CN114527117A - Method for measuring contents of glucose and xylose in sugar solution and tobacco leaching solution - Google Patents
Method for measuring contents of glucose and xylose in sugar solution and tobacco leaching solution Download PDFInfo
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- CN114527117A CN114527117A CN202210169696.5A CN202210169696A CN114527117A CN 114527117 A CN114527117 A CN 114527117A CN 202210169696 A CN202210169696 A CN 202210169696A CN 114527117 A CN114527117 A CN 114527117A
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- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 title claims abstract description 252
- 239000008103 glucose Substances 0.000 title claims abstract description 142
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 title claims abstract description 138
- 235000000346 sugar Nutrition 0.000 title claims abstract description 133
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 title claims abstract description 126
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 title claims abstract description 126
- 241000208125 Nicotiana Species 0.000 title claims abstract description 85
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 85
- 238000000034 method Methods 0.000 title claims abstract description 63
- 238000002386 leaching Methods 0.000 title claims abstract description 57
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 title claims abstract description 34
- 239000000243 solution Substances 0.000 claims abstract description 147
- 239000012086 standard solution Substances 0.000 claims abstract description 73
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 50
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 38
- 238000011161 development Methods 0.000 claims abstract description 36
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 28
- QCDYQQDYXPDABM-UHFFFAOYSA-N phloroglucinol Chemical compound OC1=CC(O)=CC(O)=C1 QCDYQQDYXPDABM-UHFFFAOYSA-N 0.000 claims abstract description 28
- 229960001553 phloroglucinol Drugs 0.000 claims abstract description 28
- 229960000583 acetic acid Drugs 0.000 claims abstract description 25
- 239000012362 glacial acetic acid Substances 0.000 claims abstract description 25
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 24
- 239000002253 acid Substances 0.000 claims abstract description 17
- 238000002371 ultraviolet--visible spectrum Methods 0.000 claims abstract description 17
- 238000002835 absorbance Methods 0.000 claims description 76
- 239000000523 sample Substances 0.000 claims description 62
- 150000002772 monosaccharides Chemical class 0.000 claims description 61
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 38
- 238000012360 testing method Methods 0.000 claims description 29
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- 238000010521 absorption reaction Methods 0.000 claims description 18
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- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 6
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- HYBBIBNJHNGZAN-UHFFFAOYSA-N furfural Chemical compound O=CC1=CC=CO1 HYBBIBNJHNGZAN-UHFFFAOYSA-N 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical class [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 4
- 239000012496 blank sample Substances 0.000 description 4
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- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- NOEGNKMFWQHSLB-UHFFFAOYSA-N 5-hydroxymethylfurfural Chemical compound OCC1=CC=C(C=O)O1 NOEGNKMFWQHSLB-UHFFFAOYSA-N 0.000 description 3
- 125000003172 aldehyde group Chemical group 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 238000004817 gas chromatography Methods 0.000 description 3
- RJGBSYZFOCAGQY-UHFFFAOYSA-N hydroxymethylfurfural Natural products COC1=CC=C(C=O)O1 RJGBSYZFOCAGQY-UHFFFAOYSA-N 0.000 description 3
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- QELUYTUMUWHWMC-UHFFFAOYSA-N edaravone Chemical compound O=C1CC(C)=NN1C1=CC=CC=C1 QELUYTUMUWHWMC-UHFFFAOYSA-N 0.000 description 2
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- 238000011160 research Methods 0.000 description 2
- 239000000779 smoke Substances 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 description 1
- MYKOKMFESWKQRX-UHFFFAOYSA-N 10h-anthracen-9-one;sulfuric acid Chemical compound OS(O)(=O)=O.C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 MYKOKMFESWKQRX-UHFFFAOYSA-N 0.000 description 1
- LWFUFLREGJMOIZ-UHFFFAOYSA-N 3,5-dinitrosalicylic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1O LWFUFLREGJMOIZ-UHFFFAOYSA-N 0.000 description 1
- AFPLNGZPBSKHHQ-UHFFFAOYSA-N Betulaprenol 9 Natural products CC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CCO AFPLNGZPBSKHHQ-UHFFFAOYSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 description 1
- 238000002485 combustion reaction Methods 0.000 description 1
- 238000013499 data model Methods 0.000 description 1
- 230000000081 effect on glucose Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 229960002715 nicotine Drugs 0.000 description 1
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 1
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- 238000003908 quality control method Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- AFPLNGZPBSKHHQ-MEGGAXOGSA-N solanesol Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CC\C(C)=C\CC\C(C)=C\CC\C(C)=C\CC\C(C)=C\CC\C(C)=C\CC\C(C)=C\CO AFPLNGZPBSKHHQ-MEGGAXOGSA-N 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 235000019605 sweet taste sensations Nutrition 0.000 description 1
- 235000018553 tannin Nutrition 0.000 description 1
- 229920001864 tannin Polymers 0.000 description 1
- 239000001648 tannin Substances 0.000 description 1
- 238000001429 visible spectrum Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4044—Concentrating samples by chemical techniques; Digestion; Chemical decomposition
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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Abstract
The patent discloses a method for measuring the contents of glucose and xylose in a sugar solution and a tobacco leaching solution, and the measuring method is an ultraviolet-visible spectrum method. When analyzing the sugar solution: (1) preparing a standard solution, (2) carrying out hydrolysis reaction; (3) carrying out color development reaction; (4) establishing a standard curve or a universal model; (5) detecting a sample; (6) and outputting the result. When analyzing the tobacco leaching liquor: (1) preparing tobacco extract; (2) carrying out hydrolysis reaction; (3) carrying out color development reaction; (4) establishing a standard curve or a universal model; (5) detecting a sample; (6) and outputting the result. The phloroglucinol color developing agent and the color developing step are positioned after the hydrolysis step, the mixed acid of concentrated hydrochloric acid and glacial acetic acid is adopted for hydrolysis, and the normal temperature condition is adopted, so that the phloroglucinol method is used for detecting glucose for the first time, the glucose and the xylose can be simultaneously detected, and the method is used for detecting the glucose and the xylose in a sugar solution and a tobacco leaching solution, and has the advantages of quick, simple and convenient operation, high detection efficiency and high accuracy.
Description
Technical Field
The invention relates to the field of quality analysis of tobacco and tobacco products, in particular to a method for measuring contents of glucose and xylose in a sugar solution and a tobacco leaching solution.
