CN114527117B - Method for measuring glucose and xylose content in sugar solution and tobacco leaching solution - Google Patents
Method for measuring glucose and xylose content in sugar solution and tobacco leaching solution Download PDFInfo
- Publication number
- CN114527117B CN114527117B CN202210169696.5A CN202210169696A CN114527117B CN 114527117 B CN114527117 B CN 114527117B CN 202210169696 A CN202210169696 A CN 202210169696A CN 114527117 B CN114527117 B CN 114527117B
- Authority
- CN
- China
- Prior art keywords
- solution
- glucose
- xylose
- sugar
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 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 254
- 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 143
- 239000008103 glucose Substances 0.000 title claims abstract description 143
- 235000000346 sugar Nutrition 0.000 title claims abstract description 129
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 title claims abstract description 127
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 title claims abstract description 127
- 241000208125 Nicotiana Species 0.000 title claims abstract description 80
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 80
- 238000000034 method Methods 0.000 title claims abstract description 60
- 238000002386 leaching Methods 0.000 title claims abstract description 49
- 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 44
- 239000000243 solution Substances 0.000 claims abstract description 159
- 239000012086 standard solution Substances 0.000 claims abstract description 73
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 50
- 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 39
- 238000001514 detection method Methods 0.000 claims abstract description 34
- 238000006243 chemical reaction Methods 0.000 claims abstract description 29
- JPYHHZQJCSQRJY-UHFFFAOYSA-N Phloroglucinol Natural products CCC=CCC=CCC=CCC=CCCCCC(=O)C1=C(O)C=C(O)C=C1O JPYHHZQJCSQRJY-UHFFFAOYSA-N 0.000 claims abstract description 25
- 229960000583 acetic acid Drugs 0.000 claims abstract description 25
- 239000012362 glacial acetic acid Substances 0.000 claims abstract description 25
- QCDYQQDYXPDABM-UHFFFAOYSA-N phloroglucinol Chemical compound OC1=CC(O)=CC(O)=C1 QCDYQQDYXPDABM-UHFFFAOYSA-N 0.000 claims abstract description 25
- 229960001553 phloroglucinol Drugs 0.000 claims abstract description 25
- 239000002253 acid Substances 0.000 claims abstract description 19
- 238000000870 ultraviolet spectroscopy Methods 0.000 claims abstract description 17
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 15
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- 238000002835 absorbance Methods 0.000 claims description 77
- 150000002772 monosaccharides Chemical class 0.000 claims description 63
- 239000000523 sample Substances 0.000 claims description 56
- 230000003595 spectral effect Effects 0.000 claims description 44
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 39
- 238000011161 development Methods 0.000 claims description 28
- 238000012360 testing method Methods 0.000 claims description 25
- 238000001228 spectrum Methods 0.000 claims description 20
- 238000010521 absorption reaction Methods 0.000 claims description 18
- 238000013507 mapping Methods 0.000 claims description 17
- 238000002211 ultraviolet spectrum Methods 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 10
- 238000005259 measurement Methods 0.000 claims description 10
- 239000012488 sample solution Substances 0.000 claims description 10
- 238000001816 cooling Methods 0.000 claims description 9
- 238000005516 engineering process Methods 0.000 claims description 9
- 239000000413 hydrolysate Substances 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- 238000012545 processing Methods 0.000 claims description 9
- 238000010790 dilution Methods 0.000 claims description 8
- 239000012895 dilution Substances 0.000 claims description 8
- 238000009835 boiling Methods 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 6
- 230000035484 reaction time Effects 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 4
- 230000010355 oscillation Effects 0.000 claims description 3
- 230000007062 hydrolysis Effects 0.000 abstract description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- 238000004587 chromatography analysis Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 238000010238 partial least squares regression Methods 0.000 description 6
- 238000004737 colorimetric analysis Methods 0.000 description 5
- 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
- 239000012496 blank sample Substances 0.000 description 4
- 150000001720 carbohydrates Chemical class 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 239000000126 substance Substances 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
- 235000019504 cigarettes Nutrition 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
- 239000000543 intermediate Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 235000019505 tobacco product Nutrition 0.000 description 3
- 238000001429 visible spectrum Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000003763 carbonization Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000010219 correlation analysis Methods 0.000 description 2
- 238000003113 dilution method Methods 0.000 description 2
- QELUYTUMUWHWMC-UHFFFAOYSA-N edaravone Chemical compound O=C1CC(C)=NN1C1=CC=CC=C1 QELUYTUMUWHWMC-UHFFFAOYSA-N 0.000 description 2
- 238000005206 flow analysis Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000779 smoke Substances 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 238000002371 ultraviolet--visible spectrum Methods 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
- 229930013930 alkaloid Natural products 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
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 238000002485 combustion reaction Methods 0.000 description 1
- 238000013499 data model Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 230000000081 effect on glucose Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 description 1
- 229960002715 nicotine Drugs 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- -1 saccharide compound Chemical class 0.000 description 1
- 230000035945 sensitivity Effects 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
- 230000001360 synchronised effect Effects 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
Classifications
-
- 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
-
- 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
-
- 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
-
- 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
Landscapes
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
Abstract
The patent discloses a method for measuring the contents of glucose and xylose in a sugar solution and a tobacco leaching solution, wherein the measuring method is an ultraviolet-visible spectrometry. When the sugar solution was analyzed: (1) standard solution preparation, (2) hydrolysis reaction; (3) a color reaction; (4) establishing a standard curve or a general model; (5) sample detection; and (6) outputting a result. When analyzing tobacco extract: (1) preparing tobacco leaching liquor; (2) hydrolysis reaction; (3) a color reaction; (4) establishing a standard curve or a general model; (5) sample detection; and (6) outputting a result. The phloroglucinol color developing agent is adopted, the color developing step is positioned after the hydrolysis step, and the mixed acid of concentrated hydrochloric acid and glacial acetic acid is adopted for hydrolysis under the normal temperature condition, so that the phloroglucinol method is firstly realized for detecting glucose, glucose and xylose can be simultaneously detected, and the method is fast and simple in operation, high in detection efficiency and high in accuracy when the method is used for detecting glucose and xylose in sugar solution and tobacco leaching liquor.
