CN103175932A - 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 of utilizing high performance liquid chromatography to measure simultaneously auxin in Leaves of Hevea Brasiliensis, branch or bark, zeatin, gibberellin and four kinds of hormones of abscisic acid.
Background technology
Caoutchouc industry for a long time take collect latex as the raw material of industry as main, develop as auxiliary take 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.Yet the above-mentioned four kinds of Endogenous Hormone Contents in Vitro of rubber tree are extremely low, and character is unstable, and rubber tree blade and bark exist Multiple components to disturb, and cause the 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, low-temperature and high-speed is centrifugal measures auxin (IAA) afterwards with Enzyme-multiplied immune technique, 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 and purification is unfavorable to the hormone protection, slightly has misoperation very easily to cause the waste of hormone to be measured in sample, and can not carry out simultaneously the mensuration of various plants endogenous hormones.Adopt the suction method to collect sample, with dislysate sample introduction on high performance liquid chromatograph of obtaining, separate and determine wherein hormone-content (Jiang Bo, Li Rihong, Jinhua, Jiang Guobin, national inventing patent CN101738441A).Substantially do not disturb normal activities in biosome though its living body sampling has, realize the advantage of living body sampling, but can't 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 that is applicable to its conjugate acid is in the alkali compounds between 2-10, is mainly amine compound.Main application is: the food safety detection melamine is analyzed; The analysis of animal sample neutral and alkali medicament residue is such as medicines such as sulfa drugs, clenobuterol hydrochlorides; The analysis of vegetables, fruit and fruit juice neutral and alkali agricultural chemicals is such as germifuge such as carbendazim, probenazoles; Biological sample: analysis of blood, urine neutral and alkali medicine etc.
High performance liquid chromatography (HPLC) is one of effective assay method of measuring plant hormone, and in tropical fruit (tree) crop lichee, coconut moderate sample successful mensuration the various plants hormone.Yet, in the stratographic analysis process, only easily occurring dragging the peak phenomenon with methyl alcohol, two kinds of organic solvents of acetonitrile, elute effect is bad, causes the error of measurement result.
Summary of the invention
The method that the purpose of this invention is to provide auxin in a kind of high-performance liquid chromatogram determination rubber tree, zeatin, gibberellin and four kinds of hormones of abscisic acid.
In a kind of high-performance liquid chromatogram determination rubber tree, the method for auxin, zeatin, gibberellin and four kinds of hormones of abscisic acid, comprise the steps:
(1) sample pre-treatments: with rubber tree tissue to be detected with liquid nitrogen grinding after, be placed in the extract extracting, centrifugal, separate obtaining water the supernatant after centrifugal, described water is crossed chromatographic column, use methanol-eluted fractions, the acquisition testing sample;
The solute of described extract is 2,6-toluene di-tert-butyl phenol, and solvent is that volumn concentration is 80% methanol aqueous solution; Described 2, the content of 6-toluene di-tert-butyl phenol in described extract is that to contain 0.1g in the described extract of every 100mL described 2,6-tert-butyl group p-cresol;
(2) high performance liquid chromatography detects: adopt the C18 reverse-phase chromatographic column, take the mixed liquor of methyl alcohol, acetonitrile and phosphate buffer as mobile phase, take 254nm as detecting wavelength, auxin, zeatin, gibberellin and abscisic acid in the testing sample that detecting step (1) obtains.
In said method, 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, and solute and concentration thereof are as follows: sodium dihydrogen phosphate 40g/L, pH3.5.Concrete compound method is as follows: the 40g sodium dihydrogen phosphate is dissolved in the 1000mL ultrapure water, regulates pH value to 3.5 with HCl.
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 said method, the described sample pre-treatments of step (1) is carried out under dark condition, keeps simultaneously low temperature (4 ℃).Described dark is that intensity of illumination is less than 20 μ mol m
-2s
-1
In said method, in step (1), the consumption of described extract specifically can be the described rubber tree tissue sample to be measured of every 0.5g (fresh weight) and adds the 15mL extract.
In said method, in step (1), describedly be placed in extract extracting, extracting 2 times.
