CN108627665A - A kind of high-throughput Continuous Flow Analysis method for nitrate in tobacco gene editor's material - Google Patents
A kind of high-throughput Continuous Flow Analysis method for nitrate in tobacco gene editor's material Download PDFInfo
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- CN108627665A CN108627665A CN201810146828.6A CN201810146828A CN108627665A CN 108627665 A CN108627665 A CN 108627665A CN 201810146828 A CN201810146828 A CN 201810146828A CN 108627665 A CN108627665 A CN 108627665A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
- G01N35/025—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations having a carousel or turntable for reaction cells or cuvettes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N2035/00178—Special arrangements of analysers
- G01N2035/00207—Handling bulk quantities of analyte
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Abstract
The invention discloses a kind of high-throughput Continuous Flow Analysis methods for nitrate in tobacco gene editor's material, include the following steps：A, it accurately weighs the sample that 0.25g contains nitrate to be placed in 25mL sample cells, excellent sample feeding pipe will be claimed to be put into successively on the sample injection disc of Continuous Flow Analysis instrument autosampler, until all piling；B, deionized water 25mL is separately added into each sample cell with liquid-transfering gun, then it is sequentially inserted into the inner sleeve of filtering sieve plate of the bottom with 0.45 μm of aperture, solution splashes out during preventing oscillation extraction, and sample disc is put on oscillator after being plugged inner sleeve, carries out oscillation extraction；C, inner sleeve is pushed after having extracted, the sample solution extracted is allowed to be entered in inner sleeve by the filtering sieve plate, sample disc is placed on the autosampler of Continuous Flow Analysis instrument again, the sample introduction needle of autosampler is only insertd at the supernatant in inner sleeve every time to draw filtered sample, is transferred in Continuous Flow Analysis instrument and is analyzed.
Description
Technical field
The invention belongs to tobacco chemistry research fields, and in particular to nitrate contains in a kind of suitable tobacco gene editor material
Measure the method for high-flux analysis of detection.
Background technology
With tobacco business " batch production breeding side of the tobacco gene group plan key special subjects based on genome editing technique
The propulsion of case " screens material by chemical composition analysis, and pass through tobacco leaf using all gene editing materials as object
Compatibility test and cigarette composition verification, determine the strong kind of industrial applicability, become the pass of tobacco business active demand
Key technology content.
The production quantities such as many harmful substances such as nitrogen oxides, hydrogen cyanide, catechol in the nitrate and flue gas in tobacco
It is related, the precursor of carcinogen N nitrosamine in nitrate or flue gas.Therefore, the nitrate in measurement tobacco has very heavy
The meaning wanted.Connected neighbor set is commonly used in the detection of nitrate in tobacco at present.Continuous Flow Analysis technology works standard
Liquid, sample, reagent are pumped into chemical analyzer in proportion, and gas is sent into timing simultaneously, make to form continuous fluid point in pipeline
Every system, the substance that participation is reacted is made to be sufficiently mixed during continuously flowing, then passes through series reaction such as mixing, adds
Heat, filtering etc., detect to obtain corresponding peak electrical finally by detector, then be calculated automatically from phase by standard curve
The concentration answered.Continuous Flow Analysis technology has many advantages, such as that fast detection speed, sample and reagent dosage are few, at home and abroad tobacco row
Industry is widely applied.
In the standard method (YC/T 296-2009) of current tobacco routine chemical components analysis, the measurement of nitrate is adopted
Sample is extracted with deionized water, is then measured again with Continuous Flow Analysis instrument.It, need to be by sample in sample pretreatment process
It weighs into ground triangular flask, extractant oscillation extraction is added, is filtered with qualitative filter paper, filtrate is then transferred to continuous flow again
In the sample feeding pipe of dynamic analyzer, subsequent measurements then could be carried out.It during the filtration process, need to be residual by solid sample in extraction flask
The mixture of slag and extracting solution is poured into bottleneck and is placed in the funnel of qualitative filter paper folding, and the extracting solution collection after filter is being received
Collect in bottle.It needs to shift sample several times in sample pretreatment process, it is not easy enough, influence the working efficiency of detection；And it shifted
The reliability of testing result may be influenced in journey because introducing accidentally splashing etc. for impurity and extracting solution.
