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A separation and quantification method of levoglucosan in biomass pyrolysis
Industrial Crops & Products 113 (2018) 266–273
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A separation and quantification method of levoglucosan in biomass pyrolysis Jun-Qi Wanga,b, Ji-Lu Zhenga, Jin-Tao Wanga,b, Zhou-Min Lua,b, a b
T
⁎
College of Forestry, Northwest A&F University, Yangling, 712100, Shaanxi, China Key Laboratory of Western Environment and Ecological Research, Ministry of Education, Northwest A&F University, Yangling, 712100, Shaanxi, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Levoglucosan Bio-oil HPLC ELSD
A rapid method for the quantification of levoglucosan in pyrolysis liquids using high-performance liquid chromatography (HPLC) was developed. The method avoids the tedious and time-consuming sample preparation required by current analytical methods In this method, levoglucosan has better separation of xylose, glucose and fructose so as to avoid interference. The calibration curve coefficient of determination on levoglucosan was > 0.990 determined by an evaporative light scattering detector (ELSD). The relative standards for the new method were ≤1.34% at low concentration and ≤2.56% at high concentration. The spiked levoglucosan recovery on the pyrolysis liquid samples were between 96.79 and 99.13%. The research demonstrates that it is possible to obtain excellent accuracy and efficiency using HPLC to quantificate levoglucosan found in fast pyrolysis bio-oils.
1. Introduction Climate change, depletion of natural resource, and scarcity of fossil fuel have triggered the awareness of people. Environmental awareness in both the public and regulatory sectors has necessitated proper treatment of waste, such as waste water, unutilized biomass etc. For some pharmaceutical waste water, it can be treated based on the TiO2 system, which can facilitate a safe ecofriendly discharge to the atmosphere. For some unutilized biomass, it can be used for on the production of green fuel with different advance processing systems in a sustainable way (Sudip et al., 2012). For waste vegetable oils, enzymatic transesterification was performed to produce biodiesel (Loprestom et al., 2015). For agricultural and forestry residues, the most available biomass materials, they can be separate into three fractions (solid, liquid and gas) after pyrolysis (Kim and Dale, 2004; Vassilev et al., 2015). To achieve maximal yields of liquid, pyrolysis at short residence times is essential. After rapid cooling of the pyrolysis vapors, a dark brown liquid is formed, called bio-oil. The liquid produced is up to 80% of the initial dry mass (Bridgwater and Peacocke, 2000). Depending on bio-oil composition, it can be categorized into acids, sugars, phenols, hydroxyaldehydes, hydroxyketones, and alcohols. The presence of these components results in a relatively low energy density (16–18 MJ/kg, which is about 40–45% of that of hydrocarbon fuels), immiscibility with hydrocarbon fuels, and storage instability. Among these ingredients, the sugar fraction is particularly interesting as a
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source of chemicals and fuels (Vassilev et al., 2015; Liu et al., 2014). One of the main components of this fraction is levoglucosan (1,6-anhydro-β-D-glucopyranose), Usually the yield of levoglucosan from the pyrolysis of lignocellulosic biomass is only a small percent, but previous studies found that infusing biomass with a weak mineral acid solution prior to pyrolysis increased levoglucosan yields from the cellulose fraction to as much as 59 wt% (Kuzhiyil et al., 2012; Wang et al., 2016). Levoglucosan can be upgraded into transportation fuels and chemicals via fermentation to alcohols, catalytic synthesis to hydrocarbons, dehydration to heterocyclic aldehydes (furfurals) and aromatics (furans), and aqueous-phase re-forming to produce alkanes as building blocks of gasoline (Helle et al., 2007). Having realized the significance of levoglucosan in applications, it is important to establish an easy and quick method to determine levoglucosan. Unfortunately, levoglucosan content in pyrolysis oil is rarely analyzed because of its reactivity and sticking tendency. In pyrolysis system, evaporation and polymerization of levoglucosan are two competing simultaneous process (Bai et al., 2013). The concentrations of levoglucosan depend on pyrolysis conditions and raw materials. It usually gives a maximum yield at around 500 °C. Previous studies usually use primary fraction to separate pyrolysis oil into less complex fractions or mixture (Vitasari et al., 2011). It is difficult to determine the concentrations of levoglucosan in bio-oil by regular analytical methods because a significant number of close related structures usually exist in bio-oil at low concentrations. What is more, the distribution coefficients of polar compounds (levoglucosan, acetol,
Corresponding author at: College of Forestry, Northwest A&F University, Yangling, 712100, China. E-mail addresses: [email protected] (J.-Q. Wang), [email protected] (J.-L. Zheng), [email protected] (J.-T. Wang), [email protected] (Z.-M. Lu).
https://doi.org/10.1016/j.indcrop.2018.01.045 Received 8 September 2017; Received in revised form 21 December 2017; Accepted 19 January 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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acetic acid and glycoladehyde) are always in the same range, which indicates poor selectivity. Thus, developing methodology for an appropriate separation of levoglucosan is very necessary. While, due to thermal and chemical instability of pyrolysis oil, solvent extraction is a better method. In comparing quantification methods, HPLC is considered the most appropriate method. It is one of the most powerful and reliable methods for less volatile, polar and unstable steroids compounds (Alsaadi et al., 2013). Refractive index and ultraviolet spectroscopic detectors have a few inherent disadvantages, such as lacking sensitivity, limiting chromatographic resolutions, depending on flow rate etc (Alsaadi et al., 2013; Yin et al., 2006). So, the introduction of ELSD brought a major advance to HPLC in the detection and quantitative analysis of levoglucosan (Lafosse and Herbreteau, 2002). Except for this experiment, other studies have investigated levoglucosan by using different detections. One study used HPLC combined with high-resolution mass spectrometry to sensitively analysis levoglucosan. The authors mention an extensive bleeding of the carbohydrate column (amino packing Waters) which is not compatible with the mass spectrometry ion source (Dye and Yttri, 2005). This bleeding is also observed when some amino columns are coupled with ELSD and this bleeding lowers the sensitivity (Lafosse and Herbreteau, 2002). Another study had shown that HPLC with aerosol charge detection and a benson BC-100 Ca2+ column can analyze levoglucosan with a lower detection limit and avoid the interference from other compounds, however, this method is only available for atmospheric samples (Dixon and Baltzell, 2006). The interferences between levoglucosan and other monosaccharides are different in bio-oils and atmospheric aerosols (Kirchgeorg et al., 2014; Piot et al., 2012; Tessini et al., 2011; You et al., 2014). Another method was investigated by using ion-exclusion high-performance liquid chromatography with low wavelength (194 nm) spectroscopic detection to analyze levoglucosan. It only had a modest detection limit and the pre-treatment was complicated (Schkolnik et al., 2005). In China, Li and Zhuang used HPLC with refractive index detection to analyze levoglucosan from cotton pyrolysis oil, while this method lack full qualitative and quantitative analysis for levoglucosan (Zhuang et al., 2001). According to our knowledge, no adequate method has been developed to analyze levoglucosan in bio-oil by using HPLC-ELSD. Previous studies (Oasmaa and Kuoppala, 2008) reported that the sugar fraction of bio-oil contains monosaccharides, anhydrosugars and anhydropolysaccharides. They indicated that it is difficult to separate and determine levoglucosan from bio-oil, thus an appropriate pretreatment of HPLC and HPLC-ELSD method in this experiment is important. The aims of this study were (i) to determine a separation method for levoglucosan in HPLC-ELSD analysis and (ii) to use response surface methodology (RSM) to optimize parameters on ELSD (Dasgupta et al., 2015; Chakraborty et al., 2012a,b), and (iii) to measure quantitatively the content of levoglucosan in bio-oil.
Table 1 Drift tube temperature (DTT), column temperature (CT) and gas flow rate (GFR) values for the three-parameter factorial designed extraction experiment. Experimental factors
Parameter level
A drift tube temperature (DTT,°C) B column temperature (CT,°C) C gas flow rate (GFR, L/min)
Low (−1)
Middle (0)
High (1)
60 10 2.5
70 15 3
80 20 3.5
Table 2 Box-Behnken experimental design matrix for levoglucosan. Run no.
A: DTT °C
B: CT, °C
C: GFR, L min−1
Peak area, mV s 106
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
60 70 80 80 60 60 80 80 70 70 80 60 70 80 80 80 80
10 20 15 15 20 15 10 15 15 15 20 15 10 10 15 20 15
3 3 3 3 3 3.5 3.5 3 3.5 2.5 2.5 2.5 3 2.5 3 3.5 3
1.85 2.63 2.59 2.59 1.92 1.90 1.95 2.56 2.77 2.90 2.25 2.25 2.55 2.26 2.56 2.27 2.56
2. Experimental Fig. 1. Schematic diagram of fluidized bed reactor (1: heater, 2: cotton straw collector, 3: screw feeder, 4: fluidized bed, 5: cyclone, 6: char collector, 7: condenser, 8: bio-oil tank).
