Article pubs.acs.org/EF
Correlation of Hydrogen Cyanide Formation with 2,5Diketopiperazine and Nitrogen Heterocyclic Compounds from Copyrolysis of Glycine and Glucose/Fructose Jufang Hao,†,‡ Jizhao Guo,‡ Fuwei Xie,‡ Qiaoling Xia,‡ and Jianping Xie*,‡ †
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, Liaoning 116023, People’s Republic of China Zhengzhou Tobacco Research Institute of China National Tobacco Corporation (CNTC), Zhengzhou, Henan 450001, People’s Republic of China
‡
ABSTRACT: It is important to understand the formation of hydrogen cyanide (HCN) for minimizing the emissions of NOx during biomass combustion. In the present study, glycine was used as the model compound of biomass N. The influences of reducing sugars (glucose and fructose) on the formation of HCN from glycine pyrolysis were investigated by analyzing the yields of HCN, 2,5-diketopiperazine (DKP), and N-heterocyclic compounds. The results indicated that the HCN yield from glycine pyrolysis decreased and the nitrogen distribution changed in the presence of glucose/fructose. Of the thermal reactions, glycine might be more likely to react with reducing sugars to form a large number of N-heterocyclic compounds rather than polymerize to form DKP. The decrease of the HCN yield from co-pyrolysis of glycine and reducing sugars is related to the reduction of DKP and the increase of N-heterocyclic compounds. Furthermore, analysis of label incorporation into HCN formation at different molar ratios of glycine/reducing sugar indicated that the contribution of reducing sugar to HCN formation might be more considerable with the increasing content of reducing sugar.
1. INTRODUCTION In recent years, biomass fuel has become one of the major choices in tackling the global energy shortage. However, biomass N can be converted to NOx (NO and NO2) during pyrolysis and combustion, which causes the formation of acid rain and photochemical smog.1 Hydrogen cyanide (HCN) is one of the most important precursors of NOx.2−4 It is generally believed that nitrogen in biomass is mainly bound in amino acids and proteins, which can release HCN during pyrolysis.5,6 Because of the complicated composition of proteins, amino acids are always used as the model compounds of biomass N to reveal the formation mechanisms of HCN.7−12 Actually, the pyrolysis process of biomass is highly complex, because as a part of biomass, amino acids can experience interactions with other components in biomass during pyrolysis.13 The Maillard reaction, taking place between the amino groups contained in amino acids or proteins and the carbonyl groups in carbohydrates,14 is an important reaction during biomass pyrolysis. The Maillard chemistry has been studied in detail to understand the formation of flavor components in food processing. However, the effect of the Maillard reaction on the HCN formation during biomass combustion or gasification has not been elucidated. As the simplest amino acid, glycine is one important precursor of HCN. On the basis of the previous studies on glycine pyrolysis, the formation of HCN through the decomposition of 2,5-diketopiperazine (DKP) is certainly an acceptable mechanism.7,9,12 However, the influence of reducing sugars on HCN formation from glycine pyrolysis has not yet been elucidated. In the present study, experiments were conducted at different molar ratios of glycine/glucose and glycine/fructose model systems. The changes of the yields of HCN, DKP, and other N-heterocyclic compounds from the © 2013 American Chemical Society
two model system were analyzed, and the correlations of the yields of HCN with DKP and other N-heterocyclic compounds were investigated in detail. Consequently, how glucose/fructose influences the HCN formation from glycine pyrolysis was explained.
