CN110286179A - The quickly high performance liquid chromatography of detection Zeaxanthin cycle pigment - Google Patents

The quickly high performance liquid chromatography of detection Zeaxanthin cycle pigment Download PDF

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CN110286179A
CN110286179A CN201910730988.XA CN201910730988A CN110286179A CN 110286179 A CN110286179 A CN 110286179A CN 201910730988 A CN201910730988 A CN 201910730988A CN 110286179 A CN110286179 A CN 110286179A
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pigment
liquid
chromatography
chromatographic
pure
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陈晓英
雷明
韦坤华
张占江
秦双双
李翠
郭晓云
李莹
韦莹
蓝祖栽
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Guangxi Botanical Garden of Medicinal Plants
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Guangxi Botanical Garden of Medicinal Plants
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G01N2030/146—Preparation by elimination of some components using membranes

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Abstract

The invention discloses a kind of high performance liquid chromatographies of quickly detection Zeaxanthin cycle pigment.Chloroplast pigment is extracted from blade to be measured first；Then chromatography is carried out to chloroplast pigment, so that 8 kinds of chloroplast pigments is efficiently and quickly separated by the way that elution requirement is arranged；The content of chloroplast pigment is finally calculated according to the content of pigment standard items and conversion factor.Xanthophyll Cycle Components are detected in method disclosed by the invention, the pigment of chloroplast pigment especially Xanthophyll Cycle Components can not only be fully extracted and separated as far as possible, it can be reduced the time-consuming with efficient liquid phase chromatographic analysis pigment simultaneously, the analysis time-consuming of a conventional sample is foreshortened into 30min or so from 40min or so, and apparent xanthophyll peak can be isolated.

Description

The quickly high performance liquid chromatography of detection Zeaxanthin cycle pigment
Technical field
The invention belongs to chromatography detection technique fields, and in particular to a kind of efficient liquid of quickly detection Zeaxanthin cycle pigment Phase chromatography.
Background technique
Xanthophyll Cycle Components pigment more be present in higher plant and green alga, it refer to violaxanthin (Violaxanthin, V), antheraxanthin (Antheraxanthin, A), zeaxanthin (Zeaxanthin, Z) mutually converting under light, dark condition Journey.Under strong light, violaxanthin carries out two step de-epoxidations under the action of Violaxanthin De and is converted into corn through antheraxanthin Huang Zhi reacts the ascorbic acid for needing reduced form and higher thylakoid membrane pH gradient；Darkling, there are oxygen, NADPH and lower Thylakoid membrane pH gradient when, reaction take a turn for the worse under the action of Cycloxygenase, zeaxanthin and antheraxanthin are again by ring Metaplasia is at violaxanthin.The wherein increase of the decylization state of oxidation (de-epoxidation state, DEPS=(A+Z)/(V+A+Z)) The enhancing for generally meaning that the superfluous luminous energy ability that dissipates is frequently necessary to calculating DEPS in practice and was dissipated with measuring plant leaf blade The ability of surplus luminous energy.
Other than 3 kinds of Xanthophyll Cycle Components pigments, 2 kinds of also other lutein-neoxathins (Neoxanthin, ) and lutein (Lutein, L) N.High performance liquid chromatography (high performance is commonly used in the measurement of this 5 kinds of lutein Liquid chromatography, HPLC), wherein method (Thayer SS, the Bj ǒ kman of Thayer and Bj ǒ kman O.1990.Leaf xanthophyll content and composition in sun and shade determined By HPLC.Photosynthesis Research, 23:331-343) it is still the measuring method that current most people uses. The measuring method can not only isolate this 5 kinds of lutein, moreover it is possible at the same isolate chlorophyll a (Chlorophyll a, Chl a), Chlorophyll b (Chlorophyll b, Chl b) and beta carotene (β-carotene).But this method is separated in HPLC It is difficult to separate the peak Z from the peak L in the process, and time-consuming for a complete HPLC analysis process, about at least about needs 40min. In view of the at high cost of HPLC, time-consuming will will increase the consuming and time and human cost of reagent and instrument.