2.1. Materials and reagents Bio-oil was produced with air-dried rice straw in a fast pyrolysis process development unit consisting of a fluidized bed operated at final pyrolysis temperature of 500 °C, holding time of 1.4 s, particle size of 600–650 μm and acid concentration of 8% (The optimum parameters based on preliminary single experiments). The reference compounds analytical grade fructose, xylose, glucose and levoglucosan were purchased from Sigma Chemical Company (Beijing, China) with a purity > 99%. Methanol and acetonitrile were purchased from Tianjin Kermel Chemical Reagent Co., Ltd (Tian Jin, China) with HPLC-grade. Ultra-pure water was produced by Milli-Q system (Millipore, Bedford, MA, USA). Standard solutions of fructose, xylose, glucose and levoglucosan were prepared at the level of 2.0 mg/mL in ultra-pure water. Working
standard solutions were prepared as needed by appropriate dilution of the stock solutions in ultra-pure water. All sample were stored at 4 °C in the refrigerator.
2.2. Sample separation Ultra-pure water (10 mL) was added to bio-oil (10 mL), and then stirred (25 °C) for 2 h. The mixture was centrifuged (15 min, 3500g, OPTIMA L-100XP, Beckman, USA) and the liquid decanted. The liquid was then added with granular activated carbon (0.5 g) and shake at an Oven Controlled Crystal Oscillator (WHY-2, China) for 12 h. Then, the mixture was filtered and combined with the filtrate. After that, Ca(OH)2 267
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Table 3 The gradient elution and isocratic elution sequence used in the HPLC-ELSD method for carbohydrate analysis using acetonitrile (ACN) and water as mobile phase. No.
Types of column
Column temperature
Mobile phase
1
XB-NH2
10 °C
Gradient elution
2 3 4
Isocratic elution
5 6 7
HILIC-D
10 °C
8 9
Gradient elution Isocratic elution
35 °C
10
Gradient elution Isocratic elution
was added to the filtrate, with proper pH adjustment to around 12. The alkaline liquid was then kept in a 60 °C water bath (HH-2 digitial constant temperature tank, China) for 3 h to remove the precipitation of organic colloid and acidic compounds. Then filter, the filtrate was collected and 10 mL of the filtrate was added to three times of 95% alcohol with continue whisking. After that, the mixture was centrifuged (3500g) for 15 min and residue from the centrifuge was dissolved in 10.0 mL water. Repeat the above alcohol washing and centrifugation processes. Finally, dissolve the centrifugal residue in the purified water (1.0 mL). The solution was filtered through 0.22 μm filters (Xinjinghua Co., Shanghai, China) before use and stored at 4 °C for further analysis.
Times (min) ACN (%) Times (min) ACN (%) Times (min) ACN (%) Times (min) ACN (%) Times (min) ACN (%) Times (min) ACN (%) Times (min) ACN (%) Times (min) ACN (%) Times (min) ACN (%) Times (min) ACN (%)
0 88 0 90 0 90 0 78 0 85 0 82 0 90 0 92 0 90 0 92
15 78 15 80 20 80 20 78 20 85 20 82 20 80 20 92 20 80 20 92
26 76.5 30 80 25 90 25 78 25 85 25 82 25 50 25 92 25 50 25 92
30 88 35 90 36 90 35 78 35 85 35 82 35 50 35 92 35 50 35 92
40 88 46 90 40 90 40 78 40 85 40 82 40 50 40 92 40 50 40 92
remained in optimal states. Table 1 shows the operational ranges of the input variables. The software Design Expert version 8.0.6.1 was used to design the Box-Behnken design, and to perform the response surface regression and the analysis of variance (P < 0.05). The complete design matrix comprising 17 experiments are listed in Table 2. Using a second order polynomial Eq. (1) to express the relation between the dependent variable Y (the peak area) and independent variables X (drift tube temperature, column temperature and gas flow rate). In Eq. (1), β0 is the intercept, βi the linear coefficient, βii the quadratic coefficient, βij the interaction coefficient and ε, the residual.
Y = β0 +
2.3. HPLC instrumentation and chromatographic conditions
∑ βi Xi + ∑ βii Xi2 + ∑ βijXiXj + ε
(1)
The mobile phase flow rate was used at 1.0 mL/min and the temperature of the chromatography oven (40 °C) was maintained as previously reported (Condezo-Hoyos et al., 2015). All aqueous solutions (include treated bio-oil samples) were filtered by 0.22 μm membrane syringe filters before being injected into the HPLC.
The HPLC apparatus was equipped with an Agilent G1329B autosampler, Agilent G1311c quaternary pump system 1260 series with online degasser, Agilent G1316A thermostatic oven, Agilent HPLC control unit 1260 series and Alltech 2000ES-ELSD. Non-UV sensitive compounds can be detected and quantified by ELSD because it is a quasi-universal detector used as mass dector (Condezo-Hoyos et al., 2015; Lafosse and Herbreteau, 2002). Data processing was carried out with the EZchrom Elite Agilent technology. The HPLC separation and quantification were made on an Ultisil® XB-NH2 column (250 mm × 4.6 mm, 5 μm particle size) (Welch, China) and a Kromasil 60-5 Hilic-D column (250 mm × 4.6 mm, 5 μm particle size) (AKZO NOBEL, Sweden) with 10 μL sample injection. The mobile phase consisted of two solvents, solvent A (water) and solvent B (acetonitrile) in gradient elution with a flow rate of 1.0 mL/min. The four carbohydrates were eluted with the increasing proportion of solvent A from 12% to 22% over 0–15 min; 22% to 23.5% over 15–26 min; then, decrease the proportion of solvent A from 23.5% to 12% over 26–30 min. The column temperature was kept at 15 °C. The drift tube temperature was set at 70 °C and nebulizer gas (air) flow rate was 3.0 L/ min.
2.5. Calibration curves, limits of detection and quantification (Nogueira et al., 2005; Yin et al., 2006) Different fructose, xylose, glucose and levoglucosan standards (high-, medium- and low concentration) were used for the constructive of calibration curves. Each standard was used three times at different concentrations, using acetonitrile-water as mobile phase, and all parameters of ELSD were set as optimal. The estimate value of limits of detection (LOD) and quantification (LOQ) were determined by different signal-to-noise (S/N). LOD was calculated as the concentration corresponding to three times the peak height of the baseline noise (S/N = 3); whereas LOQ was set as ten times the peak height (S/N = 10). Signal to noise ratio is calculated using LC Solution Software. The LOD and LOQ values were calculated experimentally from serial dilution of standard levoglucosan concentrations.
2.4. Optimization of ELSD parameters
2.6. Accuracy (Alsaadi et al., 2013; Yin et al., 2006)
To investigate the optimization parameters of ELSD detection for levoglucosan analysis, this paper used the Box-Behnken design to approach optimal design. The ELSD values were optimized with three different parameters: drift tube temperature, column temperature and gas flow rate (Dixon and Baltzell, 2006; Kao et al., 2008; Bhandari et al., 2008; Condezo-Hoyos et al., 2015). The other parameters
A recovery test was used to evaluate the accuracy of this analysis method. For the recovery experiments, three different concentrations (80, 100 and 120%) of the reference compounds were spiked in levoglucosan sample. Every sample was analyzed three times. 268
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Fig. 2. For experimental conditions of each method in bio-oil, see Table 1. (a) gradient elution on Ultisil® XB-NH2 column, (b) isocratic elution of Ultisil® XB-NH2 column, (1) = levoglucosan, (2) = xylose, (3) = fructose, (4) = glucose.
developed by comparing of retention times and peak areas of each experiment to assess its repeatability and reproducibility. The precision of the developed method was validated by repeating the procedure in duplicate on three different days with newly prepared mobile phase and samples.
2.7. Precision (Bhandari et al., 2008; Alsaadi et al., 2013) Intra- and inter-day precise measurements were carried out on three consecutive days. Each day, low and high sample concentrations of levoglucosan were independently injected in triplicate. The method was
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absorbed by active carbon, most phenols, acids, organic colloid and others still existed in the aqueous layer. Ca(OH)2 was added to remove these impurities. What is more, CaCO3 is formed by the reaction of Ca (OH)2 and the CO2 dissolved in water. CaCO3 is an absorbent, which means it can absorb impurities with good dispersity (Fangyao and Yuegang, 2004). After these steps, 95% alcohol as a non-aqueous solvent was used to wash the remained bio-oil for further extraction and purification.
