CN108508126A - The assay method of non-volatile organic acid in a kind of tobacco gene editor material - Google Patents
The assay method of non-volatile organic acid in a kind of tobacco gene editor material Download PDFInfo
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- CN108508126A CN108508126A CN201810146504.2A CN201810146504A CN108508126A CN 108508126 A CN108508126 A CN 108508126A CN 201810146504 A CN201810146504 A CN 201810146504A CN 108508126 A CN108508126 A CN 108508126A
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- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- 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
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
-
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
Abstract
The invention discloses a kind of assay methods of non-volatile organic acid in tobacco gene editor material, use following device：Sample extraction bottle, including：Outer tube (1), inner sleeve (2), catheter (4), sieve plate；Sample extraction frame (5)；Sample collection frame (6)；Turn to take out (7) and lower platen (8).Step is：1. extracting；2. filtering；3. collecting sample filtrate；4. being measured using high performance liquid chromatography.The assay method of the present invention can be used for detecting the content of non-volatile organic acid in tobacco gene editor's material, have the advantages that pre-treatment is simple and convenient, quantitative accurate, reproducible.
Description
Technical field
The invention belongs to tobacco chemistry research fields, and in particular to non-volatile in a kind of suitable tobacco gene editor material to have
The method for high-flux analysis of machine acid 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.
The organic acid being accredited out in tobacco leaf up to more than 200 is planted, with lactic acid, citric acid, malic acid, oxalic acid, succinic acid
It is important composition ingredient in tobacco for the non-volatile organic acid of representative, accounts for about 70% or more of tobacco leaf organic acid total amount.These are non-
Volatile organic acids has a major impact cigarette quality, they not only play adjustment effect to flue gas acid-base value, and can also increase
Add the non-fragrance of flue gas, make cigarette jealous more alcohol and.Therefore non-volatile organic acids comment tobacco quality in Accurate Determining tobacco
Valence has positive effect.
Organic acid assay method has gas chromatography, high performance liquid chromatography and chromatography of ions etc. in traditional tobacco,
Tobacco business standard YC/T 500-2014 just use gas chromatography to measure the organic acid in tobacco, but before gas-chromatography sample
Processing needs esterification to derive, and the operating time is long, and each chromatography time is more than 30min, cannot meet high-volume sample
The demand that product are quickly analyzed.It, will by gene editing with the startup of tobacco business tobacco gene editor's batch production breeding work
Generate tens thousand of a materials.Rapid chemical assay evaluation is carried out to the material of these magnanimity, to easy, quick high throughput
The demand of analysis method is very urgent.
Invention content
The assay method of non-volatile organic acid, chromatography time can shorten to 1min in the tobacco of the present invention.The present invention
It, can be net to non-volatile organic acid progress Rapid Extraction, filtering, Solid Phase Extraction in tobacco also by the processing unit of autonomous Design
Change and directly collect filtrate, simplifies sample pre-treatments step, extract liquor, which need not shift, can directly filter sample introduction, can be cigarette
The Accurate Determining of non-volatile organic acid provides effective ensure in grass.It is compared with current professional standard, sample analysis amount can be improved 5
Times or more.The gene editing material Chemical Screening that is established as of the method for the present invention provides quick, accurate, reliable high-throughput point
Analysis method.
The purpose of the present invention is what is be achieved by the following technical programs.
