Article pubs.acs.org/EF
Comparative Assessment of Internal Standards for Quantitative Analysis of Bio-oil Compounds by Gas Chromatography/Mass Spectrometry Using Statistical Criteria Anna Artigues,* Neus Puy, Jordi Bartrolí, and Esteve Fábregas Department of Chemistry, Universitat Autònoma de Barcelona (UAB), Edifici Cn, Campus de la UAB, 08193 Cerdanyola del Vallès, Barcelona, Spain S Supporting Information *
ABSTRACT: To better assess the potential use of bio-oil as a chemical platform and fuel, a gas chromatography/mass spectrometry (GC/MS) technique is applied to characterize qualitatively and quantitatively a pine wood bio-oil produced by fast pyrolysis. An external calibration method using three different internal standards (toluene, 1,1,3,3-tetramethoxypropane, and 1-octanol) is performed to quantify 10 target compounds of bio-oil from different chemical families [2-propen-1-ol, 2-butanone, acetic acid, furfural, 2(5H)furanone, 2,5-dimethyoxy-tetrahydrofuran, 3-methyl-1,2-cyclopentanedione, 2-methoxy-4-propyl, vanilline, and levoglucosan], which were previously selected from the qualitative analysis of the bio-oil composition. To evaluate the reliability of this method using internal standards or without them, an exhaustive precision study is performed assessing the instrumental, intraday, and interday precisions. This study results in an overall good reproducibility of the method using any internal standard or without them. Finally, a comparative assessment of the different internal standards using statistical criteria (one-way analysis of variance and relative standard deviation) is carried out, showing that any of them can be used for bio-oil quantification. Thus, this study shows the analytical method suitability for the quantification of some bio-oil compounds by GC/MS. and low thermal stability.6,7 In addition, it undergoes a process known as aging, caused by the repolymerization of part of the organic matter in the oil, which causes an increase in viscosity and water content over time.11 Because of that, bio-oil upgrading is necessary to improve these properties. Insight in the molecular composition of the crude pyrolysis oil and upgraded products is highly desirable for a number of purposes.12 It allows for determination of molecular composition, and it is a useful tool to gain insight in the molecular processes taking place during the pyrolysis processes, the aging of bio-oil, and the upgrading processes. This information is crucial for the development of efficient upgrading processes, delivering a product that meets the required product properties. Many analytical techniques have been combined to obtain an inclusive analysis of bio-oil composition. Garcia-Perez at al.13 reported and discussed the techniques used to analyze the chemical composition of bio-oil, including gas chromatography/ mass spectrometry (GC/MS) (volatile compounds), highperformance liquid chromatography (HPLC), HPLC/electrospray MS (non-volatile compounds), Fourier transform infrared (FTIR) spectroscopy, gel permeation chromatography (GPC) (molecular weight distributions), ultraviolet (UV), UV fluorescence, nuclear magnetic resonance (NMR), thermogravimetry, and solvent extraction. The bio-oil complexity nature renders essential the use of high-resolution chromatographic techniques for the chemical bio-oil composition analysis.14 Both HPLC15,16 and GC17,18 have been employed to analyze this complex mixture and to identify
1. INTRODUCTION Biomass is one of the largest primary energy resources and a promising, clean, and renewable energy source to supply the declining fossil fuel resources.1,2 Different technologies are described to convert the biomass into energy, fuels, and chemicals.3 Among them, fast pyrolysis of biomass is a thermal decomposition process that occurs in the absence of oxygen, with quick biomass decomposition and rapid vapor condensation to obtain a liquid product (known as bio-oil) with a yield as high as 75 wt %.4 Bio-oil can be used directly or as an intermediate pretreatment step to convert solid biomass into a higher energy content transportable liquid for subsequent processing for heat, power, biofuels, and chemicals.5 Bio-oil is a complex mixture of water and hundreds of organic compound products from the defragmentation of the lignin, cellulose, and hemicelluloses of biomass. These compounds are acids, aldehydes, ketones, alcohols, esters, sugars, furans, phenols, guaiacols, syringols, nitrogen-containing compounds, and large molecular oligomers.1 The chemical composition of bio-oil depends upon the nature of the biomass and the pyrolysis conditions