2. EXPERIMENTAL SECTION 2.1. Materials. Glycine and DKP, with a purity of more than 98%, were purchased from Acros Organics (Belgium). Glucose and fructose, with a purity of more than 98.5%, were purchased from Sigma Chemical Co. Isotopically labeled [13C6]-glucose and -fructose (98% enrichment) were obtained from Cambridge Isotope Laboratories, Inc. (Andover, MA). The n-alkane mixture, consisting of C7−C30 straightchain alkanes, was obtained from Supelco, Bellefonte, PA. Highperformance liquid chromatography (HPLC)-grade acetonitrile and dichloromethane were obtained from Fisher Scientific International, Inc. (Beijing, China). 2.2. Pyrolysis and Analysis of HCN. The offline pyrolysis experiments were carried out in an infrared image furnace. The pyrolysis apparatus has been previously described in detail and will be only briefly reviewed.15 The quartz tube was used as a reactor, and a sheath thermocouple was used to measure the temperature of the sample. During a typical run, the sample was stuffed into the reactor tube and heated at 10 °C/s from 40 °C up to a preset temperature and then maintained for 5 min. During the period of the rising temperature and the 5 min holding time, the generated volatile products were transported by helium carrier gas and collected by a 45 mm glass fiber filter pad and an impinger containing 30 mL of 0.1 mol/L sodium hydroxide solution. The flow rate of helium was 200 cm3/min, confirmed with a soap bubble meter on the exit of the pyrolysis apparatus (after the impinger). Received: March 11, 2013 Published: July 15, 2013 4723
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After pyrolysis, the pad was shaken with 30 mL of 0.1 mol/L sodium hydroxide solution for 30 min. A total of 1 mL of the extract was merged with the same volume of the absorption solution in the impinger, then diluted, and filtered by 0.45 μm filters. HCN was quantified by an automated continuous flow colorimetric analyzer (Bran + Luebbe, Inc., Japan) by comparison to the standard calibration. 2.3. Analysis of DKP by HPLC. For determination of the yield of DKP, the impinger contained 20 mL of deionized water. After pyrolysis, the pad was shaken with 20 mL of deionized water for 30 min. Then, the same volume of the two parts of the extract solution was merged, diluted, filtered, and subjected to HPLC to obtain the amount of DKP in the tar. The pyrolysis residue (char) was extracted by 30 mL of deionized water with ultrasonic cleaning for 30 min. The water extracts were quantified by HPLC to obtain the amount of DKP in the char by comparison to the standard calibration. The sum of the amount of DKP in the tar and char was the yield of DKP. An Agilent 1200 series liquid chromatograph was used for the analysis, using a Symmetry C18 column, 250 × 4.6 mm inner diameter, 5 μm particle size. Acetonitrile (10%) in deionized water was used as the mobile phase for isocratic elution. Ultraviolet (UV) detection was performed at 200 nm. 2.4. Analysis of the Volatile Pyrolysis Products by Gas Chromatography/Mass Spectrometry (GC/MS). For the determination of the volatile pyrolysis products, a cold trap (dry ice/ isopropanol) was connected downstream of the glass fiber filter pad to collect the pyrolysis condensate. After pyrolysis, the filter pad was placed in an amber screw cap vial, with phenylethyl acetate solution added as an internal standard, and then extracted with 20 mL of dichloromethane. The extraction solution was condensed to 2 mL for GC/MS analysis. An Agilent 7890 GC-5975 mass selective detector (MSD) and a 60 m × 0.25 mm × 0.25 μm DB-WAXETR column were used for product analysis. Temperature programming was as follows: the initial oven temperature was 40 °C, held for 2 min, heated to 240 °C at 5 °C/min, and then maintained for 10 min. The flow rate of helium carrier gas remained constant at 1 mL/min. MS was operated at 150 °C in electron impact (EI) mode (70 eV), scanning from m/z 15 to 350. The mass spectral identifications were carried out by comparison to the NIST08 mass spectral library. Furthermore, the Kovats retention indices were calculated for each peak with reference to n-alkanes, and some available authentic samples were used to identify the chromatographic peaks by matching the retention times. The products were quantified by averaging the selected ion output relative to that of the internal standard. 