Description
Technical Field
The invention relates to the field of quality analysis of tobacco and tobacco products, in particular to a method for measuring the contents of glucose and xylose in a sugar solution and tobacco leaching liquor.
Background
The sugar substances in the tobacco can be thermally cracked under the combustion condition to generate various new compounds, so that the aroma and the taste of the cigarettes are influenced, and different sugar components can also have different influences. Researches show that the glucose content in the cigarettes can enrich the aroma of the cigarettes and improve the roundness of the smoke; the fructose content is beneficial to improving the persistence and sweetness of tobacco leaves and reducing miscellaneous gases; the xylose content is favorable for improving the moist feeling and the sweet taste of the smoke, so that the aroma texture is finer and smoother. Therefore, an accurate and rapid monosaccharide analysis and detection method is an important precondition for realizing quality control analysis of the tobacco industry.
Currently, the tobacco industry sugar determination methods mainly include a Fischer-Tropsch reagent method, a continuous flow analysis method, a gas chromatography method, a high performance liquid chromatography method and the like. Wherein the total amount is determined by the Fischer-Tropsch method and the continuous flow analysis method, and the content of the single component cannot be determined; the gas chromatography can be analyzed after the saccharide compound is derivatized, and the operation is more troublesome; compared with gas chromatography, high performance liquid chromatography can be directly injected after sample pretreatment, but the problem of troublesome pretreatment exists, such as the need of solid phase extraction column preseparation, and the cost of the instrument and equipment of the method is generally higher. There is therefore a strong need in the tobacco industry to develop a simple, rapid method of monosaccharide analysis.
Ultraviolet-visible chromatography is widely applied to various industries due to the simple and rapid method, low instrument price and high detection efficiency. Under the high-temperature acidic condition, the carbohydrate can generate furfural and hydroxymethyl furfural through hydrolysis reaction, and under the action of a color reagent, the furfural derivative with characteristic absorption in the visible light region is generated. Based on this finding, in recent years, researchers have developed a series of methods for detecting reducing sugars, such as anthrone-sulfuric acid colorimetry, phenol-sulfuric acid colorimetry, 3, 5-dinitrosalicylic acid colorimetry, and the like, so that ultraviolet-visible chromatography can be used for the determination of sugars in the tobacco industry. However, due to the specificity of the raw materials, substances such as nicotine, solanesol, tannin, pigment and the like in tobacco are easily dissolved into the leaching liquid together with the sugar, so that the liquid is dark yellow to interfere with the analysis and detection results. In the early work, a certain amount of activated carbon powder is added to eliminate the interference, however, longer decoloring time (30-60 min) is needed, the activated carbon cost is higher, and in addition, the added activated carbon powder can adsorb a certain amount of components to be detected, and can also introduce additional interference and pollution, so that the accuracy of a measurement result is reduced. In recent years, research shows that the interference can be reduced by adopting a high-power dilution method, but a higher requirement is put on the accuracy of a detection method, and a mature detection method meeting the accuracy is not developed at present. In addition, in most of the existing color development methods, concentrated sulfuric acid is used, and is a strong mineral acid with high corrosiveness, high in dehydration property, and high in danger to human bodies, and sugar carbonization is caused.
The phloroglucinol method is a spectrophotometry method with higher accuracy, is mainly applied to determination of xylose at present, and has poor color development effect on glucose. In addition, the existing ultraviolet-visible chromatography method is difficult to realize simultaneous determination of more than two kinds of saccharides respectively.
Disclosure of Invention
In order to solve the technical problems that the existing phloroglucinol method has poor glucose color development effect, more than two kinds of saccharides are difficult to be measured simultaneously by the existing ultraviolet-visible chromatography, the danger of concentrated sulfuric acid to human bodies is high, the accuracy of measuring the saccharides in tobacco by the ultraviolet-visible chromatography of substances such as alkaloids in tobacco and the like interfering the measurement results is to be improved, the first aspect of the invention provides a method for measuring the contents of glucose and xylose in a sugar solution, and the second aspect of the invention provides a method for measuring the contents of glucose and xylose in a tobacco leaching solution.
The technical scheme of the invention is as follows:
according to a first technical scheme of the invention, the method for measuring the glucose and xylose contents in the sugar solution comprises the following steps:
(1) Standard solution preparation: preparing monosaccharide standard solutions with different concentrations and a sugar mixing solution, wherein the monosaccharide standard solutions are glucose standard solutions and xylose standard solutions, and the sugar mixing solution is a mixture of monosaccharide standard solutions with known concentration in equal volume and randomly mixed;
(2) 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, 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 completed;
(3) Color reaction: adding a phloroglucinol color-developing agent solution into the hydrolysate cooled in the step (2), shaking up a test tube and standing;
(4) Establishing a standard curve: sequentially treating monosaccharide standard solutions with different concentrations and sugar-mixing solutions with known concentrations in the step (1) in the steps (2) and (3), and detecting the obtained liquid by an ultraviolet-visible spectrometry to obtain a spectral absorbance signal value; for monosaccharide standard solutions, respectively establishing a glucose standard curve and a xylose standard curve according to absorbance signal values and monosaccharide contents of the monosaccharide standard solutions with different concentrations; for the mixed sugar solution, a multi-dimensional data processing technology is utilized to establish a mapping between the spectral absorbance signal value and the glucose content and xylose content, so that a general model 1 is obtained, and when the xylose concentration in the mixed sugar solution is unchanged, a mapping of the spectral absorbance signal value increment to the glucose concentration increment is established, so that a general model 2 is established;
(5) Sample detection: taking a sample sugar solution with different concentration from that used in the step (4), performing ultraviolet-visible spectrometry 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 (3) outputting results: preliminarily judging the type of sugar in the sample sugar solution according to the position of the ultraviolet spectrum characteristic absorption peak, and if the sugar is glucose, bringing the spectrum absorbance signal value obtained in the step (5) into the glucose standard curve obtained in the step (4) to obtain the glucose content; if xylose is obtained, bringing the spectral absorbance signal value obtained in the step (5) into the xylose standard curve obtained in the step (4) to obtain xylose content; and (3) if the mixture is sugar, bringing the spectral absorbance signal value obtained in the step (5) into the general model 1 and the general model 2 obtained in the step (4) to obtain the glucose and xylose contents in the sample.
