CN101701946B - Measuring method of water soluble sugar in tobacco - Google Patents
Measuring method of water soluble sugar in tobacco Download PDFInfo
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- CN101701946B CN101701946B CN2009101941694A CN200910194169A CN101701946B CN 101701946 B CN101701946 B CN 101701946B CN 2009101941694 A CN2009101941694 A CN 2009101941694A CN 200910194169 A CN200910194169 A CN 200910194169A CN 101701946 B CN101701946 B CN 101701946B
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Abstract
The invention discloses a measuring method of water soluble sugar in tobacco and establishes a method for measuring water soluble sugar in the tobacco by combining high performance liquid chromatography with differential refraction detector. The method in the invention has good linearity, the recovery rate is between 99.2%-101.7%, the degree of precision is between 0.14%-0.86%, and the detection limit of fruit sugar, glucose and cane sugar is between 0.39mu g/mL and 0.71mu g/mL. The method does not use toxic reagent, and compared with experiments, the detection result is in accordance with that of the current ion chromatography detection standard, thus being applied to rapid and batch detection of water soluble sugar in various tobacco samples.
Description
Technical field
The invention belongs to the check and analysis technical field, be specifically related to the assay method of water-soluble sugar in growing tobacco, relate in particular to the assay method of fructose in the tobacco, dextrose plus saccharose.
Background technology
Water-soluble sugar in the tobacco and the mouthfeel of tobacco, fragrance characteristic and tar growing amount are in close relations; Tobacco water-soluble sugar content and its place of production and kind all have relation, and the water-soluble sugar of measuring in the tobacco all has crucial meaning to compound design, explained hereafter, cigarette quality monitoring and reducing cigarette tar and reducing harm reduction.
Fructose, dextrose plus saccharose are water-soluble sugars important in the tobacco, and the ratio that accounts for the tobacco water-soluble sugar is very big.The liquid phase chromatography and the chromatography of ions are to measure the main method of these three kinds of sugar in the tobacco.Water-soluble sugar in the ion-chromatographic determination tobacco of YC/T 251 regulations; The chromatographic run time that comprises column equilibration is longer; Need more than the 60min; The sample preparation time is longer relatively, is unfavorable for the raising of laboratory detection efficiency, and the maintenance work that chromatographic column and detecting electrode need carry out is more.
The gloomy Walker method of the awns of YC/T 32-1996 regulation measures tobacco and the tobacco product water-soluble sugar can't be measured separately sucrose in the tobacco and fructose, can only indirect calculation draws reducing sugar (or the water-soluble total reducing sugar) content of sample.
Liu Jun etc. have set up the new method that high performance liquid chromatography is measured 7 kinds of water-soluble sugars such as rhamnose in the tobacco, wood sugar, fructose, mannose, glucose, sucrose, maltose simultaneously; Be with chromatographic column ZORBAX glycan analysis post (4.6mm * 150mm; 5 μ m); Acetonitrile: water=flow velocity was 1ml/min as moving phase in 68: 32, and detecting device is an EISD; Sun Yuan etc. adopt water-bath leaching method to extract the sugar in the tobacco; With Zorbax glycan analysis post (column temperature is 30 ℃), acetonitrile: water (80: 20, V/V) be the content of fructose, glucose, sucrose and maltose in moving phase and the EISD separation determination tobacco.Above reported method is all used EISD; And the laboratory that does not much have EISD can't be carried out the mensuration work of tobacco sugar with reference to said method; The detectability of three kinds of sugar all is greater than or equal to 1.6 μ g/mL; And need to use a large amount of poisonous and harmful reagent acetonitriles, be unfavorable for environmental protection.
Summary of the invention
The objective of the invention is deficiency, a kind of new assay method is provided, adopt high performance liquid chromatography to combine the differential refraction detection method to realize the simple rapid batch analysis of a large amount of samples to the determination techniques of water-soluble sugar in the existing tobacco.
