CN103175932B - Method for determining four hormones in rubber tree through high-efficiency liquid chromatography - Google Patents
Method for determining four hormones in rubber tree through high-efficiency liquid chromatography Download PDFInfo
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Abstract
The invention discloses a method for determining four hormones in a rubber tree through high-efficiency liquid chromatography. The method provided by the invention comprises the following steps of: (1) sample pretreatment: to be specific, grinding rubber tree tissues with liquid nitrogen, then placing in leaching liquor for extraction, centrifuging, separating a centrifuged liquid supernatant to obtain an aqueous phase, enabling the aqueous phase to pass through a chromatographic column, and eluting with methyl alcohol to obtain a sample to be tested; and (2) high-efficiency liquid chromatography detection: adopting an inverse-phase C18 chromatographic column, taking a mixed liquid of the methyl alcohol, acetonitrile and phosphate buffer as a mobile phase and 254nm as testing wavelength, and detecting auxin, zeatin, activol and abscisic acid in the sample to be tested obtained in the step (1). The method provided by the invention has the advantages that the four hormones can be detected, the extraction flow time is short, the determination accuracy rate is high, and the determination and analysis efficiency is improved. The method is a good method for researching the high-yield fast-growing mechanism of the Brazil rubber tree and the plant hormones in the cambium differentiation process.
Description
Technical field
The present invention relates to the method for four kinds of hormones in a kind of high-performance liquid chromatogram determination rubber tree, particularly a kind of method utilizing auxin, zeatin, gibberellin and abscisic acid four kinds of hormones in high performance liquid chromatography Simultaneously test Leaves of Hevea Brasiliensis, branch or bark.
Background technology
Caoutchouc industry is for a long time to collect latex as the raw material of industry, and it is auxiliary for developing with other biomass such as timber, seed.Endogenous hormones auxin (IAA) in rubber tree blade and bark, gibberellin (GA
3), the content of abscisic acid (ABA) and zeatin (ZT), activity and signal path regulation and control latex output and wood grows are grown.But the above-mentioned four kinds of Endogenous Hormone Contents in Vitro of rubber tree are extremely low, character is unstable, and rubber tree blade and bark exist Multiple components interference, cause rubber tree hormone determination to have difficulties.The method of traditional mensuration rubber tree endogenous hormones is as follows: take fresh latex with 80% methyl alcohol, the centrifugal rear Enzyme-multiplied immune technique of low-temperature and high-speed measures auxin (IAA), the content of abscisic acid (ABA) and the basic element of cell division (iPAs) (Cao Jianhua, Lin Weifu. the mensuration of 4 plant growth materials in Para rubber tree latex. tropical crops journal .2004.25(3): 1-4).Wherein the process of Extraction and separation purifying is unfavorable to hormone protection, and slightly misoperation very easily causes the waste of hormone to be measured in sample, and can not carry out the mensuration of various plants endogenous hormones simultaneously.Adopt suction method to collect sample, by the dislysate sample introduction on high performance liquid chromatograph obtained, be separated and determine hormone-content (Jiang Bo, Li Hong, Jinhua, Jiang Guobin, national inventing patent CN101738441A) wherein.Though its living body sampling has substantially do not disturb normal activities in biosome, realize the advantage of living body sampling, but cannot penetrate in old branch and bark tissue, this high megaphanerophyte of inapplicable Para rubber tree.
ProElut PXC pillar contains the agent of mixed type strong cation exchange reverse adsorption, obtained by PLS adsorbent bonding sulfonic acid group, have cation exchange and anti-phase two kinds of retained-modes concurrently, the pKa value being applicable to its conjugate acid is in the alkali compounds between 2-10, is mainly amine compound.Main application is: food safety detection melamine is analyzed; The analysis of animal sample neutral and alkali medicament residue, the such as medicine such as sulfa drugs, clenobuterol hydrochloride; The analysis of veterinary antibiotics and fruit juice neutral and alkali agricultural chemicals, the such as germifuge such as carbendazim, probenazole; Biological sample: analysis of blood, urine neutral and alkali medicine etc.
High performance liquid chromatography (HPLC) is one of effective assay method measuring plant hormone, and successfully determines various plants hormone in tropical fruit (tree) crop lichee, coconut moderate sample.But in stratographic analysis process, only easily occur dragging peak phenomenon with methyl alcohol, acetonitrile two kinds of organic solvents, elute effect is bad, causes the error of measurement result.
Summary of the invention
The object of this invention is to provide the method for auxin, zeatin, gibberellin and abscisic acid four kinds of hormones in a kind of high-performance liquid chromatogram determination rubber tree.