According to a second technical scheme of the invention, the method for measuring the content of glucose and xylose in the tobacco leaching liquor comprises the following steps:
(1) Preparing tobacco leaching liquor: oscillating and extracting a tobacco sample on a constant-temperature water bath oscillator by using water as an extractant, filtering, discarding initial filtrate, and collecting subsequent filtrate for later use to obtain tobacco leaching liquor;
(2) 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 solution, shaking uniformly, placing into a constant-temperature water bath for hydrolysis reaction, and taking out the test tube for cooling after the reaction is completed;
(3) Color reaction: adding a phloroglucinol color-developing agent solution into the hydrolysate cooled in the step (2), shaking up a test tube and standing;
(4) Establishing a standard curve: sequentially treating monosaccharide standard solutions with different concentrations and sugar-mixed solutions with known concentrations in the steps (2) and (3), and detecting the obtained liquid by an ultraviolet-visible spectrometry to obtain a spectral absorbance signal value; for monosaccharide solutions, respectively establishing a glucose standard curve and a xylose standard curve according to absorbance signal values and monosaccharide contents of the monosaccharide solutions with different concentrations; for the mixed sugar solution, a multi-dimensional data processing technology is utilized to establish a mapping between the spectral absorbance signal value and the glucose content and xylose content, so that a general model 1 is obtained, and when the xylose concentration in the mixed sugar solution is unchanged, a mapping of the spectral absorbance signal value increment to the glucose concentration increment is established, so that a general model 2 is established;
(5) Sample detection: diluting the tobacco leaching solution with unknown concentration obtained in the step (1), performing ultraviolet-visible spectrometry detection after the treatment in the steps (2) and (3), and recording the spectral absorbance signal value of the tobacco leaching solution;
(6) And (3) outputting results: preliminarily judging the type of sugar in the tobacco leaching solution according to the position of the ultraviolet spectrum characteristic absorption peak, and if the sugar is glucose, bringing the spectrum absorbance signal value obtained in the step (5) into the glucose standard curve obtained in the step (4) to obtain the content of glucose; if xylose is obtained, bringing the spectral absorbance signal value obtained in the step (5) into the xylose standard curve obtained in the step (4) to obtain xylose content; and (3) if the mixture is sugar, bringing the spectral absorbance signal value obtained in the step (5) into the general model 1 and the general model 2 obtained in the step (4) to obtain the glucose and xylose contents in the sample.
In the step (2) of the first and second embodiments, 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 is 1:3-1:6, the volume of the concentrated hydrochloric acid in the mixed acid is greater than the volume of glacial acetic acid, and preferably, the volume ratio of the concentrated hydrochloric acid to the glacial acetic acid is 2:1-5:1.
The constant temperature water bath temperature in the step (2) in the first technical scheme and the second technical scheme is boiling water bath temperature, and the reaction time is 40-80min.
When the step (3) in the first and second technical solutions is performed for the color reaction, a phloroglucinol/ethanol color reagent is adopted, and the volume ratio of the color reagent to the monosaccharide standard solution or the mixed sugar solution or the sample sugar solution or the tobacco leaching solution is 1:1, the concentration of the color developing agent is 2%, and the standing time is within 2 min.
The measurement wavelengths of the glucose standard solution, the xylose standard solution and the mixed sugar solution are 470nm, 478nm and 471 nm-477 nm respectively when the standard curves are established in the step (4) in the first technical scheme and the second technical scheme.
When the tobacco sample is 0.1±0.0001 g in the preparation of the tobacco extract in the step (1) in the second aspect, the volume of the extractant pure water is 20mL. The temperature of the constant-temperature water bath oscillator is 30+/-0.5 ℃, the rotating speed is 200rpm, and the oscillation extraction time is 30min.
In the second aspect, the dilution ratio of the tobacco leaching solution in the step (5) is determined by the spectral absorbance signal value of the tobacco leaching solution being between 0.7 and 1.0.
The beneficial technical effects of the invention are as follows:
According to the method, whether sugar solution or tobacco leaching solution is analyzed, firstly, after monosaccharide standard solutions with different concentrations and mixed sugar solutions with known concentrations are subjected to hydrolysis and chromogenic treatment, ultraviolet visible spectrum absorbance values are measured, a glucose standard curve, a xylose standard curve and mixed sugar universal models 1 and 2 are respectively established, and after the sample sugar solution and the tobacco leaching solution are subjected to the same hydrolysis and chromogenic treatment, ultraviolet visible spectrum absorbance values are measured. And primarily judging the types of sugar in the sugar solution and the tobacco leaching solution of the tested sample according to the positions of the absorption peaks of the ultraviolet spectrum characteristics. If the characteristic peak is located at 470nm, the solution almost contains glucose, and the glucose content in the sample sugar solution and the tobacco leaching solution can be obtained by substituting a glucose standard curve, so that the xylose is the same; if the characteristic peak appears between 471nm and 477nm, the solution is indicated to contain glucose and xylose at the same time, and is brought into the general models 1 and 2 to obtain the glucose and xylose contents in the sample sugar solution and the tobacco leaching solution.
The color development principle of the invention is that the furfural and the hydroxymethyl furfural in the glucose and xylose hydrolysate are complexed by phloroglucinol to generate yellow quinoid compound in the solution, and then the spectral absorbance value of the solution is measured by adopting ultraviolet visible chromatography.
The method adopts the phloroglucinol method for color development, and places the phloroglucinol color development reaction after the hydrolysis reaction, adopts concentrated hydrochloric acid to replace concentrated sulfuric acid in the hydrolysis reaction, reduces the consumption of glacial acetic acid to increase the hydrolysis reaction time, thereby increasing the hydrolysis time of glucose, completely converting glucose with a more stable structure into hydroxymethyl furfural, increasing the color development effect of phloroglucinol on glucose hydrolysis products, and reducing the color development result difference of glucose and xylose; and the color development is carried out at normal temperature, so that unstable quinoid intermediates formed by the phloroglucinol and aldehyde groups hydrolyzed by sugar are 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 glucose is greatly improved, and the sensitivity of the method for detecting glucose is remarkably improved, so that the phloroglucinol method is applied to the detection of glucose for the first time, the simultaneous determination of glucose and xylose is realized, and the rapid determination can be realized when the method is applied to the detection of glucose and xylose in sugar solutions, tobacco and tobacco products, and the operation is simple and convenient.