In said method, in step (1), the described centrifugal centrifugal 10min of 18000g that specifically can be.Described centrifuging temperature can be 4 ℃.
In said method, in step (1), separate described supernatant after centrifugal that to obtain water be to realize by the method that comprises the steps: after adding the ammoniacal liquor that is equivalent to described supernatant volume 1/300 in the described supernatant, after filtering (0.22 μ m needle type filtration device filters), use Rotary Evaporators vacuum evaporation, remove organic phase, obtain described water.Regulating described water pH with 0.1mol/L hydrochloric acid is 3.0, filters with 0.22 μ m needle type filtration device.
In said method, in step (1), described " described water is crossed chromatographic column, use methanol-eluted fractions " specifically comprises the steps:
(1) activation pillar: chromatographic column as described in activating with methyl alcohol (as 10mL) (the anti-phase reservation cation exchange column of PXC);
(2) balance pillar: (ultrapure water filters 10mL) the described chromatographic column of balance (the anti-phase reservation cation exchange column of PXC) to water through 0.22 μ m needle type filtration device;
(3) loading: described water (pH3.0 filters through 0.22 μ m needle type filtration device) is added to the described chromatographic column after balance in step (2) (the anti-phase reservation cation exchange column of PXC);
(4) drip washing: use 0.1mol/L HCL(10mL) the described chromatographic column of drip washing (the anti-phase reservation cation exchange column of PXC);
(5) wash-out: with methyl alcohol (10mL) wash-out.
In said method, the described high performance liquid chromatography of step (2) detects, and column temperature is 30 ℃.
In said method, the described high performance liquid chromatography of 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 μ m that U.S. Waters company produces.
In said method, described rubber tree is organized 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 four kinds of hormones of abscisic acid in qualitative detection rubber tree tissue, also can be used for quantitatively detecting the content of auxin, zeatin, gibberellin and four kinds of hormones of abscisic acid in the 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 items concentration (x, mg/L) return calculating, typical curve equation that must 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 four kinds of hormones of abscisic acid in high-performance liquid chromatogram determination rubber tree provided by the present invention, but one-time detection goes out four kinds of hormones, the extraction flow time is short, measures accuracy rate and accuracy high, improves and measures and analysis efficiency.The present invention is research Para rubber tree high yield fast-growing mechanism and the good method of plant hormone in forming layer atomization.
Description of drawings
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), peak 4 is abscisic acid (ABA).
Fig. 2 is the high-efficient liquid phase chromatogram of four kinds of hormone tests in the rubber tree blade.Wherein, peak 1 is zeatin (ZT), and peak 2 is gibberellin (GA
3), peak 3 is auxin (IAA), peak 4 is abscisic acid (ABA).
Fig. 3 is the high-efficient liquid phase chromatogram of four kinds of hormone tests in the rubber tree branch.Wherein, peak 1 is zeatin (ZT), and peak 2 is gibberellin (GA
3), peak 3 is auxin (IAA), 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 the rubber tree blade.
Fig. 6 is the high-efficient liquid phase chromatogram (sample-pretreating method 2) of four kinds of hormone tests in the rubber tree blade.
Fig. 7 is the high-efficient liquid phase chromatogram (sample-pretreating method 3) of four kinds of hormone tests in the rubber tree blade.
Fig. 8 is the high-efficient liquid phase chromatogram (sample-pretreating method 4) of four kinds of hormone tests in the rubber tree blade.
Fig. 9 is the high-efficient liquid phase chromatogram (mobile phase 1) of four kinds of hormone tests in the hybrid standard product.
Figure 10 is the high-efficient liquid phase chromatogram (mobile phase 2) of four kinds of hormone tests in the hybrid standard product.
Figure 11 is the high-efficient liquid phase chromatogram (mobile phase 3) of four kinds of hormone tests in the hybrid standard product.
Figure 12 is the high-efficient liquid phase chromatogram (mobile phase 4) of four kinds of hormone tests in the hybrid standard product.
