Journal of Chromatography A, 1404 (2015) 39–43
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Rapid method for determination of carbonyl groups in lignin compounds by headspace gas chromatography Jing Li a , Hui-Chao Hu b , Xin-Sheng Chai a,∗ a b
State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, Guangdong 510640, China College of Material Engineering, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
a r t i c l e
i n f o
Article history: Received 13 March 2015 Received in revised form 21 May 2015 Accepted 21 May 2015 Available online 28 May 2015 Keywords: Carbonyl groups Lignin Sodium borohydride Headspace gas chromatography
a b s t r a c t The paper reports on a novel method for rapid determination of carbonyl in lignins by headspace gas chromatography (HS-GC). The method involves the quantitative carbonyl reduction for aldehydes in 2 min at room temperature or for acetones in 30 min at 80 ◦ C by sodium borohydride solution in a closed headspace sample vial. After the reaction, the solution was acidiﬁed by injecting sulfuric acid solution and the hydrogen released to the headspace was determined by GC using thermal-conductivity detector. The results showed that with the addition of SiO2 powder, the reduction reaction of carbonyl groups can be greatly facilitated. The method has a good measurement precision (RSD < 7.74%) and accuracy (relative error <10% compared with a reference method) in the carbonyl quantiﬁcation. It is suitable to be used for rapid determination of carbonyl content in lignin and related materials. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Lignins are the natural polymers (account for 15%–25%) present in lignocellulosic materials, the most abundant renewable biomass resource. Lignins are also the promising materials that could replace synthetic polymers and thus reduce the dependence on fossil fuel sources . There are several ways to extract lignins from lignocellulosic material in industry, e.g., chemical or organosolv pulping performed at the harsh conditions . The conditions in pulping processes can affect the functional groups on lignin structures, such as carboxyl, methoxyl and carbonyl, thus it is important in selecting the proper process operation conditions to obtain the desired chemical transformation on lignin molecules for producing the advanced or high value-added materials [3–5]. Among these groups in lignins, carbonyl can be converted to acid or alcohol groups [6,7] that are important for the cross-linking reaction between phenolic compounds [8,9]. Therefore, in the lignin utilization related research and applications, the efﬁcient method for quantifying the carbonyl group in lignins is highly desired. Traditionally, the carbonyl group in lignins can be determined by titration methods, which is based on the hydrogen ions released from the oximation reaction with hydroxylamine hydrochloride [10–12], in which the reaction is performed at oxygen excluded
∗ Corresponding author. Tel.: +86 20 87113713; fax: +86 20 87113713. E-mail address: [email protected]
(X.-S. Chai). http://dx.doi.org/10.1016/j.chroma.2015.05.055 0021-9673/© 2015 Elsevier B.V. All rights reserved.
atmosphere for up to 30 h. Moreover, there is an interference reaction for the compounds containing vinyl ether group, since it can also be oximated in the reaction . Obviously, such long time and poor selectivity of the reaction in this method limits its application in many cases. The content of carbonyl groups in lignin can also be determined by differential ultra-violet spectroscopy (also called εr method), based on the differential absorption measurements before and after the carbonyl groups in lignin are reduced by borohydride (NaBH4 ) , i.e., 4-CO
+ BH4 − + 2OH− + H2 O → 4-CHOH
+ BO3 3−
The major problem of this method is the accuracy, since there is a large uncertainty in the absorption measurements. Since the residual NaBH4 in Reaction (1) can be converted to hydrogen by acidiﬁcation, i.e., BH4 − + 2H2 O → BO2 − + 4H2 ↑
a simple method based on measuring the hydrogen volume released from Reaction (2) after Reaction (1) was proposed [15,16]. At the suggested conditions, it requires 5 h to complete Reaction (1). Besides the efﬁciency, the major problem is also the accuracy because of a larger uncertainty in the gas volumetrical measurement. There are several methods available for the determination of carbonyl group in lignins using advanced instruments, which include Fourier Transform Infrared (FT-IR) spectroscopy [16,17], 19 F nuclear magnetic resonance (19 F NMR) , near-infrared Fourier
J. Li et al. / J. Chromatogr. A 1404 (2015) 39–43
transform Raman (NIR-FTR) spectroscopic technique . The major problems in these instrumental analytical methods are the precision and accuracy in the carbonyl group measurement, especially for the samples with complicated matrices. The interferences from the co-existing compounds are difﬁcult to be eliminated. Headspace gas chromatography (HS-GC) was found to be a powerful tool for indirect analysis of nonvolatile compounds that could be converted to their corresponding volatile species through chemical reactions [20–24]. Since Reaction (1) can be easily performed in a closed container (e.g., headspace sample vial), it is feasible to realize an indirect determination of carbonyl groups in lignins based on released hydrogen using HS-GC method. In this work, we developed a novel HS-GC method for rapid determination of the carbonyl groups in lignins based on the above reactions. The main focuses were to explore the reaction conditions (i.e., the addition of catalytic agent, reaction time and temperature) on the carbonyl conversion and the operation conditions for headspace equilibration in the HS-GC measurement.