In high-performance liquid chromatogram determination rubber tree, a method for auxin, zeatin, gibberellin and abscisic acid four kinds of hormones, comprises the steps:
(1) sample pre-treatments: after rubber tree tissue liquid nitrogen grinding to be detected, be placed in extract extracting, centrifugal, be separated from the supernatant after centrifugal and obtain aqueous phase, described aqueous phase is crossed chromatographic column, by methanol-eluted fractions, obtain testing sample;
The solute of described extract is 2,6-toluene di-tert-butyl phenol, solvent to be volumn concentration be 80% methanol aqueous solution; The content of described 2,6-toluene di-tert-butyl phenols in described extract is for containing 2,6-tert-butyl-p-cresol described in 0.1g in extract described in every 100mL;
(2) high performance liquid chromatography detects: adopt C18 reverse-phase chromatographic column, with the mixed liquor of methyl alcohol, acetonitrile and phosphate buffer for mobile phase, take 254nm as determined wavelength, auxin, zeatin, gibberellin and abscisic acid in the testing sample that detecting step (1) obtains.
In the above-mentioned methods, in described mobile phase, the volume ratio of described methyl alcohol, described acetonitrile and described phosphate buffer is 15:15:70; The pH of described mobile phase is 3.5.
The solvent of described phosphate buffer is water, solute and concentration as follows: sodium dihydrogen phosphate 40g/L, pH3.5.Concrete compound method is as follows: be dissolved in by 40g sodium dihydrogen phosphate in 1000mL ultrapure water, by HCl adjust ph to 3.5.
In step (1), described chromatographic column is the anti-phase reservation cation exchange column of PXC.
In one embodiment of the invention, the anti-phase reservation cation exchange column of described PXC is specially the product of Beijing Di Ma company, and its catalog number is Cat.No.:68203A.
In the above-mentioned methods, the sample pre-treatments described in step (1) is carried out under dark condition, keeps low temperature (4 DEG C) simultaneously.Described dark is that intensity of illumination is less than 20 μm of ol m
-2s
-1.
In the above-mentioned methods, in step (1), the consumption of described extract specifically can be rubber tree tissue sample (fresh weight) to be measured described in every 0.5g and adds 15mL extract.
In the above-mentioned methods, in step (1), described in be placed in extract extracting, extracting 2 times.
In the above-mentioned methods, in step (1), describedly centrifugally specifically can be the centrifugal 10min of 18000g.Described centrifuging temperature can be 4 DEG C.
In the above-mentioned methods, in step (1), describedly from the supernatant after centrifugal, be separated that to obtain aqueous phase be that method by comprising the steps realizes: add the ammoniacal liquor being equivalent to described supernatant volume 1/300 in described supernatant after, after filtering (0.22 μm of syringe filter is filtered), evaporate with rotavapor under vacuum, remove organic phase, obtain described aqueous phase.Regulate described aqueous phase pH to be 3.0 with 0.1mol/L hydrochloric acid, filter by 0.22 μm of syringe filter.
In the above-mentioned methods, in step (1), described " described aqueous phase being crossed chromatographic column, by methanol-eluted fractions ", specifically comprises the steps:
(1) pillar is activated: chromatographic column (the anti-phase reservation cation exchange column of PXC) as described in activating with methyl alcohol (as 10mL);
(2) pillar is balanced: balance described chromatographic column (the anti-phase reservation cation exchange column of PXC) with water (ultrapure water filters through 0.22 μm of syringe filter, 10mL);
(3) loading: described aqueous phase (pH3.0 filters through 0.22 μm of syringe filter) is added to the described chromatographic column (the anti-phase reservation cation exchange column of PXC) after balance in step (2);
(4) drip washing: use 0.1mol/L HCL(10mL) chromatographic column (the anti-phase reservation cation exchange column of PXC) described in drip washing;
(5) wash-out: with methyl alcohol (10mL) wash-out.
In the above-mentioned methods, the high performance liquid chromatography described in step (2) detects, and column temperature is 30 DEG C.
In the above-mentioned methods, the high performance liquid chromatography described in step (2) detects, and type of elution is isocratic elution, and flow velocity is 1mL/min, and sample size is 10 μ L.
In one embodiment of the invention, the described anti-phase C18 chromatographic column of step (2) is specially CNWSIL C18 liquid-phase chromatographic column 4.6 × 150mm5 μm that Waters, US produces.
In the above-mentioned methods, described rubber tree organizes the blade, branch or the bark that specifically can be rubber tree.In the present invention, described rubber tree is specially Para rubber tree.
Described method both can be used for auxin, zeatin, gibberellin and the abscisic acid four kinds of hormones in qualitative detection rubber tree tissue, also can be used for the content of auxin, zeatin, gibberellin and the abscisic acid four kinds of hormones quantitatively detected in rubber tree tissue.
In the present invention, when quantitatively detecting, HPLC detects the hormone standard solution of known series concentration, with peak area (Y) to standard concentration (x, mg/L) carry out recurrence to calculate, must the typical curve equation of this hormone, then according to the actual measurement peak area of this hormone in testing sample, calculate the content of this hormone in testing sample.
