CN108828078B - Method for determining inorganic anions in tobacco gene editing material - Google Patents

Method for determining inorganic anions in tobacco gene editing material Download PDF

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CN108828078B
CN108828078B CN201810214642.XA CN201810214642A CN108828078B CN 108828078 B CN108828078 B CN 108828078B CN 201810214642 A CN201810214642 A CN 201810214642A CN 108828078 B CN108828078 B CN 108828078B
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inner sleeve
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CN108828078A (en
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李晶
李天飞
刘欣
孔维松
杨叶昆
杨光宇
黄海涛
许�永
张涛
张承明
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China Tobacco Yunnan Industrial Co Ltd
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
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    • G01N2030/062—Preparation extracting sample from raw material

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Abstract

The invention discloses a method for measuring 6 inorganic anions suitable for tobacco gene editing materials, which uses a device comprising a sample extraction bottle and a sieve plate, wherein the sample extraction bottle comprises an outer sleeve (1), an inner sleeve (2), a liquid guide pipe (4) and one or more sieve plates (3) which are positioned in an opening at the lower end of the inner sleeve (2), the method comprises the following steps of ① tobacco leaf extraction, ② filtration and transfer, ③ analysis for making a chromatogram, and ④ and the peak area of a standard chromatogram can obtain the content of the inorganic anions in the tobacco gene editing materials.