Embodiment
The experimental technique that uses in following embodiment is conventional method if no special instructions.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Para rubber tree (Hevea brasiliensis) clone heat is ground 7-33-97: be Rubber Institute, Chinese Academy of Agricultural Science's kind, be documented in " Yuan Kun; Zhou Xuemei; Li Jianhui etc. the 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 Rubber Institute, Chinese Academy of Agricultural Science's country's rubber tree germplasm resource garden.
Auxin (IAA), zeatin (ZT), gibberellin (GA
3) and abscisic acid (ABA) standard items: all available from Sigma Chemical Co(St Louis, MO, USA), ZT(catalog number: Z0876), the IAA(catalog number: H8876), GA
348880), the ABA(catalog number (catalog number:: A1049).
In following embodiment, the formula of related various solution is as follows:
(1) phosphate buffer: phosphate buffer is that the 40g sodium dihydrogen phosphate is dissolved in the 1000mL ultrapure water, regulates pH value to 3.5 with HCl.Be used for preparation HPLC and detect mobile phase.
(2) extract: solute is 2,6-toluene di-tert-butyl phenol (available from Sigma company, article No. B1378), and solvent is that volumn concentration is 80% methanol aqueous solution; Described 2, the content of 6-toluene di-tert-butyl phenol in described extract is that to contain 0.1g in the described extract of every 100mL described 2, the 6-toluene di-tert-butyl phenol.
The HPLC that relates in following embodiment detects auxin (IAA), zeatin (ZT), gibberellin (GA
3) and the drafting of four kinds of hormone typical curves of abscisic acid (ABA), all specific as follows:
Chromatographic column is the CNWSIL C18 liquid-phase chromatographic column (4.6 * 150mm5 μ m) that U.S. Waters company produces, 30 ℃ of column temperatures; Mobile phase is methyl alcohol, acetonitrile, (volume ratio is 15: 15: 70 to phosphate buffer, pH3.5) isocratic elution; Flow velocity is 1mL/min; Sample size is 10 μ L; The detection wavelength is 254nm.Wherein, auxin, zeatin and abscisic acid concentration range are 0.001,0.01,0.1,0.5,1,2, the standard items of 10mg/L series concentration, and the 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 peak area (Y) of auxin (or zeatin or gibberellin or abscisic acid) standard items concentration (x to auxin (or zeatin or gibberellin or abscisic acid), mg/L) return calculating, get regression equation.Four kinds of hormone typical curve equations and related coefficient thereof are specifically as shown in table 1.
Four kinds of hormone typical curve equations of table 1 and related coefficient thereof
The pre-treating method of related rubber tree tissue sample to be measured in following embodiment, all specific as follows:
After rubber tree tissue sample to be measured (blade, branch or bark) was weighed, 0.5g sample (fresh weight) adopted 150mL liquid nitrogen fine gtinding.After grinding, add the consumption of 15ml extract according to the described rubber tree tissue sample to be measured of every 0.5g (fresh weight), at 4 ℃ of dark (intensity of illumination＜20 μ mol m
-2s
-1) repeatedly extract 2 times under condition, merge extract, 18000g4 ℃ centrifugal 10 minutes, get supernatant, after adding the ammoniacal liquor that is equivalent to described supernatant volume 50 μ L/15mL in the described supernatant, with 40 ℃ of vacuum evaporation of Rotary Evaporators (RE-52 of Shanghai Yarong Biochemical Instrument Plant Rotary Evaporators), remove organic phase, obtain water.And the pH value to 3.0 of regulating described water with 0.1mol/L watery hydrochloric acid, 0.22 μ m needle type filtration device is standby after filtering.
Adopt the 10ml methyl alcohol activation anti-phase reservation cation exchange column of PXC (Beijing ProElut PXC60mg/3mL of Di Ma company), the anti-phase reservation cation exchange column of the described PXC of ultrapure water balance that adopts 10ml to filter through 0.22 μ m needle type filtration device.Aqueous sample after filtration obtained above is added in the good pillar of balance, is the HCl drip washing of 0.1mol/L with 10ml concentration, then uses the 10ml methanol-eluted fractions.Collect eluent, to doing to the greatest extent, with the dissolving of 1mL chromatogram methyl alcohol, cross 0.2 μ m miillpore filter with 40 ℃ of vacuum evaporation of Rotary Evaporators, obtain carrying out that HPLC detects treats the loading sample.