Therefore, HPLC analysis time can be shortened by needing one kind, and can be by detection Zeaxanthin cycle that the peak Z preferably separates The high performance liquid chromatography of component pigment.
Summary of the invention
Present invention aim to address at least the above defects, and provide the advantages of illustrating later.
It is a further object of the invention that while being used for the pigment of Zeaxanthin cycle in detecting chloroplast pigment, it can Other chloroplast pigments are obtained to detect simultaneously.
Another object of the present invention is, by calculate in Zeaxanthin cycle pigment the content of each pigment and to sample into Row decylization oxidation state analysis, to measure the ability of the superfluous luminous energy of plant leaf blade dissipation.
In order to realize object of the present invention and other advantages, one kind is now provided and quickly detects Zeaxanthin cycle pigment High performance liquid chromatography.The separation and Extraction chloroplast pigment first from blade to be measured；Then chromatography point is carried out to chloroplast pigment Analysis enables 8 kinds of chloroplast pigments efficiently and quickly to separate by the way that elution requirement is arranged；Finally according to the content of pigment standard items The content of chloroplast pigment is calculated with conversion factor.Wherein, it is wrapped in chromatographic condition when carrying out chromatography to chloroplast pigment It includes: successively being eluted using A liquid and B liquid, the A liquid are as follows: Chromatographic Pure Methanol, trifluoroacetic acid aqueous solution, Tris-HCl buffer are pressed It is mixed to get according to volume ratio for 11:74:3.5, the concentration of the Tris-HCl buffer is 0.1mol/L, pH value 8.0, A liquid To elute Zeaxanthin cycle, the separating effect of Xanthophyll Cycle Components pigment and other pigments is improved；The B liquid are as follows: chromatography Pure methanol and chromatography pure ethyl acetate are mixed to get according to volume ratio for 68:32, and B liquid is to elute chlorophyll and carotenoids Element improves the separating effect of Chlorophylls and Carotenoids and other pigments.
Preferably, the method for chloroplast pigment is extracted from blade to be measured are as follows: indoor progress of the sample in duskiness It extracts, comprising the following steps: Step 1: blade to be measured is pressed 1~6cm2Area or the quality of 0.5~1.0g quantified Afterwards, it is put into mortar, while liquid nitrogen is added and is ground, obtains powder to be measured；In liquid nitrogen, the cell in blade to be measured stops life Object reaction, avoids the pigment oxidation in blade；Cell is clayed into power using grinding, it is good to break born of the same parents' effect.Step 2: will To powder to be measured be added pre-cooling chromatography pure acetone extract, obtain extracting solution；Step 3: being blown into obtained extracting solution Enter nitrogen 10min, 20min is then placed under condition of ice bath, then be centrifuged 10min under conditions of 4 DEG C, 12000g, obtains One supernatant and precipitating；Step 4: after being put into chromatography acetone and suspending again 2 times in obtained precipitating, under condition of ice bath 20min is placed, then 10min is centrifuged under the conditions of 4 DEG C, 12000g, obtains the second supernatant；Step 5: by the first supernatant Merge with the second supernatant, and be settled to 10~20mL with chromatography pure acetone, is then managed with 0.45 μm of filtering with microporous membrane to EP In, then nitrogen 10min is blown into EP pipe, and to drive the air in EP pipe away, to prevent the oxygenolysis of various pigments in sample, Obtain the sample.
Preferably, each pigment refers specifically to: neoxathin, violaxanthin, single antheraxanthin, lutein, maize Matter, chlorophyll b, chlorophyll a and beta carotene.
Preferably, the chromatographic condition specifically: use 4.6 × 250mm, 5 μm of partial sizes, the closed Zorbax of non-end ODS chromatographic column is chromatographic column；A liquid: Chromatographic Pure Methanol, trifluoroacetic acid aqueous solution and Tris-HCl buffer, volume ratio 11:74: 3.5, the concentration of the Tris-HCl buffer is 0.1mol/L, pH value 8.0；B liquid: Chromatographic Pure Methanol and chromatographically pure acetic acid second Ester, volume ratio 68:32；The flow velocity is 1.4mL/min；Applied sample amount is 20 μ L；The column temperature of chromatographic column is 30 DEG C；Detection wavelength For 440nm.
