Determination of N-Methyl morpholine in biomass pretreatment solutions by the ammonia-assisted headspace gas chromatography

Determination of N-Methyl morpholine in biomass pretreatment solutions by the ammonia-assisted headspace gas chromatography

Renewable Energy 145 (2020) 2380e2386 Contents lists available at ScienceDirect Renewable Energy journal homepage: D...

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Renewable Energy 145 (2020) 2380e2386

Contents lists available at ScienceDirect

Renewable Energy journal homepage:

Determination of N-Methyl morpholine in biomass pretreatment solutions by the ammonia-assisted headspace gas chromatography Shaokai Zhang a, b, Xun Ke a, Tong Zeng a, Yonghao Ni b, Xiaolin Luo a, *, Hui-Chao Hu a, **, Lihui Chen a, Liulian Huang a a b

College of Material Engineering, Fujian Agriculture and Forestry University, Fuzhou, 350002, China Limerick Pulp & Paper Centre, University of New Brunswick, Fredericton, P.O. Box, 4400, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 February 2019 Received in revised form 3 July 2019 Accepted 1 August 2019 Available online 2 August 2019

An ammonia-assisted headspace gas chromatography (HS-GC) method was developed to determine the N-methyl morpholine (NMM) concentration in biomass fractionation processes. The use of ammonia gas increased the detection sensitivity of NMM, and minimized the interfering effect of other substances during the analysis. Results showed that, by adding 1.5 mL ammonium hydroxide (28e30%, w/w) and a 0.125 mmol of sodium hydroxide into a 4.0 g of sample solution containing 43.3% N-methylmorpholine N-oxide (NMMO), the gas-liquid equilibration of NMM was obtained within 30 min at 80  C. The optimum sample size and the NMMO content in the sample solutions were 6.0 g and 43.3%, respectively. The present method was proved to be of high precision (RSD ¼ 4.33%), sensitivity (LOQ ¼ 71.9 mg/kg), and with recovery accuracy of 96.0e102%. The present method was rapid, accurate, which will be a powerful tool to optimize the reaction conditions in NMMO-based biomass pretreatment process for enhancing the cost effectivity. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Headspace analysis N-methyl morpholine (NMM) N-methylmorpholine N-oxide (NMMO) Biomass pretreatment Biogas

1. Introduction Biomass pretreatment technologies have vastly been studied in the biorefinery process to overcome the recalcitrant structure of lignocelluloses for converting carbohydrates and lignin to biofuels and biochemicals. Attributed to its strong polarity [1], N-methylmorpholine N-oxide (NMMO) was proved as an efficient solvent (a hydrogen bond acceptor) on lignocellulose to remedy its recalcitrant structure (mainly cellulose crystallinity), so that the production of biomethane and bioethanol can be facilitated [2e5]. The bioethanol or biomethane yield in the subsequent fermentation process can be up to 91% and 400 mL/g-cellulose (theoretical value ¼ 415 mL/g) using the NMMO/H2O pretreatment [4,5]. The high cost of solvent (NMMO) requires an extremely high recovery of NMMO, such as higher than 98% in the conventional Lyocell process (a commercial process to produce regenerated cellulose) [6]. Unfortunately, some byproducts, mainly N-methyl morpholine (NMM) (due to homolytic and heterolytic hydrolysis of NMMO), are

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (X. Luo), [email protected] (H.-C. Hu). 0960-1481/© 2019 Elsevier Ltd. All rights reserved.

