Article pubs.acs.org/EF
Development and Application of Chemical Analysis Methods for Investigation of Bio-Oils and Aqueous Phase from Hydrothermal Liquefaction of Biomass Søren Ryom Villadsen,† Line Dithmer,† Rasmus Forsberg,† Jacob Becker,† Andreas Rudolf,‡,§ Steen Brummerstedt Iversen,‡,∥ Bo Brummerstedt Iversen,† and Marianne Glasius*,† †
Department of Chemistry and iNANO, Aarhus University, Langelandsgade 140, 8000 Aarhus C, Denmark SCF Technologies A/S, Smedeholm 13B, 2730 Herlev, Denmark
‡
ABSTRACT: Hydrothermal liquefaction (HTL) is an efficient second-generation technology for conversion of biomass or biomass waste products into bio-oils for e.g. the transport sector. The product consists of a bio-oil of varying viscosity and composition, as well as aqueous and gas-phase byproducts. In order to guide optimization of the HTL process with regard to conditions, catalysts, and feedstock, there is a need for standardized methods for product analysis. We have developed analytical methods to investigate the chemical composition of bio-oils produced in a continuous HTL plant using 11 different types of feedstocks and model compounds (sucrose, cellulose, sunflower oil, casein, gluten, cellulose mixed with protein, manures, sewage sludge, and Dried Distillers Grains with Solubles, DDGS). Bio-oils and aqueous phase byproducts were analyzed directly by gaschromatography (GC) coupled with mass spectrometry (MS), while fatty acids (C10−C18) were specifically quantified using GC (with flame ionization detection) after derivatization into their methyl esters. In addition we developed and applied a new high-performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF-MS) method for detection of fatty acids (C14−C22) in the aqueous phase byproduct. Generally HTL bio-oils had a complex composition showing 100−200 separate peaks in the GC-MS chromatograms, but for most feedstocks, 30−50 peaks accounted for a large fraction of the total peak area. Grouping of identified products into chemical families provided a useful overview for characterization of the bio-oils. Fatty acids (C16 and C18) were major products from HTL of protein (gluten), sunflower oil, DDGS, sewage sludge, and some manure feedstocks, while other important compound groups in bio-oils included phenols, cyclopentenes, indoles, pyrroles, and amides. The aqueous phase contained products such as cyclopentenones, pyrazines, and fatty acids, also found in the bio-oils, which were probably present as micelles suspended in the aqueous phase.
1. INTRODUCTION
depolymerization, fragmentation, dehydration, and decarboxylation.1,6 Bio-oils from HTL have viscosities varying from liquid to tarlike and compose a very complex matrix containing small molecules of diverse polarity and boiling points, as well as large oligomeric components.4,6−10 This constitutes a challenging task for the analytical chemist, and complete identification of products cannot be achieved with a single analytical method. Gas chromatography-mass spectrometry (GC-MS) is applied in most studies of composition of HTL bio-oils4,7,11−20 due to its versatility, as well as the availability of a large library of mass spectra for compound identification. Drawbacks of GC-MS analysis include inadequate quantification of polar compounds such as sugars and fatty acids without previous derivatization of these prevalent components of bio-oils, as well as unsuitability for analysis of low-volatility oligomeric compounds. An adequately comprehensive analysis method is important in order to provide information on oil stability (since a high degree of oxygenation and unsaturation can cause polymerization) and potential toxicity, as well as predict hydrophobicity and phase distribution.
Sustainable use of energy and resources are key points in the energy system of the future, and there is a growing interest in development of sustainable energy sources based on biomass waste products rather than potential food products. Hydrothermal liquefaction (HTL) is a promising second-generation method for production of energy from biomass waste. The HTL process converts wet biomasses into crude bio-oil at elevated temperature (280−370 °C) and pressure (10−25 MPa), often involving homogeneous and/or heterogeneous catalysts to improve both the quality of the product and production yield.1,2 At these conditions water is in a subcritical phase characterized by low viscosity and high solubility of hydrophobic compounds, and it acts as both reactant and catalyst. Such diverse feedstocks as swine manure, wood residues, garbage, spent coffee grounds, and algae have been investigated, showing energy recoveries as high as 80%.1,3,4 The product may consist of four phases: a gas-phase of mainly CO2, a top-phase of bio-oil, an aqueous byproduct, and a bottomphase with mainly inorganic salts.5 Depending on oil properties (e.g., density and hydrophobicity), the oil is either just gravimetrically separated from the aqueous phase or extracted with an organic solvent. The complicated chemical reactions of biological molecules in hydrothermal processes include © 2012 American Chemical Society
Received: June 3, 2012 Revised: September 21, 2012 Published: October 2, 2012 6988
dx.doi.org/10.1021/ef300954e | Energy Fuels 2012, 26, 6988−6998
Energy & Fuels
Article