employed.6,7 This diversity of oxygenated compounds makes bio-oil a promising chemical platform for obtaining value-added chemicals,8,9 such as phenols used in the resin industry, volatile organic acids in the formation of de-icers, levoglucosan, hydroxyacetaldehyde, and some additives applied in the pharmaceutical, fiber-synthesizing, or fertilizing industry, and flavoring agents in food products.8,10 Moreover, bio-oil is a liquid fuel that contains negligible amounts of ash, and it has an energetic density 5−20 times higher than the original biomass. However, it has some properties that set up many obstacles to their application as fuel, such as corrosiveness, high viscosity, high oxygen content, © 2014 American Chemical Society
Received: March 11, 2014 Revised: May 6, 2014 Published: May 12, 2014 3908
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To obtain the chromatographic conditions specified above, a previous optimization of them is carried out. The optimized parameters were the heating rates to obtain the best peak separation with the minimum run time, the injector temperatures testing 250, 275, and 300 °C, the used liner, and the sample dilution checking 1:5, 1:10, and 1:40. 2.3. Sample Analysis. First of all, a qualitative study is carried out to indentify the bio-oil chemical compounds. With this aim, a bio-oil sample is filtered and diluted with methanol (1:10) and analyzed by GC/MS. Among the identified compounds, the most abundant compounds according to the chromatogram peak area and their added value are selected for their quantification. It is considered that almost one compound of each chemical family is selected. They are quantified by external calibration using three different internal standards: toluene, 1,1,3,3-tetramethoxypropane, and 1-octanol. Moreover, a one-way analysis of variance (ANOVA) with a confidence interval of 95% is performed to carry out a comparative assessment of internal standards. To prepare the bio-oil samples for quantitative study and the comparison between internal standards, bio-oil is filtered and diluted with methanol (1:10) and the three tested internal standards (100 mg L−1 toluene, 200 mg L−1 1,1,3,3-tetramethoxypropane, and 200 mg L−1 1-octanol) are added to the sample. The bio-oil samples are injected by triplicate. For the calibration standards, a methanol stock solution for each selected compound is prepared, and six standards with different concentrations of each compound are prepared, ranging from 20 to 70 mg L−1 for 2-propen-1-ol, from 10 to 250 mg L−1 for 2-butanone, from 2000 to 7000 mg L−1 for acetic acid, from 50 to 500 mg L−1 for furfural, from 400 to 900 mg L−1 for 2(5H)furanone, from 25 to 150 mg L−1 for 2,5-dimethoxy-tetrahydrofuran, from 300 to 800 mg L−1 for 3-methyl-1,2-cyclopentanedione, from 50 to 200 mg L−1 for 2-methoxy-4-propyl, from 500 to 1000 mg L−1 for vanillin, and from 2000 to 7000 mg L−1 for levoglucosan. In each standard, 100 mg L−1 toluene, 200 mg L−1 1,1,3,3-tetramethoxypropane, and 200 mg L−1 1-octanol are added as internal standards. The concentration range of each compound has been chosen using successive approximation until it becomes considerably slim and contains the quantified value. The standard solutions are kept in the refrigerator until use. The purities of the used reagents are ≥99.5% assay for acetic acid and toluene, ≥99% assay for 2-methoxy-4-propyl-phenol, furfural, vanillin, 2-butanone, 1,1,3,3-tetramethoxypropane, 2-propen-1-ol, 2-hydroxy-2-cyclopenen-1-one, and levoglucosan, 98% assay for 2,5-dimethoxytetrahydrofuran and 2(5H)furanone, and 95% assay for 1-octanol. 2-Butanone is purchased from Janssen Chimica; acetic acid and toluene are purchased from Panreac; 1-octanol is purchased from J.T. Baker; and the rest are purchased from Sigma-Aldrich. Apart from the quantitative analysis, a study of the precision of the method using internal standards or without them is carried out by means of an ANOVA test. This study assesses the instrumental precision, the intraday precision, and the interday precision of the method to analyze the bio-oil sample. For this purpose, three aliquots of the bio-oil vessel are sampled and analyzed by triplicate. After 10 days, another aliquot is sampled and analyzed. Each aliquot is sampled after a proper bio-oil homogenization that consists of a 10 min mixing of the sample vessel. Then, bio-oil is filtered, diluted with methanol (1:10), and analyzed by GC/MS by triplicate. A proper homogenization of the sample is crucial for a good reproducibility of the analysis and more important when the sample is as complex as bio-oil.
