CN106324241B - The assay method of Determination of Polyphenols in a kind of Litter-fall - Google Patents
The assay method of Determination of Polyphenols in a kind of Litter-fall Download PDFInfo
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- CN106324241B CN106324241B CN201610654473.2A CN201610654473A CN106324241B CN 106324241 B CN106324241 B CN 106324241B CN 201610654473 A CN201610654473 A CN 201610654473A CN 106324241 B CN106324241 B CN 106324241B
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Abstract
The present invention relates to a kind of assay methods of Determination of Polyphenols in a kind of assay method of polyphenol content more particularly to Litter-fall.Pre-treatment is carried out using ethanol solution, measured matter is quantified into oxidation using Folin-Ciocalteu reagents as oxidant after centrifugation, quantitative analysis is finally carried out using multi-function microplate reader.Present invention optimizes the determination conditions of Determination of Polyphenols in total polyphenols, particularly Litter-fall, improve accuracy, shorten minute, saved cost, are suitble to the quantitative detection of extensive sample.
Description
Technical field
The present invention relates to a kind of measure of Determination of Polyphenols in a kind of assay method of polyphenol content more particularly to Litter-fall
Method.
Background technology
Vegatable tannin is also known as plant polyphenol, is a kind of polyphenol compound being widely present in plant, in dimension Guan Zhi
Content in object is only second to cellulose, hemicellulose and lignin, is primarily present in the skin of plant, root, leaf, fruit, content can
Up to 20%.Polyphenol in Litter-fall is a kind of polymer for being difficult to decompose, and content would generally significantly affect the decomposition of Litter-fall
Speed, polyphenol content is higher, and Decomposition of leaf litter is slower.Polyphenol in Litter-fall plays in the coevolution of plant and environment
Key player, the Litter-falls such as leaf and root rich in polyphenol enter soil, can influence microbial activity in soil, mineral element
The decomposition of validity and organic nitrogen can make plant itself become the dominant population in environment.In addition, Litter-fall polyphenol can subtract
Few nutrient loss, makes the nutrient in surrounding plants environment be maintained at higher level.Determination of Polyphenols is for reason in measure Litter-fall
Solution polyphenol enters participation Nutrient Cycling in soil by Decomposition of leaf litter and leaching and is of great significance, while can be Polyphenols object
The regulatory function that matter participates in forest ecosystem provides important evidence.
At present, the assay method of common Determination of Polyphenols have permanganimetric method, Prussia's blue laws, ferrous tartrate method,
Forint phenol method, gas-chromatography and high performance liquid chromatography etc..Wherein permanganimetric method is simple and easy to do, but the more difficult discovery of titration end-point,
Measurement sensitivity is relatively low, and the reducing substances in test solution can generally be aoxidized, and selectivity is not high.Prussia's blue laws is not steady enough
Fixed, very sensitive to the reaction time, if reaction time control is inappropriate, resultant error is larger.Ion sedimentation such as tartaric acid
Ferrous method can be determined using polyphenol substituent group with some complexing of metal ion into the property of coloured chelate with the method that ion settles
Polyphenol is measured, this method overcomes the interference of protein and reducing substances in plant sample, but the range of linearity is difficult to control, no
It is very different with the polyphenol range of linearity in sample.Paper chromatography, thin layer chromatography are determined in separating effect, separating rate and accurately
Existing defects in terms of amount.Gas-liquid chromatography for plant phenolics separation determination have the advantages that speed soon, high sensitivity, but
Derivatization treatment is needed, pre-treatment step is cumbersome, and expensive, is unsuitable for the quantitative analysis of extensive sample.And Forint phenol method
(Folin-Ciocalteu methods) under alkaline condition can be by polyphenolic substance using tungsten acid in Folin-Ciocalte reagents
Quantitative oxidation, itself is reduced (W6+Become W5+) the blue compound of generation, blue compound maximum absorption wavelength is in 600-
Between 900nm, reaction solution gradation of color is directly proportional to the number rolled into a ball containing phenolic group.Due to Folin-Ciocalteu methods
Oxidation polyphenolic substance can be quantified, accuracy and reliability is guaranteed, in addition, since experimental method is simple, reduces
The trouble of pre-treatment has saved experimental cost, using multi-function microplate reader when detecting product, improves accuracy and precision
Degree, shortens minute, is suitble to the quantitative detection of extensive sample.