monitoring was applied on 172 m/z at retention time (RT) 20.1−20.5 min and 91 m/z at RT 36.25−36.60 min to record 4-bromophenol and 2-phenylethyl bromide, respectively. All MS pressures were well within the operational specifications. The MS was calibrated using heptacosa and tuned with the masses 69, 131, 219, and 414 Da to a relative peak area of 100, 36.31, 38.07, and 1.64, respectively. Compounds were tentatively identified by comparison of their mass spectra with the NIST 08 database (NIST Mass spectral search program, version 2.0f, 8.10.2008). The uncertainty of individual peak areas was estimated to within at least ±15%, based on repeated analyses over several weeks. 2.5. Derivatization of Fatty Acids and GC-FID Analysis. Fatty acids in bio-oils were derivatized to their methylesters for quantitative analysis by GC-FID. Aliquots of bio-oil (about 100 mg) were weighed directly into a sample glass and dissolved in 4 mL of dry methanol with 5% acetyl chloride. The glass was sealed, and the reaction was allowed to proceed at 65 °C for 45 min. Fatty acid methyl esters (FAMEs) were extracted in 4 mL of pentane. After evaporation of pentane using a flow of nitrogen gas, the sample was redissolved in 2.0 mL of heptane. This procedure gave acceptable recoveries (>90%) of FAMEs of C10−C18. Later, the recovery was improved for C6 and C8 FAMEs by direct extraction with 2 mL of heptane to avoid the solvent evaporation step. FAMEs were analyzed using an Agilent 7890 GC with FID detector and helium carrier gas at a flow rate of 10 mL min−1. Samples (0.2 μL) were injected with a split ratio of 5:1 at 225 °C. The column was a HP5 (30 m × 0.32 mm × 0.25 μm). The oven program was as follows: start at 45 °C, 12 °C min−1 to 200 °C, 2 °C min−1 to 216 °C, 4 °C min−1 to 230 °C, and finally 30 °C min−1 to 300 °C giving a final run time of about 30 min. 2.6. HPLC-TOF-MS Analysis of Aqueous Phase. Aliquots of aqueous phase samples were diluted 1−150 times with eluent A (95% methanol and 5% ammonium acetate) before filtration through a 0.45 μm pore size nylon Q-Max syringe filter (Frisenette, Denmark) into HPLC vials. No further adjustment of pH was applied. Samples were analyzed using a Dionex Ultimate 3000 HPLC coupled through an electrospray (ESI) inlet (operated in negative mode) to a Bruker microTOFq mass spectrometer. The following MS settings were applied: end plate offset -500 V, capillary +4000 V, inlet temperature 190 °C, nebulizer pressure 0.8 bar, dry gas flow 8.0 L min−1, funnel 1 and 2 RF 200.0 Vpp. Masses from 50−1000 m/z were analyzed. The HPLC column was a Synergi MAX-RP (particle size 4 μm, C12 bonded phase, 250 × 2.0 mm) kept at 35 °C, and the eluents were A: 95% methanol and 5% ammonium acetate and B: 60% isopropanol, 30% acetonitrile, and 10% triple-distilled water at a flow rate of 200 μL min−1. The developed HPLC method was as follows: 100% A for 10 min, change to 100% B within 4 min, 100% B for 5 min, change back to 100% A within 5 min, and kept for 6 min to a total run-time of 30 min. This final method provided maximum recovery of fatty acids as well as minimum carryover between samples. Calibration curves were prepared at 7 levels between 0.1 and 8 mg L−1 for the following fatty acids: tetradecanoic acid, hexadecanoic acid, octadecanoic acid, cis-9octadecenoic acid (oleic acid, C18:1), cis,cis-9,12-octadecadienoic acid (linoleic acid, C18:2), icosanoic acid (C20:0), and docosanoic acid (C22:0), with R2 > 0.99 for all compounds. 2.7. Supplemental Analyses. Water content in bio-oil was determined by Karl Fischer titration with a TitraLab TIM 580 (Radiometer, Denmark). Total organic carbon (TOC) was determined using a Hach-Lange TOC-X5 test kit (Hach-Lange, Denmark). Ash content was determined according to ASTM D 482. AnalyCen (Sweden) performed analysis of DDGS feedstock using standard methods for analysis of e.g. fat (Tecator AN 301, 2001), protein (Dumas method), and neutral detergent fibers, NDF (Tecator AN 304), while nitrogen-free extractives, NFE, were calculated from the other analyses. Heat value was determined according to ASTM D 240 and elemental composition according to ASTM D 5291 and 1552 at Karlshamn Kraft, Karlshamn, Sweden.
The aim of the present study was to develop and apply a set of generic methods using GC-MS, GC with flame ionization detection (FID), and HPLC-TOF-MS for investigation of chemical composition of HTL bio-oils and aqueous phase and to study composition of bio-oils from three complex feedstocks: i.e. Dried Distillers Grains with Solubles (DDGS, a waste product from bioethanol production), sewage sludge, and manure, as well as selected model compounds to derive information on the chemical process and products. HPLCTOF-MS has to our knowledge not previously been used for studying the chemical composition of the aqueous phase from HTL.
2. MATERIALS AND METHODS 2.1. Chemicals and Reagents. All solvents (acetonitrile, dichloromethane, ethanol, heptane, isopropanol, methanol, pentane, and tetrahydrofuran) were obtained in analytical grade (pro analysi p.a.) quality from Sigma-Aldrich, while NaOH (p.a., >99%), 37% HCl (p.a.), and ammonium acetate (p.a., >98%) were from Merck, 4bromophenol (practical grade) was from Struers, and 2-phenylethylbromide (p.a. >99%) was from Fluka. Standards of fatty acids (p.a., >99%) and model feedstock compounds (cellulose and proteins) were obtained from Sigma-Aldrich. Helium gas was of 99.999% purity. 2.2. Bio-Oil Production. Bio-oils were produced in a continuous process pilot scale reactor described by Toor et al.5 Briefly, the biomass feedstock was converted to crude bio-oil at subcritical conditions (350 °C and 25 MPa) in the presence of a homogeneous (K2CO3) and heterogeneous zirconia-based catalyst. Of the four resulting phases, organic compounds in top-phase bio-oil and aqueous phase were analyzed in the present study. Oil and aqueous phase were separated using a disk-stack centrifuge at atmospheric pressure.5 2.3. Sample Preparation for GC-MS Analysis. Aliquots of biooil (about 0.25 g each) were weighed directly into a sample glass and diluted with solvent to a concentration of about 15 g/L. We tested solubility and analysis of bio-oils in four solvents: tetrahydrofuran (THF), dichloromethane (DCM), ethanol, and methanol (see section 3.1). THF was the solvent of choice for analytical results reported in this work. The aqueous phase was made either alkaline or acidic before extraction in hexane followed by GC-MS analysis. The pH of 4 mL of the aqueous phase was adjusted by dropwise addition of 37% HCl until pH was below 2 or 6 M NaOH until pH was above 12, while pH was determined using a pH meter (PHM 220, MeterLab). Then 2 mL of hexane was added and the solution was stored at 5 °C, until the next day when the hexane phase was transferred to a vial for GC-MS analysis. All samples were filtered through a 0.45 μm pore size nylon Q-Max syringe filter (Frisenette, Denmark) before analysis. Quality control standards were 4-bromophenol and 2-phenylethyl bromide dissolved in THF. 30.0 μL of standard solution with 4bromophenol and 2-phenylethyl bromide both at 4.5 g/L was added to a 1.00 mL bio-oil sample just before analysis. 