individual compounds, with GC/MS being the most common technique to characterize bio-oils.13 More recently, multidimensional GC/MS analysis of pyrolytic oils is also used to increase chromatographic resolution.12,14,19,20 Computer matching of the mass spectra with a library is used to identify peaks in the GC/MS chromatogram. The use of these techniques has permitted the study of the qualitative composition of different bio-oils as well as the chemical composition of the upgraded bio-oils.13 Moreover, the relative quantification of bio-oil composition has been reported as the average percentage of the total area from each individual peak and totaling the area of compounds,21,22 a method that does not show the real concentration of the compounds because the area of the peaks is not directly proportional to the concentration of the compound. Another method present in the literature is the quantification analysis using the internal standards method,13 which is more reliable for determining the compound concentration. Despite this, further work is needed for the detailed quantification of bio-oil because, to the best of our knowledge, only a few publications address this issue.17,23,24 Even though the quantification issue is addressed in a few publications,17,23,24 an exhaustive study of method reliability has not been reported. Thus, the aim of this study is to carry out a reliable quantitative analysis of bio-oil compounds from different chemical families by means of GC/MS to reach a further characterization and better comprehension of bio-oil composition and to assess the precision of the used analytical method using internal standards or without them. With this objective, a qualitative analysis of bio-oil is carried out to identify the main compounds and to select those that are going to be quantified by means of an external calibration. Moreover, three different internal standards are tested and compared by statistical criteria to assess their suitability for the quantification analysis.
2. EXPERIMENTAL SECTION 2.1. Sample. The bio-oil analyzed in this study is purchased from BTG-BTL. It is pine wood bio-oil produced by fast pyrolysis in a rotating cone reactor. The basic physical and chemical properties of the sample, according to BTG-BTL data, are 20 wt % of water content, pH range from 2.5 to 3.5, density range from 1150 to 1200 kg m−3, and viscosity at 20 °C from 60 to 225 cSt. 2.2. GC/MS Method. The GC/MS system used to perform the qualitative and quantitative analyses is a Thermo Trace GC Ultra/MS DSQII system. The capillary column is a DB-Petro (100 m × 0.25 mm inner diameter × 0.50 μm film thickness) with helium as the carrier gas with an initial flow of 2.3 mL min−1 for 84 min, an increasing flow rate from 0.2 to 1.8 mL min−1, and kept at 1.8 mL min−1 until the end of the analysis. The oven temperature is programmed at 40 °C (4 min), a first heating rate of 1 °C min−1 to 55 °C, a second heating rate of 2 °C min−1 to 185 °C, a third heating rate of 10 °C min−1 to 250 °C, and held for 60 min. The total run time is 120 min. The injector, the ion source, and the transfer line temperatures are kept constant at 300, 230, and 280 °C, respectively. A sample volume of 1 μL is injected, applying 1:7 split mode. After a solvent delay of 8 min, a full mass spectrum is acquired. MS is operated in positive electron ionization mode, and a quantifying mass/charge ratio (m/z) range from 30 to 500 is scanned. The voltage applied to the multiplier detector is 1275 V to obtain the total ion chromatograms (TICs) in a full-scan acquisition method. A m/z for each compound has been used to produce an extracted ion chromatogram, wherein the integration of the peak area of analytes is performed. In this way, some interference with each analyte and the other compounds does not occur or are reduced. The identification of peaks is based on computer matching of the mass spectra with the National Institute of Standards and Technology (NIST) library.