Compared with the methods for detecting the content of the tobacco sugar by using the anthrone colorimetric method, the phenol sulfuric acid colorimetric method and the like described in the current literature, the method has more excellent accuracy, has obvious experimental phenomenon on 0.01mmol/L glucose and xylose, and can realize the accurate detection of low-concentration sugar.
Based on the method, for a sample solution containing glucose and xylose simultaneously, the method adopts a multidimensional data processing technology to analyze through a two-dimensional correlation matrix, specifically, utilizes chemometrics software to analyze Partial Least Squares Regression (PLSR), establishes a mapping between a spectral absorbance signal value and glucose content and xylose content, thereby establishing a data model auxiliary, namely a mixed sugar universal model, and calculates glucose and xylose concentration so as to realize synchronous quantitative determination of the glucose and xylose content in a sugar solution and tobacco leaching liquor. And for a sample containing only monosaccharide, the monosaccharide concentration can be obtained by bringing a glucose standard curve and a xylose standard curve.
The acid used in the hydrolysis step is concentrated hydrochloric acid and glacial acetic acid, so that a milder reaction environment can be provided compared with the concentrated sulfuric acid with strong dewatering property, the problem of sugar carbonization caused by the over-strong water absorption of the concentrated sulfuric acid is avoided, the danger of the concentrated hydrochloric acid and the glacial acetic acid to a human body is much smaller than that of the concentrated sulfuric acid, and the safety problem of an experimenter in the operation process can be ensured.
The method for detecting the glucose and xylose content in the tobacco leaching liquor adopts a high-power dilution method to eliminate the color interference brought by the tobacco leaching liquor, and the subsequent detection method with high accuracy is realized by adopting the phloroglucinol ultraviolet visible chromatography on the basis. The dilution factor of the leaching solution of the tobacco sample is determined by the spectral absorbance signal value of the tobacco leaching solution being between 0.7 and 1.0. Preferably, the dilution factor of the leaching solution 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 an ultraviolet/visible spectrum characteristic absorption peak of a single glucose, single xylose, mixed sugar solution in examples 1 and 2; wherein the concentration of glucose and xylose is 0.4mmol/L; the mixed sugar A is 0.2mmol/L glucose+0.2 mmol/L xylose; the mixed sugar A is 0.1mmol/L glucose+0.1 mmol/L xylose;
FIG. 2 is a graph of absorbance signal values versus glucose concentration standard for the ultraviolet/visible spectrum in examples 1 and 2;
FIG. 3 is a graph of absorbance signal values in the ultraviolet/visible spectrum versus xylose concentration standard curve in 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 example 1 and example 2;
Fig. 5 shows the deviation between the predicted value and the measured value of the general model 1 in example 1 and example 2;
fig. 6 is a correlation analysis of predicted data and measured data obtained by the general model 2 in examples 1 and 2;
Fig. 7 shows the deviation between the predicted value and the measured value of the general model 2 in example 1 and example 2.
Detailed Description
In order to more clearly illustrate the technical scheme of the invention, the invention is further specifically described below with reference to the accompanying drawings 1-7. The present invention is not limited to the following embodiments.
A method for determining the glucose and xylose content of a sugar solution comprising the steps of:
(1) Standard solution preparation: 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 concentration in equal volume and randomly mixed.
(2) 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, shaking uniformly, placing into a constant-temperature water bath kettle for hydrolysis reaction, taking out the test tube for cooling after the reaction is finished, and carrying out the same hydrolysis reaction with equal volume of deionized water to obtain a blank sample.
In the step, concentrated hydrochloric acid is adopted to replace concentrated sulfuric acid, and the consumption 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 the volume of the glacial acetic acid, preferably, the volume ratio of the concentrated hydrochloric acid to the glacial acetic acid is 2:1-5:1, 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 boiling water bath temperature, and the reaction time is 40-80min.
(3) Color reaction: and (3) adding the phloroglucinol color-developing agent solution into the hydrolysate cooled in the step (2), shaking up a test tube and standing.
Separating the color development step from the hydrolysis step, and placing the color development reaction after hydrolysis to increase the hydrolysis time of glucose, thereby reducing the difference of color development results of the hydrolysis products of glucose and xylose and the color development of phloroglucinol; and the color development is carried out at normal temperature, so that unstable quinoid intermediates formed by the phloroglucinol and aldehyde groups hydrolyzed by sugar are prevented from being easily decomposed at high temperature, and the color development effect is improved.
The volume ratio of the color reagent to the sugar solution is 1:1, the concentration of the color developing agent is 2%, and the standing time is within 2 min.
(4) Establishing a standard curve: sequentially treating glucose standard solutions with different concentrations, xylose standard solutions and mixed sugar solutions with known concentrations in the step (1) in the steps (2) and (3), and detecting the obtained liquid by an ultraviolet-visible spectrometry to obtain a spectral absorbance signal value;
for monosaccharide standard solutions, respectively establishing a glucose standard curve and a xylose standard curve according to absorbance signal values and monosaccharide contents of the monosaccharide standard solutions with different concentrations;
And for the mixed sugar solution, a multi-dimensional data processing technology is utilized to establish a mapping between the spectral absorbance signal value and the glucose content and xylose content, so that a general model 1 is obtained, and when the xylose concentration in the mixed sugar solution is unchanged, a mapping of the spectral absorbance signal value increment to the glucose concentration increment is established, so that a general model 2 is established.
The measurement wavelengths of the glucose standard solution, the xylose standard solution and the mixed sugar solution are 470nm, 478nm and 471 nm-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), performing ultraviolet-visible spectrometry detection after the treatment in the steps (2) and (3), 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 (3) outputting results: preliminarily judging the type of sugar in the sample sugar solution according to the position of the ultraviolet spectrum characteristic absorption peak, and if the sugar is glucose, bringing the spectrum absorbance signal value obtained in the step (5) into the glucose standard curve obtained in the step (4) to obtain the glucose content; if xylose is obtained, bringing the spectral absorbance signal value obtained in the step (5) into the xylose standard curve obtained in the step (4) to obtain xylose content; and (3) if the mixture is sugar, bringing the spectral absorbance signal value obtained in the step (5) into the general model 1 and the general model 2 obtained in the step (4) to obtain the glucose and xylose contents in the sample.
