Rapid Method for Simultaneous Determination of the Acetic Acid and

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Rapid Method for Simultaneous Determination of the Acetic Acid and Furfural Contents in Lignocellulosic Hydrolysate by Full Evaporation Headspace Gas Chromatography Hui-Chao Hu, Xue-Fang Yang, Ting-Ting He, Liu-Lian Huang,* and Li-Hui Chen* College of Material Engineering, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, People’s Republic of China ABSTRACT: This paper presents a high-throughout method for simultaneous determination of acetic acid and furfural in hydrothermal hydrolysate of lignocellulose using a full evaporation headspace gas chromatographic technique. In this method, the sample was acidified to pH 1.0−3.5 to a complete acetic acid release from the medium, which also avoids the risk for an acidized furfural decomposition at an elevated temperature. With a maximum sampling volume of 5 μL, full evaporation for both acetic acid and furfural at the headspace equilibration temperature (100 °C) can be achieved in 3 min. The results showed that the present method has a good precision [relative standard deviation (RSD) < 4.67%] and accuracy (apparent recoveries of 95.0−104.8% for acetic acid and 95.4−104.3% for furfural. The limits of quantitation in the acetic acid and furfural detection were 0.339 and 0.208 μg, respectively. The present method is simple, rapid, and accurate, which is suitable for rapid analysis of acetic acid and furfural in lignocellulosic hydrolysate for research of biotechnology screening and biofuel production.

1. INTRODUCTION In the biorefinery process aiming at converting lignocellulosic material into biomaterial or biofuels, hydrothermal pretreatment processes play a pivotal role for the subsequent production of cellulosic polymers and/or ethanol fermented from carbohydrates. This is because the destruction on the recalcitrant plant cell wall is an important step to the separation of hemicelluloses from biomass. During the pretreatment processes, such as alkaline, hot water, and dilute acid prehydrolysis, the o-acetyl groups located mainly on hemicelluloses are converted to acetic acid through the deacetylation reaction,1 while xylose and arabinose can be degraded to furfural at the intensive conditions (high temperature, long time, and stronger acidity of the medium).2 Because of the considerable amount of acetic acid and furfural in the hydrolysates, their inhibition effects on the subsequent ethanol or xylitol fermentation are very significant.3,4 As a result, the detoxification before fermentation or a process to recovery these species must be introduced, which no doubt increases the cost in the biorefinery.1,3 Therefore, a better understanding of the levels of acetic acid and furfural formed during the hydrothermal hydrolysis pretreatment is very important in the process control. Clearly, a rapid and accurate for simultaneous quantification of acetic acid and furfural contents in the process effluents is highly desired in the biorefinery-related laboratory research and industrial application. The separation-based methods have been widely used in the composition analysis of lignocellulosic hydrolysate, which include gas chromatography (GC),5,6 ion chromatography (IC), 7,8 and high-performance liquid chromatography (HPLC).9,10 To avoid the contamination and/or damage of the non-volatile species (e.g., sugars and organic and inorganic salts) in the hydrolytes to the GC injection port and columns for direct GC analysis, solvent extraction is a typical procedure for pretreating the sample in the direct GC method. For subsequent analysis of acetic acid, a complicated derivatization © 2015 American Chemical Society

procedure must also be performed, otherwise a significant error will be produced as a result of the formation of acetic acid dimmers in the solvent system.11,12 To overcome this problem, a water-phase derivatization step using benzyl bromide as a derivatization reagent was proposed, in which the derivatization products was determined by a headspace solid-phase microextraction (HS-SPME)-based GC measurement.13 However, the derivarization procedure is not only still very complicated but also time-consuming (at least 210 min). Moreover, they could introduce significant errors in the quantification analysis for hydrolysate, because of the very low yield of derivarization (4%) and the interference of benzyl alchohol on fiber−gasphase partitioning in the water-phase derivatization.13 IC is regarded as a very effective method in the analysis of sugar species.7,8 However, it failed to simultaneously quantify furfural in the hydrolyates, because its molecule dose not have the ionizable group, such as the hydroxyl group in sugars. The HPLC technique was found to be the only method that could simultaneously determine the contents of acetic acid and furfural.9,14 However, the composition complexity in hydrolysate, e.g., the interference from levulinic acid14 and baseline fluctuation caused by unknown compounds,15 affects the accuracy in the acetic acid quantification. Moreover, because the tiny particles, organic acids, and inorganic salts in hydrolyates can cause a serious problem in the HPLC system, the sample neutralization, filtration, and ash removal are mandatory. On the other hand, a longer running time is required in HPLC measurement, to completely wash out all of the species from the separation column. All of these make the method less effective and efficient. To develop a high-throughput testing technique, an atmospheric pressure chemical ionization mass spectrometry Received: September 12, 2015 Revised: October 15, 2015 Published: October 15, 2015 7428