3. RESULTS AND DISCUSSION 3.1. Qualitative Analysis. First, a bio-oil analysis for the identification of its chemical compounds is necessary to select the most abundant bio-oils according to the chromatogram area and those that might have an interest as added-value products for a further quantification study. A representative GC/MS TIC is provided in Figure 1. Bio-oil TICs are very complex, and there are overlapping peaks. These interferences hinder a proper integration of the selected 3909
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in peak areas, which means a good repeatability of the method and no significant effect of bio-oil sampling whenever a good homogenization of the bio-oil vessel is carried out. Finally, the interday precision is determined to evaluate the influence of the analyzed bio-oil samples in different days. After 10 days elapsed between analyses, different aliquots are analyzed. Thus, an ANOVA test between the peak area values obtained in days 1 and 2 is calculated. The analysis showed no significant differences between days, except for furfural, 2(5H)furanone, and 2,5-dimethoxy-tetrahydrofuran. Therefore, a good interday precision is achieved for the majority of the compound, and for those whose precision is not so good, a good quantification analysis should be carried out if the calibration curve is performed the same day of the sample analysis because it achieved a good intraday precision. Second, to assess the precision of the method using an internal standard, the area ratio relative to the three internal standards (toluene, 1,1,3,3-tetramethoxypropane, and 1-octanol) of each compound and it RSD for all of the aliquots are calculated. The area ratio is the area of the selected compound relative to the area of the internal standard. In Table 3, the selected m/z and the retention time for the internal standard peak integration are shown. These results are shown in Tables S.1, S.2, and S.3 of the Supporting Information. With the obtained data, the influence of the use of the three internal standards in the method precision is studied. The use of an internal standard does not have a high influence in the method precision because the results are similar to those obtained with the integrated area. The instrumental and intraday precisions are good for all compounds and using either of the internal standards. The interday precision is good for all compounds, except furfural, 2(5H)furanone, and 2,5-dimethoxy-tetrahydrofuran, when toluene or 1,1,3,3tetramethoxypropane are used. However, when 1-octanol is used, there is no good interday precision for any compound, which mean that 1-octanol is more sensitive to changes in the column with time. Moreover, the highest method precision is expected when an internal standard is used because it should reduce the effect of the instrumental drift (possible deviation in the injection volume and possible variations in the performance of the detectors) in the final result. Nonetheless, this fact is not very noticeable in our results because there are not several instrumental drifts during the analysis. Although a good precision is achieved for all compounds, there are two considerations that are necessary to take into account. The first consideration is the levoglucosan boiling point (380 °C). As a result, levoglucosan is not completely volatilized. However, good precision is obtained for this compound when the analysis is obtained, which can be explained by the fact that the volatilized fraction is always the same. It is necessary to take into account that non-volatilized levoglucosan is retained in the liner glass wool reducing its lifetime and the use of a glass wool liner is indispensable to prevent it from reaching into the column. The second consideration is that there is a double peak of 2,5-dimethoxy-tetrahydrofuran in the TIC. It is possible that they correspond to a two different isomers of this compound, and it is no possible to distinguish them with the software and library used. 3.3. Quantitative Analysis. For the purpose of quantification, an external calibration method is followed using 10 standards, chosen between the major bio-oil compounds according to the peak area from different chemical families of
Figure 1. TIC of the bio-oil.
