CN111505167B - High performance liquid chromatography method for simultaneously determining cellulose, hemicellulose and lignin in tobacco and tobacco products - Google Patents
High performance liquid chromatography method for simultaneously determining cellulose, hemicellulose and lignin in tobacco and tobacco products Download PDFInfo
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Abstract
A high performance liquid chromatography method for simultaneously determining cellulose, hemicellulose and lignin in tobacco and tobacco products comprises the following steps: s1, crushing and uniformly mixing tobacco and tobacco products, sieving, then adding the crushed and mixed tobacco and tobacco products into a G3 sand core hopper for pickling and filtering to obtain a first product; s2, washing and drying the first product, hydrolyzing with sulfuric acid solution, and filtering to obtain supernatant and filter residues; s3, washing filter residues, drying and weighing to obtain a first residue with the mass of m1; s4, weighing the first residue after ashing treatment, wherein the mass of the obtained second residue is m2; analyzing the content of glucose, xylose, arabinose, galactose and mannose in the supernatant by high performance liquid chromatography to obtain cellulose and hemicellulose content; the lignin content is obtained according to (m 1-m 2). The method has the advantages of simple pretreatment, accurate result, short analysis time, capability of simultaneously detecting the contents of cellulose, hemicellulose and lignin in the tobacco and tobacco products, batch, rapidness, accuracy and the like.
Description
Technical Field
The invention belongs to the technical field of tobacco and chemistry, and particularly relates to a high performance liquid chromatography method for simultaneously determining cellulose, hemicellulose and lignin in tobacco and tobacco products.
Background
The cellulose is a linear polysaccharide formed by connecting 1000-10000D-glucopyranosyl groups through beta-1, 4 glycosidic bonds, the basic unit is cellobiosyl groups, and chains are combined together along a long axis to form a microfiber bundle, so that the cellulose has high stability and chemical degradation resistance. Hemicellulose components are complex and include many high molecular sugars such as pentose and hexose, and hemicellulose is insoluble in water, but unlike cellulose, hemicellulose has the property of being soluble in dilute alkali and easily hydrolyzed by acid. Lignin is not sugar, is mainly a phenolic three-dimensional reticular high polymer formed by polymerizing p-coumarin alcohol, coniferyl alcohol and sinapyl alcohol 3-type phenylpropane monomers, is mainly positioned between cellulose fibers, and plays roles in resisting compression and improving mechanical strength of tissues.
Cellulose, hemicellulose and lignin are the major components of the tobacco cell wall, and account for more than about 20% of the total dry matter in flue-cured tobacco leaves, and the content in tobacco stems exceeds 40%. The substances have different effects on the physical properties of the tobacco leaves, the processing properties of the tobacco leaves are determined to a certain extent, cellulose is a main structural component of the tobacco leaves, the integrity of the tobacco leaves is determined, the effects on adjusting the combustibility of the tobacco leaves and tobacco products are better, the firepower of the tobacco leaves can be increased, but when the cellulose content is high, the tissue of the tobacco leaves is rough and is easy to break. On the other hand, the substances can generate more lower aldehydes during thermal cracking, and can generate irritating cough during burning and sucking, especially in low-grade tobacco leaves, the cellulose, hemicellulose and lignin are high in content, the smoke has strong irritation, choking, astringent mouth and heavy dry and burnt gas and woody gas, and the taste, coordination and safety of the tobacco are directly influenced, so that the smoke is a negative factor influencing the internal quality of the tobacco.
Therefore, the method has important practical significance in accurately measuring the contents of cellulose, hemicellulose and lignin in the tobacco from the viewpoints of the sensory quality of the cigarettes, the safety evaluation of tobacco products and the like. Since the nineties of the last century, domestic researchers have established methods for measuring these components by weight.
Zhang Huailing and the like are prepared by extracting a sample with benzene-alcohol rope for 48 hours, extracting with hot water for 3-4 times, separating substances such as lipid, sugar, starch, protein, pectin and the like, drying, weighing, replacing the solvent for multiple times in the experimental process, and carrying out suction filtration and cleaning for multiple times, so that the experimental operation is complicated and the experimental period is long; guo Xiaoyi et al measured hemicellulose, cellulose, acid washed lignin (ADL) and crude cellulose content in 31 tobacco samples using a cellulose meter. In the method, the sample does not need to undergo degreasing process, but washing and filtering are important pretreatment processes, the washing steps are more, and the types of reagents used in the test process are still more than 10; the application number CN201410520615.7 uses acetic acid and nitric acid mixed solution to destroy the outer coating of cellulose, then uses sulfuric acid and potassium dichromate to hydrolyze the cellulose under a heating state, uses sodium thiosulfate to titrate excessive potassium dichromate, uses potassium iodide and starch solution as indicators to calculate the amount of the potassium dichromate, and converts the amount into the content of cellulose.
