Article Cite This: Energy Fuels 2017, 31, 13769−13774
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Determination of Furfural and 5‑Hydroxymethylfurfural in Biomass Hydrolysate by High-Performance Liquid Chromatography Jun Li,† Youjie Xu,† Meng Zhang,‡ and Donghai Wang*,† †
Department of Biological and Agricultural Engineering and ‡Department of Industrial and Manufacturing Systems Engineering, Kansas State University, Manhattan, Kansas 66506, United States ABSTRACT: A high-performance liquid chromatography (HPLC) method for determining furfural and 5-hydroxymethylfurfural (HMF) in biomass hydrolysate was developed. The extraction procedure used n-butanol as the solvent to collect furfural and HMF from biomass hydrolysate. The C18 column and ultraviolet−visible detector were used as the separation and detection units for determination of the furfural and HMF, respectively. The new method performed well with wide linear ranges of 0.01− 0.5 mg/mL (R2 > 0.9994), low limits of detection and quantitation (less than 0.003 and 0.009 mg/mL), and good recoveries (98.34−100.21% for furfural and 98.78−101.45% for HMF). In comparison to the widely accepted National Renewable Energy Laboratory (NREL) method, it did not differ significantly in determining furfural and HMF in biomass hydrolysate but reduced the running time to less 28 min (from the pretreatment to the finish of analysis), almost half of the 55 min running time for the NREL method. Moreover, if large amounts of samples need to be analyzed, the time-saving advantage of the current method is more obvious as a result of having the capacity of batch extraction.
1. INTRODUCTION The increasing demand for clean energy and climate change enhances the need to develop alternative energy resources.1 Bioethanol as an option of renewable energy has attracted much interest of scholars working in the field. Currently, most bioethanol is produced from cereal crops, such as corn and grain sorghum. Although the high ethanol yield and fermentation efficiency are obtained using those starch-based crops, using large amounts of grains for bioethanol production would compete with human food and animal feedstock.2 The biofuel sector struggles to overcome the “food versus fuel” controversy as a result of limited natural resources, particularly productive agricultural lands and usable freshwater for production. Lignocellulosic biomass exists widely in the natural world and has great potential as a renewable resource for production of biofuels.3 Unlike grains, however, it cannot be used to produce ethanol directly through saccharification and fermentation as a result of the complex nature of chemical structures among cellulose, hemicellulose, and lignin.4 Pretreatment is usually carried out as an essential step to disrupt the chemical linkages, facilitate the separation of cellulose, hemicellulose, and lignin, and increase the access of the enzyme to cellulose.5 Although various pretreatment methods have been proposed, a majority are still in the laboratory stage and only a few of them have been applied in industrial production of bioethanol. Currently, the dilute acid pretreatment method has been industrialized, and the liquid hot water (LHW) pretreatment method has also received much attention because no chemicals are needed in the process of pretreatment.4,6−8 These two pretreatment methods are both performed at high temperatures. Acid pretreatment at high temperatures results in the formation of sugar degradation products, such as furfural and 5hydroxymethylfurfural (HMF).9 The acetic group released from hemicellulose increases the acid strength of biomass hydrolysate, in turn causing the formation of furfural and HMF.10 © 2017 American Chemical Society
Furfural is produced from pentoses released mainly from hemicellulose, and HMF is produced from hexoses released mainly from cellulose.11 The formation of furfural and HMF not only reduces fermentable sugars for ethanol production but also lowers the enzyme and yeast activities if the contents of furfural and HMF are higher than the minimum threshold values that enzyme and yeast can tolerate during saccharification and fermentation, which also results in low ethanol fermentation efficiency.12 Thus, it is crucial to monitor the contents of furfural and HMF in biomass hydrolysate after pretreatment. Currently, the most acceptable detection technique for furfural and HMF is high-performance liquid chromatography (HPLC), employing an organic acid column to separate them through the ion-exchange function. The running time of this method is up to 50−55 min, and the peak widths of furfural and HMF are up to 5 and 4 min, respectively, resulting in inaccurate