compound peaks and, therefore, a proper quantification of them. Because of that, it is crucial to choose a m/z for each compound that reduces or eliminates these interferences, allowing for a faultless integration. From the TIC, a total of 46 compounds among the more than 200 detected are identified by a probability match of >800 by comparison to spectra from the NIST mass spectral library. The identified compounds, classified into chemical families, are listed in Table 1. In this bio-oil sample, the chemical family distribution is divided into mostly ketones, followed by sugar, acids, alcohols and phenols, aldehydes, and other chemical families. The most abundant compounds in bio-oil, according to the peak area in the chromatogram, are levoglucosan, acetic acid, 1-hydroxy-2-propanone, 2-methoxy-4-methyl-phenol, and hydroxy-acetaldehyde. From the identified compounds, almost one compound of each chemical family is selected for it quantification. These compounds are those with a high abundance according to the chromatogram area in the bio-oil sample, with no interference within other peaks and those that might have an interest as added-value products. Thus, 2-propen-1-ol, 2-butanone, acetic acid, furfural, 2(5H)furanone, 2,5-dimethyoxy-tetrahydrofuran, 3-methyl-1,2-cyclopentanedione, 2-methoxy-4-propyl, vanilline, and levoglucosan are the selected compounds to carry out the quantitative analysis of the bio-oil. 3.2. Method Precision. To ensure that the method is suitable for the quantification of bio-oil compounds, a study is performed of the precision of the method considering the instrumental precision, the intraday precision, and the interday precision for the selected compounds. First of all, the precision of the method is assessed without using an internal standard. In Table 2, the average of the integrated area of each selected compound and it relative standard deviation (RSD) for each aliquot sampled are shown. The instrumental precision is evaluated by a sequence of repeated injections of the same aliquot and calculating the RSD of the three replicates of each aliquot for each compound. The calculated RSDs are less than 10% for all compounds and for all of the aliquots, indicating a good instrumental precision of the runs. Thus, it can be concluded that the method presents a good instrumental precision. Then, the repeatability or intraday precision is assessed, which expresses the precision under the same operating conditions over a short period of time. For this purpose, an ANOVA test for each compound with a confidence interval of 95% between the three different analyzed aliquots in the same day is calculated. The analysis showed no significant differences 3910
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Table 1. Identified Compounds in Bio-oil Classified into Chemical Families and Ordered by Retention Timea compounds acids and esters methyl acetate formic acid acetic acid alcohols and phenols 2-propen-1-ol phenol 2-methyl-phenol 4-methyl-phenol guaiacol 2,4-dimethyl-phenol 2-methoxy-5-methyl-phenol 2-methoxy-4-methyl-phenol 4-(2-propenyl)-phenol 3-methyl-1,2-benezenediol 4-ethyl-2-methoxy-phenol 4-methyl-1,2 benzenediol 2-methoxy-4-vinyl-phenol eugenol 2-methoxy-4-propyl-phenol 4-ethyl-3-benzenediol 2-methoxy-4-(1-propenyl)-phenol 2-methoxy-4-(1-propenyl)-phenol (E) aldehydes hydroxy-acetaldehyde furfural 2,3-dihydrocybenzaldehyde 5-(hydroxymethyl)-2-furancarboxaldehyde vanillin ketones 2,3-butanedione 2-butanone 1-hydroxy-2-propanone 1-hydroxy-2-butanone 2-cyclopenten-1-one 1-(acetyloxy)-2-propanone 2(5H)furanone 3-methyl-2,5-furandione 2,5-hexanedione 1,2-cyclopentanedione 4-methyl-5H-furan-2-one 3-methyl-1,2-cyclopentanedione 3-ethyl-2-hydroxy-2-cyclopenten-1-one 1-(4-hydroxy-3-methoxyphenyl)-2-propanone acetovanilline sugar levoglucosan others 1,2-cyclopentadiene 2-methoxy-1,3-dioxolane 2,5-dimethoxy-tetrahydrofuran maltol a
m/z
RT (min)
area
RSD (%)
43 46 60
9.83 10.09 14.3
25107780 26595133 271893330
1 4 5
57 94 108 107 124 107 123 123 134 124 137 124 150 164 137 123 164 164
10.97 47.06 53.44 55.13 56.2 61.17 63.64 64.72 68.93 69.51 71.2 71.66 73.46 76.62 77.47 78.09 79.98 82.68
3498326 8258576 2751230 6057931 40169907 3222899 2493568 62247801 380423 1529871 37938360 2356650 7353951 19243155 21334633 1894554 7480418 11300945
2 9 0.3 3 0.9 6 5 9 9 6 3 6 9 4 4 5 10 7
32 95 138 126 151
11.03 31.41 62.33 65.84 78.43
54555915 30352159 1622250 6460226 32255730
5 9 8 6 7
43 43 43 57 82 43 55 68 99 98 69 112 126 137 151
11.71 12.27 15.63 24.69 31.06 34.41 37.47 39.99 40.13 40.71 50.04 50.53 58.47 86.45 84.3
23045428 5315364 183129067 33003194 8154790 8354572 52226497 5810536 738240 5894607 14194017 35261242 1455932 30959234 36592941
0.3 6 8 6 6 1 6 6 6 6 1 4 7 4 5
60
84.42
374833565
6
66 73 101 126
10.3 36.16 37.76 57.6
2237965 19030850 1651204 4656036
1 4 3 5
m/z, quantifying mass/charge ratio; RT, retention time; RSD, relative standard deviation.