A method for determining the glucose and xylose content of a tobacco extract, comprising the steps of:
(1) Preparing tobacco leaching liquor: oscillating and extracting a tobacco sample on a constant-temperature water bath oscillator by using water as an extractant, filtering, discarding initial filtrate, and collecting subsequent filtrate for later use to obtain tobacco leaching liquor;
the volume of pure water was 20mL at 0.1.+ -. 0.0001g for the tobacco sample. The temperature of the constant-temperature water bath oscillator is 30+/-0.5 ℃, the rotating speed is 200rpm, and the oscillation extraction time is 30min.
(2) 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 solution, shaking uniformly, placing into a constant-temperature water bath for hydrolysis reaction, taking out the test tube for cooling after the reaction is finished, and carrying out the same hydrolysis reaction with equal volume of deionized water to obtain a blank sample;
In the step, concentrated hydrochloric acid is adopted to replace concentrated sulfuric acid, and the consumption 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 the volume of the glacial acetic acid, preferably, the volume ratio of the concentrated hydrochloric acid to the glacial acetic acid is 2:1-5:1, 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 boiling water bath temperature, and the reaction time is 40-80min.
(3) Color reaction: adding a phloroglucinol color-developing agent solution into the hydrolysate cooled in the step (2), shaking up a test tube and standing;
Separating the color development step from the hydrolysis step, and placing the color development reaction after the hydrolysis to increase the reaction time, thereby increasing the difference of the color development results of the hydrolysis products of glucose and xylose and the color development of phloroglucinol; and the color development is carried out at normal temperature, so that unstable quinoid intermediates formed by the color development of phloroglucinol and the aldehyde group formed by hydrolysis of sugar are prevented from being easily decomposed at high temperature, and the color development effect is improved.
The volume ratio of the color reagent to the sugar solution is 1:1, the concentration of the color developing agent is 2%, and the standing time is within 2 min.
(4) Establishing a standard curve: sequentially treating glucose standard solutions with different concentrations, xylose standard solutions and mixed sugar solutions with known concentrations in the steps (2) and (3), and detecting the obtained liquid by an ultraviolet-visible spectrometry to obtain a spectral absorbance signal value;
for monosaccharide standard solutions, respectively establishing a glucose standard curve and a xylose standard curve according to absorbance signal values and monosaccharide contents of the monosaccharide standard solutions with different concentrations;
For the mixed sugar solution, a multi-dimensional data processing technology is utilized to establish a mapping between the spectral absorbance signal value and the glucose content and xylose content, so that a general model 1 is obtained, and when the xylose concentration in the mixed sugar solution is unchanged, a mapping of the spectral absorbance signal value increment to the glucose concentration increment is established, so that a general model 2 is established;
The measurement wavelengths of the glucose standard solution, the xylose standard solution and the mixed sugar solution are 470nm, 478nm and 471 nm-477 nm respectively when the standard curve is established.
(5) Sample detection: diluting the tobacco leaching solution with unknown concentration obtained in the step (1), performing ultraviolet-visible spectrometry detection after the treatment in the steps (2) and (3), and recording the spectral absorbance signal value of the tobacco leaching solution.
The dilution of the tobacco leaching solution is based on the dilution of the final absorbance between 0.7 and 1.0. The tobacco extract was diluted 5, 10, 15, 20 times by comparison and the final absorbance reached the optimal range at 20 times dilution. Different varieties of raw materials or different optimal dilution factors should be based on proper final absorbance.
(6) And (3) outputting results: preliminarily judging the type of sugar in the tobacco leaching solution according to the position of the ultraviolet spectrum characteristic absorption peak, and if the sugar is glucose, bringing the spectrum absorbance signal value obtained in the step (5) into the glucose standard curve obtained in the step (4) to obtain the glucose content; if xylose is obtained, bringing the spectral absorbance signal value obtained in the step (5) into the xylose standard curve obtained in the step (4) to obtain xylose content; and (3) if the mixture is sugar, bringing the spectral absorbance signal value obtained in the step (5) into the general model 1 and the general model 2 obtained in the step (4) to obtain the glucose and xylose contents in the sample.
The following are specific examples.
Example 1 method for determining glucose and xylose content in self-made monosaccharide, mixed sugar solution
(1) Standard solution preparation: accurately preparing 10.00mmol/L glucose and xylose stock mother solution, and diluting to obtain different monosaccharide standard solutions with concentration ranges of 0.01-2 mmol/L; mixing the prepared glucose standard solution with xylose standard solution at equal volume randomly to obtain mixed sugar solution with known concentration;
(2) 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 of 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;
(3) Color reaction: transferring 1mL of 2% concentration phloroglucinol/ethanol developer solution, adding the solution into the cooled hydrolysate in the step (2), shaking the test tube rapidly, and standing for 1min;
(4) Establishing a standard curve: carrying out ultraviolet-visible spectrometry detection on the obtained liquid after the glucose standard solution, the xylose standard solution and the mixed sugar solution prepared in the step (1) with different concentrations are treated in the steps (2) and (3), 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 measured 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;
For monosaccharide standard solutions, respectively establishing standard curves according to the obtained spectrum absorbance signal values, glucose content and xylose content of the monosaccharide standard solutions with different concentrations to obtain a glucose standard curve: y= 1.4138x-0.0196, correlation coefficient R 2 =0.9961, see fig. 2; xylose standard curve: y=4.3425x+0.0191, correlation coefficient R 2 =0.9960, see fig. 3;
for detecting glucose and xylose with the concentration of 0.01mmol/L, the final absorbance can reach 0.14 and 0.41, the experimental phenomenon is obvious, and the accurate detection of low-concentration sugar can be realized;
For the mixed sugar solution, performing Partial Least Squares Regression (PLSR) analysis by using a multidimensional data processing technology, namely chemometrics software, and establishing a mapping between a spectrum absorbance signal value and glucose content and xylose content, thereby obtaining a general model 1, wherein Y= 5.6823X 1+1.1474X2 +0.0571 (Y is absorbance, X 1 is xylose concentration and X 2 is glucose concentration), as shown in figures 4 and 5; and when the xylose concentration in the mixed sugar solution is unchanged, mapping of the spectrum absorbance signal value increment to the glucose concentration increment is established, and a general model 2 is established, wherein Y= 0.9517X 3 +0.0558 (Y is the absorbance increment and X 3 is the glucose concentration increment), so that the influence of xylose is eliminated, and the method is shown in figures 6 and 7. Calculating glucose content by the general model 2, and substituting the glucose content into the general model 1 to obtain xylose content;
(5) Sample detection: taking 1mL of a sample sugar solution with different concentration from that used in the step (4), wherein the sample sugar solution is any one of a glucose solution, a xylose solution and a mixed sugar solution, performing ultraviolet-visible spectrometry detection after the treatment of the steps (2) and (3), and recording a spectral absorbance signal value of the sample;
(6) And (3) outputting results: and (3) comparing the spectral absorbance signal value obtained in the step (5) with the general model or the monosaccharide standard curve obtained in the step (4), namely, carrying the spectral absorbance signal value into the corresponding general models 1 and 2 or the corresponding monosaccharide standard curve to obtain the glucose and/or xylose content in the sample.