quantitation, especially when their contents are low. A HPLC method using an evaporative light scattering detector (ELSD) for the detection of furfural and HMF was also researched.13 However, sulfuric acid in the mobile phase is nonvolatile and causes somewhat damage to the ELSD after a long time run. Subsequently, a mass spectrometry method14 using atmospheric pressure chemical ionization and a headspace gas chromatography method15 for the detection of furfural in biomass hydrolysate are reported. However, nonvolatile HMF cannot be detected, and the mass spectrometry method is affected largely by the pH value of biomass hydrolysate.14 Chi et al.16 reported using an ultraviolet (UV)−visible photometry method for the detection of furfural and HMF, and the strongly toxic sodium borohydride was employed as the reducing agent. Received: September 19, 2017 Revised: November 15, 2017 Published: November 25, 2017 13769
DOI: 10.1021/acs.energyfuels.7b02827 Energy Fuels 2017, 31, 13769−13774
Article
Energy & Fuels Table 1. Pretreatment Conditions of Corn Stover experimental number
sample weight (g)
water volume (mL)
1 2 3 4 5 6
5.0 5.0 5.0 5.0 5.0 5.0
50 50 50
acid concentration (%, w/w)
5% 2% 0.5%
Dong et al.17 reported a liquid chromatography method with an UV detector and a C18 column for the determination of furfural and HMF, and biomass hydrolysate was directly injected to analyze the contents of furfural and HMF without any pretreatment. The concentration ranges of dilute sulfuric acid used to pretreat biomass are usually 0.5−5% (w/v),18 and thus, the final biomass hydrolysate is a strong acid solution, which exceeds the tolerance range (2−8) of the C18 column to the pH value. Also, the needle and needle seat are easy to become somewhat damaged when strong acid solutions are injected frequently. Extraction is one of the most common pretreatment methods capable of selectively collecting the desired analytes from complicated samples, equivalent to rough preseparation. Meanwhile, the interferences from other undesired components coexisting in the complicated samples can be avoided or reduced as much as possible when the analytes are detected, which would improve the analytical precision and accuracy. For this study, the extraction procedure was employed to collect furfural and HMF from partially neutralized biomass hydrolysate before HPLC analysis using a C18 column as the separation unit. Different extracting solvents were investigated, and the extraction procedure was optimized. The ratio of the mobile phase was also investigated to obtain the desired resolution and analytical time.
acid volume (mL)
temperature (°C)
time (min)
50 50 50
190 210 230 120 165 210
30 20 10 30 20 10
and dissolved with n-butanol saturated with water. Then, sufficient nbutanol saturated with distilled water was added to achieve a solution volume of 100 mL. Finally, the flasks were shaken until homogeneous and clear solutions were formed. All stock solutions were stored in a refrigerator (4 °C) and away from light for a maximum of 1 month. Before use, standard working solutions of variable concentrations (0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.20, and 0.50 mg/mL) were prepared daily by diluting appropriate volumes of the stock solutions in n-butanol saturated with distilled water. 2.4. Biomass Hydrolysate Preparation. A total of 5.0 g of ground corn stover was weighed into a 75 mL stainless-steel reactor, and 50 mL of distilled water or dilute sulfuric acid solution was added to the reactor. The reactor was screwed with a wrench and shaken upside down for a while to wet biomass. After the sand reached a designed temperature, the reactor was submerged into the boiling sand for different reaction times. Once the reaction was over, the reactor was transferred immediately to approximately 5 °C water to quench the hydrolysis reaction of biomass as soon as possible. The specific experimental design is shown in Table 1. The slurries were filtrated with a Buchner funnel loaded with 2-fold filter paper to collect biomass hydrolysates. Biomass hydrolysates were frozen in a refrigerator until analysis. After biomass hydrolysates were collected, the solids were washed with distilled water of 100 mL to remove toxic inhibitors adhering to the surface of pretreated biomass and then dried at 45 °C overnight in preparation for further analysis. 