First of all, the area, the area ratio relative to toluene, the area ratio relative to 1,1,3,3-tetramethoxypropane, and the area ratio relative to 1-octanol are plotted against the concentration of the selected compounds in the standards. Thus, the calibration curve data are fitted to a linear least-squares regression model to obtain four calibration equations for each compound
the qualitative analysis of the bio-oil [2-propen-1-ol, 2-butanone, acetic acid, furfural, 2(5H)furanone, 2,5-dimethyoxy-tetrahydrofuran, 3-methyl-1,2-cyclopentanedione, 2-methoxy-4-propyl, vanilline, and levoglucosan] (see section 3.1). Moreover, toluene, 1,1,3,3-tetramethoxypropane, and 1-octanol are used as internal standards. 3911
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Table 2. Average of the Peak Area of Each Selected Compound and Its RSDa day 1 aliquot 1
2-propen-1-ol 2-butanone acetic acid furfural 2(5H)furanone 2,5-dimethyoxy-tetrahydrofuran 2-hydoxy-3-methyl-2-cyclopenten-1-one 2-methoxy-4-propyl-phenol vanilline levogucosan a
aliquot 2
day 2
RT (min)
m/z
average (n = 3)
RSD (%)
average (n = 3)
RSD (%)
average (n = 3)
RSD (%)
average (n = 3)
RSD (%)
10.97 12.27 14.30 31.41 37.46 39.08 50.53 77.47 78.43 84.42
57 43 60 95 55 101 112 137 151 60
2868224 5372496 220655592 23433492 32392937 22649091 36330149 24474339 37897482 411103389
6 3 7 9 6 9 6 2 1 0.2
2899068 5256107 211479931 23808535 31922756 23359202 36652257 24876406 38924515 429381572
1 3 1 5 2 1 5 1 3 2
2816769 5012921 208922210 19678893 30017943 24373241 34115182 25085703 38877889 424954928
1 8 2 6 2 1 4 1 1 0.4
2700082 5066275 223463513 13911930 23334018 11368326 35287319 25596683 40521856 404529876
3 3 2 7 1 10 1 3 5 2
m/z, quantifying mass/charge ratio; RT, retention time; RSD, relative standard deviation.
Table 3. Retention Time and the m/z for Internal Standard Peak Integrationa
a
aliquot 3
internal standards
RT (min)
m/z
toluene 1,1,3,3-tetramethoxypropane 1-octanol
26.52 51.57 55.31
91 75 164
coefficient around 0.99 for all of the selected compounds and using any of the internal standards, except for the vanillin calibration curve using an area ratio relative to toluene and 1-octanol and furfural and 2(5H)furanone calibration curves using an area ratio relative to 1-octanol. Thus, it is considered that a good calibration of each selected compound is achieved. Then, the bio-oil samples with the added internal standards are analyzed. The area ratios relative to toluene, 1,1,3,3tetramethoxypropane, and 1-octanol are calculated, and the concentration of each compound is calculated using the corresponding calibration equations (Table 5). The quantitative analysis of all of the selected compounds is achieved. With regard to the concentrations calculated using
m/z, quantifying mass/charge ratio; RT, retention time.
(Table 4) depending upon the internal standard used or it absence. In Figure 2, the calibration curves using the peak area (without an internal standard) of all compounds are shown. With regard to the calibration (Table 4), an acceptable linearity of the data is reached, obtaining calibration curves with a correlation
Table 4. Calibration Equation for Each Compound Obtained to Fit the Area, Area Ratio Relative to Toluene, Area Ratio Relative to 1,1,3,3-Tetramethoxypropane, and Area Ratio Relative to 1-Octanol into a Linear Least Squares Regression Model 2-propen-1-ol
2-butanone
acetic acid
furfural
2(5H)furanone
y = 54122x − 209308 0.9972
y = 41909x − 1 × 107 0.9955
y = 39352x − 2 × 106 0.9808
y = 59456x − 2 × 107 0.9977
equation y = 0.0028x − 0.0182 y = 0.0014x − 0.001 R2 0.9712 0.9983 Area Ratio Relative to 1,1,3,3-Tetramethoxypropane
y = 0.0011x − 0.0152 0.9745
y = 0.001x − 0.05 0.9857
y = 0.0015x − 0.3525 0.993
equation y = 0.0015x − 0.0127 R2 0.9946 Area Ratio Relative to 1-Octanol
y = 0.0006x − 0.1779 0.9962
y = 0.0005x − 0.0291 0.9807
y = 0.0008x − 0.2188 0.993
y = 0.0038x − 0.0159 y = 0.003x − 0.945 y = 0.0027x − 0.1476 0.9849 0.9694 0.9396 2-hydroxy-2-cyclopenten-1-one 2-methoxy-4-propyl-phenol vanillin
y = 0.0038x − 0.6445 0.9268 levoglucosan
Area without Internal Standards equation y = 107234x − 868316 R2 0.9919 Area Ratio Relative to Toluene
equation R2
y = 0.0075x − 0.0656 0.9712 2,5-dimethoxytetrahydrofuran
y = 0.0007x − 0.0036 0.9948
Area without Internal Standards equation y = 100317x − 546084 R2 0.9995 Area Ratio Relative to Toluene
y = 79644x − 1 × 107 0.9878
y = 393366x − 4 × 106 0.9992