The method of auxin, zeatin, gibberellin and abscisic acid four kinds of hormones in high-performance liquid chromatogram determination rubber tree provided by the present invention, one-time detection can go out four kinds of hormones, extraction flow time is short, measure accuracy rate and accuracy high, improve measure and analysis efficiency.The present invention is that research Para rubber tree high yield fast-growing mechanism and plant hormone are forming the good method in layer atomization.
Accompanying drawing explanation
Fig. 1 is the high-efficient liquid phase chromatogram of four kinds of hormone hybrid standard product.Wherein, peak 1 is zeatin (ZT), and peak 2 is gibberellin (GA
3), peak 3 is auxin (IAA), and peak 4 is abscisic acid (ABA).
Fig. 2 is the high-efficient liquid phase chromatogram of four kinds of hormone tests in rubber tree blade.Wherein, peak 1 is zeatin (ZT), and peak 2 is gibberellin (GA
3), peak 3 is auxin (IAA), and peak 4 is abscisic acid (ABA).
Fig. 3 is the high-efficient liquid phase chromatogram of four kinds of hormone tests in rubber tree branch.Wherein, peak 1 is zeatin (ZT), and peak 2 is gibberellin (GA
3), peak 3 is auxin (IAA), and peak 4 is abscisic acid (ABA).
Fig. 4 is the high-efficient liquid phase chromatogram of four kinds of hormone tests in rubber tree bark.Wherein, peak 1 is zeatin (ZT), and peak 2 is auxin (IAA), and peak 3 is abscisic acid (ABA).
Fig. 5 is the high-efficient liquid phase chromatogram (sample-pretreating method 1) of four kinds of hormone tests in rubber tree blade.
Fig. 6 is the high-efficient liquid phase chromatogram (sample-pretreating method 2) of four kinds of hormone tests in rubber tree blade.
Fig. 7 is the high-efficient liquid phase chromatogram (sample-pretreating method 3) of four kinds of hormone tests in rubber tree blade.
Fig. 8 is the high-efficient liquid phase chromatogram (sample-pretreating method 4) of four kinds of hormone tests in rubber tree blade.
Fig. 9 is the high-efficient liquid phase chromatogram (mobile phase 1) of four kinds of hormone tests in hybrid standard product.
Figure 10 is the high-efficient liquid phase chromatogram (mobile phase 2) of four kinds of hormone tests in hybrid standard product.
Figure 11 is the high-efficient liquid phase chromatogram (mobile phase 3) of four kinds of hormone tests in hybrid standard product.
Figure 12 is the high-efficient liquid phase chromatogram (mobile phase 4) of four kinds of hormone tests in hybrid standard product.
Embodiment
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Para rubber tree (Hevea brasiliensis) clone heat grinds 7-33-97: be Rubber Institute, Chinese Academy of Agricultural Science's kind, be documented in " Yuan Kun; Zhou Xuemei; Li Jianhui etc. dead skin control agent is on the physiological impact of dead skin latex of panama rubber tree. hubei agricultural science; 17 phases in 2011 " in a literary composition, provided by country of Rubber Institute, Chinese Academy of Agricultural Science rubber tree germplasm resource garden.
Auxin (IAA), zeatin (ZT), gibberellin (GA
3) and abscisic acid (ABA) standard items: all purchased from Sigma Chemical Co(St Louis, MO, USA), ZT(catalog number: Z0876), IAA(catalog number: H8876), GA
3(catalog number: 48880), ABA(catalog number: A1049).
The formula of various solution involved in following embodiment is as follows:
(1) phosphate buffer: phosphate buffer is dissolved in 1000mL ultrapure water by 40g sodium dihydrogen phosphate, by HCl adjust ph to 3.5.Mobile phase is detected for preparing HPLC.
(2) extract: solute is 2,6-toluene di-tert-butyl phenol (available from Sigma, article No. B1378), solvent to be volumn concentration be 80% methanol aqueous solution; The content of described 2,6-toluene di-tert-butyl phenols in described extract is for containing 2,6-toluene di-tert-butyl phenols described in 0.1g in extract described in every 100mL.
The HPLC related in following embodiment detects auxin (IAA), zeatin (ZT), gibberellin (GA
3) and the drafting of abscisic acid (ABA) four kinds of hormone typical curves, all specific as follows:
Chromatographic column is the CNWSIL C18 liquid-phase chromatographic column (4.6 × 150mm5 μm) that Waters, US produces, column temperature 30 DEG C; Mobile phase is methyl alcohol, acetonitrile, phosphate buffer (volume ratio is 15: 15: 70, pH3.5) isocratic elution; Flow velocity is 1mL/min; Sample size is 10 μ L; Determined wavelength is 254nm.Wherein, auxin, zeatin and abscisic acid concentration range be 0.001,0.01,0.1,0.5,1,2, the standard items series concentration of 10mg/L, gibberellin concentration range is 1.07,2.14,5.35,10.7,21.4,107,214, the standard items series concentration of 856mg/L.Under these conditions after liquid chromatograph detects, with the standard concentration (x of the peak area (Y) of auxin (or zeatin or gibberellin or abscisic acid) to auxin (or zeatin or gibberellin or abscisic acid), mg/L) carry out recurrence to calculate, obtain regression equation.Four kinds of hormone typical curve equations and related coefficient specifically as shown in table 1.
