Determination of Furfural and 5-Hydroxymethylfurfural in Biomass

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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 Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.7b02827 • Publication Date (Web): 25 Nov 2017 Downloaded from http://pubs.acs.org on November 26, 2017

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Determination of furfural and 5-hydroxymethylfurfural in biomass hydrolysate by high-performance liquid chromatography

Jun Lia, Youjie Xua, Meng Zhangb, Donghai Wanga,* a

Department of Biological and Agricultural Engineering, Kansas State University, Manhattan, KS 66506, US

b

Department of Industrial and Manufacturing Systems Engineering, Kansas State University, Manhattan, KS 66506, US.

* Corresponding author. Telephone: 785-532-2919, Fax: 785-532-5825, email address: [email protected]

Co-authors’ email addresses: [email protected] (Jun Li), [email protected] (Youjie Xu), [email protected] (Meng Zhang)

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Abstract A high-performance liquid chromatography (HPLC) method for determining furfural and 5hydroxymethylfurfural (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, 98.78-101.45% for HMF). Compared to the widely accepted 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 current method is more obvious due to having the capacity of batch extraction. Keywords: Biomass hydrolysate; Furfural; 5-hydroxymethylfurfural; n-butanol extraction; HPLC -UV detector; C18 column


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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 of bioethanol are produced from cereal crops, such as corn and grain sorghum. Although the high ethanol yield and fermentation efficiency are obtained by using those starch-based crops, using large amounts of grains for bioethanol production would compete with human food and animal feedstock.2 The biofuels sector struggles to overcome the “food vs. fuel” controversy due to 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 renewable resource for production of biofuels.3 Unlike grains, however, it cannot be used to produce ethanol directly through the saccharification and fermentation due to 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 stage of laboratory and only 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 5-hydroxymethylfurfural (HMF).9 The acetic group released from hemicellulose increases the acid strength of biomass hydrolysate, in turn causing


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the formation of furfural and HMF.10 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 HFM are higher than the minimum threshold values that enzyme and yeast can tolerate during the 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 highperformance 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 width 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, the sulfuric acid in 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. Dong et al.17 reported that a liquid chromatography method with UV detector and C18 column for the determination of furfural and HMF, and the biomass hydrolysate was directly injected to analyze the contents of furfural and HMF without any pretreatment. The concentration ranges of dilute


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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 C18 column to the pH value. Also, the needle and needle seat are easy to get 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 like a 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 mobile phase was also investigated to obtain the desired resolution and analytical time.

2. Experimental 2.1. Chemicals and materials Furfural (purity >98%) and HMF (purity >97%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 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, USA). Corn stover was harvested from the Kansas State University Research Farm (Manhattan, KS, USA). Corn stover was ground to < 1 mm particle size using a SM 2000 cutting


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mill (Retsch Inc. Newton, PA, USA) and then the samples were sealed in plastic bags with zippers and stored at room temperature before use. 2.2. Apparatus The HPLC spectra were measured with a G1362A refractive index detector (RID) and an ion exchange column or an G1315D ultraviolet (UV)/visible detector and a C18 column (Agilent, Santa Clara, CA). A sandbath (Techne Inc., Princeton, NJ) with a temperature controller and a reactor (Swagelok, Kansas City Valve & Fitting Co., KS, USA) made of 316 L stainless steel with a measured internal volume of 75 mL (outside diameter of 38.1 mm, length of 125 mm, and wall thickness of 2.4 mm) were used to prepare biomass hydrolysates. The vortex mixer (Fisher Scientific, Inc., Ward Hill, MA), P250D ultrasonic apparatus (ETL testing laboratories Inc., Cortland, NY), Allegra 6R centrifuge (Beckman Coulter Inc., Brea, CA), and SHB-III vacuum pump (Zhengzhou Great Wall Scientific Industrial and Trade CO., LTD., Zhengzhou, China) were also used in the experiments. 2.3. Preparation and storage of furfural and HMF standard solutions To prepare standard stock solutions of furfural and HMF, 0.10 g (accurately weighed to 0.1 mg, similarly hereafter) of each of them was individually weighted into a 100-mL amber volumetric flask and dissolved with n-butanol saturated with water. Then, sufficient n-butanol saturated with distilled water was added to achieve a solution volume of 100 mL. Finally, the flasks were shaken until homogenous and clear solutions were formed. All stock solutions were stored in a refrigerator (4 °C) and away from light for a maximum of one 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.


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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 to a designed temperature, the reactor was submerged into the boiling sand for different reaction time. 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 Buchner funnel loaded with two-fold filter papers to collect biomass hydrolysates. The biomass hydrolysates were frozen in a refrigerator until analysis. After collecting biomass hydrolysates, 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 samples 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 3,000 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


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NREL method19: the method proposed by the National Renewable Energy Laboratory (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 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; the column and RID detector temperature was 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 XDBC18 (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 comparing their retention time to those of standards. Data were collected and processed using Instrument B.04.02 chemstation (Agilent).