y = 45854x + 3 × 106 0.9517
y = 68187x + 3 × 107 0.9947
equation y = 0.0026x − 0.0064 y = 0.0021x − 0.3354 R2 0.9926 0.9633 Area Ratio Relative to 1,1,3,3-Tetramethoxypropane
y = 0.0101x − 0.0338 0.98977
y = 0.0011x + 0.1663 0.8644
y = 0.0017x + 1.3391 0.996
equation y = 0.0014x − 0.009 R2 0.9993 Area Ratio Relative to 1-Octanol
y = 0.0011x − 0.2018 0.9885
y = 0.0049x − 0.129 0.9983
y = 0.0006x + 0.0148 0.9625
y = 0.0009x + 0.3194 0.9889
y = 0.007x − 0.0455 0.9818
y = 0.0056x − 1.0373 0.9934
y = 0.0273x − 0.265 0.98
y = 0.0033x + 0.0467 0.936
y = 0.0048x + 1.5972 0.9615
equation R2
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Figure 2. Calibration curves using the peak area of (a) acetic acid (□) and levoglucosan (■), (b) 2-methoxy-4-propyl-phenol (×), furfural (▲), 2,5-dimethoxytetrahydrofuran (△), 2-hydroxy-3-methyl-2-cyclopenten-1-one (○), and vanilline (●), and (c) 2-propen-1-ol (⊗), 2-butanone (◆), and 2(5H)furanone (◇).
concentration calculated using the area ratio relative to toluene is 5.8 ± 0.8 wt %, which is higher than the calculated concentration using 1-octanol (5.2 ± 0.6 wt %), and this is higher than the calculated concentration using 1,1,3,3tetramethoxypropane (4.3 ± 0.3 wt %). Because of that, a one-way ANOVA is performed to compare them and see if there are significant differences between the calculated concentrations of each compound with a confidence interval of 95%. The concentration average of each compound in the studied bio-oil and its confidence interval of a 95% confidence level are also calculated (Table 5). The ANOVA shows that the calculated concentrations are significantly different in all cases, although the RSD values are below 10% for all compounds, and among them, seven have a RSD below 5%. This can be explained by the fact that the differences detected by ANOVA are more noticeable when there is a good instrumental precision. In conclusion, despite the significant differences detected by ANOVA, these differences are not so important when the data are compared.
the peak area without considering the internal standards, acetic acid and levoglucosan are the most concentrated compounds, with concentrations as high as 4.1 ± 0.2 and 3.8 ± 0.4 wt %, respectively. 2(5H)Furanone, vanilline, and 2-hydroxy-2cyclopenten-1-one have substantial concentrations of 0.7 ± 0.1, 0.53 ± 0.08, and 0.43 ± 0.07 wt %. Less concentrated compounds are furfural (0.18 ± 0.02 wt %) and 2,5dimethoxytetrahydrofuran (0.08 ± 0.02 wt %), followed by 2-butanone (0.07 ± 0.01 wt %), 2-methoxy-4-propyl-phenol (0.054 ± 0.008 wt %), and 2-propen-1-ol (0.028 ± 0.005 wt %). From the quantification results, a comparison between internal standards is carried out. Once more, it is observed that the use of an internal standard does not mean a higher precision of the measurements. However, it permits the detection of possible anomalous points. Furthermore, it is observed that all internal standards tested can be used for the quantification analysis because they do not have interferences with other compounds, and if so, they are eliminated by m/z. Thus, four concentrations for each compound are obtained from the quantification without an internal standard and with each of them. From these results, it is observed that the calculated concentration of each compound is greater when the area ratio relative to toluene is used for their calculation in comparison to the calculated concentration with the area ratio relative to 1-octanol, and this is greater than the calculated concentration with the area ratio relative to 1,1,3,3tetramethoxypropane. Taking acetic acid as an example, the
4. CONCLUSION In this work, a quantitative analysis of some target compounds of a pine wood bio-oil by means of GC/MS, including the most representative compounds of each chemical family present in bio-oil (acids, alcohols, phenols, ketones, aldehydes, and sugars), is achieved. The quantified compounds are 2-propen-1-ol, 2-butanone, acetic acid, furfural, 2(5H)furanone, 3913
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5±1 9 0.70 ± 0.10 4 0.12 ± 0.02 5
■
0.10 ± 0.03 9 0.23 ± 0.02 3
0.73 ± 0.06 3
0.53 ± 0.10 6
ASSOCIATED CONTENT
* Supporting Information S
Average of the area ratio relative to toluene (Table S.1), 1,1,3,3tetramethoxypropane (Table S.2), and 1-octanol (Table S.3) of each selected compound and its RSD. This material is available free of charge via the Internet at http://pubs.acs.org.
