Article pubs.acs.org/EF
Quantification of Sugar Compounds and Uronic Acids in Enzymatic Hydrolysates of Lignocellulose Using High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection Qiulong Hu,†,‡ Lin Tan,† Zhou Heng,† Xiaojun Su,† Tingting Zhang,† Ziwei Jiang,† and Xingyao Xiong*,† †
Hunan Agricultural University, Changsha 410128, China Hunan Food Quality Supervision and Inspection Institute, Changsha 410002, China
‡
ABSTRACT: A method was developed for simultaneous determination of seven sugar compounds and two uronic acids by high-performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) using a CarboPac PA20 column under a gradient elution (mobile phase: H2O/200 mM NaOH/100 mM NaAc) at 0.4 mL/min. Under these conditions, D-(−)-arabinose, D-(+)-galactose, D-(+)-glucose, D-(+)-mannose, D-(+)-xylose, D-(−)-fructose, D-(+)-cellobiose, Dgalacturonic acid, and D-glucuronic acid were all separated in 46 min. The calibration curve, detection limits, reproducibility and accuracy were also determined. Furthermore, seven sugar compounds and two uronic acids were all detected in the enzymatic hydrolysates of different lignocellulosic materials treated by different pretreatment processes. Our results demonstrate that this method was feasible for sugar compounds separation without the need for derivatization and organic solvent, and it also has the advantages of having low interference, high sensitivity, low detection limit of picomol, and excellent reproducibility and accuracy.
1. INTRODUCTION Lignocellulosic biomass is the most abundant and cheapest renewable biomass, with an estimated annual production of 1 × 1010 Mt worldwide.1 Making use of abundant lignocellulosic biomass to produce fuel ethanol reflects multidimensional advantages of being economical, practical, and environmental friendly. Furthermore, fuel ethanol as a potential alternative energy source offers a broad market prospect and may play an important role in solving the energy problem. Finding an effective way to hydrolyze lignocellulosic biomass to fermentable soluble sugars is a key step to produce fuel ethanol.2−5 Lignocellulose is mainly composed of cellulose, hemicellulose, and lignin. Cellulose is straight chain polysaccharides made from D-glucose units connected by β-1,4 glycosidic bonds, which can be hydrolyzed into glucose and cellobiose; hemicellulose is heteropolysaccharide polymerized by many different monosaccharides, which can be hydrolyzed mainly into xylose, glucose, mannose, arabinose, fructose, and galactose; lignin can be degraded into many aromatic compounds. However, these hydrolyzed products are not stable and can further hydrolyze into acetic acid, furfural, hydroxymethylfurfural, uronic acid, and aromatic resins. The commonly used methods for sugar determination are gas chromatography (GC),6 high performance liquid chromatography (HPLC),7 and capillary electrophoresis (CE).8 Since sugar compounds have low volatility, strong polarity, and high boiling point, it is necessary to derivatize them into the corresponding derivatives for efficient GC analysis,9−14 which greatly restricts the practical application of the GC method. Alternatively, HPLC is used more often, but sample preparation is more troublesome, and the sugar compounds do not absorb in the ultraviolet (UV) region. Thus, a refractive index detector must be used for detection. However, the mobile phase and extraction solvents are incompatible, which can lead to interference and complicate the analytical results.7 CE is a © 2012 American Chemical Society
relatively new technology that takes advantage of the different migration rates of charged particles under a high-voltage field for sample separation. Sugar compounds are neither charged nor UV active and fluorescent. Therefore, sugar compounds can not be analyzed directly by CE, but need to be converted into charged particles or derived into compounds with chromophores or fluorophores prior to separation,8 which also makes the method quite complicated. For the determination of uronic acids, acid derivatives of sugars, colorimetry with carbazole sulfate15,16 and m-hydroxy biphenyl is often employed, but these methods have some defects, resulting in interference and inaccuracy in uronic acid determination.17 The separation of sugar compounds and uronic acids could be achieved by CE, but the sensitivity is limited by injection volume and the detection limit of UV-detectors at >12 mg/L.18 A new method using high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) has been developed for the quantification of sugars.19−21 The HPAEC-PAD technique employs an alkaline condition, where affinity between sugar and a stationary phase occurs. Under alkaline conditions, weak-acidic sugars are present as anions, which can be separated and detected by anion-exchange column with PAD .This method has the advantages of no derivatization, simplicity, high sensitivity, organic solvent free, and extremely low detection limit at the picomol level.22−24 D-(−)-arabinose, D-(+)-galactose, D-(+)-glucose, D-(+)-mannose, D-(+)-xylose, D-(−)-fructose, D-(+)-cellobiose, D-galacturonic acid, and D-glucuronic acid were the major products of enzymatic hydrolysates of lignocellulose; several sugar compounds had been often determined in the enzymatic hydrolysates of lignocellulose by gas chromatography and high Received: January 10, 2012 Revised: April 11, 2012 Published: April 11, 2012 2942