Background
The carbohydrate in tobacco can be thermally cracked under the combustion condition to generate various new compounds, thereby affecting the aroma and the taste of the cigarette, and different sugar components can also have different effects. Researches show that the glucose content in the cigarette can enrich the aroma of the cigarette and improve the smoothness of the smoke; the fructose content is beneficial to improving the ductility and sweetness of the tobacco leaves and reducing the miscellaneous gas; the xylose content is beneficial to improving the moistening feeling of the smoke and the sweet taste of the taste, so that the aroma texture is more exquisite and smooth. Therefore, an accurate and rapid monosaccharide analysis and detection method is an important premise for realizing quality control analysis in the tobacco industry.
At present, the sugar determination method in the tobacco industry mainly comprises a furin reagent method, a continuous flow analysis method, a gas chromatography, a high performance liquid chromatography and the like. Wherein the measured contents of the furin reagent method and the continuous flow analysis method are total amounts, and the content of a single component cannot be measured; the gas chromatography needs to be analyzed after the derivatization of the carbohydrate, and the operation is troublesome; compared with gas chromatography, high performance liquid chromatography can be directly used for sample introduction after sample pretreatment, but the problem of troublesome pretreatment is also existed, for example, a solid phase extraction column is needed for pre-separation, and the method has generally higher instrument and equipment cost. Therefore, in the tobacco industry, there is an urgent need to develop a simple and rapid monosaccharide analysis method.
The ultraviolet-visible chromatography is widely applied to various industries due to the simplicity and rapidness of the method, the cheap price of instruments and the high detection efficiency. Under the high-temperature acidic condition, the saccharide substance can generate furfural and hydroxymethyl furfural through hydrolysis reaction, and can generate a furfural derivative with characteristic absorption in a visible light region under the action of a color developing agent. Based on the findings, in recent years, researchers developed a series of reducing sugar detection methods such as anthrone-sulfuric acid colorimetric method, phenol-sulfuric acid colorimetric method, and 3, 5-dinitrosalicylic acid colorimetric method, so that the ultraviolet-visible chromatography can be used for sugar determination in the tobacco industry. However, due to the particularity of the raw materials, nicotine, solanesol, tannin, pigments and other substances in the tobacco are easily dissolved into the extraction liquid together with the saccharides, so that the liquid is dark yellow and the analysis and detection results are interfered. In the early work, a certain amount of activated carbon powder is added to eliminate the interference, however, a long decoloring time (30-60 min) is needed, the cost of the activated carbon is high, and in addition, the added activated carbon powder not only adsorbs a certain amount of components to be measured, but also introduces additional interference and pollution, and the accuracy of the measurement result is reduced. In recent years, researches show that the interference can be reduced by adopting a high-power dilution method, but higher requirements are put on the accuracy of the detection method, and a mature detection method meeting the accuracy is not developed at present. In addition, concentrated sulfuric acid is used in most of the existing color development methods, and is a strong mineral acid with high corrosivity, the dehydration property is strong, the sugar carbonization problem is caused, and the concentrated sulfuric acid has high danger to a human body.
The phloroglucinol method is a spectrophotometric method with higher accuracy, is mainly applied to the determination of xylose at present, and has poor color development effect on glucose. In addition, the existing ultraviolet-visible chromatography is difficult to realize the simultaneous determination of more than two saccharides respectively.
Disclosure of Invention
In order to solve the technical problems that the existing phloroglucinol method has poor glucose color development effect, the existing ultraviolet-visible chromatography is difficult to realize simultaneous determination of more than two sugars, the concentrated sulfuric acid has high risk to human bodies, and the accuracy of determination of the sugars in the tobacco by the ultraviolet-visible chromatography is to be improved.
The technical scheme of the invention is as follows:
the first technical scheme of the invention is a method for measuring the contents of glucose and xylose in a sugar solution, which comprises the following steps:
(1) preparation of standard solution: preparing monosaccharide standard solutions with different concentrations and a mixed sugar solution, wherein the monosaccharide standard solutions are glucose standard solutions and xylose standard solutions, and the mixed sugar solution is a mixture formed by randomly mixing monosaccharide standard solutions with known concentrations in equal volumes;
(2) and (3) hydrolysis reaction: adding mixed acid of concentrated hydrochloric acid and glacial acetic acid into a test tube containing a monosaccharide standard solution or a mixed sugar solution, shaking uniformly, putting into a constant-temperature water bath kettle for hydrolysis reaction, and taking out the test tube for cooling after the reaction is finished;
(3) and (3) color development reaction: adding phloroglucinol color developing agent solution into the hydrolysate cooled in the step (2), shaking up the test tube and standing;
(4) establishing a standard curve: sequentially carrying out the treatment of the steps (2) and (3) on monosaccharide standard solutions with different concentrations in the step (1) and mixed sugar solutions with known concentrations, and carrying out ultraviolet-visible spectrum detection on the obtained liquid to obtain a spectrum absorbance signal value; respectively establishing a glucose standard curve and a xylose standard curve for the monosaccharide standard solution according to the absorbance signal values and the monosaccharide contents of the monosaccharide standard solutions with different concentrations; for the mixed sugar solution, establishing mapping between a spectral absorbance signal value and glucose content and xylose content by using a multidimensional data processing technology so as to obtain a universal model 1, then establishing mapping of a spectral absorbance signal value increment to a glucose concentration increment when the xylose concentration in the mixed sugar solution is unchanged, and establishing a universal model 2;
(5) sample detection: taking a sample sugar solution with different concentration from that used in the step (4), carrying out ultraviolet-visible spectroscopy detection after the treatment of the steps (2) and (3), and recording a spectral absorbance signal value of the sample, wherein the sample sugar solution is any one of a glucose solution, a xylose solution and a mixed sugar solution;
(6) and (4) outputting a result: preliminarily judging the type of sugar in the sugar solution according to the position of the characteristic absorption peak of the ultraviolet spectrum, and if the sugar is glucose, substituting the spectral absorbance signal value obtained in the step (5) into the glucose standard curve obtained in the step (4) to obtain the content of the glucose; if the xylose is obtained, substituting the spectral absorbance signal value obtained in the step (5) into the xylose standard curve obtained in the step (4) to obtain the xylose content; and (4) if the sample is mixed sugar, substituting the spectral absorbance signal value obtained in the step (5) into the universal model 1 and the universal model 2 obtained in the step (4) to obtain the content of glucose and xylose in the sample.
