CN110174361A - A kind of measurement method of total reducing sugar - Google Patents

A kind of measurement method of total reducing sugar Download PDF

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Publication number
CN110174361A
CN110174361A CN201910369015.8A CN201910369015A CN110174361A CN 110174361 A CN110174361 A CN 110174361A CN 201910369015 A CN201910369015 A CN 201910369015A CN 110174361 A CN110174361 A CN 110174361A
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solution
unknown
reducing sugar
measurement method
total
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张辞海
彭黔荣
胡芸
刘娜
段凯
张佳芸
黄锡娟
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China Tobacco Guizhou Industrial Co Ltd
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/314Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/33Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/314Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
    • G01N2021/3148Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths using three or more wavelengths
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/314Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
    • G01N2021/3155Measuring in two spectral ranges, e.g. UV and visible

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  • Physics & Mathematics (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

The present invention relates to total reducing sugar detection technique fields, in particular to a kind of measurement method of total reducing sugar includes the following steps: the preparation of 1) unknown sugared sample solution: soluble solids carbohydrate sample being taken to be configured to solution of the concentration in the section 0.01-0.07g/L with ultrapure water;2) unknown sugared sample solution, acid solution the total sugar content measurement of unknown sugared sample solution: are sequentially added in test tube, it is placed in ice water bath environment after mixing, it is placed in room temperature environment, then measures its absorbance at maximum absorption band with ultraviolet-visual spectrometer;3) total sugar content of unknown sugared sample solution calculates: the total sugar content in soluble solids sugar-like sheet is calculated using the configuration proportion of standard curve regression equation and unknown sugared sample solution;Solve the problems, such as that accuracy is low, the reaction time is long for color developing agent healthhazard and detection.