Table 4 Analysis of the model variances. Variance
Sum of squares
df
Mean square
F value
P (prob > F)
Model A B C AB AC BC A2 B2 C2 Residual Lack of Fit Pure Error Cor total
1.66 0.56 0.024 0.017 2.445E-003 0.010 0.030 0.82 0.39 0.032 8.043E-003 6.96E-003 1.080E-003 1.87
9 1 1 1 1 1 1 1 1 1 7 3 4 16
0.18 0.56 0.024 0.017 2.445E-003 0.010 0.030 0.82 0.39 0.032 1.149E-003 2.321E-003 2.700E-004
160.34 488.86 21.27 14.85 2.13 9.08 25.79 716.79 342.30 27.99
< 0.0001 < 0.0001 0.0025 0.0063 0.1880 0.0196 0.0014 < 0.0001 < 0.0001 0.0011
8.60
0.0323
3.2. Method development 3.2.1. Chromatography column and mobile phase selection Levoglucosan can be described as a pyranose ring with two ether groups and three hydroxyl groups (Bennett et al., 2009). The structure of levoglucosan shows that it is soluble in water and insoluble in nonhydroxylic solvent. What is more, levoglucosan is present in low concentrations of bio-oil, between 3 and 6% depending on pyrolysis conditions and the raw materials (Tessini et al., 2011). The choice of a suitable chromatography column and mobile phase is the key to separation. To effectively separate levoglucosan from the other three main carbohydrates (fructose, xylose and glucose) in the bio-oil sample, two different types of columns (Ultisil® XB-NH2 column, Kromasil 60-5 Hilic-D column) with different mobile phase systems were tested. In the Kromasil 60-5 Hilic-D column, acetonitrile-water was used in both gradient and isocratic elution to obtain a better separation of the four carbohydrates (Table 3). Other conditions, such as an increase or reduction in the flow rate or adjustment of the pH value of mobile phases were also taken, but in all cases, the separation of levoglucosan was not good. What is more, the influence of concentration of water in the mobile phase was observed, and the separation and retention time of the carbohydrates in Kromasil 60-5 Hilic-D column was also changed. It is worthwhile to note that both peak interferance and broadening effects still strongly remained on the Hilic-D column, whether in gradient elution or isocratic elution. What is more, when column temperature changed from 10 °C to 35 °C, the separation of the carbohydrates was not obviously changed in the Hilic-D column. After that, the Ultisil® XB-NH2 column was designed to retain and separate fructose, xylose, glucose and levoglucosan. Many kinds of mobile phases were generally used in the NH2 column. Different compounds require different mobile phases due to their chemical properties (Yang et al., 2012). In this study, acetonitrile-water was chosen as the mobile phase. The resolutions of investigated compounds (fructose, xylose, glucose and levoglucosan) were tested with different elution programs. The method 3 and method 4 had a peak overlapping not only in fructose but also in glucose (Fig. 2a, b). While the elution programs method 2, method 5 and method 6 can separate the four carbohydrates but with a wider peak (Fig. 2a, b). Wider peak meant lower column efficiency (Dixon and Baltzell, 2006). Furthermore, when the water content is below 10% (v/v), the chromatographic peaks became broad and asymmetric (Fig. 2b). Therefore, in the next experiments, water content in mobile phase should be increased. By testing several solvent
2.8. Statistical analysis Statistics of the compositional data of levoglucosan was conducted with the aid of SPSS (SPSS17 for Windows, SPSS Inc., Chicago, IL, USA). Analysis of variance was used to statistically analyze the results. The relative standard deviation (RSD) was used to assess intra- and interday precisions by three replicates per sample and carried out by the same operator. Differences were considered to be statistically significant when p < 0.05. 3. Results and discussion 3.1. Sample separation In the previous bio-oil pyrolysis system, pyrolysis temperature, particle size, reaction time and other parameters were considered the key factors (Abnisa et al., 2011; Isa et al., 2011; Lazzari et al., 2016). In this experiment, in order to obtain the bio-oil with high content levoglucosan, the fluidized bed fast pyrolysis reactor with the condenser system used for production of the bio-oil was shown in Fig. 1. The amount of rice straw samples used in this experiment was 500 g. Sugar within the bio-oil matrix is difficult to analyze because the large number of variations that can occur and potential interference with other bio-oil components (Rover et al., 2013). Previous studies also showed, levoglucosan has a weak hydrogen bond interaction with water, which leads to higher aqueous concentration (Vitasari et al., 2011). Thus, according to Nicole M. Bennett et al. (2009), 10 mL high purity water was initially presented in 10 mL bio-oil, stirred for 2 h and then centrifuged for 15 min at 3500g. Most levoglucosan would mix with water. Other viscous constituents, which were not dissolved in water, as lignin-derived compounds would sink to the bottom. Active carbon was added to absorb no hydrogen bonded substances (Huber et al., 2006) which still present in bio-oil, like pigments and aromatic compounds. In addition to these high molecular weight compounds
Fig. 3. 3-D surface plot showing the interaction of DTT, CT and GFR on the ELSD response of levoglucosan standard. DTT = drift tube temperature; CT = column temperature; GFR = gas flow rate.
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Fig. 4. Calibration curves for fructose, xylose, glucose and levoglucosan.
significant for the levoglucosan analyzed. In ELSD, increasing the column temperature to 30 °C lead to a better separation. In a general way, sugar analysis is preferred at a higher temperature in order to obtain better column efficiency while increasing the column temperature too much also can decrease S/N ratio (Fig. 3). At high temperature, the S/N is reduced because of the increase of baseline, which also leads to a greater column bleed (Dixon and Baltzell, 2006). A column temperature of 15.08 °C was selected based on the Box-Behnken response surface design and used in the following analyses. In the HPLC-ELSD analysis, the drift tube temperature is also an important parameter (Kao et al., 2008). Solvent cannot be completely evaporated at low temperatures while at high temperatures, the detector response will also be decreased, because the partial vaporization of the analytes leads to a decrease in particle size (Fig. 3). The S/N ratio was improved as the drift tube temperature was increased (Lecoeur et al., 2015; Man Ki et al., 1996). However, when the drift tube temperature was increased more than 70 °C, a substantial loss of analytes was observed (the detector response decreased). Thus, based on the Box-Behnken response surface design, the optimal drift tube temperature was 70.46 °C. Regarding gas flow, lower or higher rates can cause a decreases in the ELSD response. The higher the gas flow rate, the smaller the ELSD response value (Fig. 3). While, a lower gas flow rate produces larger droplets, resulting in spike and noisy signals in HPLC analysis. On the other hand, a higher gas flow rate forms smaller droplets, which results in less effective light scattering. Then there will be a decrease in ELSD response value (Bhandari et al., 2008; Condezo-Hoyos et al., 2015; Man Ki et al., 1996). Thus, in this study, based on the Box-Behnken response surface design, the optimal gas flow rate was determined to be 2.92 L/min. In view of the practicability, the following ELSD parameters were set to 15 °C column temperature, 70 °C drift tube temperature and 3.0 L/min gas flow rate. The experimental ELSD parameter changes, trends of this work are quite comparable to others. Previous studies showed similar tendencies of these three parameters. For example, levoglucosan separated from atmospheric aerosols and was detected with an aerosol charge detection (Dixon and Baltzell, 2006). When the column temperature was below or above the optimal value (50 °C), the response value would decrease. Drift tube temperatures of 35 °C and 145 °C were set as optimal values for plasticizers and ginseng saponins (Lecoeur et al., 2015; Man Ki et al., 1996). Neither increasing nor decreasing the optimal temperature could result in obtaining a maximal S/N ratio. Optimal gas flow rate of 40 mm rotameter units and 2.0 SLM respectively for ginseng saponins and P. kurroa were investigated (Bhandari et al., 2008; Man Ki et al., 1996). Their experiments showed that, lower or higher gas flow rate could not obtain the best response of ELSD.
Table 5 The intra-day and inter-day precision of the proposed method. No.
Intra-day (n = 6)
Inter-day (n = 6)
High concentration
Low concentration
tR
Area
tR
Area
1 2 3 4 5 6 RSD% 1
9.04 9.06 9.05 9.05 9.06 9.04 0.13 9.04 ± 0.01
70753 69478 71298 69914 71205 70996 0.93 70753(ave.)
9.08 9.09 9.09 9.09 9.07 9.08 0.06 9.08 ± 0.01
2
9.02 ± 0.01
69478(ave.)
9.09 ± 0.01
3
8.98 ± 0.01
72298(ave.)
9.09 ± 0.01
4
8.99 ± 0.01
69502(ave.)
9.08 ± 0.01
5
8.99 ± 0.01
70128(ave.)
9.07 ± 0.01
6
8.96 ± 0.01
69392(ave.)
9.09 ± 0.01
RSD%
0.44
1.60
0.47
39023 38038 38257 38145 39756 39145 1.34 39942 (ave.) 39327 (ave.) 38639 (ave.) 38826 (ave.) 38063 (ave.) 37108 (ave.) 2.56
Ave. means the average of the retention time and peak area in one day.
systems, proper proportion was selected (method 1), results showed that the Ultisil® XB-NH2 column can provide good separation of fructose, xylose, glucose and levoglucosan from the bio-oil, the whole elution time was within 24 min and without any interfering peaks (Fig. 2a). In this study, the retention times were 20.53 min for fructose, 16.24 min for xylose, 23.84 min for glucose and 9.08 min for levoglucosan. 3.2.2. ELSD parameters optimization The drift tube temperature, column temperature and gas flow rate should be optimized to obtain a maximum of ELSD response. Thus, this study used RSM to obtain the maximum ELSD response to analyze levoglucosan. In order to optimize ELSD, a Box-Behnken response surface design with three central points was used. The independent variables were determined as: drift tube temperature (DTT), column temperature (CT) and gas flow rate (GFR). The peak area (response) was used for the dependent variable as in the previous reports (Condezo-Hoyos et al., 2015). In this study, the Box-Behnken design had been shown to be more efficient than the central composite design (Ferreira et al., 2007). Based on single-factor experiments, three levels of each parameter were determined (Table 2). According to the data (Table 4) subjected to the analysis of variance (ANOVA) of regression model (P < 0.0001) and the value of R2 (0.9961) demonstrated that the quadratic model is
3.3. Method validation The developed HPLC-ELSD method showed good sensitivities and 271
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Table 6 Recovery of levoglucosan by spiking method. No.
