CN108427008A - A kind of high-throughput Continuous Flow Analysis method for total alkaloid in tobacco gene editor's material - Google Patents
A kind of high-throughput Continuous Flow Analysis method for total alkaloid in tobacco gene editor's material Download PDFInfo
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- CN108427008A CN108427008A CN201810146539.6A CN201810146539A CN108427008A CN 108427008 A CN108427008 A CN 108427008A CN 201810146539 A CN201810146539 A CN 201810146539A CN 108427008 A CN108427008 A CN 108427008A
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- sample
- inner sleeve
- continuous flow
- flow analysis
- autosampler
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Classifications
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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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
Abstract
The invention discloses a kind of high-throughput Continuous Flow Analysis methods for total alkaloid in tobacco gene editor's material, include the following steps：A, it accurately weighs the sample that 0.25g contains total alkaloid 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 total alkaloid in a kind of suitable tobacco gene editor material
The method for high-flux analysis of content 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.
Tobacco total alkaloid refers to alkaloid all in tobacco, and nicotiana alkaloids is most important with nicotine, it accounts for tobacco life
95% or more of alkaloids total amount, followed by nornicotine (nornicotine), anabasine etc., therefore measuring containing for tobacco total alkaloid
When amount, often in terms of nicotine.Excessively high nicotine content can seriously affect quality of tobacco, and it is available in cigarette composition to reduce tobacco leaf
Property.By detecting nicotine content and tobacco leaf thickness, the relationship of planting density, nicotine content of tobacco leaves can be reduced in production, is carried
The availability of high tobacco leaf.Nicotine content is also medically important indicator, can alleviate and treat a variety of diseases, as Parkinson, Ah
Wurz sea is write from memory disease, neurasthenia, amnesia etc..
Connected neighbor set is commonly used in the detection of total alkaloid 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 468-2013) of current tobacco routine chemical components analysis, the measurement of total alkaloid
Sample is extracted using water, is then measured at 460nm wavelength with Continuous Flow Analysis instrument spectrophotometric detector again.Before sample
In processing procedure, sample need to be weighed into ground triangular flask, extractant oscillation extraction is added, is filtered with qualitative filter paper, filtered
Solution afterwards is transferred in the sample feeding pipe of Continuous Flow Analysis instrument, then could carry out subsequent measurements.During the filtration process, it needs
The mixture of solid sample residue and extracting solution in triangular flask is poured into bottleneck to be placed in the funnel of qualitative filter paper folding, it will
Extracting solution after filter is collected in receiving flask.It needs to shift sample several times in sample pretreatment process, it is not easy enough, influence detection
Working efficiency；And testing result may be influenced because introducing accidentally splashing etc. for impurity and extracting solution in transfer process
Reliability.
Gene editing batch production breeding at least will produce tens thousand of editor's materials, at present the day sample of total alkaloid sample analysis
Product 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
Lag will influence whether the progress of entire batch production breeding, therefore to accurately and reliably high-throughput in gene editing breeding process
The demand of rapid analysis method is very urgent.
Invention content
The purpose of the present invention is to provide a kind of gene editing material total alkaloid measure quick sample pretreatment method,
Include the following steps：
A, it accurately weighs the sample that 0.25g contains total alkaloid to be placed in 25mL sample cells, certainly from Continuous Flow Analysis instrument
Sample injection disc is removed on dynamic injector, excellent sample feeding pipe will be claimed to be put into successively on Continuous Flow Analysis instrument sample injection disc, until should
All pile sample cell in 100 sample cell positions in sample injection disc；
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 total alkaloid flow analysis continuous mode, tobacco need to be broken into fine powder and be extracted, normal
Fine powder meeting partial gelatinization blocks filter paper in rule method sample pretreatment process, and the rate of filtration is slower, and in improved processing method
In, 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 and upper layer
The stillness of night contacts, and can effectively avoid the sieve plate in filter process from blocking in this way, and faster, filter operation is easier real the rate of filtration
It is existing.
When sample analysis, by tobacco business standard YC/T 468-2013, (measurement of tobacco and tobacco product total alkaloid connects
(potassium rhodanide) method is moved in afterflow) selected condition is measured.It is first worked curve with nicotine standard solution when measurement, sample is pressed
Sequence is placed on autosampler, and the titer of an intermediate concentration is put after 10 samples as quality control (sample introduction
Frequency is 1 sample per minute)；Analysis contains from total alkaloid in sample is calculated on working curve after hours, according to peak height
Amount.
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.