Gene editing batch production breeding at least will produce tens thousand of editor's materials, the day sample of nitrate sample analysis at present
Treating capacity only has 60 or so, and the routine chemical components analysis for completing tens thousand of a materials needs very long period, analysis result it is stagnant
The progress of entire batch production breeding will be influenced whether afterwards, therefore to accurately and reliably high-throughput fast in gene editing breeding process
The demand of fast analysis method is very urgent.
Invention content
The purpose of the present invention is to provide the quick sample pretreatment method that a kind of gene editing material nitrate measures, packets
Include following steps：
A, accurately the sample that 0.25g contains nitrate is weighed to be placed in 25mL sample cells, it is automatic from Continuous Flow Analysis instrument
Sample injection disc is removed on injector, excellent sample feeding pipe will be claimed to be put into successively on Continuous Flow Analysis instrument sample injection disc, until should be into
All pile sample cell in 100 sample cell positions in sample dish；
B, it is separately added into deionized water 25mL into each sample cell with the liquid-transfering gun of 25mL, is then sequentially inserted into bottom belt
There is the inner sleeve of the filtering sieve plate in 0.45 μm of aperture, solution splashes out during preventing oscillation extraction, is plugged after inner sleeve sample
Disk is put on oscillator, and oscillation extraction is carried out by the oscillation extraction condition that standard method is selected；
C, the inner sleeve of sample cell is pushed after having extracted, the sample solution extracted is allowed to enter by the filtering sieve plate
It is placed on the autosampler of Continuous Flow Analysis instrument into inner sleeve, then sample disc, the sample introduction needle of autosampler is every
To draw filtered sample at the secondary supernatant only insertd into inner sleeve, it is transferred in Continuous Flow Analysis instrument and is analyzed.
Preferably, sealing ring is carried at the top of the sample cell, the inner sleeve is inserted into the sealing before oscillation extraction
In circle, extract liquor is then added, inner sleeve is stuck in the oral area of sample cell prevents extract liquor in oscillatory process from splashing out, but inner sleeve
The filtering sieve plate of bottom is not contacted with sample；Inner sleeve is pushed after sample extraction is complete, extract liquor, which is just filtered by sieve plate, to be entered
Into inner sleeve, autosampler can extract sample directly from inner sleeve and be analyzed.
In order to reduce transfer workload, inventor have also been devised with the matched oscillator of the sample disc of autosampler, can
Sample disc is removed from autosampler, fills direct oscillation extraction after a disk sample, inner sleeve filtering, entire sample after having extracted
Product disk is put on autosampler and is analyzed again.Whole operation flow need not shift specific sample, before sample
Processing procedure is greatly simplified, and sample analysis efficiency is compared with current tobacco business standard method and several times can be improved.And
The reduction of transfer step can effectively prevent the error in sample pre-treatments operating process, greatly reduce the introducing of human error.
In addition, in conventional tobacco nitrate flow analysis continuous mode, tobacco need to be broken into fine powder and be extracted, in routine
Fine powder meeting partial gelatinization blocks filter paper in method sample pretreatment process, and the rate of filtration is slower, and in improved processing method,
It slightly places after the completion of oscillation extraction, under tobacco sample powder is just sunk to, sieve plate when filtering in inner sleeve is only clear with upper layer
Night contacts, and can effectively avoid the sieve plate in filter process from blocking in this way, and faster, filter operation is easier to realize the rate of filtration.
When sample analysis, by tobacco business standard YC/T 269-2008, (measurement of tobacco and tobacco product nitrate is continuous
Flow method) selected condition is measured.It is first worked curve with sulfuric acid sodium standard solution when measurement, sample is placed in order
On autosampler, the titer of an intermediate concentration is put after 10 samples, and as quality control, (sample introduction frequency is every two
1 sample of minute)；Analysis after hours, according to peak height from working curve calculate sample in nitrate content.
It is higher than conventional method to the uniformity requirement of sample since connected neighbor set sample volume is small, the present invention
In test influence of the sample comminution grain size to measurement result, the results showed that：Sample is not sieved, the precision of measurement result compared with
Difference, with the increase for crossing grit number, the precision of sample measurement result increases, and when mesh number reaches 60 mesh or more, sample measures knot
The precision of fruit tends towards stability, and the relative standard deviation (RSD) of measurement is 1% hereinafter, therefore selection crushes simultaneously in the present invention
Cross 60 sieves.