2.5. Online Pyrolysis (Py)−GC/MS Analysis for the Mixture of Glycine and Labeled Glucose/Fructose. To examine further the origin of HCN from co-pyrolysis of glycine and glucose/fructose, the isotopic-labeled [13C6]-glucose and -fructose were used. Different molar ratios of glycine/glucose and glycine/fructose were pyrolyzed by online Py−GC/MS, and the proportion of isotopic-labeled molecules in HCN was analyzed. The pyrolysis was performed using a CDS Pyroprobe 5200 pyrolyzer. The experimental conditions during online pyrolysis were chosen to match the conditions of the offline pyrolysis. During pyrolysis, the pyrolysate were transported into the heated GC injection port by helium carrier gas, which passed through the interface continuously. The interface was maintained at 280 °C to prevent condensation of pyrolysis products during pyrolysis. The pyrolysis vapors were analyzed by GC/MS (Agilent 7890 GC-5975 MSD), and a DB-WAXETR capillary column (60 m × 0.25 mm × 0.25 μm) was used. The injector temperature was kept at 280 °C. Helium carrier gas flow remained constant at 1 mL/min. The oven temperature was programmed from 50 °C (2 min) to 240 °C (10 min) with the heating rate of 5 °C/min. MS was operated at 150 °C in EI mode (70 eV), scanning from m/z 15 to 350. To obtain reliable results, all experiments were repeated 3 times or more.
3. RESULTS AND DISCUSSION 3.1. Changes of the HCN Yield from Co-pyrolysis of Glycine and Glucose/Fructose at Different Heating Temperatures. A total of 0.5 mmol of glycine and equimolar binary mixtures of glycine and glucose/fructose were pyrolyzed at the set temperature from 400 to 900 °C. The yield of HCN was increased with the heating temperature in the presence or absence of glucose and fructose, especially from 400 to 800 °C, as shown in Figure 1. Johnson et al.7 and Li et al.12 have
Figure 1. Yields of HCN from glycine and equimolar binary mixtures of glycine with glucose/fructose at different heating temperatures.
suggested that HCN is formed at high temperatures, which is in accordance with the present results. On the other hand, the equimolar binary mixtures of glycine and glucose/fructose produced similar amounts of HCN at all of the heating temperatures, which decreased sharply in comparison to that from glycine at the corresponding temperature. For glycine alone, the yield of HCN increased strikingly from 700 to 800 °C, while it increased more slowly in the presence of glucose/ fructose. On the basis of the above results, the following experiments were carried out at 800 °C. 3.2. Changes of the HCN Yield from Co-pyrolysis of Different Molar Ratios of Glycine to Glucose/Fructose. A total of 0.5 mmol of glycine and the mixtures of glycine and glucose/fructose with different molar ratios were pyrolyzed at 800 °C, and the yields of HCN were shown in Figure 2. According to Figure 2, the yield of HCN decreased after glucose/fructose was added. When the glycine/glucose (fructose) ratios changed from 1:0 to 1:1, the yield of HCN
Figure 2. Yields of HCN from co-pyrolysis of different molar ratios of glycine to glucose/fructose. 4724
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gradually decreased with the increase of the glucose/fructose content. However, when the content of glucose/fructose was greater than glycine, the yield of HCN was slightly increased. In comparison to the two model mixtures, there was no significant difference between both mixtures by statistical analysis (p > 0.05). 3.3. Analysis of the Relationship between HCN and DKP from Co-pyrolysis of Different Molar Ratios of Glycine to Glucose/Fructose. Previous studies on the pyrolysis mechanisms of amino acids have indicated that DKP is a critical intermediate to generate HCN.7,9,12 Dehydration is considered to be a primary decomposition mode of α-amino acids, involving a double-dehydration reaction yielding DKP. DKP can cleave through either pathway A or B to form HCN, as shown in eq 1. Therefore, the yields of DKP from co-pyrolysis of different molar ratios of glycine to glucose/fructose at low temperatures were analyzed (Figure 3), and the relationship between the amounts of HCN and DKP was investigated, as shown in Figure 4.