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performance liquid chromatography.25−27 In addition, most reports on HPAEC-PAD analysis for saccharides contain a limited number of sugar compounds and no uronic acid.28−30 Up to now, there is no report of the simultaneous determination of seven main sugar compounds and two uronic acids in enzymatic hydrolysates of different lignocellulose by high-performance anion-exchange chromatography with pulsed amperometric detection. In this study, we used HAPEC-PAD to analyze seven sugar compounds and two uronic acids in hydrolysates from the enzymatic hydrolysis of different lignocellulose samples. The linearity, repeatability, and accuracy of the method was also determined. Our results demonstrate that the method is able to separate and quantitate the sugars and uronic acids in the hydrolysates using a CarboPac PA20 column (150 × 3 mm i.d.). Establishing the analysis method of sugar compounds and uronic acid in hydrolysate of lignocellulose has great significance; the applicability of the method will supply the evaluation methods and standards for some promising pretreatment technology and saccharification efficiency in research of fuel ethanol.
analytical sequence. No degradation of standard compounds was found during the course of the analytical week. The calibrations were performed by duplicate analysis of seven standard mixtures at different concentration levels. Calibration curves were constructed by plotting peak area vs analyte amount injected. Calibration functions were calculated using curve fitting by means of linear regression analysis. The limit of detection (LOD) was determined as the minimum concentration that was visible on the chromatogram and produced a peak height at least three times the signal-to-noise (S/N) ratio, The noise level was measured using the PeakNet6.7 software for a 1-min interval of a blank injection, corresponding to the same retention time as the analyte of interest. The mobile phase consisted of a 200 mM NaOH solution and a 100 mM NaAc solution. Both mobile phase solutions were prepared in Milli-Q water and then filtered through 0.22 μm nylon filters. Moreover, the mobile phase solutions were stored under nitrogen at 6−8 psi to prevent absorption of carbon dioxide from the atmosphere. 2.4. Sample Preparations. The steam explosion pretreatment was conducted using an apparatus (Hebi Zhengdao Heavy Machine Factory, China). Steam explosion temperature was 220 °C and the retention time was 5 min. Each batch of about 200 g of straw was put into the steam chamber. Once the desired retention time and temperature were reached, the steam pressure was transiently released. The exploded material was recovered and dried at 80 °C for 24 h in a constant temperature oven, and it was stored for subsequent enzymatic hydrolysis. The straw samples were irradiated with 2 kGy/h dose rate by a Co-60γ source in sealed polyethylene bags at room temperature. After the irradiation dose achieved 1600 kGy, all the treated samples were stored in sealed flasks at room temperature in the dark. Lignocellulose biomass (reed straw, corn straw, and rice straw) treated with steam explosion (220 °C, 5 min) and Co-60γ irradiation (1600 kGy) were enzymatically hydrolyzed in 50.0 mL sodium acetate buffer (0.05 M, pH 4.8) with the substrate loading of 1.00 g sample. The enzyme loading of cellulase was 100 UI/g substrate. The hydrolysis runs were all performed in 150 mL screw flasks in the incubator at 150 rpm and heated at 50 °C for 60 h. The resulting hydrolysate was deactivated by heating in a 100 °C water bath for 15 min. The hydrolyzed samples of each sample type were prepared in duplicate. The enzymatic hydrolysate was transferred quantitatively to a 200 mL flask with Milli-Q water. Further dilution of each sample was carried out prior to analysis to bring sample concentrations into the linear range of the pulsed amperometric detector. Final dilution factors for enzymatic hydrolysate were 1:4000. The diluted samples was centrifuged at a speed of 6000 r/min for 10 min, and the supernatant was filtered through a 0.22 μm nylon membrane, Finally, the hydrolysates sample can be directly injected onto the column. All analytical determinations were performed in duplicate, and average results are shown. 2.5. Anion-Exchange Chromatographic Method. Seven sugar compounds and two uronic acids were separated on the CarboPac PA20 column. The solvents used in the gradient elution system were water, 200 mM NaOH, and 100 mM NaAc. The flow rate was adjusted to 0.4 mL/min, and the column temperature was set to 30 °C. The injection volume was 10 μL. The quadruple waveform for the pulsedamperometric detection of the sugars is shown in Table 1.