tR/min
Initial content (μg)
Area
Sample Added (μg)
Content after adding (μg)
Average recovery (%)
RSD (%)
Standard (low) Standard (high) Sample 80% added 100% added 120% added
9.03 9.03 9.04 9.03 9.04 9.04
9.08 36.32 10.9 10.9 10.9 10.9
18532 72447 26532 39685 42641 48431
– – – 8.72 10.9 13.08
– – – 19.45 21.1 23.43
– – – 99.13 96.79 97.71
– – – 0.79 0.80 1.14
Table 7 Separation and quantification of levoglucosan in bio-oil by HPLC-ELSD. No (low concentration/high concentration)
Levoglucosan 18.16/36.32 μg/mL Fructose 18.98/37.96 μg/mL Xylose 10.0/20.0 μg/mL Glucose 19.7/39.4 μg/mL
Standard reference
Bio-oil sample
Content (mg/ml)
High concentration
Low concentration
tR/min
Area
tR/min
Area
tR/min
Area
9.08 20.53 16.24 23.84
74289 189843 73764 383389
9.09 20.56 16.22 23.93
39011 86863 43695 190212
9.11 – – –
67811 – – –
0.033 – – –
“–” means not detected. Journal：
•
recovery of levoglucosan was found between 96.79 and 99.13%, and the average RSD was below 1.14% (Table 6). Fructose, xylose and glucose were tested for verifying the good separation of levoglucosan by this analysis method. These three carbohydrates have had no effect on the quantification and separation.
linear responses to the studied carbohydrates (fructose, xylose, glucose and levoglucosan), which were good enough to quantify them. Calibration curves were obtained by injecting each standard solution (10 μL) of fructose (44.4, 88.8, 222, 444, 666 and 888 μg/mL), xylose (68.8, 137.6, 344, 688, 1032 and 1376 μg/mL), glucose (20.0, 40.0, 100, 200, 300 and 400 μg/mL) and levoglucosan (8.8, 17.6, 44, 88, 132 and 176 μg/mL) in triplicate. The linearity was evaluated based on the correlations between the nature logarithm of peak areas and the nature logarithm of concentration of the four analytes. The linear relationship followed the equation Y = aX + b, as shown in Fig. 4. All of the R2-values for the regression lines were greater than 0.980. The results of these four analytes correspond well to other researches (Dixon and Baltzell, 2006; Nogueira et al., 2005; Wan and Yu, 2006). Levoglucosan was identified by comparing its retention time with the standard one. The detection limits were estimated as the concentration providing a signal three times or ten times higher than the standard deviation of the background noise. LOD and LOQ for levoglucosan were calculated experimentally from serial dilution. Therefore, successive dilutions were dertermined to find the suitable concentration of levoglucosan on the measurement without confusion with background noise. LOD was 3.632 μg/mL and LOQ was 9.08 μg/ mL for levoglucosan. This showed that this analysis method had a lower LOD, which was sensitive enough to detect levogluocsan. The precision of this method was determined in consideration of the RSD of levoglucosan at two different concentrations: low (0.05 mg/mL) and high (0.2 mg/mL). In order to detect intra-day variation with precision, the low and high concentrations of levoglucosan were injected in six times within one day. For inter-day precision, the two concentrations were injected in triplicates on six different days (Table 5). At the higher concentration level, the RSD of peak area and retention time were 0.13% and 0.93% for intra-day precision and 0.44% and 1.6% for inter-day precision, respectively. At the lower concentration level, the RSD of peak area and retention time were 0.47% and 2.56% for interday precision and 0.06% and 1.34% for intra-day precision, respectively. These values demonstrated this method is of good precision. Lower RSD values of levoglucosan in inter-day and intra-day meant better repeatability and reproducibility. The accuracy of the method was determined by investigating the recovery of levoglucosan at three levels (i.e. 8.72 μg, 10.9 μg and 13.08 μg) under the valid HPLC method. Results showed that, the
3.4. Extraction method validation An optimal extraction should include a single step or multiple steps to remove one or more unwanted components (Kakiyama et al., 2006). In this paper, unwanted components were removed by precipitation and extraction. Previous studies showed that fructose, xylose, glucose and levoglucosan are abundant in the sugar fraction of bio-oil (Tessini et al., 2011). Thus, in this work a simple method was established for separating and quantifying levoglucosan in bio-oil and fractions thereof, by using the HPLC-ELSD technique. As shown in Table 7, HPLC showed that with the treatment shown in Section 2.2, fructose, xylose and glucose were undetected (below the limit of detection), while the content of levoglucosan in the rice straw bio-oil was 0.033 mg/mL. The content of levoglucosan determined by this study is quite comparable to others (Tessini et al., 2011). Therefore, this pre-treatment was suited for determination of levoglucosan in bio-oil. 4. Conclusion We have described a separation method of levoglucosan from bio-oil and established an easy and quick method to determine levoglucosan through the HPLC method with Alltech 2000ES-ELSD, Ultisil® XB-NH2 column and acetonitrile-water as the mobile phase. The optimal ELSD parameters for separating fructose, xylose, glucose and levoglucosan was achieved at 15 °C column temperature and by adjusting ELSD setting at 70 °C drift tube temperature and 3.0 L/min gas flow rate. The method for levoglucosan quantification is a fast-reliable method with low LOD, high sensitivity, precision and good accuracy. Recovery for levoglucosan was in the range of 96.79–99.13%, while LOD was 3.632 μg/mL and LOQ was 9.08 μg/mL. The content of levoglucosan in the rice straw bio-oil was 0.033 mg/mL. The main advantage of this method is the easier sample extraction and no need of derivatization. What is more, the optimal conditions established with low LOD for high sensitive detection of levoglucosan would be easily applied to any other 272
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Total water-soluble sugars quantification in bio-oil using the phenol–sulfuric acid assay. J. Anal. Appl. Pyrolysis 104, 194–201. Schkolnik, G., Falkovich, A.H., Rudich, Y., Maenhaut, W., Artaxo, P., 2005. New analytical method for the determination of levoglucosan, polyhydroxy compounds, and 2methylerythritol and its application to smoke and rainwater samples. Environ. Sci. Technol. 39, 2744–2752. Sudip, C., Varun, A., Debolina, M., Koris, A., 2012. Biomass to biofuel: a review on production technology. Asia-Pacific J. Chem. Eng. 7, S254–S262. Tessini, C., Vega, M., Müller, N., Bustamante, L., von Baer, D., Berg, A., Mardones, C., 2011. High performance thin layer chromatography determination of cellobiosan and levoglucosan in bio-oil obtained by fast pyrolysis of sawdust. J. Chromatogr. A 1218, 3811–3815. Vassilev, S.V., Vassileva, C.G., Vassilev, V.S., 2015. Advantages and disadvantages of composition and properties of biomass in comparison with coal: an overview. Fuel 158, 330–350. Vitasari, C.R., Meindersma, G.W., de Haan, A.B., 2011. Water extraction of pyrolysis oil: the first step for the recovery of renewable chemicals. Bioresour. Technol. 102, 7204–7210. Wan, E.C.H., Yu, J.Z., 2006. Determination of sugar compounds in atmospheric aerosols by liquid chromatography combined with positive electrospray ionization mass spectrometry. J. Chromatogr. A 1107, 175–181. Wang, J.Q., Wei, Q., Zheng, J.L., Zhu, M.Q., 2016. Effect of pyrolysis conditions on levoglucosan yield from cotton straw and optimization of levoglucosan extraction from bio-oil. J. Anal. Appl. Pyrolysis 122, 294–303. Yang, G.D., Gao, R., Wang, Y., Li, J.C., Hu, Y.C., Kang, D.J., Li, Y.H., Li, H.L., Geng, G.X., Wang, J.H., 2012. Determination of swainsonine in the endophytic Undifilum fungi by high-performance liquid chromatography with evaporative light-scattering detector. Toxicon 60, 44–49. Yin, J., Yang, G., Wang, S., Chen, Y., 2006. Purification and determination of stachyose in Chinese artichoke (Stachys Sieboldii Miq.) by high-performance liquid chromatography with evaporative light scattering detection. Talanta 70, 208–212. You, C., Yao, T.D., Gao, S.P., Gong, P., Zhao, H.B., 2014. Simultaneous determination of levoglucosan, mannosan and galactosan at trace levels in snow samples by GC/MS. Chromatographia 77, 969–974. Zhuang, X.L., Zhang, H.X., Yang, J.Z., Qi, H.Y., 2001. Preparation of levoglucosan by pyrolysis of cellulose and its citric acid fermentation. Bioresour. Technol. 79, 63–66.
