CN108489778A - A kind of high-throughput Continuous Flow Analysis method for potassium content in tobacco gene editor's material - Google Patents
A kind of high-throughput Continuous Flow Analysis method for potassium content in tobacco gene editor's material Download PDFInfo
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- CN108489778A CN108489778A CN201810146830.3A CN201810146830A CN108489778A CN 108489778 A CN108489778 A CN 108489778A CN 201810146830 A CN201810146830 A CN 201810146830A CN 108489778 A CN108489778 A CN 108489778A
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- 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
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- 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
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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- 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
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- 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
- G01N1/40—Concentrating samples
- G01N1/4055—Concentrating samples by solubility techniques
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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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
- G01N35/1065—Multiple transfer devices
- G01N35/1074—Multiple transfer devices arranged in a two-dimensional array
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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
- G01N35/1095—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices for supplying the samples to flow-through analysers
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- 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
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
- G01N2001/2866—Grinding or homogeneising
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- 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
- G01N1/40—Concentrating samples
- G01N1/4055—Concentrating samples by solubility techniques
- G01N2001/4061—Solvent extraction
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Abstract
The invention discloses a kind of high-throughput Continuous Flow Analysis methods for potassium content in tobacco gene editor's material, including step：A, it accurately weighs the sample that 0.25g contains potassium 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 potassium content is examined in a kind of suitable tobacco gene editor material
The method for high-flux analysis of survey.
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.
Potassium is routine chemical components important in tobacco, is one of the factor for influencing tobacco leaf important quality, helps to improve
The flammability of tobacco leaf, safety, fragrance value and the perfume quantity of tower height tobacco leaf.The common continuous flowing point of the detection of Potassium in Tobacco at present
Analysis method.Standard working solution, sample, reagent are pumped into chemical analyzer by Continuous Flow Analysis technology in proportion, and timing simultaneously
Feeding gas makes to form continuous fluid separating system in pipeline, keeps the substance that participation is reacted abundant during continuous flowing
Mixing, then such as mixes, heats, filters by series reaction, detects to obtain corresponding peak value electricity finally by detector
Signal, then corresponding concentration is calculated automatically from by standard curve.Continuous Flow Analysis technology fast, sample with detection speed
The advantages that few with reagent dosage, at home and abroad tobacco business is widely applied.
In the standard method (YC/T 217-2007) of current tobacco routine chemical components analysis, the measurement of potassium is all made of
Water extracts sample, burns to extract liquor, obtains its concentration according to the radiation intensity that electro-optical system detects, and then calculate sample
Potassium content in product.The index of Continuous Flow Analysis is used compared to some other, the measurement of potassium need not introduce reagent, relatively easy,
But in sample pretreatment process, sample need to be weighed into ground triangular flask, extractant oscillation extraction is added, uses qualitative filter paper
Filtrate, is then transferred in the sample feeding pipe of Continuous Flow Analysis instrument, then could carry out subsequent measurements by filtering again.In filtering
In the process, the mixture of solid sample residue and extracting solution in extraction flask need to be poured into bottleneck and be placed with qualitative filter paper folding
In funnel, the 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 the working efficiency of detection；And it may be influenced because introducing accidentally splashing etc. for impurity and extracting solution in transfer process
The reliability of testing result.
Gene editing batch production breeding at least will produce tens thousand of editor's materials, at present the day sample treatment of potassium sample analysis
Amount only 60 or so, the routine chemical components analysis for completing tens thousand of a materials need very long period, and the lag of analysis result will
It influences whether the progress of entire batch production breeding, therefore quickly divides accurately and reliably high-throughput in gene editing breeding process
The demand of analysis method is very urgent.
Invention content
The purpose of the present invention is to provide a kind of high-throughput Continuous Flow Analysis method of gene editing material potassium content, packets
Include following steps：
A, it accurately weighs the sample that 0.25g contains potassium 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 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 potassium 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.
When sample analysis, by tobacco business standard YC/T 217-2007, (measurement of tobacco and tobacco product potassium continuously flows
Method) selected condition is measured.It is first worked curve with potassium chloride standard solution when measurement, sample is placed in automatically in order
On injector, the titer of an intermediate concentration is put after 10 samples, and as quality control, (sample introduction frequency is 1 per minute
Sample)；Analysis after hours, according to peak height from working curve calculate sample in potassium 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.
Choose flue-cured tobacco, Turkish tobaccos, burley tobaccos, thin slice and finished cigarettes sample, respectively in the present inventive method with tobacco row
Industry standard YC/T 217-2007 (the measurement continuous flow method of tobacco and tobacco product potassium) are measured, and are carried out to result
Compare (table 1).The method of the present invention measurement result and at present with tobacco business standard method measurement result without significant difference, analysis
As a result 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 targetedly devises for deficiency present in current Potassium in Tobacco Continuous Flow Analysis quadrat method
Quick sample pretreatment method.In improving analysis method, whole operation flow need not shift sample, sample pretreatment process
It is greatly simplified.Using more than two sample disc alternate treatments (a disk sample introduction, a disk pre-treatment), can it is continual into
Row analysis, daily 2 people can handle 8-10 disk samples, and routinely 60 samples of sample introduction speed/h is calculated, and can analyze 400 or more daily
Sample, sample analysis efficiency are compared with current tobacco business standard method and several times can be improved, and actual sample analysis result and
Industry standard methods are consistent at present.The present invention is that the high flux screening for magnanimity editor's material that gene batch production breeding generates carries
High-throughput rapid analysis method has been supplied, the process that potassium is analyzed in gene editing material screening process is greatly accelerated.