And the type of sugar in the sample solution can be primarily judged according to the ultraviolet spectrum characteristic absorption peak position. If the characteristic peak is located at 470nm, the solution almost contains glucose, and the glucose content in the sample solution can be obtained by substituting a glucose standard curve, so that the xylose is the same; if the characteristic peak appears between 471nm and 477nm, the sample solution contains glucose and xylose, and is brought into the general models 1 and 2;
(7) Measurement results: the results of the monosaccharide solutions according to the monosaccharide standard curve are shown in Table 1; the results of the determination of the sugar-mixed solution 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 result.
TABLE 1 determination of monosaccharide solutions based on monosaccharide standard curves
TABLE 2 determination of sugar-mixed solution according to general model
Example 2 determination of glucose and xylose content in tobacco products
(1) Preparing tobacco leaching solution: 0.1+ -0.0001 g of tobacco sample was weighed into a 50mL triangle bottle with plug, 20mL of pure water was added, the plug was covered, and extraction was performed on a constant temperature water bath shaker at 30+ -0.5℃with shaking at 200rpm for 30min. 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 tobacco leaching liquor.
(2) 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 monosaccharide standard solution and the mixed sugar solution were at the same concentration as in example 1;
(3) Color reaction: transferring 1mL of 2% concentration phloroglucinol/ethanol developer solution, adding the solution into the cooled hydrolysate in the step (2), shaking the test tube rapidly, and standing for 1min;
(4) Establishing a standard curve: carrying out ultraviolet-visible spectrometry detection on the prepared glucose standard solution, xylose standard solution and mixed sugar solution of the glucose standard solution and xylose standard solution with different concentrations after the steps (2) and (3) are processed, wherein a 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 measured 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;
For monosaccharide standard solutions, respectively establishing standard curves according to the obtained spectrum absorbance signal values, glucose content and xylose content of the monosaccharide standard solutions with different concentrations to obtain a glucose standard curve: y= 1.4138x-0.0196, correlation coefficient R 2 =0.9961, see fig. 2; xylose standard curve: y=4.3425x+0.0191, correlation coefficient R 2 =0.9960, see fig. 3;
for detecting glucose and xylose with the concentration of 0.01mmol/L, the final absorbance can reach 0.14 and 0.41, the experimental phenomenon is obvious, and the accurate detection of low-concentration sugar can be realized;
For the mixed sugar solution, performing Partial Least Squares Regression (PLSR) analysis by using a multidimensional data processing technology, namely chemometrics software, and establishing a mapping between a spectrum absorbance signal value and glucose content and xylose content, thereby obtaining a general model 1, wherein Y= 5.6823X 1+1.1474X2 +0.0571 (Y is absorbance, X 1 is xylose concentration and X 2 is glucose concentration), as shown in figures 4 and 5; and when the xylose concentration in the mixed sugar solution is unchanged, mapping of the spectrum absorbance signal value increment to the glucose concentration increment is established, and a general model 2 is established, wherein Y= 0.9517X 3 +0.0558 (Y is the absorbance increment and X 3 is the glucose concentration increment), so that the influence of xylose is eliminated, and the method is shown in figures 6 and 7. Calculating glucose content by the general model 2, and substituting the glucose content into the general model 1 to obtain xylose content;
(5) Sample detection: and (3) diluting the prepared tobacco sample solution in the step (1), namely the tobacco leaching solution, 20 times to faint yellow, transferring lmL to a 25mL colorimetric tube with a plug, adding 4mL of concentrated hydrochloric acid and lmL glacial acetic acid, shaking uniformly, then placing into a boiling constant-temperature water bath 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 of 2% concentration phloroglucinol/ethanol color-developing agent solution, shaking uniformly the test tube rapidly, standing for 1min, then detecting by an ultraviolet-visible spectrometry, and recording the spectral absorbance signal value of the sample.
(6) And (3) outputting results: substituting the spectral absorbance signal value obtained in the step (5) into the general models 1 and 2, or into a glucose standard curve or a xylose standard curve, calculating to obtain the glucose and/or xylose content in the sample, and recording the numerical value.
And the type of sugar in the sample solution can be primarily judged according to the ultraviolet spectrum characteristic absorption peak position. If the characteristic peak is located at 470nm, the solution almost contains glucose, and the glucose content in the sample solution can be obtained by substituting a glucose standard curve, so that the xylose is the same; if the characteristic peak appears between 471nm and 477nm, the sample solution contains glucose and xylose, and is brought into the general models 1 and 2;
(7) Measurement results: the experimental results are shown in Table 3.
In order to verify the accuracy of the method, a conventional 1-phenyl-3-methyl-5-pyrazolone (English full name 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 sugar content determination results by the method and HPLC
Note that: a, respectively taking tobacco sheets with different brands at home as samples 1-4; sample 5 is an unknown cut tobacco sample; sample 6 is a domestic tobacco stalk raw material; sample 7 is the domestic reconstituted tobacco of a certain brand by a papermaking method.
B, the substance is not detected.