In a second technical scheme of the invention, the method for determining the contents of glucose and xylose in the tobacco leaching solution comprises the following steps:
(1) preparing a tobacco leaching solution: oscillating and extracting a tobacco sample on a constant-temperature water bath oscillator by using water as an extracting agent, filtering, discarding initial filtrate, and collecting subsequent filtrate for later use to obtain tobacco leaching liquor;
(2) and (3) hydrolysis reaction: adding mixed acid of concentrated hydrochloric acid and glacial acetic acid into a test tube containing monosaccharide standard solution or mixed sugar solution or tobacco leaching liquor, shaking uniformly, placing into a constant-temperature water bath kettle for hydrolysis reaction, and taking out the test tube for cooling after the reaction is finished;
(3) and (3) color development reaction: adding phloroglucinol color developing agent solution into the hydrolysate cooled in the step (2), shaking up the test tube and standing;
(4) establishing a standard curve: processing monosaccharide standard solutions with different concentrations and mixed sugar solutions with known concentrations in the steps (2) and (3) in sequence, and detecting the obtained liquid by using an ultraviolet-visible spectrum method to obtain a spectrum absorbance signal value; respectively establishing a glucose standard curve and a xylose standard curve for the monosaccharide solution according to the absorbance signal values and the monosaccharide contents of the monosaccharide solutions with different concentrations; for the mixed sugar solution, establishing mapping between a spectral absorbance signal value and glucose content and xylose content by using a multidimensional data processing technology so as to obtain a universal model 1, then establishing mapping of a spectral absorbance signal value increment to a glucose concentration increment when the xylose concentration in the mixed sugar solution is unchanged, and establishing a universal model 2;
(5) sample detection: diluting the tobacco leaching liquor with unknown concentration obtained in the step (1), processing the tobacco leaching liquor in the steps (2) and (3), detecting the tobacco leaching liquor by using an ultraviolet-visible spectrum method, and recording a spectral absorbance signal value of the tobacco leaching liquor;
(6) and (4) outputting a result: preliminarily judging the type of sugar in the tobacco leaching liquor according to the position of the ultraviolet spectrum characteristic absorption peak, and if the type of sugar is glucose, substituting the spectral absorbance signal value obtained in the step (5) into the glucose standard curve obtained in the step (4) to obtain the content of the glucose; if the xylose is obtained, substituting the spectral absorbance signal value obtained in the step (5) into the xylose standard curve obtained in the step (4) to obtain the xylose content; and (4) if the sample is mixed sugar, substituting the spectral absorbance signal value obtained in the step (5) into the universal model 1 and the universal model 2 obtained in the step (4) to obtain the content of glucose and xylose in the sample.
In the first technical scheme and the second technical scheme, the volume ratio of the monosaccharide standard solution or the mixed sugar solution or the sample sugar solution or the tobacco leaching solution to the mixed acid in the step (2) is 1:3-1:6, the volume of concentrated hydrochloric acid in the mixed acid is larger than that of glacial acetic acid, and preferably, the volume ratio of the concentrated hydrochloric acid to the glacial acetic acid is 2:1-5: 1.
In the first technical scheme and the second technical scheme, the temperature of the constant-temperature water bath in the step (2) is the temperature of a boiling water bath, and the reaction time is 40-80 min.
In the first technical scheme and the second technical scheme, when the color development reaction is performed in the step (3), phloroglucinol/ethanol color developing agent is adopted, and the volume ratio of the color developing agent to the monosaccharide standard solution or mixed sugar solution or sample sugar solution or tobacco leaching solution is 1: 1, the concentration of the color developing agent is 2 percent, and the standing time is within 2 min.
The measurement wavelengths for the glucose standard solution, the xylose standard solution and the mixed sugar solution when the standard curves are established in the step (4) in the first and second embodiments are 470nm, 478nm and 471nm to 477nm, respectively.
When the tobacco leachate is prepared in step (1) in the second technical scheme, the volume of the pure water serving as the extracting agent is 20mL when the tobacco sample is 0.1 +/-0.0001 g. The temperature of the constant temperature water bath oscillator is 30 +/-0.5 ℃, the rotating speed is 200rpm, and the oscillation extraction time is 30 min.
In the second technical scheme, the dilution factor of the tobacco leaching solution in the step (5) is determined by that the spectral absorbance signal value of the tobacco leaching solution is between 0.7 and 1.0.
The invention has the beneficial technical effects that:
according to the method, no matter sugar solution or tobacco leaching liquor is analyzed, firstly, monosaccharide standard solutions with different concentrations and mixed sugar solution with known concentrations are subjected to hydrolysis and color development treatment, then the ultraviolet-visible spectrum absorbance value is measured, a glucose standard curve, a xylose standard curve and mixed sugar universal models 1 and 2 are respectively established, and on the basis, the ultraviolet-visible spectrum absorbance value is measured after the same hydrolysis and color development treatment is respectively carried out on the sample sugar solution and the tobacco leaching liquor. And preliminarily judging the types of sugar in the sugar solution of the sample to be detected and the tobacco leaching liquor according to the characteristic absorption peak position of the ultraviolet spectrum. If the characteristic peak is positioned at 470nm, the solution almost only contains glucose, and the glucose content in the sample sugar solution and the tobacco leaching solution can be obtained by substituting into a glucose standard curve, and the xylose is the same; if the characteristic peak appears between 471nm and 477nm, the solution contains glucose and xylose at the same time, and the glucose and xylose contents in the sample sugar solution and the tobacco leaching liquor are obtained by substituting the solution into the universal models 1 and 2.
The color development principle of the invention is that phloroglucinol is complexed with glucose and furfural and hydroxymethyl furfural in xylose hydrolysate to generate yellow quinoid compound in the solution, and then the ultraviolet-visible chromatography is adopted to determine the spectral absorbance value of the solution.