Fig. 1. Chromatogram in a sample analysis.
NaBH4 to release hydrogen. The vials were then immediately put into the headspace autosampler for HS-GC measurement.
2. Materials and methods
3. Results and discussion
3.1. Nitrogen purging in sample preparation
All chemicals used in the experiment were of analytical grade and received from commercial sources. The standard model lignin compound solutions: acetovanillone (9.99 mmol/L), acetosyringone (9.62 mol/mL), syringealdehyde (10.5 mol/mL) and p-hydroxybenzaldehyde (9.26 mol/mL) solutions were prepared by dissolving the corresponding amount of these compounds in 0.1 mol/L NaOH solution, respectively. A standard NaBH4 stock solution was prepared by adding 0.1004 g of NaBH4 to 100 mL of 0.1 mol/L sodium hydroxide solution. A set of standard NaBH4 solutions with various concentrations were prepared by mixing the standard NaBH4 stock solution and 0.1 mol/L NaOH solution. The given grams of SiO2 (<200 meshes, analytical grade, Shanghai Lingfeng Chemical Reagent Co., Ltd.) were added in the reduction reaction. Lignin samples, labeled as L1–L4, were obtained through acid precipitation from four different kraft pulp mills. Lignin solutions were prepared by adding the given grams of the corresponding lignin samples to 0.1 mol/L NaOH solution.
According to Reactions (1) and (2), hydrogen is the only volatile species from these reactions in the headspace sample vial. However, oxygen involved in the sample vial may react with both carbonyl and NaBH4 at elevated temperature [16,25]. Therefore, it is necessary to purge the sample vial by an inert gas (e.g., nitrogen) before the reactions. Such a purging procedure can also effectively avoid the methanol formation from the demethoxylation of lignins due to the presence of oxygen . The GC column used in this work is not favorable for the methanol (also a volatile species) separation. In Fig. 1, it shows a chromatogram from HS-GC measurement on a sample after reduction and acidiﬁcation. As observed, hydrogen is the only volatile species from the reactions detected by GC.
2.2. Apparatus and operations All measurements were carried out using an automatic headspace sampler (DANI HS 86.50, Italy) and a GC system (Agilent 7890A, USA) equipped with a TCD and a GS-Q capillary column (30 m × 0.53 mm, J&W Scientiﬁc, US) operating at a temperature of 200 ◦ C with nitrogen carrier gas (ﬂow rate = 10.3 mL/min). Headspace operating conditions were as follows: the equilibration temp. = 40 ◦ C; needle and sampling coil temp. = 50 ◦ C; transfer line temp. = 60 ◦ C; vial pressurization = 0.2 min; and sample loop ﬁll time = 0.2. The volume of the headspace sample loop is 4.0 mL.
3.2. The conditions for carbonyl groups reduction 3.2.1. Dosage of NaBH4 As shown in Reaction (1), an excess amount of NaBH4 is essential for a complete conversion of carbonyl groups to hydroxyl groups. Fig. 2 shows the concentration of NaBH4 required to achieve conversion equilibrium for a given amount of carbonyl groups in a lignin compound at an alkaline medium (0.1 mol/L NaOH). It can be seen that the minimum NaBH4 concentration required for the complete reaction on such a sample (carbonyl groups = 9.26 mol) should be greater than 0.5 g/L.
2.3. Sample preparation and measurement procedures 1 mL of lignin solution was mixed with SiO2 (0–10 g) and 1 mL of NaBH4 solution in a headspace test vial (21.6 mL) ﬁlled with nitrogen, and then the vial was sealed with a septum. For aldehydes, the vials were placed in room temperature for various periods of time (range of 0.5–5 min). For acetones, the vials were placed in water bath and heated for 0–60 min at 80 ◦ C, and then cooled to temperature, and 1 mL of 0.8 mol/L sulfuric acid was injected into the sealed vial by a syringe to neutralize the alkaline medium and react with
Fig. 2. Effect of NaBH4 addition on carbonyl group conversion. *Lignin solution: 1.154 g/L of p-hydroxybenzaldehyde solution (i.e., carbonyl content: 9.26 mol).
J. Li et al. / J. Chromatogr. A 1404 (2015) 39–43
Fig. 3. Effect of SiO2 dosage on reduction reaction.