2.4. Composition Analysis by GC-MS. Analysis was performed using a Perkin-Elmer Autosystem XL gas chromatograph coupled with a Perkin-Elmer Turbomass 500 mass spectrometer and equipped with Perkin-Elmer Turbomass software package. The GC injection port was operated at 250 °C in 1:19 split mode with the carrier gas helium at 1 mL/min, and the injection volume was 2.5 μL. The column was a Zebron ZB-5 (60 m × 0.25 mm × 0.25 μm) with a 5%-phenyl, 95% dimethylpolysiloxane stationary phase. The column oven program started at 70 °C which was held for 1 min and progressing at 1 °C/min to 100 °C (hold time 5 min), then 4 °C per min until 250 °C (hold time 1 min), and finally 15 °C/min until 300 °C (hold time 5 min), giving a total run-time of 82.8 min. The GC interface inlet line temperature and the ion source were kept at 180 °C. Ionization was performed using an electron impact (EI) source in positive ion mode with an electron energy of 70 eV. The MS was set to scan mode (50−300 m/z) and single ion 6989
dx.doi.org/10.1021/ef300954e | Energy Fuels 2012, 26, 6988−6998
Energy & Fuels
Article
Figure 1. GC-MS chromatogram of bio-oil sample (cellulose + protein feedstock) dissolved in four different solvents (red line: tetrahydrofuran, green line: methanol, purple line: ethanol, and black line: dichloromethane).
3. RESULTS AND DISCUSSION 3.1. Development of the GC-MS Method. We investigated four solvents for dilution of bio-oil before analysis, in order to identify the most suitable one(s). Based on preliminary investigations of bio-oil composition these four polar solvents were chosen: tetrahydrofuran (THF), dichloromethane (DCM), ethanol, and methanol. THF and DCM are both polar aprotic solvents with intermediate dielectric constants (7.6 and 9.1, respectively), while ethanol and methanol are polar protic solvents with higher dielectric constants (24 and 33, respectively). A bio-oil sample, prepared from HTL of a feedstock with protein and cellulose, was diluted in the four solvents and analyzed by GC-MS. The results (Figure 1 and Table 1) show
is the peaks at about 11.5 min, which are only observed in the THF chromatogram. They are due to formation of butyrolactone and 4-hydroxybutanoic acid from reaction between THF and water either during storage or in the sample. Despite formation of these two artifact peaks, THF was preferred rather than DCM as solvent for bio-oils in the present study, to avoid the use of chlorinated solvent and the rise in background signal observed with DCM. In previous studies, bio-oil has typically been diluted in methanol, acetone, DCM, and chloroform (see Table 2) before GC-MS analysis. Valdez et al.4 investigated recovery of bio-oil from crude algal bio-oil using four nonpolar and three polar solvents and found that extraction with chlorinated polar solvents (chloroform and DCM) gave higher yields of compounds which could be identified by GC-MS analysis. Table 2 presents an overview of GC-MS methods applied in the present as well as former studies of bio-oil composition. Regarding type of column, nine out of thirteen studies listed in Table 2 have applied a 5% phenyl-95% dimethylpolysiloxane column (e.g., HP-5 and similar), which is known as a nonpolar column for general purposes. One study used a 100% dimethylpolysiloxane column (HP-1, nonpolar),12 while three studies of bio-oil from HTL of microalgae used more polar column phases (Rtx-1701 and Stabilwax).15,16,19 Most studies have applied a 30 m column, while the present and three other studies4,15,16 have chosen a longer column (50 or 60 m) to increase the chromatographic resolution. Film thicknesses are between 0.1 and 0.5 μm (see Table 2). Helium has been used as carrier gas in the present as well as most previous studies, at flow rates from 0.9 to 20 mL/min e.g. refs 4,9,17. Table 2 shows that the applied oven temperature settings and increase rates vary considerably between studies. In the present work we have developed a stepwise temperature gradient to achieve optimal separation of chromatographic peaks within a reasonable analysis time. Here we have used a set of injection standards, 4bromophenol and 2-phenylethyl bromide, to correct for variations in injection and analysis, which is of special importance in quantitative studies of bio-oil composition. We chose brominated standards to ensure that the compounds were not already present in the bio-oil samples.
Table 1. Data Analysis of GC-MS Chromatograms of Bio-Oil Sample Diluted in Four Different Solvents (See Figure 1)a analyzed number of peaks
analyzed peak area
THF DCM MeOH EtOH
total peak area (area)
(area)
29,795,650 34,225,518 13,143,705 13,753,901
20,868,944 21,924,863 5,101,601 5,249,066
(%)
total number of peaks (#)
(#)
(%)
70.0 64.1 38.8 38.2
87 103 79 79
18 20 7 6
20.7 19.4 8.9 7.6
a
THF is tetrahydrofuran, DCM is dichloromethane, EtOH is ethanol, and MeOH is methanol.
quite good overall similarity between results obtained with different solvents, but a closer look also reveals some distinct differences. Chromatograms of bio-oil in ethanol and methanol are almost identical but give rise to fewer and smaller chromatographic peaks, from which fewer compounds are identified. DCM gives the largest number of peaks and the largest peak area, but the chromatogram also contains unresolved late-eluting compounds leading to a rise in background noise. Dilution in THF gives almost the same number of peaks and size of peak area but without the background noise observed using DCM. One distinct difference 6990
dx.doi.org/10.1021/ef300954e | Energy Fuels 2012, 26, 6988−6998
Energy & Fuels
Article
Table 2. Overview of Solvents and GC-MS Methods for Bio-Oil Analysis in the Present Work and Former Studiesb feedstock
process
solvent
inlet
column
tstart (°C)
hold (min)
ratea (°C/min)
tend (°C)
hold (min)
300
5
present work
290
0
9
reference
DDGS, cellulose, protein, manure and more
HTL
THF
split, 250 °C
ZB-5, 60 m × 0.25 mm × 0.25 μm
70
1
woody biomass
fast pyrolysis pyrolysis
MeOH
1
40
1
6
260
12
27
HTL
n.s.
n.s.