inevitably formed in the biomass pretreatment process and NMMO recovery process [6,7]. Therefore, it is necessary to develop an efficient analytical method to accurately quantify NMM content in biomass pretreatment solutions for process optimization and stabilizer screening to save chemical costs. For this purpose, several methods have been reported in the literature. They are based on either capillary electrophoresis (CE) [8e10], nuclear magnetic resonance (NMR) [6,11], high performance liquid chromatography (HPLC) [12e14], or gas chromatography (GC) [15e17]. For the CE-based method, the electrolyte is composed of 4-methylbenzyamine, 2-hudroxy-2-methylpropanoic acid, and 18-crown-6 [10]. Due to the instability of electrolytes, it is necessary to replace the electrolyte every 24 h to avoid the interference of its decomposition products on NMM quantification. Furthermore, its concentrated solutions could not be stored for long due to the same instability issue. Thus, it is desirable to develop new electrolyte systems with high stability and high separation efficiency. The NMR technique is suitable to qualitatively determine the main components in reaction mixtures or products, while the accurate measurements of minor constituents (content < 5%) with NMR are challenging [6]. For the direct HPLC and GC analysis, the matrix effect of biomass pretreatment solution by NMMO will cause a serious problem. The oxidation of NMMO (up to 86.7% in

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sample solution) will result in damage on HPLC tubing and packing materials and filler of the column, thus negatively affecting the chromatographic separation of NMM. Furthermore, the lowvolatile NMMO and cellulose degradation products will result in a serious deterioration of the GC injection port and capillary column. The tedious liquid-liquid extraction procedure is mandatory prior to HPLC and GC analysis [6]. The headspace (HS) analysis technique is a commercial and high efficient gas-liquid extraction technique [18]. All or part of analytes are steadily transferred from the sample solution to the gaseous phase in a closed sample vial at a given equilibrium temperature. The analytes in gaseous phase can be sampled with the commercial HS sampler, transferred to, and quantified by GC [19e23]. We believe that the HS-GC analysis technology is suited to analyze NMM in the NMMO/H2O solvent system. However, challenges remain that include: 1) how to eliminate the interference of amines, that could be adsorbed on the HS sampler tubing, on the NMM chromatographic separation and accurate quantification, 2) how to protect NMMO and NMM from thermal degradation, 3) how to enhance the detection sensitivity of HS-GC on NMM. In this study, we developed a new HS-GC method using an ammonia-assisted in-situ flushing technique to accurately quantify the NMM concentration in biomass pretreatment solutions. The scope of the present study includes: 1) Examine the in-situ flushing effect of ammonia on adsorbed amines in the HS tubing; 2) Determine the dosages of ammonium hydroxide and sodium hydroxide for this purpose; 3) Optimize the equilibration conditions (temperature and time), NMMO content, and detection solution size. Furthermore, the accuracy and reproducibility of the present method is validated. The present method will be a useful tool for process optimization and new stabilizer screening for the NMMO pretreatment processes. 2. Materials and methods 2.1. Chemicals and materials N-methyl morpholine (>99.0%, GC), ammonium hydroxide (28e30%, ACS), copper sulfate pentahydrate (99%, AR), and sodium hydroxide (96%, AR) were purchased from Aladdin Reagent (Shanghai, China). N-methylmorpholine N-oxide (97%, GR) was obtained from Xianding Biotechnology Co. Ltd (Shanghai, China). A standard solution of N-methyl morpholine (50 g/L) was prepared by dissolving 5.000 g of N-methyl morpholine (NMM) in N-methylmorpholine N-oxide solution (43.3%) to make 100 mL with volumetric flask. The eucalyptus woodchips were provided by Qingshan Paper Industry in China. The air-dried eucalyptus woodchips were grinded with a centrifugal grinding mill (Retsch ZM 200, Germany), and then was screened by a set of sieves. The woodmeal with powder sizes from 32 to 100 mm was used for the NMMO-based pretreatment. Its chemical components were measured by Tappi Standard Testing Method (T17wd-70, T19wd-71, T13wd-74), which were 45.8% for cellulose, 22.7% for pentosan, and 27.9% for lignin, respectively. 2.2. Apparatus and operations The static headspace sampler (Thermo Fisher Scientific TriPlus™ 300, Italy) and a gas chromatograph (Agilent GC 7890B, US) equipped with a flame ionization detector was used to conduct the