Because the method adopts the phloroglucinol method for color development, the phloroglucinol color development reaction is placed after the hydrolysis reaction, concentrated hydrochloric acid is adopted to replace concentrated sulfuric acid in the hydrolysis reaction, the dosage of glacial acetic acid is reduced, so that the hydrolysis reaction time is increased, the hydrolysis time of glucose is increased, the glucose with a more stable structure is completely converted into hydroxymethyl furfural, the color development effect of phloroglucinol on a hydrolysis product of the glucose is increased, and the color development result difference of the glucose and xylose is reduced; and the color development is carried out at normal temperature, so that the unstable quinoid intermediate formed by phloroglucinol and aldehyde group hydrolyzed from sugar during color development is prevented from being easily decomposed at high temperature, and the color development effect is improved. The final result shows that the color development effect of the glucose is greatly improved, and the sensitivity of the method for detecting the glucose is obviously improved, so that the phloroglucinol method is applied to the detection of the glucose for the first time, the simultaneous determination of the glucose and the xylose is realized, and the rapid determination can be realized when the method is applied to the detection of the glucose and the xylose in sugar solution, tobacco and tobacco products, and the operation is simple and convenient.
Compared with similar methods for detecting the content of the sugar in the tobacco, such as an anthrone colorimetric method, a phenol-sulfuric acid colorimetric method and the like which are recorded in the existing literature, the method has more excellent accuracy, still has more obvious experimental phenomena for 0.01mmol/L glucose and xylose, and can realize accurate detection of the sugar with low concentration.
On the basis, for a sample solution containing glucose and xylose simultaneously, the method adopts a multidimensional data processing technology to carry out two-dimensional correlation matrix analysis, specifically, Partial Least Squares Regression (PLSR) analysis is carried out by using chemometrics software, and mapping between a spectral absorbance signal value and glucose content and xylose content is established, so that a data model auxiliary, namely a general mixed sugar model is established, and the concentrations of the glucose and the xylose are calculated, so that the contents of the glucose and the xylose in the sugar solution and the tobacco leaching solution are synchronously and quantitatively determined. For the sample containing only monosaccharide, the monosaccharide concentration can be obtained by substituting the glucose standard curve and the xylose standard curve.
The acid used in the hydrolysis step is concentrated hydrochloric acid and glacial acetic acid, and compared with concentrated sulfuric acid with strong dehydration property, the method can provide a milder reaction environment, avoid the problem of sugar carbonization caused by the over-strong water absorption of the concentrated sulfuric acid, and ensure the safety problem of the operation process of experimenters because the concentrated hydrochloric acid and the glacial acetic acid have much less danger to the human body than the concentrated sulfuric acid.
According to the method for detecting the contents of glucose and xylose in the tobacco leaching liquor, the color interference caused by the tobacco leaching liquor is eliminated by adopting a high-power dilution method, and on the basis, the phloroglucinol ultraviolet-visible chromatography is adopted to realize a high-accuracy subsequent detection method. And the dilution times of the leaching liquor of the tobacco sample are determined by the fact that the spectral absorbance signal value of the tobacco leaching liquor is between 0.7 and 1.0. Preferably, the dilution ratio of the leaching liquor in the step (5) is 20 times, the color of the sample is changed from orange yellow to light yellow, and the detection result in the step (4) is not affected finally.
Drawings
FIG. 1 is the characteristic absorption peak of the ultraviolet/visible spectrum of a single glucose, single xylose and mixed sugar solution in example 1 and example 2; wherein the concentration of glucose and xylose is 0.4 mmol/L; the mixed sugar A is 0.2mmol/L glucose +0.2mmol/L xylose; the mixed sugar A is 0.1mmol/L glucose +0.1mmol/L xylose;
FIG. 2 is a standard curve of absorbance signal values of UV/Vis spectrum versus glucose concentration in example 1 and example 2;
FIG. 3 is a standard curve of absorbance signal values of UV/Vis spectrum versus xylose concentration for examples 1 and 2;
FIG. 4 is a correlation analysis of the predicted data and the measured data obtained by the general model 1 in examples 1 and 2;
FIG. 5 shows the deviation between the predicted value and the measured value of the general model 1 in examples 1 and 2;
FIG. 6 is a correlation analysis of the predicted data and the measured data obtained by the general model 2 in example 1 and example 2;
fig. 7 shows the deviation between the predicted value and the measured value of the generic model 2 in examples 1 and 2.
Detailed Description
In order to more clearly illustrate the technical solution of the present invention, the present invention will be described in further detail with reference to fig. 1 to 7. The present invention is not limited to the following embodiments.
A method for determining the content of glucose and xylose in a sugar solution comprises the following steps:
(1) preparation of standard solution: preparing monosaccharide standard solutions with different concentrations and a mixed sugar solution, wherein the monosaccharide standard solutions are glucose standard solutions and xylose standard solutions, and the mixed sugar solution is a mixture of monosaccharide standard solutions with known concentrations in equal volumes and mixed randomly.
(2) And (3) hydrolysis reaction: adding mixed acid of concentrated hydrochloric acid and glacial acetic acid into a test tube containing a monosaccharide standard solution or a mixed sugar solution, shaking uniformly, putting into a constant-temperature water bath kettle for hydrolysis reaction, taking out the test tube after the reaction is finished, cooling, and carrying out the same hydrolysis reaction by using deionized water with the same volume as the test tube to obtain a blank sample.
In the step, concentrated sulfuric acid is replaced by concentrated hydrochloric acid, and the dosage of glacial acetic acid is reduced. Specifically, the volume ratio of the sugar-containing solution to the mixed acid is 1:3-1:6, the volume of the concentrated hydrochloric acid in the mixed acid is larger than that of the glacial acetic acid, preferably, the volume ratio of the concentrated hydrochloric acid to the glacial acetic acid is 2:1-5:1, and more preferably, the volume ratio of the concentrated hydrochloric acid to the glacial acetic acid is 4: 1. The constant-temperature water bath temperature is the boiling water bath temperature, and the reaction time is 40-80 min.
(3) And (3) color development reaction: and (3) adding phloroglucinol color developing agent solution into the hydrolysate cooled in the step (2), shaking up the test tube and standing.
The color development step and the hydrolysis step are separated, and the color development reaction is carried out after hydrolysis to increase the hydrolysis time of the glucose, so as to reduce the difference of the color development results of the color development of the hydrolysis products of the glucose and the xylose and the phloroglucinol; and the color development is carried out at normal temperature, so that the unstable quinoid intermediate formed by phloroglucinol and aldehyde group hydrolyzed from sugar during color development is prevented from being easily decomposed at high temperature, and the color development effect is improved.