40
10
5
300
10
12
acetone
1.5
20
320
5−75
11
40
3
5
300
8
14
microalgae
HTL
CHCl3
70
2
10
300
10
7
microalgae
HTL
DCM
splitless, 250 °C splitless, 280 °C split, 250 °C split
45
macroalgae
liquefaction HTL
80
2
3
300
10
,1720
microalgae
HTL
DCM
n.s.
40
2
n.s.
250
30
,1615
microalgae
HTL
DCM
n.s.
40
2
60
260
10
19
microalgae
HTL
7 different solvents
split, 300 °C
HP-5MS, 30 m × 0.25 mm × 0.25 μm HP-5, 30 m × 0.25 mm × 0.5 μm HP-1, 25 m × 0.32 mm × 0.17 μm Rxi-5Sil MS, 30 m × 0.25 mm HP-5MS, 30 m × 0.25 mm × 0.25 μm VF-5, 30 m × 0.2 mm × 0.33 μm DB-5HT, 30 m × 0.25 mm × 0.10 μm Rtx 1701, 60 m × 0.32 mm × 0.25 μm Rtx Stabilwa×, 30 m × 0.32 mm × 0.25 μm HB-5MS, 50m × 0.20 mm × 0.33 μm
40
MeOH
split, 250 °C 250 °C
1→100 4→250 15→300 3
35
5
1→50 3→300
300
15
4
pine chips sawdust, rice husk, lignin and cellulose swine manure
a
DCM
Rates written as e.g. 1→100 means temperature increase of 1 °C/min until 100 °C. bn.s. means not specified in the reference.
Figure 2. GC-MS total ion chromatogram (TIC) of HTL bio-oil from cellulose feedstock. Numbers refer to peaks listed in Table 3.
3.2. Composition of HTL Bio-Oil from Cellulose. Cellulose was investigated as a model compound, since it is a major component of many types of biomass. Cellulose is a polysaccharide of β-1,4-linked D-glucose units. The specific feedstock had an elemental composition of 44% C, 6% H, and 49% O and thus did not contain nitrogen and sulfur. GC-MS chromatograms of bio-oils produced from cellulose contained almost 160 different compounds (Figure 2). Interestingly, there were no major products, but a multitude of minor products each contributing 0.5−1% of the total peak area (Table 3). Identified products were primarily cyclic ketones, phenols, indanones, and aromatic compounds. In contrast, fast pyrolysis of cellulose in the presence of water has been reported to yield especially furanes (furaldehydes) and derivatives hereof, furfural and pyranones12,21 through dehydration of saccharides.22 3.3. Composition of HTL Bio-Oil from Protein. Protein is a major component in many waste biomasses such as residues from meat production, biological fermentation/cultivation, and from algae cultivation, while levels are low in plant biomass. Protein consists of one or several peptide-chains made up from amino acids held together by peptide bonds. The two proteins tested are gluten and casein, of which casein is a group of phosphoproteins derived from milk, while gluten is a proteincomposite found in e.g. wheat. They have relatively similar
structures and contain high levels of proline and glutamic acid. The gluten feedstock also contains a small amount of starch. The elemental composition of protein feedstocks was 52% C, 7% H, 23% O, 16% N, and 0.8% S. In comparison with other biomass components, proteins are thus characterized by rather low oxygen but high nitrogen content. The chromatogram of bio-oil from the protein casein (Figure 3) is complex with 226 peaks of which many products are in low yields. The products are primarily substituted phenols, indoles, fatty acids, and amides. Only 16% of the peaks were analyzed, and many of the unidentified compounds seem to contain an aromatic system with oxygen and nitrogen containing substituents. 37 peaks were identified, constituting 71% of the total peak area. HTL bio-oils produced from gluten contained a much higher degree of fatty acids, which could be caused by impurities in the feedstock. Biller et al.15 also observed phenols, indole, pyrroles, and piperidine as major products from HTL of protein feedstocks. 3.4. Composition of HTL Bio-Oil from DDGS. DDGS is a waste product from bioethanol production, and its suitability as feedstock for bio-oil production was investigated. The specific DDGS used for the HTL bio-oils contained 5.9 ± 0.6% fat, 33 ± 2% protein, 30 ± 3% neutral detergent fibers, NDF (lignin, cellulose, and hemicellulose), and 48 ± 5% NFE (sugars, starch, hemicellulose, and pectins). The elemental composition was 6991
dx.doi.org/10.1021/ef300954e | Energy Fuels 2012, 26, 6988−6998
Energy & Fuels
Article
Table 3. GC-MS Analysis of Major Chemical Composition of HTL Bio-Oil from Cellulose #
rt
area%
compound
groupa
#
RT
area%
compound
1 2 3 4 5 6 7 8 9