HS-GC measurement. The headspace sampler operated at the followed conditions: oven temperature ¼ 80  C, loop temperature ¼ 150  C, transfer line temperature ¼ 155  C, carrier gas pressure ¼ 100 kPa, auxiliary gas pressure ¼ 350 kPa, pressurizing time ¼ 0.5 min, pressure equilibration time ¼ 0.2 min, loop filling time ¼ 0.5 min, loop equilibration time ¼ 0.2 min, injection time with standard mode ¼ 0.5 min, purge time ¼ 5min, purge flow ¼ 300 mL/min. The injection port in the GC operated at 300  C with a single taper liner (Agilent 5183e4711, USA), splitless injection, 1.5 min of purging time, 60 mL/min of purge flow, and a 10 psi of pressure. RTX-5 capillary column (30 m  0.32 mm  1.0 mm, RESTEK, Germany) ran with gradient temperatures: 40  C for a  16 min, elevating to 200  C as 120 C/min and keeping 2 min,   cooling to 40 C as 120 C/min and keeping 1.0 min. The FID operated at 300  C with a 30 mL/min of hydrogen flow and a 400 mL/ min of air flow. 2.3. Biomass pretreatment with NMMO/H2O A specific mass of NMMO, water, and Eucalyptus wood powder (5.0 g) or dissolving pulp (5.0 g) or copper sulfate solution, was added in a 250-mL three-necked flask to obtain a NMMO/H2O pretreatment solution (NMMO 86.7%, 100 g). After stirring about 15 min with a mechanical agitator and PTFE blade in an 80  C thermostatic oil bath, the reaction system was purged with nitrogen at 30 mL/min of gas flow for 5 min. Then, the degradation reaction was conducted at 110  C for different times with 200 rpm agitation speed. After the biomass pretreatment reaction, a 100 g of deionized water was added to the flask to stop the reaction. The well-mixed solution was stored in a refrigerator at 4  C until the HSGC measurements. 2.4. Sample preparation and HS-GC measurement A 4.0 g of diluted biomass pretreatment solution (containing about 43.3% of NMMO) and 0.5 g of NaOH solution (0.25 mol/L) were added into a 21-mL headspace vial. The sample vial was purged with nitrogen at 30 mL/min of gas flow for 1.5 min, and then it was immediately sealed with a PTFE/butyl rubber pad and magnetic aluminum/iron cap. This purging procedure was verified that it can completely drive off the oxygen in sample vial. Finally, a 1.5 mL of ammonium hydroxide was injected into the sealed sample vial using a 3.0-mL syringe coupled with a micro needle. The well-mixed sample solution was incubated at 80  C for 30min in a headspace sampler, and then the gas sample in the headspace of the sample vial was sampled and measured by GC-FID. 3. Results and discussion 3.1. Ammonia-assisted desorption of NMM In the biomass pretreatment solution, there are many species of impurities and NMMO-related degradation products. With the traditional HS-GC measurement method, it was identified that there are some interefering substances presented in detection solution, which resulted a vital effect on the separation of NMM peak. Considering the presence of different amine substances in the detection solution, we proposed that the interfering substance can be removed using basic gas. In here, ammonia gas was used as a flushing reagent to eliminate the effect of amines on NMM quantification. As shown in Fig. 1, the interfering substances can be flushed out of the HS sampler by adding ammonium hydroxide in each single test. Consequently, their interferences on NMM quantification can be eliminated. In addition, Figs. 1 and 2, show that the GC responses of NMM increased with ammonium hydroxide


S. Zhang et al. / Renewable Energy 145 (2020) 2380e2386

Fig. 1. Chromatogram for sample solutions with or without ammonia addition.