Preferably, the elution program of above-mentioned chromatography is equal are as follows: first elutes 10min with 100%A liquid；Then exist It is transformed into B liquid in 4min, then elutes 8min with 100%B liquid；Finally chromatographic column 8min is balanced with A liquid.A liquid and B liquid can be preferably By Xanthophyll Cycle Components pigment, Chlorophylls and Carotenoids elution separation.
Preferably, the efficient liquid phase chromatographic analysis is further comprising the steps of: S1: Chromatographic Pure Methanol, trifluoroacetic acid aqueous solution, And pure water, it is prepared with the volume ratio of 11:74:3.5, obtains C liquid；S2: after elution program, first with C liquid with 0.5- The flow velocity of 1mL/min rinses at least 40min to chromatographic column；Use Chromatographic Pure Methanol with the flow velocity of 0.5-1mL/min to chromatographic column again At least 40min is rinsed, Tris-HCl buffer remaining in chromatographic column and pollutant are completely removed, is reduced to chromatographic column Damage.
Preferably, the efficient liquid phase chromatographic analysis is further comprising the steps of: S1: by Chromatographic Pure Methanol, chromatographically pure second Nitrile and pure water carry out preparing C liquid with the volume ratio of 11:74:3.5；After S2, elution program, first with C liquid with 0.5-1mL/ The flow velocity of min rinses at least 40min to chromatographic column；Again with Chromatographic Pure Methanol with the flow velocity of 0.1-0.2mL/min to chromatographic column into Row rinses overnight.Further Tris-HCl buffer remaining in chromatographic column and pollutant are completely removed, reduced to chromatographic column Damage, to extend the service life of chromatographic column.
Preferably, the available pure water containing 5% Chromatographic Pure Methanol of the C liquid replaces, and the pure water is using preceding use 0.22 μm of miillpore filter is filtered, and avoids bringing impurity in chromatographic column into, and influences the efficiency and accuracy of chromatography.
Preferably, the preparation method of the chromatography peak figure and conversion factor of the pigment standard items are as follows: utilize neoxathin, purple Huang Zhi, lutein, chlorophyll b, chlorophyll a and beta carotene configure to obtain the standard solution of assorted element；The color of standard items Spectrum elution process: are as follows: first 10min is eluted with 100%A liquid；Then it is transformed into B liquid in 4min, then is eluted with 100%B liquid 8min；Finally chromatographic column 8min is balanced with A liquid.Flow velocity is 1.4mL/min, 20 μ L of standard items applied sample amount, and the temperature of chromatographic column is It 30 DEG C, is detected at 440nm, obtains the chromatography peak figure of pigment standard items；It calculates transforming factor: passing through spectrophotometric first The light absorption value and percentage extinction coefficient measuredThe concentration of pigment standard solution is calculated, then multiplied by applied sample amount Obtain the content of the pigment standard items for chromatography, the ratio of the pigment content and its chromatographic peak area be convert because Son；Wherein, the percentage that neoxathin, violaxanthin, lutein, chlorophyll b, chlorophyll a and beta carotene standard items use disappears Backscatter extinction logarithmic ratioIt is respectively as follows: 2243,2500,2550,518,840,2500g/100mL；The calculation formula of conversion factor are as follows:Pigment content=applied sample amount × pigment standard solution is dense Degree；Conversion factor=pigment content/correspondence pigment standard solution chromatographic peak area；Wherein, single antheraxanthin and jade The conversion factor of cream-coloured matter calculates resulting conversion factor using lutein.