Description

A kind of measurement method of total reducing sugar
Technical field
The present invention relates to total reducing sugar detection technique field, in particular to a kind of measurement method of total reducing sugar.
Background technique
The concentration of total reducing sugar in solution is detected in each research field, such as petrochemical industry, medicine, food field are all particularly significant 's.At present there are many kinds of the analysis methods of detection total reducing sugar, such as chromatography, capillary electrophoresis, infra-red sepectrometry, nuclear magnetic resonance Method, evaporative light-scattering spectroscopic methodology, colorimetric method etc..But these methods have cost more and greatly, excessively rely on expensive instrument, time-consuming, inspection The disadvantages of surveying high operation requirements.
Colorimetric method becomes detection always because of the advantages that its operation is relatively easy, testing cost is cheap, detecting instrument is relatively cheap The common method of sugar;The basic principle is that the product and the reaction of specific color developing agent after saccharide compound is hydrolyzed generate to have and add lustre to The derivative compound of group, this derivative can generate absorption at the Vis scan wave band of uv-vis spectra scanner Peak.Phenol-sulphate acid method is hydrolyzed dewatered in concentrated sulfuric acid as the most widely used colorimetric method using saccharide compound Product can generate a kind of coloured compound with phenol reactant, and this compound has maximum absorption band, but the party at 490nm Method still has following problems: 1) color developing agent phenol used in has very big harm to the health of human body, and phenol steam is to skin Skin, eyes and respiratory system are all corrosive, and contact phenol will cause scytitis or second level very to skin repeatedly or for a long time To burning for three-level;Same sucking phenol will cause pulmonary edema for a long time or repeatedly, influence central nervous system.2) reaction time Too long, the time for generally requiring 30min or more could fully reacting.3) accuracy is low;Make standard curve with glucose, and it is practical Carbohydrate complicated composition in the sample of life, not only contain glucose, also contain acid sugar, due to the phenol sulfuric acid of various carbohydrates The molar absorptivity rate difference of the substance generated after derivatization is larger, and especially neutral sugar and acid sugar is the most significant, so cause The resultant error of detection is larger.
Patent publication No. CN105510257A discloses a kind of method for measuring total sugar content in shenxiong glucose injection, 1) reference substance solution preparation step: precision weighs glucose control product 10mg, sets in 100ml measuring bottle, adds water to make to dissolve and dilutes To scale, the reference substance solution to get 0.1mg/ml is shaken up;2) preparation step of test solution: precision measures ginseng rhizome of chuanxiong grape Sugared injection 0.5ml, sets in 25ml volumetric flask, adds water 5.0ml, then plus 2.0ml hydrochloric acid, shake up;It hydrolyzes 1 hour, puts at 95 DEG C It is cold, scale is added water to, is shaken up;The accurate 1.0ml that measures is set in 10ml measuring bottle again, is diluted with water to scale, is shaken up to get test sample Solution;3) sulfuric acid solution preparation steps: 760ml sulfuric acid is diluted with water 1000ml to obtain the final product;4) Anthrone Sulphuric acid solution prepares step It is rapid: 0.2g anthrone being taken to be dissolved in 100mL sulfuric acid solution.The same day, which prepares, to be used;5) determination step: precision measures test solution 1.0mL is set in 10mL tool plug test tube, is added Anthrone Sulphuric acid solution 8.0mL prepared by step 4) after adding water 1ml, is dipped in rapidly It is cooling in ice-water bath, then in 100 DEG C of water-baths, accurately boil l0min taking-up, be cooled to room temperature to obtain measurement test sample with ice-water bath Solution;Precision is measured for reference substance solution 1.0mL, is set in 10mL tool plug test tube, adds anthracene prepared by step 4) after adding water 1ml Ketone-sulfuric acid solution 8.0mL, is dipped in rapidly in ice-water bath cooling, then in 100 DEG C of water-baths, accurately boils l0min taking-up, uses ice Water-bath cooling to room temperature must measure reference substance solution;Its absorption value is measured in 626nm with ultraviolet-uisible spectrophotometer, calculates ginseng Total sugar content in rhizome of chuanxiong glucose injection.But it also belongs to poisonous substance using anthrone, still without solve it is toxic harm and The problem of multiple types sugar part influences the accuracy of measurement.
(Su Yushun et al. is in 2011 for document " application of the UV-VIS spectrophotometry in plant polyose assay " Year deliver) and document " progress of uronic acid and neutral sugar analyzing detecting method in plant polyose " (Chen Hui et al. in Deliver within 2011) all illustrate that the middle resulting result difference of different color developing agents is larger, and material molecule is to different wave length and spy The radiation absorption degree of given wavelength is different and different material molecule is also different to the radiation absorption degree of certain wave strong point, Thus illustrate that the use of color developing agent and sample multicomponent will affect the accuracy that total sugar content detects.
Summary of the invention
In order to solve the above technical problems, The present invention provides a kind of measurement methods of total reducing sugar.
It is realized especially by following technical scheme:
A kind of measurement method of total reducing sugar, includes the following steps:
1) preparation of unknown sugared sample solution: soluble solids carbohydrate sample is taken to be configured to concentration in 0.01- with ultrapure water Solution in the section 0.07g/L;
2) the total sugar content measurement of unknown sugared sample solution: sequentially adding unknown sugared sample solution, acid solution in test tube, It places in ice water bath environment, is placed in room temperature environment after mixing, then inhaled with ultraviolet-visual spectrometer in maximum It receives and records its absorbance at peak;
3) total sugar content of unknown sugared sample solution calculates: utilizing standard curve regression equation and unknown sugared sample solution Configuration proportion calculate soluble solids sugar-like sheet in total sugar content.
The work of the ultraviolet-visual spectrometer is from wavelength be 200 to wavelength be 800nm carry out full scan, in maximum inhale It receives and records its absorbance at peak.
The wavelength of the maximum absorption band is 315nm.
The determination method of the maximum absorption band, includes the following steps:
1. glucose standards solution is prepared: taking dextrose standard sample dry to constant weight, be first configured to mother liquor with ultrapure water, so Afterwards with mother liquor at the standard solution of more concentration gradients;
2. the determination of maximum absorption band: sequentially adding glucose standards solution, acid solution in test tube, exist after mixing It places in ice water bath environment, is placed in room temperature environment, is then scanned with ultraviolet-visual spectrometer, in maximum absorption band Place's record absorbance.
The standard curve regression equation is to calculate gained according to absorbance and corresponding concentration of standard solution.
The standard curve regression equation: Y=0.0762X-0.0017.
The concentration of the mother liquor is 0.2g/L.
The concentration of standard solution of more concentration gradients is followed successively by 0.01,0.03,0.05,0.07g/L.
The temperature of the drying is 103-107 DEG C.
The standing time is 4-6min.
The utility model has the advantages that