Disclosure of Invention
Aiming at the problems, the invention aims to provide the high-performance liquid chromatography method for simultaneously measuring the cellulose, hemicellulose and lignin in the tobacco and the tobacco products, which has the advantages of simple pretreatment, accurate result, short analysis time, capability of simultaneously detecting the content of the cellulose, hemicellulose and lignin in the tobacco and the tobacco products, batch, rapidness, accuracy and the like.
In order to achieve the above object, the technical scheme of the present invention is as follows:
a high performance liquid chromatography method for simultaneously determining cellulose, hemicellulose and lignin in tobacco and tobacco products, comprising the following steps:
(1) Crushing and uniformly mixing tobacco and tobacco products, sieving, adding the crushed and mixed tobacco and tobacco products into a G3 sand core funnel, and filtering and pickling to obtain a first product;
(2) Washing the first product to be neutral, drying, hydrolyzing with a sulfuric acid solution with gradient concentration, and filtering to obtain a supernatant and filter residues;
(3) Washing filter residues to be neutral, and drying and weighing to obtain a first residue with the mass of m1;
(4) Weighing the first residue after ashing treatment, wherein the mass of the obtained second residue is m2;
analyzing the content of glucose, xylose, arabinose, galactose and mannose in the supernatant by adopting high performance liquid chromatography to further obtain the content of cellulose and hemicellulose;
the lignin content is obtained from the poor quality (m 1-m 2) of the first and second residues.
As a further preferred aspect of the present invention, the pickling process comprises the steps of:
a. adding an acetic acid aqueous solution into the G3 sand core funnel for pickling to obtain a pickled product;
b. washing the acid-washed product to be neutral, and adding an ethanol-saturated NaCl solution for washing and removing impurities to obtain a first product.
As a further preferred aspect of the invention, the sulfuric acid hydrolysis process comprises the steps of:
s1, adding 70-75% sulfuric acid solution to carry out ultrasonic hydrolysis;
s2, adding distilled water to dilute the sulfuric acid solution to 14-16% (w/w), then continuing to hydrolyze, and filtering to obtain supernatant and filter residues.
As a further preferable mode of the invention, the supernatant is subjected to high performance liquid chromatography detection after centrifugation, dilution and fine filtration, and the dilution factor is 100-1000 times.
As a further preferred aspect of the present invention, the fine filtration membrane comprises: one of 0.22 μm and 0.45 μm.
As a further preferred aspect of the present invention, the ultrasonic hydrolysis conditions include: the time is 0.8-1.2 h, and the temperature is 28-32 ℃.
As a further preferable mode of the invention, the drying temperature in the step (2) is 100-120 ℃, and the drying temperature in the step (3) is 100-110 ℃.
In a further preferred aspect of the present invention, the sieving in the step (1) is performed by passing through a 40 to 60 mesh sieve.
As a further preferred aspect of the present invention, the calculation of the content of cellulose, hemicellulose, lignin comprises:
cellulose:
hemicellulose:
lignin:
（C glucose 、C Arabinose (Arabic sugar) 、C Xylose 、C Galactose 、C Mannose The contents of the components detected by high performance liquid chromatography are 0.9 and 0.88 as conversion coefficients, and V is the volume of the solution during acidolysis of sulfuric acid).
As a further preferred aspect of the present invention, the conditions for high performance liquid chromatography include:
mobile phase: chromatographic grade deionized water is filtered by a 0.2 mu m filter membrane; flow rate: 0.8mL/min; sample injection amount: 10 μl.
In summary, the invention has the following beneficial effects:
1. according to the method, the acid hydrolysis sample is adopted and then is directly measured by adopting the high performance liquid chromatography, so that the operation is simple and convenient, the consistency of the result is good, the measurement result is accurate, less solvent is used in the experimental process, the experimenter can be effectively prevented from being exposed to the environment of the organic solvent for a long time, and the method is environment-friendly.
2. The instrument and the equipment used by the invention are laboratory conventional analysis equipment, and the method is easy to popularize and has lower detection cost.