The content of auxin, zeatin, gibberellin and four kinds of hormones of abscisic acid 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 obtains testing sample with reference to mentioned above carrying out.
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 μ m) that U.S. Waters company produces, 30 ℃ of column temperatures; Mobile phase is methyl alcohol, acetonitrile, (volume ratio is 15: 15: 70 to phosphate buffer, pH3.5) isocratic elution; Flow velocity is 1mL/min; Sample size is 10 μ L; The detection wavelength is 254nm.
(1) utilize four kinds of hormone hybrid standard product to measure each hormone and go out the honeybee time
Accurately take respectively ZT, IAA, GA and each 5mg of ABA standard items, (volume ratio of methyl alcohol, acetonitrile and phosphate buffer is the mixed liquor of 15: 15: 70 to use respectively mobile phase, pH3.5) be settled 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, the final concentration that makes ZT is that the final concentration of 0.1 μ g/mL, IAA is that the final concentration of 0.1 μ g/mL, GA is that the final concentration of 0.1 μ g/mL, ABA is 0.1 μ g/mL.Detect mixed standard solution according to above-mentioned chromatographic condition.The experiment triplicate.
Chromatogram as shown in Figure 1, the retention time of zeatin (ZT) is 2.166min, the retention time of gibberellin (GA3) is 3.871min, the retention time of auxin (IAA) is 8.133min, the retention time of abscisic acid (ABA) is 13.405min.Annotate: all carry out standard specimen at every turn before testing sample being detected in following examples and measure, the retention time that the contrast standard specimen is measured is come the respective components in qualitative testing sample, due to experimental error, each standard specimen retention time of measuring can have with the result of Fig. 1 small difference, all in the experimental error allowed band.
(2) high performance liquid chromatography detects four kinds of hormone-contents in testing sample
Content according to auxin, zeatin, gibberellin and abscisic acid in the testing sample for preparing in above-mentioned chromatographic condition detecting step 1.Qualitative with the standard specimen appearance time, external standard method peak area quantification (namely calculating hormone-content according to peak area detected value and corresponding typical curve).Experiment triplicate, quantitative result are got the mean value that repeats three times.
Chromatogram as shown in Figure 2, the hormone-content that further grinds in the 7-33-97 blade according to standard curve determination Para rubber tree (Hevea brasiliensis) clone heat is as follows: 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 is measured
(1) sample pre-treatments
After pulverizing the blade that picks 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 evenly, weighing becomes four 0.1g aliquots, each 0.1g aliquot adds a kind of 10 μ g in auxin, zeatin, gibberellin and four kinds of hormone standard items of abscisic acid, standing 30min after mixing, then carry out sample pre-treatments (with reference to mentioned above carrying out), obtain testing sample (mark-on sample).Simultaneously with the equivalent blade powder that do not add four kinds of hormone standard models as blank (not mark-on sample).
(2) high performance liquid chromatography detects
According to the content of auxin, zeatin, gibberellin and abscisic acid in the testing sample for preparing in the detecting step of chromatographic condition described in step 2 (1), calculate average recovery rate.Experiment repeats 6 times, and quantitative result is got the mean value that repeats 6 times.
The recovery=(mark-on Specimen Determination value-not 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 has confirmed 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 four kinds of hormones of abscisic acid in embodiment 2, high-performance liquid chromatogram determination Para rubber tree branch
1, the collection of Para rubber tree branch and pre-treatment
The Para rubber tree branch picks up from Para rubber tree (Hevea brasiliensis) the clone heat of planting in Chinese Academy of Tropical Agricultural Sciences testing field and grinds 7-33-97.Its sample pre-treatments obtains testing sample with reference to mentioned above carrying out.