2.5. Extraction Procedure. A total of 1.0 g of each of biomass hydrolysate sample was weighed into a 10 mL test tube; an appropriate amount of sodium carbonate was added to neutralize the sulfuric acid in biomass hydrolysate; and then 4 mL of n-butanol saturated with distilled water was added. The mixture was agitated for 2 min using a vortex mixer and then centrifuged at 3000 rpm for 2 min at room temperature. The supernatant was quantitatively transferred to a 10 mL volumetric flask. The extraction procedure was repeated twice, adding 3 mL of n-butanol saturated with distilled water each time. Finally, sufficient n-butanol saturated with distilled water was added to achieve a 10 mL solution. The solution was filtered through a 0.45 μm membrane before HPLC analysis. 2.6. HPLC Analysis of Furfural and HMF. The National Renewable Energy Laboratory (NREL) method:19 the method proposed by the NREL for the determination of sugars, byproducts, and degradation products in liquid fraction process samples was used to detect the contents of furfural and HMF in biomass hydrolysates. The injection volume for each sample was 20 μL; the column was an Aminex HPX-87H ion-exclusion column (7.8 × 300 mm, Bio-Rad); the solvent system was water containing 0.005 M sulfuric acid; the flow rate was 0.6 mL/min; and the column and refractive index detector (RID) temperatures were set at 60 and 45 °C, respectively. The mobile phase was degassed for 15 min by an ultrasonic apparatus before use. The identification of compounds was achieved by comparing their retention time to those of standards. Data were collected and processed using OpenLAB CDS C.01.05 ChemStation (Agilent). New method: the injection volume for each sample was 5 μL; the column was Eclipe XDB-C18 (4.6 × 150 mm, 5 μm, Agilent); the solvent system was water with 20% methanol (v/v); the flow rate was 0.8 mL/min; the dual detection wavelengths were 268 nm for furfural and 277 nm for HMF; and the column temperature was 35 °C. The mobile phase was degassed for 15 min by an ultrasonic apparatus before use. The identification of compounds was achieved by
2. EXPERIMENTAL SECTION 2.1. Chemicals and Materials. Furfural (purity of >98%) and HMF (purity of >97%) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Ultrapure water (HPLC grade), methanol (HPLC grade), n-butanol (HPLC grade), acetic acid (HPLC grade), diethyl ether (HPLC grade), ethyl acetate (HPLC grade), and 72% sulfuric acid (w/w) were purchased from Thermo Fisher Scientific Chemicals, Inc. (Ward Hill, MA, U.S.A.). Corn stover was harvested from the Kansas State University Research Farm (Manhattan, KS, U.S.A.). Corn stover was ground to 0.05). The slight reduction of recoveries of furfural and HMF might be due to the long-time exposure to air and light, resulting in some decomposition. Thus, to improve the labor efficiency and save pretreatment
10cm′ m
where w (mg/mL) is the content of furfural or HMF in biomass hydrolysate, c (mg/mL) is the content of furfural or HMF detected by HPLC with a C18 column and an UV detector as the separation and detection units, m (g) is the weight of biomass hydrolysate used to extract furfural and HMF, m′ (g) is the weight of 1 mL of biomass hydrolysate used to extract furfural and HMF, 10 is the volume of nbutanol used for the extraction of furfural and HMF. 2.8. Statistical Analyses. All experiments were performed at least in duplicate. All data were presented as the mean ± standard deviation (SD). The significance of the differences was assessed using a one-way analysis of variance (ANOVA). Data were analyzed using SPSS software for Windows (version 16.0, SPSS, Inc., Chicago, IL, U.S.A.). Differences were considered significant when the p value was 0.9994). The instrumental LOD was estimated and defined as the average response (n = 3) of the lower concentration level of the calibration curve for furfural or HMF plus 3 times the standard deviation.24 The instrumental LOQ was taken as 10/3.3 times the LOD.25 Results showed that the LOD and LOQ of furfural were below 0.003 and 0.009 mg/mL, respectively, and the LOD and LOQ of HMF were below 0.002 and 0.007 mg/mL, respectively, which were both lower than those reported by Dong et al.17 3.6. Intra- and Interday Precision and Accuracy of Furfural and HMF. The precision of the HPLC system using the C18 column as the separation unit was demonstrated by intra- and interday variation studies.26 In the intraday studies, six repeated injections of furfural or HMF standard solutions (0.02, 0.10, and 0.50 mg/mL) were made in 1 day and its relative standard deviation (RSD) and bias were calculated. In the interday variation studies, three repeated injections of
Table 2. Values of the Intraday Precision and Accuracy of 0.02 mg/mL of Furfural and HMF Dissolved in Diethyl Ether, Ethyl Acetate, or n-Butanol diethyl ether a
a
n-butanol
ethyl acetate a
analyte
RSD (%)
bias (%)
RSD (%)
bias (%)
RSDa (%)
bias (%)
furfural HMF
0.44 2.00
−0.13 2.51
0.56 0.96
−0.84 −1.04
0.47 0.22
−2.21 −1.98
RSD means relative standard deviation.