13.9 14.5 15.7 16.4 16.6 18.9 21.6 22.3 23.6 24.5 24.7 30.1 31.2
1.2 2.4 0.8 1.3 5.7 1.4 2.2 2.8 1.2 3.8 1.0 0.8 3.2
5 5 2 5 5 8 2 5 2 5 5 2 9
1 2 3
15.46 16.71 21.6
6.5 2.2 0.9
4 5 6
21.96 23.46 27.36 28.39 29.68 30.17 31.14
0.6 6.9 0.9 1.9 0.9 0.6 1.0
12
37.6
0.9
33.46 44.98 48.54 49.63
6.5 3.9 3.7 2.6
13 14 15 16 17
39.6 42.7 43.8 44.7 45.7 47.9 49.4
0.6 1.8 1.0 0.9 2.2 0.9 1.5
49.94 50.07 54.2 54.47 58.44
1.6 1.3 1.5 2.5 1.0
51.5 51.7 52.1 54.9 59.6 59.7 60.2
0.8 1.6 0.7 0.6 1.1 0.8 1.4
3-ethylcyclopentanone 3-methyl-2-cyclopenten-1-one phenol 3,4-dimethyl-2-cyclopenten-1-one 2,3-dimethyl-2-cyclopenten-1-one isopropylidenecyclohexane (uncertain) m-cresol (major) and unknown (minor) 2,3,4-trimethyl-2-cyclopenten-1-one p-cresol unidentified (possibly cyclopentenone) 3,5,5-trimethyl-2-cyclopenten-1-one o-ethylphenol unidentified (complex alkane/alkene system) 4,5,6,6a-tetrahydro-2(1H)-pentalenone and unidentified bornane 2,3,4,5,6,7-hexahydro-1H-inden-1-one 1-indanone 4-ethyl-3,4-dimethyl-2-cyclohexen-1-one 2-methyl-1-indanone possibly dimethylbenzaldehyde possibly 7a-methyl-1,4,5,6,7,7a-hexahydro2H-inden 3,3-dimethyl-1-indanone possibly 2,4,6-trimethylbenzaldehyde possibly 1-isopropenyl-3-isopropylbenzene possibly 2,3,4,5-tetramethylbenzaldehyde 3-allylsalicylaldehyde 3-ethyl-1,2,4,5-tetramethylbenzene possibly 1-ethyl-2,3,4,5,6pentamethylbenzene possibly 1-ethyl-3,5-diisopropylbenzene unidentified analyzed peaks # analyzed peaks %
62.77 62.9 14 67.85 68.48 15 72.01 16 72.82 17 73.53 total #peaks analyzed area %
0.8 0.7 2.0 1.0 2.3 0.8 0.7 226 71
phenol 2,4-dimethyl-3-ethylpyrrole m-cresol (minor) and pyrrole + 5 Me (major) pyrrole + 5 Me p-cresol phenylethyl alcohol 1,3,4,5,6,7-hexahydro-2H-inden-2-one possibly 4-methylphenyl carbamate o-ethylphenol m-ethylphenol and 2,3-dimethyl-1Hpyrrole (50/50) p-ethylphenol indole possibly N-butyl-2-pyrrolidinone complex compound possibly containing adamantane structure similar to above skatole N-ethylindole and unknown (50/50) 2,3-dimethylindole and unknown (50/50) 2,3,5-trimethylindole (minor) and unknown (major) 1-phenethylpiperidine tetradecanoic acid hexadecanoic acid Aribin: 9H-pyrido[3,4-b]indole. 1-methyl C18 fatty acid and unidentified comp. hexadecanamide N-methylhexadecanamide analyzed peaks # analyzed peaks %
10 11
18 19
20 21 22
61.2 62.5 total #peaks analyzed area % a
Table 4. GC-MS Analysis of Major Chemical Composition of HTL Bio-Oil from Protein (Casein)
0.6 1.0 158 76
7
8 9
9 8 4 4 5 4 8 4
10 11 12
13
4 8 8 8 8 8 8 8 9 51 32
a
groupa 2 1 1 1 8 4 2 2 2+1 2 3 9 9 9 3 3 9 9 8 6 6 3 6+9 7 7 37 16
Group numbers refer to chemical families listed in Table 8.
9-octadecenoic acid (oleic acid, C18:1), and cis,cis-9,12octadecadienoic acid (linoleic acid, C18:2), which dominate the chromatograms and constitute about 65% of the total peak area. Fatty acids make up a large fraction owing to their high thermal stability once formed. Derivatization of fatty acids into their methyl esters could improve the quantitative analysis of fatty acids and is discussed in section 3.7. A number of nitrogen-containing compounds originating from the protein fraction, such as pyrroles and indoles, were found as well as cellulose degradation products, such as furan derivates and cyclopentenones. Substituted phenols were found in the DDGS-oil; however, these were also seen in the degradation of proteins and
Group numbers refer to chemical families listed in Table 8.
46% C, 7% H, 39% O, 7% N, and 0.8% S, and DDGS thus has some similarities with cellulose, except that DDGS also contains nitrogen and sulfur compounds but less oxygen. Three DDGS HTL bio-oil samples were analyzed, giving quite similar chromatograms and composition. Figure 4 shows one of the TIC chromatograms. About 108 chromatographic peaks were identified, and 93% of the overall area was contained within the largest 29 peaks (Table 5). This is due to large peaks from fatty acids, primarily hexadecanoic acid, cis-
Figure 3. GC-MS TIC of HTL bio-oil from protein feedstock. Numbers refer to peaks listed in Table 4. 6992
dx.doi.org/10.1021/ef300954e | Energy Fuels 2012, 26, 6988−6998
Energy & Fuels
Article
Figure 4. GC-MS TIC of HTL bio-oil from DDGS feedstock. Numbers refer to Table 5.