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 3, the hormone-content that further grinds in the 7-33-97 branch according to standard curve determination Para rubber tree (Hevea brasiliensis) clone heat is as follows: 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 four kinds of hormones of abscisic acid in embodiment 3, high-performance liquid chromatogram determination Para rubber tree bark
1, the collection of Para rubber tree bark and pre-treatment
The Para rubber tree bark picks up from Para rubber tree (Hevea brasiliensis) the clone heat of planting in Chinese Academy of Tropical Agricultural Sciences testing field and grinds 7-33-97.Its sample pre-treatments obtains testing sample with reference to mentioned above carrying out.
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 4, the hormone-content that further grinds in the 7-33-97 bark according to standard curve determination Para rubber tree (Hevea brasiliensis) clone heat is as follows: 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 Examples 1, different sample-pretreating methods
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 (Hevea brasiliensis) 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 to be measured (blade) was weighed, 0.1g sample (fresh weight) adopted 15mL liquid nitrogen fine gtinding.After grinding, add the consumption of the pre-cold methanol of 15ml80% according to the described rubber tree tissue sample to be measured of every 0.1g (fresh weight), at 4 ℃ of dark (intensity of illumination＜20 μ mol m
-2s
-1) extract under condition, 18000g4 ℃ centrifugal 10 minutes, get supernatant, filter, filtrate is transferred pH to 3.0 with 0.1mol/L HCl, with the ethyl acetate extraction of equivalent 3 times, merges the ester phase; With 40 ℃ of evaporated in vacuo of Rotary Evaporators (RE-52 of Shanghai Yarong Biochemical Instrument Plant Rotary Evaporators), the acetic acid that adds pH3.5 washs cucurbit 2 times, and each 2mL merges washing lotion.
Washing lotion is crossed the anti-phase reservation cation exchange column of PXC (Beijing ProElut PXC60mg/3mL of Di Ma company) (pillar first activates with 10mL methyl alcohol, then uses the 10mL water balance), uses the 10mL10% methanol wash, and 4mL100% acetonitrile wash-out is used in the 5mL water washing at last.Collect eluent, to doing to the greatest extent, with the dissolving of 1mL chromatogram methyl alcohol, obtain carrying out that HPLC detects treats the loading sample with 40 ℃ of vacuum evaporation of Rotary Evaporators.
(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 through liquid chromatographic detection, can't detect four kinds of hormones to be measured as shown in Figure 5.
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 (Hevea brasiliensis) 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 to be measured (blade) was weighed, 0.1g sample (fresh weight) adopted 15mL liquid nitrogen fine gtinding.After grinding, add the consumption of the pre-cold methanol of 15ml80% according to the described rubber tree tissue sample to be measured of every 0.1g (fresh weight), at 4 ℃ of dark (intensity of illumination＜20 μ mol m
-2s
-1) extract under condition, 18000g4 ℃ centrifugal 10 minutes, get supernatant, filter; With 40 ℃ of evaporated in vacuo of Rotary Evaporators (RE-52 of Shanghai Yarong Biochemical Instrument Plant Rotary Evaporators), the acetic acid that adds pH3.5 washs cucurbit 2 times, and each 2mL merges washing lotion.
Washing lotion is crossed the anti-phase reservation cation exchange column of PXC (Beijing ProElut PXC60mg/3mL of Di Ma company) (pillar first activates with 10mL methyl alcohol, then uses the 10mL water balance), uses the 10mL10% methanol wash, and 4mL100% acetonitrile wash-out is used in the 5mL water washing at last.Collect eluent, to doing to the greatest extent, with the dissolving of 1mL chromatogram methyl alcohol, obtain carrying out that HPLC detects treats the loading sample with 40 ℃ of lower vacuum evaporation of Rotary Evaporators.
(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 through liquid chromatographic detection, can't detect four kinds of hormones to be measured as shown in Figure 6.
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 (Hevea brasiliensis) 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 to be measured (blade) was weighed, 0.1g sample (fresh weight) adopted 15mL liquid nitrogen fine gtinding.After grinding, add the consumption of the pre-cold methanol of 15ml80% according to the described rubber tree tissue sample to be measured of every 0.1g (fresh weight), at 4 ℃ of dark (intensity of illumination＜20 μ mol m
-2s
-1) extract under condition, 18000g4 ℃ centrifugal 10 minutes, get supernatant, filter, filtrate is transferred pH to 3.0 with 0.1mol/L HCl; With 40 ℃ of evaporated in vacuo of Rotary Evaporators (RE-52 of Shanghai Yarong Biochemical Instrument Plant Rotary Evaporators), the acetic acid that adds pH3.5 washs cucurbit 2 times, and each 2mL merges washing lotion.