The object of the invention is achieved through following technical scheme:
Provide one grow tobacco in the assay method of water-soluble sugar, adopt high-efficiency liquid chromatography method for detecting, specifically may further comprise the steps:
(1) with extract solvent to the tobacco sample extraction after filter the filtrating that obtains as analyte sample fluid;
(2) adopt high performance liquid chromatography to combine differential refraction detector to carry out the assay determination of sample water-soluble sugar.
Above-mentioned steps (2) be to use with the Zeo-karb be filler chromatographic column, as moving phase the water-soluble sugar in the tobacco is carried out efficient liquid phase chromatographic analysis with pure water and measures.
The preferred Metacarb 67C of said cation-exchange chromatography post chromatographic column.
The present invention provides preferred chromatographic condition: the Metacarb 67C of Varian company chromatographic column (300mm * 6.5mm), Metacarb 67C pre-column post core.Sample size: 20 μ L; Flow velocity: 0.5mL/min; The termination of pumping time: 40min; Column oven: 80 ℃; Differential refraction detector (RID) optical element temperature: 40 ℃; Moving phase: water, constant gradient wash-out.
The invention has the beneficial effects as follows:
The inventive method is used water-soluble sugar in the high-performance liquid chromatography method tobacco be combined with differential refraction detector, employing be the laboratory common instrument, do not use EISD to carry out the mensuration work of tobacco sugar.
The detectability of the inventive method is between 0.39 μ g/mL～0.71 μ g/mL, and compared with prior art, detectability is lower, and the fructose, the dextrose plus saccharose that help the low sample of sugar content detect.
The present invention uses with Zeo-karb and the water-soluble sugar in the tobacco is analyzed as moving phase with pure water simultaneously as the chromatographic column of filler; Avoided using poisonous and hazardous organic solvent; Only use cheap pure water just can well measure to the water-soluble sugar in the tobacco as moving phase; Highly sensitive, accurate, easy; Satisfy to fructose, dextrose plus saccharose in the various tobacco samples fast, accurately, the batch detection requirement, reach that detectability is low, good reproducibility, sample pre-treatments are simple, detect the low effect of cost, and can accomplish analysis faster a large amount of samples.
Description of drawings
Fig. 1 fructose, dextrose plus saccharose standard colors spectrogram
Fig. 2 tobacco sample chromatogram
Embodiment
Below in conjunction with accompanying drawing and specific embodiment further explain the present invention.
The instrument and the reagent that use in the embodiment of the invention are following:
Instrument and reagent
Instrument: 1200HPLC is equipped with G1378B degasser, G1311A quaternary pump, G1329A automatic sampler, G1316A column oven and G1362A differential refraction detector (U.S. Agilent company); Milli-Q ElementA10 water purification machine (U.S. Millipore company); Fuhua HY-5 cyclotron oscillation device (homemade, Jintan City's Fuhua Electrical Appliances Co., Ltd), German Sartorious electronic balance CP 224S.
Reagent: sucrose, D (-)-fructose, D-(+)-DEXTROSE ANHYDROUS (U.S. Sigma-Aldrich company); NaOH (Belgian Pure Chemical Analysis Co., Ltd.), acetonitrile (99.9%GC, German MERCK company); Milli-Q level water, by Milliore Element A10 preparation, if without special instruction, experimental water is Milli-Q level water in the present embodiment.
The selection of embodiment 1 efficient liquid phase chromatographic analysis chromatographic condition
(1) selection of chromatographic column
Experiment has contrasted surveys the sugar Metacarb 67C commonly used and the cation-exchange chromatography post of two kinds of calcium patterns of Sugar-PAK I.
The moving phase of Sugar-PAK I chromatographic column is the Ca EDTA of 0.001M, and analysis time is greater than 1 day the time, and needs with heating arrangement the moving phase temperature maintenance at 70～90 ℃, to prevent CO
2Dissolve in Deng gas, operation is trouble, and follows a small amount of evaporation of moisture during heating; The concentration of Ca EDTA in moving phase increases, and the retention time of sugar to be measured is changed, and RID is responsive to the change in concentration of moving phase; Cause the fluctuation of baseline, thereby influence the constant of testing conditions.