Table 1 four kinds of hormone typical curve equations and related coefficient thereof
The pre-treating method of rubber tree tissue sample to be measured involved in following embodiment, all specific as follows:
After being weighed by rubber tree tissue sample to be measured (blade, branch or bark), 0.5g sample (fresh weight) adopts 150mL liquid nitrogen fine gtinding.After grinding, add the consumption of 15ml extract according to rubber tree tissue sample (fresh weight) to be measured described in every 0.5g, at 4 DEG C of dark (intensity of illumination <20 μm of ol m
-2s
-1) repeatedly extract 2 times under condition, merge extract, 18000g4 DEG C centrifugal 10 minutes, get supernatant, add the ammoniacal liquor being equivalent to described supernatant volume 50 μ L/15mL in described supernatant after, with Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant RE-52 Rotary Evaporators) 40 DEG C of vacuum evaporation, remove organic phase, obtain aqueous phase.And regulating the pH value of described aqueous phase to 3.0 with 0.1mol/L watery hydrochloric acid, 0.22 μm of syringe filter is for subsequent use after filtering.
Adopt the 10ml methyl alcohol activation anti-phase reservation cation exchange column of PXC (Beijing Di Ma company ProElut PXC60mg/3mL), adopt 10ml to balance the anti-phase reservation cation exchange column of described PXC through the ultrapure water that 0.22 μm of syringe filter is filtered.Aqueous sample after filtration obtained above is added in the pillar balanced, is the HCl drip washing of 0.1mol/L by 10ml concentration, then uses 10ml methanol-eluted fractions.Collect eluent, with Rotary Evaporators 40 DEG C of vacuum evaporation to the greatest extent dry, dissolve, cross 0.2 μm of miillpore filter with 1mL chromatogram methyl alcohol, what obtain carrying out HPLC detection treats loading sample.
The content of auxin, zeatin, gibberellin and abscisic acid four kinds of hormones in embodiment 1, high-performance liquid chromatogram determination Leaves of Hevea Brasiliensis
1, the collection of Leaves of Hevea Brasiliensis and pre-treatment
Leaves of Hevea Brasiliensis picks up from Para rubber tree (Heveabrasiliensis) the clone heat of planting in Chinese Academy of Tropical Agricultural Sciences testing field and grinds 7-33-97.Its sample pre-treatments is carried out with reference to mentioned above, obtains testing sample.
2, high performance liquid chromatography detects four kinds of hormone-contents in testing sample
Chromatographic condition: chromatographic column is the CNWSIL C18 liquid-phase chromatographic column (4.6 × 150mm5 μm) that Waters, US produces, column temperature 30 DEG C; Mobile phase is methyl alcohol, acetonitrile, phosphate buffer (volume ratio is 15: 15: 70, pH3.5) isocratic elution; Flow velocity is 1mL/min; Sample size is 10 μ L; Determined wavelength is 254nm.
(1) utilize four kinds of hormone hybrid standard product to measure each hormone and go out the honeybee time
Accurately take ZT, IAA respectively, the each 5mg of GA and ABA standard items, (volume ratio of methyl alcohol, acetonitrile and phosphate buffer is the mixed liquor of 15: 15: 70 to use mobile phase respectively, pH3.5) 50ml is settled to, be mixed with 4 kinds of standard solution that final concentration is 100mg/L, again above-mentioned 4 kinds of standard solution are hybridly prepared into mixed standard solution according to a certain percentage, making the final concentration of ZT be the final concentration of 0.1 μ g/mL, IAA to be the final concentration of 0.1 μ g/mL, GA to be the final concentration of 0.1 μ g/mL, ABA is 0.1 μ g/mL.Mixed standard solution is detected according to above-mentioned chromatographic condition.Experiment in triplicate.
As shown in Figure 1, the retention time of zeatin (ZT) is 2.166min to chromatogram, and the retention time of gibberellin (GA3) is 3.871min, and the retention time of auxin (IAA) is 8.133min, and the retention time of abscisic acid (ABA) is 13.405min.Note: all carry out standard specimen mensuration before at every turn testing sample being detected in following examples, the retention time that contrast standard specimen measures carrys out the respective components in qualitative testing sample, due to experimental error, the standard specimen retention time of each mensuration can have small difference with the result of Fig. 1, all in experimental error allowed band.
(2) high performance liquid chromatography detects four kinds of hormone-contents in testing sample
According to the content of auxin, zeatin, gibberellin and abscisic acid in the testing sample prepared in above-mentioned chromatographic condition detecting step 1.Qualitative with standard specimen appearance time, external standard method peak area quantification (namely calculating hormone-content according to peak area detected value and corresponding typical curve).In triplicate, quantitative result gets three mean values repeated in experiment.