DOI: 10.1021/acs.energyfuels.5b02072 Energy Fuels 2015, 29, 7428−7432

Article

Energy & Fuels

Clara, CA) equipped with a flame ionization detector (FID) operated at 300 °C. The flow rates of hydrogen and air were 30 and 400 mL/ min, respectively. A HP-5 capillary column (30 m length × 0.32 mm inner diameter, 0.25 μm film thickness, Agilent J&W, Santa Clara, CA) was operated with nitrogen carrier gas and a 180 °C of column temperature and 2.8 min for each run. The injection port of the GC system was operated using 250 °C of temperature, splitless mode, and 20 psi of pressure. Headspace operating conditions were as follows: equilibration temperature, 110 °C; needle and sampling coil temperature, 115 °C; transfer temperature, 120 °C; pressurization time, 0.2 min; sample loop filling time, 0.2 min; transfer time, 0.2 min; sample loop volume, 0.5 mL; carrier pressure, 1.0 bar; and vial pressure, 1.5 bar. 2.3. Sample Preparation and Measurement. A total of 20 μL of sulfuric acid solution (∼2.0 mol/L) was added to a 1.5 mL sample vial containing 1 mL of supernatant liquor of hydrolysate or standard solution of acetic acid and furfural to obtain a pH value ranging from 2.0 to 3.5, and it was sealed with a rubber pad and plastic cap. After mixing it with shaking, 4 μL of acidified solution was accurately injected into a headspace sample vial (21.6 mL), which was immediately sealed with a polytetrafluoroethylene (PTFE)/butyl septum and aluminum cap. Then, the headspace vial was placed in the headspace sampler and was equilibrated at 100 °C for 3 min. Finally, part of the gaseous phase in the headspace sample vial was automatically withdraw and analyzed by GC−FID.

(APCI−MS) incorporated with a headspace analysis method has been proposed.16 Although the method can rapidly determine several species of interest (e.g., formic acid, acetic acid, and furfural) in the hydrolysates, the poor repeatability and, thus, poor accuracy for acetic acid quantification was observed. Clearly, the interference from the other organic compounds in hydrolysates on the quantitative ionization of acetic acid in the APCI−MS measurement is still very significant without using GC or HPLC column separation. Therefore, the accuracy of the APCI−MS method cannot satisfy the requirement in biorefinery research. The other major concern is that APCI−MS is very expensive in both the instrument investment and its maintenance. Therefore, it is necessary to develop an alternative method that can also efficiently determine these species of interest in the hydrolysates based on the less expensive instruments. Because of the volatility of acetic acid and furfural, a headspace-analysis-based GC (HS−GC) method can conduct testing on the hydrolysates without sample pretreatment procedures. However, acetic acid cannot be determined by the conventional HS−GC method based on vapor−liquid equilibration, because of the strong interaction with liquid/ gaseous water and the effect of the ionic strength in the hydrolysates.17 Attributed to the full solvent vaporization, the volatile species can be determined by a full evaporation headspace GC (FE HS−GC) technique.18 In general, a very short time is required in FE headspace equilibration (within 3 min).19,20 Because most of commercial headspace samplers can perform an automatic batch sample test, an efficient analysis of the volatile species of interest in the hydrolysates can be realized using a FE HS−GC technique. Although the previous work has been developed for determination of furfural in biomass hydrolysate,20 it failed to determine acetic acid as a result of the presence of acetate and molecular association of acetic acid with vapor water.17 Therefore, the challenges in the simultaneous determination of acetic acid and furfural are to overcome the formation of acetic acid dimmer and molecular association of acetic acid with vapor water17 and to prevent the risk of furfural decomposition in the acidified hydrolysate21,22 during headspace equilibration at the full evaporation conditions. In the present work, we developed a FE HS−GC method to rapidly and simultaneously determine the contents of acetic acid and furfural in the hydrolysates from lignocellulose hydrothermal pretreatment processes. The major efforts were on the sample preparation condition for converting acetate ion in hydrolysate into acetic acid and equilibration temperature and time of the headspace sampler for the full evaporation of acetic acid from the liquid to gaseous phase and eliminating the effect of hydration of acetic acid in the gaseous phase on HS− GC measurement.