3. The method has the advantages of higher labeling recovery rate, better repeatability of the measurement result, shorter time consumption of the whole measurement process, high sensitivity, complete satisfaction of analysis requirements on recovery rate and repeatability, capability of simultaneously obtaining the contents of three components by one measurement, and suitability for simultaneous, rapid and batch measurement of cellulose, hemicellulose and lignin in tobacco and tobacco products.
Drawings
FIG. 1 is a high performance liquid chromatogram of monosaccharide components after hydrolysis of cellulose and hemicellulose in a tobacco sample according to the invention.
Detailed Description
1. Material preparation
1. Instrument and reagent
(1) Instrument: high performance liquid chromatograph (Agilent 1200), KQ-500DE ultrasonic generator (Kunshan ultrasonic instruments Co., ltd.), electronic balance (METTER TOLEDOO Co., ltd., sensing 0.1. 0.1 mg), DHG-9240A oven (Shanghai-Hengsu scientific instruments Co., ltd.), muffle (Shanghai Soxhlet instruments Co., ltd.), sigma 3-18K centrifuge (Germany Sigma Co.), SHZ-D (III) circulating water vacuum pump (Kunzea Yingshi instruments Co., ltd.), KQB-30 glass instrument air dryer (Shanghai Yishen instruments Co., ltd.), G3 sand core funnel; 0.45 mu m water phase filter membrane; pH test paper and glass instrument are commonly used in laboratories.
(2) Reagent: anhydrous glucose (AR, national pharmaceutical group chemical company); xylose (national pharmaceutical group chemical reagent limited, BR); arabinose (AR, national pharmaceutical group chemical company); galactose (BR, south kyo chemical agents inc); mannose (AR, shanghai aladine Biochemical technologies Co., ltd.); glacial acetic acid (AR, national pharmaceutical systems chemical); 98% sulfuric acid (AR, south Beijing chemical Co., ltd.).
2. Solution preparation
(1) 5% glacial acetic acid solution: 48mL of glacial acetic acid was measured, and the volume was diluted to 1000mL with distilled water.
（2）72%H 2 SO 4 Solution: 665mL98% H 2 SO 4 Add to 300mL distilled water. After cooling to room temperature, transfer to 1000mL volumetric flask, add distilled water to volume to 1000mL.
(3) Glucose, xylose, arabinose, galactose and mannose mixed standard stock solution: accurately weighing 0.1g (accurate to 0.0001 g) of glucose, xylose, arabinose, galactose and mannose samples into a 100mL volumetric flask, dissolving with distilled water and fixing the volume to 100mL.
(4) Glucose, xylose, arabinose, galactose and mannose mixed standard solution: 0.1mL, 0.2mL, 0.5mL, 1mL and 2mL are sequentially removed from a glucose, xylose, arabinose, galactose and mannose mixed standard stock solution, respectively put into a 100mL volumetric flask, and distilled water is used for diluting and fixing the volume to 100mL, so as to obtain 1-5-grade mixed standard solutions with the concentrations of 1,2,5, 10 and 20mg/L respectively.
2. High performance liquid chromatography conditions
Mobile phase: chromatographic grade deionized water is filtered by a 0.2 mu m filter membrane;
flow rate: 0.8mL/min;
column temperature: 88 ℃;
detector temperature: 88 ℃;
a detector: a differential refractive detector;
sample injection amount: 10 μl;
run time: 30min.
3. Calculation formula of cellulose, hemicellulose and lignin
Cellulose:
hemicellulose:
lignin:
(the contents of glucose, arabinose, xylose, galactose and mannose are detected by high performance liquid chromatography, the conversion coefficients are 0.9 and 0.88, V is the volume of the solution during sulfuric acid acidolysis, m1 is the mass of the first residue after drying, and m2 is the mass of the second residue after ashing treatment).
4. Method recovery measurement
Preparation examples 1 to 4
The compositions of examples 1 to 4 are shown in Table 1 below:
TABLE 1 recovery experiments amounts of ingredients
The processing steps are as follows:
(1) Respectively weighing four groups of tobacco powder samples which are sieved by a 40-mesh sieve, placing the tobacco powder samples in four G3 sand core funnels, and respectively and sequentially adding cellulose, hemicellulose and lignin into each group of tobacco powder samples to be uniformly mixed;
(2) Respectively adding 30mL of 5% acetic acid solution into the four groups of samples, ultrasonically extracting for 30min, removing filtrate, and washing filter residues with distilled water until the pH value is neutral;
(3) Continuously adding 25mL of 80% ethanol-saturated NaCl solution, performing ultrasonic treatment at 85 ℃ for 30min, removing filtrate, and washing filter residues with distilled water until the pH value is neutral;
(4) The funnel and filter residues are put into a baking oven to be dried at 120 ℃, and 72 percent of H with different capacities is respectively added 2 SO 4 Carrying out ultrasonic hydrolysis on the solution at 30 ℃ for 1h;
(5) Respectively adding distilled water with different capacities to dilute the sulfuric acid solution to 15.4% (w/w), uniformly mixing, placing in a water bath at 100 ℃ for hydrolysis for 1.5h, cooling the solution to room temperature, taking a supernatant filtering membrane, and detecting the contents of glucose, xylose, arabinose, galactose and mannose in the supernatant by adopting a high performance liquid chromatograph.