Fig. 2. Effect of ammonia addition on GC signals of NMM and interfering substances.

addition, which indicates NMM was also absorbed on the sampling tubes in the HS sampler (all tubes are surface-coated by silylated polymers), and that also can be flushed into GC system to increase the detection sensitivity. Finally, to maximize the detection sensitivity of NMM and prevent the gas leakage from sample vial, we selected a 1.5 mL of NH3$H2O (28%e30%, w/w) as the assisting reagent for HS-GC measurement of NMM.

the NaOH addition ranging from 0 to 0.25 mmol in the 6.0 g of testing solution, the GC response of ammonia gas increased firstly and then leveled off. Furthermore, the increase of ammonia gas led to an increase in the GC peak area of NMM. At higher than 0.125 mmol NaOH charge, the effect of sodium hydroxide is negligible. Therefore, we selected 0.125 mmol, i.e., a 0.5 mL of NaOH solution with a 0.25 mol/L of concentration, as the addition amount of sodium hydroxide.

3.2. Effect of alkali addition 3.3. Conditions for headspace equilibration In this work, sodium hydroxide was used as a stabilizer to minimize the NMMO degradation reactions. Fig. 3 shows that, with

Fig. 4 shows the GC response of NMM as equilibrium time with

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Fig. 3. Variation of GC signal of NMM, NH3 and interfering substance on alkaline content.

Fig. 4. Dependence of NMM's GC response on equilibrium temperature and time.



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different HS equilibration temperatures. A higher equilibrium temperature will produce a higher GC response, and the increase of oven temperature from 60 to 80  C will triple the GC signal of NMM. Moreover, the gas-liquid equilibration is obtained within 20 min. However, at a 90  C oven temperature, the GC signal of NMM increased first and then decreased over time, which is attributed to the NMM degradation (high temperature and strong alkaline conditions). For obtaining higher detection sensitivity and accuracy, we selected 80  C and 20 min as the equilibration conditions in the HS sampler's oven. 3.4. Effect of the selected measurement conditions on NMMO degradation

in the liquid phase but also on the phase ratio of liquid on vapor. Fig. 6 shows the effect of testing solution volume on the NMM's GC response for a 21.0-mL headspace vial, and all chemicals for each solution volume were proportionally scaled up or down according to their mass in 6.0 g of testing solution. The results show that as the solution volume increases, the GC signal of NMM has a rapid increase in the initial stage and then levels off. With a sample mass larger than 6.0 g, a further increase in the testing solution volume has negligible effect on the NMM's GC signal. Therefore, the volume of testing solution was selected as 6.0 g, i.e., using 4.0 g of sample solution, 1.5 g of ammonium hydroxide (28%e30%), and 0.5 g of sodium hydroxide (0.25 mol/L) for each testing. 3.7. Method calibration, precision and validation

The stability of NMMO under the alkaline environment in the present method was verified. Fig. 4 shows that NMMO/H2O solutions (without pretreatment) produce a tiny GC signal of NMM at the testing temperature (80  C) with different times (0e40min), indicative of very small amount of NMM in NMMO. However, the NMM GC signal did not increase with reaction time, even with the addition of a 15 mg/kg of cupric ion. Therefore, with the selected measurement conditions, the present method will not overestimate NMM content in the biomass pretreatment solution. 3.5. Matrix effect of biomass pretreatment solution We prepared different NMMO/H2O solutions (CNMMO ¼ 0e43.3%, w/w) with the equal NMM content (3.00 g/L) to investigate the effect of NMMO concentration in sample solutions on NMM's GC response. The results in Fig. 5 show that the increased NMMO concentration results in increases in the ammonia gas and NMM concentrations in the gaseous phase, and more pronounced increases for NMM than for ammonia. The latter can be explained by the fact that ammonia gas has a flushing effect on NMM as noted in Section 3.1. Hence, we recommend to dilute the NMMO content of biomass pretreatment solution to 43.3% before NMM determination. 3.6. Selection of detection solution volume Due to the phase transfer of NMM from liquid to gaseous phase in volatilization, the real concentration of NMM in liquid after equilibration decreased, and thus its equilibrium concentration in the gaseous phase is dependent not only on its initial concentration