Using the method for the present invention, the reaction time is shortened under conditions of not using color developing agent, reduces the detection of total reducing sugar Error, the present invention do not use color developing agent, improve detection safety and health, are reacted merely with acid and sugar, are contracted significantly In the short reaction time, influence of the color developing agent to accuracy of detection is also reduced, and overcomes neutral sugar and acid sugar is mutually dry The problem of disturbing and then increasing detection error.
Detailed description of the invention
Fig. 1: sugar juice concentration comparison chart (a is 1 measurement result of comparative example, and b is 1 measurement result of embodiment);
Fig. 2: reaction time comparison diagram (solid expression control methods is as a result, hollow expression the method for the present invention result).
Specific embodiment
Specific embodiments of the present invention will be described in further detail below.For those of skill in the art For member, from detailed description of the invention, the features and advantages of the present invention be will be evident.
Embodiment 1
A kind of measurement method of total reducing sugar, includes the following steps:
1) preparation of unknown sugared sample solution: soluble solids carbohydrate sample is taken to be configured to concentration in 0.01- with ultrapure water Solution in the section 0.07g/L;
2) 1mL unknown sugared sample solution, 5mL the total sugar content measurement of unknown sugared sample solution: are sequentially added in test tube Concentrated sulfuric acid solution places 5min in ice water bath environment after mixing, and 5min is placed in room temperature environment, then with ultraviolet Visible spectrophotometer from wavelength be 200 to wavelength be 800nm carry out full scan, its absorbance is recorded at 315nm;
3) total sugar content of unknown sugared sample solution calculates: utilizing standard curve regression equation and unknown sugared sample solution Configuration proportion calculate soluble solids sugar-like sheet in total sugar content.
Embodiment 2
A kind of measurement method of total reducing sugar, includes the following steps:
1) preparation of unknown sugared sample solution: soluble solids carbohydrate sample is taken to be configured to concentration in 0.01- with ultrapure water Solution in the section 0.07g/L;
2) 1mL unknown sugared sample solution, 5mL the total sugar content measurement of unknown sugared sample solution: are sequentially added in test tube Concentrated sulfuric acid solution places 4min in ice water bath environment after mixing, and 6min is placed in room temperature environment, then with ultraviolet Visible spectrophotometer from wavelength be 200 to wavelength be 800nm carry out full scan, its absorbance is recorded at 350nm;
3) total sugar content of unknown sugared sample solution calculates: utilizing standard curve regression equation and unknown sugared sample solution Configuration proportion calculate soluble solids sugar-like sheet in total sugar content.
Embodiment 3
A kind of measurement method of total reducing sugar, includes the following steps:
1) preparation of unknown sugared sample solution: soluble solids carbohydrate sample is taken to be configured to concentration in 0.01- with ultrapure water Solution in the section 0.07g/L;
2) 1mL unknown sugared sample solution, 5mL the total sugar content measurement of unknown sugared sample solution: are sequentially added in test tube Concentrated sulfuric acid solution places 4min in ice water bath environment after mixing, and 4min is placed in room temperature environment, then with ultraviolet Visible spectrophotometer from wavelength be 200 to wavelength be 800nm carry out full scan, its absorbance is recorded at 300nm;
3) total sugar content of unknown sugared sample solution calculates: utilizing standard curve regression equation and unknown sugared sample solution Configuration proportion calculate soluble solids sugar-like sheet in total sugar content.
Comparative example 1
A kind of measurement method of total reducing sugar, includes the following steps:
1) preparation of unknown sugared sample solution: soluble solids carbohydrate sample is taken to be configured to concentration in 0.01- with ultrapure water Solution in the section 0.07g/L;
2) 1mL unknown sugared sample solution, 1mL the total sugar content measurement of unknown sugared sample solution: are sequentially added in test tube After the phenol solution of 5% concentration mixes, 5mL concentrated sulfuric acid solution is added, 10min vortex 30s is placed after mixing, in ice water 20min is placed in bath environment, be then 200 from wavelength with ultraviolet-visual spectrometer to wavelength is that 800nm carries out full scan, in Its absorbance is recorded at 490nm;
3) total sugar content of unknown sugared sample solution calculates: utilizing standard curve regression equation and unknown sugared sample solution Configuration proportion calculate soluble solids sugar-like sheet in total sugar content;
Test example 1
In order to check the error condition of the method for the present invention, glucose that the present inventor's compound concentration is 0.03g/L, fructose, Sucrose, starch, glucan 1 (dextran), glucan 2 (actigum), xanthan gum, polygalacturonic acid standard solution, so Embodiment 1 is respectively adopted in various sugar juices afterwards and the method for comparative example 1 measures its absorbance value, the relative error of two methods Mean value comparing result is as shown in table 1:
Table 1
Sugar type True value (g/L) Phenol-sulphate acid method relative error mean value (%) This method relative error mean value (%)
Neutral sugar 0.03 6.5 2.8
Acid sugar 0.03 14.4 7.5
Meanwhile the present inventor also prepares glucose, fructose, sucrose, the starch, Portugal of 0.01g/L, 0.05g/L, 0.07g/L Glycan 1 (dextran), glucan 2 (actigum), xanthan gum, polygalacturonic acid standard solution, be respectively adopted embodiment 1 with And the method for comparative example 1 records its absorbance value, draws sugar juice concentration comparison chart, as (a is 1 measurement result of comparative example, b to Fig. 1 For 1 measurement result of embodiment) shown in.
The acquisition of method veracity and precision data: pass through extinction of the test various concentration known sample under this method Angle value is brought into, and the standard curve that this method is established calculates measured value.And by true value and measured value be calculated standard deviation, The results are shown in Table 2 for the rate of recovery:
Table 2
Test example 2
In order to check influence of the reaction time to test result after acid adding, the present inventor prepares the Portugal 0.01g/L and 0.07g/L Grape sugar juice, respectively as follows:
Control methods: sequentially adding the phenol solution of 1mL glucose standards solution, 5% concentration of 1mL in test tube, mixes After add 5mL concentrated sulfuric acid solution, place after mixing, then be placed in water bath and place, then use uv-vis spectra Instrument is scanned, and absorbance is recorded at maximum absorption band;
The method of the present invention: 1mL glucose standards solution, 5mL concentrated sulfuric acid solution are sequentially added in test tube, after mixing It places in ice water bath environment, is placed in room temperature environment, is then scanned with ultraviolet-visual spectrometer, in absorption maximum Absorbance is recorded at peak;
Wherein, after acid adding the reaction time be 5min, 15min, 30min, 45min, 75min, 105min, 135min, 225min, and reaction time comparison diagram is drawn according to this, as (solid expression control methods is as a result, hollow expression the method for the present invention by Fig. 2 As a result shown in).
It should be appreciated that although the present invention has carried out clear explanation by above embodiments, without departing substantially from the present invention Spirit and its essence in the case where, person of ordinary skill in the field make in accordance with the present invention it is various it is corresponding variation and Amendment, but these corresponding variations and modifications all should belong to scope of protection of the claims of the invention.

