Method for determining ethylene content in wheat by gas chromatography

Abstract

The invention discloses a method for determining ethylene content in wheat by gas chromatography, and relates to the technical field of biological detection.The method is characterized by comprising the steps that a wheat grain sample is prepared and placed in a glass bottle with a plug under certain conditions, and gas to be detected is generated; extracting a to-be-detected gas sample from the glass bottle with the plug, and sequentially injecting the to-be-detected gas sample into the gas chromatograph to obtain the ethylene peak area of the to-be-detected gas; sequentially injecting the standard gas into a gas chromatograph, and drawing a calibration curve to obtain the ethylene peak area of the standard gas; and calculating the ratio of the ethylene peak area of the to-be-detected gas to the ethylene peak area of the standard gas to obtain the ethylene content in the to-be-detected gas sample. The determination method provided by the invention can realize detection with lower quantitation limit and wider linear range, and has the characteristics of high sensitivity, high flux, rapidness, convenience, accuracy and reliability, the detection time of one sample is only 1-2min, and the method is suitable for detecting the plant hormone ethylene in wheat grains and seedlings.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and more specifically to a method for determining the ethylene content in wheat by gas chromatography. Background Art

[0002] Plant hormones, also known as endogenous plant hormones or natural plant hormones, are organic compounds that are induced by plant cells in response to specific environmental signals and can regulate (promote or inhibit) plant physiological processes at low concentrations. They regulate plant growth, development and differentiation separately or in a coordinated manner in terms of cell division and growth, tissue and organ differentiation, flowering and fruiting, maturity and aging, dormancy and germination, and in vitro tissue culture.

[0003] There are six major types of hormones known to be produced in plants, namely auxins, gibberellins (GAs), cytokinins (CTKs), abscisic acid (ABA), ethylene (ETH) and brassnosteroids (BRs). Although the content of these hormones in plants is very small, they play a very important role. They are indispensable substances for plant life activities and are of great significance in regulating various growth and development processes of plants and their responses to the environment.

[0004] The content of ethylene in plants is extremely low and it is flammable and explosive. The instrument used for detection must have a highly selective sensor to distinguish the interference of other gases. Gas chromatography (GC) is currently one of the most widely used and reliable methods for determining ethylene content; however, there is currently no method specifically proposed for common wheat to accurately detect the ethylene content in wheat, and there is also a lack of relevant reports on the accurate detection of ethylene content in wheat in the existing technology.

[0005] Therefore, the present invention aims to provide a method for measuring ethylene content in wheat by gas chromatography to solve the above problems. Summary of the invention

[0006] The purpose of the present invention is to provide a method for determining the ethylene content in wheat by gas chromatography. The present invention utilizes the difference in the distribution coefficients of different substances between the gas phase and the stationary phase to separate the mixture through a chromatographic column, and then quantitatively detects the separated ethylene through a flame ionization detector (FID). The method has the advantages of high sensitivity, high resolution, good repeatability, etc., and can accurately determine the ethylene content in a complex matrix.

[0007] The above technical purpose of the present invention is achieved through the following technical scheme: A method for determining the ethylene content in wheat by gas chromatography, comprising the following steps:

[0008] S1. Prepare wheat grain samples. Remove quantitative wheat grains in the development period from the ear, weigh them, place them in a 15 mL stoppered glass bottle, seal them and place them in a 22°C greenhouse for 48 hours to generate the gas to be tested.

[0009] S2. Inject 0.5 mL of 1-2 standard gases of different concentrations into the gas chromatograph in sequence, measure the response value of each concentration point in the standard series, draw a calibration curve with the concentration of the concentration point as the horizontal axis and the peak area of ​​each concentration point as the vertical axis, and obtain the ethylene peak area of ​​the standard gas.

[0010] S3. Draw 0.5 mL of the gas sample to be tested from the stoppered glass bottle and inject it, then add 0.5 mL of water to make up the volume.

[0011] S4. Inject 0.5 mL of the gas sample to be tested into the gas chromatograph in sequence to obtain the ethylene peak area of ​​the gas to be tested.

[0012] S5. Calculate the ratio of the ethylene peak area of ​​the gas to be tested to the ethylene peak area of ​​the standard gas to obtain the ethylene content in the gas sample to be tested.

[0013] The present invention is further configured as follows: the wheat grain sample is replaced by a common wheat seedling sample, 10 single-leaf seedlings planted for 7 days are weighed, placed in a 50 mL stoppered centrifuge tube, and an appropriate amount of water is added to the centrifuge tube to cover the roots of the plant, and the tube is sealed in a 22°C greenhouse and left to stand for 24 hours to generate the gas to be tested.