3.7.1. Calibration The calibration was established using a set of NMMO/H2O solutions (CNMMO ¼ 43.3%, w/w) with different NMM contents (C ¼ 0.1e10.0 g/kg). The mixtures of these standard solutions (4.0 g) with sodium hydroxide solution (0.25 mol/L, 0.5 mL) and ammonium hydroxide (28%e30%, 1.5 mL), were equilibrated in HS sampler at 80  C for 30 min, and then the vapor in headspace vial was sampled by HS sampler and its NMM's content was determined by GC. Each sample solution was tested five times, and their averages of GC peak area (A) were plotted against the NMM concentration of these solutions (C, g/kg). A liner correlation with high coefficient (R2 ¼ 0.996) and very low deviation of slope and intercept in the calibration curve, as shown in Eq. (1), indicate that the present method has good performance. However, it should be noted that the calibration curve would have to be re-established for sample solutions with different NMMO concentrations.

  A ¼ 8:75ð ± 20:8Þ þ 2739ð±39:5ÞC n ¼ 7; R2 ¼ 0:996


3.7.2. Precision and limitation of quantification The precision of the present method was verified by the quintuple measurement of a diluted (1:1, w/w) sample solutions (86.7% NMMO solution containing a 5 mg/kg of cupric ion, reacted at 110  C for 3 h), and the relative standard deviation was 1.56%. In another batch measurement, the RSD of all samples ranged from 1.56% to 7.19%, with an average of 4.33%. In addition, the limitation of quantification (LOQ) of the present method was calculated as 71.9 mg/kg by Eq. (2) [24] with the coefficient in Eq. (1). These results indicate that the present method can satisfy the quantification requirement in the NMMO- based biomass pretreatment processes.


a þ 10jDaj s


In which a, s, and Da was the intercept, slope, and standard deviation of intercept at 95% of confidence.

Fig. 5. Effect of NMMO concentration on GC response of NMM and NH3.

3.7.3. Method validation The present method was validated based on the recovery test. Different amounts of NMM (0.1e7.4 g/kg) were added into a set of diluted (1:1, w/w) sample solution (86.7% NMMO solution containing a 10 mg/kg of cupric ion, reacted at 110  C for 2.5 h). Subsequently, these samples were subjected to HS-GC analysis using the method developed above. The net GC response of added NMM was obtained by subtracting the GC response of the unspiked sample, and was calibrated using Eq. (1). Finally, the added and measured NMM amount is listed in Table 1. The NMM recovery ranged from 96.0% to 102%, indicating that the present method can

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Fig. 6. Effect of detection solution volume on NMM detection.

Table 1 Recovery validation.

Table 2 Contents of NMM in pretreated NMMO/H2O solutionsa.

Sample no.

NMM, g/kg Added


1 2 3 4 5

0.10 0.90 1.90 4.90 7.40

0.0960 ± 0.0012a 0.898 ± 0.013 1.89 ± 0.05 4.99 ± 0.10 7.48 ± 0.09


Recovery, %

Cupric ion, mg/kg

Time, h

GC signal

Content in solution, g/kg Diluted


96.0 99.8 99.5 102 101


0.5 1 2 3

152 ± 6 263 ± 12 1266 ± 91 2620 ± 41

0.052 ± 0.002 0.093 ± 0.004 0.459 ± 0.033 0.954 ± 0.015

0.104 ± 0.004 0.185 ± 0.009 0.917 ± 0.066 1.91 ± 0.03

0.00 5.00 10.0 15.0


630 ± 27 1961 ± 104 4105 ± 45 5908 ± 399

0.227 ± 0.010 0.713 ± 0.038 1.49 ± 0.02 2.15 ± 0.15

0.454 ± 0.019 1.43 ± 0.076 2.99 ± 0.33 4.31 ± 0.29

Standard deviation of NMM content at 95% of confidence.

accurately quantify NMM's content in biomass pretreatment solutions.

