The Liquefaction Mechanism Research of Straw Fiber Polyhydric Alcohol
植物纤维液化的目的是最大限度地将固态天然高分子化合物转化为液态的小分子有机物质加以利用。对植物纤维多羟基醇液化机理的研究主要有两种方法，一种是以结构简单的模型化合物为研究对象，研究其反应产物结构及变化过程，从而推断实际的天然高分子液化反应机理，如Yamada T [1~3]以5-羟甲基呋喃(HMF)等为模型化合物研究纤维素的液化产物及反应机理。这种方法使反应物和产物结构相对简单，易于检出，但毕竟不能等同于植物纤维整体液化时的实际过程，到目前为止，还未见上述机理在植物纤维整体液化中的验证报道。并且木质素、半纤维素的多羟基醇液化机理目前尚不清楚。另一种是直接以全植物纤维为研究对象，如梁凌云利用FTIR通过对纤维素、木质素以及玉米秸秆的液化混合产物及残渣分析研究液化机理，Zhang T 通过红外分析、凝胶渗透色谱和元素分析，对蔗渣整体液化的产物进行定性分析。这种方法研究结果更接近于实际液化过程，但由于液化产物成分十分复杂，其分离和结构鉴定十分困难，因此大多只能定性地描述液化过程的相对变化，而未能对降解产物进行分离和定量分析，具有较大的局限性。
The purpose of plant fibers liquefaction is to furthest transform solid natural polymer compound into liquid micromolecule organics to use. There are two research methods to probe into the liquefaction mechanism of straw fiber polyhydric alcohol, one method is to take simple model compound as research object, and then probe into the structure and change process of reaction product, and then infer the liquefaction mechanism of actual natural polymer, such as Yamada T [1~3] took 5 - (HMF) and etc, as model compounds to research the liquefied products and reaction mechanism of cellulose. This method makes the reactants and products structure relatively simple and easy to detect, however, it is not equal to the actual process of the overall liquefaction of plant fiber after all, so far, no verification report of such mechanism has been made on the overall liquefaction of plant fiber. At present, the liquefaction mechanism of lignin and hemi-cellulosic polyhydric alcohol is not clear either. The other method is to directly take whole plant fiber as object, for instance, Liang Lingyun  once used FTIR to research the liquefaction mechanism through the analysis of the cellulose, lignin, and the liquefied mixture and residue of corn stalks, Zhang T  conducted qualitative analysis of overall liquefaction products of bagasse through infrared analysis, gel permeation chromatography and elemental analysis. The results of this method is closer to the actual liquefaction process, nevertheless, the composition of liquefaction product is very complex, and it is also difficult to separate and identify structure, hence, we can only qualitatively describe the relative change of liquefaction process, but we have great limitations to separate and make quantitatively analyze the degradation products.
To this end, this paper adopted straw fiber as raw material, and reaction product would be step by step extracted from the liquefied mixed products of wheat-straw polyhydroxy-alcohol through organic solvent extraction and column chromatography methods, and then I conducted purification and structural identification to key products. Based on this, I could deduce the liquefaction reaction mechanism of cellulose and lignin in wheat-straw.
1 Test Portion
1.1 Materials Reagents and Equipments
N-butyl alcohol, chloroform and methanol, which were all industrial raw materials, so the reduced pressure distillation should be conducted through rotary evaporator before their use; other reagents were analytically pure;
Rotary Evaporator: RE5002 type, Gongyi YuHua Instrument Co., Ltd;
Automatic Potentiometric Titrator: Mine magnetism ZD-2-type, Shanghai Precision & Scientific Instrument Co., Ltd.;
Equinox55 Fourier Transform Infrared Spectroscopy (FTIR) analyzer, produced by Germany Bruker Company, was adopted in infrared analysis; solid samples were tested by KBr pellet method while ATR method for liquid samples;
BRUKER DRX500MHz superconducting NMR was adopted in nuclear magnetic resonance (1H-NMR, 13C-NMR, DEPT); the solvent was methanol; and TMS internal standard method was used within the test.
质谱分析采用API QSTAR PULSAR质谱仪，分别采用FAB-MS 和ESI-MS法测试，溶剂为甲醇。喷雾电压4000V；雾化气0.2Mpa；质量扫描范围：0～12000Da。
API QSTAR PULSAR mass spectrometer was used in mass spectrum analysis; FAB-MS and ESI-MS methods were respectively used in testing, with methanol as the solvent. Spray voltage: 4000V; atomization gas: 0.2Mpa; quality scanning range: 0 ~ 12000Da.