2.7. Calculation of contents of furfural and HMF The amounts of furfural and HMF in the hydrolysates were calculated using: w=

10 × ܿ × ݉′ ݉

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 C18 column and UV detector as the 8 ACS Paragon Plus Environment

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separation and detection units; m (g) is the weight of biomass hydrolysate used to extract furfural and HMF; and m’ (g) is the weight of 1 mL of biomass hydrolysate used to extract furfural and HMF; 10 is the volume of n-butanol 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 rel. 16.0, SPSS Inc., Chicago, IL, USA). Differences were considered significant when the p value was < 0.05.

3. Results and discussion 3.1. Wavelength screening The UV absorption spectra of furfural and HMF are shown in Fig.1. Their maximum characteristic absorption wavelengths were 268 and 277 nm, respectively, which had several nm fluctuations compared to previous reportsError!

Bookmark not defined.17

, possibly due to instrument

difference. To obtain the best analytical sensitivity, 268 and 277 nm were selected as the detection wavelengths of furfural and HMF, respectively, namely, dual wavelength detection.

3.2. Selection and optimization of mobile phase Generally, the most common solvents used in the reverse-phase (RP)-HPLC system are water, methanol, acetonitrile, etc. Due to the high toxicity of acetonitrile, methanol is generally preferred for use in the RP-HPLC system under the premise that the separation and detection


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effects have no or little difference. From the experimental results, there was no difference in the separation effect of furfural and HMF when methanol/water or acetonitrile/water were used as a mobile phase (Fig. 2). Thus, methanol and water were employed as the mobile phase of RPHPLC system. Initially, the straightforward isocratic elution conditions were attempted. The volume ratios of methanol to water of 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, and 35:65 (v/v) were tested. When methanol added in mobile phase was more than 25%, although both peaks of furfural and HMF were narrow and symmetric, the baseline separation of furfural and HMF were incomplete due to the high volume ratio of methanol weakening the retention ability of the stationary phase of C18 column to furfural and HMF. When the volume ratio of methanol was less than 15%, although there was good separation between furfural and HMF, the retention time of them was extended from 3.68 and 4.93 to 7.42 and 8.64 min with the decrease of volume ratio of methanol from 15 to 5. Thus, the volume ratio of methanol to water of 20:80 (v/v) was finally employed as the mobile phase with good resolution and short retention time (Fig. 2A). The retention time of furfural and HMF were 4.32 and 3.21 min, respectively.

3.3. Selection of extraction solvent To extract furfural and HMF from water-based biomass hydrolysate, solvents insoluble or having a very limited solubility in water can be considered as the suitable extraction solvents. Considering the solubility of furfural and HMF in extraction solvents, three types of medium polar solvents, n-butanol, diethyl ether, and ethyl acetate, attracted our attention after the screening to common solvents. From the view of inter-day precision and accuracy (Table 2) of furfural and HMF dissolved individually in diethyl ether, ethyl acetate, and n-butanol, they all


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can be considered as the extraction solvent. However, diethyl ether has a low boiling point (34.6 °C) and is very volatile, especially when samples are centrifuged at a high speed, resulting in reduced extraction efficiency and increased diethyl ether consumption during the extraction process. Compared to ethyl acetate having a limited solubility in water (8.3 g/ 100 mL water) at room temperature, a boiling point of 77 °C20, and a cut-off wavelength of 260 nm21, n-butanol also has a limited solubility in water (7.4 g/100 mL water) at room temperature but a higher boiling point of 117.7 °C22, a lower cut-off wavelength of 210 nm21, which determines that nbutanol is the more suitable solvent for the extraction of furfural and HMF from the water-based biomass hydrolysate. Also, n-butanol is one of the fermentation products when biomass is used to produce bioethanol through the industrial fermentation, which gives n-butanol another advantage that it could be readily available in the ethanol plants. In addition, because water has an approximately 20% solubility (w/w) in n-butanol, n-butanol needs to be saturated with water before use.

3.4. Optimization of extraction procedure The extraction procedure usually follows the principle of multi-repeated extraction with a small amount of extraction solvents each time. Three types of extraction procedures, including three repeated extractions (4, 3, and 3 mL of n-butanol each time, respectively), four repeated extractions (3, 3, 2, and 2 mL of n-butanol each time, respectively), and five repeated extractions (2, 2, 2, 2, and 2 mL of n-butanol each time, respectively), were investigated with the fixed extraction solvent of 10 mL and 1.0 g of biomass hydrolysate. Results (Table 3) showed that the recoveries of furfural and HMF were in the range of 96.44-100.21% and 97.84-98.78%, respectively, and there was no significant difference in recoveries of furfural among three types


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of extraction procedures as well as recoveries of HMF (p >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 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 due to the release of 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 C18 column to pH. After extraction, a large amount of acetic acid remained in the water-based biomass hydrolysate and only a small amount of acetic acid was coextracted, thus having no effect on the C18 column. Therefore, no addition of sodium carbonate was needed to neutralize acetic acid in biomass hydrolysate. The corn stover hydrolysate from the dilute acid pretreatment had a very low pH value. For example, when the acid concentration used to pretreat corn stover was 0.5% (w/w), the pH value of pretreated corn stover hydrolysate was approximately 1, which was smaller than the tolerance range of C18 column to pH. Thus, to extend the lifetime of C18 column and reduce the damage to the needle and needle seat, approximate amount of sodium carbonate was added to biomass hydrolysate to neutralize the dilute acid before extraction.