(6) The four groups of residual solutions obtained are respectively filtered, filter residues are respectively washed to be neutral by distilled water, the residues are respectively dried in an oven at 105 ℃ for 2 hours, then weighed, the mass is marked as m1 (1, 2,3, 4), then the obtained product is placed in a muffle furnace for ashing treatment for 3 hours, and the obtained product is completely cooled and weighed again, and the mass is marked as m2 (1, 2,3, 4).
The cellulose, hemicellulose and lignin contents of examples 1-4 were calculated according to the formula and the recovery rates of the methods were calculated, and the results are shown in tables 2-4:
TABLE 2 recovery of cellulose content detection methods
TABLE 3 recovery rate of hemicellulose content detection method
TABLE 4 recovery of lignin content detection method
The data in tables 2-4 show that the detection methods for cellulose, hemicellulose and lignin in tobacco and tobacco products established by the invention have recovery rates of more than 95%, and the accuracy of the method is higher.
5. Method reproducibility assay
Preparation examples 5-10, wherein the addition amounts of the components of the samples are the same, and the samples are respectively placed in six G3 sand core funnels, and the processing steps are as follows:
(1) Respectively adding 30mL of 5% acetic acid solution into six groups of samples, ultrasonically extracting for 30min, removing filtrate, and washing filter residues with distilled water until the pH value is neutral;
(3) Continuously adding 25mL of 80% ethanol-saturated NaCl solution, performing ultrasonic treatment at 85 ℃ for 30min, removing filtrate, and washing filter residues with distilled water until the pH value is neutral;
(4) The funnel and filter residues are put into a baking oven to be dried at 120 ℃, and 5ml of 72% H is added respectively 2 SO 4 Carrying out ultrasonic hydrolysis on the solution at 30 ℃ for 1h;
(5) Adding 29.6. 29.6mL distilled water to dilute the sulfuric acid solution to 15.4% (w/w), mixing uniformly, placing in a water bath at 100deg.C for hydrolysis for 1.5 hr, cooling the solution to room temperature, collecting supernatant, filtering, and detecting glucose, xylose, arabinose, galactose and mannose content by high performance liquid chromatograph.
(6) The six groups of residual solutions obtained were respectively filtered, the filter residues were respectively washed with distilled water to neutrality, the residues were respectively dried in an oven at 105 ℃ for 2 hours, then weighed, the mass was designated as m1 (5, 6,7,8,9, 10), then placed in a muffle furnace for ashing treatment for 3 hours, completely cooled and weighed again, and the mass was designated as m2 (5, 6,7,8,9, 10).
The relative standard deviation of the methods of calculating the cellulose, hemicellulose and lignin contents of examples 5-10 respectively according to the formula is shown in Table 5:
TABLE 5 repeatability of the methods for detecting the content of cellulose, hemicellulose and lignin
6. Detection of different tobacco leaf positions
Preparation examples 11 to 13, the processing steps are as follows:
(1) Sample impurity removal: respectively weighing 0.5G of 40-mesh upper (example 11), middle (example 12) and lower (example 13) tobacco powder samples, placing in a G3 sand core funnel, adding 30mL of 5% acetic acid solution, ultrasonically extracting for 30min, removing filtrate, and washing filter residues with distilled water until the pH value is neutral; 25mL of 80% ethanol-saturated NaCl solution was added to the funnel containing the filter residue, sonicated at 85℃for 30min, the filtrate was removed, and the filter residue was washed with distilled water to neutral pH.
(2) Determination of cellulose and hemicellulose: after the funnel and filter residue were dried in an oven at 120deg.C, 5mL of 72% H was added to the funnel 2 SO 4 Carrying out ultrasonic hydrolysis on the solution at 30 ℃ for 1h; adding 29.6mL of distilled water into the funnel, diluting the sulfuric acid solution to 15.4% (w/w), uniformly mixing, placing into a water bath kettle at 100 ℃ for hydrolysis for 1.5h, cooling the solution to room temperature, taking a supernatant, filtering, and detecting the contents of glucose, xylose, arabinose, galactose and mannose by adopting a high performance liquid chromatograph.