Metacarb 67C only need use pure water as moving phase, convenient changes moving phase at any time, and the retention time of sugar to be measured can be consistent for a long time, and the relative standard deviation of three kinds of sugared retention times in 123 days is seen shown in the table 1 less than 0.07%.
The regenerative operation of Sugar-PAK I chromatographic column is also frequent than Metacarb 67C.Easy to use, the analytical effect of Metacarb 67C and favorable reproducibility are elected to be experiment and use.
Table 1 sucrose, fructose and the glucose retention time on Metacarb 67C chromatographic column
(2) column temperature and analysis time condition
When column temperature is 80C～90 ℃, help reducing obtaining preferable post when Metacarb 67C chromatographic column post is pressed and imitate.Experimental result is seen accompanying drawing 1 with shown in the accompanying drawing 2, and 1 is sucrose among the figure, and 2 is glucose, and 3 is fructose, and column temperature is made as 80 ℃, and glucose, fructose and sucrose separate totally with contiguous assorted peak, and retention time is in 12min.
For other materials in the abundant wash-out tobacco sample, be 40min with the chromatographic run time set.
The chromatographic condition that the present invention confirms is: Metacarb 67C chromatographic column (300mm * 6.5mm), Metacarb 67C pre-column post core (Varian company).Sample size: 20 μ L; Flow velocity: 0.5mL/min; The termination of pumping time: 40min; Column oven: 80 ℃; Differential refraction detector (RID) optical element temperature: 40 ℃; Moving phase: water, constant gradient wash-out.
The determination experiment of water-soluble sugar in embodiment 2 tobacco samples
Use this method and tobacco sample is carried out the mensuration of water-soluble sugar.
(1) sample pre-treatments
Take by weighing the 1g tobacco powder, be accurate to 0.0001g, place the 100mL triangular flask, add the 50mL0.01M sodium hydroxide solution, tool plug oscillation extraction 60min, to be measured behind 0.45 μ m water membrane filtration;
(2) preparation of standard solution
Take by weighing glucose 0.25g, fructose 0.25g, sucrose 0.15g, be accurate to 0.0001g, place the 100mL volumetric flask, the water constant volume gets the standard reserving solution that glucose, fructose and sucrose concentration are respectively 2500 μ g/mL, 2500 μ g/mL and 1500 μ g/mL.Be diluted with water to needed concentration more step by step.
(3) efficient liquid phase chromatographic analysis
Chromatographic condition: the Metacarb 67C of Varian company chromatographic column (300mm * 6.5mm), Metacarb 67C pre-column post core.Sample size: 20 μ L; Flow velocity: 0.5mL/min; The termination of pumping time: 40min; Column oven: 80 ℃; Differential refraction detector (RID) optical element temperature: 40 ℃; Moving phase: water, constant gradient wash-out.
Measuring the result sees shown in the table 2.
The fructose of table 2 actual sample, dextrose plus saccharose content (%, w/w)
Can be known that by table 2 most of tobaccos and tobacco product are the highest with fructose and glucose content, cane sugar content is relatively low.
Typical curve linearity, detection limit and method precision
The employing external standard method is quantitative.(μ g/mL) is horizontal ordinate x with concentration, and peak area carries out linear regression for ordinate y.With signal to noise ratio (S/N ratio) is 3, confirms detection limit with the method for stepwise dilution low concentration standard solution.According to this inventive method tobacco sample A is carried out 5 replicate determinations, get method precision, represent that with RSD the result sees shown in the table 3.
The linear equation of table 3 typical curve, related coefficient, detection limit and method precision
Visible by table 3, the linearity of the inventive method is good, and the facies relationship number average is greater than 0.9999; The precision of fructose, dextrose plus saccharose is between 0.14%～0.86%, and the precision of method is good; The detection limit of method is low, can satisfy the detection requirement to all kinds of tobacco sample fructose, dextrose plus saccharose.