Adopting phloroglucinol/ethanol color developing agent, wherein the volume ratio of the color developing agent to the sugar solution is 1: 1, the concentration of the color developing agent is 2 percent, and the standing time is within 2 min.
(4) Establishing a standard curve: sequentially treating the glucose standard solution, the xylose standard solution and the mixed sugar solution with known concentration in the step (1) in the steps (2) and (3), and detecting the obtained liquid by using an ultraviolet-visible spectrum method to obtain a spectral absorbance signal value;
respectively establishing a glucose standard curve and a xylose standard curve for the monosaccharide standard solution according to the absorbance signal values and the monosaccharide contents of the monosaccharide standard solutions with different concentrations;
and for the mixed sugar solution, establishing a mapping between the spectral absorbance signal value and the glucose content and xylose content by using a multidimensional data processing technology so as to obtain a universal model 1, and then establishing a mapping between the spectral absorbance signal value increment and the glucose concentration increment when the xylose concentration in the mixed sugar solution is unchanged so as to establish a universal model 2.
The measurement wavelengths of the glucose standard solution, the xylose standard solution and the mixed sugar solution are 470nm, 478nm and 471-477 nm respectively when the standard curve is established.
(5) Sample detection: and (3) taking a sample sugar solution with different concentration from that used in the step (4), treating the sample sugar solution in the steps (2) and (3), detecting by using an ultraviolet-visible spectroscopy, and recording a spectral absorbance signal value of the sample, wherein the sample sugar solution can be any one of a glucose solution, a xylose solution and a mixed sugar solution.
(6) And (4) outputting a result: preliminarily judging the type of sugar in the sugar solution according to the position of the ultraviolet spectrum characteristic absorption peak, and if the type of sugar is glucose, substituting the spectral absorbance signal value obtained in the step (5) into the glucose standard curve obtained in the step (4) to obtain the content of the glucose; if the xylose is obtained, substituting the spectral absorbance signal value obtained in the step (5) into the xylose standard curve obtained in the step (4) to obtain the xylose content; and (4) if the sample is mixed sugar, substituting the spectral absorbance signal value obtained in the step (5) into the universal model 1 and the universal model 2 obtained in the step (4) to obtain the content of glucose and xylose in the sample.
A method for determining the content of glucose and xylose in a tobacco leaching solution comprises the following steps:
(1) preparing a tobacco leaching solution: oscillating and extracting a tobacco sample on a constant-temperature water bath oscillator by using water as an extracting agent, filtering, discarding initial filtrate, and collecting subsequent filtrate for later use to obtain tobacco leaching liquor;
when the tobacco sample is 0.1 +/-0.0001 g, the volume of the pure water is 20 mL. The temperature of the constant temperature water bath oscillator is 30 +/-0.5 ℃, the rotating speed is 200rpm, and the oscillation extraction time is 30 min.
(2) And (3) hydrolysis reaction: adding mixed acid of concentrated hydrochloric acid and glacial acetic acid into a test tube containing a monosaccharide standard solution or a mixed sugar solution or a tobacco leaching liquor, shaking uniformly, placing the test tube into a constant-temperature water bath kettle for hydrolysis reaction, taking out the test tube after the reaction is finished, cooling, and carrying out the same hydrolysis reaction by using deionized water with the same volume as the volume of the test tube to obtain a blank sample;
in the step, concentrated sulfuric acid is replaced by concentrated hydrochloric acid, and the dosage of glacial acetic acid is reduced. Specifically, the volume ratio of the sugar-containing solution to the mixed acid is 1:3-1:6, the volume of the concentrated hydrochloric acid in the mixed acid is larger than that of the glacial acetic acid, preferably, the volume ratio of the concentrated hydrochloric acid to the glacial acetic acid is 2:1-5:1, and more preferably, the volume ratio of the concentrated hydrochloric acid to the glacial acetic acid is 4: 1. The constant-temperature water bath temperature is the boiling water bath temperature, and the reaction time is 40-80 min.
(3) And (3) color development reaction: adding phloroglucinol color developing agent solution into the hydrolysate cooled in the step (2), shaking up the test tube and standing;
the color development step and the hydrolysis step are separated, and the color development reaction is carried out after hydrolysis to increase the reaction time, so that the color development result difference of the color development of the hydrolysis products of glucose and xylose and phloroglucinol is increased; and the coloration is carried out at normal temperature, so that the coloration of phloroglucinol and aldehyde groups hydrolyzed from sugar are prevented from forming unstable quinoid intermediate, the quinoid intermediate is easy to decompose at high temperature, and the coloration effect is improved.
Adopting phloroglucinol/ethanol color developing agent, wherein the volume ratio of the color developing agent to the sugar solution is 1: 1, the concentration of the color developing agent is 2 percent, and the standing time is within 2 min.
(4) Establishing a standard curve: after glucose standard solutions with different concentrations, xylose standard solutions and mixed sugar solutions with known concentrations are sequentially processed in the steps (2) and (3), detecting the obtained liquid by using an ultraviolet-visible spectrum method to obtain a spectrum absorbance signal value;
respectively establishing a glucose standard curve and a xylose standard curve for the monosaccharide standard solution according to the absorbance signal values and the monosaccharide contents of the monosaccharide standard solutions with different concentrations;
for the mixed sugar solution, establishing mapping between a spectral absorbance signal value and glucose content and xylose content by using a multidimensional data processing technology so as to obtain a universal model 1, then establishing mapping of a spectral absorbance signal value increment to a glucose concentration increment when the xylose concentration in the mixed sugar solution is unchanged, and establishing a universal model 2;
the measurement wavelengths of the glucose standard solution, the xylose standard solution and the mixed sugar solution are 470nm, 478nm and 471-477 nm respectively when the standard curve is established.
(5) Sample detection: diluting the tobacco leaching liquor with unknown concentration obtained in the step (1), processing the diluted tobacco leaching liquor through the steps (2) and (3), detecting the tobacco leaching liquor through an ultraviolet-visible spectrum method, and recording a spectral absorbance signal value of the tobacco leaching liquor.
The dilution factor of the tobacco leaching liquor is based on the dilution factor that the final absorbance is between 0.7 and 1.0. The tobacco leaching solution is diluted by 5, 10, 15 and 20 times in comparison, and the final absorbance reaches the optimal range when the tobacco leaching solution is diluted by 20 times. Different varieties of raw materials or different optimal dilution times are determined according to the appropriate final absorbance.