Washing lotion is crossed the anti-phase reservation cation exchange column of PXC (Beijing ProElut PXC60mg/3mL of Di Ma company) (pillar first activates with 10mL methyl alcohol, then uses the 10mL water balance), uses the 10mL10% methanol wash, and 4mL100% acetonitrile wash-out is used in the 5mL water washing at last.Collect eluent, to doing to the greatest extent, with the dissolving of 1mL chromatogram methyl alcohol, cross 0.22 μ m organic phase pin type filter with 40 ℃ of lower vacuum evaporation of Rotary Evaporators, obtain carrying out that HPLC detects treats the 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 through liquid chromatographic detection, can't detect four kinds of hormones to be measured as shown in Figure 7.
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 (Hevea brasiliensis) 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 to be measured (blade) was weighed, 0.1g sample (fresh weight) adopted 15mL liquid nitrogen fine gtinding.After grinding, add the consumption of the pre-cold methanol of 15ml80% according to the described rubber tree tissue sample to be measured of every 0.1g (fresh weight), at 4 ℃ of dark (intensity of illumination＜20 μ mol m
-2s
-1) extract under condition, 18000g4 ℃ centrifugal 10 minutes, get supernatant, filter.
Washing lotion is crossed the anti-phase reservation cation exchange column of PXC (Beijing ProElut PXC60mg/3mL of Di Ma company), and (pillar is first with the activation of 5mL methyl alcohol, use again the 5mL water balance), after loading, with 10mL0.1mol/L HCl drip washing, use the 10mL methanol-eluted fractions, to not having methyl alcohol remaining, obtain water with 40 ℃ of lower vacuum evaporation of Rotary Evaporators.And the pH value to 3.0 of regulating described water with 0.1mol/L watery hydrochloric acid, with isopyknic ethyl acetate extraction 3 times, merging ester phase; With 40 ℃ of evaporated in vacuo of Rotary Evaporators (RE-52 of Shanghai Yarong Biochemical Instrument Plant Rotary Evaporators), with the dissolving of 1mL chromatogram methyl alcohol, cross 0.22 μ m organic phase pin type filter, obtain carrying out that HPLC detects treats the 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 through liquid chromatographic detection, can't detect four kinds of hormones to be measured as shown in Figure 8.
The comparison of Comparative Examples 2, different mobile phases
One, mobile phase 1
(1) preparation of standard items mixed solution
Accurately take respectively ZT, IAA, GA and each 5mg of ABA standard items, with 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, the final concentration that makes ZT is that the final concentration of 0.1 μ g/mL, IAA is that the final concentration of 0.1 μ g/mL, GA is that the final concentration of 0.1 μ g/mL, ABA is 0.1 μ g/mL.
(2) high performance liquid chromatography detects the hybrid standard product
Chromatographic condition: chromatographic column is the CNWSIL C18 liquid-phase chromatographic column (4.6 * 150mm5 μ m) that U.S. Waters company produces, 30 ℃ of column temperatures; Mobile phase is methyl alcohol, 0.5%(volume fraction) (volume ratio is 50: 50 to acetic acid, pH3.5) isocratic elution; Flow velocity is 1mL/min; Sample size is 10 μ L; The detection wavelength is 254nm.
Chromatogram can not effectively separate four kinds of hormones to be measured as shown in Figure 9.
Two, mobile phase 2
(1) preparation of standard items mixed solution
With (1) in step 1.
(2) high performance liquid chromatography detects the hybrid standard product
Chromatographic condition: chromatographic column is the CNWSIL C18 liquid-phase chromatographic column (4.6 * 150mm5 μ m) that U.S. Waters company produces, 30 ℃ of column temperatures; Mobile phase is methyl alcohol, 0.5%(volume fraction) (volume ratio is 40: 60 to acetic acid, pH3.5) isocratic elution; Flow velocity is 1mL/min; Sample size is 10 μ L; The detection wavelength is 254nm.