types of bio-oil. This research has demonstrated the HPLC-ELSD method gave highly reproducible results while providing a reliable standardized test method for quantification of levoglucosan in bio-oil. This means this method is effective in levoglucosan analysis, and may provide an economic detection method for levoglucosan conversion mass production. Acknowledgements This study was carried out based on the University’s Construction of Agricultural Science and Technology Promotion mode (XTG2017014) which was supported by treasury. This study also received assistance from the Key Laboratory of Western Environment and Ecological Research. The authors wish to thank Ms. Janet Calhoun (The Ohio State University) for critical review. References Abnisa, F., Daud, W., Husin, W.N.W., Sahu, J.N., 2011. Utilization possibilities of palm shell as a source of biomass energy in Malaysia by producing bio-oil in pyrolysis process. Biomass Bioenergy 35, 1863–1872. Alsaadi, M.M., Christine Carter, K., Mullen, A.B., 2013. High performance liquid chromatography with evaporative light scattering detection for the characterisation of a vesicular delivery system during stability studies. J. Chromatogr. A 1320, 80–85. Bai, X., Johnston, P., Sadula, S., Brown, R.C., 2013. Role of levoglucosan physiochemistry in cellulose pyrolysis. J. Anal. Appl. Pyrolysis 99, 58–65. Bennett, N.M., Helle, S.S., Duff, S.J.B., 2009. Extraction and hydrolysis of levoglucosan from pyrolysis oil. Bioresour. Technol. 100, 6059–6063. Bhandari, P., Kumar, N., Singh, B., Kaul, V.K., 2008. Simultaneous determination of sugars and picrosides in Picrorhiza species using ultrasonic extraction and high-performance liquid chromatography with evaporative light scattering detection. J. Chromatogr. A 1194, 257–261. Bridgwater, A.V., Peacocke, G.V.C., 2000. Fast pyrolysis processes for biomass. Renew. Sustain. Energy Rev. 4, 1–73. Chakraborty, S., Aggarwal, V., Mukherjee, D., Andras, K., 2012a. Biomass to biofuel: a review on production technology. Asia-Pac. J. Chem. Eng. 7, S254–S262. Chakraborty, S., Drioli, E., Giornoa, L., 2012b. Development of a two separate phase submerged biocatalytic membrane reactor for the production of fatty acids and glycerol from residual vegetable oil streams. Biomass Bioenergy 46, 574–583. Condezo-Hoyos, L., Pérez-López, E., Rupérez, P., 2015. Improved evaporative light scattering detection for carbohydrate analysis. Food Chem. 180, 265–271. Dasgupta, J., Singh, M., Sikder, J., Padarthi, V., Chakraborty, S., Curcio, S., 2015. Response surface-optimized removal of Reactive Red 120 dye from its aqueous solutions using polyethyleneimine enhanced ultrafiltration. Ecotoxicol. Environ. Saf. 121, 271–278. Dixon, R.W., Baltzell, G., 2006. Determination of levoglucosan in atmospheric aerosols using high performance liquid chromatography with aerosol charge detection. J. Chromatogr. A 1109, 214–221. Dye, C., Yttri, K.E., 2005. Determination of monosaccharide anhydrides in atmospheric aerosols by use of high-performance liquid chromatography combined with highresolution mass spectrometry. Anal. Chem. 77, 1853–1858. Fangyao, N., Yuegang, H., 2004. Study on sugar juice clarification using over-fine calcium carbonate. Guangxi J. Light Ind. 17–19. Ferreira, S.L.C., Bruns, R.E., Ferreira, H.S., Matos, G.D., David, J.M., Brandão, G.C., da Silva, E.G.P., Portugal, L.A., dos Reis, P.S., Souza, A.S., dos Santos, W.N.L., 2007. BoxBehnken design: an alternative for the optimization of analytical methods. Anal. Chim. Acta 597, 179–186. Helle, S., Bennett, N.M., Lau, K., Matsui, J.H., Duff, S.J.B., 2007. A kinetic model for production of glucose by hydrolysis of levoglucosan and cellobiosan from pyrolysis oil. Carbohydr. Res. 342, 2365–2370. Huber, G.W., Iborra, S., Corma, A., 2006. Synthesis of transportation fuels from biomass: chemistry, catalysts, and engineering. Chem. Rev. 106, 4044–4098. Isa, K.M., Daud, S., Hamidin, N., Ismail, K., Saad, S.A., Kasim, F.H., 2011. Thermogravimetric analysis and the optimisation of bio-oil yield from fixed-bed pyrolysis of rice husk using response surface methodology (RSM). Ind. Crops Prod. 33, 481–487. Kakiyama, G., Hosoda, A., Iida, T., Fujimoto, Y., Goto, T., Mano, N., Goto, J., Nambara, T., 2006. A direct method for the separation and quantification of bile acid acyl glycosides by high-performance liquid chromatography with an evaporative light scattering detector. J. Chromatogr. A 1125, 112–116.
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Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
A separation and quantification method of levoglucosan in biomass pyrolysis Jun-Qi Wanga,b, Ji-Lu Zhenga, Jin-Tao Wanga,b, Zhou-Min Lua,b, a b
T
⁎
College of Forestry, Northwest A&F University, Yangling, 712100, Shaanxi, China Key Laboratory of Western Environment and Ecological Research, Ministry of Education, Northwest A&F University, Yangling, 712100, Shaanxi, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Levoglucosan Bio-oil HPLC ELSD
A rapid method for the quantification of levoglucosan in pyrolysis liquids using high-performance liquid chromatography (HPLC) was developed. The method avoids the tedious and time-consuming sample preparation required by current analytical methods In this method, levoglucosan has better separation of xylose, glucose and fructose so as to avoid interference. The calibration curve coefficient of determination on levoglucosan was > 0.990 determined by an evaporative light scattering detector (ELSD). The relative standards for the new method were ≤1.34% at low concentration and ≤2.56% at high concentration. The spiked levoglucosan recovery on the pyrolysis liquid samples were between 96.79 and 99.13%. The research demonstrates that it is possible to obtain excellent accuracy and efficiency using HPLC to quantificate levoglucosan found in fast pyrolysis bio-oils.
1. Introduction Climate change, depletion of natural resource, and scarcity of fossil fuel have triggered the awareness of people. Environmental awareness in both the public and regulatory sectors has necessitated proper treatment of waste, such as waste water, unutilized biomass etc. For some pharmaceutical waste water, it can be treated based on the TiO2 system, which can facilitate a safe ecofriendly discharge to the atmosphere. For some unutilized biomass, it can be used for on the production of green fuel with different advance processing systems in a sustainable way (Sudip et al., 2012). For waste vegetable oils, enzymatic transesterification was performed to produce biodiesel (Loprestom et al., 2015). For agricultural and forestry residues, the most available biomass materials, they can be separate into three fractions (solid, liquid and gas) after pyrolysis (Kim and Dale, 2004; Vassilev et al., 2015). To achieve maximal yields of liquid, pyrolysis at short residence times is essential. After rapid cooling of the pyrolysis vapors, a dark brown liquid is formed, called bio-oil. The liquid produced is up to 80% of the initial dry mass (Bridgwater and Peacocke, 2000). Depending on bio-oil composition, it can be categorized into acids, sugars, phenols, hydroxyaldehydes, hydroxyketones, and alcohols. The presence of these components results in a relatively low energy density (16–18 MJ/kg, which is about 40–45% of that of hydrocarbon fuels), immiscibility with hydrocarbon fuels, and storage instability. Among these ingredients, the sugar fraction is particularly interesting as a
⁎
source of chemicals and fuels (Vassilev et al., 2015; Liu et al., 2014). One of the main components of this fraction is levoglucosan (1,6-anhydro-β-D-glucopyranose), Usually the yield of levoglucosan from the pyrolysis of lignocellulosic biomass is only a small percent, but previous studies found that infusing biomass with a weak mineral acid solution prior to pyrolysis increased levoglucosan yields from the cellulose fraction to as much as 59 wt% (Kuzhiyil et al., 2012; Wang et al., 2016). Levoglucosan can be upgraded into transportation fuels and chemicals via fermentation to alcohols, catalytic synthesis to hydrocarbons, dehydration to heterocyclic aldehydes (furfurals) and aromatics (furans), and aqueous-phase re-forming to produce alkanes as building blocks of gasoline (Helle et al., 2007). Having realized the significance of levoglucosan in applications, it is important to establish an easy and quick method to determine levoglucosan. Unfortunately, levoglucosan content in pyrolysis oil is rarely analyzed because of its reactivity and sticking tendency. In pyrolysis system, evaporation and polymerization of levoglucosan are two competing simultaneous process (Bai et al., 2013). The concentrations of levoglucosan depend on pyrolysis conditions and raw materials. It usually gives a maximum yield at around 500 °C. Previous studies usually use primary fraction to separate pyrolysis oil into less complex fractions or mixture (Vitasari et al., 2011). It is difficult to determine the concentrations of levoglucosan in bio-oil by regular analytical methods because a significant number of close related structures usually exist in bio-oil at low concentrations. What is more, the distribution coefficients of polar compounds (levoglucosan, acetol,
Corresponding author at: College of Forestry, Northwest A&F University, Yangling, 712100, China. E-mail addresses: [email protected] (J.-Q. Wang), [email protected] (J.-L. Zheng), [email protected] (J.-T. Wang), [email protected] (Z.-M. Lu).