3.2.2. Dosage of catalyst As mentioned above, Reaction (1) is a very slow process even at an elevated temperature . However, the reaction can be greatly accelerated when adding 30 m/m of SiO2 solid (as a catalyst) . Since the reaction is conducted in a closed headspace sample vial and a pressurized sampling mode is used in the given autoheadspace sampler, such large amount of SiO2 powder in the vial increases the risk of clogging in the headspace sampling channels. Therefore, it is necessary to reduce the amount of SiO2 in the reaction. In Fig. 3, it shows the effect of SiO2 dosage on the reaction. The GC signal is proportional to the hydrogen released from the acidiﬁcation after the reduction reaction, in which the smaller signal indicates the less hydrogen is detected and thus more amount of carbonyl group is reduced. As shown in Fig. 3, when the amount of SiO2 added is greater than 5 g, the dosage effect on the reaction at the given condition becomes less signiﬁcant. Therefore, we chose to add 5 g of SiO2 in the reaction. 3.2.3. Reaction time In Fig. 4, it shows the time effect on the complete conversion (reduction) of an aromatic aldehyde (p-hydroxybenzaldehyde) or ketone compound (acetovanillone). It is clear that the conversion reaction on the carbonyl of aldehyde group is only in 2 min, which is much easier than that of ketone group (in 30 min) at the same conditions. Therefore, we chose 30 min as the reaction time to ensure all carbonyl groups in the compounds are reacted. The reaction time required in the present conditions is much shorter than the conventional carbonyl determination method reported previously [16,28].
Fig. 5. Effect of headspace equilibrium temperature.
Since the reaction on carbonyl in ketone groups is much slower than that of aldehyde groups, a short reaction time can be selected to the determination of aldehyde groups individually when it is needed. 3.2.4. Acidiﬁcation of the medium from reduction reaction and HS-GC testing The carbonyl reduction (Reaction (1)) must be performed in an alkaline medium (using NaOH). In order to determine the amount of residual NaBH4 after Reaction (1), the solution was acidiﬁed by adding a sufﬁcient amount of sulfuric acid. In the present method, a 1 mL of sulfuric acid solution (0.8 mol/L) was injected into the sealed headspace sample vial by a syringe. The residual NaBH4 in the solution can instantly decomposed to hydrogen gas and thus be measured by HS-GC. 3.3. Conditions for headspace equilibration It was observed that the hydrogen gas is instantly release to headspace after acidiﬁcation. In order to minimize the amount of hydrogen remaining in the liquid medium, it is favorable to perform the headspace equilibration at the temperatures higher than the room temperature. Fig. 5 shows that the temperature effect on the hydrogen measurement and it indicates that the complete equilibration on the hydrogen release can be achieved in 3 min at 40 ◦ C. To ensure the headspace equilibration, we chose 40 ◦ C and 10 min for the headspace equilibration in the present method. 3.4. Method calibration, precision, and validation. A simple external standard calibration can be employed in the present method, in which the calibration was performed on the basis of the addition of different volumes (0–1000 L) of p-hydroxybenzaldehyde solution (9.26 mol/mL) in a set of headspace sample vials. The samples were tested by HS-GC after acidiﬁcation. According to the GC signals (peak areas) from HS-GC Table 1 Repeatability testing for carbonyl determination in the lignin model compounds and lignin sample. Sample
Fig. 4. Effect of reaction time on the conversion of (a) ketone and (b) aromatic aldehyde compound.
Carbonyl content, mmol/g p-Hydroxybenzaldehyde
1 2 3 4 Average
7.27 8.67 7.56 7.82 7.83
6.22 5.98 6.59 6.42 6.30
1.01 1.07 1.09 0.96 1.03
J. Li et al. / J. Chromatogr. A 1404 (2015) 39–43
Table 2 Method comparison for the carbonyl analysis in lignin model compounds. Sample
Acetovanillone Acetosyringone Syringaldehyde p-Hydroxybenzaldehyde a
Relative error, %a
Carbonyl content, mmol/g Calculated
6.02 5.10 5.31 8.19
5.92 – 5.46 8.02
6.30 4.68 5.45 7.83
4.69 −8.20 2.55 −4.40
6.41 – −0.18 −2.37
Data comparison between the reference and the present HS-GC method.
Table 3 Method comparison for the carbonyl analysis in lignin samples. Sample ID
Carbonyl content, mmol/g
L1 L2 L3 L4 a
Nonea 0.83 0.98 1.16
0.16 0.91 1.03 1.22
Relative error, %
– 9.53 5.09 5.18
Note: the carbonyl content was not detected.