Flue-cured tobacco, burley tobaccos, Turkish tobaccos, reconstituted tobacco and finished cigarettes sample are chosen, respectively in the present inventive method and cigarette
Careless professional standard YC/T 296-2009 (the measurement continuous flow method of tobacco and tobacco product nitrate) are measured, and to knot
Fruit compares (table 1).The method of the present invention measurement result and at present with tobacco business standard method measurement result without notable
Difference, analysis result are accurate and reliable.
The comparison (%) of 1 the method for the present invention of table and current standard
Beneficial effects of the present invention
Project team is directed in current tobacco deficiency present in nitrate Continuous Flow Analysis quadrat method, targetedly sets
Quick sample pretreatment method is counted.In improving analysis method, whole operation flow need not shift sample, sample pre-treatments
Process is greatly simplified.It, can be uninterrupted using more than two sample disc alternate treatments (a disk sample introduction, a disk pre-treatment)
Carry out analyze, 8-10 disk samples can be handled daily, routinely 60 samples of sample introduction speed/h calculate, and can analyze 400 or more daily
Sample, sample analysis efficiency is compared with current tobacco business standard method and several times can be improved, and actual sample analysis result
It is consistent with current industry standard methods.The present invention is the high flux screening for magnanimity editor's material that gene batch production breeding generates
High-throughput rapid analysis method is provided, the process of nitrate analysis in gene editing material screening process is greatly accelerated.
In conventional tobacco nitrate flow analysis continuous mode, tobacco need to be broken into fine powder and be extracted, in conventional method
Fine powder meeting partial gelatinization blocks filter paper in sample pretreatment process, and the rate of filtration is slower, and in improved processing method, oscillation
It is slightly placed after the completion of extraction, under tobacco sample powder just sinks to, sieve plate when filtering in inner sleeve only connects with supernatant
It touches, can effectively avoid the sieve plate in filter process from blocking in this way, faster, filter operation is easier to realize the rate of filtration.
Description of the drawings
Fig. 1 is the sample injection disc used in the present invention.
Fig. 2 is the sample feeding pipe used in the present invention and the schematic diagram of inner sleeve.Wherein a figures are sample feeding pipes；B figures are inner sleeves
Pipe；C figures are schematic diagram when inner sleeve is inserted at the top of sample feeding pipe；D figures are the schematic diagram that inner sleeve presses downwards；E figures are inner sleeve
Pipe is pressed onto bottom and completes the schematic diagram for being inserted into sampling probe Aspirate supernatant after original position is filtered again.
Specific implementation mode
The present invention is further described by following specific examples, but does not limit the present invention.
Embodiment 1：The measurement of nitrate in Virginian-type cigarette
1) sample pre-treatments：First by shredded tobacco baking, it is broken that cell wall is carried out to tobacco and tobacco product with high speed whirlwind mill
Then wall crosses 60 mesh screens, be sealed as standby sample.By YC/T296-2009 standardized test specimen moisture content, by following processing：
A, 0.25g samples accurately are weighed in the improved sample feeding pipes of 25mL, from Continuous Flow Analysis instrument autosampler
Sample injection disc is removed, the 100 sample feeding pipe positions that excellent sample feeding pipe will be claimed to pile Continuous Flow Analysis instrument sample injection disc successively；
B, it is separately added into deionized water 25mL into each sample cell with the liquid-transfering gun of 25mL, is then sequentially inserted into bottom belt
There is the inner sleeve of the filtering sieve plate in 0.45 μm of aperture, solution splashes out during preventing oscillation extraction, is plugged after inner sleeve sample
Disk is put on oscillator, and oscillation extraction is carried out by the oscillation extraction condition that standard method is selected；
C, the inner sleeve of sample cell is pushed after having extracted, the sample solution extracted is allowed to enter by the filtering sieve plate
It is placed on the autosampler of Continuous Flow Analysis instrument into inner sleeve, then sample disc, the sample introduction needle of autosampler is every
To draw filtered sample at the secondary supernatant only insertd into inner sleeve, it is transferred in Continuous Flow Analysis instrument and is analyzed.
2) according to standard method YC/T 296-2009 (the measurement continuous flow method of tobacco and tobacco product nitrate) correlations
Regulation completes the subsequent measurements work of nitrate in sample.
It is 0.25% to measure nitrate content in the Virginian-type cigarette sample.
Embodiment 2：The measurement of nitrate in blended type cigarette sample
The process in embodiment 1 is repeated, the measurement of nitrate in blended type cigarette sample is carried out, measures nitric acid in the sample
Salt content is 1.08%.