(6) And (4) outputting a result: preliminarily judging the type of sugar in the tobacco leaching liquor according to the position of the ultraviolet spectrum characteristic absorption peak, and if the sugar is glucose, substituting the spectral absorbance signal value obtained in the step (5) into the glucose standard curve obtained in the step (4) to obtain the content of the glucose; if the xylose is obtained, substituting the spectral absorbance signal value obtained in the step (5) into the xylose standard curve obtained in the step (4) to obtain the xylose content; and (4) if the sample is mixed sugar, substituting the spectral absorbance signal value obtained in the step (5) into the universal model 1 and the universal model 2 obtained in the step (4) to obtain the content of glucose and xylose in the sample.
The following are specific examples.
Example 1 method for determining glucose and xylose content in self-made monosaccharide and mixed sugar solution
(1) Preparation of standard solution: accurately preparing 10.00mmol/L of glucose and xylose stock mother liquor, and diluting to obtain different monosaccharide standard solutions with concentration ranges of 0.01-2 mmol/L; randomly mixing the prepared glucose standard solution and the xylose standard solution in equal volume to obtain a mixed sugar solution with known concentration;
(2) and (3) hydrolysis reaction: transferring lmL the monosaccharide standard solution or the mixed sugar solution prepared in the step (1) into a 25mL colorimetric tube with a plug, adding 4mL concentrated hydrochloric acid and lmL glacial acetic acid, shaking uniformly, placing into a boiling constant-temperature water bath kettle for reaction for 40min, immediately taking out the test tube after the reaction is finished, placing into cold water for cooling for 5min, and performing the same hydrolysis reaction by lmL deionized water to obtain a blank sample;
(3) and (3) color development reaction: transferring 1mL of 2% phloroglucinol/ethanol color developing agent solution, adding into the hydrolysate cooled in the step (2), quickly shaking the test tube uniformly and standing for 1 min;
(4) establishing a standard curve: after the glucose standard solution and the xylose standard solution with different concentrations prepared in the step (1) and the mixed sugar solution of the glucose standard solution and the xylose standard solution are processed in the steps (2) and (3), carrying out ultraviolet-visible spectrum detection on the obtained liquid, wherein a specific spectrum is shown in a figure 1;
as can be seen from FIG. 1, if the measured solution contains only glucose, the characteristic absorption peak of the spectrum appears at 470 nm; if the tested solution only contains xylose, the characteristic absorption peak of the spectrum appears at 478 nm; if both the two are present, the characteristic absorption peak of the spectrum appears between 471nm and 477nm, and the specific position is related to the relative content of glucose and xylose;
and for the monosaccharide standard solution, respectively establishing standard curves according to the spectral absorbance signal values of the obtained monosaccharide standard solutions with different concentrations, the glucose content and the xylose content to obtain a glucose standard curve: y1.4138 x-0.0196 and correlation coefficient R20.9961, see fig. 2; xylose standard curve: y4.3425 x +0.0191 and correlation coefficient R20.9960, see fig. 3;
for detecting 0.01mmol/L glucose and xylose, the final absorbance can reach 0.14 and 0.41, and the method still has a relatively obvious experimental phenomenon and can realize accurate detection of low-concentration sugar;
for the mixed sugar solution, a multidimensional data processing technology, namely chemometrics software is used for performing Partial Least Squares Regression (PLSR) analysis, and a mapping between a spectral absorbance signal value and glucose content and xylose content is established, so that a general model 1 is obtained, wherein Y is 5.6823X1+1.1474X2+0.0571(Y is absorbance, X1Is the xylose concentration, X2Glucose concentration) as shown in figures 4 and 5; then establishing the mapping of the increment of the spectral absorbance signal value to the increment of the glucose concentration when the xylose concentration in the mixed sugar solution is not changed, and establishing a general model 2, wherein Y is 0.9517X3+0.0558(Y is the increase in absorbance, X3Glucose concentration increase) to eliminate the effect of xylose, see fig. 6 and 7. Calculating to obtain the glucose content by the general model 2, and substituting the glucose content into the general model 1 to obtain the xylose content;
(5) sample detection: respectively taking 1mL of sample sugar solution with different concentration from that used in the step (4), taking the sample sugar solution each time to be any one of glucose solution, xylose solution and mixed sugar solution, carrying out ultraviolet-visible spectrum detection after the treatment of the steps (2) and (3), and recording the spectral absorbance signal value of the sample;
(6) and (4) outputting a result: and (3) comparing the spectral absorbance signal value obtained in the step (5) with the universal model or the monosaccharide standard curve obtained in the step (4), namely, bringing the values into the corresponding universal models 1 and 2 or the corresponding monosaccharide standard curve to obtain the content of glucose and/or xylose in the sample.
The type of sugar in the sample solution can be preliminarily judged according to the position of the characteristic absorption peak of the ultraviolet spectrum. If the characteristic peak is positioned at 470nm, the solution almost only contains glucose, and the glucose content in the sample solution can be obtained by substituting into a glucose standard curve, and the xylose is the same; if the characteristic peak appears between 471nm and 477nm, the sample solution contains glucose and xylose at the same time and is brought into the general models 1 and 2;
(7) and (3) measuring results: the results of the monosaccharide solution measurements based on the monosaccharide standard curve are shown in Table 1; the results of the measurements obtained for the mixed sugar solutions according to the general model are shown in Table 2. As can be seen from tables 1 and 2, the absolute value of the relative deviation between the measured value and the theoretical value is within 5%, which shows that the detection method has accurate and reliable results.
TABLE 1 measurement results of monosaccharide solutions from monosaccharide standard curves
TABLE 2 measurement results of the mixed sugar solution according to the general model
Example 2 determination of glucose and xylose content in tobacco products
(1) Preparing a tobacco leaching solution: weighing 0.1 + -0.0001 g of tobacco sample into a 50mL triangular flask with a plug, adding 20mL of pure water, covering the flask with the plug, and extracting for 30min by shaking at 200rpm on a constant temperature water bath shaker at 30 + -0.5 deg.C. And after extraction is finished, filtering by using quick qualitative filter paper, discarding 2-3 mL of filtrate obtained by initial filtration, and collecting subsequent liquid for later use to obtain a tobacco sample solution, namely a tobacco leaching liquor.