[0014] The present invention is further configured as follows: drawing the calibration curve in step S2 specifically draws the calibration curve using a standard gas at one concentration point, and the calibration curve passes through the origin.

[0015] The present invention is further configured as follows: the gas chromatograph specifically uses an Agilent chromatographic column with a catalog number of 113-4332, the length of the chromatographic column is 30m, the specification is 7 inches, the inner diameter is 0.32mm, and the chromatographic column maintains a constant flow mode with a flow rate of 4mL / min.

[0016] The present invention is further configured as follows: the temperature of the front injection port of the chromatographic column is 150° C., the injection mode is split injection, the split ratio is 3:1, the pressure is 16.817 psi, and the septum purge is 3 mL / min.

[0017] The present invention is further configured as follows: a hydrogen flame ionization detector is specifically used in the gas chromatograph, the operating temperature of the detector is 250° C., and it has high sensitivity and a temperature range of up to 400° C.

[0018] The present invention is further configured as follows: the detector includes high-purity hydrogen for detection, high-purity air for adjusting sensitivity, high-purity nitrogen for carrier gas, high-purity helium for tracing and leak detection, and tail gas for eliminating extra-column effects, the flow rate of the high-purity hydrogen is 40mL / min, the flow rate of the high-purity air is 350mL / min, and the flow rate of the tail gas is 25mL / min.

[0019] The present invention is further configured as follows: in step S2, the standard gas uses certified ethylene gas, and the concentration of the ethylene gas is 1 ppm.

[0020] The present invention is further configured as follows: the gas chromatograph specifically uses an Agilent injection port liner with item number 5190-2295, the injection port liner has a volume of 870uL and an inner diameter of 1mm, the injection port liner has ultra-high inertness, can be diverted, has low pressure drop, and is provided with glass wool, and liners of different volumes can be replaced according to test requirements.

[0021] In summary, the present invention has the following beneficial effects:

[0022] 1. The present invention utilizes the difference in the distribution coefficients of different substances between the gas phase and the stationary phase to separate the mixture through a chromatographic column, and then quantitatively detects the separated ethylene through a flame ionization detector FID. It has the advantages of high sensitivity, high resolution, good repeatability, etc., and can accurately determine the ethylene content in a complex matrix;

[0023] 2. The method for determining the ethylene content in wheat by gas chromatography provided by the present invention can achieve a lower limit of quantification and a wider linear range of detection, and has the characteristics of high sensitivity, high throughput, rapidity, convenience, accuracy and reliability. The detection time of a sample is only 1-2 minutes, and it is suitable for the detection of plant hormone ethylene in wheat grains and seedlings;

[0024] 3. The method for determining the ethylene content in wheat by gas chromatography of the present invention is simple to operate and has high sensitivity. The detection limit of ethylene detected by the method of the present invention can reach below 0.1ppm. The splitter can also be backflushed, thereby shortening the analysis cycle and extending the life of the chromatographic column. The septum purge gas can not only take away the pollutants decomposed by the septum but also eliminate the phenomenon of secondary injection. At the same time, the automation performance of the instrument shortens the analysis time and can reduce the cost of each sample analysis to a very low level.

[0025] 4. The gas chromatography method of the present invention for determining the ethylene content in wheat is fast and convenient, does not require complicated pre-treatment of the sample, greatly reduces the decomposition of ethylene during the pre-treatment process, and avoids the distortion of the plant hormone ethylene content. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the ethylene standard gas chromatogram in Example 1 of the present invention;

[0027] Figure 2 is a calibration curve diagram under ethylene standard gas in Example 1 of the present invention;

[0028] Figure 3 This is a chromatogram of ethylene in the gas sample to be tested in Example 1 of the present invention. DETAILED DESCRIPTION

[0029] The following is combined with Figure 1-3 The present invention is described in further detail.

[0030] Embodiment 1: A method for determining ethylene content in wheat by gas chromatography, comprising the following steps:

[0031] S1. Prepare wheat grain samples. Take an appropriate amount of wheat grains in the development period from the ear, weigh them, put them in a 15 mL stoppered glass bottle, seal them and place them in a 22°C greenhouse for 48 hours to generate the gas to be tested for the determination of ethylene content.