benzofuranes, phenols/cresols, and general methylation of other compounds.23,24 Bayerbach et al.23 studied bio-oil from fast pyrolysis of beech and have empirically and theoretically determined several guaiacols, syringols, and catechols as products of lignin, in line with our findings of substitued phenols. 3.5. Composition of HTL Bio-Oil from Manure. Manure has a complex chemical composition, and primary components of dairy cattle manure can be 16.5% protein, 22% cellulose, 12.5% hemicellulose, and 14% lignin,25 but considerable variations, depending on type and handling procedures, are expected. Three types of swine manure feedstocks were tested: 1: liquid manure (slurry) + whey + recirculated concentrate of the waterphase, 2: liquid manure + whey, and 3: liquid manure. Chromatogram and data from analysis of bio-oil produced from manure-2 is found in Figure 5 and Table 6. The chromatogram contains about 132 peaks, of which 38 peaks, making up 68% of the total peak area, were analyzed in details. Some of the major identified products, each constituting 2−5.5% of the peak area, were substituted cyclopenten-1-ones, substituted cyclohexanone, and ethylphenol, as well as different guaiacol, indenone, and indole derivatives, which are expected as breakdown products of lignin and cellulose. Each of the fatty acids hexadecanoic acid, oleic acid, and linoleic acid only constituted 1−2% of the peak area. In contrast, bio-oils from manure feedstocks 1 and 3 contained 36% and 27% fatty acids, respectively. Other major products of these feedstocks were phenols, indoles, and cyclopentenones, as also observed for manure-2. Xiu et al.11 also found hexadecanoic acid and oleic acid as major products of HTL of swine manure, in addition to amides and substituted phenols. The difference in yield of fatty acids can be attributed to dissimilarities in feedstock and process conditions. 3.6. Comparison of HTL Bio-Oils from Different Feedstocks. In addition to feedstocks described in sections 3.2−3.5 the comparison includes bio-oils from sucrose, sewage plant sludge, a mix of cellulose and protein, and sunflower oil.
Table 5. GC-MS Analysis of Major Composition of HTL Bio-Oil from DDGS #
RT/ min
area%
1 2 3
13.4 15.6 16.8
1.0 1.3 1.2
4 5 6 7 8 9 10 11
21.7 22.1 22.5 23.6 25.0 27.5 29.3 31.4
1.5 1.9 0.5 1.3 1.0 0.4 0.3 0.7
12 13 14 15 16
33.6 45.0 50.1 54.2 54.5 56.9 17 58.0 18 62.9 19 67.9 20 72.0 21 72.9 22 73.5 23 74.5 total #peaks analyzed area % a
1.2 0.8 1.2 0.5 0.5 0.3 0.5 0.4 18.2 47.0 4.8 1.2 0.8 108 93
compound N-butylpyrrole phenol 2,4-dimethyl-3-ethylpyrrole + others (minor) pyrrole + 5 Me (+ m-cresol (minor)) pyrrole + 5 Me 2,3,4-trimethyl-2-cyclopenten-1-one p-cresol 3,4,4-trimethyl-2-cyclopenten-1-one unidentified 5-ethyl-2-furaldehyde 3,4-dimethylphenol + other (complex pyrrole) p-ethylphenol indole skatole N-ethylindole 2,3-dimethylindole indole trace - unknown subst. 1,2,3,7-tetramethylindole 1-phenylethylpiperidine hexadecanoic acid oleic (minor) and linoleic acid (major) hexadecanamide N-methylhexadecanamide N,N-dimethylhexadecanamide analyzed peaks # analyzed peaks %
groupa 1 2 1 1 1 5 2 5 9 8 2 2 3 3 3 3 3 3 8 6 6 7 7 7 29 27
Group numbers refer to chemical families listed in Table 8.
cellulose but might in the case of DDGS also originate from the lignin fraction. Lignin is a complex polymer, primarily composed of the monomers p-coumaryl alcohol, coniferyl alchol, and sinapyl alcohol. Studies of the degradation of lignin using fast pyrolysis identified degradation products such as
Figure 5. GC-MS TIC of HTL bio-oil from manure feedstock. Numbers refer to Table 6. 6993
dx.doi.org/10.1021/ef300954e | Energy Fuels 2012, 26, 6988−6998
Energy & Fuels
Article
(containing originally 15−16% N) during HTL, while no obvious change was observed for the other feedstocks. High nitrogen levels will affect combustion properties of bio-oils negatively. The bio-oils contained 1−11% water, except bio-oil from sewage sludge that contained 19% water. The high polarity of bio-oils compared to fossil oils increase water solubility. Most bio-oils contained less than 1% ash (Table 7). TOC of the aqueous phase was 9.5−31 g/L (geometric mean 24 ± 6 g/L), indicating that variations in chemical composition and separation conditions affect the efficiency of separation of oil from the aqueous phase. Based on quantification using 20 standards of major products, a rough estimate of the concentrations of compounds detected by GC-MS yields an identification of approximately 9−25% of the total mass (data not shown). Altogether, across feedstocks only 13 ± 5% in bio-oil from cellulose and protein mixture to 44 ± 20% in bio-oil from sludge could be identified as water, ash, or compounds detectable by GC-MS. Our results are thus comparable to those of Valdez et al.4 where about 35% of the compounds in bio-oil from algae feedstock could be analyzed by GC-MS. This very rough estimate suggests that a large fraction (87− 56%) of the bio-oil constituents cannot be identified directly by conventional GC-MS analysis. There is thus a need for additional analytical tools for identification of constituents of HTL bio-oils. In order to compare chemical composition of bio-oils from different feedstocks, it is convenient to group compounds into chemical families of compounds with structural similarities in order to reduce the complexity.26 Most previous