3. RESULTS AND DISCUSSION 3.1. Chromatogram for Acetic Acid and Furfural in Hydrolysate. Figure 1 shows a GC chromatogram from a full

Figure 1. Chromatogram of hydrolysate analysis (three GC testing cycles).

evaporation headspace measurement on a hydrolysate. It can be seen that both acetic acid and furfural can be well-separated from the other volatile species coexisting in the hydrolysate and the testing is completed within 3 min at the present GC conditions. Clearly, GC can provide much more efficient analysis for these species than that of HPLC.14 Because the equilibration time in the full evaporation headspace analysis is usually very short,19,20 the FE HS−GC method for the determination of acetic acid and furfural is efficient. With the overlapping heating function in the headspace autosampler, the total experiment time in FE HS−GC could be less than 3 min. 3.2. Effect of pH in Hydrolysate on the Acetic Acid and Furfural Measurement. Because acetic acid can associate with metal ions in the hydrolysate to form the nonvolatile salts, it will lead to a negative error in the quantification

2. EXPERIMENTAL SECTION 2.1. Chemicals and Samples. All chemicals used in the experiment were of analytical grade and purchased from Aladdin Reagents (Shanghai, China). A standard solution containing acetic acid (10.54 g/L) and furfural (11.98 g/L) was prepared by placing about 100 μL of acetic acid and furfural in a 100 mL volumetric flask and diluting with deionized water to volume. Several hydrolysates of bamboo were obtained from a set of lab-scale bamboo hydrothermal pretreatments. 2.2. Apparatus and Operations. All measurements were carried out using an automatic headspace sampler (DANI HS 86.50 Plus, Cologno Monzese, Italy) and a GC system (Agilent GC 7890B, Santa 7429

DOI: 10.1021/acs.energyfuels.5b02072 Energy Fuels 2015, 29, 7428−7432

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Energy & Fuels measurement. Therefore, the sample should be acidified to minimize such an effect. Figure 2 shows the effect of pH in the

Figure 3. Effect of the equilibrium temperature and time on the GC signal of acetic acid and furfural. Figure 2. Effect of pH on acetic acid releasing from hydrolysate.

sample on the GC signal (proportional to the species concentration) of acetic acid in the headspace at a given equilibration condition. It is clear that a maximum GC signal for acetic acid can be obtained when the pH in the sample is below 3.5. However, the stronger acidity in the hydrolysate might have the risk for the acid-catalyzed furfural decomposition and/or the polymerization with the functional groups in lignins.21,22 Therefore, the effect of pH on furfural at the given conditions was also investigated. As shown in Figure 2, the variations of the GC signal of furfural in the hydrolysates in a pH range from 0.74 to 7.5 were basically the same, with no significant variation observed. Therefore, the pH of hydrolysate should be adjusted to 0.74−3.5 to realize the simultaneous determination of acetic acid and furfural by a FE HS−GC method. 3.3. Conditions for Full Evaporation of Acetic Acid and Furfural. The key feature in the FE HS−GC measurement is that the analyte is nearly completely released to the vapor phase (from the sample). The effects of headspace equilibration temperature and time and sample size will affect the completeness of mass transfer and should be addressed. 3.3.1. Effect of the Temperature and Time on the Full Evaporation Equilibrium of the Species. Figure 3 shows the dependence of the GC signal of acetic acid and furfural (in headspace) on the equilibrium time at various temperatures. It can be seen that the headspace equilibrium for furfural can be quickly achieved within 1 min at 100 °C, and a higher temperature is helpful to speed up the headspace equilibrium for acetic acid. Therefore, we chose the headspace equilibration at 100 °C for 3 min for the rest of the study. 3.3.2. Maximum Permissible Sample Size. Because the full evaporation is conducted in a closed vial, the sample size is a critical factor for the nearly complete release of acetic acid and furfural from the liquid sample to the vapor phase. As shown in Figure 4, a linear relationship between the GC signal and furfural can be obtained when the sample size is within 10 μL, while it is only 5 μL for acetic acid. This indicates that acetic acid will associate with a water molecule in the vapor phase when the sample size (water) is larger than 5 μL for a headspace vial with 21.6 mL of capacity used in present paper.

Figure 4. Effect of the sample size on the GC signal of acetic acid and furfural.

To perform a simultaneous measurement for acetic acid and furfural, a sample size of 5 μL was chosen in the present method. Although a smaller sample size will sacrifice the detection sensitivity of the method, it is not a problem for the GC detector (FID) to measure the contents of acetic acid and furfural found in the hydrolysates in biorefinery research. 3.4. Method Calibration, Precision, and Validation. The conventional external standard calibration can be applied in the FE HS−GC method. In the present work, the calibration was conducted by adding 5 μL of acidified standard solution of acetic acid and furfural with various concentrations (0−10 g/L) to a series of headspace vials, which was detected using the present FE HS−GC method with 100 °C of equilibrium temperature and 3 min of equilibrium time. Thereby, the following standard calibration curves are established, i.e. AA = 4.89(± 0.05)mA + 0.240(± 0.142)