Invention content
Present invention solves the technical problem that be how the content of total polyphenols and to content in rapid and accurate determination Litter-fall
The factors such as reagent concentration and dosage, Pretreatment, reaction system, detection device, Detection wavelength during measure optimize.
To solve the above problems, the present invention provides a kind of assay method of Determination of Polyphenols in Litter-fall, using certain body
The ethanol solution of product and concentration carries out pre-treatment, using Folin-Ciocalteu reagents as oxidant by measured matter after centrifugation
Quantitative oxidation, finally carries out quantitative analysis using multi-function microplate reader.Specifically comprise the steps of：
(1) it by after Litter-fall impurity elimination, dries and crushes rapidly in 55 DEG C of -65 DEG C of baking ovens, cross 70-80 mesh sieve；
(2) powder after 0.08g-0.1g steps (1) sieving is weighed, it is 70%-95% to add in 0.8mL-1mL volume fractions
Ethanol solution, seal after mixing and put in constant temperature oscillation case, vibrate 3-4 hours under room temperature in 200rpm-300rpm, then
Mixture is protected from light culture 12-16h；
(3) mixture after culture is put in supercentrifuge, 12min- is centrifuged under the conditions of 10000rpm-12000rpm
16min, supernatant are moved in new test tube, and 4 DEG C of sealing is kept in dark place for use；
(4) it draws on the supernatant to enzyme mark orifice plate that 10 μ L-15 μ L steps (3) obtain, adds in 200 μ L-300 μ L mass point
Na of the number for 1.5%-2.5%2CO3Solution reacts at room temperature 4-6min after mixing, and then adding in 10 μ L-15 μ L volume fractions is
Folin-Ciocalteu the reagents of 45%-55% are stored at room temperature 25-35min after mixing, then by enzyme mark orifice plate with multi-functional
Microplate reader surveys absorbance value at OD650nm-OD760nm, to be not added with powder as blank control；
(5) content of total polyphenols in Litter-fall is estimated by standard curve.
Further, the supernatant interior operation for carrying out step (4) in 0-3 days that the step (3) obtains.
Further, standard curve determination method is：It is accurate to weigh the dry gallic acid reference substance 0.1g to constant weight, it puts
In brown beaker plus distillation water dissolution postposition enters in 100mL brown volumetric flasks, and distilled water is added to be diluted to scale, is shaken up, is obtained
The reference substance solution of a concentration of 1mg/mL.Respectively it is accurate draw gallic acid reference substance solution 0,0.25,0.5,1.0,2.0,
3.0th, 4.0 and 5.0mL is placed in 5mL brown volumetric flasks, and respectively plus distilled water is diluted to scale, shakes up, therefrom accurate to draw 10 μ L-
On the 15 above-mentioned solution of μ L to enzyme mark orifice plate, the Na that 200 μ L-300 μ L mass fractions are 1.5%-2.5% is added in2CO3Solution, mixing
After react at room temperature 4-6min, then add in 10 μ L-15 μ L volume fractions be 45%-55% Folin-Ciocalteu reagents, mix
25-35min is stored at room temperature after even, then absorbance value at OD650nm-OD760nm is measured with multi-function microplate reader, with light absorption value
For ordinate, concentration (mg/mL) is abscissa, draws standard curve.
A kind of purposes of the assay method of above-mentioned Determination of Polyphenols is the content for measuring total polyphenols in Forest Litter.
It is dense including extracting solution present invention optimizes the determination condition of Determination of Polyphenols in total polyphenols, particularly Litter-fall
Degree and dosage, extraction time, concentration of lye and dosage, Folin-Ciocalteu reagent concentrations and dosage, reaction temperature are timely
Between, the factors such as colorimetric device, Detection wavelength, establish Folin-Ciocalteu methods and measure the suitable of Determination of Polyphenols in Litter-fall
Suitable condition.The present invention used in preceding processing 0.5mL-1mL volume fractions be 70%-95% ethanol solution as polyphenol chemical combination
The extracting solution of object, and extraction 3-4 hours is vibrated, the ethanol solution of the concentration and volume can well extract polyphenolic substance
Go out, and method is simple, as a result accurately, good reproducibility and stability.The color development system of the present invention is set on enzyme mark orifice plate,
Used sample amount and amount of reagent are few, can accurately be drawn with liquid-transfering gun, are also beneficial to the quick detection in later stage, while can be greatly
Experimental cost is saved, shortens minute.Using multi-function microplate reader when the present invention measures absorbance value, stability is high, and pole
Big improves accuracy and precision, shortens minute.