Chromatogram is as shown in Figure 2, as follows according to the hot hormone-content ground in 7-33-97 blade of standard curve determination Para rubber tree (Hevea brasiliensis) clone further: the content of zeatin (ZT) is 21.6 μ g/g fresh weights; The content of gibberellin (GA3) is 42.2 μ g/g fresh weights; The content of auxin (IAA) is 0.0033 μ g/g fresh weight; The content of abscisic acid (ABA) is 0.084 μ g/g fresh weight.
3, the sample recovery of standard addition of four kinds of hormones measures
(1) sample pre-treatments
The blade picking up from Para rubber tree (Hevea brasiliensis) the clone heat of planting in Chinese Academy of Tropical Agricultural Sciences testing field and grind 7-33-97 is pulverized evenly and weighs into four 0.1g aliquots, each 0.1g aliquot adds the one 10 μ g in auxin, zeatin, gibberellin and abscisic acid four kinds of hormone standard items, 30min is left standstill after mixing, then carry out sample pre-treatments (carrying out with reference to mentioned above), obtain testing sample (mark-on sample).Simultaneously not add the equivalent blade powder of four kinds of hormone standard models as blank (non-mark-on sample).
(2) high performance liquid chromatography detects
According to the content of auxin, zeatin, gibberellin and abscisic acid in the testing sample prepared in the detecting step of chromatographic condition described in step 2 (1), calculate average recovery rate.Experiment repetition 6 times, quantitative result gets 6 mean values repeated.
The recovery=(mark-on Specimen Determination value-non-mark-on Specimen Determination value)/add scalar × 100%
Result is as shown in table 2, can find out, adds the average recovery rate after standard items: GA, 105.2%; ZT, 85.6%; IAA, 90.9%; ABA, 109.1%.This result confirms that high performance liquid chromatography detects validity and the accuracy of four kinds of these analytical approachs of hormone-content in testing sample.
Table 2 adds average recovery rate measurement result after each standard items
The content of auxin, zeatin, gibberellin and abscisic acid four kinds of hormones in embodiment 2, high-performance liquid chromatogram determination Para rubber tree branch
1, the collection of Para rubber tree branch and pre-treatment
Para rubber tree branch picks up from Para rubber tree (Heveabrasiliensis) the clone heat of planting in Chinese Academy of Tropical Agricultural Sciences testing field and grinds 7-33-97.Its sample pre-treatments is carried out with reference to mentioned above, obtains testing sample.
2, high performance liquid chromatography detects four kinds of hormone-contents in testing sample
Method of operating is with step 2 in embodiment 1.
Chromatogram is as shown in Figure 3, as follows according to the hot hormone-content ground in 7-33-97 branch of standard curve determination Para rubber tree (Hevea brasiliensis) clone further: the content of zeatin (ZT) is 1581.6 μ g/g fresh weights; The content of gibberellin (GA3) is 5.067 μ g/g fresh weights; The content of auxin (IAA) is 0.118 μ g/g fresh weight; The content of abscisic acid (ABA) is 65.856 μ g/g fresh weights.
The content of auxin, zeatin, gibberellin and abscisic acid four kinds of hormones in embodiment 3, high-performance liquid chromatogram determination Para rubber tree bark
1, the collection of Para rubber tree bark and pre-treatment
Para rubber tree bark picks up from Para rubber tree (Heveabrasiliensis) the clone heat of planting in Chinese Academy of Tropical Agricultural Sciences testing field and grinds 7-33-97.Its sample pre-treatments is carried out with reference to mentioned above, obtains testing sample.
2, high performance liquid chromatography detects four kinds of hormone-contents in testing sample
Method of operating is with step 2 in embodiment 1.
Chromatogram is as shown in Figure 4, as follows according to the hot hormone-content ground in 7-33-97 bark of standard curve determination Para rubber tree (Hevea brasiliensis) clone further: the content of zeatin (ZT) is 102.9 μ g/g fresh weights; The content of gibberellin (GA3) is 0 μ g/g fresh weight; The content of auxin (IAA) is 0.022 μ g/g fresh weight; The content of abscisic acid (ABA) is 0.45 μ g/g fresh weight.
The comparison of comparative example 1, different sample-pretreating method
One, sample-pretreating method 1
(1) collection of Leaves of Hevea Brasiliensis and pre-treatment
Leaves of Hevea Brasiliensis picks up from Para rubber tree (Heveabrasiliensis) the clone heat of planting in Chinese Academy of Tropical Agricultural Sciences testing field and grinds 7-33-97.Its sample-pretreating method is as follows:
After rubber tree tissue sample (blade) to be measured is weighed, 0.1g sample (fresh weight) adopts 15mL liquid nitrogen fine gtinding.After grinding, add the consumption of the pre-cold methanol of 15ml80% according to rubber tree tissue sample (fresh weight) to be measured described in every 0.1g, at 4 DEG C of dark (intensity of illumination <20 μm of ol m
-2s
-1) extract under condition, 18000g4 DEG C is centrifugal 10 minutes, gets supernatant, filters, and filtrate adjusts pH to 3.0 with 0.1mol/L HCl, with the extraction into ethyl acetate 3 times of equivalent, merges ester phase; By Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant RE-52 Rotary Evaporators) 40 DEG C of evaporated in vacuo, the acetic acid adding pH3.5 washs cucurbit 2 times, each 2mL, merges washing lotion.