3.5. Linearity, limit of detection (LOD), and limit of quantitation (LOQ) of furfural and HMF The calibration curves of furfural and HMF were constructed by plotting the measured peak areas versus corresponding concentrations, respectively. Excellent linearity was obtained within the concentration range of 0.01-0.50 mg/mL for furfural and HMF (R2 > 0.9994).


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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 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 inter-day precision and accuracy of furfural and HMF The precision of the HPLC system using C18 column as the separation unit was demonstrated by intra- and inter-day 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 one day and its relative standard deviation (RSD) and bias were calculated. In the inter-day variation studies, three repeated injections of furfural or HMF standard solutions (0.02, 0.10, and 0.50 mg/mL) were performed on three consecutive days and its RSD and bias were also calculated. Results showed that intra- and inter-day precisions of furfural and HMF with the HPLC system using C18 column as the separation unit were acceptable, with its RSD lower than 0.52% and bias lower than 3.06% (Table 4).

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


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and 1.00, 2.00, and 5.00 mg/mL (n = 6) for HMF, respectively, 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, 98.78-101.45% for HMF, with the maximum RSD less than 1.23%, respectively.

3.8. Application and comparison To verify the difference of results obtained from the current method and 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.

4. Conclusions 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 coexisting 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.


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5. Acknowledgements The authors also acknowledge the National Science Foundation for partially supporting this work through Award Number: 1562671. This is no. 18-100-J of the Kansas Agricultural Experiment Station.


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Solvent

cut-off

wavelengths,

https://www.chem.fsu.edu/~shatruk/docs/Solvent-UV-

cutoffs.pdf., 2017. (22) Ethyl acetate, Wikipedia, https://en.wikipedia.org/wiki/Ethyl_acetate, 2017 (23) Davison, B. H.; Lee, J. W.; Finkelstein, M.; McMillan, J. D. Biotechnology for fuels and chemicals, in: Negro, M. J.; Manzanares, P.; Ballesteros, I.; Oliva, J. M.; Cabañas, A.; Ballesteros, M. (Eds.), Hydrothermal pretreatment conditions to enhance ethanol production from poplar biomass, Humana Press, 2003, pp. 87-100. (24) Li, J.; Bi, Y. L.; Liu, W.; Sun, S. S. J. Agric. Food Chem. 2015, 63, 8584-8591. (25) Li; J.; Bi, Y. L.; Sun, S. S.; Peng, D. Food Chem. 2017, 234, 205-211. (26) Chen, M.; Hu, X. J.; Tai, Z. G.; Qin, H.; Tang, H. N.; Liu, M. S.; Yang. Y. L. Food Anal. Method. 2013, 6, 28-35.


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Figure Captions Fig. 1. The ultraviolet absorption spectra of furfural and 5-hydroxymethylfurfural. Fig. 2. HPLC chromatograms of furfural and 5-hydroxymethylfurfural (HMF) using methanol/water (profile A) or acetonitrile/water (profile B) as mobile phase.


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Table 1 Pretreatment conditions of corn stover. Experimental

Sample weight

Water volume

Acid concentration

Acid volume

Temperature

Time

number

(g)

(mL)

(%, w/w)

(mL)

(°C)

(min)

1

5.0

50

/

/

190

30

2

5.0

50

/

/

210

20

3

5.0

50

/

/

230

10

4

5.0

/

5%

50

120

30

5

5.0

/

2%

50

165

20

6

5.0

/

0.5%

50

210

10


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Table 2 Values of the intraday precision and accuracy of 0.02 mg/mL of furfural and 5hydroxymethylfurfural (HMF) dissolved in diethyl ether, ethyl acetate, or n-butanol. Diethyl ether

Ethyl acetate

n-butanol

Analytes RSDa (%)

Bias (%)

RSDa (%)

Bias (%)

RSDa (%)

Bias (%)

Furfural

0.44

-0.13

0.56

-0.84

0.47

-2.21

HMF

2.00

2.51

0.96

-1.04

0.22

-1.98

a

RSD means relative standard deviation.


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Table 3 The effect of different types of extraction procedure on the recovery and precision of spiked furfural and 5-hydroxymethylfurfural (HMF) from acidized water. Recovery (%) Extraction procedure Furfural

HMF

4/3/3

100.21 ± 0.74a

98.78 ± 0.85a

3/3/2/2

98.56 ± 1.67ab

98.28 ± 0.24a

2/2/2/2/2

96.44 ± 1.48b

97.84 ± 0.22a

In each column, means with different lower-case letters are significantly different at p

Determination of Furfural and 5-Hydroxymethylfurfural in Biomass

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