measurement on these samples, a standard calibration curve was obtained, that is A0 − An = 9365.74(±157.04) × m − 12.27(±8.58) (n = 6 and R2 = 0.999)
where A0 and An represent the GC signal of hydrogen from the sample with and without lignin compound, respectively, m represents the absolute amount (in mmol) of carbonyl groups in the headspace sample vial, respectively. The limit of quantiﬁcation (LOQ) in the present method is 7.85 × 10−3 mmol, which is calculated by the equation below, LOQ =
a + 10 × a s
where a, a and s represent the intercept, uncertainty of the intercept, and the slope in Eq. (3), respectively. The carbonyl content (C, mmol/g) in lignin compound can be calculated by the following equation, A0 − An + 12.27 C= 9365.74w
where w is the weight (in g) of the lignin sample added in the headspace sample vial. The repeatability testing of the present method was studied. The results show that the relative standard deviation (RSD) in four measurements is 7.74, 4.17 and 5.72% for p-hydroxybenzaldehyde, acetovanillone, and lignin sample respectively (see Table 1). The errors are associated with the uncertainties in both sample preparation and HS-GC measurements. The validation of the method was performed by using the data from the model lignin compounds with known carbonyl content or measured by the traditional oximation method  as the references. As listed in Table 2, the results of the carbonyl content of these samples tested by the present method match well with the theoretical amount calculated (relative error < 8.20%). The differences between the data measured by the oximation method and the present HS-GC method are within 7.0%. Different from the model lignin compounds, the molecular weights of lignins in the natural resources and deligniﬁcation processes are much higher. For these samples, the oximation method was used to validate the present HS-GC method. As shown in Table 3, the relative difference between the carbonyl contents of these samples measured by the present method and the oximation method are less than 10%. Therefore, the present HS-GC method is
justiﬁable to be used for the determination of carbonyl content in the lignin compounds in industrial related research and applications. It is also noticed that the sensitivity of the oximation method is lower than that of the present method, since it cannot be applicable to a sample with low carbonyl content, e.g., 0.16 mmol/g in this case. Therefore, the present method has the advantage not only on its efﬁciency but also sensitivity over the traditional oximation method in the quantiﬁcation of carbonyl content in lignins. 4. Conclusions A novel HS-GC technique for rapid determination of carbonyl groups in lignin compounds has been developed. Compared with the traditional oximation method, the present method is not only more efﬁcient (reaction time < 30 min) but also has good measurement precision (RSD < 7.74%) and accuracy (relative error < 10%). Therefore, it is more suitable to be used for rapid determination of carbonyl content in lignins or their enriched materials in the industrial related research and applications. Acknowledgements This work were supported by the National key Basic Research Program of China (973: 2012CB214705) and the Scientiﬁc Research Special Project of Provincial Higher Education Institutions of The Education Department of Fujian Province (JK2014015). References  A. Pinkert, D.F. Goeke, K.N. Marsh, S. Pang, Extracting wood lignin without dissolving or degrading cellulose: investigations on the use of food additivederived ionic liquids, Green Chem. 13 (2011) 3124–3136.  H. Sixta, Raw material for pulp, in: Handbook of Pulp, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2006.  J.W. Green, The Carbohydrates, Academic Press, New York, 1980.  A. Corma, S. Iborra, A. Velty, Chemical routes for the transformation of biomass into chemicals, Chem. Rev. 107 (2007) 2411–2502.  R.C. Sun, J. Tomkinson, Comparative study of lignins isolated by alkali and ultrasound-assisted alkali extractions from wheat straw, Ultrason. Sonochem. 9 (2002) 85–93.  J. Zakzeski, P. Bruijnincx, A. Jongerius, B. Weckhuysen, The catalytic valorization of lignin for the production of renewable chemicals, Chem. Rev. 110 (2010) 3552–3599.  W. Partenheimer, The aerobic oxidative cleavage of lignin to produce hydroxyaromatic benzaldehydes and carboxylic acids via metal/bromide catalysts in acetic acid/water mixtures, Adv. Synth. Catal. 351 (2009) 456–466.  M. Saisu, T. Sato, M. Watanabe, T. Adschiri, K. Arai, Conversion of lignin with supercritical water–phenol mixtures, Energy Fuels 17 (2003) 922–928.  J.L. Gardon, B. Leopold, Manufacture of lignin derivatives, US Patent 2934531, 1960.  E. Adler, J. Gierer, The alkylation of lignin with alcoholic hydrochloric acid, Acta Chem. Scand. 9 (1955) 84–93.  S.Y. Lin, C.W. Dence, Methods in Lignin Chemistry, Springer Series in Wood Science, Germany, 1992.  J. Gierer, B. Lenz, Reaction of lignin during sulphate cooking, Svensk. Papperstidn 68 (1965) 334–338.  O. Faix, B. Andersons, G. Zakis, Determination of carbonyl groups of six round robin lignins by modiﬁed oximation and FTIR spectroscopy, Holzforschung 52 (1998) 268–274.  E. Adler, J. Marton, Zur Kenntnis Der Carbonylgruppen im Lignin: I, Acta Chem. Scand. 13 (1959) 75–96.
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