Choose flue-cured tobacco, burley tobaccos, Turkish tobaccos, respectively in the present inventive method with tobacco business standard YC/T468-2013
(measurement of tobacco and tobacco product total alkaloid continuously flows (potassium rhodanide) method) is measured, three mark-ons for growing tobacco sample
The rate of recovery is 99.07%, 99.09% and 98.33%, the results are shown in Table 1；Three grow tobacco sample measure precision be 2.78%,
2.64% and 3.87%, it the results are shown in Table 2；The method of the present invention measurement result and at present with tobacco business standard method measurement result
It is shown in Table 3.Result is compared, no significant difference, analysis result is accurate and reliable.
1 flue-cured tobacco of table, burley tobaccos, Turkish tobaccos recovery of standard addition
2 flue-cured tobacco of table, burley tobaccos, Turkish tobaccos measure precision (%)
The comparison (%) of 3 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 total alkaloid Continuous Flow Analysis quadrat method, targetedly
Devise quick sample pretreatment method.In improving analysis method, whole operation flow need not shift sample, before sample
Reason process is greatly simplified.Using more than two sample disc alternate treatments (a disk sample introduction, a disk pre-treatment), can not between
Disconnected analyze, and can handle 8-10 disk samples daily, and routinely 60 samples of sample introduction speed/h calculate, can analyze daily 400 with
On sample, sample analysis efficiency compares with current tobacco business standard method and several times can be improved, and actual sample analysis knot
Fruit and current industry standard methods are consistent.The present invention is the high-throughput sieve for magnanimity editor's material that gene batch production breeding generates
Choosing provides high-throughput rapid analysis method, greatly accelerate that total alkaloid in gene editing material screening process analyzes into
Journey.
In conventional tobacco total alkaloid flow analysis continuous mode, tobacco need to be broken into fine powder and be extracted, in conventional side
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 shakes
It swings and slightly places after the completion of extraction, under tobacco sample powder just sinks to, sieve plate when filtering in inner sleeve is only and supernatant
Contact, 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.
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 total alkaloid in flue-cured tobacco sample A
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, then press 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 468-2013 (the measurement continuous flow method of tobacco and tobacco product total alkaloid) phase
Close the subsequent measurements work that regulation completes total alkaloid in sample.
It is 1.75% to measure total alkaloid content in the Virginian-type cigarette sample.
Embodiment 2：The measurement of total alkaloid in finished cigarettes sample B
The process in embodiment 1 is repeated, the measurement of total alkaloid in finished cigarettes sample B is carried out, is measured total in the sample
Plant alkali content is 4.25%.
Claims (2)
1. a kind of high-throughput Continuous Flow Analysis method for total alkaloid in tobacco gene editor's material, which is characterized in that
Include the following steps：
A, accurate to weigh the sample that 0.25g contains total alkaloid and be placed in 25mL sample cells, from Continuous Flow Analysis instrument automatically into
Sample injection disc is removed on sample 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 introduction
All pile sample cell in 100 sample cell positions in disk；
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.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810146539.6A CN108427008A (en) | 2018-02-12 | 2018-02-12 | A kind of high-throughput Continuous Flow Analysis method for total alkaloid in tobacco gene editor's material |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810146539.6A CN108427008A (en) | 2018-02-12 | 2018-02-12 | A kind of high-throughput Continuous Flow Analysis method for total alkaloid in tobacco gene editor's material |
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| CN108427008A true CN108427008A (en) | 2018-08-21 |
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ID=63156889
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| CN201810146539.6A Pending CN108427008A (en) | 2018-02-12 | 2018-02-12 | A kind of high-throughput Continuous Flow Analysis method for total alkaloid in tobacco gene editor's material |
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Citations (4)
| 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 |
| CN104132937A (en) * | 2014-07-08 | 2014-11-05 | 国家烟草质量监督检验中心 | Continuous flow method for measuring total alkaloid in tobacco or tobacco products |
| CN106932462A (en) * | 2017-02-24 | 2017-07-07 | 云南中烟工业有限责任公司 | A kind of method of quick measure Determination of Nicotine Content of 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 CN201810146539.6A patent/CN108427008A/en active Pending
Patent Citations (4)
| 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 |
| CN104132937A (en) * | 2014-07-08 | 2014-11-05 | 国家烟草质量监督检验中心 | Continuous flow method for measuring total alkaloid in tobacco or tobacco products |
| CN106932462A (en) * | 2017-02-24 | 2017-07-07 | 云南中烟工业有限责任公司 | A kind of method of quick measure Determination of Nicotine Content of 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 |
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 468-2013 烟草及烟草制品 总植物碱的测定 连续流动（硫氰酸钾）法》", 30 June 2013, 中国标准出版社 * |
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Application publication date: 20180821 |