The assay method of non-volatile organic acid, uses following device in a kind of tobacco gene editor material：
Sample extraction bottle, including：Outer tube 1, sealed bottom and to be flat, oral area has a sealing ring 11；Inner sleeve
2, outer diameter is adapted with the internal diameter of the outer tube 1, length be more than the outer tube 1 length；Catheter 4, is located at
In the inner sleeve 2, lower end has an extension mouth and the lower end inner wall of the inner sleeve 2 to be sealedly and fixedly connected, and described interior
2 upper port of casing is fixedly connected to form compression face 21, and upper part is stretched out outside the inner sleeve 2, and back bending is downward；Sieve plate 3, position
In in the lower ending opening of the inner sleeve 2, for one or more；Sample extraction frame 5 inside has several sample extraction bottle putting holes
51, for placing the sample extraction bottle；Sample collection frame 6 inside has several sample bottle putting holes 61 and several waste collections
Bottle putting hole 62, for placing sample bottle 610 and waste collection bottle 620；Turn to take out 7, is located at 6 central part of sample collection frame；
The sample collection frame 6 and the sample extraction frame 5 can surround the pumping 7 that turns and rotate respectively；Lower platen 8, surface is extended with
Several push-down heads 81；
This approach includes the following steps：
1. accurately weighing the tobacco powder sample of 0.25g in outer tube, the extractant of 25mL is added, is inserted into inner sleeve simultaneously
It is placed on the sample extraction frame and carries out ultrasonic extraction 30min；
2. being fixed using shaft, the nozzle of the catheter is made to be directed at waste collection bottle 620, push inner sleeve by
Pressure surface 21 simultaneously makes extraction solution pass through after filtering sieve plate 3 filters to enter in catheter, and pass out into waste collection bottle 620
In, initial filtrate 2-3mL enters in waste collection bottle 620；
3. centered on shaft, sample extraction frame is rotated, the downward back bending nozzle of the catheter is made to be directed at the sample
610 bottlenecks of bottle continue the compression face 21 for pushing inner sleeve, 1-1.5mL sample filtrates are transferred in sample bottle 610, are cooled to
Room temperature obtains sample filtrate to be analyzed；
4. being measured using efficient liquid phase chromatographic analysis, quantified using peak area method, contrast sample tobacco gene
Edit the peak area method of the peak area and standard solution working curve of non-volatile organic acid in material, you can determine non-in tobacco
The content of volatilization organic acid.
Preferably, non-volatile organic acid is lactic acid, citric acid, malic acid, oxalic acid and succinic acid.
Preferably, the step sodium hydroxide solution that 1. used extractant is 1v/v%.
Preferably, 2. the aperture for obtaining sieve plate (3) is 0.45 μm to step；The sieve plate (3) is two pieces, two pieces of sieve plates
Between be filled with solid phase extraction filler.
Preferably, the solid phase extraction filler is MCI-GEL reversed-phase resin fillers.
Preferably, 4. the liquid phase chromatogram condition is step：Chromatographic column：WatersAcquity UPLCTM BEH C18
(2.1 × 50mm, 1.7 μm) analytical column；Mobile phase：The acetonitrile of 5v/v%；Flow velocity：0.5mL/min；Sample size：2.0μL；Column temperature：
35℃；Detector：Diode array detector, organic acid Detection wavelength are 210nm.
Preferably, the also phosphoric acid containing 0.01moL/L in the mobile phase.
The sample extraction bottle that the method for the present invention uses integrates extraction, filtering, transfer, and sample extracts in outer tube 1
After taking；Inner sleeve 2 is firmly pushed at compression face 21, extract liquor enters after being filtered by sieve plate 3 in catheter 4, and
The downward mouth of back bending from catheter 4 exits into sample bottle, obtains sample filtrate and carries out chromatography.This process sample
Extraction, filtering, transfer are integrated, and avoid experimental error caused by operation.
1 oral area of outer tube dress fixes inner sleeve 2 there are one sealing ring 11 when for extracting, with prevent extract liquor from splashing out with
And it is buffered when sample filtering.
In the present invention, 4 diameter of catheter is preferably 1-2mm.
It is as follows using multiple sample extraction bottle operating process of apparatus of the present invention：Several sample extraction bottles (such as eight) are put
In the extraction flask putting hole 51 for entering sample extraction frame 5, it is respectively put into sample and carries out extracting operation；Start the part solution collected
(such as 2ml) is abandoned, therefore is started first respectively the waste collection bottle 620 on the downward back bending of catheter 4 alignment sample collection frame 6
Oral area, lower platen 8 is covered, push-down head 81 is made to be respectively aligned to compression face 21, the liquid in waste collection bottle 620 reaches requirement
Afterwards, rotation sample extraction frame 5 or sample collection frame 6 make the downward back bending of catheter 4 be directed at the oral area of sample bottle 610, continue to
Lower platen 8 is pushed, the fluid sample in eight sample extraction bottles enters respective drain after being filtered respectively by respective sieve plate 3
It in pipe 4, enters back into sample bottle 610, continues to push the bottom that the lower platen 8 makes inner sleeve 2 be depressed into outer tube 1, then institute
There is the liquid in sample extraction bottle to be transferred to respectively in sample bottle 610 by respective catheter 4, completes sample transfer behaviour
Make.Sample in sample bottle 610 can be respectively used to analyze.