[0032] S2. Inject 0.5 mL of 1 ppm standard gas into the gas chromatograph in sequence with a split ratio of 3:1. Use the concentration of the concentration point as the horizontal coordinate and the peak area of ​​each concentration point as the vertical coordinate to draw a calibration curve to obtain the ethylene peak area of ​​the standard gas; wherein the calibration curve drawn with the standard gas at one concentration point passes through the origin.

[0033] S3. Then draw 0.5 mL of the gas sample to be tested from the stoppered glass bottle for sampling, and then add 0.5 mL of water to make up the volume. Repeat the measurement 3 times for each bottle.

[0034] S4. Inject 0.5 mL of the gas sample to be tested into the gas chromatograph in sequence, and the ethylene peak area of ​​the gas to be tested can be directly measured by the gas chromatograph.

[0035] The peak area corresponding to the 1 ppm concentration point in this embodiment is shown in Table 1:

[0036] Table 11 Peak area corresponding to ppm concentration point

[0037] Concentration point (ppm) 1 Response value (peak area) 16192.37

[0038] The ethylene calibration curve constructed from the data in Table 1 is as follows: Figure 2 As shown, the correlation coefficient R = 1.0 (Note: when setting the calibration curve, if the standard gas has one level, the origin cannot be ignored (the origin is included or forced); if the standard gas has two levels, the origin can be ignored).

[0039] The gas chromatogram of 1 ppm ethylene standard gas in this embodiment is as follows Figure 1 shown.

[0040] S5. Calculate the ratio of the ethylene peak area of ​​the gas to be tested to the ethylene peak area of ​​the standard gas to obtain the ethylene content in the gas sample to be tested.

[0041] In the calculation process, since the concentration point unit of the calibration curve is ppm, the result of the sample calculation is ppm. If you want to change the unit to nL / g / h, convert it according to the ethylene release rate formula:

[0042] Ethylene release rate = peak area / constant / volume / t / w

[0043] The unit of ethylene release rate is nL / g / h, the constant represents the ethylene content constant obtained from the curve made using the ethylene standard sample, V represents the container volume, t represents the closed culture time after covering, and w represents the fresh weight of the sample.

[0044] When the determination result is less than 1nL / g / h, retain to two decimal places; when the result is greater than or equal to 1nL / g / h, retain three significant figures; take three times the signal-to-noise ratio S / N as the detection limit of the method (LOD, LOD = 3S / N), take ten times the signal-to-noise ratio S / N as the quantification limit of the method (LOQ, LOQ = 10S / N), calculate the detection limit and quantification limit of ethylene in wheat grains, and the specific ethylene standard curve related parameters, LOD and LOQ related information are shown in Table 2:

[0045] Table 2 Information about ethylene standard curve, detection limit and quantification limit

[0046] Compound Name Linear equations Correlation coefficient Detection limit Limit of Quantitation Ethylene Y=16192.372510.x 1.0 553.8 1846

[0047] In this embodiment, a 7890B gas chromatograph and a hydrogen flame ionization detector (FID) from Agilent Technologies, Inc. of the United States are specifically used, wherein the chromatographic column is a 113-4332 capillary column, length: 30m, specification: 7 inches, inner diameter: 0.32mm, and the chromatographic column adopts a constant flow mode, flow rate: 4mL / min, and is maintained for 2.5min.

[0048] Front inlet temperature: 150°C; injection mode: split injection; split ratio: 3:1; injection volume: 0.5 mL; pressure: 16.817 psi; septum purge: 3 mL / min.

[0049] The flame ionization detector (FID) has an operating temperature of 250°C and has a high sensitivity and a temperature range of up to 400°C.

[0050] The detector includes high-purity hydrogen for detection, high-purity air for adjusting sensitivity, high-purity nitrogen (purity ≥99.999%) for carrier gas, high-purity helium (purity ≥99.999%) for tracing and leak detection, and tail gas for eliminating extra-column effects. The flow rate of high-purity hydrogen (purity ≥99.999%) is 40 mL / min, the flow rate of high-purity air (purity ≥99.999%) is 350 mL / min, and the flow rate of tail gas is 25 mL / min. Among them, high-purity helium (purity ≥99.999%), high-purity nitrogen (purity ≥99.999%) and high-purity hydrogen (purity ≥99.999%) are measured on a gas chromatograph, and there is no ethylene peak or other impurity peaks.

[0051] The ethylene standard gas in this embodiment is: ethylene standard gas with a certificate is purchased as needed. The concentration of the standard gas used in this embodiment is 1 ppm.