Advantages of the present invention:
1, make 8 kinds of chloroplast pigments by the way that elution requirement is arranged: neoxathin, violaxanthin, single antheraxanthin, leaf are yellow Element, zeaxanthin, chlorophyll b, chlorophyll a and beta carotene can be separated efficiently and quickly；And reduce 8 kinds as far as possible The oxidational losses of chloroplast pigment regulates and controls chromatographic condition and chromatographic program, efficiently separates out apparent xanthophyll absorption peak.
2, the process for improving chromatography is improved, the time of chromatography is shortened, thereby reduces the consumption of reagent, instrument Take and manpower, time cost.
3, by chromatography, the content for the Xanthophyll Cycle Components being calculated, and DEPS is calculated with this, is planted with measuring The ability of the superfluous luminous energy of object blade dissipation.
Detailed description of the invention
Fig. 1-2 is the chromatogram of pigment standard items neoxathin；
Fig. 3-4 is the chromatogram of pigment standard items violaxanthin；
Fig. 5-6 is the chromatogram of pigment standard items lutein；
Fig. 7-8 is the chromatogram of pigment standard items chlorophyll b；
Fig. 9-10 is the chromatogram of pigment standard items chlorophyll a；
Figure 11 is the chromatogram of pigment standard items beta carotene；
Figure 12 is the chromatogram of the hybrid generation 1-12 wheat samples in the small 54 × capital 411 of laying down of cross combination；
Figure 13 is the hybrid generation 1-12 wheat samples in the small 54 × capital 411 of laying down of cross combination in Leaf Senescence middle period Huang The content change diagram of element circulation pigment；
Figure 14 is hybrid generation 1-12 wheat samples decylization in Leaf Senescence in the small 54 × capital 411 of laying down of cross combination State of oxidation figure；
Figure 15 is hybrid generation 1-12 wheat samples disleaf in Leaf Senescence in the small 54 × capital 411 of laying down of cross combination Flavine recycles other 5 kinds of pigment content variation diagrams outside pigment；
Figure 16 is that the male parent capital 411 in the small 54 × capital 411 of laying down of cross combination recycles color in Leaf Senescence Lutein The content change diagram of element；
Figure 17 is that decylization of the male parent capital 411 in Leaf Senescence in the small 54 × capital 411 of laying down of cross combination aoxidizes shape State figure；
Figure 18 is that Zeaxanthin cycle is removed in the male parent capital 411 in the small 54 × capital 411 of laying down of cross combination in Leaf Senescence Other 5 kinds of pigment content variation diagrams outside pigment.
Specific embodiment
The present invention is described in further detail below, to enable those skilled in the art's refer to the instruction text being capable of evidence To implement.
Embodiment 1
The chromatography peak figure and conversion factor of pigment standard items:
1, pigment standard items Lutein (including neoxathin Neoxanthin, violaxanthin Violaxanthin, lutein Lutein it) is prepared by laboratory, chlorophyll (Chlorophyll b, Chlorophyll a) and beta carotene are purchased from Sigma company.The chromatography straight alcohol that lutein volume fraction is 100% is dissolved, chlorophyll volume fraction is 80% Chromatographically pure acetone solution, the chromatographically pure n-hexane dissolution that it is 100% with volume fraction that beta carotene, which is used, configuration obtain each color The standard solution of element.
2, the chromatography eluant process of standard items: 10min first is eluted with 100%A liquid；Then B liquid is transformed into 4min, then 8min is eluted with 100%B liquid；Finally chromatographic column 8min is balanced with A liquid.Flow velocity is 1.4mL/min, and applied sample amount is 20 μ L, elution Temperature is 30 DEG C, and 440nm detects absorption value, obtains the chromatography peak figure of pigment standard items.
3, transforming factor: the light absorption value and percentage extinction coefficient measured first by spectrophotometer is calculatedIt calculates The concentration of pigment standard solution is obtained, the content of the pigment standard items for chromatography is then obtained multiplied by applied sample amount, The ratio of the pigment content and its chromatographic peak area is conversion factor；Wherein, neoxathin, violaxanthin, lutein, chlorophyll b, The percentage extinction coefficient that chlorophyll a and beta carotene standard items useBe respectively as follows: 2243,2500,2550,518, 840,2500g/100mL；The calculation formula of conversion factor are as follows: Pigment content=applied sample amount × pigment standard solution concentration；Conversion factor=pigment content/correspondence pigment standard solution Chromatographic peak area.
