calculatedaccording to the following equations.

AOPGAE = AOPsample × 100GAE

(6)

AOPlignin = AOPsample × 100ASL + AIL

(7)

2.4. Lignin purification step

With the aim of establishing the influence that hemicellulosescontent had on the antioxidant capacity of the analyzed lignin sam-ples, a strong acid hydrolysis treatment (1:10 (w/v) with 72% (v/v)sulphuric acid at 30 ◦C for 1 h) followed by a mild hydrolysis (dilu-tion up to 4% (v/v) sulphuric acid and heating at 100 ◦C for 4 h)were carried out to purify the lignin samples [15] removing theacid-soluble components. After the treatment, the acid insolublefractions were collected by filtration, vacuum dried at 50 ◦C for 24 hand weighed for dissolution rate determination. The obtained acidinsoluble lignin samples were analyzed by ATR-IR, and their antiox-idant behaviour determined by the methods described in Section2.3.

3. Results and discussion

3.1. Yield of lignin recovered from the different appliedtreatments

The yield of lignin sample obtained from each fractionationand after secondary processes (differential precipitation, ultrafil-tration and purification) is indicated in Fig. 2. The results indicatedthat the applied autohydrolysis process achieved a very low ligninyield (1.2 g/100 g of treated raw material, RM), whereas organo-solv processes dissolved higher lignin amounts (between 7.1 and10.1 g/100 g of RM). The total yield of the two studied alkalineprocesses results from the sum of those obtained after secondarytreatments. In this way, under the assessed conditions, the S/L ratioof 1:18 may allow obtaining 13.8 g of lignin/100 g of raw material,whereas with a S/L of 1:10 more than 9 g of lignin sample couldbe removed from 100 g of apple tree pruning residues. However,the alkaline samples are usually obtained with high impurity, asobserved later in the results of the present work.

In agreement with a previous work [25], the amount of alkalinelignin obtained by differential precipitation varied in function ofthe pH, obtaining up to a 20% of precipitate when the black liquoris acidified until pH 6–5, and about 80% of lignin recovery whenprecipitation is carried out between pH 5 and 2. In this way, theS1.1 sample showed a yield of 5.5 g/100 g RM, and S1.2 represents8.3 g/100 g of RM.

On the other hand, the ultrafiltration yields showed that morethan 45% of alkaline sample obtained with a S/L of 1:10, was recov-ered in the filtrate of more than 300 kDa (S2.2 sample, 4.2 g/100 gRM) and that this yield decreased as the used membrane cut-offwas smaller (from 1.7 to 0.7 g of alkaline sample/100 g of RM forS2.2 and S2.6 samples, respectively).

The applied purification process caused a drastic decrease inthe yield of some lignin samples. This result was expected, sincethe purpose of the applied treatment was to remove the hemicel-lulosic contamination present in the samples. In this way, very lowyields were observed for alkaline lignins, for which up to 94.3% ofthe sample was dissolved in some cases. The organosolv and auto-hydrolysis samples showed higher yields before purification, withless than 40% of dissolution, suggesting a lower contamination rate.

3.2. Influence of obtaining process on composition andphysicochemical properties of the analyzed lignins

3.2.1. Chemical composition of lignin samplesThe results of chemical composition of the analyzed samples

are shown in Table 2. It was clearly noticeable that the fraction-ation processes and the subsequently applied treatments greatlyinfluenced the composition of the lignin samples, particularly theash, AIL and xylose content.

Alkali obtained precipitates showed very high ash contents (upto 55% (w/w) of the total sample) whereas autohydrolysis andorganosolv lignins presented low inorganic impurities (below 7%,w/w). Differential precipitation reduced the ash content in alka-line samples. The S1.1 sample, obtained by precipitation until pH5, presented 5% more ashes than S1.2, which was precipitated afterthe previous one, i.e. from pH 5 to 2. The purifying effect of theultrafiltration process resulted more significant, which agrees withthe results obtained in previous work [18]. The ash content in theultrafiltrated samples (from S2.2 to S2.6) decreased as the cut-offof the used ceramic membranes was smaller, until values close tothose observed for some organosolv lignins.

The soluble lignin contents of the analyzed samples resultedsimilar (in the range of 1.61 ± 0.52% (w/w) of ASL), being in agree-ment with those found by other authors [13,26]. High acid insolublelignin contents (to over than 87%, w/w) were found in the sam-ples obtained by autohydrolysis (HYD), organosolv processes (AC1,AC2, AC3, ET1, ET2) and ultrafiltration with cut-offs under 50 kDa(S2.5 and S2.6). On the other hand, low acid insoluble lignin frac-tions were detected in alkaline samples. It can be concluded thatthe solvents, reaction conditions and techniques used during ligninisolation affected the presence of impurities in the samples andtherefore the real lignin concentration. Generally, for samplesobtained by fractionation with water or organic solvents the lignincontent resulted higher. The effect of the reaction time could beevaluated for the acetic acid extracted samples; it was observed thatprolonged fractionation treatment resulted in an increase of AIL.The effect of the used solid to liquid ratio during apple tree pruningfractionation could be assessed for S1.2 and S2.1 alkaline samples,because both were sequentially precipitated. However, the S/L ratioused for S1.2 obtaining was almost twice and its AIL content almostdoubled the insoluble lignin content. The AIL fraction was improvedwith the ultrafiltration process, resulting in more lignin enrichedsamples when low cut-offs were used.