1.2 Experimental methods and procedures
1.2.1 WS liquefaction
Take the wheat-straw with 2 to 12 mesh fiber of about 10% moisture content, liquid-solid ratio 5:1, material confect PEG400: EG: concentrated H2SO4=80∶20∶3, liquefaction temperature 160 ℃, liquefaction time 60min. After reaction, take three bottles into cold bath to terminate reaction and get mixed liquefaction products (LWS).After liquefaction, conduct separation and composition analysis of mixed liquefaction products.
1.2.2 LWS Separation
Firstly, remove the liquefaction residue: use 20 times the quality of 80/20 (v/v) dioxane aqueous solution LWS to dissolve liquefied products, and filter them after full stir and solution. Separate the residue, and use rotary evaporator to conduct reduced pressure distillation to filtrate to constant weight in 80 ℃, then recycle dioxane and get mixed liquid A.
Further separate mixed liquid A. Select n-butyl alcohol as extractant, and extract liquefaction organic product from A and get nigger-brown liquid, and then conduct further separation by column chromatography, and get compound 1 and compound 2 through multi-stage separation and purification. Finally, respectively carry through NMR and mass spectrometry to compounds 1 and 2.
2 Results and Discussion
2.1 Structural Analysis of Compound 1
化合物1的核磁共振（13C-NMR，DEPT）谱表明存在5个碳信号，包括位于δC 209.7和174.5的酮羰基和酯羰基，2个CH2位于δC 38.6，δ28.7。这些信号与yamada T用EC液化α-纤维素时得到的产物信号十分相似，表明化合物1含有乙酰丙酸 (levulinic acid)片段（如图1），它的FAB-MS在m/z 115的碎片离子峰也证明含有乙酰丙酸片段。而在δC 60~90多个CH2峰表明化合物所连接的R基团含有多个CH2基团。
The NMR spectra of compound 1 (13C-NMR, DEPT) indicated that there were five carbon signals, including ketone carbonyl and ester carbonyl in δC 209.7 and 174.5, and two CH2 at δC38.6 and δ28.7. These signals extremely resembled the product signals obtained when yamada T  used EC to liquefy α-cellulose, indicating that compound 1 contained the levulinic acid fragment (Figure 1), and its FAB-MS at m / z 115 fragment ion peaks also shown the existing of levulinic acid fragment.While many CH2 peaks in δC60 ~ 90 indicated that the R-perssad connected by compounds contained multiple CH2 perssads.
为进一步确定该化合物中“—R”的结构，分别采用FAB-MS 和EI-MS对化合物1进行质谱分析，EI-MS在m/z 363出现准分子离子峰，其余碎片离子峰值均相差44 (m/z: 363，407，451等)，恰好为1个乙二醇单体（—CH2CH2O—）的相对分子量，表明分子中存在多个乙二醇单元。结合在13C-NMR谱中δ60-70的多个亚甲基(CH2)峰，可推断化合物1为乙酰丙酸及其不同聚合度的乙二醇酯，结构如图2所示。
In order to further determine the “-R" structure in such compound, FAB-MS and EI-MS MS were respectively used to conduct mass spectrum analysis of compound 1, EI-MS appeared quasi-molecular ion peaks at m/z 363, while the peaks discrepancy among remaining fragment ion was 44 (m/z: 363,407,451, etc.), and exactly right, the relative molecular weight of a glycol monomer (-CH2CH2O-) indicated that multiple ethylene glycol units existed in the molecule. Combined with the multiple methylene (CH2) peaks of δ60-70 in 13C-NMR spectra, compound 1 can be inferred as levulinic acid and ethylene glycol esters with different polymerization degree. See its structure in Figure 2.
2.2 Structural Analysis of Compound 2
The NMR spectrum of compound 2 13C-NMR (DEPT) is shown in Figure 3.