Figure 4. Correlation between the yield of HCN and DKP: (a) DKP at 400 °C in the glycine/glucose system, (b) DKP at 600 °C in the glycine/glucose system, (c) DKP at 400 °C in the glycine/fructose system, and (d) DKP at 600 °C in the glycine/fructose system.
Indeed, when the contents of reducing sugars were less than glycine in glycine/glucose and glycine/fructose model systems, the yields of HCN significantly decreased. The formation of HCN from glycine/glucose and glycine/fructose model systems was highly correlated with DKP when the molar ratios of glycine to glucose/fructose were more than 1. When the molar ratios changed from 1:0 to 1:1, the coefficients of correlation between the yield of HCN from the glycine/glucose model system and the yield of DKP at 400 and 600 °C were 0.9677 and 0.9382, respectively. Correspondingly, the coefficients of correlation in the glycine/fructose model system were 0.9391 and 0.9164, demonstrating that DKP plays a role in HCN formation and the decrease of the HCN yield from co-pyrolysis of glycine and reducing sugar is related to the reduction of DKP when the molar ratios of glycine to glucose/fructose were more than 1. However, when the molar ratios of glycine to glucose/ fructose decreased to 1:2, the DKP yield still decreased, while the HCN yield increased. This is presumably due to the formation of HCN mainly through other pathways rather than DKP; that is, DKP did not play a great role on the formation of HCN when the glycine/sugar ratio was 1:2. 3.4. Analysis of the Relationship between HCN and Other N-Heterocyclic Compounds from Co-pyrolysis of Different Molar Ratios of Glycine and Glucose/Fructose. The reactions between amino acids and reducing sugars played an important role on food processing and attracted widespread attention.16−25 The previous studies mainly focused on browning reaction conditions20,21 and the formation mechanisms of specific products.22−25 In the present study, we are currently investigating the nitrogen distribution during pyrolysis of glycine and glucose/fructose mixtures to explain
Figure 3. Yields of DKP from co-pyrolysis of different molar ratios of glycine to glucose/fructose at low temperatures: (a) glycine/glucose model system and (b) glycine/fructose model system.
As shown in Figure 3, the yield of DKP decreased with the increasing amount of reducing sugars in both models. This could be due to the addition of sugars diluting the glycine sample and restraining the intermolecular polymerization to form DKP; in addition, the reaction of glycine with reducing sugars will form other products. 4725
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glucose. In comparison to the glycine/glucose model system, most of the products showed similar changes from co-pyrolysis of glycine and fructose (Figure 6), while some differences also
the role of N-heterocyclic compounds on HCN formation. The pyrolysis products were quantified by averaging the selected ion output relative to the internal standard. Although the exact yield of products could not be determined, it is able to have a primary estimate of the yield changes at different molar ratios of glycine to glucose/fructose. Glycine formed mainly nitriles and amides at 800 °C, whereas when co-pyrolyzed with glucose/fructose, a lot of nitrogenous compounds were generated, which included substituted pyrroles, pyridines, and imidazoles. Furthermore, there were lots of products not observed from glycine alone, such as a number of substituted pyrazines and pyrazoles. Most of the heterocyclic compounds have been reported in Maillard model systems, such as pyrroles, pyridines, imidazoles, and pyrazines, formed through α-dicarbonyl and α-hydroxycarbonyl intermediates. However, β-dicarbonyl moieties were necessary to form a N−N bond in pyrazole formation.25 The changes of the yield of pyrolysis products from the glycine/glucose model system with different molar ratios were shown in Figure 5. When the glycine/glucose ratios changed
Figure 6. Changes of the yield of pyrolysis products from the glycine/ fructose model system with different molar ratios.