The hemicelluloses content in the analyzed samples resultedclearly depending on the fractionation process. In this way, alkaliextracted samples showed higher contents of polysaccharides,mainly constituted of xylose, suggesting that during the alka-line process high amounts of lignin–carbohydrate-complexes weredissolved. Lower values of monomeric sugars in autohydrolysisand organosolv lignin samples, together with higher ASL and AILcontents, indicated a higher lignin-dissolving selectivity of these



Table 2Chemical composition of the analyzed lignins.

Sample H A AIL ASL GLU XYL ARA

HYD 2.85 1.03 87.37 ± 1.07 2.28 ± 0.13 2.47 ± 0.46 2.32 ± 0.35 0.70 ± 0.02AC1 7.46 2.64 62.09 ± 0.99 1.28 ± 0.05 2.30 ± 0.32 1.41 ± 0.17 n/dAC2 6.92 4.65 71.65 ± 1.50 1.12 ± 0.02 2.04 ± 0.08 1.48 ± 0.08 n/dAC3 2.91 2.97 82.58 ± 1.03 0.96 ± 0.02 0.80 ± 0.02 1.20 ± 0.01 n/dET1 3.34 7.07 66.01 ± 3.34 1.05 ± 0.09 2.63 ± 0.38 1.67 ± 0.16 0.44 ± 0.00ET2 4.69 2.72 64.50 ± 4.73 0.92 ± 0.04 2.74 ± 0.45 1.80 ± 0.29 0.46 ± 0.02S1.1 4.15 45.28 4.87 ± 0.85 1.67 ± 0.05 3.09 ± 0.13 18.70 ± 0.97 1.34 ± 0.02S1.2 4.93 40.89 12.96 ± 1.15 1.45 ± 0.03 2.19 ± 0.03 17.64 ± 0.63 1.58 ± 0.01S2.1 0.47 55.02 20.33 ± 0.34 1.77 ± 0.01 3.20 ± 0.08 8.79 ± 0.09 1.18 ± 0.01S2.2 1.44 47.11 21.82 ± 4.08 1.75 ± 0.01 2.24 ± 0.02 20.67 ± 0.23 2.55 ± 0.01S2.3 2.25 37.50 25.48 ± 0.31 2.21 ± 0.02 2.27 ± 0.02 20.90 ± 0.32 1.04 ± 0.02S2.4 2.09 14.88 56.48 ± 2.06 2.60 ± 0.07 1.30 ± 0.00 15.62 ± 0.33 1.11 ± 0.01S2.5 1.51 12.50 78.69 ± 0.31 1.72 ± 0.01 1.71 ± 0.08 1.61 ± 0.09 0.23 ± 0.02S2.6 2.15 9.12 82.44 ± 3.94 1.81 ± 0.02 2.03 ± 0.06 0.77 ± 0.12 0.30 ± 0.01

n/d: not detected.H: moisture, A: ash, AIL: acid insoluble lignin, ASL: acid soluble lignin, GLU: glucose, XYL: xylose, ARA: arabinose.

processes [11,27]. The nature of the solvent also influenced the wayhow the hemicelluloses were extracted from the raw material, andtherefore it should affect the carbohydrate fraction composition ofthe precipitated lignin samples. Thus, the fractionation with waterand ethanol–water improved the glucoxylans and arabinoxylansextraction, while the use of acetic acid as solvent, as reported inprevious works [27], showed a low selectivity of this organic acidagainst arabinoxylans (no arabinose was detected in the by HPLCdetermination).

The application of ultrafiltration and differential precipitationalso improved the samples purity. The hemicelluloses, generally oflarge size, prevent sample permeation; hence as the membranecut-off was smaller the sugars content in lignin became lower.Similarly, a previous precipitation (up to pH 5 o 4) eliminatedmore lignin–carbohydrate complexes and consequently the pre-cipitated samples (S1.2 and S2.1) resulted with less contaminationdue polysaccharides content.