Description

Method for determining inorganic anions in tobacco gene editing material
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a method for determining inorganic anions in tobacco gene editing materials.
Background
Inorganic anions in tobacco have important effects on the growth, development and quality of tobacco. Such as NO3 -Is the main source of nitrogen element, Cl, in the tobacco growth process-The moisture absorption of the leaves is enhanced, and the combustibility of the tobacco leaves is all influenced; SO (SO)4 2-And PO4 3-During the tobacco growth process withAssimilation of proteins, sugars and phospholipids, NO3 -And NO2 -The content is an important index reflecting nitrogen assimilation and the generation of tobacco-specific nitrosamines. Therefore, the accurate determination of the inorganic anions in the tobacco has important significance for the growth, development and quality evaluation of the tobacco.
The inorganic anions are generally determined by chromogenic, titration and ion chromatography. The ion chromatography is simple, convenient, rapid and less in interference, and is widely applied to the determination of inorganic anions of tobacco in recent years. However, the background of the tobacco sample matrix is complex, and in the reported method, the sample is purified before ion chromatography to obtain a better effect, and the sample needs to be transferred for many times in the sample purification process, so that the operation steps are multiple. At present, the tobacco industry starts the industrial breeding work of tobacco gene editing, and the growth, development and quality of the edited material are known through the determination of inorganic anions and are important contents for material screening. However, in the gene editing factory breeding process, tens of thousands of materials are produced by gene editing. The conventional method is difficult to meet the requirements for carrying out chemical analysis evaluation on the massive materials.
In the process of evaluating the quality of tobacco gene editing materials, a simpler and faster analysis method needs to be found, which can be operated in batch and high-throughput manner and realize rapid, efficient and accurate determination.
Disclosure of Invention
The invention aims to provide a method for measuring inorganic anions of a tobacco gene editing material aiming at the defects of the prior art and provides method support for screening and evaluating tobacco gene breeding.
The method integrates an ion chromatographic analysis method with an online solid-phase extraction and purification function, and can effectively realize high-flux analysis and determination of inorganic anions in tobacco gene editing materials.
The purpose of the invention is realized by the following technical scheme.
A method for measuring inorganic anions in tobacco gene editing materials, which uses the following device:
a sample extraction vial comprising: the bottom of the outer sleeve 1 is sealed and is flat, and the opening part of the outer sleeve is provided with a sealing ring 11; the outer diameter of the inner sleeve 2 is matched with the inner diameter of the outer sleeve 1, and the length of the inner sleeve is greater than that of the outer sleeve 1; the liquid guide pipe 4 is positioned in the inner sleeve 2, the lower end of the liquid guide pipe is provided with an expansion port which is fixedly connected with the inner wall of the lower end of the inner sleeve 2 in a sealing way, the liquid guide pipe is fixedly connected with the upper end port of the inner sleeve 2 to form a pressure receiving surface 21, and the upper part of the liquid guide pipe extends out of the inner sleeve 2 and bends downwards; the sieve plates 3 are positioned in the lower end opening of the inner sleeve 2 and are one or more;
the method comprises the following steps:
① freezing tobacco leaf at-80 deg.C, oven drying at 40 deg.C, pulverizing, and sieving with 40 mesh sieve;
② weighing 0.25g of the powder obtained in step 1 into the outer sleeve 1, adding 25m L extracting agent, inserting a part of the inner sleeve 2 into the outer sleeve 1 and sealing by the sealing ring 11, and then performing ultrasonic extraction for 20min at a certain temperature;
③, applying downward force on the pressure surface 21, filtering the extraction solution in the outer sleeve 1 through the sieve plate 3, then entering the liquid guide tube 4, flowing out of the liquid guide tube 4, entering a sample collection bottle, cooling to room temperature to obtain a sample filtrate to be analyzed, collecting the sample filtrate 1-1.5m L, and performing on-line purification ion chromatography;
④, quantifying by adopting a peak area method, and comparing the peak area of the inorganic anions in the sample with a standard working curve to determine the content of the inorganic anions in the tobacco gene editing material.
The anion determined by the invention is fluoride (F)-) Chloride ion (Cl)-) Nitrite ion (NO)2 -) Phosphate radical ion (PO)4 3-) Nitrate ion (NO)3 -) And sulfate ion (SO)4 2-)。
Preferably, the extraction reagent of step ② is water.
Preferably, the heated extraction temperature of step ② is 50 ℃.
Preferably, the sieve plate of step ③ has a pore size of 0.45 μm.
Preferably, the on-line purification ion chromatography described in step ③ is to add an on-line solid phase extraction purification column before the ion chromatography analysis column, the extraction column packing is MCI-GE L reverse resin, the specification of the on-line solid phase extraction column is 4 × 10mm, the packing particle size is 5.0 μm, and the extraction capacity is 30 mg.
Preferably, the online purifying ion chromatography condition of step ③ is that an online solid phase extraction purifying column is connected to the front end of an ion chromatography analysis column, the ion chromatography condition is IonPac-AS22(250mm × 4.6.6 mm i.d.,4.0 μm), the column temperature is 35 ℃, the column flow rate is 1.5m L/min, the sample injection amount is 50 μ L, and the mobile phase is 4.5 mmol/L Na2CO3/1.4mmol/L NaHCO3An aqueous solution; isocratic elution; and detecting by a conductivity detector.
In the technical scheme of the invention, the online solid phase extraction purification column can be continuously fed with a sample for 500 times, after the sample is fed for more than 500 times, the purification column is reversely connected to the end of a liquid chromatography pump, and the purification column can be reused after backwashing is carried out for 1h at the flow rate of 1.0m L/min by using 100% methanol.
Compared with the prior art, the invention has the beneficial effects that:
① the method for determining inorganic anions in tobacco gene editing material of the invention compares fluorine ions (F) in different materials or gene editing materials with normal tobacco samples-) Chloride ion (Cl)-) Nitrite ion (NO)2 -) Phosphate radical ion (PO)4 3-) Nitrate ion (NO)3 -) And sulfate ion (SO)4 2-) The content of (b) can be used for judging and evaluating the quality of different tobacco leaves.