Embodiment 2
The high performance liquid chromatography of the quick detection Zeaxanthin cycle pigment of sample to be tested, comprising the following steps:
Step 1: in the indoor extraction chloroplast pigment of duskiness:
S1, blade to be measured is pressed into 2cm2Area carry out it is quantitative after, be put into mortar, while liquid nitrogen is added and is ground, obtain To powder to be measured.
S2, the chromatography pure acetone that pre-cooling is added into obtained powder to be measured extract, and obtain extracting solution.
S3, be blown into nitrogen 10min into obtained extracting solution, then place 20min under condition of ice bath, then 4 DEG C, It is centrifuged 10min under conditions of 12000g, obtains the first supernatant and precipitating.
S4, after being put into chromatography acetone and suspending again 2 times in obtained precipitating, 20min is placed under condition of ice bath, so It is centrifuged 10min under the conditions of 4 DEG C, 12000g afterwards, obtains the second supernatant.
S5, the first supernatant and the second supernatant are merged, and is settled to 15mL with chromatography acetone, it is then micro- with 0.45 μm Hole membrane filtration is blown into nitrogen 10min into EP pipe, then into EP pipe to get the sample is arrived.
Step 2: efficient liquid phase chromatographic analysis, chromatographic apparatus uses Waters 600controller, 996 photoelectricity, two pole Pipe array detector.
S1, efficient liquid phase chromatographic analysis prepare:
1, the Tris-HCl that Chromatographic Pure Methanol, trifluoroacetic acid aqueous solution and concentration are 0.1mol/L, pH value is 8.0 is buffered Liquid is prepared with the volume ratio of 11:74:3.5, obtains A liquid.
2, it by Chromatographic Pure Methanol and chromatography pure ethyl acetate, is prepared with the volume ratio of 68:32, obtains B liquid.
3, it by Chromatographic Pure Methanol, trifluoroacetic acid aqueous solution and pure water, is prepared with the volume ratio of 11:74:3.5, obtains C Liquid.
4, prepare chromatographic column: with the closed Zorbax ODS chromatography of non-end that partial size is 5 μm, size is 4.6 × 250mm Column is as chromatographic column.
S2, the chromatography peak figure and conversion factor that each pigment standard items are obtained using embodiment 1.
The efficient liquid phase chromatographic analysis of S3, sample to be tested: 10min first is eluted with 100%A liquid；Then it is converted in 4min 8min is eluted to B liquid, then with 100%B liquid；Finally chromatographic column 8min is balanced with A liquid.Flow velocity is 1.4mL/min, applied sample amount 20 μ L, eluting temperature are 30 DEG C, and 440nm detects absorption value.
Step 3: calculating pigment content: calculating 8 kinds of chloroplast pigments according to the content of pigment standard items and conversion factor Content, pigment content=conversion factor × correspondence colour component chromatographic peak area.
Step 4: at least 40min is first rinsed to chromatographic column with the flow velocity of 0.5-1mL/min with C liquid after measurement, then At least 40min is rinsed to chromatographic column with the flow velocity of 0.5-1mL/min with Chromatographic Pure Methanol.
Embodiment 3
The high performance liquid chromatography of the quick detection Zeaxanthin cycle pigment of sample to be tested, comprising the following steps:
Step 1: in the indoor extraction chloroplast pigment of duskiness:
S1, after carrying out blade to be measured quantitatively by the quality of 0.5g, it is put into mortar, while liquid nitrogen is added and is ground, obtained To powder to be measured.
S2, the chromatography pure acetone that pre-cooling is added into obtained powder to be measured extract, and obtain extracting solution.
S3, be blown into nitrogen 10min into obtained extracting solution, then place 20min under condition of ice bath, then 4 DEG C, It is centrifuged 10min under conditions of 12000g, obtains the first supernatant and precipitating.