(2) And (3) hydrolysis reaction: transferring lmL prepared monosaccharide standard solution or mixed sugar solution or tobacco leaching solution into a 25mL colorimetric tube with a plug, adding 4mL concentrated hydrochloric acid and lmL glacial acetic acid, shaking uniformly, placing into a boiling constant-temperature water bath kettle for reaction for 40min, immediately taking out the test tube after the reaction is finished, placing into cold water for cooling for 5min, and carrying out the same hydrolysis reaction with lmL deionized water to obtain a blank sample;
the concentrations of the monosaccharide standard solution and the mixed sugar solution are the same as those of the embodiment 1;
(3) and (3) color development reaction: transferring 1mL of 2% phloroglucinol/ethanol color developing agent solution, adding into the hydrolysate cooled in the step (2), quickly shaking the test tube uniformly and standing for 1 min;
(4) establishing a standard curve: after the glucose standard solution and the xylose standard solution with different concentrations and the mixed sugar solution of the glucose standard solution and the xylose standard solution are processed in the steps (2) and (3), carrying out ultraviolet visible spectrum detection on the obtained liquid, wherein the specific spectrogram is shown in figure 1;
as can be seen from FIG. 1, if the measured solution contains only glucose, the characteristic absorption peak of the spectrum appears at 470 nm; if the tested solution only contains xylose, the characteristic absorption peak of the spectrum appears at 478 nm; if both the two are present, the characteristic absorption peak of the spectrum appears between 471nm and 477nm, and the specific position is related to the relative content of glucose and xylose;
and for the monosaccharide standard solution, respectively establishing standard curves according to the spectral absorbance signal values of the obtained monosaccharide standard solutions with different concentrations, the glucose content and the xylose content to obtain a glucose standard curve: y1.4138 x-0.0196 and correlation coefficient R20.9961, see fig. 2; xylose standard curve: y4.3425 x +0.0191 and correlation coefficient R20.9960, see fig. 3;
for detecting 0.01mmol/L glucose and xylose, the final absorbance can reach 0.14 and 0.41, and the method still has a relatively obvious experimental phenomenon and can realize accurate detection of low-concentration sugar;
for the mixed sugar solution, a multidimensional data processing technology, namely chemometrics software is used for performing Partial Least Squares Regression (PLSR) analysis, and a mapping between a spectral absorbance signal value and glucose content and xylose content is established, so that a general model 1 is obtained, wherein Y is 5.6823X1+1.1474X2+0.0571(Y is absorbance, X1Is the xylose concentration, X2Glucose concentration) as shown in figures 4 and 5; then establishing the mapping of the increment of the spectral absorbance signal value to the increment of the glucose concentration when the xylose concentration in the mixed sugar solution is not changed, and establishing a general model 2, wherein Y is 0.9517X3+0.0558(Y is the increase in absorbance, X3Glucose concentration increase) to eliminate the effect of xylose, see fig. 6 and 7. Calculating to obtain the glucose content by the universal model 2, and substituting the glucose content into the universal model 1 to obtain the xylose content;
(5) sample detection: diluting the tobacco sample solution prepared in the step (1), namely the tobacco leaching liquor, by 20 times to light yellow, transferring lmL into a 25mL colorimetric tube with a plug, adding 4mL concentrated hydrochloric acid and lmL glacial acetic acid, shaking up, placing into a boiling constant-temperature water bath kettle for reaction for 40min, immediately taking out the test tube after the reaction is finished, placing into cold water for cooling for 5min, adding 1mL phloroglucinol/ethanol developer solution with the concentration of 2%, quickly shaking up the test tube, standing for 1min, then carrying out ultraviolet visible spectroscopy detection, and recording the spectral absorbance signal value of the sample.
(6) And (4) outputting a result: and (3) substituting the spectral absorbance signal value obtained in the step (5) into the universal models 1 and 2 or a glucose standard curve or a xylose standard curve, calculating to obtain the content of glucose and/or xylose in the sample, and recording the numerical value.
The type of sugar in the sample solution can be preliminarily judged according to the position of the characteristic absorption peak of the ultraviolet spectrum. If the characteristic peak is positioned at 470nm, the solution almost only contains glucose, and the glucose content in the sample solution can be obtained by substituting into a glucose standard curve, and the xylose is the same; if the characteristic peak appears between 471nm and 477nm, the sample solution contains glucose and xylose at the same time and is brought into the general models 1 and 2;
(7) and (3) measuring results: the results are shown in Table 3.
In order to verify the accuracy of the method, a conventional 1-phenyl-3-methyl-5-pyrazolone (1-phenyl-3-methyl-5-pyrazolone, PMP for short) derivatization High Performance Liquid Chromatography (HPLC) method is adopted for comparison, and the experimental results are shown in Table 3.
TABLE 3 comparison of the results of the determination of the content of the mixed sugar by the present method with those by high performance liquid chromatography
Note: a, samples 1-4 are respectively tobacco sheets of different domestic brands; sample 5 is an unknown cigarette tobacco sample; sample 6 is a tobacco stalk raw material in China; sample 7 is a reconstituted tobacco of a certain brand of paper-making process in China.
b indicates that the substance was not detected.
As can be seen from Table 3, the absolute value of the relative deviation between the detection results of the method and the HPLC method is within 10%, and the detection results of the two methods have no significant difference. Therefore, the method has better consistency and high accuracy with the measurement result of the HPLC method. While representative examples and test examples of the present invention have been described in detail, the present invention is not limited to the details of the foregoing embodiments, and various modifications may be made within the scope of the technical idea of the present invention, and modifications and combinations apparent to those skilled in the art are within the scope of the present invention.