As can be seen from Table 3, the absolute value of the relative deviation between the method and the HPLC detection result is within 10%, and the measurement results of the two methods have no significant difference. Therefore, the method has better consistency with the measurement result of the HPLC method and high accuracy. While the present invention has been described in detail with reference to the exemplary embodiments and test examples, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present invention within the scope of the technical concept of the present invention, and it is obvious to those skilled in the art that the modifications and combinations are all within the scope of the present invention.
Claims (8)
1. A method for determining the glucose and xylose content of a sugar solution, comprising the steps of:
(1) Standard solution preparation: preparing monosaccharide standard solutions with different concentrations and a sugar mixing solution, wherein the monosaccharide standard solutions are glucose standard solutions and xylose standard solutions, and the sugar mixing solution is a mixture of monosaccharide standard solutions with known concentration in equal volume and randomly mixed;
(2) Hydrolysis reaction: adding mixed acid of concentrated hydrochloric acid and glacial acetic acid into a test tube of a monosaccharide standard solution or a mixed sugar solution, 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 completed;
(3) Color reaction: adding a phloroglucinol color-developing agent solution into the hydrolysate cooled in the step (2), shaking up a test tube and standing;
(4) Establishing a standard curve: sequentially treating monosaccharide standard solutions with different concentrations and sugar-mixing solutions with known concentrations in the step (1) in the steps (2) and (3), and detecting the obtained liquid by an ultraviolet-visible spectrometry to obtain a spectral absorbance signal value; for monosaccharide standard solutions, respectively establishing a glucose standard curve and a xylose standard curve according to absorbance signal values and monosaccharide contents of the monosaccharide standard solutions with different concentrations; for the mixed sugar solution, a multi-dimensional data processing technology is utilized to establish a mapping between the spectral absorbance signal value and the glucose content and xylose content, so that a first general model is obtained, and when the xylose concentration in the mixed sugar solution is unchanged, a mapping of the spectral absorbance signal value increment to the glucose concentration increment is established, so that a second general model is established;
(5) Sample detection: taking a sample sugar solution with different concentration from that used in the step (4), performing ultraviolet-visible spectrometry 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 or a xylose solution or a mixed sugar solution;
(6) And (3) outputting results: preliminarily judging the type of sugar in the sample solution according to the position of the ultraviolet spectrum characteristic absorption peak, and if the sugar is glucose, bringing the spectrum absorbance signal value obtained in the step (5) into the glucose standard curve obtained in the step (4) to obtain the glucose content; if xylose is obtained, bringing the spectral absorbance signal value obtained in the step (5) into the xylose standard curve obtained in the step (4) to obtain xylose content; if the mixture is sugar, the spectrum absorbance signal value obtained in the step (5) is brought into the first general model and the second general model obtained in the step (4), so that the glucose and xylose contents in the sample are obtained;
The volume ratio of the monosaccharide standard solution or the mixed sugar solution or the sample sugar solution to the mixed acid is 1:3-1:6, and the volume ratio of the concentrated hydrochloric acid to the glacial acetic acid in the mixed acid is 2:1-5:1; when the color development reaction is carried out, the volume ratio of the color development agent to the monosaccharide standard solution or the mixed sugar solution or the sample sugar solution is 1:1, the concentration of the color-developing agent is 2%.
2. A method for determining the glucose and xylose content of a tobacco extract, comprising the steps of:
(1) Preparing tobacco leaching liquor: oscillating and extracting a tobacco sample on a constant-temperature water bath oscillator by using water as an extractant, filtering, discarding initial filtrate, and collecting subsequent filtrate for later use to obtain tobacco leaching liquor;
(2) Hydrolysis reaction: adding mixed acid of concentrated hydrochloric acid and glacial acetic acid into a test tube of monosaccharide standard solution or mixed sugar solution or tobacco leaching solution, shaking uniformly, placing into a constant-temperature water bath for hydrolysis reaction, and taking out the test tube for cooling after the reaction is completed;
(3) Color reaction: adding a phloroglucinol color-developing agent solution into the hydrolysate cooled in the step (2), shaking up a test tube and standing;
(4) Establishing a standard curve: sequentially treating monosaccharide standard solutions with different concentrations and sugar-mixed solutions with known concentrations in the steps (2) and (3), and detecting the obtained liquid by an ultraviolet-visible spectrometry to obtain a spectral absorbance signal value; for monosaccharide solutions, respectively establishing a glucose standard curve and a xylose standard curve according to absorbance signal values and monosaccharide contents of the monosaccharide solutions with different concentrations; for the mixed sugar solution, a multi-dimensional data processing technology is utilized to establish a mapping between the spectral absorbance signal value and the glucose content and xylose content, so that a first general model is obtained, and when the xylose concentration in the mixed sugar solution is unchanged, a mapping of the spectral absorbance signal value increment to the glucose concentration increment is established, so that a second general model is established;
(5) Sample detection: diluting the tobacco leaching solution with unknown concentration obtained in the step (1), performing ultraviolet-visible spectrometry detection after the treatment in the steps (2) and (3), and recording the spectral absorbance signal value of a tobacco leaching solution sample;
(6) And (3) outputting results: preliminarily judging the type of sugar in the tobacco leaching solution sample according to the position of the ultraviolet spectrum characteristic absorption peak, and if the sugar is glucose, bringing the spectrum absorbance signal value obtained in the step (5) into the glucose standard curve obtained in the step (4) to obtain the content of glucose; if xylose is obtained, bringing the spectral absorbance signal value obtained in the step (5) into the xylose standard curve obtained in the step (4) to obtain xylose content; if the mixture is sugar, the spectrum absorbance signal value obtained in the step (5) is brought into the first general model and the second general model obtained in the step (4), so that the glucose and xylose contents in the sample are obtained;
Wherein the volume ratio of the monosaccharide standard solution or the mixed sugar solution or the tobacco leaching solution to the mixed acid is 1:3-1:6, and the volume ratio of the concentrated hydrochloric acid to the glacial acetic acid in the mixed acid is 2:1-5:1; when the color development reaction is carried out, the volume ratio of the color development agent to the monosaccharide standard solution or the mixed sugar solution or the tobacco leaching solution is 1:1, the concentration of the color-developing agent is 2%.