existed; that is, the yields of imidazoles and pyrazines were increased monotonically with the content of fructose. All of the above results exhibited that the pyrolytic ways of glycine were greatly changed by glucose/fructose. The total yields of N-heterocyclic compounds from pyrolysis of the two model systems at different molar ratios were shown in Figure 7. The two model systems exhibited a similar change trend. The total yield of N-heterocyclic compounds gradually increased with the content of sugars when the glycine/sugar ratios changed from 1:0 to 1:1, while they decreased when the glycine/sugar ratio was 1:2. This is contrary to the change of the HCN yield. It is clear that the formation of HCN from
Figure 5. Changes of the yield of pyrolysis products from the glycine/ glucose model system with different molar ratios.
from 1:0 to 1:1, the yields of many components, such as pyrroles, pyridines, imidazoles, pyrazines, and pyrazoles, were increased monotonically with the content of glucose, while the yields of these products were strikingly decreased when the glycine/glucose ratio was 1:2. This is probably due to the fact that 0.5 mmol of glycine interacted with equimolar glucose to form a great deal of N-heterocyclic compounds, and the Nheterocyclic compounds could further react with the rest of glucose or the pyrolyzate of glucose. Another interesting effect of glucose on the glycine pyrolysis was that the yields of pyrolysis products, which do not contain nitrogen, such as ketones, furans, and acids, increased with the content of
Figure 7. Total yield of N-heterocyclic compounds from co-pyrolysis of glycine and glucose/fructose. 4726
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more important with the content of reducing sugars increasing. When the glycine/sugar ratios changed from 1:0 to 1:1, the yield of HCN decreased with the reduction of the DKP yield, although the proportion of HCN formed through the participation of glucose/fructose increased; therefore, DKP played a leading role on the HCN formation. However, when the glycine/sugar ratio was 1:2, most of the proportion of HCN was from other N-heterocyclic compound cleavage and the HCN yield was not reduced as the DKP yield decreased; therefore, the N-heterocyclic compounds played a major role on the HCN formation.
glycine/glucose and glycine/fructose was highly correlated with the total yield of N-heterocyclic compounds (Figure 8). The
4. CONCLUSION The current study on the correlation of HCN formation with DKP and other N-heterocyclic compounds from co-pyrolysis of glycine and glucose/fructose provides several interesting findings. The presence of glucose/fructose reduced the yield of HCN from pyrolysis of glycine and influenced the nitrogen distribution significantly. Of the thermal reactions, glycine might be more likely to react with reducing sugars to form a large number of N-heterocyclic compounds rather than polymerize to form DKP. The decrease of the HCN yield from co-pyrolysis of glycine and reducing sugars is related to the reduction of DKP and the increase of N-heterocyclic compounds. To the best of our knowledge, this is the first report to elucidate how reducing sugars influence the formation of HCN from pyrolysis of glycine by analyzing the correlation of HCN formation with DKP and other N-heterocyclic compounds from co-pyrolysis of glycine and reducing sugars.
Figure 8. Correlation between the yield of HCN and N-heterocyclic compounds: (a) glycine/glucose model system and (b) glycine/ fructose model system.
coefficients of correlation between the yield of HCN and Nheterocyclic compounds from glycine/glucose and glycine/ fructose were 0.8594 and 0.9209, respectively, demonstrating that the reduction of the HCN yield in the presence of glycine/ fructose is related to the increase of N-heterocyclic compounds. 3.5. Analysis of the Participation of Glucose/Fructose in HCN Formation. To gain additional insight into the formation mechanism of HCN, the isotopic-labeled 13C6glucose and -fructose were used and the proportion of produced isotopic-labeled HCN at different molar ratios was analyzed (Figure 9). As Figure 9 shows, the proportion of 13C
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*Telephone/Fax: +86-371-67672103. E-mail: ztridicp@126. com. Notes
The authors declare no competing financial interest.
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Figure 9. Proportion of 13C isotope incorporation in HCN formation from co-pyrolysis of glycine and isotopic-labeled 13C6-glucose/fructose.
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