https://doi.org/10.1016/j.indcrop.2018.01.045 Received 8 September 2017; Received in revised form 21 December 2017; Accepted 19 January 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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acetic acid and glycoladehyde) are always in the same range, which indicates poor selectivity. Thus, developing methodology for an appropriate separation of levoglucosan is very necessary. While, due to thermal and chemical instability of pyrolysis oil, solvent extraction is a better method. In comparing quantification methods, HPLC is considered the most appropriate method. It is one of the most powerful and reliable methods for less volatile, polar and unstable steroids compounds (Alsaadi et al., 2013). Refractive index and ultraviolet spectroscopic detectors have a few inherent disadvantages, such as lacking sensitivity, limiting chromatographic resolutions, depending on flow rate etc (Alsaadi et al., 2013; Yin et al., 2006). So, the introduction of ELSD brought a major advance to HPLC in the detection and quantitative analysis of levoglucosan (Lafosse and Herbreteau, 2002). Except for this experiment, other studies have investigated levoglucosan by using different detections. One study used HPLC combined with high-resolution mass spectrometry to sensitively analysis levoglucosan. The authors mention an extensive bleeding of the carbohydrate column (amino packing Waters) which is not compatible with the mass spectrometry ion source (Dye and Yttri, 2005). This bleeding is also observed when some amino columns are coupled with ELSD and this bleeding lowers the sensitivity (Lafosse and Herbreteau, 2002). Another study had shown that HPLC with aerosol charge detection and a benson BC-100 Ca2+ column can analyze levoglucosan with a lower detection limit and avoid the interference from other compounds, however, this method is only available for atmospheric samples (Dixon and Baltzell, 2006). The interferences between levoglucosan and other monosaccharides are different in bio-oils and atmospheric aerosols (Kirchgeorg et al., 2014; Piot et al., 2012; Tessini et al., 2011; You et al., 2014). Another method was investigated by using ion-exclusion high-performance liquid chromatography with low wavelength (194 nm) spectroscopic detection to analyze levoglucosan. It only had a modest detection limit and the pre-treatment was complicated (Schkolnik et al., 2005). In China, Li and Zhuang used HPLC with refractive index detection to analyze levoglucosan from cotton pyrolysis oil, while this method lack full qualitative and quantitative analysis for levoglucosan (Zhuang et al., 2001). According to our knowledge, no adequate method has been developed to analyze levoglucosan in bio-oil by using HPLC-ELSD. Previous studies (Oasmaa and Kuoppala, 2008) reported that the sugar fraction of bio-oil contains monosaccharides, anhydrosugars and anhydropolysaccharides. They indicated that it is difficult to separate and determine levoglucosan from bio-oil, thus an appropriate pretreatment of HPLC and HPLC-ELSD method in this experiment is important. The aims of this study were (i) to determine a separation method for levoglucosan in HPLC-ELSD analysis and (ii) to use response surface methodology (RSM) to optimize parameters on ELSD (Dasgupta et al., 2015; Chakraborty et al., 2012a,b), and (iii) to measure quantitatively the content of levoglucosan in bio-oil.
Table 1 Drift tube temperature (DTT), column temperature (CT) and gas flow rate (GFR) values for the three-parameter factorial designed extraction experiment. Experimental factors
Parameter level
A drift tube temperature (DTT,°C) B column temperature (CT,°C) C gas flow rate (GFR, L/min)
Low (−1)
Middle (0)
High (1)
60 10 2.5
70 15 3
80 20 3.5
Table 2 Box-Behnken experimental design matrix for levoglucosan. Run no.
A: DTT °C
B: CT, °C
C: GFR, L min−1
Peak area, mV s 106
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
60 70 80 80 60 60 80 80 70 70 80 60 70 80 80 80 80
10 20 15 15 20 15 10 15 15 15 20 15 10 10 15 20 15
3 3 3 3 3 3.5 3.5 3 3.5 2.5 2.5 2.5 3 2.5 3 3.5 3
1.85 2.63 2.59 2.59 1.92 1.90 1.95 2.56 2.77 2.90 2.25 2.25 2.55 2.26 2.56 2.27 2.56
2. Experimental Fig. 1. Schematic diagram of fluidized bed reactor (1: heater, 2: cotton straw collector, 3: screw feeder, 4: fluidized bed, 5: cyclone, 6: char collector, 7: condenser, 8: bio-oil tank).
2.1. Materials and reagents Bio-oil was produced with air-dried rice straw in a fast pyrolysis process development unit consisting of a fluidized bed operated at final pyrolysis temperature of 500 °C, holding time of 1.4 s, particle size of 600–650 μm and acid concentration of 8% (The optimum parameters based on preliminary single experiments). The reference compounds analytical grade fructose, xylose, glucose and levoglucosan were purchased from Sigma Chemical Company (Beijing, China) with a purity > 99%. Methanol and acetonitrile were purchased from Tianjin Kermel Chemical Reagent Co., Ltd (Tian Jin, China) with HPLC-grade. Ultra-pure water was produced by Milli-Q system (Millipore, Bedford, MA, USA). Standard solutions of fructose, xylose, glucose and levoglucosan were prepared at the level of 2.0 mg/mL in ultra-pure water. Working
standard solutions were prepared as needed by appropriate dilution of the stock solutions in ultra-pure water. All sample were stored at 4 °C in the refrigerator.
2.2. Sample separation Ultra-pure water (10 mL) was added to bio-oil (10 mL), and then stirred (25 °C) for 2 h. The mixture was centrifuged (15 min, 3500g, OPTIMA L-100XP, Beckman, USA) and the liquid decanted. The liquid was then added with granular activated carbon (0.5 g) and shake at an Oven Controlled Crystal Oscillator (WHY-2, China) for 12 h. Then, the mixture was filtered and combined with the filtrate. After that, Ca(OH)2 267
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Table 3 The gradient elution and isocratic elution sequence used in the HPLC-ELSD method for carbohydrate analysis using acetonitrile (ACN) and water as mobile phase. No.
Types of column
Column temperature
Mobile phase
1
XB-NH2
10 °C
Gradient elution
2 3 4
Isocratic elution
5 6 7
HILIC-D
10 °C
8 9
Gradient elution Isocratic elution
35 °C
10
Gradient elution Isocratic elution
was added to the filtrate, with proper pH adjustment to around 12. The alkaline liquid was then kept in a 60 °C water bath (HH-2 digitial constant temperature tank, China) for 3 h to remove the precipitation of organic colloid and acidic compounds. Then filter, the filtrate was collected and 10 mL of the filtrate was added to three times of 95% alcohol with continue whisking. After that, the mixture was centrifuged (3500g) for 15 min and residue from the centrifuge was dissolved in 10.0 mL water. Repeat the above alcohol washing and centrifugation processes. Finally, dissolve the centrifugal residue in the purified water (1.0 mL). The solution was filtered through 0.22 μm filters (Xinjinghua Co., Shanghai, China) before use and stored at 4 °C for further analysis.
Times (min) ACN (%) Times (min) ACN (%) Times (min) ACN (%) Times (min) ACN (%) Times (min) ACN (%) Times (min) ACN (%) Times (min) ACN (%) Times (min) ACN (%) Times (min) ACN (%) Times (min) ACN (%)
0 88 0 90 0 90 0 78 0 85 0 82 0 90 0 92 0 90 0 92
15 78 15 80 20 80 20 78 20 85 20 82 20 80 20 92 20 80 20 92
26 76.5 30 80 25 90 25 78 25 85 25 82 25 50 25 92 25 50 25 92
30 88 35 90 36 90 35 78 35 85 35 82 35 50 35 92 35 50 35 92
40 88 46 90 40 90 40 78 40 85 40 82 40 50 40 92 40 50 40 92
remained in optimal states. Table 1 shows the operational ranges of the input variables. The software Design Expert version 8.0.6.1 was used to design the Box-Behnken design, and to perform the response surface regression and the analysis of variance (P < 0.05). The complete design matrix comprising 17 experiments are listed in Table 2. Using a second order polynomial Eq. (1) to express the relation between the dependent variable Y (the peak area) and independent variables X (drift tube temperature, column temperature and gas flow rate). In Eq. (1), β0 is the intercept, βi the linear coefficient, βii the quadratic coefficient, βij the interaction coefficient and ε, the residual.
Y = β0 +
2.3. HPLC instrumentation and chromatographic conditions
∑ βi Xi + ∑ βii Xi2 + ∑ βijXiXj + ε
(1)
The mobile phase flow rate was used at 1.0 mL/min and the temperature of the chromatography oven (40 °C) was maintained as previously reported (Condezo-Hoyos et al., 2015). All aqueous solutions (include treated bio-oil samples) were filtered by 0.22 μm membrane syringe filters before being injected into the HPLC.
The HPLC apparatus was equipped with an Agilent G1329B autosampler, Agilent G1311c quaternary pump system 1260 series with online degasser, Agilent G1316A thermostatic oven, Agilent HPLC control unit 1260 series and Alltech 2000ES-ELSD. Non-UV sensitive compounds can be detected and quantified by ELSD because it is a quasi-universal detector used as mass dector (Condezo-Hoyos et al., 2015; Lafosse and Herbreteau, 2002). Data processing was carried out with the EZchrom Elite Agilent technology. The HPLC separation and quantification were made on an Ultisil® XB-NH2 column (250 mm × 4.6 mm, 5 μm particle size) (Welch, China) and a Kromasil 60-5 Hilic-D column (250 mm × 4.6 mm, 5 μm particle size) (AKZO NOBEL, Sweden) with 10 μL sample injection. The mobile phase consisted of two solvents, solvent A (water) and solvent B (acetonitrile) in gradient elution with a flow rate of 1.0 mL/min. The four carbohydrates were eluted with the increasing proportion of solvent A from 12% to 22% over 0–15 min; 22% to 23.5% over 15–26 min; then, decrease the proportion of solvent A from 23.5% to 12% over 26–30 min. The column temperature was kept at 15 °C. The drift tube temperature was set at 70 °C and nebulizer gas (air) flow rate was 3.0 L/ min.