The method recovery
Get tobacco sample, with the mean value measured for its parallel 5 times foundation as the original content that calculates its fructose, dextrose plus saccharose, add the standard of two concentration levels of height and carry out recovery test, experimental result is seen shown in the table 4.
Table 4 method recovery experimental result
Visible by table 4, the recovery of fructose, dextrose plus saccharose is between 99.2%～101.7%, and the result is desirable.
The test data contrast of embodiment 3 the inventive method and ion chromatographic method
This method and chromatography of ions YC/T 251 methods have been carried out the contrast of detection data, and testing result is seen table 5.
The test data comparison of table 5 this method (LC) and ion chromatographic method (IC) (%, w/w)
Visible by table 5, the relative average debiation of the test number of the inventive method and YC/T 251 methods is all less than 4.1%, and the consistance between method is better.
In sum; The inventive method is linear good, r >=0.9999, and the recovery is between 99.2%～101.7%; Precision is between 0.14%～0.86%, and the detection limit of fructose, dextrose plus saccharose (LOD) is respectively 0.60,0.71 and 0.39 μ g/mL (S/N=3: 1).
Through the experiment contrast, the testing result of this method and the chromatography of ions is consistent.And the inventive method detectability is low, good reproducibility, and the recovery is desirable, and sample pre-treatments is simple, and analysis efficiency is enhanced about more than once than YC/T 251 methods, effectively raises breadboard daily detection efficiency, can accomplish the analysis to a large amount of samples faster.
Claims (1)
- One grow tobacco in the assay method of water-soluble sugar, it is characterized in that adopting high performance liquid chromatography, may further comprise the steps:(1) with extract solvent to the tobacco sample extraction after filter the filtrating that obtains as analyte sample fluid;(2) adopt high performance liquid chromatography to combine differential refraction detector to carry out the assay determination of sample water-soluble sugar;Wherein, the said extraction solvent of step (1) is the 0.01M sodium hydroxide solution;The said high performance liquid chromatography chromatographic condition of step (2) is Metacarb 67C chromatographic column, Metacarb 67C pre-column post core; Sample size is 20 μ L; Flow velocity is 0.5mL/min; The termination of pumping time is 40min; Column oven is 80 ℃; Differential refraction detector optical element temperature is 40 ℃; Moving phase is water, the constant gradient wash-out.
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| CN105116088A (en) * | 2015-08-19 | 2015-12-02 | 汤臣倍健股份有限公司 | Detection method for mannose content in dendrobium officinale |
| CN106596758A (en) * | 2016-12-04 | 2017-04-26 | 中国水稻研究所 | Method for simultaneously testing multiple free sugars in rice |
| CN108508103A (en) * | 2018-02-12 | 2018-09-07 | 云南中烟工业有限责任公司 | A method of measuring water-soluble sugar in tobacco |
| CN109030656A (en) * | 2018-08-17 | 2018-12-18 | 华仁药业股份有限公司 | Detection method of the glucose in relation to substance in acetate Multiple electrolytes injection |
| CN110836931A (en) * | 2019-10-10 | 2020-02-25 | 国家烟草质量监督检验中心 | Detection method for saccharides in heating non-combustible tobacco products |
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Non-Patent Citations (3)
| Title |
|---|
| CORESTA.DETERMINATION OF 1,2-PROPYLENE GLYCOL, GLYCEROL AND SORBITOL IN TOBACCO AND TOBACCO PRODUCTS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC).《CORESTA RECOMMENDED METHOD N° 61》.2005,第1－10页. * |
| 周元清等.高效液相色谱法同时测定烟草中的糖和烟碱.《http://www.chemistrymag.org/cji/2006/082009nc.htm》.2006,第8卷(第2期),全文. * |
| 施红林等.高效液相色谱法测定烟草中的糖.《分析化学》.2002,第30卷(第3期),第384页. * |
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