3.2.2. Infrared spectroscopy analysisIn Fig. 3 the infrared detailed spectra of the analyzed puri-

fied and non-purified lignin samples are shown. The vibrationbands of typical functional groups associated to the lignin struc-ture were found: aromatic and aliphatic hydroxyl (3400 cm−1),aliphatic and aromatic methyl and methylene (2930, 2850 and1455 cm−1) and non-conjugated carbonyl groups (1700 cm−1). Thebands at 1600, 1510 and 1425 cm−1 were attributed to the aromaticskeleton vibrations, and the existing peaks at 1160 and 1035 cm−1

to the OH stretching of secondary and primary alcohol respec-tively. Two of the main monomeric constituents of the lignin werealso detected with more or less intensity in their vibration bands:syringyl (1330, 1115 and 825 cm−1) and guaiacyl type units (1215,1025 and 855 cm−1).

For non-purified lignins it could be observed that in autohy-drolysis and organosolv samples the intensity of the aromatic andphenolic type group bands (such as aromatic skeleton, guaiacyl andsyringyl units) was higher than those detected in alkaline sam-ples. On the other hand, typical broad bands (1160 and 1035 cm−1)associated to hemicelluloses [27,28] were present in most of sodalignins. This confirmed that higher impurities due hemicellulosic-lignin bonds were present in alkaline samples, as shown by thechemical characterization discussed above.

The nature of the used solvent in the obtaining of non-alkalinelignins, i.e. autohydrolysis and organosolv samples, had slight influ-ence on their structure. Thus, the use of acetic acid caused greatercontent of carbonyl type bonds in the lignin structure (1700 cm−1),whereas a weak band at 1685 cm−1, attributed by Łojewski et al.

[29] to the presence of enol or diketone groups, was observed inET1 and ET2 samples.

Evident differences were not found during spectral analysiswhen different ways for lignin precipitation were used (for ET1 andET2 or S1.1 and S1.2 samples). However the spectra of the ligninsobtained from ultrafiltration process (from S2.2 to S2.6) showedan improvement in their structure, in terms of less hemicellulosiccontent and more intense aromatic functional groups, as the cut-offwas smaller, supporting the results of chemical characterization.

The purifying effect of the acid hydrolysis on the alkaline sam-ples was evident. The treatment with sulphuric acid resulted in anincrease of the intensity of the bands associated to the aromaticskeleton of lignin (1600, 1510 and 1425 cm−1), and in a decreaseof the broad band around 1100 cm−1 related to hemicellulosiclinks, confirming the elimination of the polysaccharides in the sam-ple. However the severity of the purification step caused a highertransformation on those lignins that contained less hemicellulosiccontamination, i.e. on autohydrolysis (HYD), organosolv (AC andET samples) and some ultrafiltrated lignins (S2.5 and S2.6). In thesesamples the intensity of guaiacyl and syringyl bands decreased andthat of carbonyl group increased, suggesting that the purificationby acid hydrolysis resulted in the oxidation or degradation of somephenolic groups.

3.2.3. Thermal behaviour of lignin samplesIn Table 3 the thermal properties (glass transition Tg and max-

imum degradation temperatures DTmax and residue after thermaldegradation) are shown.

The Tg of the lignin usually varies from 80 to 180 ◦C dependingon several factors [13,30] as its origin and the conditions ormethodologies applied during its isolation. In the present work,glass transitions between 110 and 135 ◦C were observed. Tg resultsvery sensitive to variations in the composition (impurities) andthe structure (crosslinking and molecular weight) [13,14] andtherefore it cannot be considered as an inherent and characteristicproperty to lignin type, although it is possible to assess the effectthat treatments have on this feature. The autohydrolysis andethanol-organosolv samples (HYD, ET1 and ET2) presented thehighest Tg values (133, 130 and 134 ◦C, respectively). For AC1,AC2 and AC3 samples, Tg decreased with increasing fractionationtime. The effect of differential precipitation was not found to bedeterminant for S1.1 and S1.2 samples, which showed similarTg (119 and 115 ◦C, respectively). However, a difference in Tg ofalmost 10 ◦C was observed between S1.1 and S2.1, which wereobtained for fractionation processes with different solid to liquidratio (1:10 and 1:18). The glass transition significantly varied



Fig. 3. ATR-IR detailed spectra (1800–850 cm−1) of (a) autohydrolysis and organosolv and (b) alkaline lignins (P means purified sample).

Table 3Thermal properties (glass transition temperature, temperature of maximum degradation and residue) and molecular weight distributions (average molecular weight andpolydispersity) of the analyzed lignins.