(n = 7,

2

R = 0.998)

(1)

AF = 11.2(± 0.1)mF − 0.440(± 0.189) 2

R = 0.999) 7430

(n = 7, (2)

DOI: 10.1021/acs.energyfuels.5b02072 Energy Fuels 2015, 29, 7428−7432

Article

Energy & Fuels where AA, AF, mA, and mF represent the GC signals of acetic acid and furfural and their absolute masses (in micrograms) added in the headspace vial, respectively. The concentration (C) of acetic acid and furfural in hydrolysate can be calculated by eq 3. The limit of quantification (LOQ) of the present method for acetic acid can be calculated by eq 423 and is 0.339 and 0.208 μg for acetic acid and furfural, respectively A−a C= sV0 (3) LOQ =

a + 10|Δa| s

Table 2. Method Validation added (μg)

(4)

temperature (°C)

time (min)

160

20 40 60 80 100 120 20 40 60 80 100 120 20 40 60 80 100 120

170

180

acetic acid

CF (mg/L)

RSD (%)

CA (mg/L)

RSD (%)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.40 1.29 4.67 2.89 3.62 2.82 4.19 4.44 4.21 3.80 4.18 1.59 3.33 1.22 0.93 0.37 1.35 0.48

287 572 732 953 1280 1440 352 659 1040 1580 2000 2170 518 1090 1780 2660 3280 4060

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.71 1.49 4.08 4.04 4.08 2.71 1.69 3.61 3.90 3.06 3.43 1.25 4.51 0.19 0.52 0.06 0.51 0.11

15.2 46.8 93.4 189 360 566 69.1 108 243 494 878 1350 104 415 1160 2200 2620 2100

0.51 0.57 0.87 0.70 4.85 2.71 2.90 4.81 10.2 18.8 36.7 21.5 3.53 5.36 54.2 63.6 84.6 87.6

13.0 1.08 3.79 0.54 6.51 1.63 5.94 23.8 40.4 48.6 68.7 27.1 19.2 12.3 72.4 107 134 110

sample number

acetic acid

furfural

acetic acid

furfural

acetic acid

furfural

1 2 3 4 5

5.27 10.5 31.6 42.2 52.7

5.99 12.0 35.9 47.9 59.9

5.15 11.0 32.8 40.1 55.2

5.84 12.5 37.2 45.7 61.2

97.7 105 104 95.0 105

97.5 104 103 95.4 102

4. CONCLUSION A FE HS−GC method for simultaneous determination of acetic acid and furfural in lignocellulosic hydrolysate was developed. By adjustment of the pH level of hydrolysate to the range of 1.0−3.5 and selection of a high temperature (100 °C) and very small sample size (5 μL), acetic acid and furfural were fully transferred to the vapor phase within 3 min. This method is simple, accurate, and with a high throughput, which is a valuable tool to control the degradation products during the prehydrolysis process of biomass.

Table 1. Determination of Furfural and Acetic Acid in Hydrolysates by the Present Method furfural

apparent recovery (%)

method is suitable to determine acetic acid and furfural contents in lignocellulosic hydrolysate.

where A, a, Δa, s, and V0 represent the GC signal of acetic acid or furfural, absolute value of the intercept, relative standard deviation of the intercept, slope of standard curves, and hydrolysate volume added in the headspace vial, respectively. The precision of the present method was evaluated by triplicate measurement of hydrolysate for 16 samples. As shown in Table 1, the relative standard deviations (RSDs) for both

prehydrolysis conditions

measured (μg)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors acknowledge the Demonstration Project of Forest Science and Technology supported by the Central Finance and Fujian Provincial Department of Science and Technology (2015J05018) for sponsoring this research.

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REFERENCES

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acetic acid and furfural were less than 4.67%, which is much lower than those in the ACPI−MS method16 and can satisfy the precision requirement of those two species for biorefinery research. To verify the use of this present method in practice, a set of sample solutions were prepared by accurately adding various masses of acetic acid and furfural (0−60 μg) to a hydrolysate sample, in which the GC signals of acetic acid and furfural were determined using this FE HS−GC method. The net contribution from added acetic acid and furfural was obtained by deducting the GC signal of the reference sample (original sample). Finally, the actual measured values for the added acetic acid and furfural were calculated by eq 3. As shown in Table 2, the apparent recovery24 of acetic acid and furfural using the present method ranged from 95.0 to 104.8% and from 95.4 to 104.3%, respectively, which indicates that the present 7431

DOI: 10.1021/acs.energyfuels.5b02072 Energy Fuels 2015, 29, 7428−7432

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DOI: 10.1021/acs.energyfuels.5b02072 Energy Fuels 2015, 29, 7428−7432