Chromatogram can not effectively separate four kinds of hormones to be measured as shown in figure 10.
Three, mobile phase 3
(1) preparation of standard items mixed solution
With (1) in step 1.
(2) high performance liquid chromatography detects the hybrid standard product
Chromatographic condition: chromatographic column is the CNWSIL C18 liquid-phase chromatographic column (4.6 * 150mm5 μ m) that U.S. Waters company produces, 30 ℃ of column temperatures; Mobile phase is acetonitrile, 0.5%(volume fraction) (volume ratio is 50: 50 to acetic acid, pH3.5) isocratic elution; Flow velocity is 1mL/min; Sample size is 10 μ L; The detection wavelength is 254nm.
Chromatogram can not effectively separate four kinds of hormones to be measured as shown in figure 11.
Four, mobile phase 4
(1) preparation of standard items mixed solution
With (1) in step 1.
(2) high performance liquid chromatography detects the hybrid standard product
Chromatographic condition: chromatographic column is the CNWSIL C18 liquid-phase chromatographic column (4.6 * 150mm5 μ m) that U.S. Waters company produces, 30 ℃ of column temperatures; Mobile phase is methyl alcohol, acetonitrile, 0.5%(volume fraction) and acetic acid (volume ratio is 30:10: 60, and pH3.5) isocratic elution; Flow velocity is 1mL/min; Sample size is 10 μ L; The detection wavelength is 254nm.
Chromatogram can not effectively separate four kinds of hormones to be measured as shown in figure 12.
Claims (10)
1. the method for auxin, zeatin, gibberellin and four kinds of hormones of abscisic acid in a high-performance liquid chromatogram determination rubber tree, comprise the steps:
(1) sample pre-treatments: after rubber tree tissue to be detected is ground, be placed in the extract extracting, centrifugal, separate obtaining water the supernatant after centrifugal, described water is crossed chromatographic column, use methanol-eluted fractions, obtain testing sample;
The solute of described extract is 2,6-toluene di-tert-butyl phenol, and solvent is that volumn concentration is 80% methanol aqueous solution; Described 2, the content of 6-toluene di-tert-butyl phenol in described extract is that to contain 0.1g in the described extract of every 100mL described 2, the 6-toluene di-tert-butyl phenol;
(2) high performance liquid chromatography detects: adopt the C18 reverse-phase chromatographic column, take the mixed liquor of methyl alcohol, acetonitrile and phosphate buffer as mobile phase, take 254nm as detecting wavelength, auxin, zeatin, gibberellin and abscisic acid in the testing sample that detecting step (1) obtains.
2. method according to claim 1, it is characterized in that: 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.
3. method according to claim 1 and 2, it is characterized in that: in step (1), described chromatographic column is the anti-phase reservation cation exchange column of PXC.
4. arbitrary described method according to claim 1-3, it is characterized in that: the described sample pre-treatments of step (1) is carried out under dark condition.
5. arbitrary described method according to claim 1-4, it is characterized in that: in step (1), the consumption of described extract is that the described rubber tree tissue sample to be measured of every 0.5 gram adds the described extract of 15ml.
6. arbitrary described method according to claim 1-5, it is characterized in that: in step (1), separate described supernatant after centrifugal that to obtain water be to obtain by the method that comprises the steps: adding volume in the described supernatant is the ammoniacal liquor of described supernatant volume 1/300, collect water, obtain described water.
7. arbitrary described method according to claim 1-6, it is characterized in that: the described high performance liquid chromatography of step (2) detects, and column temperature is 30 ℃.
8. arbitrary described method according to claim 1-7, it is characterized in that: the described high performance liquid chromatography of step (2) detects, and type of elution is isocratic elution, and flow velocity is 1mL/min, and sample size is 10 μ L.
9. arbitrary described method according to claim 1-8, it is characterized in that: described rubber tree is organized as blade, branch or the bark of rubber tree.
10. method according to claim 9, it is characterized in that: described rubber tree is Para rubber tree.
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| 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 |
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