Washing lotion crosses the anti-phase reservation cation exchange column of PXC (Beijing Di Ma company ProElut PXC60mg/3mL) (pillar first with the activation of 10mL methyl alcohol, then uses 10mL water balance), uses 10mL10% methanol wash, 5mL water washing, finally uses 4mL100% acetonitrile.Collect eluent, with Rotary Evaporators 40 DEG C of vacuum evaporation to the greatest extent dry, dissolve with 1mL chromatogram methyl alcohol, what obtain carrying out HPLC detection treats loading sample.
(2) high performance liquid chromatography detects four kinds of hormone-contents in testing sample
Method of operating is with step 2 in embodiment 1.
Chromatogram as shown in Figure 5, through liquid chromatographic detection, cannot detect four kinds of hormones to be measured.
Two, sample-pretreating method 2
(1) collection of Leaves of Hevea Brasiliensis and pre-treatment
Leaves of Hevea Brasiliensis picks up from Para rubber tree (Heveabrasiliensis) the clone heat of planting in Chinese Academy of Tropical Agricultural Sciences testing field and grinds 7-33-97.Its sample-pretreating method is as follows:
After rubber tree tissue sample (blade) to be measured is weighed, 0.1g sample (fresh weight) adopts 15mL liquid nitrogen fine gtinding.After grinding, add the consumption of the pre-cold methanol of 15ml80% according to rubber tree tissue sample (fresh weight) to be measured described in every 0.1g, at 4 DEG C of dark (intensity of illumination <20 μm of ol m
-2s
-1) extract under condition, 18000g4 DEG C is centrifugal 10 minutes, gets supernatant, filters; By Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant RE-52 Rotary Evaporators) 40 DEG C of evaporated in vacuo, the acetic acid adding pH3.5 washs cucurbit 2 times, each 2mL, merges washing lotion.
Washing lotion crosses the anti-phase reservation cation exchange column of PXC (Beijing Di Ma company ProElut PXC60mg/3mL) (pillar first with the activation of 10mL methyl alcohol, then uses 10mL water balance), uses 10mL10% methanol wash, 5mL water washing, finally uses 4mL100% acetonitrile.Collect eluent, with vacuum evaporation at Rotary Evaporators 40 DEG C to the greatest extent dry, dissolve with 1mL chromatogram methyl alcohol, what obtain carrying out HPLC detection treats loading sample.
(2) high performance liquid chromatography detects four kinds of hormone-contents in testing sample
Method of operating is with step 2 in embodiment 1.
Chromatogram as shown in Figure 6, through liquid chromatographic detection, cannot detect four kinds of hormones to be measured.
Three, sample-pretreating method 3
(1) collection of Leaves of Hevea Brasiliensis and pre-treatment
Leaves of Hevea Brasiliensis picks up from Para rubber tree (Heveabrasiliensis) the clone heat of planting in Chinese Academy of Tropical Agricultural Sciences testing field and grinds 7-33-97.Its sample-pretreating method is as follows:
After rubber tree tissue sample (blade) to be measured is weighed, 0.1g sample (fresh weight) adopts 15mL liquid nitrogen fine gtinding.After grinding, add the consumption of the pre-cold methanol of 15ml80% according to rubber tree tissue sample (fresh weight) to be measured described in every 0.1g, at 4 DEG C of dark (intensity of illumination <20 μm of ol m
-2s
-1) extract under condition, 18000g4 DEG C is centrifugal 10 minutes, gets supernatant, filters, and filtrate adjusts pH to 3.0 with 0.1mol/L HCl; By Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant RE-52 Rotary Evaporators) 40 DEG C of evaporated in vacuo, the acetic acid adding pH3.5 washs cucurbit 2 times, each 2mL, merges washing lotion.
Washing lotion crosses the anti-phase reservation cation exchange column of PXC (Beijing Di Ma company ProElut PXC60mg/3mL) (pillar first with the activation of 10mL methyl alcohol, then uses 10mL water balance), uses 10mL10% methanol wash, 5mL water washing, finally uses 4mL100% acetonitrile.Collect eluent, with vacuum evaporation at Rotary Evaporators 40 DEG C to the greatest extent dry, dissolve, cross 0.22 μm of organic phase pin type filter with 1mL chromatogram methyl alcohol, what obtain carrying out HPLC detection treats loading sample.