studies have applied 4−8 chemical families (in addition to the groups “unknown” and “others”) often including phenols, fatty acids, sugars, and volatile ketones and aldehydes.3,7,9,26−29 In order to cover the diversity of the samples in the present study, we have applied seven groups here, namely pyrroles, phenols, indoles, indanones, cyclopentenes, fatty acids, and amides. Pyrroles are here defined as being composed of a fivemembered ring with one nitrogen and two double bonds sustaining an aromatic system; phenols are composed of a benzene structure substituted with an alcohol or ester and aliphatic substituents; indoles are composed of a pyrrole structure fused to a benzene molecule; and fatty acids and amides are composed of a long hydrocarbon chain with either a terminal acid or amide group. Table 8 presents the composition of bio-oils divided into chemical families, which gives an overview of products from different feedstocks. HTL products of both disaccharide and polysaccharides (sucrose and cellulose) are primarily cyclopentenes (formed by dehydration of saccharides22), phenols, and indanones, in accordance with previous studies.12,21 Fatty acids are dominant species in most of the analyzed biooils, except from sucrose, cellulose, casein, and one manure feedstock. Analysis of bio-oil made from sunflower oil indicates that fatty acids may be formed by hydrolysis of glycerides in the HTL process. Sunflower oil mainly consists of triglycerides, while bio-oil from sunflower oil consists of 95% fatty acids, which documents that the process effectively hydrolyzes triglycerides into free fatty acids. Biller et al.15 also observed fatty acids as major products from HTL of sunflower oil. Pyrroles seem to form from HTL of proteins and give high contributions in some protein, DDGS, and manure samples. Interestingly, bio-oil from mixed cellulose and protein feedstock
Table 6. GC-MS Analysis of Major Composition of HTL Bio-Oil from Manure (Feedstock 2) #
RT
area%
compound
groupa
1 2 3 4 5
12.8 14.5 15.8 16.4 16.6 19.5 21.8 22.2 23.3 23.9 24.5 24.7 25.8 26.0
1.1 2.3 1.7 1.2 3.2 1.0 1.0 2.1 1.5 2.1 3.1 1.2 1.4 1.0
8 5 2 5 5 9 2 5 5 2 5 5 8 8
13 14 15
28.9 30.4 31.1
3.5 0.8 1.0
16
31.3
1.4
17
33.8 39.5
5.5 0.9
1-cyclohexylethanol 3-methyl-2-cyclopenten-1-one phenol 3,4-dimethyl-2-cyclopenten-1-one 2,3-dimethyl-2-cyclopenten-1-one unidentified o-cresol 2,3,4-trimethyl-2-cyclopenten-1-one 3-ethyl-2-cyclopenten-1-one p-cresol and 1-(3-thienyl)ethanone 3,5,5-trimethyl-2-cyclopenten-1-one 3,4,4-trimethyl-2-cyclopenten-1-one 5-ethyl-2-furaldehyde 5-ethyl-2-furaldehyde (similar spectra isomers?) possibly 2-ethylidenecyclohexanone o-ethylphenol and unknown (50/50) 2,3,4,5-tetramethyl-2-cyclopenten-1-one (major) and unknown (minor) 2,2,5,5-tetramethyl-3-cyclopenten-1-one and 3,4-dimethylphenol p-ethylphenol cyclopentene + 4 Me or cyclophexanone + 3 Me (?) cyclohexanone + 4 Me (?) 2,3,4,5,6,7-hexahydro-1H-inden-1-one phenol + unidentified p-ethylguaiacol unidentified (multiple peaks) p-propylguaiacol skatole unknown (multiple peaks) 2,3-dimethylindole 1,2,3-trimethylindole indole + 3 Me and unknown 2,3,5-trimethylindole unidentified hexadecanoic acid oleic acid linoleic acid unidentified hexadecanamide analyzed peaks # analyzed peaks %
6 7 8 9 10 11 12
41.3 42.7 43.4 19 44.0 47.2 20 49.2 21 50.1 52.7 22 54.5 23 55.8 57.0 24 58.0 58.6 25 67.8 26 71.9 27 72.4 72.5 28 72.8 total #peaks analyzed area % 18
a
0.9 2.4 1.1 5.6 2.1 3.3 1.3 1.5 1.6 1.9 0.9 1.2 0.9 1.5 1.8 1.3 0.9 1.1 132 68
9 2 5 5 2 5 5 4 2 2 9 2 3 9 3 3 3 3 9 6 6 6 9 7 38 29
Group numbers refer to chemical families listed in Table 8.
Results from elemental analysis of bio-oils (Table 7) show that irrespective of feedstock, all bio-oils are composed of 76− 82% carbon and 2−13% oxygen. Interestingly, the feedstocks cellulose, protein, and DDGS were originally composed of 23− 49% oxygen, and the oxygen content has thus been significantly reduced during the HTL process (possibly due to formation of CO2), improving the fuel quality of bio-oil compared to feedstock. In addition, the heat value was improved from 16 MJ kg−1 in cellulose and DDGS and 24 MJ kg−1 in protein feedstocks to 35 ± 1 MJ kg−1 in bio-oils. Nitrogen content in manure bio-oil was 2−3%, while it was 5−8% in bio-oils from DDGS, sewage sludge, and protein, so nitrogen was thus removed from protein in these feedstocks 6994
dx.doi.org/10.1021/ef300954e | Energy Fuels 2012, 26, 6988−6998
Energy & Fuels
Article
Table 7. Results from Elemental Analysis of HTL Bio-Oils from Different Feedstocks Including Ash and Water Content, Lower Heat Value, As Well as TOC of the Aqueous Byproducta
a
feedstock
heat value (MJ/kg)
C%
H%
O%
N%
S%
ash %
water %
TOC of aqueous phase g/L
sucrose cellulose 1 cellulose 2 protein (casein) protein (gluten 1) protein (gluten 2) cellulose + protein sludge DDGS manure-1 manure-2 manure-3
33.8 34.2 34.2 35.8 36.9 35.7 36.9 34.5 35.5 35.2 35.7 35.5
79.2 79.6 79.2 78.5 78.5 76.4 80.2 80.6 77.1 80.8 81.6 79.2
7.7 8.3 8.2 9.5 9.8 9.7 9.4 7.6 9.4 9.5 8.6 9.2
12.9 11.9 12.4 1.6 3.8 6.1 2.7 3.9 6.3 6.3 6.0 8.8
0 0.1 0.1 9.3 6.9 6.7 6.9 7.8 6.8 2.8 3.3 2.3
0 0.1 0.1 1.1 0.9 1 0.8 0.1 0.5 0.6 0.7 0.5
0.3 0.1 n.a. n.a. 0.1 0.3 n.a. n.a. 0.7 0.9 0.9 1.3
5.4 7.2 9.6 4.2 4.5 11.1 4.3 18.7 3.8 4.8 4.3 3.5
21.8 28 31 9.5 27 21 26 25.2 26.1 29 30 23
The elementary composition is for water-free oil.