It is test sample that the present invention, which selects the Forest Litter that richness is higher, representative in subtropical forest, fortune
Each sample Determination of Polyphenols is determined with detection method of the present invention, with permanganimetric method, Prussia's blue laws, wine
The ferrous method of stone acid is compared, and error is smaller, and stability and precision are greatly improved, and particularly pre-treatment step is molten using ethyl alcohol
Liquid extracts, and destructive small, extraction is abundant, and simple operation, suitable for the quantitative analysis of extensive sample.With needing derivatization treatment
Red, orange, green, blue, yellow (ROGBY) is compared, and experimental method and step are simple and easy to do, of low cost, and reproducibility is high.
Description of the drawings
Fig. 1 is the canonical plotting of embodiment 1；
Fig. 2 is the canonical plotting of embodiment 2；
Fig. 3 is the canonical plotting of embodiment 3；
Fig. 4 is the canonical plotting of embodiment 4.
Specific embodiment
Purpose, technical scheme and advantage to make the embodiment of the present invention are clearer, below in conjunction with the embodiment of the present invention,
Technical solution in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is the present invention one
Divide embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making
All other embodiments obtained under the premise of creative work, shall fall within the protection scope of the present invention.
Embodiment 1：
The assay method of Determination of Polyphenols in a kind of Litter-fall, using ethanol solution carry out pre-treatment, with Folin-
Measured matter is quantified oxidation by Ciocalteu reagents, finally carries out quantitative analysis using multi-function microplate reader.By following steps group
Into：
(1) it by after Litter-fall impurity elimination, dries and crushes rapidly in 55 DEG C of baking ovens, cross 70 mesh sieve；
(2) powder after 0.08g steps (1) sieving is weighed, the ethanol solution that 0.8mL volume fractions are 70% is added in, mixes
Sealing is put in constant temperature oscillation case after closing uniformly, is vibrated under room temperature 3 hours in 250rpm, and then mixture, which is protected from light, cultivates 16h；
(3) mixture after culture is put in supercentrifuge, 15min is centrifuged under the conditions of 10000rpm, supernatant moves to
In new test tube, for use, the operation of progress step (4) in 0-3 days is kept in dark place in 4 DEG C of sealing；
(4) it draws on the supernatant to enzyme mark orifice plate that 15 μ L steps (3) obtain, it is 2.5% to add in 300 μ L mass fractions
Na2CO3Solution reacts at room temperature 6min after mixing, then adds in the Folin-Ciocalteu reagents that 15 μ L volume fractions are 55%,
35min is stored at room temperature after mixing, enzyme mark orifice plate multi-function microplate reader is then surveyed into absorbance value at OD760nm, to be not added with
Powder is as blank control；
(5) content of total polyphenols in Litter-fall is estimated by standard curve.
Standard curve determination method is：It is accurate to weigh the dry gallic acid reference substance 0.1g to constant weight, it is placed in brown burning
In cup plus distillation water dissolution postposition enters in 100mL brown volumetric flasks, and distilled water is added to be diluted to scale, is shaken up, is obtained a concentration of
The reference substance solution of 1mg/mL.It is accurate respectively to draw 0,0.25,0.5,1.0,2.0,3.0,4.0 and of gallic acid reference substance solution
5.0mL is placed in 5mL brown volumetric flasks, and respectively plus distilled water is diluted to scale, shakes up, therefrom accurate to draw the 15 above-mentioned solution of μ L extremely
On enzyme mark orifice plate, the Na that 300 μ L mass fractions are 2.5% is added in2CO3Solution reacts at room temperature 6min after mixing, then adds in 15 μ
L volume fractions are 55% Folin-Ciocalteu reagents, are stored at room temperature 35min after mixing, are then surveyed with multi-function microplate reader
Determine absorbance value at OD760nm, using light absorption value as ordinate, concentration (mg/mL) is abscissa, draws standard curve.
The purposes of the assay method of Determination of Polyphenols is for measuring locust tree (Robinia in a kind of above-mentioned Litter-fall
Pseudoacacia) in Litter-fall total polyphenols content.According to standard curve such as Fig. 1, the Determination of Polyphenols measured is
79.4mg·g-1.And using the Determination of Polyphenols that Prussia's blue laws measures as 64.5mgg-1, and in Prussia's blue laws continuous mode
Solution is susceptible to layering, and the inhomogenous of system causes light absorption value reading unstable, this proves that the method for the present invention has high precision
Property and stability, while the time used in the present invention about for 24 hours, compared to red, orange, green, blue, yellow (ROGBY), substantially reduce experimental period, save
Manpower and materials and experimental cost.