Claims (2)
1. a kind of high-throughput Continuous Flow Analysis method for nitrate in tobacco gene editor's material, which is characterized in that packet
Include following steps：
A, it accurately weighs the sample that 0.25g contains nitrate to be placed in 25mL sample cells, from Continuous Flow Analysis instrument automatic sampling
Sample injection disc is removed on device, excellent sample feeding pipe will be claimed to be put into successively on Continuous Flow Analysis instrument sample injection disc, until by the sample injection disc
In 100 sample cell positions all pile sample cell；
B, it is separately added into deionized water 25mL into each sample cell with the liquid-transfering gun of 25mL, is then sequentially inserted into bottom and carries
The inner sleeve of the filtering sieve plate in 0.45 μm of aperture, solution splashes out during preventing oscillation extraction, is plugged after inner sleeve sample disc
It is put on oscillator, oscillation extraction is carried out by the oscillation extraction condition that standard method is selected；
C, the inner sleeve of sample cell is pushed after having extracted, allows the sample solution extracted to be entered by the filtering sieve plate interior
In casing, then sample disc is placed on the autosampler of Continuous Flow Analysis instrument, the sample introduction needle of autosampler is every time only
To draw filtered sample at the supernatant being inserted into inner sleeve, it is transferred in Continuous Flow Analysis instrument and is analyzed.
2. high throughput Continuous Flow Analysis method according to claim 1, which is characterized in that carried at the top of the sample cell
Sealing ring, the inner sleeve are inserted into before oscillation extraction in the sealing ring, and extract liquor is then added, and inner sleeve is stuck in sample
The oral area of quality control prevents extract liquor in oscillatory process from splashing out, but the filtering sieve plate of inner sleeve bottom of the tube is not contacted with sample；Sample extracts
Inner sleeve is pushed after taking, extract liquor is just entered by sieve plate filtering in inner sleeve, and autosampler can be directly from inner sleeve
Sample is extracted in pipe to be analyzed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810146828.6A CN108627665A (en) | 2018-02-12 | 2018-02-12 | A kind of high-throughput Continuous Flow Analysis method for nitrate in tobacco gene editor's material |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810146828.6A CN108627665A (en) | 2018-02-12 | 2018-02-12 | A kind of high-throughput Continuous Flow Analysis method for nitrate in tobacco gene editor's material |
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| Publication Number | Publication Date |
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| CN108627665A true CN108627665A (en) | 2018-10-09 |
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| CN201810146828.6A Pending CN108627665A (en) | 2018-02-12 | 2018-02-12 | A kind of high-throughput Continuous Flow Analysis method for nitrate in tobacco gene editor's material |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN203264748U (en) * | 2013-05-24 | 2013-11-06 | 中国热带农业科学院分析测试中心 | Filter cover of soil sample effective state analysis centrifugal tube |
| CN107179417A (en) * | 2017-06-29 | 2017-09-19 | 中烟施伟策（云南）再造烟叶有限公司 | Alliance Continuous Flow Analysis instrument Microflow module is used for the method for measuring of nitrate content in reconstituted tobacco |
| CN107199063A (en) * | 2017-05-19 | 2017-09-26 | 黑龙江谱尼测试科技有限公司 | A kind of novel filter-type colorimetric cylinder |
-
2018
- 2018-02-12 CN CN201810146828.6A patent/CN108627665A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN203264748U (en) * | 2013-05-24 | 2013-11-06 | 中国热带农业科学院分析测试中心 | Filter cover of soil sample effective state analysis centrifugal tube |
| CN107199063A (en) * | 2017-05-19 | 2017-09-26 | 黑龙江谱尼测试科技有限公司 | A kind of novel filter-type colorimetric cylinder |
| CN107179417A (en) * | 2017-06-29 | 2017-09-19 | 中烟施伟策（云南）再造烟叶有限公司 | Alliance Continuous Flow Analysis instrument Microflow module is used for the method for measuring of nitrate content in reconstituted tobacco |
Non-Patent Citations (1)
| Title |
|---|
| 国家烟草专卖局: "《中华人民共和国烟草行业标准 YC/T 296-2009 烟草及烟草制品 硝酸盐的测定 连续流动法》", 31 May 2009, 中国标准出版社 * |
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Application publication date: 20181009 |
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