S4, after being put into chromatography acetone and suspending again 2 times in obtained precipitating, 20min is placed under condition of ice bath, so It is centrifuged 10min under the conditions of 4 DEG C, 12000g afterwards, obtains the second supernatant.
S5, the first supernatant and the second supernatant are merged, and is settled to 15mL with chromatography pure acetone, then with 0.45 μm Filtering with microporous membrane is blown into nitrogen 10min into EP pipe, then into EP pipe to get the sample is arrived.
Step 2: efficient liquid phase chromatographic analysis, chromatographic apparatus uses Waters 600controller, 996 photoelectricity, two pole Pipe array detector.
S1, efficient liquid phase chromatographic analysis prepare:
1, the Tris-HCl that Chromatographic Pure Methanol, trifluoroacetic acid aqueous solution and concentration are 0.1mol/L, pH value is 8.0 is buffered Liquid is prepared with the volume ratio of 11:74:3.5, obtains A liquid.
2, it by Chromatographic Pure Methanol and chromatography pure ethyl acetate, is prepared with the volume ratio of 68:32, obtains B liquid.
3, it by Chromatographic Pure Methanol, trifluoroacetic acid aqueous solution and pure water, is prepared with the volume ratio of 11:74:3.5, obtains C Liquid.
4, prepare chromatographic column: with the closed Zorbax ODS chromatography of non-end that partial size is 5 μm, size is 4.6 × 250mm Column is as chromatographic column.
The chromatography peak figure and conversion factor of S2, each pigment standard items obtained using embodiment 1.
The efficient liquid phase chromatographic analysis of S3, sample to be tested: 10min first is eluted with 100%A liquid；Then it is converted in 4min 8min is eluted to B liquid, then with 100%B liquid；Finally chromatographic column 8min is balanced with A liquid.Flow velocity is 1.4mL/min, applied sample amount 20 μ L, eluting temperature are 30 DEG C, and 440nm detects absorption value.
Step 3: calculating the content of each pigment in chloroplast pigment: according to the content of pigment standard items and conversion factor meter Calculate the content of 8 kinds of chloroplast pigments, pigment content=conversion factor × correspondence colour component chromatographic peak area.
Step 4: at least 40min is first rinsed to chromatographic column with the flow velocity of 0.5-1mL/min with C liquid after measurement, then Chromatographic column is carried out with the flow velocity of 0.1-0.2mL/min with Chromatographic Pure Methanol to stay overnight flushing.
Data analysis:
1, pigment standard items are detected in the method for embodiment 1, obtained chromatographic parameter such as table 1:
2, the conversion factor of each pigment standard items is calculated in the mean value of 1 parameters obtained of Example, such as table 2:
3, the 0th, 7,13,17,20,25,30 day noon 12:00-15:00 respectively after spending be small to cross combination to lay down 54 The blade of hybrid generation 1-12 wheat samples in × capital 411 is sampled as sample 1, and with the progress of the method for embodiment 2 Detection, obtained chromatogram is as shown in Fig. 12, the content of pigment is then calculated, as a result as shown in attached drawing 13, attached drawing 15.
From attached drawing 12 as it can be seen that the chromatographic peak of 8 kinds of pigments in sample 1 separates, effect is good, and the peak type of various pigments is clear It is clear, complete.
4, the 0th, 7,13,17,20,25,30 day noon 12:00-15:00 respectively after spending be small to cross combination to lay down 54 The blade of 411 wheat samples of male parent capital in × capital 411 is sampled as sample 2, and is examined in the method for embodiment 2 It surveys, and calculates the content of pigment, as a result as shown in attached drawing 16, attached drawing 18.
5, the decylization state of oxidation of sample 1 is calculated, as a result as shown in Fig. 14；Calculate the decylization state of oxidation of sample 2, knot Fruit is as shown in Fig. 17.Wherein, the decylization state of oxidation (DEPS=(A+Z)/(V+A+Z)), A, Z, V are respectively antheraxanthin, jade Cream-coloured matter, the content of violaxanthin.