Embodiment 2：
The assay method of Determination of Polyphenols in a kind of Litter-fall, using ethanol solution carry out pre-treatment, with Folin-
Measured matter is quantified oxidation by Ciocalteu reagents, finally carries out quantitative analysis using multi-function microplate reader.By following steps group
Into：
(1) it by after Litter-fall impurity elimination, dries and crushes rapidly in 60 DEG C of baking ovens, cross 70 mesh sieve；
(2) powder after 0.09g steps (1) sieving is weighed, the ethanol solution that 0.9mL volume fractions are 80% is added in, mixes
Sealing is put in constant temperature oscillation case after even, is vibrated under room temperature 4 hours in 220rpm, and then mixture, which is protected from light, cultivates 14h；
(3) mixture after culture is put in supercentrifuge, 16min is centrifuged under the conditions of 11000rpm, supernatant moves to
In new test tube, for use, the operation of progress step (4) in obtained supernatant 0-3 days is kept in dark place in 4 DEG C of sealing；
(4) it draws on the supernatant to enzyme mark orifice plate that 13 μ L steps (3) obtain, it is 2.0% to add in 250 μ L mass fractions
Na2CO3Solution reacts at room temperature 5min after mixing, then adds in the Folin-Ciocalteu reagents that 13 μ L volume fractions are 50%,
30min is stored at room temperature after mixing, enzyme mark orifice plate multi-function microplate reader is then surveyed into absorbance value at OD690nm, to be not added with
Powder is as blank control；
(5) content of total polyphenols in Litter-fall is estimated by standard curve.
Standard curve determination method is：It is accurate to weigh the dry gallic acid reference substance 0.1g to constant weight, it is placed in brown burning
In cup plus distillation water dissolution postposition enters in 100mL brown volumetric flasks, and distilled water is added to be diluted to scale, is shaken up, is obtained a concentration of
The reference substance solution of 1mg/mL.It is accurate respectively to draw 0,0.25,0.5,1.0,2.0,3.0,4.0 and of gallic acid reference substance solution
5.0mL is placed in 5mL brown volumetric flasks, and respectively plus distilled water is diluted to scale, shakes up, therefrom accurate to draw the 13 above-mentioned solution of μ L extremely
On enzyme mark orifice plate, the Na that 250 μ L mass fractions are 2.0% is added in2CO3Solution reacts at room temperature 5min after mixing, then adds in 13 μ
L volume fractions are 50% Folin-Ciocalteu reagents, are stored at room temperature 30min after mixing, are then surveyed with multi-function microplate reader
Determine absorbance value at OD690nm, using light absorption value as ordinate, concentration (mg/mL) is abscissa, draws standard curve.
The purposes of the assay method of Determination of Polyphenols is for measuring sweetgum (Liquidambar in a kind of above-mentioned Litter-fall
Formosana) in Litter-fall total polyphenols content.According to standard curve such as Fig. 2, the Determination of Polyphenols measured is 270mgg-1。
And using the Determination of Polyphenols that Prussia's blue laws measures as 239.6mgg-1, and light absorption value reading in Prussia's blue laws continuous mode
Unstable, this proves that the method for the present invention has high accuracy and stability, while the time used in the present invention is about for 24 hours, compared to
Red, orange, green, blue, yellow (ROGBY) substantially reduces experimental period, saves manpower and materials and experimental cost.
Embodiment 3：
The assay method of Determination of Polyphenols in a kind of Litter-fall, using ethanol solution carry out pre-treatment, with Folin-
Measured matter is quantified oxidation by Ciocalteu reagents, finally carries out quantitative analysis using multi-function microplate reader.By following steps group
Into：
(1) it by after Litter-fall impurity elimination, dries and crushes rapidly in 65 DEG C of baking ovens, cross 80 mesh sieve；
(2) powder after 0.1g steps (1) sieving is weighed, 1mL volume fractions are added in as 95% ethanol solution, after mixing
Sealing is put in constant temperature oscillation case, is vibrated under room temperature 3.5 hours in 300rpm, and then mixture, which is protected from light, cultivates 12h；
(3) mixture after culture is put in supercentrifuge, 12min is centrifuged under the conditions of 12000rpm, supernatant moves to
In new test tube, for use, the operation of progress step (4) in obtained supernatant 0-3 days is kept in dark place in 4 DEG C of sealing；
(4) it draws on the supernatant to enzyme mark orifice plate that 10 μ L steps (3) obtain, it is 2.0% to add in 200 μ L mass fractions
Na2CO3Solution reacts at room temperature 4min after mixing, then adds in the Folin-Ciocalteu reagents that 10 μ L volume fractions are 45%,
25min is stored at room temperature after mixing, enzyme mark orifice plate multi-function microplate reader is then surveyed into absorbance value at OD650nm, to be not added with
Powder is as blank control；
(5) content of total polyphenols in Litter-fall is estimated by standard curve.