Sample Tg (◦C) DTmax (◦C) Residue at 600 ◦C (%, w/w) Mw Mw/Mn

HYD 133 346 47.97 4263 2.09AC1 118 337 35.82 16,391 5.20AC2 116 348 35.89 13,348 4.85AC3 111 352 39.89 7175 3.56ET1 130 351 37.10 8810 3.15ET2 134 353 39.47 8294 4.09S1.1 119 290 42.73 5068 2.92S1.2 115 245 60.89 8829 3.70S2.1 126 210 72.16 23,868 6.08S2.2 128 232 55.34 5141 2.60S2.3 117 253 54.84 5431 2.90S2.4 116 257 42.35 4192 2.35S2.5 113 375 47.19 5273 2.54S2.6 112 383 42.78 4018 2.56



among the ultrafiltrated lignins, showing a decrease of the Tg asthe membrane cut-off becomes smaller [12].

The final residue and the DTmax values obtained in TG analy-sis could be related to the composition of the analyzed sample. Inthe literature, the temperature ranges of the lignocellulosic com-ponents degradation have been reported [18,28], establishing thathemicelluloses usually degrade between 200 and 300 ◦C, whereaslignin thermal decomposition usually occurs up to 500 ◦C. Thus, theDTmax values showed in Table 3 confirmed the high hemicellulosesamounts in most of alkaline samples and higher purity for HYD, AC,ET, S2.5 and S2.6 lignin samples, supporting the results obtained inthe previous chemical characterization. High final residues couldbe related not only to high inorganic contents but also with highpolysaccharides contamination and high complexity of the ligninstructure, because the pyrolysis of hemicelluloses and lignin ininert atmosphere generally involves the formation of char carboncompounds [18,28].

3.2.4. Molecular weight distribution of lignin samplesThe average molecular weight results must be relatively consid-

ered because polystyrene standards were used for SEC calibration[31]. Table 3 shows the average molecular weight Mw and polydis-persity Mw/Mn of the samples obtained by different methods. Themolecular weight distribution of the samples was analyzed fromthe IR detector response, which not only detects the lignin, butalso molecules that do not have the aromatic structure detectableby UV detector [22,23]. Therefore, the results represent the dis-tribution of the whole sample obtained. It can be observed thatthe obtaining process (i.e. solvent type, temperature, duration oftreatment, etc.) severely affected the molecular distribution of thesamples. Considering the non-ultrafiltrated lignin fractions, thelowest both average molecular weight and polydispersity valuescorresponded to HYD sample (4263 g/mol and 2.09, respectively)and the highest Mw and Mw/Mn to S2.1 lignin (23,868 g/mol and6.08, respectively). This suggested that the fractionation of lig-nocellulose in acidic media provides lignin fractions with lowersize and narrower distribution, probably due to the less contentof linked carbohydrates in the lignin fractions obtained at neutralor acidic pH. The Mw observed for ET1 and ET2 samples (180 ◦C,90 min) was found to be more than twice that for HYD lignin(180 ◦C, 30 min). On the other hand, the molecular size of the ace-tosolv fractions extracted at 150 ◦C, samples AC1, AC2 and AC3,resulted smaller as the fractionation process was longer (16,391,13,348 and 7175 g/mol for 30, 45 and 75 min, respectively). Respectto the effect of the obtaining conditions have on the lignin size,Zhang et al. [32] claimed that higher temperatures and longer res-idence times increase the degree of cleavage of ether bonds in thelignin macromolecule, and therefore lower size lignins could beobtained when the extraction process becomes severer. This factwas observed between the acetosolv samples, but also when the Mw

of AC1 and HYD lignins is compared; both samples were obtainedafter 30 min of treatment, but the first at a lower temperaturethan the second one (150 and 180 ◦C, respectively) and also highermolecular weight. However, when comparing ET and AC samplesthe above mentioned behaviour was not observed, and aceto-solv lignins showed higher molecular weight although they wereobtained at lower temperature and shorter times than the ethanol-organosolv samples. This suggested that not only the temperatureand treatment length affect the lignin size distribution, but alsothe acidic or basic nature of the employed solvent. Some authors[6,11,26] reported that under strongly acidic conditions lignin couldsuffers repolymerization/self-condensation processes because theformation of highly reactive and unstable carbocations. This factcould explain the higher Mw observed for the acetosolv sam-ples when comparing with autohydrolysis and ethanol-organosolvones.

A noticeable difference was found between the molecular distri-butions observed for S1.1, S1.2 and S2.1 alkaline lignins (5068, 8829and 23,868 g/mol, respectively). As discussed above, the solid to liq-uid ratio used during the fractionation of the raw material resultsa very important factor to consider in the obtaining of lignin andthe evaluation of its properties. According to the obtained Mw datathe higher S/L ratio of 1:10 used during the isolation of S2.1 samplepromoted condensation mechanisms that resulted in higher Mw

than the observed for the S1.1 and S1.2 lignin fractions obtained atS/L of 1:18. The molecular weight distributions observed in ultrafil-trated samples (from S2.2 to S2.6) showed a negligible influence ofthe membrane cut-off. This fact suggested that the lignins fractionsunder the molecular size of 300 kDa present an almost homogenousmolecular weight and the ultrafiltration process did not achievea noticeable fractionation of lignin of different molecular weightdistribution, and that mostly affected the purity of the samples interms of sugars and ash content., which resulted less hemicellulosiccontaminated as the cut-off was smaller. Only remarkable differ-ences in Mw and Mw/Mn were detected between S2.1 rough ligninsample and those ultrafiltrated.