2. MATERIALS AND METHODS 2.1. Materials. Reed (Phragmites communis Trin) straw was collected in the summer from the embankment of the Liuyang River in Hunan Province, China; Corn (hybrid corn of Yinhe 14) and rice (Indica hybrid rice of Shanyou63) straws were harvested in autumn from the suburb of Chang-sha, Hunan Province, China. The air-dried reed straw, corn straw, and rice straw (the moisture contents of straws are all lower than 10.0%) were cut into small sections and screened to attain a length of 3−4 cm for steam explosion pretreatment. The 3−4 cm straws were then crushed with a high-speed (24 000 rpm) pulverizer for 10 min, the powder samples after sieving with a 80 mesh screen were kept in sealed polyethylene bags for Co-60γ irradiation pretreatment. All reagents employed were of analytical-grade unless stated differently. D-(−)-arabinose, D-(+)-galactose, D-(+)-glucose, D(+)-mannose, D-(+)-xylose, D-(−)-fructose, D-(+)-cellobiose, D-galacturonic acid, and D-glucuronic acid were all purchased from Sigma (St. Louis, MO). Sodium hydroxide solution (50% w/w) and sodium azide were purchased from Beijing Chemical Corporation (Beijing, China). Anhydrous sodium acetate was obtained from Dionex (Sunnyvale, CA). The water used to prepare the standard solutions and to extract the samples was 18.2 MΩ·cm (Milli-Q, Millipore, U.S.A.) water. Cellulase was obtained from Wuxi Xuemei enzyme preparation technology Co., Ltd. of China (40 000 IU/g). 2.2. Instrumentation. The development and validation of the method were performed on a Dionex ICS-3000 system equipped with a quaternary pump and degasser (SP), a column compartment (DC), an electrochemical detector and gold electrode (ED), an eluent organizer (EO) unit, and an AS40 autosampler (Dionex, Sunnyvale, CA). The waveform used for pulsed amperometric detection was the standard quadruple potential for analysis (Dionex Technical Note 21). The separation was carried out on a CarboPac PA20 column (150 × 3 mm i.d., Dionex) . PeakNet 6.7 (Chromeleon) chromatography software (Dionex) was used for system control and data analysis. 2.3. Preparation of Standard Solutions. The mixed standard stock solutions of seven sugar compounds and two uronic acids at 1.000 g/L were made by dissolving appropriate amounts of each standard sugar compound and uronic acid in water. To prevent microorganisms from changing the concentration, the mixed standard solution containing 20 mg/L NaN3 was used to prepare standard solutions at low concentration by diluting the corresponding stock solutions. The mixed stock solutions were kept at −18 °C for a maximum of 6 months. The mixed standard solutions were freshly prepared at the beginning of each analytical week and stored at 5 °C for a maximum of 1 week. A calibration was performed for each
3. RESULTS AND DISCUSSION 3.1. Separation of Seven Sugar Compounds and Two Uronic Acids. Sugar compounds are weakly acidic organic compounds, and their pKa values are generally in the range of 12−14.31 In alkaline conditions, sugar compounds can become anionic, which can be separated on an anion-exchange CarboPac PA20 column using a low concentration of NaOH mobile phase solution. In addition, the column’s pellicular resins are stable in the pH range of 0−14. Table 2 presents the molecular formulas, molar masses, pKa values, and chemical structures of the seven sugar compounds and two uronic acids. In general, the elution sequence of the sugar compounds 2943
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ability, NaAc with strong elution ability was chosen, so the separation of sugar compounds and uronic acids can be improved by optimizing the gradient elution of NaOH and NaAc.9 Table 3 shows the optimal gradient conditions obtained using H2O/200 mM NaOH/100 mM NaAc as the mobile phase at a flow rate of 0.4 mL/min. Seven sugar compounds and two uronic acids can achieve a good separation on the CarboPac PA20 column. The total separation time excluding washing steps was about 46 min; the HAPEC-PAD chromatogram for the mixed standard solution (20 mg/L) separation is shown in Figure 1. After each run, the column was flushed with 200 mM NaOH for 4 min to remove any possible carbonate from the column and then equilibrated with 2.4 mM NaOH for 10 min to ensure reproducibility of the retention time. 3.2. Calibration, Detection Limits, and Reproducibility. The calibration curve of the sugar compounds and uronic acids was determinated using the optimal separation conditions. The regression equation, coefficient (r2), limit of detection, linear range, and average relative standard deviation (RSD) are listed in Table 4. The limit of detection (LOD) was determined as the minimum concentration that was visible on the chromatogram and produced a peak height at least three times the signal-to-noise ratio. The relative standard deviations (RSD) of peak areas were determined from ten consecutive analyses of 10 mg/L mixed standard solutions. A calibration curve was prepared for each enzymatic hydrolysate of lignocellulose from seven different concentrations of sugar compounds and uronic acids. For D-arabinose, D-(+)-galactose, D-(+)-glucose, D-(+)-xylose, and D-(+)-cellobiose, the correlation coefficient was more than 0.999; for D(+)-mannose, D-(−)-fructose, D-galacturonic acid, and D-
Table 1. Quadruple Waveform of Pulsed-Ampere Detector time (s)
potentiala (v)
0 0.20 0.40 0.41 0.42 0.43 0.44 0.50
0.10 0.10 0.10 −2.00 −2.00 0.60 −0.10 −0.10
integration (start/end) start end
a
Potentials applied to Au working electrode and referenced vs glass/ Ag/AgCl combination electrode.