The assay method of the present invention：
(1) standard curve and detection of the invention are limited to：Using the series of 5 kinds of non-volatile organic acids of selected chromatographic condition pair
Standard solution is measured, and using each component peak area as ordinate (Y), corresponding each component mass concentration is drawn for abscissa (X)
Standard curve, obtains regression equation and related coefficient, and detection limit takes continuous 10 sample introductions of minimum concentration standard solution, calculating to work as noise
Corresponding standard detection limit, quantitative limit is determined with concentration corresponding to signal-to-noise ratio S/N=10 when than S/N=3.
Table 1：Regression equation, related coefficient and the detection limit of 5 kinds of non-volatile organic acids
(2) repeatability of the invention and recovery of standard addition
Sample introduction is analyzed measures by selected sample pre-treatments condition processing, and by selected chromatographic condition for sample, a copy of it
On the basis of, another be added known quantity non-volatile organic acid standard specimen (set be similar to actual sample content 0.5,1.0,2.0 times
Three additive amounts), the rate of recovery is calculated by the amount of measuring divided by Standard entertion amount that standard is added.Obtain returning for each non-volatile organic acid
For yield between 93.8-98.5%, the illustration method rate of recovery is very high.
3 kinds of cigarettes samples are taken, interior parallel determination 7 times respectively on the same day on the same day and not by selected experiment condition,
Calculate the relative standard deviation of 7 parallel determinations.The result shows that the in a few days RSD of 5 kinds of non-volatile organic acid measurement results exists
Between 1.5-2.2%, RSD illustrates that the method precision of the present invention is excellent between 1.9-2.6% in the daytime.
Beneficial effects of the present invention are：
(1) a kind of novel sample processing device of the invention, can collect extraction, filtering, Solid phase extraction and transfer
It is integrated, sample transfer is not needed in entire sample pretreatment process can obtain filtered sample to be analysed, pre-treatment behaviour
It is simplified, may increase the link of introducing error also can accordingly be reduced, and the precision of analysis result is improved.Normal
When rule method sample pre-treatments operate, in syringe filters filter process, tobacco fine powder can block filtering head, the rate of filtration compared with
Slowly；And the processing method of the present invention, it is slightly placed after the completion of extraction, under tobacco sample powder just sinks to, inner sleeve when filtering
In sieve plate only contacted with supernatant, can effectively avoid the sieve plate in filter process from blocking in this way, the rate of filtration faster, mistake
Filter operation is easier to realize.
(2) in assay method of the invention, sample injection volume only 2.0 μ L, sample with the sodium hydroxide medium of 1v/v% into
It mixes with flowing after sample and is neutralized by mobile phase quickly；Therefore, color will not be caused with regard to direct injected not adjusting sample pH
Spectral peak modification is omitted and adjusts pH after sample is extracted with sodium hydroxide again to acid step.
(3) method detection time of the invention is short.Lactic acid, lemon in assay method measurement tobacco using the present invention
Acid, malic acid, oxalic acid and succinic acid, only need 1min or so.
(4) present invention have the advantages that operation accurately, high sensitivity and reproducible.
Description of the drawings
Fig. 1 is the outer tube schematic diagram of the present invention；
Fig. 2 is the inner sleeve schematic diagram of the present invention；
Inner sleeve is inserted into housing spout part schematic diagram when Fig. 3 is the extraction mixing of the present invention；
Inner sleeve is inserted into outer tube schematic internal view when Fig. 4 is the extract liquor filtering of the present invention, transfer；
Fig. 5 is the sample extraction frame and sample collection frame schematic diagram of the present invention；
Fig. 6 is that the sample extraction bottle of the present invention is placed on sample extraction frame and sample collection bottle is placed on sample receipts
Collect the schematic diagram of sample transfer step when on frame；
Fig. 7 is the transparent lower platen schematic diagram of the present invention；
Fig. 8 is standard solution chromatogram；
Fig. 9 is the sample solution chromatogram of embodiment 1.