3.2.5. Chemical structure of lignin samples by 13C NMRIn the 13C NMR spectra of evaluated samples, typical lignin sig-

nals were found [14,27,34]. Fig. 4 shows the 13C NMR spectra ofsome of the analyzed lignin fractions (HYD, AC3, S2.1 and S2.6).Between 177 and 168 ppm the signal attributed to aliphatic COORappeared. The intensity of this signal was more evident in AC1,AC2 and AC3 lignins than HYD, ET1 and ET2 samples, probablydue to acetylation processes during the fractionation of raw mate-rial. In the aromatic region, between 100 and 162 ppm, commonsignals associated with lignin forming units, i.e. syringyl (S), guaia-cyl (G) and p-coumaric acid (PC), were observed. The S units gavesignal around 153, 147, 137, 134 and 105 ppm. Typical chemicalshifts related to G units were found at 147 ppm and 120 ppm, andthe presence of PC units was reflected by the signal at 116 ppm.Between 50 and 105 ppm most of the signals are related to polysac-charide contamination and lignins inter unit linkages. The �, �and � carbons in �-O-4 linkages were found around 85, 70 and60 ppm respectively. The presence of methoxy groups was veri-fied by the intense signal founded between 57 and 54 ppm. Theregion comprised between 15 and 34 ppm represents �-methyland � and �-methylene groups in the n-propyl side chain of thelignin.

In order to better evaluate the variation in the aromatic/aliphaticstructure of obtained lignin samples and the quantity of certainfunctional groups and structural units, the relative abundance(integral) of several groups of signals was determined taking theintegral of aromatic region from 162 to 100 ppm as reference(assuming that it contains six aromatic carbons). The regions whoseintensities were evaluated were those related to aliphatic frac-tion (100–15 ppm), oxygenated aromatic carbons (162–145 ppm),condensated aromatic carbons (145–120 ppm), protonated aro-matic carbons (120–100 ppm), hemicelluloses (80–60 ppm) andthe amount of � and �-carbons involved in �-O-4 linkages (88–85and 62–58 ppm). The results of this estimation are shown in Table 4.As it can be observed, the relative abundance of the evaluatedcluster clearly depended on the lignin obtaining process. The ratioof aliphatic/aromatic fraction resulted quite higher in the alkalinelignins (from 3.63 to 5.60) than those obtained by autohydrolysisor organosolv processes (between 1.03 and 1.77). On other hand,the proportion of O CAr found in the alkaline fractions resultedlower compared with the organosolv and autohydrolysis ligninsO CAr amount. Both results could indicate a lower phenolic con-tent in soda lignins than in the other samples. The increase of C CArand the decrease of H CAr and �-O-4 linkages were founded by ElHage et al. [14] as indicative of condensation mechanisms due the



Fig. 4. 13C NMR spectra of some of the analyzed lignin samples (HYD, AC3, S2.1 and S2.6).

increase on the severity of the fractionation. In the present study,most of the alkaline samples presented higher proportion of �-O-4 linkages and protonated aromatic carbons as well as very highratios of aliphatic structures. Therefore, a more condensed and ram-ified structure should be expected in those lignin fractions obtainedafter soda process. Also the relative intensity of the signal rangeattributed to hemicelluloses resulted higher in alkaline samples,between 0.32 and 3.27 respect to aromatic moiety, while the cor-responding value to autohydrolysis and organosolv fractions did

not reach a ratio greater than 0.13. These results agreed with thoseobtained by lignin chemical characterization.

3.3. Antiradical behaviour of lignin samples and referencecompounds

As well explained in [24], even though commonly used for theestimation of total phenolics content, the F–C assay can be consid-ered as an antioxidant capacity method based on a single electron

Table 4Relative abundance per aromatic unit of several moieties found in 13C NMR in the spectra of the analyzed lignin samples.