[0052] The injection port liner in this embodiment has a product number of 5190-2295, a volume of 870uL, an inner diameter of 1mm, has ultra-high inertness, can be split, has low pressure drop, and is provided with glass wool. Liners of different volumes can be replaced according to test requirements.

[0053] Example 2: The difference between this example and Example 1 is that the wheat grain samples are replaced by common wheat seedling samples, 10 single-leaf seedlings planted for 7 days are weighed, placed in a 50 mL stoppered centrifuge tube, and an appropriate amount of water is added to the centrifuge tube to cover the roots of the plants. The tube is sealed and placed in a greenhouse at 22°C for 24 hours to generate the gas to be tested, which can be used to determine the ethylene content in the common wheat seedling samples.

[0054] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A method for determining ethylene content in wheat by gas chromatography, characterized in that: The following steps are involved: S1. Prepare wheat grain samples. Take the wheat grains in the quantitative development period from the ears, weigh them, put them in a 15 mL stoppered glass bottle, seal them and place them in a 22°C greenhouse for 48 hours to generate the gas to be tested; S2. Inject 0.5 mL of 1-2 different concentrations of standard gas into the gas chromatograph in sequence, measure the response value of each standard series concentration point, draw a calibration curve with the concentration of the concentration point as the abscissa and the peak area of ​​each concentration point as the ordinate, and obtain the ethylene peak area of ​​the standard gas; S3, draw 0.5mL of the gas sample to be tested from the stoppered glass bottle, and then add 0.5mL of water to make up the volume; S4, injecting 0.5 mL of the gas sample to be tested into the gas chromatograph in sequence to obtain the ethylene peak area of ​​the gas to be tested; S5. Calculate the ratio of the ethylene peak area of ​​the gas to be tested to the ethylene peak area of ​​the standard gas to obtain the ethylene content in the gas sample to be tested.

2. The method for determining ethylene content in wheat by gas chromatography according to claim 1, characterized in that: The wheat grain samples were replaced by common wheat seedling samples. Ten single-leaf seedlings planted for 7 days were weighed and placed in a 50-mL stoppered centrifuge tube. An appropriate amount of water was added to the centrifuge tube to cover the roots of the plants. The tube was sealed and placed in a 22°C greenhouse for 24 hours to generate the gas to be tested.

3. The method for determining ethylene content in wheat by gas chromatography according to claim 1, characterized in that: Drawing the calibration curve in step S2 specifically involves drawing the calibration curve using a standard gas at one concentration point, and the calibration curve passes through the origin.

4. The method for determining ethylene content in wheat by gas chromatography according to claim 1, characterized in that: The gas chromatograph specifically uses an Agilent chromatographic column with a catalog number of 113-4332, the length of the chromatographic column is 30m, the specification is 7 inches, the inner diameter is 0.32mm, and the chromatographic column maintains a constant flow mode with a flow rate of 4mL / min.

5. The method for determining ethylene content in wheat by gas chromatography according to claim 4, characterized in that: The temperature of the inlet of the chromatographic column was 150° C., the injection method was split injection, the split ratio was 3:1, the pressure was 16.817 psi, and the septum purge was 3 mL / min.

6. The method for determining ethylene content in wheat by gas chromatography according to claim 1, characterized in that: The gas chromatograph specifically uses a hydrogen flame ionization detector, which has an operating temperature of 250° C., high sensitivity and a temperature range of up to 400° C.

7. The method for determining ethylene content in wheat by gas chromatography according to claim 6, characterized in that: The detector includes high-purity hydrogen for detection, high-purity air for adjusting sensitivity, high-purity nitrogen for carrier gas, high-purity helium for tracing and leak detection, and tail gas for eliminating extra-column effects. The flow rate of the high-purity hydrogen is 40 mL / min, the flow rate of the high-purity air is 350 mL / min, and the flow rate of the tail gas is 25 mL / min.

8. The method for determining ethylene content in wheat by gas chromatography according to claim 1, characterized in that: The standard gas in step S2 uses certified ethylene gas, and the concentration of the ethylene gas is 1 ppm.

9. The method for determining ethylene content in wheat by gas chromatography according to claim 1, characterized in that: The gas chromatograph specifically uses an Agilent injection port liner with item number 5190-2295, the injection port liner has a volume of 870uL and an inner diameter of 1mm, and the injection port liner has ultra-high inertness, can be split, has low pressure drop, and is provided with glass wool, and liners of different volumes can be replaced according to test requirements.

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