From testing result it is found that the blade of the hybrid generation 1-12 in the small 54 × capital 411 of laying down of 1 cross combination of sample is in aging The decline of later period pigment content；But the decylization state of oxidation increase, show blade can the aging later period start optical protection mechanism with Dissipate superfluous luminous energy.
The blade in the male parent capital 411 in the small 54 × capital 411 of laying down of 2 cross combination of sample aging later period pigment content sharply under Drop；It is compared with attached drawing 17, the increase of the decylization state of oxidation in male parent capital 411 is also apparently higher than filial generation 1-12, shows and filial generation 1- 12 compare, and early ageing occurs in the 411 blade later period of male parent capital.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed With.It can be applied to various suitable the field of the invention completely.It for those skilled in the art, can be easily Realize other modification.Therefore, without departing from the general concept defined in the claims and the equivalent scope, the present invention and unlimited In specific details.

Claims (9)

1. a kind of high performance liquid chromatography of quickly detection Zeaxanthin cycle pigment, it is characterised in that:
Chloroplast pigment is extracted from blade to be measured, chromatography is carried out to chloroplast pigment, makes 8 kinds by the way that elution requirement is arranged Chloroplast pigment can be separated efficiently and quickly, finally calculate chloroplast pigment according to the content of pigment standard items and conversion factor Content；
It wherein, include: successively to be eluted using A liquid and B liquid in chromatographic condition when carrying out chromatography to chloroplast pigment, The A liquid are as follows: Chromatographic Pure Methanol, trifluoroacetic acid aqueous solution, Tris-HCl buffer are mixed to get according to volume ratio for 11:74:3.5, The concentration of the Tris-HCl buffer is 0.1mol/L, pH value 8.0；The B liquid are as follows: Chromatographic Pure Methanol and chromatographically pure acetic acid Ethyl ester is mixed to get according to volume ratio for 68:32.
2. the high performance liquid chromatography of quickly detection Zeaxanthin cycle pigment as described in claim 1, it is characterised in that: to The method for extracting chloroplast pigment is surveyed in blade are as follows: the sample is extracted in the interior of duskiness, comprising the following steps:
Step 1: blade to be measured is pressed 1~6cm2Area or 0.5~1.0g quality carry out it is quantitative after, be put into mortar, simultaneously Liquid nitrogen is added to pulverize；
Step 2: the chromatography pure acetone for adding pre-cooling extracts, extracting solution is obtained；
Step 3: be blown into nitrogen 10min into extracting solution, then place 20min under condition of ice bath, then in 4 DEG C, 12000g Under the conditions of be centrifuged 10min, obtain the first supernatant and precipitating；
Step 4: 20min is placed under condition of ice bath after being put into chromatography pure acetone and suspending again 2 times in obtained precipitating, Then it is centrifuged 10min under the conditions of 4 DEG C, 12000g, obtains the second supernatant；
Step 5: the first supernatant and the second supernatant are merged, and it is settled to 10~20mL with chromatography pure acetone, then used 0.45 μm of filtering with microporous membrane is blown into nitrogen 10min into EP pipe, then into EP pipe to get the sample is arrived.
3. the high performance liquid chromatography of quickly detection Zeaxanthin cycle pigment as described in claim 1, it is characterised in that: described Each pigment refers specifically to: neoxathin, violaxanthin, single antheraxanthin, lutein, zeaxanthin, chlorophyll b, chlorophyll a and β- Carrotene.
4. the high performance liquid chromatography of quickly detection Zeaxanthin cycle pigment as described in claim 1, it is characterised in that: described Chromatographic condition specifically:
Using 4.6 × 250mm, 5 μm of partial sizes, the closed Zorbax ODS chromatographic column of non-end is chromatographic column；
A liquid: Chromatographic Pure Methanol, trifluoroacetic acid aqueous solution and Tris-HCl buffer, volume ratio 11:74:3.5, the Tris- The concentration of HCl buffer is 0.1mol/L, pH value 8.0；
B liquid: Chromatographic Pure Methanol and chromatography pure ethyl acetate, volume ratio 68:32；
Flow velocity: 1.4mL/min；
Applied sample amount: 20 μ L；
Chromatogram column temperature: 30 DEG C；
Detection wavelength: 440nm.