Sample Aliphatic carbons O CAr C CAr H CAr Hemicelluloses C� and C� in �-O-4 linkages

HYD 1.03 0.18 0.28 0.54 0.13 0.05AC1 1.40 0.20 0.25 0.55 0.01 0.02AC2 1.77 0.16 0.32 0.52 0.02 0.01AC3 1.62 0.22 0.31 0.47 0.01 0.01ET1 1.61 0.25 0.23 0.52 0.06 0.14ET2 1.70 0.18 0.26 0.55 0.09 0.07S1.1 4.38 0.04 0.13 0.83 2.43 0.08S1.2 5.26 0.00 0.07 0.93 2.76 0.14S2.1 3.63 0.04 0.25 0.71 1.93 0.06S2.2 5.60 0.00 0.02 0.98 3.27 0.22S2.3 4.85 0.03 0.09 0.88 2.51 0.10S2.4 4.98 0.14 0.22 0.64 1.30 0.27S2.5 3.79 0.14 0.31 0.54 0.32 0.20S2.6 5.26 0.17 0.34 0.49 0.43 0.23



Table 5Antioxidant capacity (TPC and AOP) and specific antioxidant power of the analyzed lignins (P means purified sample).

Sample TPC AOP (%) Specific AOP

cGAE (mg/L) GAE (%) AOPsample AOPGAE AOPlignin

HYD 751.18 ± 24.00 38.13 ± 1.22 89.73 ± 0.45 2.28 5.97 2.54AC1 569.70 ± 1.75 27.39 ± 0.08 90.90 ± 0.45 2.19 7.98 3.45AC2 590.91 ± 2.02 29.11 ± 0.10 93.19 ± 0.29 2.30 7.88 3.15AC3 683.16 ± 0.58 33.99 ± 0.03 97.96 ± 0.03 2.44 7.17 2.92ET2 610.10 ± 4.63 30.35 ± 0.23 89.61 ± 1.28 2.23 7.34 3.32ET1 653.87 ± 2.10 32.21 ± 0.10 87.44 ± 3.23 2.15 6.69 3.29S1.1 87.54 ± 1.54 4.25 ± 0.07 23.37 ± 0.20 0.57 13.35 8.67S1.2 219.53 ± 2.10 10.61 ± 0.10 41.68 ± 2.32 1.01 9.49 6.98S2.1 167.68 ± 1.75 7.76 ± 0.08 26.69 ± 0.05 0.62 7.96 2.80S2.2 87.88 ± 1.75 4.09 ± 0.08 12.72 ± 0.65 0.30 7.24 1.26S2.3 142.76 ± 1.54 6.83 ± 0.07 23.27 ± 0.17 0.56 8.15 2.01S2.4 293.60 ± 2.54 15.13 ± 0.13 37.48 ± 1.04 0.97 6.38 1.63S2.5 473.74 ± 2.02 23.69 ± 0.10 63.94 ± 0.11 1.60 6.75 1.99S2.6 730.98 ± 1.54 29.24 ± 0.06 87.72 ± 1.09 1.75 6.00 2.08PHYD 501.68 ± 3.09 23.33 ± 0.14 54.59 ± 3.19 1.27 5.44 –PAC1 222.90 ± 0.58 10.46 ± 0.03 28.95 ± 0.49 0.68 6.49 –PAC2 300.34 ± 2.10 13.41 ± 0.09 41.84 ± 0.13 0.93 6.97 –PAC3 283.16 ± 0.58 12.64 ± 0.03 40.80 ± 0.02 0.91 7.20 –PET1 360.74 ± 5.79 18.44 ± 0.30 58.67 ± 0.51 1.50 8.13 –PET2 333.33 ± 2.67 13.39 ± 0.11 49.23 ± 0.58 0.99 7.38 –PS1.1 369.33 ± 1.21 17.50 ± 0.06 40.93 ± 0.75 0.97 5.54 –PS1.2 451.48 ± 2.40 21.71 ± 0.12 71.89 ± 0.85 1.73 7.96 –PS2.1 506.73 ± 2.10 22.03 ± 0.09 47.69 ± 0.14 1.04 4.71 –PS2.2 447.47 ± 1.01 20.11 ± 0.05 48.64 ± 0.57 1.09 5.44 –PS2.3 386.87 ± 1.01 17.67 ± 0.05 32.30 ± 0.69 0.74 4.17 –PS2.4 253.54 ± 2.67 12.43 ± 0.13 22.33 ± 0.54 0.55 4.40 –PS2.5 358.89 ± 4.83 17.46 ± 0.23 48.98 ± 1.41 1.19 6.82 –PS2.6 335.59 ± 1.92 15.76 ± 0.09 60.30 ± 1.19 1.42 8.98 –

transfer (SET) mechanism. As it concerns the ABTS assay, eventhough it is usually classified as a SET reaction, the radical in factmay be neutralized either by direct reduction via electron trans-fers or by radical quenching via H atom transfer (HAT). Based onthis premise, the results obtained from the two tests have beeninterpreted as reported in the following.

3.3.1. F–C assayThe results of the F–C assay are showed in Table 5. It can be

observed that the fractionation conditions decidedly affected theTPC in lignin samples. In general, mild acidic conditions favouredhigher content of phenolic groups in lignin [25]. Severe treatments(very long, at high temperature or at extremely basic conditions)promoted the dissolving of more components from the raw mate-rial [2,6], and therefore the lignin samples could content moreimpurities and less phenolics amounts. Considering the mechanismof the F–C assay, the lower values probably indicated a degradationof phenolic compounds during the fractionation process [1].