Standard curve determination method is：It is accurate to weigh the dry gallic acid reference substance 0.1g to constant weight, it is placed in brown burning
In cup plus distillation water dissolution postposition enters in 100mL brown volumetric flasks, and distilled water is added to be diluted to scale, is shaken up, is obtained a concentration of
The reference substance solution of 1mg/mL.It is accurate respectively to draw 0,0.25,0.5,1.0,2.0,3.0,4.0 and of gallic acid reference substance solution
5.0mL is placed in 5mL brown volumetric flasks, and respectively plus distilled water is diluted to scale, shakes up, therefrom accurate to draw the 10 above-mentioned solution of μ L extremely
On enzyme mark orifice plate, the Na that 200 μ L mass fractions are 2.0% is added in2CO3Solution reacts at room temperature 4min after mixing, then adds in 10 μ
L volume fractions are 45% Folin-Ciocalteu reagents, are stored at room temperature 25min after mixing, are then surveyed with multi-function microplate reader
Determine absorbance value at OD650nm, using light absorption value as ordinate, concentration (mg/mL) is abscissa, draws standard curve.
The purposes of the assay method of Determination of Polyphenols is for measuring masson pine (Pinus in a kind of above-mentioned Litter-fall
Massoniana) in Litter-fall total polyphenols content.According to standard curve such as Fig. 3, the Determination of Polyphenols measured is
192.97mg·g-1.And using the Determination of Polyphenols that Prussia's blue laws measures as 169.5mgg-1, and Prussia's blue laws measured
Solution is susceptible to layering in journey, and light absorption value reading is unstable, this proves that the method for the present invention has high accuracy and stability, together
When the time used in the present invention be about 21h, compared to red, orange, green, blue, yellow (ROGBY), substantially reduce experimental period, save manpower and materials and
Experimental cost.
Embodiment 4：
The assay method of Determination of Polyphenols in a kind of Litter-fall, using ethanol solution carry out pre-treatment, with Folin-
Measured matter is quantified oxidation by Ciocalteu reagents, finally carries out quantitative analysis using multi-function microplate reader.By following steps group
Into：
(1) it by after Litter-fall impurity elimination, dries and crushes rapidly in 60 DEG C of baking ovens, cross 80 mesh sieve；
(2) powder after 0.1g steps (1) sieving is weighed, 1mL volume fractions are added in as 90% ethanol solution, after mixing
Sealing is put in constant temperature oscillation case, is vibrated under room temperature 4 hours in 300rpm, and then mixture, which is protected from light, cultivates 15h；
(3) mixture after culture is put in supercentrifuge, 16min is centrifuged under the conditions of 11000rpm, supernatant moves to
In new test tube, for use, the operation of progress step (4) in obtained supernatant 0-3 days is kept in dark place in 4 DEG C of sealing；
(4) it draws on the supernatant to enzyme mark orifice plate that 12 μ L steps (3) obtain, it is 1.5% to add in 250 μ L mass fractions
Na2CO3Solution reacts at room temperature 5min after mixing, then adds in the Folin-Ciocalteu reagents that 12 μ L volume fractions are 50%,
30min is stored at room temperature after mixing, enzyme mark orifice plate multi-function microplate reader is then surveyed into absorbance value at OD720nm, to be not added with
Powder is as blank control；
(5) content of total polyphenols in Litter-fall is estimated by standard curve.