Table 3. Effect of Different Types of the Extraction Procedure on the Recovery and Precision of Spiked Furfural and HMF from Acidized Watera recovery (%) extraction procedure
furfural
HMF
4/3/3 3/3/2/2 2/2/2/2/2
100.21 ± 0.74 a 98.56 ± 1.67 ab 96.44 ± 1.48 b
98.78 ± 0.85 a 98.28 ± 0.24 a 97.84 ± 0.22 a
a
In each column, means with different letters are significantly different at p < 0.05.
time, the three repeated extractions, namely, 4, 3, and 3 mL of n-butanol each time, was chosen to extract furfural and HMF from biomass hydrolysate. The corn stover hydrolysate from the LHW pretreatment had a pH value of approximate 3.3 as a result of the release of the acetic group from hemicellulose, which was in accordance with the fact that the pH range of biomass hydrolysate pretreated by LHW is usually 3−4.23 It was in the tolerance ranges of the C18 column to pH. After extraction, a large amount of acetic acid remained in water-based biomass hydrolysate and only a small amount of acetic acid was coextracted, thus having no effect on the C18 column. Therefore, 13772
DOI: 10.1021/acs.energyfuels.7b02827 Energy Fuels 2017, 31, 13769−13774
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Energy & Fuels
the NREL method, corn stover was pretreated under different conditions to prepare biomass hydrolysates. The pretreatment conditions and the results obtained from two detection methods are shown in Tables 1 and 6, respectively. Results showed that there was no significant difference (p > 0.05) between contents of furfural obtained from the two detection methods as well as contents of HMF, indicating that the current method was feasible in detecting furfural and HMF in biomass hydrolysate.
furfural or HMF standard solutions (0.02, 0.10, and 0.50 mg/ mL) were performed on 3 consecutive days and its RSD and bias were also calculated. Results showed that intra- and interday precisions of furfural and HMF with the HPLC system using the C18 column as the separation unit were acceptable, with its RSD lower than 0.52% and bias lower than 3.06% (Table 4). Table 4. Values of the Intra- and Interday Precision and Accuracy of Furfural and HMF Dissolved in n-Butanol intraday analyte
nominal concentration (mg/mL)
RSDa (%)
bias (%)
RSDa (%)
bias (%)
0.02 0.1 0.5 0.02 0.1 0.5
0.47 0.40 0.32 0.22 0.25 0.09
−2.21 1.69 0.58 −1.98 2.84 0.31
0.16 0.28 0.47 0.52 0.20 0.28
−3.06 1.91 0.56 −1.83 2.49 0.74
furfural
HMF
a
4. CONCLUSION A HPLC method for the detection of furfural and HMF in biomass hydrolysate was developed with good resolution, good and wide linear ranges, low LODs and LOQs, and high recoveries. The pretreatment to biomass hydrolysate, namely, extraction procedure before HPLC analysis, removed a large number of undesired components co-existing in biomass hydrolysate and, thus, largely reduced their interferences to the quantitative detection of furfural and HMF. The entire running time to complete each sample from the pretreatment to the finish of analysis was shortened to less than 28 min.
interday
RSD means relative standard deviation.
■
3.7. Recoveries and Precision of Furfural and HMF. Recoveries of furfural and HMF were determined using water spiking with them. Considering the limited solubility of furfural and HMF in water, one drop (approximately 0.006 g) of acetic acid was added to water to ensure their complete dissolution. The recovery experiments were carried out at three levels of 1.00, 5.00, and 10.00 mg/mL (n = 6) for furfural and 1.00, 2.00, and 5.00 mg/mL (n = 6) for HMF, and the percentage recoveries were calculated and presented in Table 5. Recoveries of furfural and HMF in acidized water were in the ranges of 98.34−100.21% for furfural and 98.78−101.45% for HMF, with the maximum RSD less than 1.23%.
Corresponding Author
*Telephone: 785-532-2919. Fax: 785-532-5825. E-mail:
[email protected]. ORCID
Donghai Wang: 0000-0001-9293-1387 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The authors also acknowledge the National Science Foundation for partially supporting this work through Award 1562671. This is 18-100-J of the Kansas Agricultural Experiment Station.
Table 5. Recovery and Precision of Spiked Furfural and HMF from Acidized Water analyte
added (mg/mL)
furfural
1 5 10 1 2 5
HMF
a
98.34 100.21 99.35 101.45 100.42 98.78
± ± ± ± ± ±
■
RSDa (%)
recovery (%) 0.38 0.74 0.77 1.24 0.73 0.85
AUTHOR INFORMATION
0.39 0.73 0.78 1.23 0.73 0.86
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Table 6. Comparison of Contents of Furfural and HMF in Pretreated Corn Stover Hydrolysate Detected by the Current and NREL Methodsa contents of furfural (mg/mL) experimental number 1 2 3 4 5 6 a
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DOI: 10.1021/acs.energyfuels.7b02827 Energy Fuels 2017, 31, 13769−13774
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DOI: 10.1021/acs.energyfuels.7b02827 Energy Fuels 2017, 31, 13769−13774