Autohydrolysis and organosolv lignins showed TPC very closeor even higher than those obtained for the reference compoundscatechin and Trolox® (28.12% and 26.32% of GAE, respectively).The alkaline samples, not ultrafiltrated or ultrafiltrated above50 kDa showed the lowest TPC. Ultrafiltration, and also differentialprecipitation, had a favourable effect on the TPC of alkaline sam-ples, probably due to the improved purity of final samples. Thisbehaviour must be directly related to the purity of the samplesand to the fractionation effect of the membrane technology, whichallows obtaining not only different sized lignins but also sampleswith different chemical structure [18,19,33].

The acid hydrolysis employed for the removal of hemicellu-losic fraction clearly affected the GAE content in all analyzed ligninsamples. As shown by the spectroscopic analysis, the purificationtreatment dissolved the lignin linked carbohydrates and allowedobtaining acid insoluble lignin samples. For highly contaminatedlignins (most of the alkaline samples) this has a positive effect onthe TPC which increased from 2 to 5 times. However, as confirmed

with the ATR-IR spectra, the purification process applied to autohy-drolysis, organosolv and small cut-off ultrafiltrated samples, whichpresented lower hemicellulosic impurities, caused a decrease intheir GAE content, suggesting a degradation of phenolic compoundsduring the severe acid hydrolysis. This reduction effect was morepronounced (up to 2 times) for the purer lignins (AC1, AC2, AC3and ET2). Therefore, the purification process should be modifiedaccording to the contamination degree of the lignin sample (usingshorter purification treatments or with less concentrated acids),because the conditions that achieved good results for very contam-inated samples could cause a degradation of phenolic groups inmore pure lignins. This behaviour can be noticed in Fig. 5a, wherethe phenolic content for all analyzed samples (gallic acid, catechin,Trolox® and lignins) is shown.

3.3.2. ABTS assayIn Table 5 the AOP values of analyzed lignin samples are shown

with a clear influence of the fractionation method, that confirmingthe conclusions of other authors [2,4,6,7].

Autohydrolysis and organosolv lignins showed AOP valuesclose and even above 90%, while alkaline samples presented inmost cases an antiradical power below 40%, except for the samplesultrafiltrated under 50 kDa. Similarly to what observed for the TPC,a variation in the antioxidant activity of the lignins was observedwhen they were purified, confirming that this step improves prop-erties only of alkaline lignins. A linear and positive relationshipbetween TPC and AOP for all the analyzed lignin samples wasobserved (Fig. 5b). The organosolv and autohydrolysis samplespresented higher phenolic contents and better antioxidant powerthan the alkaline lignins, but this behaviour resulted less favourablewhen acid purification was performed. Most of the soda ligninsamples showed better antioxidant activity after purification step.In this way, an increase in AOP values between 1 and 4 times wasachieved by applying purification to alkaline lignins, whereas fororganosolv and autohydrolysis samples a worsening up to 3 timeswas observed. These results confirmed the direct relationship



Fig. 5. Antioxidant behaviour of the tested samples: (a) variation of TPC (GAE%) with sample concentration; (b) influence of GAE% on the antioxidant power of the analyzedlignin samples; correlation of AOP% with the concentration of reference compounds, for (c) autohydrolysis and organosolv lignins, and (d) for alkaline lignins.

between GAE content and AOP, being in agreement with otherauthors [1,9,10].

Several authors reported how some factors could interferein the activity of antioxidant agents. It has been detailed [9,10]that free radical scavenging and antioxidant activity of phenolicsmainly depends on the number and position of hydrogen-donatinghydroxyl groups on the aromatic ring of the phenolic molecules,but it is also affected by other factors, such as the presence of otherproton donating groups that could reduce or increase their activity.

For example, it was discussed that flavonoids show worse antioxi-dant activity in their glycoside form [10]. Other authors [3] arguedthat saccharides could derive to endiol reduction agents in alkalinemedium, which act as easily reducing compounds.