Standard curve determination method is：It is accurate to weigh the dry gallic acid reference substance 0.1g to constant weight, it is placed in brown burning
In cup plus distillation water dissolution postposition enters in 100mL brown volumetric flasks, and distilled water is added to be diluted to scale, is shaken up, is obtained a concentration of
The reference substance solution of 1mg/mL.It is accurate respectively to draw 0,0.25,0.5,1.0,2.0,3.0,4.0 and of gallic acid reference substance solution
5.0mL is placed in 5mL brown volumetric flasks, and respectively plus distilled water is diluted to scale, shakes up, therefrom accurate to draw the 12 above-mentioned solution of μ L extremely
On enzyme mark orifice plate, the Na that 250 μ L mass fractions are 1.5% is added in2CO3Solution reacts at room temperature 5min after mixing, then adds in 12 μ
L volume fractions are 50% Folin-Ciocalteu reagents, are stored at room temperature 30min after mixing, are then surveyed with multi-function microplate reader
Determine absorbance value at OD720nm, using light absorption value as ordinate, concentration (mg/mL) is abscissa, draws standard curve.
The purposes of the assay method of Determination of Polyphenols is for measuring Quercus acutissima (Quercus in a kind of above-mentioned Litter-fall
Acutissima) in Litter-fall total polyphenols content.According to standard curve such as Fig. 4, the Determination of Polyphenols measured is
206.37mg·g-1.And using the Determination of Polyphenols that Prussia's blue laws measures as 176.7mgg-1, and Prussia's blue laws measured
There is layering in solution in journey, and the inhomogenous of system causes light absorption value reading unstable, this proves that the method for the present invention has Gao Zhun
True property and stability, while the time used in the present invention is about for 24 hours, compared to red, orange, green, blue, yellow (ROGBY), substantially reduces experimental period, saves
Manpower and materials and experimental cost are saved.
Claims (3)
1. the assay method of Determination of Polyphenols in a kind of Litter-fall, which is characterized in that comprise the steps of：
(1) it by after Litter-fall impurity elimination, dries and crushes rapidly in 55 DEG C of -65 DEG C of baking ovens, cross 70-80 mesh sieve；
(2) powder after 0.08g-0.1g steps (1) sieving is weighed, adds in the second that 0.8mL-1mL volume fractions are 70%-95%
Alcoholic solution is sealed after mixing and is put in constant temperature oscillation case, vibrates 3-4 hours in 200rpm-300rpm, then mix under room temperature
Object is protected from light culture 12-16h；
(3) mixture after culture is put in supercentrifuge, 12min-16min is centrifuged under the conditions of 10000rpm-12000rpm,
Supernatant is moved in new test tube, and 4 DEG C of sealing is kept in dark place for use；
(4) it draws on the supernatant to enzyme mark orifice plate that 10 μ L-15 μ L steps (3) obtain, adding in 200 μ L-300 μ L mass fractions is
The Na of 1.5%-2.5%2CO3Solution reacts at room temperature 4-6min after mixing, then adds in 10 μ L-15 μ L volume fractions as 45%-
55% Folin-Ciocalteu reagents are stored at room temperature 25-35min after mixing, then by enzyme mark orifice plate multi-function microplate reader
Absorbance value is surveyed at OD650nm-OD760nm, to be not added with powder as blank control；
(5) content of total polyphenols in Litter-fall is estimated by standard curve.
2. the assay method of Determination of Polyphenols in a kind of Litter-fall as described in claim 1, it is characterised in that：The step
(3) supernatant obtained carries out the operation of step (4) preserving in 0-3 days.
3. the assay method of Determination of Polyphenols in a kind of Litter-fall as described in claim 1, it is characterised in that：In step (5)
Standard curve determination method is：It is accurate to weigh the dry gallic acid reference substance 0.1g to constant weight, it is placed in brown beaker plus steams
After distilled water dissolving in merging 100mL brown volumetric flasks, distilled water is added to be diluted to scale, is shaken up, obtains pair of a concentration of 1mg/mL
According to product solution；0,0.25,0.5,1.0,2.0,3.0,4.0 and 5.0mL of gallic acid reference substance solution is accurately drawn respectively to be placed in
In 5mL brown volumetric flasks, respectively plus distilled water is diluted to scale, shakes up, and therefrom the accurate 10 μ above-mentioned solution of L-15 μ L of drawing are to enzyme mark
On orifice plate, the Na that 200 μ L-300 μ L mass fractions are 1.5%-2.5% is added in2CO3Solution reacts at room temperature 4-6min after mixing,
Then Folin-Ciocalteu the reagents that 10 μ L-15 μ L volume fractions are 45%-55% are added in, 25- is stored at room temperature after mixing
Then 35min measures absorbance value at OD650nm-OD760nm with multi-function microplate reader, a concentration of using light absorption value as ordinate
Abscissa draws standard curve.
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