Description of the drawings：1- outer tubes；2- inner sleeves；21- compression faces；3- sieve plates；4- catheters；5- sample extraction framves；51-
Sample extraction bottle putting hole；6- sample collection framves；61- sample bottle putting holes；62- waste collection bottle putting holes；610- samples are received
Collect bottle；620- waste collection bottles；7- shafts；8- lower platens；81- push-down heads；82- handles；11- sealing rings；12- samples.
Specific implementation mode
The present invention is further described by following specific examples, but does not limit the present invention.
Embodiment 1：Non-volatile organic acid content testing in tobacco sample A
1, instrument and reagent
Lactic acid, citric acid, malic acid, oxalic acid and succinic acid standard items (purity >=98%) (Sigma Co., USA)；Hydrogen-oxygen
Change sodium (analysis is pure)；Phosphoric acid (analysis is pure)；Acetonitrile (chromatographically pure) is purchased from Merck companies of Germany；Water be quartzy sub-boiling distillation water simultaneously
It is handled with Milli-Q50 ultra-pure water instrument.
Waters Acquity type ultra performance liquid chromatography systems, equipped with vacuum degassing machine, binary gradient pump, automatic sampling
Device, thermocolumn case, diode array detector and Empower-2 chromatographic work stations (Waters, US)；AE240 electronics point
Analyse balance (accurate 0.0001g, Mettler company)；Electric heating constant temperature ultrasound bath pot (Changzhou Nuo Ji Instrument Ltd.)；
Milli-Q50 ultra-pure waters instrument (Millipore companies of the U.S.).
2, standard solution is prepared
Within the scope of 0.8-500 μ g/mL with 1% sodium hydroxide solution making lactic acid, citric acid, malic acid, oxalic acid and
The standard solution of at least five concentration of succinic acid standard items.
3, the processing of sample
Sample treatment is carried out using the device of the invention, it is one that can collect extraction, filtering, Solid phase extraction and transfer
Body, specific device structure are shown in attached drawing 1-7.
Sample pretreatment process：
(1) it extracts：Tobacco sample crushes, and crosses 40 mesh sieve, accurately weighs sample 0.25g in outer tube (Fig. 1), use 25mL
Liquid-transfering gun be added 25mL 1% sodium hydroxide solution, then the inner sleeve of extraction flask be stuck in outer tube oral area (figure
3).Sample disc is put into ultrasonic extraction 30min in constant-temperature ultrasonic water-bath after installing.
(2) filtering transfer：After having extracted, the inner sleeve of extraction flask is pressed downward, extraction solution is just by filtering sieve plate simultaneously
It is entered in catheter by the solid phase extraction filler between two layers of sieve plate, and is flowed out from catheter, reach sample filtering and solid phase
The effect (Fig. 4 and Fig. 5) of extracting and purifying.Extract liquor is collected from catheter, initial 2-3mL is collected into waste collection bottle,
It discards.Then use-sample collection bottle directly collects the sample extraction liquid of 1-1.5mL, for liquid-phase chromatographic analysis.
4, chromatographic condition
Chromatographic column：Waters Acquity UPLCTM BEH C18 (2.1 × 50mm, 1.7 μm) analytical column；
Mobile phase：The acetonitrile (phosphoric acid for including 0.01moL/L) of 5v/v%；
Flow velocity：0.5mL/min；
Sample size：2.0μL；
Column temperature：35℃；
Using qualitative, the quantified by external standard method with standard specimen comparison retention time.
5, assay method
Regression analysis is carried out with the chromatographic peak area of object and its respective concentration, standard curve is obtained, sees Fig. 8.To system
The sample A got ready is measured, and measures the chromatographic peak area of detection object, is substituted into standard curve, is respectively obtained in sample
5 kinds of non-volatile organic acid contents such as lactic acid, citric acid, malic acid, oxalic acid and succinic acid, are shown in Fig. 9.In fig.9,1- oxalic acid；2-
Malic acid；3- lactic acid；4- citric acids；5- succinic acids.
Embodiment 2：Non-volatile organic acid content testing in tobacco sample B
Specific steps are as described in Example 1, select another tobacco sample B, measure 5 kinds of non-volatile organic acid contents and are shown in Table 2.