Claims (10)
1. A method for measuring the contents of glucose and xylose in a sugar solution is characterized by comprising the following steps:
(1) preparation of standard solution: preparing monosaccharide standard solutions with different concentrations and a mixed sugar solution, wherein the monosaccharide standard solutions are glucose standard solutions and xylose standard solutions, and the mixed sugar solution is a mixture formed by randomly mixing monosaccharide standard solutions with known concentrations in equal volumes;
(2) and (3) hydrolysis reaction: adding mixed acid of concentrated hydrochloric acid and glacial acetic acid into a test tube of the monosaccharide standard solution or the mixed sugar solution, shaking uniformly, putting into a constant-temperature water bath kettle for hydrolysis reaction, and taking out the test tube for cooling after the reaction is finished;
(3) and (3) color development reaction: adding phloroglucinol color developing agent solution into the hydrolysate cooled in the step (2), shaking up the test tube and standing;
(4) establishing a standard curve: processing monosaccharide standard solutions with different concentrations in the step (1) and mixed sugar solutions with known concentrations in the steps (2) and (3) in sequence, and detecting the obtained liquid by using an ultraviolet-visible spectrum method to obtain a spectral absorbance signal value; respectively establishing a glucose standard curve and a xylose standard curve for the monosaccharide standard solution according to the absorbance signal values and the monosaccharide contents of the monosaccharide standard solutions with different concentrations; for the mixed sugar solution, establishing mapping between a spectral absorbance signal value and glucose content and xylose content by using a multidimensional data processing technology so as to obtain a universal model 1, then establishing mapping of a spectral absorbance signal value increment to a glucose concentration increment when the xylose concentration in the mixed sugar solution is unchanged, and establishing a universal model 2;
(5) sample detection: taking a sample sugar solution with different concentration from that used in the step (4), carrying out ultraviolet-visible spectroscopy detection after the treatment of the steps (2) and (3), and recording a spectral absorbance signal value of the sample, wherein the sample sugar solution is a glucose solution, a xylose solution or a mixed sugar solution;
(6) and (4) outputting a result: preliminarily judging the type of sugar in the sample solution according to the position of the characteristic absorption peak of the ultraviolet spectrum, and if the sugar is glucose, substituting the spectral absorbance signal value obtained in the step (5) into the glucose standard curve obtained in the step (4) to obtain the content of the glucose; if the xylose is obtained, substituting the spectral absorbance signal value obtained in the step (5) into the xylose standard curve obtained in the step (4) to obtain the xylose content; and (4) if the sample is mixed sugar, substituting the spectral absorbance signal value obtained in the step (5) into the universal model 1 and the universal model 2 obtained in the step (4) to obtain the contents of glucose and xylose in the sample.
2. A method for determining the contents of glucose and xylose in a tobacco leaching solution is characterized by comprising the following steps:
(1) preparing a tobacco leaching solution: oscillating and extracting a tobacco sample on a constant-temperature water bath oscillator by using water as an extracting agent, filtering, discarding initial filtrate, and collecting subsequent filtrate for later use to obtain tobacco leaching liquor;
(2) and (3) hydrolysis reaction: adding mixed acid of concentrated hydrochloric acid and glacial acetic acid into a test tube of the monosaccharide standard solution or the mixed sugar solution or the tobacco leaching liquor, shaking uniformly, putting the test tube into a constant-temperature water bath kettle for hydrolysis reaction, and taking out the test tube for cooling after the reaction is finished;
(3) and (3) color development reaction: adding phloroglucinol color developing agent solution into the hydrolysate cooled in the step (2), shaking up the test tube and standing;
(4) establishing a standard curve: sequentially carrying out the steps (2) and (3) on monosaccharide standard solutions with different concentrations and mixed sugar solutions with known concentrations, and carrying out ultraviolet-visible spectrum detection on the obtained liquid to obtain a spectrum absorbance signal value; respectively establishing a glucose standard curve and a xylose standard curve for the monosaccharide solution according to the absorbance signal values and the monosaccharide contents of the monosaccharide solutions with different concentrations; for the mixed sugar solution, mapping between a spectral absorbance signal value and glucose content and xylose content is established by using a multidimensional data processing technology, so that a universal model 1 is obtained, mapping of a spectral absorbance signal value increment to a glucose concentration increment is established when the xylose concentration in the mixed sugar solution is unchanged, and a universal model 2 is established;
(5) sample detection: diluting the tobacco leaching liquor with unknown concentration obtained in the step (1), processing the tobacco leaching liquor through the steps (2) and (3), detecting the tobacco leaching liquor by using an ultraviolet-visible spectrum method, and recording a spectral absorbance signal value of a tobacco leaching liquor sample;
(6) and (4) outputting a result: preliminarily judging the type of sugar in the tobacco leaching liquor sample according to the position of the ultraviolet spectrum characteristic absorption peak, and substituting the spectral absorbance signal value obtained in the step (5) into the glucose standard curve obtained in the step (4) if the sugar is glucose, so as to obtain the content of the glucose; if the xylose is obtained, substituting the spectral absorbance signal value obtained in the step (5) into the xylose standard curve obtained in the step (4) to obtain the xylose content; and (4) if the sample is mixed sugar, substituting the spectral absorbance signal value obtained in the step (5) into the universal model 1 and the universal model 2 obtained in the step (4) to obtain the content of glucose and xylose in the sample.
3. The method according to claim 1 or 2, wherein the volume ratio of the monosaccharide standard solution or mixed sugar solution or sample sugar solution or tobacco leaching solution to the mixed acid in the step (2) is 1:3-1:6, and the volume of the concentrated hydrochloric acid in the mixed acid is larger than that of the glacial acetic acid.
4. The method according to claim 3, characterized in that the volume ratio of concentrated hydrochloric acid to glacial acetic acid is from 2:1 to 5: 1.
5. The method according to any one of claims 1 to 4, wherein the constant temperature water bath temperature in the step (2) is a boiling water bath temperature, and the reaction time is 40 to 80 min.
6. The method according to any one of claims 1 to 5, wherein the chromogenic reaction in step (3) is carried out by using phloroglucinol/ethanol developer, and the volume ratio of the developer to the monosaccharide standard solution or mixed sugar solution or sample sugar solution or tobacco leaching solution is 1: 1, the concentration of the color developing agent is 2 percent, and the standing time is within 2 min.
7. The method according to any one of claims 1 to 6, wherein the standard curves established in step (4) are measured at wavelengths of 470nm, 478nm, 471nm and 477nm for the glucose standard solution, the xylose standard solution and the mixed sugar solution, respectively.
8. The method according to claim 2, wherein when the tobacco extract is prepared in step (1), the volume of the extractant pure water is 20mL when the tobacco sample is 0.1 ± 0.0001 g.
9. The method according to claim 2 or 8, wherein the temperature of the thermostatic waterbath oscillator in the step (1) is 30 ± 0.5 ℃, the rotating speed is 200rpm, and the oscillation extraction time is 30 min.
10. The method according to claim 2, wherein the dilution factor of the tobacco extract in step (5) is determined by the value of the spectral absorbance signal of the tobacco extract being between 0.7 and 1.0.
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