Plant endogenous hormones play an extremely important role in regulating the growth and development of plants, and they show obvious physiological effects in very small amounts. However, due to the low concentration of endogenous hormones in plants, and the complexity of plant components, many interfering components, and easy to be destroyed during separation, the analysis and determination of hormones have brought many difficulties.
The enzyme immunoassay has high sensitivity but low reproducibility in the detection of plant endogenous hormones, while high performance liquid chromatography (HPLC) is suitable for the determination and separation of compounds with different properties, and has the characteristics of high sensitivity, selectivity, reproducibility and fast analysis speed, which is a more ideal method for the analysis of endogenous hormones in plants. This experiment introduces the extraction and determination of four hormones: IAA, GA, ZR and ABA.
Principle
High Performance Liquid Chromatography (HPLC) is based on the classical chromatography and the theory of gas chromatography, which has the advantages of high pressure, high speed, high efficiency and high sensitivity, etc. The separation system in the chromatograph includes the stationary phase and the mobile phase. The separation system in the chromatograph includes the stationary phase and the mobile phase. When the components dissolved in the mobile phase pass through the stationary phase, due to the different sizes and strengths of the interactions with the stationary phase (adsorption, partitioning, ionic attraction, exclusion, affinity), the retention time in the stationary phase is different, and thus the components are successively discharged from the stationary phase and enter the detector for detection.
Operation method
Determination of endogenous hormones in plants by high performance liquid chromatography
Principle
High Performance Liquid Chromatography (HPLC) is based on the classical chromatography and the theory of gas chromatography, which has the advantages of high pressure, high speed, high efficiency and high sensitivity, etc. The separation system in the chromatograph includes the stationary phase and the mobile phase. The separation system in the chromatograph includes the stationary phase and the mobile phase. When the components dissolved in the mobile phase pass through the stationary phase, due to the different sizes and strengths of the interactions with the stationary phase (adsorption, partitioning, ionic attraction, exclusion, affinity), the retention time in the stationary phase is different, and thus the components are successively discharged from the stationary phase and enter the detector for detection.
Materials and Instruments
Material: Move The basic process of determining the endogenous hormone content of plants by high performance liquid chromatography (HPLC) can be divided into the following steps: 1. Treatment of test materials: Weigh 1~2 g of sample, grind it into a pulp under an ice bath, add 20 mL of 80% pre-cooled methanol, seal it with plastic wrap, and cold soak it in the refrigerator at 4 °C overnight. The extract was filtered, 10 mL of methanol washed twice in the mortar, filtered and combined with the extract, and evaporated under reduced pressure at 40 °C until no methanol remained. The remaining aqueous phase was completely transferred to a triangular flask. Extract with 30 mL of petroleum ether to decolorize twice, and discard the ether phase. Add 0.01 g of PVPP (crosslinked polyvinylpyrrolidone), sonicate for 30 min, filter and extract with 30 mL of ethyl acetate three times, combine the ester phases, and evaporate to dryness at 40 °C under reduced pressure. Dissolve the residue with methanol (chromatographically pure) and make up to 2 mL, filter through 0.45 microporous membrane to obtain the solution to be measured, and store in the refrigerator at 4 °C. 2. Start the chromatograph, and then set up the chromatograph. 2. Start the chromatograph as follows: (1) Turn on the power of the computer and each module of the chromatography. (2) Double click "Instrument Online" on the desktop to enter the online interface. (3) Manually open the flushing valve at the pump, right-click the "pump" icon, adjust the flow rate to 5 mL - min-1, open all the pumps, and the system starts to flush until there are no bubbles in the pipeline (from the solvent bottle to the inlet of the pump) (generally 5~10 min), then switch the channel and continue to flush as directed by the processor physiological feast test until all the channels to be used are free of bubbles, and set the flow rate to 0 mL - min-1, and set the flow rate to 0 mL - min-1, then manually tighten the flushing valve. Manually tighten the flushing valve. (4) Click "Method" and "New Method" to create a new method. (4) Right-click the icon of "Pump", select the appropriate ratio of mobile phase according to the method requirements, set the flow rate at 1.0 mL - min-1 (5) Right-click the icon of "Detector", set the wavelength at 254 nm, turn on the detector, and wait for the baseline to stabilize for about 10 minutes before measurement. 3. Measurement (1) Drawing of standard curve: Take a certain amount of growth hormone, erythromycin, zeatin and abscisic acid standards, dissolve them in methanol (chromatographic purity) and prepare them into solutions with different concentrations, click the "Run Control" menu, select "Run Method", inject 10-30 ptL of samples into the manual or autosampler, and then analyze them with liquid-phase detection. If the peak areas of the four hormones show a good linear relationship with the concentration within a certain concentration range, a standard curve can be drawn. (2) Determination: Take the same amount of the sample to be tested and inject the sample. After the sample peaks are all out, rinse the sample for 30 min, then the next sample can be done. (3) Characterization: The peak in the sample with the same retention time as the standard is the hormone peak of the sample. 4. (4) After getting all the sample chromatograms, you can turn off the machine. The shutdown steps are as follows: (1) Before shutting down, flush the system fully with methanol for about 30 min (the column should be stored in methanol or ethanol eventually). (2) Exit the chemistry workstation, close the pumps and other windows, and shut down the computer. (3) Turn off the power switch of each module of the liquid chromatograph. 5. Calculate the content of each hormone in the sample by the peak area of the sample and the standard curve. Caveat 1. Upper Column Pressure Limit: The upper column pressure limit for established liquid phase methods should be less than 3800 psi. 2. Use of mobile phase: Any mobile phase must be filtered twice through a 0.45 μm membrane, and the pH and mobile phase selection must be within the limits of the column. 3. Sample Handling: Samples must be filtered through a 0.45 μm membrane prior to injection and must meet the requirements for pH, molecular weight, solubility in the mobile phase, and purity. For more product details, please visit Aladdin Scientific website.
Leaves of plants such as wheat or rice
Reagents:
100% methanol (chromatographically pure), 80% methanol (analytically pure), petroleum cool, ethyl acetate, growth hormone, gibberellin, zeatin, abscisic acid standards
Materials:
Agilent 1200 liquid chromatograph, mortar and pestle, Brinell's funnel, rotary evaporation drier, dispensing funnel, filter paper, micro syringe, 0.45 μm microporous filter membrane.