它的1H-NMR谱位于δH 7.65 (1H，d，J = 15.5 Hz), 6.39 (1H，d，J = 15.5 Hz)的信号表明分子中含有一个反式双键，而位于δH 7.05 (1H，d，J = 9.8Hz)，6.82 (1H，d，J = 9.8Hz)表明分子中含有一个顺式双键；它的13C-NMR (DEPT)谱表明两个双皱起相应的碳信号位于δC 111.7，146.9和 127.6, 130.4。它的13C-NMR (DEPT)谱位于δC 172.5和δC 110 -140的多个CH峰分别表明分子中含有一个α-β不饱和酯羰基和芳环。1H-NMR谱位于δ7.17 (1H，s)，δ7.07 (1H，d，J = 8.0 Hz)和6.80 (1H，d，J = 8.0Hz)的质子信号峰表明分子中含有1,3,4-三取代苯环；而位于δ131.1 (×2)和116.8(×2) 表明分子中含有一个对羟基取代的苯环。这些数据表明化合物2为含芳环的小分子化合物，由于液化试剂中不含芳香族化合物，因此该化合物只可能是由木质素降解液化而来。
The signal of its 1H-NMR spectrum at δH 7.65 (1H, d, J = 15.5 Hz), 6.39 (1H, d, J = 15.5 Hz) indicated that trans double bond was contained in the molecule, while the signal at δH7.05 (1H, d, J = 9.8Hz), 6.82 (1H, d, J = 9.8Hz) showed that the molecule contained a cis-form double bond; its 13C-NMR (DEPT) spectrum showed the corresponding carbon signal of two pairs of pucker at δC 111.7,146.9 and 127.6, 130.4. Multiple CH peaks of its 13C-NMR (DEPT) spectrum at δC 172.5 and δC 110 -140 respectively showed that one α-β unsaturated ester carbonyl and the aromatic ring were contained in molecule. The proton signal peak of 1H-NMR spectrum at δ7.17 (1H, s), δ7.07 (1H, d, J = 8.0 Hz) and 6.80 (1H, d, J = 8.0Hz) indicated that the molecule contained 1, 3, 4 - tri-substituted benzene ring; while those at δ131.1 (× 2) and 116.8 (× 2) showed that one p-hydroxy substitutional benzene ring were contained in the molecule. These data indicated that compound 2 was micromolecule compound with aromatic rings. Because of no aromatic compound in liquefaction reagent, so such compound could only be degenerated and liquefied from lignin.
另外，化合物2的EI-MS图中m/z为 325，369，413等处出现准分子离子峰，与化合物1相似，各组分的相对分子质量也相差44的整数倍，并且在13C-NMR，DEPT谱图中δ60~90之间存在大量亚甲基峰，说明该化合物也存在重复的乙二醇单元。而FAB-MS在m/z为325 [M-R]+, 207 [C11H11O4]+, 179 [C10H11O3]+等碎片峰表明分子中存在片段A-C，因此，该化合物结构鉴定为如图4 (见表1)。
In addition, the EI-MS of compound 2 appeared quasi-molecular ion peak when m/z in the figure were 325, 369, 413, etc., which was similar to compound 1, and the relative molecular mass of each component is also a difference of an integer multiple of 44, and a large number of methylene peaks existed betweenδ60 ~ 90 in 13C- NMR, DEPT spectra, indicating that the compound also contained repeated ethylene glycol units. While the FAB-MS with the m/z at 325 [MR] +, 207 [C11H11O4] +, 179 [C10H11O3] + and other fragment peaks indicated that fragment A-C existed in the molecule, therefore, the structural identification of such compound was shown in Figure 4 (see table 1 .)
2.3 Liquefaction Reaction Mechanism of Wheat-straw Cellulose
从上面的分析发现，化合物1即乙酰丙酸乙二醇酯与Yamada T用α-纤维素作为模型在聚乙二醇和乙二醇中液化时得到的产物非常吻合，对于该化合物的产生机制，Yamada T认为首先是纤维素与液化试剂发生醇解反应，其β—O—4键断裂并与液化试剂相连接形成葡萄糖苷，然后生成5—羟甲基呋喃（HMF）衍生物，HMF可开环形成乙酰丙酸乙二醇酯，但过多的HMF也可发生缩聚反应生成不溶性残渣。对该反应历程本文简称为纤维素的“醇解—酯化机理”。利用该机理可以很好地解释麦秆液化过程中的一些现象：如羟值随着液化时间的延长一直下，液化过程中液化残渣的量先减少后逐步升高的实验现象。因此，说明麦秆纤维素的液化可能存在醇解—酯化反应。
From the above analysis, we could see that compound 1, levulinic acid glycol ester, was consistent with the product obtained in the experiment that Yamada T used α-cellulose as a model to liquefy polyethylene glycol and ethylene glycol, as for the generation mechanism of this compound, Yamada T  believed that the first reaction was the alcoholysis reaction between cellulose and liquefaction reagents, of which, the β-O-4 bond broke and connected with the liquefaction reagents to form glucoside, and then generated 5 - Hydroxymethyl Furan (HMF) derivatives, HMF could open the ring to form levulinic acid glycol ester, but too much HMF might also occur polycondensation reaction and generate insoluble residue. This reaction course was referred to as cellulose’s “alcohol solution - esterification mechanism” for short in this paper. We can well explain some phenomena  in the process of straw liquefaction with this mechanism: if the hydroxyl value descends along with the liquefaction time, the amount of liquefied residue in liquefaction process will gradually increase and then reduce. Therefore, the liquefaction of wheat-straw cellulose is potential to have alcohol solution - esterification reaction.