Claims (9)

1. a kind of measurement method of total reducing sugar, which comprises the steps of:
1) preparation of unknown sugared sample solution: soluble solids carbohydrate sample is taken to be configured to concentration in 0.01- with ultrapure water Solution in the section 0.07g/L;
2) unknown sugared sample solution, acid solution, mixing the total sugar content measurement of unknown sugared sample solution: are sequentially added in test tube It places in ice water bath environment after uniformly, is placed in room temperature environment, then with ultraviolet-visual spectrometer in maximum absorption band Place records its absorbance;
3) total sugar content of unknown sugared sample solution calculates: utilizing matching for standard curve regression equation and unknown sugared sample solution Ratio processed calculates the total sugar content in soluble solids sugar-like sheet.
2. the measurement method of total reducing sugar as described in claim 1, which is characterized in that the work of the ultraviolet-visual spectrometer is from wave A length of 200 carry out full scan to wavelength for 800nm, its absorbance is recorded at maximum absorption band.
3. the measurement method of total reducing sugar as described in claim 1, which is characterized in that the determination method of the maximum absorption band, including Following steps:
1. glucose standards solution is prepared: taking dextrose standard sample dry to constant weight, be first configured to mother liquor with ultrapure water, then use Mother liquor at more concentration gradients standard solution;
2. the determination of maximum absorption band: glucose standards solution, acid solution are sequentially added in test tube, after mixing in ice water It is placed in bath environment, places in room temperature environment, be then scanned with ultraviolet-visual spectrometer, remembered at maximum absorption band Record absorbance.
4. the measurement method of total reducing sugar as described in claim 1, which is characterized in that the standard curve regression equation is according to extinction Degree and corresponding concentration of standard solution calculate gained.
5. the measurement method of total reducing sugar as described in claim 1, which is characterized in that the standard curve regression equation: Y= 0.0762X-0.0017。
6. the measurement method of total reducing sugar as described in claim 1, which is characterized in that the concentration of the mother liquor is 0.2g/L.
7. the measurement method of total reducing sugar as claimed in claim 3, which is characterized in that the concentration of standard solution of more concentration gradients according to It is secondary be 0.01,0.03,0.05,0.07g/L.
8. the measurement method of total reducing sugar as claimed in claim 3, which is characterized in that the temperature of the drying is 103-107 DEG C.
9. the measurement method of total reducing sugar as claimed in claim 1 or 3, which is characterized in that the standing time is 4-6min.
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Publication number Priority date Publication date Assignee Title
CN112986231A (en) * 2021-03-08 2021-06-18 青岛农业大学 High-throughput method for measuring content of tremella polysaccharide

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