(3) Measurement of lignin: after the remaining solution was filtered, the residue was washed with distilled water to neutrality, the residue was dried in an oven at 105 ℃ for 2 hours, then weighed, the mass was designated as m1, and then placed in a muffle furnace for ashing treatment for 3 hours, completely cooled, and weighed again, the mass was designated as m2.
The cellulose, hemicellulose and lignin contents were calculated separately according to the formula and the repeatability of the process was calculated, the results are shown in table 6:
TABLE 6 percentage of cellulose, hemicellulose and lignin in tobacco leaves at different locations
Note that: examples 11 to 13 correspond to the tobacco sample positions A-B2F, B-C3F, C-X2F, respectively.
In summary, the data in tables 2-6 show that the method adopted by the invention has higher labeling recovery rate, better repeatability of the measurement result, high sensitivity, and the recovery rate and the repeatability completely meet the analysis requirement; the contents of three components can be obtained simultaneously, and the method is suitable for simultaneous, rapid and batch measurement of cellulose, hemicellulose and lignin in tobacco and tobacco products.
The specific embodiments described herein are offered by way of example only to illustrate the spirit of the invention. Those skilled in the art may make various modifications or additions to the described embodiments or substitutions thereof without departing from the spirit of the invention or exceeding the scope of the invention as defined in the accompanying claims.
Claims (5)
1. A high performance liquid chromatography method for simultaneously measuring cellulose, hemicellulose and lignin in tobacco and tobacco products, which is characterized by comprising the following steps:
(1) Respectively weighing 0.5G of 40-mesh upper, middle and lower tobacco powder samples, placing in a G3 funnel, adding 30mL of 5% acetic acid solution, ultrasonically extracting for 30min, removing filtrate, washing the filter residue with distilled water until the pH value is neutral, adding 25mL of 80% ethanol-saturated NaCl solution into the funnel containing the filter residue, ultrasonically extracting for 30min at 85 ℃, removing filtrate, and washing the filter residue with distilled water until the pH value is neutral;
(2) The funnel and filter residues are put into an oven for drying at 120 ℃, and then are hydrolyzed by sulfuric acid solution with gradient concentration: s1 to the funnel was added 5mL72% H 2 SO 4 Carrying out ultrasonic hydrolysis on the solution at 30 ℃ for 1h; s2, adding 29.6mL of distilled water into a funnel, diluting the sulfuric acid solution to 15.4%, uniformly mixing and then placing inHydrolyzing in a water bath at 100 ℃, and filtering to obtain supernatant and filter residues;
(3) Washing filter residues with distilled water to be neutral, and then drying and weighing to obtain first residue with the mass of m1;
(4) Weighing the first residue after ashing treatment, wherein the mass of the obtained second residue is m2;
analyzing the content of glucose, xylose, arabinose, galactose and mannose in the supernatant by adopting high performance liquid chromatography to further obtain the content of cellulose and hemicellulose;
the lignin content was obtained from the above-mentioned mass differences of m1 and m2.
2. The method for simultaneously measuring cellulose, hemicellulose and lignin in tobacco and tobacco products according to claim 1, wherein the supernatant is subjected to high performance liquid chromatography detection after centrifugation, dilution and fine filtration, and the dilution factor is 100-1000 times.
3. The method according to claim 2, wherein the fine filtration membrane comprises: one of 0.22 μm and 0.45 μm.
4. The method for simultaneously determining cellulose, hemicellulose and lignin in tobacco and tobacco products according to claim 1, wherein the calculating the content of cellulose, hemicellulose and lignin comprises:
cellulose:
hemicellulose:
；
lignin:
C glucose 、C Arabinose (Arabic sugar) 、C Xylose 、C Galactose 、C Mannose The content detected by high performance liquid chromatography is 0.9 and 0.88 as conversion coefficients, and V is the volume of the solution during acidolysis of sulfuric acid.
5. The method according to claim 2, wherein the conditions of the high performance liquid chromatography for simultaneous determination of cellulose, hemicellulose and lignin in tobacco and tobacco products comprise:
mobile phase: chromatographic grade deionized water is filtered by a 0.2 mu m filter membrane; flow rate: 0.8mL/min; sample injection amount: 10 μl.
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