Also in [7] the antioxidant activity of lignins obtained from Acan-thopanax senticosus remainder by organosolv (ethanol and acetic)and kraft treatments was correlated with the TPC of the samples.However, from Fig. 5b, it is clear that the TPC is not the only fac-tor determining the scavenging activity of the lignin since samples



with similar amount of phenolics did not showed similar antiox-idant power. For example, AC2 and S2.6 contained comparableGAE contents (29.11% and 29.24%, respectively) but their ABTSinhibitions were different (93.19% and 87.72%, respectively). Fur-thermore, it has been reported how the AOP evaluated with theABTS assay increases up to a maximum value as a function of phe-nolics concentration which is not always linear [2]. This meansthat if the assay is carried out on a unique sample concentration,an over- or underestimation of the real AOP might occur. In thepresent study, the variation of AOP with sample concentration wasalso studied for a better evaluation of the antioxidant quality orefficiency of the tested compounds (Fig. 5c). As observed, gallicacid, Trolox® and catechin reached their maximum AOP values forsmall amounts (from 100 to 650 mg/L of sample). Between 1600and 2000 mg/L of most of the organosolv and autohydrolysis ligninswere necessary to obtain the same antioxidant effect. It resultedremarkable that almost all obtained samples showed very simi-lar antioxidant capacity regardless to the used isolation process.Similarly to that observed above, the purification of these ligninsresulted in a decline in their AOP values, requiring from 2 to 4times more sample to reach the results observed for non-purifiedsamples. This confirmed that suggested above, that acid purifica-tion treatment achieved not only to decrease the hemicellulosiccontent but also to partially modify the lignin structure, declin-ing its GAE content and its reducing capacity. Similarly, Fig. 5dshows the AOP behaviour of the analyzed alkaline lignins. The sam-ples obtained by soda processes covered a broad range of AOP andappeared highly affected by isolation and secondary treatments.The inhibition observed for S1.1 and S1.2 samples indicated thatthe different precipitation pH used not only improved the ligninpurity but also the antioxidant behaviour. Similarly, an enhance-ment of AOP% was achieved by the use of ultrafiltration process, asshowed the higher activity of alkaline lignin S2.6 (obtained from<15 kDa liquor) compared with the initial rough lignin S2.1.

According to these findings, it is suggested that for a betterdefinition of lignin antiradical activity, the specific antioxidantpower respect to sample (AOPsample), total phenolic (AOPGAE) andlignin contents (AOPlignin) should be calculated and considered(Table 5). The AOPsample, can be considered as an indicator of ligninpurity since a strong (r2 0.914) and positive linear correlationbetween total lignin content (AIL + ASL) and this parameter wasfound (Fig. 6). The most pure lignin AC3, which contained a 4.82%of inorganic and hemicellulosic impurities, showed an AOPsampleof 2.44%/�g of sample, whereas the >300 kDa lignin sample S2.2

Fig. 6. Variation of AOPsample with the sum of inorganics and hemicellulosic impu-rities content (%, w/w).

presented the lower AOPsample value (0.30%/�g of sample) andthe highest contamination (71.53%). The parameter AOPGAE can beused to denote the quality or inhibition efficacy of the phenolicspresent in the samples. The calculated values help to understandFig. 5b. The autohydrolysis and organosolv samples exhibited val-ues between 5.90%/�g GAE and 7.98%/�g GAE, whereas AOPGAEof alkaline lignins were found in a wider range (from 6.00%/�gGAE to 13.35%/�g GAE). These results suggested that the pheno-lic structure of most of the analyzed samples had similar quality orinhibition efficiency, except for S1.1 and S1.2 samples that showedthe highest values (13.35 and 9.49%/�g GAE, respectively). Accord-ing to the definition of the AOPGAE parameter, these two alkalinesamples seemed to content low phenolic structure but with avery effective radical inhibition capacity. This could be due to ahigher content of functional groups that improves the phenolicreducing activity, the presence of non-phenolic components withantiradical scavenging activity, or a higher oxidation state of thesesamples [9,10]. The latter might appear in contradiction with previ-ous comments, however some intermediates of phenolic oxidation(quinones) sometimes show an increased activity and a differenceresponse of the Folin reducing power and the ABTS power to oxi-dation can be shown by different phenolic compounds [1].

4. Conclusions

In the present work the validity of lignin as antiradical scav-enger was proved. Many of the analyzed samples reached the 100%of reduction of the ABTS radical, and accordingly they could replacesome commercial antioxidants. However the required quantityof sample to reach a certain effect was strongly affected by theobtaining methods. Thermal behaviour, structural determinationand molecular distribution analyses demonstrated that the use ofwater and organic solvents should be preferred instead alkalineones since lignin samples with higher purity, total phenolic contentand antioxidant activity could be achieved. It was found that theimprovement of the antioxidant power of the alkaline lignin sam-ples could be possible by using secondary intensification process asultrafiltration, differential precipitation or, purification techniquesto remove hemicellulosic impurities.

Acknowledgements

The authors would like to thank the Department of Agricul-ture, Fishing and Food of the Basque Government (scholarship ofyoung researchers training) and the Spanish Ministry of Science andInnovation and the Italian Ministry of Instruction, University andResearch (Italy–Spain Integrated Action, project IT2009-0054) andDiputación Foral de Gipuzkoa (OF 53/2011) for supporting finan-cially this work.

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