separated on an anion exchange column is related to the pKa value. Under the same condition, the rate of elution increases with the increase of pKa value. The order of separating sugar compounds on anion exchange separation column is successively monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Thus, D-(+)-cellobiose was sequentially separated following D-(−)-arabinose, D-(+)-galactose, D(+)-glucose, D-(+)-mannose, D-(+)-xylose, D-(−)-fructose. Uronic acids are the acidic derivatives of the corresponding monosaccharide, in which the presence of carboxylic groups makes them more acidic and have lower pKa values; thus, the retention time of uronic acids is longer than the parent sugars. The anionic OH− of NaOH not only plays the role of the elution ion, but its strong alkali property also can cause amperometric detection to have a high sensitivity. However, the retention ability of sugar compounds reduces with increasing NaOH concentration. Since uronic acids have strong retention
Table 2. Molecular Formulas, Molar Masses, pKa Values,18,32 and Chemical Structures of the Seven Sugar Compounds and Two Uronic Acids (in Water, 25 °C)
a
pKa: the logarithmic measure of the acid dissociation constant . bNA: Not available. 2944
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Table 3. HAPEC-PAD Chromatographic Gradient Elution Conditions for the Separation of Sugar Compounds and Uronic Acids time (min)
φ(H2O) (%)
φ(200 m mol/LN aOH) (%)
φ(100 Mmo l/LN aAc) (%)
0.0 18.0 20.0 30.0 30.1 46.0 46.1 50.0 50.1 60.0
98.8 98.8 50.0 50.0 0.0 0.0 0.0 0.0 98.8 98.8
1.2 1.2 50.0 50.0 0.0 0.0 100.0 100.0 1.2 1.2
0 0 0 0 100 100 0 0 0 0
curve
5 5 5 5
Table 5. Recoveries of Sugar Compounds and Uronic Acids sugar compounds and uronic acids D-(−)-arabinose D-(+)-galactose D-(+)-glucose D-(+)-mannose D-(+)-xylose
contents (mg/L)
spiked value (mg/L)
found(n = 3, mg/L)
recovery (%)
0.73 0.54 38.42 0.84 18.31
10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00
10.41 9.79 49.40 10.24 27.38 8.49 22.42 11.59 9.45
96.8 92.5 109.8 94.0 90.7 84.9 108.9 97.8 88.9
D-(−)-fructose D-(+)-cellobiose D-galacturonic
acid D-glucuronic acid
Figure 1. HAPEC-PAD chromatogram for the separation of seven sugar compounds (20 mg/L) and two uronic acids standard solution (20 mg/L). 1. D-(−)-arabinose, 8.535 min; 2. D-(+)-galactose, 10.518 min; 3. D-(+)-glucose, 12.418 min; 4. D-(+)-xylose, 15.001 min; 5. D(+)-mannose, 16.285 min; 6. D-(−)-fructose, 18.418 min; 7. D(+)-cellobiose, 30.701 min; 8. D-galacturonic acid, 41.018 min; 9. Dglucuronic acid, 44.318 min.
11.52 1.81 0.56
recovery results were obtained for seven sugar compounds and two uronic acids. For concentration >10 mg/L, the recovery were found from 90.7% to 109.8%; for concentration