Table 8. Chemical Composition of HTL Bio-Oils from 11 Different Types of Feedstocks and Model Compounds Listed As Percentages of Total Chromatographic Peak Areaa feedstock sucrose cellulose sunflower oil proteincasein proteingluten cellulose + protein DDGS manure-1 manure-2 manure-3 sewage sludge a
number of samples
1. pyrrole
2. phenols
3. indoles
4. indanones
5. cyclopentenes
7. amides
8. other
9. unidentified
total
1 2 1
0.0 0.0 0.0
9.4 6.5 ± 1.9 0.0
0.0 0.0 0.0
1.7 6.5 ± 1.0 0.0
14.5 21.1 ± 2.8 0.0
1.9 0.0 95.2
0.0 0.0 0.0
13.6 8.9 ± 5.2 1.6
32.5 21.9 ± 10.6 2.4
73.6 64.8 ± 1.6 99.1
1
4.2
21.9
7.7
1.9
0.0
3.9
1.5
1.7
28.5
71.3
2
8.2 ± 5.0
7.2 ± 2.7
6.1 ± 3.8
0.9 ± 0.5
0.0
35.6 ± 4.2
9.2 ± 2.6
3.1 ± 1.2
15.5 ± 4.9
85.6 ± 3.0
1
23.2
3.4
4.7
0.9
0.7
11.9
1.6
4.0
11.1
61.4
3 1 1 1 1
5.7 ± 1.3 4.8 0.0 3.2 0.6
4.4 ± 0.2 10.8 21.2 15.8 0.2
3.3 ± 0.5 7.3 6.9 6.5 0.5
0.2 ± 0.2 0.0 2.4 1.3 0.0
2.0 ± 0.5 4.4 18.6 14.9 0.0
62.1 ± 4.8 36.4 4.6 27.0 57.4
6.0 ± 0.7 1.7 1.1 0.0 21.9
1.1 ± 0.6 2.4 3.4 1.9 5.0
4.8 ± 0.7 9.4 9.9 2.4 8.2
89.5 ± 3.3 77.3 68.1 72.9 93.8
6. fatty acids
The group “unidentified” is included in the column “total”. Numbers refers to group numbers listed in Tables 3−6.
shows a relatively high content of pyrroles. Hydrolysis of proteins will give amino acids, while cellulose yields carbohydrates which may combine in a Maillard reaction1,30 to form e.g. pyrroles instead of cyclopentenes and could potentially cause processing difficulties.30 Amides identified in the bio-oil samples generally have C16 hydrocarbon chains and are also found as N-methylated and Ndimethylated derivatives. Highest relative peak area of amides was observed in bio-oil from sewage sludge. 3.7. Characterization and Quantification of Fatty Acids. In order to improve the qualitative and quantitative investigation of fatty acid composition, a subset of samples were derivatized to obtain the fatty acid methyl ester (FAME) derivatives. The GC-FID analysis of FAMEs showed that the fatty acids in bio-oil samples were primarily hexadecanoic acid, octadecanoic acid, cis-9-octadecenoic acid, and cis,cis-9,12octadecadienoic acid. Only bio-oils from casein and sewage sludge contained decanoic acid, dodecanoic acid, and tetradecanoic acid. As described above, fatty acids seem to be formed from hydrolysis of glycerides during the HTL process. A total of 3−18% fatty acids (w/w) was detected, with highest yields from sewage sludge and DDGS feedstocks, in accordance with results from GC-MS analysis (see Table 9). Deviations
Table 9. Concentrations of Fatty Acids (% Fatty Acid w/w) in HTL Bio-Oils Determined by GC-FID Analysis of FAME Derivatives feedstock
C10
C12
C14
C16
C 18:0
C 18:1
C 18:2
total
protein (casein) protein (gluten 1) protein (gluten 2) cellulose + protein sludge DDGS-1 DDGS-3
0.1
0.2
0.4
1.2
0.5
0.6
0.7
3.8
n.d.
n.d.
n.d.
2.5
0.2
1.7
2.3
6.7
n.d.
n.d.
n.d.
2.9
0.2
2.3
3.4
8.8
n.d.
n.d.
n.d.
1.5
0.1
0.9
0.8
3.3
0.4 n.d. n.d.
0.8 n.d. n.d.
2.2 n.d. n.d.
7.7 2.3 2.9
2.3 0.3 0.3
2.2 1.9 2.2
2.6 1.9 2.7
18.2 6.4 8.1
between results in Table 9 and in previous sections are due to e.g. differences between normalization to total oil or total chromatographic peak area, as well as uncertainty related to use of peak area compared to authentic standards. 3.8. Analysis of Aqueous Phase from HTL of DDGS. The aqueous phase is composed of 0.9−3% organic compounds (see TOC analysis data in Table 7), and it is 6995
dx.doi.org/10.1021/ef300954e | Energy Fuels 2012, 26, 6988−6998
Energy & Fuels
Article
Figure 6. HPLC-TOF-MS chromatogram (BPC) of an aqueous phase sample obtained from HTL liquefaction of DDGS. Compounds eluting at 3− 6 min are carboxylate derivatives of pyrazine and cyclopentenone, while hydroxylated fatty acids elute at 6−15 min.
Table 10. Results (Relative Peak Areas) from GC-Ms Analysis of Hexane-Extractable Compounds from Aqueous Phase Byproduct of HTL of DDGS pyrazine/pyridine
cyclopentanones
cyclopentenones
acids
amides
furans
unknown
other
13.8 45.7
1.2 0
41.4 30.8
30.8 0
0.0 12.5
3.5 3.6
2.1 0.8
1.4 0
pH 12
Table 11. Concentrations of Fatty Acids (mg/L) in Aqueous Phase from HTL, Determined by HPLC-TOF-MS feedstocka
C14
C16
C18:0
C18:1
C18:2
C20
C22
total
sucrose protein (casein) protein (gluten 2) DDGS-1 DDGS-2 DDGS-4 DDGS-5 DDGS-6 DDGS-7 sludge-2
0.1 0.3 0.6 2.7