② the determination method also provides an ion chromatography method integrating on-line solid phase extraction purification function, the tobacco sample inorganic anion analysis process, the sample extract will contain a small amount of wax, pigment, polyphenol, fatty substance and other interference components, these components can not be eluted by the mobile phase of the ion chromatography, it is easy to remain on the chromatographic column to pollute the chromatographic column, and then affect the analysis result, the sample pretreatment using solid phase extraction can get better analysis effect, but the introduction of solid phase extraction increases the sample pretreatment operation steps, the ion chromatographic column is connected with MCI-GE L reverse resin filler on-line solid phase extraction column, when the sample passes the solid phase extraction column along with the mobile phase, the small polar component is enriched on the solid phase extraction column, can not be eluted by the mobile phase of the ion chromatography, the inorganic anion to be measured is not retained on the reverse phase solid phase extraction column, and can enter the ion chromatographic column after passing the solid phase extraction column, thus the on-line solid phase extraction purification effect can be achieved, the sample determination process does not need to be carried out separately, the sample can be removed by on-line extraction interference components, the sample in the on-phase extraction column, the sample can be removed, the sample can be transferred into the ion chromatography column, the sample, the test method provides accurate quality, the advantages of simple and the tobacco sample, the method, the advantages of simple and the accurate tobacco sample pretreatment of the method.
③ the determination method of the invention selects an on-line solid phase extraction column which is connected with MCI-GE L reverse resin filler before an ion chromatographic column, the MCI-GE L reverse resin matrix is polystyrene and divinyl copolymer, has similar retention behavior with C18, but has wider pH range than C18, can be stably used in the pH range of 0-14, and can be stably used for a long time under the condition of alkaline mobile phase of the ion chromatography, successfully solves the defects that the conventional solid phase extraction material (C18 and the like) has narrow pH range, is easy to decompose under the alkaline condition and cannot be suitable for the alkaline mobile phase of the ion chromatography, and the MCI-GE L reverse resin can be directly extracted in a solid phase without activation, and has good compatibility with the on-line solid phase extraction method.
④ the determination method of the invention optimizes the ion chromatography conditions and optimizes the conditions to obtain fluorine ion (F)-) Chloride ion (Cl)-) Nitrite ion (NO)2 -) Phosphate radical ion (PO)4 3-) Nitrate ion (NO)3 -) And sulfate ion (SO)4 2-) The chromatographic analysis time of 6 inorganic anion ions is only 10min,compared with the existing tobacco industry standard YC/T248-2008, the analysis time is shortened by more than 3 times. Further greatly improving the analysis efficiency of the sample.
⑤ the device used in the invention can directly extract, filter and collect the sample to be analyzed without transferring the sample in the sample pretreatment process, effectively avoids the experimental error caused by operation, and has the advantages of simple and convenient treatment and good effect.
Drawings
FIG. 1 is a schematic view of an outer sleeve according to the present invention;
FIG. 2 is a schematic illustration of the inner casing of the present invention;
FIG. 3 is a schematic view of the inner sleeve inserted into the outer sleeve during extraction mixing according to the present invention;
FIG. 4 is a schematic view of the inner cannula inserted into the outer cannula during filtration and transfer of the extract liquid of the present invention;
FIG. 5 is a schematic illustration of a sample transfer step of the present invention with one sample extraction vial positioned on the sample extraction rack and one sample collection vial positioned on the sample collection rack;
FIG. 6 shows 6 inorganic anion standard solutions.
FIG. 7 is a chromatogram of 6 inorganic anions in tobacco gene editing material.
Description of the drawings: 1-outer sleeve; 2-inner sleeve; 21-a pressure-bearing surface; 3, a sieve plate; 4-a catheter; 5-sample extraction rack; 51-sample extraction bottle placing hole; 6-sample collection rack; 61-sample bottle placement holes; 62-waste liquid collecting bottle placing holes; 610-a sample collection vial; 620-waste liquid collection bottle; 11-a sealing ring; 12-sample.
Detailed Description
The technical solutions of the present invention are described in detail below with reference to embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available by purchase.
Example 1
Taking 4 finished tobacco leaves as investigation samples, determining the content of inorganic anions in the tobacco leaves, and comprising the following steps:
① sample pretreatment, selecting 4 finished tobacco leaves currently used as objects to be inspected, drying at 40 ℃ in an oven, crushing and sieving with a 40-mesh sieve, accurately weighing 0.25g of sample powder in an outer sleeve, adding 25m L water as an extracting agent, fixing an inner sleeve of an extraction bottle at the opening of the outer sleeve by using a sealing ring, placing the outer sleeve after installation, heating and ultrasonically treating at 50 ℃ for 20min, forcibly pressing the inner sleeve of the extraction bottle after extraction is completed, enabling an extraction solution to enter a liquid guide pipe through a filtering sieve plate and flow out of the liquid guide pipe to achieve the effect of filtering the sample, collecting 1-1.5m L of extraction liquid from the return bend of the liquid guide pipe to the lower opening, and performing online purification ion chromatography analysis.
② ion chromatographic analysis, adding an on-line solid phase extraction purification column before the ion chromatographic analysis column, wherein the extraction column filler is MCI-GE L, the extraction capacity is 30mg, the ion chromatographic conditions are IonPac-AS22(250mm × 4.6.6 mm i.d.,4.0 μm), the column temperature is 35 ℃, the column flow rate is 1.5m L/min, the sample injection amount is 50 μ L, and the mobile phase is 4.5 mmol/L Na2CO3/1.4mmol/LNaHCO3An aqueous solution; isocratic elution; and detecting by a conductivity detector.
And quantifying by adopting a peak area method, and comparing the peak area of the inorganic anions in the sample with a standard working curve to determine the content of the inorganic anions in the tobacco gene editing material.
The 4 finished tobacco leaves were tested and analyzed simultaneously using industry standard methods, and the results of the two analyses are compared in table 2.
TABLE 1 tobacco leaf product inorganic anion analysis results (mg/g)
Figure BDA0001598234040000051
Example 2
Selecting tobacco seedlings of 5 material samples of a gene editing and breeding factory as objects to be investigated, and freeze-drying the samples at-80 ℃. Sample treatment and ion chromatography were the same as in example 1. The results and evaluations of the sample analysis are shown in Table 2.
TABLE 2 tobacco leaf product inorganic anion analysis results (mg/g)
Sample (I) F- Cl- NO2 - PO4 3- NO3 - SO4 2- Evaluation of
Sample No. 1 1.33 7.524 - 5.23 0.404 13.4 Is normal
Sample No. 2 0.22 3.47 0.0208 2.18 3.14 5.28 Is abnormal
Sample No. 3 0.968 8.47 - 6.82 0.463 12.2 Is normal
Sample No. 4 0.428 4.18 0.0187 3.81 0.266 6.59 Is abnormal
Sample No. 5 3.59 5.22 0.215 9.22 0.628 25.7 Is abnormal
Example 3
5 kinds of white remainder tobacco finished tobacco leaves are used as a test sample, the content of 6 kinds of inorganic anions is measured, the measuring steps are the same as those in the example 1, and the content of the inorganic anions in the sample conforms to the range of the content of the inorganic anions in the high-quality white remainder tobacco.
Example 4
The content of 6 inorganic anions in 5 types of finished aromatic tobacco leaves is measured by taking the finished aromatic tobacco leaves as a test sample, the measuring steps are the same as those in the example 1, and the content of the inorganic anions in the sample conforms to the range of the content of the inorganic anions in the high-quality aromatic tobacco.