Detection conditions: diluted twofold, diluted solution ¼ 4.0 g, NaOH (0.25 mol/ L) ¼ 0.5 mL, ammonium hydroxide (28e30%) ¼ 1.5 mL. a Reaction conditions: temperature ¼ 110  C, NMMO ¼ 86.7%.

3.8. Method application The present method has been applied to quantify NMM's concentration in biomass pretreatment solution (a 5.0 g of biomass, reaction temperature ¼ 90e120  C, reaction time ¼ 1.0e5.0 h) and the reacted solution of NMMO/H2O without biomass (cupric ion ¼ 0e15 mg/kg, reaction time ¼ 0.5e3.0 h at 110  C). All samples were diluted by two times, and each of the diluted sample solutions had quintuple measurements in accordance with the present method. The NMM content in the reacted solution (CNMM) was calculated by Eq. (3), in which ‘D’ is the dilution times. As shown in Table 2, the cupric ion content has a significant effect on NMMO degradation, and the formed NMM jumped almost ten times

(0.454e4.31 g/kg) by adding 15 mg/kg of cupric ion in solvent. In addition, the NMM content increased significantly with reaction time, which indicates the NMMO degradation is promoted by the reaction byproducts. Table 3 shows that, during the pretreatment of Eucalyptus woodmeal, significant amounts of NMM were generated. After the pretreatment at 110  C for 5 h or at 120  C for 3 h, up to 10.4 or 7.02 g/kg NMM was generated, which indicated significant amount of NMMO (1.21%e0.813%) was degraded in biomass pretreatment process. That was attributed to the presence of various metal ions in lignocellulosic biomass. Thus, it is necessary to optimize the pretreatment process to minimize the NMMO


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Table 3 Contents of NMM in pretreatment solution of Eucalyptus wood powdera. Reaction temp., oC


90 100 110 120

[3] Time, h

GC signal

Content in solution, g/kg Diluted



1.0 2.0 3.0 5.0

460 ± 16 582 ± 23 1555 ± 69 3563 ± 46

0.165 ± 0.006 0.209 ± 0.008 0.564 ± 0.025 1.298 ± 0.017

1.32 ± 0.04 1.67 ± 0.07 4.52 ± 0.20 10.4 ± 0.14


1078 ± 7 1204 ± 15 1555 ± 69 2411 ± 91

0.390 ± 0.002 0.436 ± 0.005 0.564 ± 0.025 0.877 ± 0.033

3.12 ± 0.02 3.49 ± 0.04 4.52 ± 0.20 7.02 ± 0.27

Detection conditions: diluted eight times, diluted solution ¼ 4.0 g, NaOH (0.25 mol/ L) ¼ 0.5 mL, ammonium hydroxide (28e30%) ¼ 1.5 mL. a Reaction conditions: NMMO content in solution ¼ 86.7%, ratio of solid to liquid ¼ 5 g/100 g.







CNMM ¼ DðA  aÞ=s




4. Conclusion A new method based on headspace GC (HS-GC) for determination of N-methyl-morpholine (NMM) concentration in biomass pretreatment solutions was developed. The use of ammonia gas minimizes the interference of other amines during the analysis, while the NMM detection sensitivity is enhanced. The optimum conditions are as follows: 4.0 g of sample solution, 1.5 mL ammonium hydroxide (28e30%, w/w), 0.125 mmol of sodium hydroxide, gas-liquid equilibration conditions of 80  C for 30 min. The present method offers high detection accuracy, high precision and high sensitivity, which provides a powerful tool to optimize reaction conditions and screen the stabilizer for biogas and biofuel production. This method will find many applications, including the optimization of NMMO-based biomass pretreatment process. Acknowledgments The authors acknowledge the National Natural Science Foundation of China (31700507 and 21576105), the Fujian Provincial Department of Science and Technology (2015J05018), the National Key Research and Development Program of China (2017YFB0307900), the FAFU’s Fund for Distinguished Young Scholars (XJQ201601) for sponsoring this research. The authors acknowledge Prof. Xin-Sheng Chai's help for improving this paper's quality.







[18] [19]




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