2.5. Calibration curves, limits of detection and quantification (Nogueira et al., 2005; Yin et al., 2006) Different fructose, xylose, glucose and levoglucosan standards (high-, medium- and low concentration) were used for the constructive of calibration curves. Each standard was used three times at different concentrations, using acetonitrile-water as mobile phase, and all parameters of ELSD were set as optimal. The estimate value of limits of detection (LOD) and quantification (LOQ) were determined by different signal-to-noise (S/N). LOD was calculated as the concentration corresponding to three times the peak height of the baseline noise (S/N = 3); whereas LOQ was set as ten times the peak height (S/N = 10). Signal to noise ratio is calculated using LC Solution Software. The LOD and LOQ values were calculated experimentally from serial dilution of standard levoglucosan concentrations.
2.4. Optimization of ELSD parameters
2.6. Accuracy (Alsaadi et al., 2013; Yin et al., 2006)
To investigate the optimization parameters of ELSD detection for levoglucosan analysis, this paper used the Box-Behnken design to approach optimal design. The ELSD values were optimized with three different parameters: drift tube temperature, column temperature and gas flow rate (Dixon and Baltzell, 2006; Kao et al., 2008; Bhandari et al., 2008; Condezo-Hoyos et al., 2015). The other parameters
A recovery test was used to evaluate the accuracy of this analysis method. For the recovery experiments, three different concentrations (80, 100 and 120%) of the reference compounds were spiked in levoglucosan sample. Every sample was analyzed three times. 268
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Fig. 2. For experimental conditions of each method in bio-oil, see Table 1. (a) gradient elution on Ultisil® XB-NH2 column, (b) isocratic elution of Ultisil® XB-NH2 column, (1) = levoglucosan, (2) = xylose, (3) = fructose, (4) = glucose.
developed by comparing of retention times and peak areas of each experiment to assess its repeatability and reproducibility. The precision of the developed method was validated by repeating the procedure in duplicate on three different days with newly prepared mobile phase and samples.
2.7. Precision (Bhandari et al., 2008; Alsaadi et al., 2013) Intra- and inter-day precise measurements were carried out on three consecutive days. Each day, low and high sample concentrations of levoglucosan were independently injected in triplicate. The method was
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absorbed by active carbon, most phenols, acids, organic colloid and others still existed in the aqueous layer. Ca(OH)2 was added to remove these impurities. What is more, CaCO3 is formed by the reaction of Ca (OH)2 and the CO2 dissolved in water. CaCO3 is an absorbent, which means it can absorb impurities with good dispersity (Fangyao and Yuegang, 2004). After these steps, 95% alcohol as a non-aqueous solvent was used to wash the remained bio-oil for further extraction and purification.
Table 4 Analysis of the model variances. Variance
Sum of squares
df
Mean square
F value
P (prob > F)
Model A B C AB AC BC A2 B2 C2 Residual Lack of Fit Pure Error Cor total
1.66 0.56 0.024 0.017 2.445E-003 0.010 0.030 0.82 0.39 0.032 8.043E-003 6.96E-003 1.080E-003 1.87
9 1 1 1 1 1 1 1 1 1 7 3 4 16
0.18 0.56 0.024 0.017 2.445E-003 0.010 0.030 0.82 0.39 0.032 1.149E-003 2.321E-003 2.700E-004
160.34 488.86 21.27 14.85 2.13 9.08 25.79 716.79 342.30 27.99
< 0.0001 < 0.0001 0.0025 0.0063 0.1880 0.0196 0.0014 < 0.0001 < 0.0001 0.0011
8.60
0.0323
3.2. Method development 3.2.1. Chromatography column and mobile phase selection Levoglucosan can be described as a pyranose ring with two ether groups and three hydroxyl groups (Bennett et al., 2009). The structure of levoglucosan shows that it is soluble in water and insoluble in nonhydroxylic solvent. What is more, levoglucosan is present in low concentrations of bio-oil, between 3 and 6% depending on pyrolysis conditions and the raw materials (Tessini et al., 2011). The choice of a suitable chromatography column and mobile phase is the key to separation. To effectively separate levoglucosan from the other three main carbohydrates (fructose, xylose and glucose) in the bio-oil sample, two different types of columns (Ultisil® XB-NH2 column, Kromasil 60-5 Hilic-D column) with different mobile phase systems were tested. In the Kromasil 60-5 Hilic-D column, acetonitrile-water was used in both gradient and isocratic elution to obtain a better separation of the four carbohydrates (Table 3). Other conditions, such as an increase or reduction in the flow rate or adjustment of the pH value of mobile phases were also taken, but in all cases, the separation of levoglucosan was not good. What is more, the influence of concentration of water in the mobile phase was observed, and the separation and retention time of the carbohydrates in Kromasil 60-5 Hilic-D column was also changed. It is worthwhile to note that both peak interferance and broadening effects still strongly remained on the Hilic-D column, whether in gradient elution or isocratic elution. What is more, when column temperature changed from 10 °C to 35 °C, the separation of the carbohydrates was not obviously changed in the Hilic-D column. After that, the Ultisil® XB-NH2 column was designed to retain and separate fructose, xylose, glucose and levoglucosan. Many kinds of mobile phases were generally used in the NH2 column. Different compounds require different mobile phases due to their chemical properties (Yang et al., 2012). In this study, acetonitrile-water was chosen as the mobile phase. The resolutions of investigated compounds (fructose, xylose, glucose and levoglucosan) were tested with different elution programs. The method 3 and method 4 had a peak overlapping not only in fructose but also in glucose (Fig. 2a, b). While the elution programs method 2, method 5 and method 6 can separate the four carbohydrates but with a wider peak (Fig. 2a, b). Wider peak meant lower column efficiency (Dixon and Baltzell, 2006). Furthermore, when the water content is below 10% (v/v), the chromatographic peaks became broad and asymmetric (Fig. 2b). Therefore, in the next experiments, water content in mobile phase should be increased. By testing several solvent
2.8. Statistical analysis Statistics of the compositional data of levoglucosan was conducted with the aid of SPSS (SPSS17 for Windows, SPSS Inc., Chicago, IL, USA). Analysis of variance was used to statistically analyze the results. The relative standard deviation (RSD) was used to assess intra- and interday precisions by three replicates per sample and carried out by the same operator. Differences were considered to be statistically significant when p < 0.05. 3. Results and discussion 3.1. Sample separation In the previous bio-oil pyrolysis system, pyrolysis temperature, particle size, reaction time and other parameters were considered the key factors (Abnisa et al., 2011; Isa et al., 2011; Lazzari et al., 2016). In this experiment, in order to obtain the bio-oil with high content levoglucosan, the fluidized bed fast pyrolysis reactor with the condenser system used for production of the bio-oil was shown in Fig. 1. The amount of rice straw samples used in this experiment was 500 g. Sugar within the bio-oil matrix is difficult to analyze because the large number of variations that can occur and potential interference with other bio-oil components (Rover et al., 2013). Previous studies also showed, levoglucosan has a weak hydrogen bond interaction with water, which leads to higher aqueous concentration (Vitasari et al., 2011). Thus, according to Nicole M. Bennett et al. (2009), 10 mL high purity water was initially presented in 10 mL bio-oil, stirred for 2 h and then centrifuged for 15 min at 3500g. Most levoglucosan would mix with water. Other viscous constituents, which were not dissolved in water, as lignin-derived compounds would sink to the bottom. Active carbon was added to absorb no hydrogen bonded substances (Huber et al., 2006) which still present in bio-oil, like pigments and aromatic compounds. In addition to these high molecular weight compounds
Fig. 3. 3-D surface plot showing the interaction of DTT, CT and GFR on the ELSD response of levoglucosan standard. DTT = drift tube temperature; CT = column temperature; GFR = gas flow rate.
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Fig. 4. Calibration curves for fructose, xylose, glucose and levoglucosan.
significant for the levoglucosan analyzed. In ELSD, increasing the column temperature to 30 °C lead to a better separation. In a general way, sugar analysis is preferred at a higher temperature in order to obtain better column efficiency while increasing the column temperature too much also can decrease S/N ratio (Fig. 3). At high temperature, the S/N is reduced because of the increase of baseline, which also leads to a greater column bleed (Dixon and Baltzell, 2006). A column temperature of 15.08 °C was selected based on the Box-Behnken response surface design and used in the following analyses. In the HPLC-ELSD analysis, the drift tube temperature is also an important parameter (Kao et al., 2008). Solvent cannot be completely evaporated at low temperatures while at high temperatures, the detector response will also be decreased, because the partial vaporization of the analytes leads to a decrease in particle size (Fig. 3). The S/N ratio was improved as the drift tube temperature was increased (Lecoeur et al., 2015; Man Ki et al., 1996). However, when the drift tube temperature was increased more than 70 °C, a substantial loss of analytes was observed (the detector response decreased). Thus, based on the Box-Behnken response surface design, the optimal drift tube temperature was 70.46 °C. Regarding gas flow, lower or higher rates can cause a decreases in the ELSD response. The higher the gas flow rate, the smaller the ELSD response value (Fig. 3). While, a lower gas flow rate produces larger droplets, resulting in spike and noisy signals in HPLC analysis. On the other hand, a higher gas flow rate forms smaller droplets, which results in less effective light scattering. Then there will be a decrease in ELSD response value (Bhandari et al., 2008; Condezo-Hoyos et al., 2015; Man Ki et al., 1996). Thus, in this study, based on the Box-Behnken response surface design, the optimal gas flow rate was determined to be 2.92 L/min. In view of the practicability, the following ELSD parameters were set to 15 °C column temperature, 70 °C drift tube temperature and 3.0 L/min gas flow rate. The experimental ELSD parameter changes, trends of this work are quite comparable to others. Previous studies showed similar tendencies of these three parameters. For example, levoglucosan separated from atmospheric aerosols and was detected with an aerosol charge detection (Dixon and Baltzell, 2006). When the column temperature was below or above the optimal value (50 °C), the response value would decrease. Drift tube temperatures of 35 °C and 145 °C were set as optimal values for plasticizers and ginseng saponins (Lecoeur et al., 2015; Man Ki et al., 1996). Neither increasing nor decreasing the optimal temperature could result in obtaining a maximal S/N ratio. Optimal gas flow rate of 40 mm rotameter units and 2.0 SLM respectively for ginseng saponins and P. kurroa were investigated (Bhandari et al., 2008; Man Ki et al., 1996). Their experiments showed that, lower or higher gas flow rate could not obtain the best response of ELSD.