(2) high performance liquid chromatography detects four kinds of hormone-contents in testing sample
Method of operating is with step 2 in embodiment 1.
Chromatogram as shown in Figure 7, through liquid chromatographic detection, cannot detect four kinds of hormones to be measured.
Four, sample-pretreating method 4
(1) collection of Leaves of Hevea Brasiliensis and pre-treatment
Leaves of Hevea Brasiliensis picks up from Para rubber tree (Heveabrasiliensis) the clone heat of planting in Chinese Academy of Tropical Agricultural Sciences testing field and grinds 7-33-97.Its sample-pretreating method is as follows:
After rubber tree tissue sample (blade) to be measured is weighed, 0.1g sample (fresh weight) adopts 15mL liquid nitrogen fine gtinding.After grinding, add the consumption of the pre-cold methanol of 15ml80% according to rubber tree tissue sample (fresh weight) to be measured described in every 0.1g, at 4 DEG C of dark (intensity of illumination <20 μm of ol m
-2s
-1) extract under condition, 18000g4 DEG C is centrifugal 10 minutes, gets supernatant, filters.
Washing lotion crosses the anti-phase reservation cation exchange column of PXC (Beijing Di Ma company ProElut PXC60mg/3mL), and (pillar is first with the activation of 5mL methyl alcohol, use 5mL water balance again), after loading, with 10mL0.1mol/L HCl drip washing, use 10mL methanol-eluted fractions, with vacuum evaporation at Rotary Evaporators 40 DEG C to not having methyl alcohol remaining, obtain aqueous phase.And regulate the pH value of described aqueous phase to 3.0 with 0.1mol/L watery hydrochloric acid, with isopyknic extraction into ethyl acetate 3 times, merge ester phase; By Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant RE-52 Rotary Evaporators) 40 DEG C of evaporated in vacuo, dissolve with 1mL chromatogram methyl alcohol, cross 0.22 μm of organic phase pin type filter, what obtain carrying out HPLC detection treats loading sample.
(2) high performance liquid chromatography detects four kinds of hormone-contents in testing sample
Method of operating is with step 2 in embodiment 1.
Chromatogram as shown in Figure 8, through liquid chromatographic detection, cannot detect four kinds of hormones to be measured.
The comparison of comparative example 2, different mobile phase
One, mobile phase 1
(1) preparation of standard items mixed solution
Accurately take ZT, IAA respectively, the each 5mg of GA and ABA standard items, by methanol constant volume to 50ml, be mixed with 4 kinds of standard solution that final concentration is 100mg/L, again above-mentioned 4 kinds of standard solution are hybridly prepared into mixed standard solution according to a certain percentage, making the final concentration of ZT be the final concentration of 0.1 μ g/mL, IAA to be the final concentration of 0.1 μ g/mL, GA to be the final concentration of 0.1 μ g/mL, ABA is 0.1 μ g/mL.
(2) high performance liquid chromatography detects hybrid standard product
Chromatographic condition: chromatographic column is the CNWSIL C18 liquid-phase chromatographic column (4.6 × 150mm5 μm) that Waters, US produces, column temperature 30 DEG C; Mobile phase is methyl alcohol, 0.5%(volume fraction) acetic acid (volume ratio is 50: 50, pH3.5) isocratic elution; Flow velocity is 1mL/min; Sample size is 10 μ L; Determined wavelength is 254nm.
Four kinds of hormones to be measured as shown in Figure 9, can not effectively separate by chromatogram.
Two, mobile phase 2
(1) preparation of standard items mixed solution
With (1) in step one.
(2) high performance liquid chromatography detects hybrid standard product
Chromatographic condition: chromatographic column is the CNWSIL C18 liquid-phase chromatographic column (4.6 × 150mm5 μm) that Waters, US produces, column temperature 30 DEG C; Mobile phase is methyl alcohol, 0.5%(volume fraction) acetic acid (volume ratio is 40: 60, pH3.5) isocratic elution; Flow velocity is 1mL/min; Sample size is 10 μ L; Determined wavelength is 254nm.
Four kinds of hormones to be measured as shown in Figure 10, can not effectively separate by chromatogram.
Three, mobile phase 3
(1) preparation of standard items mixed solution
With (1) in step one.
(2) high performance liquid chromatography detects hybrid standard product
Chromatographic condition: chromatographic column is the CNWSIL C18 liquid-phase chromatographic column (4.6 × 150mm5 μm) that Waters, US produces, column temperature 30 DEG C; Mobile phase is acetonitrile, 0.5%(volume fraction) acetic acid (volume ratio is 50: 50, pH3.5) isocratic elution; Flow velocity is 1mL/min; Sample size is 10 μ L; Determined wavelength is 254nm.
Four kinds of hormones to be measured as shown in figure 11, can not effectively separate by chromatogram.
Four, mobile phase 4
(1) preparation of standard items mixed solution
With (1) in step one.