Claims (7)

1. A method for measuring inorganic anions in tobacco gene editing material, which is characterized by using the following devices:
a sample extraction vial comprising: the bottom of the outer sleeve (1) is sealed and is flat, and the opening part of the outer sleeve is provided with a sealing ring (11); the outer diameter of the inner sleeve (2) is matched with the inner diameter of the outer sleeve (1), and the length of the inner sleeve is greater than that of the outer sleeve (1); the liquid guide pipe (4) is positioned in the inner sleeve (2), the lower end of the liquid guide pipe is provided with an expansion port which is fixedly connected with the inner wall of the lower end of the inner sleeve (2) in a sealing way, the liquid guide pipe is fixedly connected with the upper end port of the inner sleeve (2) to form a pressed surface (21), and the upper part of the liquid guide pipe extends out of the outer part of the inner sleeve (2) and is bent downwards; the sieve plates (3) are positioned in the lower end opening of the inner sleeve (2) and are one or more;
the method comprises the following steps:
① freezing tobacco leaf at-80 deg.C, oven drying at 40 deg.C, pulverizing, and sieving with 40 mesh sieve;
② weighing 0.25g of the powder obtained in step ① in the outer sleeve (1), adding 25m L of extracting agent, inserting a part of the inner sleeve (2) into the outer sleeve (1) and sealing by the sealing ring (11), and then carrying out ultrasonic extraction for 20min at a certain temperature;
③, applying a downward force on the pressure surface (21), filtering the extraction solution in the outer sleeve (1) through the sieve plate (3), then entering the liquid guide tube (4), flowing out of the liquid guide tube (4), entering a sample collection bottle, cooling to room temperature to obtain a sample filtrate to be analyzed, collecting the sample filtrate 1-1.5m L, and performing on-line purification ion chromatography, wherein the on-line purification ion chromatography comprises adding an on-line solid phase extraction purification column in front of an ion chromatography column, and the extraction column filler is MCI-GE L reversed phase resin;
④, quantifying by adopting a peak area method, and comparing the peak area of the inorganic anions in the sample with a standard working curve to determine the content of the inorganic anions in the tobacco gene editing material.
2. The method according to claim 1, wherein the inorganic anion is a fluoride ion (F)-) Chloride ion (Cl)-) Nitrite ion (NO)2 -) Phosphate ion (P0)4 3-) Nitrate ion (NO)3 -) And sulfate ion (SO)4 2-）。
3. The method of claim 1, wherein the extractant of step ② is water.
4. The method of claim 1, wherein the temperature of step ② is 50 ℃.
5. The assay method according to claim 1, wherein the sieve plate of step ③ has a pore size of 0.45 μm.
6. The determination method according to claim 1, wherein the extraction column packing of the step ③ is MCI-GE L reversed phase resin, the specification of the on-line solid phase extraction column is 4 × 10mm, the grain size of the packing is 5.0 μm, and the extraction capacity is 30 mg.
7. The method of claim 1, wherein the step ③ is performedThe on-line purification ion chromatographic analysis conditions comprise that an on-line solid phase extraction purification column is connected to the front end of the ion chromatographic analysis column, the ion chromatographic conditions comprise IonPac-AS22250mm × 4.6.6 mm i.d.,4.0 mu m, the column temperature is 35 ℃, the column flow rate is 1.5m L/min, the sample injection amount is 50 mu L, and the mobile phase is 4.5 mmol/L Na2CO3/1.4 mmol/L NaHCO3An aqueous solution; isocratic elution; and detecting by a conductivity detector.
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