The content of 5 kinds of non-volatile organic acids in table 2, tobacco sample B
| Compound | Content (mg/g) |
| Lactic acid | 5.30 |
| Citric acid | 7.01 |
| Malic acid | 34.45 |
| Oxalic acid | 8.11 |
| Succinic acid | 2.22 |
Embodiment 3：Non-volatile organic acid content testing in tobacco sample C
Specific steps are as described in Example 1, select another tobacco sample C, measure 5 kinds of non-volatile organic acid contents and are shown in Table 3.
The content of 5 kinds of non-volatile organic acids in table 3, tobacco sample C
| Compound | Content (mg/g) |
| Lactic acid | 3.21 |
| Citric acid | 19.6 |
| Malic acid | 48.7 |
| Oxalic acid | 12.3 |
| Succinic acid | 5.21 |
Claims (6)
1. the assay method of non-volatile organic acid in a kind of tobacco gene editor material, which is characterized in that use following device：
Sample extraction bottle, including：Outer tube (1), sealed bottom and to be flat, oral area has a sealing ring (11)；Inner sleeve
(2), outer diameter is adapted with the internal diameter of the outer tube (1), length be more than the outer tube (1) length；Catheter
(4), it is located in the inner sleeve (2), lower end has the lower end inner wall of an extension mouth and the inner sleeve (2) to seal fixed connect
It connecing, compression face (21) is fixedly connected to form with the inner sleeve (2) upper port, upper part stretches out inner sleeve (2) outside,
And back bending is downward；Sieve plate (3) is located in the lower ending opening of the inner sleeve (2), for one or more；Sample extraction frame (5),
There are several sample extraction bottle putting holes (51) in it, for placing the sample extraction bottle；Sample collection frame (6), if inside having
Dry-eye disease bottle putting hole (61) and several waste collection bottle putting holes (62), for placing sample bottle (610) and waste collection bottle
(620)；Turn to take out (7), is located at sample collection frame (6) central part；The sample collection frame (6) and the sample extraction frame
(5) pumping (7) that turns can be surrounded respectively to rotate；Lower platen (8), surface are extended with several push-down heads (81)；
This approach includes the following steps：
1. accurately weighing the tobacco powder sample of 0.25g in outer tube, the extractant of 25mL is added, be inserted into inner sleeve and places
Ultrasonic extraction 35min is carried out on the sample extraction frame (5)；
2. being fixed using shaft, so that the nozzle of the catheter is directed at waste collection bottle (620), push the compression of inner sleeve
Face (21) simultaneously makes extraction solution pass through after filtering sieve plate (3) filters to enter in catheter, the initial filtrate 2-3mL that passes out into
Enter in waste collection bottle (620)；
3. centered on shaft, sample extraction frame is rotated, the downward back bending nozzle of the catheter is made to be directed at the sample bottle
(610) bottleneck continues the compression face (21) for pushing inner sleeve, 1-1.5mL sample filtrates is transferred in sample bottle (610), are obtained
To sample filtrate to be analyzed；
4. being measured using efficient liquid phase chromatographic analysis, quantified using peak area method, contrast sample tobacco gene editor
The peak area method of the peak area of non-volatile organic acid and standard solution working curve in material, you can determining non-volatile in tobacco has
The content of machine acid.
2. according to the method described in claim 1, it is characterized in that, the step sodium hydroxide that 1. used extractant is 1v/v%
Solution.
3. according to the method described in claim 1, it is characterized in that, step 2. the aperture for obtaining sieve plate (3) is 0.45 μm；
The sieve plate (3) is two pieces, and solid phase extraction filler is filled between two pieces of sieve plates.
4. according to the method described in claim 1, it is characterized in that, the solid phase extraction filler is filled out for MCI-GEL reversed-phase resins
Material.
5. according to the method described in claim 1, it is characterized in that, step 4. the high-efficient liquid phase chromatogram condition is：Chromatography
Column：WatersAcquity UPLCTM BEH C18 (2.1 × 50mm, 1.7 μm) analytical column；Mobile phase：The acetonitrile of 5v/v%；Stream
Speed：0.5mL/min；Sample size：2.0μL；Column temperature：35℃；Detector：Diode array detector；Organic acid Detection wavelength is
210nm。
6. according to the method described in claim 5, it is characterized in that, the also phosphoric acid containing 0.01moL/L in the mobile phase.
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| CN105784455A (en) * | 2016-05-16 | 2016-07-20 | 张起香 | Extraction structure and liquid pretreatment device |
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2018
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