4.9 ± 0.3 2
■
AUTHOR INFORMATION
Corresponding Author
The authors declare no competing financial interest.
■
Confidence interval at 95% of the confidence level.
0.09 ± 0.01 5 0.032 ± 0.003 3 concentration ± CIa (wt %) RSD (%)
Notes
REFERENCES
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a
5±1 9 0.7 ± 0.2 9 0.03 ± 0.02 9 0.58 ± 0.03 2 0.11 ± 0.02 6 0.7 ± 0.1 7 0.24 ± 0.1 3 5.2 ± 0.6 5 0.034 ± 0.004 4 concentration ± CIa (wt %) RSD (%) Concentration Average
0.090 ± 0.004 2
4.5 ± 0.6 5 0.66 ± 0.09 6 0.060 ± 0.009 6 0.51 ± 0.06 5 0.09 ± 0.02 10 0.7 ± 0.1 8 0.21 ± 0.03 2 4.3 ± 0.3 2 0.03 ± 0.005 6
0.08 ± 0.01 5
2,5-dimethyoxy-tetrahydrofuran, 3-methyl-1,2-cyclopentanedione, 2-methoxy-4-propyl, vanilline, and levoglucosan. An acceptable linearity is achieved when the obtained data from the prepared standards are fitted to a linear least-squares regression model for all compounds, allowing for the quantification of all of them. With regard to the three internal standards (toluene, 1,1,3,3tetramethoxypropane, or 1-octanol), each of them can be used for quantification, although 1-octanol is more sensitive to changes in the column with time in comparison to the other internal standards. The comparison between the different internal standards by means of one-way ANOVA shows that the calculated concentration of each compound depends upon the internal standard used, although these differences are not remarkable, as observed from the RSD of the concentration average for each compound. Thus, any of the internal standards evaluated can be used for the quantitative analysis of bio-oil. Moreover, the analytical method used using internal standards or without them presents a good reproducibility, which is supported by the good instrumental, intraday, and interday precisions achieved. This fact confirms the analytical method suitability for bio-oil quantification. Thus, the obtained results permit having further knowledge of a proper quantification of bio-oil chemical composition. They provide crucial information for the development and assessment of new upgrading processes and the evaluation of the viability of the value-added product extraction from the biooil. Besides, a better comprehension of the effect of pyrolysis conditions and biomass type on the bio-oil composition as well as the reactions that take place during the upgrading processes may be achieved.
*Telephone: +34-93-5812118. Fax: +34-581-2477. E-mail:
[email protected].
concentration ± CIa (wt %) RSD (%) Area Ratio Relative to 1-Octanol
6±4 7 0.9 ± 0.2 10 0.11 ± 0.08 8 5.8 ± 0.8 2 0.10 ± 0.04 4 0.036 ± 0.001 concentration ± CIa (wt %) RSD (%) 0.4 Area Ratio Relative to 1,1,3,3-Tetramethoxypropane
0.7 ± 0.1 8 0.18 ± 0.02 1 4.1 ± 0.2 1 0.07 ± 0.01 7 concentration ± CIa (wt %) 0.028 ± 0.005 RSD (%) 7 Area Ratio Relative to Toluene
0.27 ± 0.02 3
0.8 ± 0.1 2
0.43 ± 0.07 7 0.08 ± 0.02 12
0.6 ± 0.3 5
0.07 ± 0.01 6
Article
0.30 ± 0.05 8
3.8 ± 0.4 4 0.53 ± 0.08 6
vanillin 2-methoxy-4-propyl-phenol 2-hydroxy-2-cyclopenten-1-one 2,5-dimethoxytetrahydrofuran 2(5H)furanone furfural acetic acid 2-butanone 2-propen-1-ol Area without Internal Standards
Table 5. Quantitative Analysis of Bio-oil by the GC/MS Method by Means of the Area, the Area Ratio Relative to Toluene, the Area Ratio Relative to 1,1,3,3Tetramethoxypropane, and the Area Ratio Relative to 1-Octanol
levoglucosan
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