Table 5 The intra-day and inter-day precision of the proposed method. No.
Intra-day (n = 6)
Inter-day (n = 6)
High concentration
Low concentration
tR
Area
tR
Area
1 2 3 4 5 6 RSD% 1
9.04 9.06 9.05 9.05 9.06 9.04 0.13 9.04 ± 0.01
70753 69478 71298 69914 71205 70996 0.93 70753(ave.)
9.08 9.09 9.09 9.09 9.07 9.08 0.06 9.08 ± 0.01
2
9.02 ± 0.01
69478(ave.)
9.09 ± 0.01
3
8.98 ± 0.01
72298(ave.)
9.09 ± 0.01
4
8.99 ± 0.01
69502(ave.)
9.08 ± 0.01
5
8.99 ± 0.01
70128(ave.)
9.07 ± 0.01
6
8.96 ± 0.01
69392(ave.)
9.09 ± 0.01
RSD%
0.44
1.60
0.47
39023 38038 38257 38145 39756 39145 1.34 39942 (ave.) 39327 (ave.) 38639 (ave.) 38826 (ave.) 38063 (ave.) 37108 (ave.) 2.56
Ave. means the average of the retention time and peak area in one day.
systems, proper proportion was selected (method 1), results showed that the Ultisil® XB-NH2 column can provide good separation of fructose, xylose, glucose and levoglucosan from the bio-oil, the whole elution time was within 24 min and without any interfering peaks (Fig. 2a). In this study, the retention times were 20.53 min for fructose, 16.24 min for xylose, 23.84 min for glucose and 9.08 min for levoglucosan. 3.2.2. ELSD parameters optimization The drift tube temperature, column temperature and gas flow rate should be optimized to obtain a maximum of ELSD response. Thus, this study used RSM to obtain the maximum ELSD response to analyze levoglucosan. In order to optimize ELSD, a Box-Behnken response surface design with three central points was used. The independent variables were determined as: drift tube temperature (DTT), column temperature (CT) and gas flow rate (GFR). The peak area (response) was used for the dependent variable as in the previous reports (Condezo-Hoyos et al., 2015). In this study, the Box-Behnken design had been shown to be more efficient than the central composite design (Ferreira et al., 2007). Based on single-factor experiments, three levels of each parameter were determined (Table 2). According to the data (Table 4) subjected to the analysis of variance (ANOVA) of regression model (P < 0.0001) and the value of R2 (0.9961) demonstrated that the quadratic model is
3.3. Method validation The developed HPLC-ELSD method showed good sensitivities and 271
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Table 6 Recovery of levoglucosan by spiking method. No.
tR/min
Initial content (μg)
Area
Sample Added (μg)
Content after adding (μg)
Average recovery (%)
RSD (%)
Standard (low) Standard (high) Sample 80% added 100% added 120% added
9.03 9.03 9.04 9.03 9.04 9.04
9.08 36.32 10.9 10.9 10.9 10.9
18532 72447 26532 39685 42641 48431
– – – 8.72 10.9 13.08
– – – 19.45 21.1 23.43
– – – 99.13 96.79 97.71
– – – 0.79 0.80 1.14
Table 7 Separation and quantification of levoglucosan in bio-oil by HPLC-ELSD. No (low concentration/high concentration)
Levoglucosan 18.16/36.32 μg/mL Fructose 18.98/37.96 μg/mL Xylose 10.0/20.0 μg/mL Glucose 19.7/39.4 μg/mL
Standard reference
Bio-oil sample
Content (mg/ml)
High concentration
Low concentration
tR/min
Area
tR/min
Area
tR/min
Area
9.08 20.53 16.24 23.84
74289 189843 73764 383389
9.09 20.56 16.22 23.93
39011 86863 43695 190212
9.11 – – –
67811 – – –
0.033 – – –
“–” means not detected. Journal：
•
recovery of levoglucosan was found between 96.79 and 99.13%, and the average RSD was below 1.14% (Table 6). Fructose, xylose and glucose were tested for verifying the good separation of levoglucosan by this analysis method. These three carbohydrates have had no effect on the quantification and separation.
linear responses to the studied carbohydrates (fructose, xylose, glucose and levoglucosan), which were good enough to quantify them. Calibration curves were obtained by injecting each standard solution (10 μL) of fructose (44.4, 88.8, 222, 444, 666 and 888 μg/mL), xylose (68.8, 137.6, 344, 688, 1032 and 1376 μg/mL), glucose (20.0, 40.0, 100, 200, 300 and 400 μg/mL) and levoglucosan (8.8, 17.6, 44, 88, 132 and 176 μg/mL) in triplicate. The linearity was evaluated based on the correlations between the nature logarithm of peak areas and the nature logarithm of concentration of the four analytes. The linear relationship followed the equation Y = aX + b, as shown in Fig. 4. All of the R2-values for the regression lines were greater than 0.980. The results of these four analytes correspond well to other researches (Dixon and Baltzell, 2006; Nogueira et al., 2005; Wan and Yu, 2006). Levoglucosan was identified by comparing its retention time with the standard one. The detection limits were estimated as the concentration providing a signal three times or ten times higher than the standard deviation of the background noise. LOD and LOQ for levoglucosan were calculated experimentally from serial dilution. Therefore, successive dilutions were dertermined to find the suitable concentration of levoglucosan on the measurement without confusion with background noise. LOD was 3.632 μg/mL and LOQ was 9.08 μg/ mL for levoglucosan. This showed that this analysis method had a lower LOD, which was sensitive enough to detect levogluocsan. The precision of this method was determined in consideration of the RSD of levoglucosan at two different concentrations: low (0.05 mg/mL) and high (0.2 mg/mL). In order to detect intra-day variation with precision, the low and high concentrations of levoglucosan were injected in six times within one day. For inter-day precision, the two concentrations were injected in triplicates on six different days (Table 5). At the higher concentration level, the RSD of peak area and retention time were 0.13% and 0.93% for intra-day precision and 0.44% and 1.6% for inter-day precision, respectively. At the lower concentration level, the RSD of peak area and retention time were 0.47% and 2.56% for interday precision and 0.06% and 1.34% for intra-day precision, respectively. These values demonstrated this method is of good precision. Lower RSD values of levoglucosan in inter-day and intra-day meant better repeatability and reproducibility. The accuracy of the method was determined by investigating the recovery of levoglucosan at three levels (i.e. 8.72 μg, 10.9 μg and 13.08 μg) under the valid HPLC method. Results showed that, the
3.4. Extraction method validation An optimal extraction should include a single step or multiple steps to remove one or more unwanted components (Kakiyama et al., 2006). In this paper, unwanted components were removed by precipitation and extraction. Previous studies showed that fructose, xylose, glucose and levoglucosan are abundant in the sugar fraction of bio-oil (Tessini et al., 2011). Thus, in this work a simple method was established for separating and quantifying levoglucosan in bio-oil and fractions thereof, by using the HPLC-ELSD technique. As shown in Table 7, HPLC showed that with the treatment shown in Section 2.2, fructose, xylose and glucose were undetected (below the limit of detection), while the content of levoglucosan in the rice straw bio-oil was 0.033 mg/mL. The content of levoglucosan determined by this study is quite comparable to others (Tessini et al., 2011). Therefore, this pre-treatment was suited for determination of levoglucosan in bio-oil. 4. Conclusion We have described a separation method of levoglucosan from bio-oil and established an easy and quick method to determine levoglucosan through the HPLC method with Alltech 2000ES-ELSD, Ultisil® XB-NH2 column and acetonitrile-water as the mobile phase. The optimal ELSD parameters for separating fructose, xylose, glucose and levoglucosan was achieved at 15 °C column temperature and by adjusting ELSD setting at 70 °C drift tube temperature and 3.0 L/min gas flow rate. The method for levoglucosan quantification is a fast-reliable method with low LOD, high sensitivity, precision and good accuracy. Recovery for levoglucosan was in the range of 96.79–99.13%, while LOD was 3.632 μg/mL and LOQ was 9.08 μg/mL. The content of levoglucosan in the rice straw bio-oil was 0.033 mg/mL. The main advantage of this method is the easier sample extraction and no need of derivatization. What is more, the optimal conditions established with low LOD for high sensitive detection of levoglucosan would be easily applied to any other 272
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