5. the high performance liquid chromatography of quickly detection Zeaxanthin cycle pigment as described in claim 1, which is characterized in that above-mentioned The elution program of chromatography is equal are as follows:
First 10min is eluted with 100%A liquid；Then it is transformed into B liquid in 4min, then elutes 8min with 100%B liquid；Finally use A Liquid balances chromatographic column 8min.
6. the high performance liquid chromatography of quickly detection Zeaxanthin cycle pigment as claimed in claim 5, which is characterized in that described Efficient liquid phase chromatographic analysis is further comprising the steps of:
S1: Chromatographic Pure Methanol, trifluoroacetic acid aqueous solution, pure water are prepared with the volume ratio of 11:74:3.5, obtain C liquid；
S2: after elution program, at least 40min is first rinsed to chromatographic column with the flow velocity of 0.5-1mL/min with C liquid；Color is used again It composes pure methanol and at least 40min is rinsed to chromatographic column with the flow velocity of 0.5-1mL/min.
7. the high performance liquid chromatography of quickly detection Zeaxanthin cycle pigment as claimed in claim 5, which is characterized in that described Efficient liquid phase chromatographic analysis is further comprising the steps of:
S1: Chromatographic Pure Methanol, trifluoroacetic acid aqueous solution and pure water are carried out to prepare C liquid with the volume ratio of 11:74:3.5；
After S2, elution program, at least 40min is first rinsed to chromatographic column with the flow velocity of 0.5-1mL/min with C liquid；Color is used again Pure methanol is composed chromatographic column is carried out with the flow velocity of 0.1-0.2mL/min to stay overnight flushing.
8. the high performance liquid chromatography of quick detection Zeaxanthin cycle pigment as claimed in claims 6 or 7, which is characterized in that The available pure water containing 5% Chromatographic Pure Methanol of the C liquid replaces, and the pure water was carried out using preceding with 0.22 μm of miillpore filter Filter.
9. the high performance liquid chromatography of quickly detection Zeaxanthin cycle pigment as described in claim 1, which is characterized in that described The chromatography peak figure of pigment standard items and the preparation method of conversion factor are as follows:
The chromatography straight alcohol dissolution for being 100% by lutein volume fraction, chlorophyll a and chlorophyll b are with volume fraction 80% chromatographically pure acetone solution, the chromatographically pure n-hexane dissolution that beta carotene volume fraction is 100%, configuration obtain each The standard solution of a pigment；
The chromatography eluant process of standard items: 10min first is eluted with 100%A liquid；Then it is transformed into B liquid in 4min, then uses 100%B liquid elutes 8min；Finally chromatographic column 8min is balanced with A liquid；Flow velocity is 1.4mL/min, and chromatogram column temperature is 30 DEG C, mark Quasi- 20 μ L of product applied sample amount, is detected at 440nm, obtains the chromatography peak figure of pigment standard items；
Calculate transforming factor: the light absorption value and percentage extinction coefficient measured first by spectrophotometerPigment is calculated The concentration of standard solution, then obtains the content of the pigment standard items for chromatography multiplied by applied sample amount, which contains Amount and the ratio of its chromatographic peak area are conversion factor；
Wherein, the percentage that neoxathin, violaxanthin, lutein, chlorophyll b, chlorophyll a and beta carotene standard items use Extinction coefficientIt is respectively as follows: 2243,2500,2550,518,840,2500g/100mL；
The calculation formula of conversion factor is as follows:
Pigment content=applied sample amount × pigment standard solution concentration；
Conversion factor=pigment content/correspondence pigment standard solution chromatographic peak area；
Wherein, the conversion factor of single antheraxanthin and zeaxanthin calculates resulting conversion factor using lutein.
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