In conventional tobacco potassium flow analysis continuous mode, tobacco need to be broken into fine powder and be extracted, in conventional method sample
Fine powder meeting partial gelatinization blocks filter paper in pretreatment process, and the rate of filtration is slower, and in improved processing method, oscillation extraction
It slightly places after the completion, under tobacco sample powder just sinks to, sieve plate when filtering in inner sleeve is only contacted with supernatant, this
Sample can effectively avoid the sieve plate in filter process from blocking, 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 potassium in flue-cured tobacco sample
1) sample pre-treatments：Tobacco leaf (or pipe tobacco) is dried first, tobacco and tobacco product are carried out with high speed whirlwind mill
Then cell wall broken wall crosses 60 mesh screens, be sealed as standby sample.By YC/T31-1996 standardized test specimen moisture content.Then
By following processing：
A, it accurately weighs the sample that 0.25g contains potassium 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 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 217-2007 (the measurement continuous flow method of tobacco and tobacco product potassium) relevant regulations
Complete the subsequent measurements work of potassium in sample.
It is 2.18% to measure potassium content in flue-cured tobacco sample.
Embodiment 2：The measurement of potassium in burley tobaccos sample
The process in embodiment 1 is repeated, the measurement of potassium in burley tobaccos sample is carried out, it is 1.32% to measure potassium content.
Claims (2)
1. a kind of high-throughput Continuous Flow Analysis method for potassium content 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 potassium to be placed in 25mL sample cells, from Continuous Flow Analysis instrument autosampler
Sample injection disc is removed, excellent sample feeding pipe will be claimed to be put into successively on Continuous Flow Analysis instrument sample injection disc, until will be in the sample injection disc
All pile sample cell in 100 sample cell positions；
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 |
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| CN201810146830.3A CN108489778A (en) | 2018-02-12 | 2018-02-12 | A kind of high-throughput Continuous Flow Analysis method for potassium content in tobacco gene editor's material |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810146830.3A CN108489778A (en) | 2018-02-12 | 2018-02-12 | A kind of high-throughput Continuous Flow Analysis method for potassium content in tobacco gene editor's material |
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101279146A (en) * | 2007-12-28 | 2008-10-08 | 河南理工大学 | Sample-pretreating method for novel continuous flow-solid phase micro-extraction and extractor thereof |
| 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 |
| WO2013171667A1 (en) * | 2012-05-15 | 2013-11-21 | Pompeo Moscetta | Device and method for the determination and monitoring of water toxicity |
| CN105510108A (en) * | 2016-01-11 | 2016-04-20 | 云南中烟工业有限责任公司 | Extraction bottle for extracting and separating total particulate matter in cigarette smoke |
| CN206027158U (en) * | 2016-09-21 | 2017-03-22 | 天津安邦键合科技有限公司 | Processing apparatus before extraction module and liquid |
| CN106908564A (en) * | 2017-02-28 | 2017-06-30 | 云南省烟草质量监督检测站 | Lithium, sodium, ammonia, potassium, magnesium, the method for calcium content in smoke-free tobacco product are determined simultaneously |
| CN106908513A (en) * | 2017-03-09 | 2017-06-30 | 浙江大学 | It is a kind of for 96 hole high flux solid-phase extraction devices of mass spectral analysis and its application |
| 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 |
| CN206710167U (en) * | 2017-04-21 | 2017-12-05 | 广州瑞森生物科技股份有限公司 | A kind of residual detection Microwave Extraction pipe of medicine |
-
2018
- 2018-02-12 CN CN201810146830.3A patent/CN108489778A/en active Pending
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| CN101279146A (en) * | 2007-12-28 | 2008-10-08 | 河南理工大学 | Sample-pretreating method for novel continuous flow-solid phase micro-extraction and extractor thereof |
| WO2013171667A1 (en) * | 2012-05-15 | 2013-11-21 | Pompeo Moscetta | Device and method for the determination and monitoring of water toxicity |
| 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 |
| CN105510108A (en) * | 2016-01-11 | 2016-04-20 | 云南中烟工业有限责任公司 | Extraction bottle for extracting and separating total particulate matter in cigarette smoke |
| CN206027158U (en) * | 2016-09-21 | 2017-03-22 | 天津安邦键合科技有限公司 | Processing apparatus before extraction module and liquid |
| CN106908564A (en) * | 2017-02-28 | 2017-06-30 | 云南省烟草质量监督检测站 | Lithium, sodium, ammonia, potassium, magnesium, the method for calcium content in smoke-free tobacco product are determined simultaneously |
| CN106908513A (en) * | 2017-03-09 | 2017-06-30 | 浙江大学 | It is a kind of for 96 hole high flux solid-phase extraction devices of mass spectral analysis and its application |
| CN206710167U (en) * | 2017-04-21 | 2017-12-05 | 广州瑞森生物科技股份有限公司 | A kind of residual detection Microwave Extraction pipe of medicine |
| 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 |
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| Title |
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| 国家烟草专卖局: "《中华人民共和国烟草行业标准-YC/T 217-2007 烟草及烟草制品 钾的测定 连续流动法》", 1 May 2007, 中国标准出版社 * |
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Application publication date: 20180904 |