However, just alcohol solution - esterification mechanism was hard to explain the phenomenon that the acid value increased and then decreased in reaction process. In addition, in the liquefaction experiment, I could also find that the presence of a bit of water could effectively improve the liquefaction rate of wheat straw fibers, indicating that except for alcohol solution, hydrolysis reaction also existed in reaction process. Literature  showed that the cellulose could occur hydrolysis and generate single sugar (glucose) in high temperature with acid catalysis, and then dehydrated to form 5 – hydroxymethyl furfural, and then further decarboxylate to a platform compounds with high activity - levulinic acid , because of a large number of alcohols in liquefaction reagents, so the generated levulinic acid could easily occur esterification and generate ester with ester with sulfuric acid-catalyzed. Thus, this paper made another hypothesis of cellulose degradation mechanism, shown in Figure 5, and here it was referred as a cellulose’s "Hydrolysis - Esterification" mechanism: in the initial stage of reaction, cellulose hydrolyzed with a small amount of water in reactant under the help of acid catalysis, and then generated a certain amount of levulinic acid and formic acid, therefore, the acid value gradually increased, then these levulinic acid would occur esterification with ethylene glycol and polyethylene glycol to generate acetyl propionate, and this process would also consume hydroxyl and carboxyl perssads, accordingly, the acid value and hydroxyl value would simultaneously decrease. Under the effect of acid, the water with small molecules would be easier to combine with the molecular chain in cellulose than ethylene glycol and polyethylene glycol, resulting in the molecular strand breaking, thus a small amount of water molecules can play acatalysis in fibre liquefaction, so as to improve the liquefaction reaction efficiency.
2.4 Liquefaction Reaction Mechanism of Lignin
As mentioned above, compound 2 was the product of lignin degenerated and liquefied in polyethylene glycol. Since compound 2 contained a benzene ring and a 1, 3, 4-3 substituted benzene ring and one 1, 4 - p-substituted benzene ring. By comparing to the three structure units of lignin, we could find that the corresponding structure was to p-coumaric alcohol structure and mustard alcohol structure. Through the degradation experiment on lignin model compounds, Evtuguin  believed that the cleavage of β-O-4 bond played the dominant role in the lignin model of phenolic perssad. Therefore, we could infer that when straw liquefied, the cleavage of β-O-4 bond would firstly occur in lignin high polymer and produce a large number of small molecule monomers, of which guaiacyl lignin would degenerate into p-coumaric alcohol structure mustard alcohol structure, while the hydroxymethyl phenyl lignin would degenerate into mustard alcohol structure. Compound 2 was the reaction product of these two structural units. The generation of ester bond indicated there were esterification reactions occurring in carboxylic acids and alcohols, namely, it was generated through the esterification reactions between the acid oxidized from mustard alcohol structure alcohol units and p-coumaric alcohol. The acidity of the phenolic hydroxyl corresponding to mustard alcohol was on a bit of strong but less steric hindrance, so it was easy to be combined with glycol or polyethylene glycol to form ethers, accordingly, the liquefaction and degeneration mechanism of lignin in straw was speculated as follows (Figure 6.)
This paper carried on research on the liquefaction mechanism of wheat straw through conducting multistage-separation and structure elucidation on the products of wheat straw fiber polyhydroxy alcohols liquefaction, and then got the following conclusions:
(1) Use extraction and column chromatography methods, and repeated separation and purification to separate compound 1 and compound 2 from the liquefaction mixture.
(2) Through the NMR and MS analysis of compound 1, this paper inferred its structure was the levulinic acid and the ethylene glycol esters with different polymerization degree, and which was degenerated from straw cellulose; through the NMR and MS analysis of compound 2, this paper identified the lignin in straw was degradation products of polyhydric alcohols, and compound 2 contained two molecules of benzene, which was the ester compound containing the structural units of guaiacyl lignin and hydroxyphenyl lignin.
(3) According to the structure of compound 1, this paper verified that cellulose alcohol solution - esterification process existed in the polyhydric alcohol liquefaction of the straw fiber; meanwhile, by combining with the changes of hydroxyl value, acid value and residual rate in liquefaction process, this paper supplemented the degradation mechanism of cellulose and proposed hydrolysis-esterification process in cellulose liquefaction.
(4) According to the structure of compound 2, this paper extrapolated the degradation mechanism of lignin: put forward that lignin firstly degenerated into p-coumaric alcohol, sinapinic alcohol and other structural elements under the action of acid, water and alcohol, followed by oxidation and esterification; meanwhile, phenolic hydroxyl will form ether with liquefaction reagents polyol.