(2) high performance liquid chromatography detects hybrid standard product
Chromatographic condition: chromatographic column is the CNWSIL C18 liquid-phase chromatographic column (4.6 × 150mm5 μm) that Waters, US produces, column temperature 30 DEG C; Mobile phase is methyl alcohol, acetonitrile, 0.5%(volume fraction) acetic acid (volume ratio is 30:10: 60, pH3.5) isocratic elution; Flow velocity is 1mL/min; Sample size is 10 μ L; Determined wavelength is 254nm.
Four kinds of hormones to be measured as shown in figure 12, can not effectively separate by chromatogram.
Claims (7)
1. the method for auxin, zeatin, gibberellin and abscisic acid four kinds of hormones in high-performance liquid chromatogram determination rubber tree, comprises the steps:
(1) sample pre-treatments: after rubber tree tissue grinder to be detected, be placed in extract extracting, centrifugal, add the ammoniacal liquor being equivalent to described supernatant 1/300 volume in the supernatant after centrifugal after, with Rotary Evaporators 40 DEG C of vacuum evaporation, remove organic phase, obtain aqueous phase, and regulate the pH value of described aqueous phase to 3.0 with 0.1mol/L watery hydrochloric acid, 0.22 μm of syringe filter crosses chromatographic column after filtering, by methanol-eluted fractions, obtain testing sample;
Described chromatographic column is the anti-phase reservation cation exchange column of PXC;
The solute of described extract is 2,6-toluene di-tert-butyl phenol, solvent to be volumn concentration be 80% methanol aqueous solution; The content of described 2,6-toluene di-tert-butyl phenols in described extract is for containing 2,6-toluene di-tert-butyl phenols described in 0.1g in extract described in every 100mL;
(2) high performance liquid chromatography detects: adopt C18 reverse-phase chromatographic column, with the mixed liquor of methyl alcohol, acetonitrile and phosphate buffer for mobile phase, take 254nm as determined wavelength, auxin, zeatin, gibberellin and abscisic acid in the testing sample that detecting step (1) obtains;
In described mobile phase, the volume ratio of described methyl alcohol, described acetonitrile and described phosphate buffer is 15:15:70; The pH of described mobile phase is 3.5.
2. method according to claim 1, is characterized in that: the sample pre-treatments described in step (1) is carried out under dark condition.
3. method according to claim 1, is characterized in that: in step (1), and the consumption of described extract adds extract described in 15ml for every 0.5 gram of described rubber tree tissue sample to be measured.
4. method according to claim 1, is characterized in that: the high performance liquid chromatography described in step (2) detects, and column temperature is 30 DEG C.
5. method according to claim 1, is characterized in that: the high performance liquid chromatography described in step (2) detects, and type of elution is isocratic elution, and flow velocity is 1mL/min, and sample size is 10 μ L.
6. according to described method arbitrary in claim 1-5, it is characterized in that: described rubber tree is organized as the blade of rubber tree, branch or bark.
7. method according to claim 6, is characterized in that: described rubber tree is Para rubber tree.
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| CN103529156B (en) * | 2013-09-29 | 2014-12-10 | 福建省农业科学院作物研究所 | Method for extracting and measuring ABA (abscisic acid) of strawberry |
| CN104820031B (en) * | 2015-04-28 | 2016-09-07 | 青岛康大分析检测有限公司 | A kind of probenazole and the method for quick of intermediate thereof |
| CN104897843B (en) * | 2015-06-24 | 2017-01-11 | 南京信息工程大学 | Method for measuring content of endogenous hormones of burgeons of tea tree |
| MX2018001594A (en) | 2015-08-10 | 2018-09-26 | Beem Biologics Inc | Compositions and their use for pest control and to induce plant hormone and gene regulation for improved plant production and defense. |
| CN106404507A (en) * | 2016-08-31 | 2017-02-15 | 广东省农业科学院农产品公共监测中心 | Leaf vegetable gibberellins residue detection sample pretreatment method and detection method |
| CN106442792B (en) * | 2016-10-14 | 2019-04-26 | 广西壮族自治区农业科学院甘蔗研究所（中国农业科学院甘蔗研究中心） | The HPLC detection method of GA content in a kind of Sugarcane Leaves |
| CN107064391B (en) * | 2017-03-28 | 2020-02-04 | 北京林业大学 | Method for determining zeatin in magnolia subgenus plant |
| CN107202724B (en) * | 2017-07-11 | 2020-08-28 | 安徽宏远职业卫生技术服务有限公司 | Sample pretreatment method for detecting preservative in fruit juice |
| CN108344824A (en) * | 2018-03-27 | 2018-07-31 | 福建省热带作物科学研究所 | A kind of highly effective extraction method measured suitable for xylophyta endogenous hormones |
| CN114793675A (en) * | 2022-03-24 | 2022-07-29 | 甘肃农业大学 | Method for rapidly screening optimal concentration of methyl jasmonate for relieving salt stress of corn seedlings |
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