3. The method according to any one of claims 1 or 2, characterized in that the constant temperature water bath temperature in step (2) is a boiling water bath temperature and the reaction time is 40-80min.
4. The method according to any one of claims 1 or 2, characterized in that the standing time of step (3) is within 2 min.
5. The method according to any one of claims 1 or 2, wherein the measurement wavelength for the glucose standard solution, the xylose standard solution and the mixed sugar solution in the step (4) is 470nm, 478nm, 471nm to 477nm, respectively.
6. The method of claim 2, wherein the volume of extractant water is 20mL when the tobacco sample is 0.1 ± 0.0001g when the tobacco extract is prepared in step (1).
7. The method according to claim 2 or 6, wherein the temperature of the thermostatic water bath oscillator in the step (1) is 30.+ -. 0.5 ℃ and the rotation speed is 200rpm, and the oscillation extraction time is 30min.
8. A method according to claim 2, characterized in that the dilution factor of the tobacco extract in step (5) is determined by the spectral absorbance signal value of the tobacco extract being between 0.7 and 1.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210169696.5A CN114527117B (en) | 2022-02-23 | 2022-02-23 | Method for measuring glucose and xylose content in sugar solution and tobacco leaching solution |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210169696.5A CN114527117B (en) | 2022-02-23 | 2022-02-23 | Method for measuring glucose and xylose content in sugar solution and tobacco leaching solution |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114527117A CN114527117A (en) | 2022-05-24 |
| CN114527117B true CN114527117B (en) | 2024-06-18 |
Family
ID=81624450
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210169696.5A Active CN114527117B (en) | 2022-02-23 | 2022-02-23 | Method for measuring glucose and xylose content in sugar solution and tobacco leaching solution |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114527117B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111157472A (en) * | 2020-01-07 | 2020-05-15 | 云南省烟草质量监督检测站 | Method for rapidly determining content of glucose in tobacco by using enzyme-labeling instrument |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3964871A (en) * | 1974-12-18 | 1976-06-22 | Becton, Dickinson And Company | Method and device for detecting glucose |
| CN101493420B (en) * | 2009-02-24 | 2011-03-30 | 华南理工大学 | Method for rapidly measuring pentoses and hexose content in hemicellulose extract |
| CN102384907B (en) * | 2011-08-12 | 2013-04-03 | 河南科技大学 | Method by utilize vanillin-sulfuric acid colorimetry to measure glabridin content |
| CN110174362B (en) * | 2019-05-05 | 2024-05-03 | 贵州中烟工业有限责任公司 | Method for detecting content of neutral sugar and acidic sugar |
| CN110530806B (en) * | 2019-08-22 | 2022-03-29 | 中国林业科学研究院林产化学工业研究所 | Method for rapidly determining content of pentosan and cellulose in broad-leaved wood raw material |
| CN112557385A (en) * | 2020-12-07 | 2021-03-26 | 云南省烟草质量监督检测站 | Gray scale analysis method for rapidly determining glucose content in tobacco and tobacco products |
-
2022
- 2022-02-23 CN CN202210169696.5A patent/CN114527117B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| Analysis of selected alkaloids and sugars in tobacco extract;Z. Svob Troje et al.;《Journal of Chromatography A》;19971231;第101-107页 * |
| 高效液相色谱法测定烟草中的淀粉含量;王淑华 等;《烟草科技》;20041231(第7期);第31-33页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114527117A (en) | 2022-05-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Dalzell et al. | A rapid method for the measurement of Leucaena spp proanthocyanidins by the proanthocyanidin (butanol/HCl) assay | |
| Powell Gaines | Automated determination of reducing sugars, total sugars, and starch in plant tissue from one weighed sample | |
| CN114527117B (en) | Method for measuring glucose and xylose content in sugar solution and tobacco leaching solution | |
| CN110632203A (en) | Synchronous and rapid detection of vitamin A and vitamin D3And vitamin E | |
| Brownley Jr et al. | Browning of spray‐processed lactose | |
| Rocha et al. | Occurrence of furfuraldehydes during the processing of Quercus suber L. cork. Simultaneous determination of furfural, 5-hydroxymethylfurfural and 5-methylfurfural and their relation with cork polysaccharides | |
| Megarrity | An automated colorimetric method for mimosine in Leucaena leaves | |
| CN108241033A (en) | The method of 6 quality index content of material and application in a kind of quick detection Radix Ophiopogonis alcohol extract | |
| CN115791753A (en) | Dual-mode method for rapidly detecting nicotine | |
| Alamo et al. | Determination of titratable acidity and ascorbic acid in fruit juices in continuous-flow systems | |
| CN111257447B (en) | Method for analyzing stable hydrogen isotope ratio of non-exchangeable hydrogen in total sugar of food | |
| CN107860770B (en) | Method for measuring content of amino nitrogen in water extract of tobacco and tobacco products | |
| Lyons et al. | Assessment of procedures for the determination of nitrate and nitrite in vegetable extracts | |
| CN115452816A (en) | Method for detecting nitrite by ratio absorbance colorimetric method | |
| Li et al. | Rapid analysis of alcohol content during the green jujube wine fermentation by FT-NIR | |
| CN114487197A (en) | Method for detecting content of peanut lycopene in food and application thereof | |
| DiLuzio et al. | Rapid Near Infra-red Reflectance Analysis (NIRA) of Mainstream Smoke Collected on Cambridge Filter Pads | |
| Gutfinger et al. | Determination of benzoic and sorbic acids in orange juice | |
| CN115389666A (en) | Method for efficiently and simultaneously detecting ergothioneine and ectoine in cosmetics | |
| Smith | Quantifying carbohydrate fractions in forage plants | |
| Casassas et al. | Spectrophotometric determination of furosemide with phloroglucinol | |
| Wahba | A simple micro colorimetric method for the determination of lactose in milk | |
| Dong-Qun et al. | Determination of germanium in human serum by electrothermal atomic absorption spectrometry using a chemical modifier | |
| CN114814009B (en) | Analysis method simultaneously applicable to phenolic compounds in novel tobacco products and metabolites of phenolic compounds in urine | |
| EP1324016B1 (en) | Colorimetric method for determining the color of wine from the grape |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |