CN105699552A - Sample liquid purification treatment method for detecting squalene contained in cigarette smoke based on high performance liquid chromatography - Google Patents
Sample liquid purification treatment method for detecting squalene contained in cigarette smoke based on high performance liquid chromatography Download PDFInfo
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- G—PHYSICS
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- 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
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- G01N30/06—Preparation
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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
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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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Abstract
The invention discloses a sample liquid purification treatment method for detecting squalene contained in cigarette smoke based on high performance liquid chromatography. The method comprises the steps that smoke solid particle phase substances are collected and extracted with chloroform, and obtained extraction liquid is subjected to rotary evaporation to a dry state; a product is dissolved with normal hexane and then washed with normal hexane, the liquid is subjected to rotary evaporation to a dry state, and then the product is subjected to column chromatographic separation treatment; the treated product is eluted with chloroform, elution liquid is collected and subjected to rotary evaporation to a dry state, the product is made into a constant volume with chromatographic acetonitrile and then passes through a 0.22-micrometer filter membrane, and obtained filter liquid is sample liquid to be detected, wherein an N-primary secondary amine solid-phase extraction column is adopted for column chromatographic separation. Based on the sample pre-treatment method, the high performance liquid chromatography for squalene contained in cigarette smoke can be established, chromatographic peak separation is good, and the method has the advantages of being accurate, fast, high in sensitivity, good in reproducibility, high in recycling rate and the like.
Description
Technical field
The present invention relates to the detection technique field of composition in cigarette smoke, detect the sample liquid purifying treatment method of Squalene contained by cigarette smoke more particularly, to a kind of high performance liquid chromatography。
Background technology
Squalene is find from the liver oil of shark at first, and within 1914, being named as Squalene, its chemical name is 2,6,10,15,19,23--hexamethyl--2,6,10,14,18,22--tetracosa carbon six alkene, belong to open chain triterpenoid compound。
Squalene has superoxide dismutase (SOD) activity in raising human body, enhancing body non-oxidizability and immunocompetence, improves the different physiological roles such as sexual function, defying age, resisting fatigue, antitumor, is a kind of avirulent bioactive substance with the effect of preventing and curing diseases。Research recently finds that human body is also had Detoxication, the fat-soluble toxins in removable tissue by Squalene。There are some researches show: add a certain amount of Squalene in tobacco shred, it is possible to decrease several harmful substances and free-radical contents in flue gas, it was shown that Squalene has uniqueness and drops evil effect, developing Squalene at present has become scientific and technological circle and the focus of tobacco research。
In April, 2014, State Tobacco Monopoly Bureau issues about the notice (domestic cigarettes do comprehensive [2014] No. 218) accelerating Tar technological achievement popularization and application, proposition to strengthen the application dynamics of Harm reduction techniques technology key special subjects achievement in research, and Counter-techniques measure will be taked to accelerate Tar paces。In order to coordinate State Bureau to accelerate the very important decision of Tar paces, the applicant is completing on China Tobacco Corporation 2013 year science and technology general project " the ecological accumulation of Guangxi main flume Natural antioxidant Squalene and harm reduction research " (middle cigarette does [2013] No. 149) Research foundation, in the urgent need to setting up the detection method of a kind of feasible main flume Squalene content being suitable to standard。
But, at present both at home and abroad but without the detection method that in the flue gas being referred to, especially Squalene is qualitative and quantitative in main flume, therefore, the detection method setting up Squalene in flue-cured tobacco qualitative and quantitative at tobacco business is forward-looking, is also very necessary。And in flue gas in the detection of Squalene, the testing result obtaining accurate science is played key effect by good sample-pretreating method。Under the testing conditions determined, different extractant is adopted to extract Squalene in sample, the factors such as its extraction efficiency, extraction operation used time are all the key factors that must take into, it is again interactional relation between each factor, has no the sample pre-treatments research report detecting Squalene in Nicotiana tabacum L. based on high performance liquid chromatography at present。
Summary of the invention
The technical problem to be solved in the present invention is to fill up the detection technique deficiency of Squalene in existing flue gas, it is more particularly suitable in high performance liquid chromatography detection flue gas the deficiency of sample-pretreating method of Squalene, it is provided that a kind of detect the sample liquid purifying treatment method of Squalene contained by cigarette smoke based on high performance liquid chromatography。
Technical scheme is achieved by the following technical programs:
There is provided a kind of and detect the sample liquid purifying treatment method of Squalene contained by cigarette smoke based on high performance liquid chromatography, comprise the following steps:
S11. collect the solid granule phase substance of flue gas and extract: smoking cigarette, adopting filter disc to collect the solid granule phase substance of flue gas；
S12. extract: the solid granule phase substance cambridge filter of flue gas will be have collected and put in conical flask, and add extractant, mechanical shaking extraction under room temperature, obtain extracting solution；
S13. purifying: take step S12 gained extracting solution, the solution obtained after reprocessing, then through post, chloroform eluting, is collected eluent, rotary evaporated to dryness, with chromatograph acetonitrile constant volume, is crossed 0.22 μm of filter membrane and namely obtain analyte sample fluid；
Wherein, extractant described in step S12 is chloroform；
Described in step S13, reprocessing refers to step S12 gained extracting solution rotary evaporated to dryness, with n-hexane dissolution, then washs with normal hexane；
Crossing post described in step S13 is adopt N-propyl group ethylenediamine solid phase solid-phase extraction column。
Preferably, smoking cigarette described in step S11 is according to GB/T16450 standard operation。Filter disc described in step S11 is the cambridge filter of Φ 92mm。
Preferably, mechanical shaking extraction described in step S12 is with 130 turns/min vibration 20min with shaking table。
Preferably, described in step S13, the number of times of washing is 3 times。
Preferably, the dress column method of N-propyl group ethylenediamine solid phase solid-phase extraction column described in step S13, for weighing 0.3gN-propyl group ethylenediamine solid-phase adsorbent dress post, adds 1.0g anhydrous sodium sulfate above it。
Based on described sample-pretreating method, present invention simultaneously provides the method for contained Squalene in a kind of high performance liquid chromatography detection main flume, with methanol and acetonitrile for mobile phase, adopt the column chromatography separation technology of the high-effective fixed phase being filled with particle diameter 5 μm, accurately draw 10 μ L analyte sample fluids injection high performance liquid chromatographs and carry out chromatographic isolation, with ultraviolet absorption detector, detect the absorption value of Squalene in 210nm wavelength place, adopt external standard method, according to its content of absworption peak areal calculation。
In described high performance liquid chromatography detection main flume, the method for contained Squalene comprises the following steps:
S1. sample obtains sample liquid through pre-treatment；
S2. preparing standard solution, Criterion curve；
S3. detection: take sample liquid liquid and inject high performance liquid chromatograph detection；
S4. external standard method is adopted, according to absworption peak areal calculation Squalene content。
The testing conditions of described detection is:
(1) high performance liquid chromatograph HPLC (Agilent1100) joins UV-detector；
(2) chromatographic column: C18 post, 5 μm, 4.6 × 250mm；
(3) column temperature: 20 DEG C；
(4) mobile phase type: methanol: acetonitrile (V:V=70:30)；
(5) flow rate of mobile phase: 0.8mL/min；
(6) detection wavelength is: 210nm；
(7) sample size: 10 μ L。
The method of preparing standard solution described in step S2 is accurately to weigh 0.1g Squalene standard substance, is accurate to 0.0001g, is settled to 100mL and is configured to the Squalene standard solution of 1000mg/L after dissolving with acetonitrile (chromatographically pure), and fully vibration shakes up。Joined standard solution is placed in 4 DEG C of preservations, stand-by。
Described in step S2, the method for the foundation of Squalene standard curve is: adopt serial dilutions, is configured to Squalene standard solution that concentration is 0.002,0.01,0.05,0.2,0.5mg/mL respectively in high performance liquid chromatography sample detection；With sample introduction concentration for abscissa, peak area is vertical coordinate Criterion curve, obtains the equation of linear regression of Squalene concentration and respective peaks area。
The present invention is the detection of Squalene suitable in cigarette mainstream flue gas。
The method have the advantages that
The present invention is directed to cigarette smoke to establish and a kind of be suitable to the sample-pretreating method of Squalene in high performance liquid chromatography detection Nicotiana tabacum L., adopt chloroform as Extraction solvent, with n-hexane dissolution and washing after rotary evaporated to dryness, ensured higher extraction efficiency。Purify then through N-propyl group ethylenediamine solid phase solid-phase extraction column science, remove impurity well。Based on described sample-pretreating method, can setting up the high-efficiency liquid chromatography method for detecting of Squalene in flue gas, chromatographic peak separates better, has accurate, quick, highly sensitive, favorable reproducibility, response rate advantages of higher。
Accompanying drawing explanation
Fig. 1 Squalene canonical plotting。
Fig. 2 Squalene standard sample chromatogram (1mg/L Squalene standard specimen, peak area=41.9)。
Fig. 3 Squalene standard sample chromatogram (5mg/L Squalene standard specimen, peak area=213.3)。
Fig. 4 chloroform extracted solution is not added with Squalene blank sample chromatogram。
Fig. 5 chloroform extracted solution adds 40mg/kg (being scaled tobacco content) Squalene sample chromatogram figure。
Fig. 6 is without purified treatment effect chromatogram (blank)。
Fig. 7 is without purified treatment effect chromatogram (adding 40mg/kg Squalene sample)。
The chromatogram (blank) of Fig. 8 sample after PSA column purification。
The chromatogram (adding 40mg/kg Squalene sample) of Fig. 9 sample after PSA column purification。
Figure 10 chloroform extracted solution directly crosses chromatogram after post processes。
Figure 11 chloroform extracted solution is then through crossing chromatogram after post processes after n-hexane dissolution。
The chromatogram (mobile phase methanol: acetonitrile, 5mg/kg Squalene standard specimen) of Figure 12 C8 post detection sample。
The chromatogram (mobile phase methanol: acetonitrile, blank) of Figure 13 C8 post detection sample。
The chromatogram (mobile phase methanol: acetonitrile adds the sample of 40mg/kg Squalene) of Figure 14 C8 post detection sample。
The chromatogram (mobile phase acetonitrile: water, 5mg/kg Squalene standard specimen) of Figure 15 C8 post detection sample。
The chromatogram (mobile phase acetonitrile: water, blank) of Figure 16 C8 post detection sample。
The chromatogram (mobile phase acetonitrile: water adds the sample of 40mg/kg Squalene) of Figure 17 C8 post detection sample。
The chromatogram (mobile phase methanol: acetonitrile, 75:25, flow velocity 1.0mL/min, column temperature 30 DEG C, wavelength 210nm, 5mg/kg Squalene standard specimen) of Figure 18 C18 post detection sample。
The chromatogram (mobile phase methanol: acetonitrile, 75:25, flow velocity 1.0mL/min, column temperature 30 DEG C, wavelength 210nm, blank) of Figure 19 C18 post detection sample。
The chromatogram (mobile phase methanol: acetonitrile, 75:25, flow velocity 1.0mL/min, column temperature 30 DEG C, wavelength 210nm, add the sample of 40mg/kg Squalene) of Figure 20 C18 post detection sample。
The chromatogram (mobile phase methanol: acetonitrile, 70:30, flow velocity 0.8mL/min, column temperature 20 DEG C, wavelength 210nm, blank) of Figure 21 C18 post detection sample。
The chromatogram (mobile phase mobile phase methanol: acetonitrile, 70:30, flow velocity 0.8mL/min, column temperature 20 DEG C, wavelength 210nm, add the sample of 40mg/kg Squalene) of Figure 22 C18 post detection sample。
Detailed description of the invention
The inventive method is further illustrated below in conjunction with the drawings and specific embodiments。Following embodiment and accompanying drawing being merely cited for property explanation, it is impossible to be interpreted as limitation of the present invention。Unless stated otherwise, the reagent raw material used in following embodiment is reagent raw material that is conventional commercial or that be either commercially available, and unless stated otherwise, the method and apparatus used in following embodiment is method and apparatus commonly used in the art。For convenience of description, the instrument used in the embodiment of the present invention and reagent illustrate as follows, but therefore do not limit the present invention。
Instrument:
(1) high performance liquid chromatograph HPLC (Agilent1100), is equipped with: degassed pump, quaternary pump, automatic sampler, column oven and UV-detector (Agilent company of the U.S.)
(2) full temperature control shaking table (high honour Instrument Ltd. of Community of Jin Tan County city)
(3) electronic balance (BSA2245, d=0.1mg, Sai Duolisi scientific instrument company limited)
(4) rotary vacuum evaporator: ZFQ-3 type (Shanghai Yarong Biochemical Instrument Plant)
(5) vacuum filtration pump: SHZ-D III type (Yu Hua instrument plant of Gongyi City)
(6)MS3basic vortex instrument (MS3BS25, IKA)
(7) RM200A smoking machine (Germany)
(8) solid-phase extraction column (varian)
(9) the general glass drying oven of various laboratorys
Reagent:
(1) 99.8% Squalene reference substance (medicine inspecting institute of China)
(2) methanol (chromatographically pure)
(3) acetonitrile (chromatographically pure)
(4) normal hexane (analytical pure)
(5) chloroform (chloroform, analytical pure)
(6) petroleum ether (analytical pure)
(7) 0.22 μm of organic filter membranes
(8) cleanser: PSA (Welchrom), silica gel (200-300 order, Haiyang Chemical Plant, Qingdao)
(9) commodity cigarette (randomness extracts from Guangxi China Tobacco Industrial LCC's product)
Embodiment 1
1. the preparation of Squalene standard solution
Accurately weighing 0.1g Squalene standard substance, be accurate to 0.0001g, be settled to 100mL and be configured to 1000mg/L Squalene standard solution after dissolving with acetonitrile (chromatographically pure), fully vibration shakes up。Joined standard solution is placed in 4 DEG C of preservations。
2. the foundation of standard curve
Adopt serial dilutions, respectively compound concentration be 0.5,1,2,5,10mg/L Squalene standard solution detect in high performance liquid chromatography；With sample introduction concentration for abscissa, peak area is vertical coordinate Criterion curve, as shown in Figure 1。Obtain the equation of linear regression of Squalene concentration and respective peaks area, y=44.61x-3.2993, R2=0.9996。Standard angle zamene chromatogram is shown in shown in Fig. 2 and Fig. 3。
3. liquid phase chromatogram condition
(1) high performance liquid chromatograph HPLC (Agilent1100) joins UV-detector
(2) chromatographic column: C18 post, 5 μm, 4.6 × 250mm
(3) column temperature: 20 DEG C
(4) mobile phase type: methanol: acetonitrile (V:V=70:30)
(5) flow rate of mobile phase: 0.8mL/min
(6) detection wavelength is: 210nm
(7) sample size: 10 μ L
4. sample size calculates
Sample Squalene concentration (mg/L) can calculate according to Squalene standard curve and equation of linear regression。
C: Squalene content in cigarette mainstream smoke condensate, mg/kg；
C0: the actually measured Squalene concentration mg/L of sample；
V1: filter disc extracting liquid volume, mL；
V2: pipette extracting liquid volume, mL；
V3: final constant volume, mL；
M: suction cigarette tobacco shred gross weight, kg。
5. the solid granule phase substance of flue gas is collected
According to GB/T16450 standard, smoking cigarette, often group suction 10, every cigarette shreds weight 0.5g, use the suction of RM200A smoking machine。Φ 92mm cambridge filter is put in 250mL conical flask, adds 1000mg/L Squalene titer 0.2mL, adds such as different extractant 200mL respectively, then the amount adding Squalene in sample solution is 1mg/L, is scaled tobacco content then for 40mg/kg。With shaking table with 130 turns/min vibration 20min under room temperature, 4 DEG C preserve or to be measured。Detect according to aforementioned testing conditions。
6. result and analysis
6.1 different extractant extraction efficiencies compare
Test compares the extraction effect of different extractants, the present embodiment illustrates using normal hexane, petroleum ether, chloroform as extractant, wherein normal hexane and the petroleum ether response rate are relatively low, lower than 70%, the chloroform response rate is higher, the response rate, more than 70%, reaches response rate requirement, addition and the response rate and all represents with tobacco content。Different extractant extraction efficiencies are shown in Table 1。
Table 1: the extraction efficiency of different extractants compares
The chloroform response rate is higher, reaches 77%；Normal hexane and petroleum ether are minimum, lower than 30%。Different extracting solution flue gases add and are not added with the chromatogram of Squalene, see shown in Fig. 4～5。
The comparison of 6.2 cleanser clean-up effects
6.2.1 sample-pretreating method
(1) without purified treatment extracting solution as liquid to be measured
Take chloroform extracted solution 10mL, rotary evaporated to dryness, 2mL chromatograph acetonitrile constant volume, cross 0.22 μm of filter membrane, to be measured or 4 DEG C of preservations。
Measure effect without purified treatment to see shown in Fig. 6 and Fig. 7。Sample is more without purified treatment impurity, and object is had interference, causes the object can not be accurately qualitative and quantitative, and chromatographic column is seriously polluted, and long-time analysis meeting makes chromatographic column post effect reduce, serious meeting blocking chromatographic column。
(2) extracting solution through reprocessing after as liquid to be measured
When chloroform cooks extractant, with chloroform drip washing SPE post, eluting impurity is more, therefore selects 10mL chloroform extracted solution rotary evaporated to dryness, uses 3mL n-hexane dissolution, divides three washings with 10mL normal hexane, obtain hexane solution, hexane solution is crossed post。Shown in clean-up effect See Figure 10 (extracting solution directly crosses post) and Figure 11 (crossing post after n-hexane dissolution)。
(3) purified dose of process
Adopt PSA (N-propyl group ethylenediamine solid-phase adsorbent) Solid-Phase Extraction (PSA) post
Dress post: weigh 0.3gPSA, carries out dress post, and top adds about 1.0g anhydrous sodium sulfate (preventing solution from rinsing PSA, to cause uneven)。
Loading: take extracting solution 10mL, carried out post。
Eluting: with 3mL extractant eluting。
Collect eluent, rotary evaporated to dryness, 2mL chromatograph acetonitrile constant volume, cross 0.22 μm of filter membrane, to be measured or 4 DEG C of preservations。The eluent collected, is all measured by aforementioned liquid phase chromatogram condition。
By drawing after above purified treatment, non-purified sample impurity peaks is more, and impurity and object are not completely separated。Sample reduces substantially through PSA column purification rear impurity, it is possible to accurately qualitative, clean-up effect is shown in shown in Fig. 8 and 9。
Can be drawn by above-mentioned experimental result, after crossing post, sample good separating effect。Crossing normal hexane and the petroleum ether response rate after post relatively low, the chloroform response rate is up to 70%。
The comparative experiments of the different testing conditions of embodiment 2
In order to improve analysis efficiency, experiment devises different chromatographic column difference mobile phase and the different proportion impact on separating effect。For exempting to repeat one by one, it is below that example illustrates for C18 post and C8 post。
One, C8 post:
(1), methanol: acetonitrile different proportion has:
(1), methanol: acetonitrile ratio is 70:30, flow velocity 1.0mL/min, column temperature 30 DEG C, wavelength 210nm。
Methanol and acetonitrile do mobile phase, and object appearance time is at about 9min, and appearance time is too early, and impurity serious interference, separating effect is undesirable。
(2), mobile phase ratio and chromatographic condition are adjusted
Methanol: acetonitrile ratio is 5:95, flow velocity 0.7mL/min, column temperature 35 DEG C, wavelength 210nm。
Analysis result shows, in object appearance time, impurity effect is serious, and impurity peaks is overlapping with target peak, not up to separation requirement, affects object quantitative。Analyze result to see shown in Figure 12,13,14。
(2), with C8 post separation methanol and acetonitrile as mobile phase and different proportion time, the separating effect of target peak is undesirable, impurity serious interference。Now do mobile phase with acetonitrile and water and the ratio that adjusts is easily separated。
Acetonitrile: water, different proportion has: 96:4,95:5,94:6,93:7,92:8, flow velocity 1.0mL/min, column temperature 30 DEG C。
Mobile phase acetonitrile: water, ratio 94:6, flow velocity 1.0mL/min, column temperature 30 DEG C。
Doing mobile phase with acetonitrile and water, object peak area significantly increases, and blank group is inconspicuous with sample interpolation group peak area difference。Separated analysis verification is learnt, does mobile phase, impurity and object with acetonitrile and water and goes out peak simultaneously, and chromatographic peak is overlapping。
Representative chromatogram is shown in shown in Figure 15,16,17。
Summing up through great many of experiments relative analysis, it has been found that use C8 post, when acetonitrile and water are as mobile phase and different proportion, blank and interpolation sample peak area are all abnormal high than C18 post。Same sample C18 post, methanol and acetonitrile do mobile phase, go out peak place at object and two fused peakss occur, it is impossible to being kept completely separate, it is almost identical that two peak-to-peak areas record peak area with C8 post。So, to use C8 post, acetonitrile and water to do mobile phase, object and impurity and be likely to go out peak simultaneously, chromatographic peak is completely overlapped。
Two, C18 post:
With C8 post separate targets thing not up to separation requirement, it is impossible to impurity peaks and target peak are kept completely separate and come, owing to impurity disturbs, object can not be accurately qualitative and quantitative。The present embodiment adopts C18 post with acetonitrile: water, methanol: acetonitrile does the embodiment that mobile phase is easily separated and illustrates the inventive method。
(1), acetonitrile: water, different proportion has: 95:5, flow velocity 1.5mL/min, column temperature 30 DEG C。
C18 post uses acetonitrile and water as mobile phase, and object appearance time is more than 90min, and analysis time is long。
(2), methanol: the impact on appearance time of the acetonitrile different proportion, design has: 75:25,70:30,60:40,40:60,30:70 etc.。
1, methanol and acetonitrile are mobile phase, flow velocity 1.0mL/min, column temperature 30 DEG C, wavelength 210nm。
Methanol and acetonitrile do mobile phase, with multiple ratio at flow velocity 1.0mL/min, column temperature 30 DEG C, under wavelength 210nm, are easily separated, and object separating effect is all not up to requiring, impurity peaks jamming target thing is qualitative and quantitative。
Separating effect is in Table 2:
Table 2. methanol and acetonitrile do mobile phase, and different proportion separating effect compares:
| Methanol: acetonitrile | Separating effect |
| 75:25 | Object and impurity are not kept completely separate, and there is overlap at two peaks |
| 70:30 | Object and impurity are not kept completely separate, and there is overlap at two peaks |
| 60:40 | Object and impurity are not kept completely separate, and there is overlap at two peaks |
| 40:60 | Object and impurity are not kept completely separate, and there is overlap at two peaks |
| 30:70 | Object and impurity are not kept completely separate, and there is overlap at two peaks |
Representative chromatogram, is shown in shown in Figure 18, Figure 19, Figure 20。
2, flow velocity and column temperature are adjusted:
Sum up through great many of experiments and find, at mobile phase methanol: acetonitrile=70:30, flow velocity 0.8mL/min, column temperature 20 DEG C, when wavelength 210nm, separating effect significantly improves, object can with magazins' layout, separating degree R > 0.9, can object have been carried out accurately qualitative and quantitative。Representative chromatogram is shown in shown in Figure 21 and Figure 22。
The present invention is directed to the detection of the Squalene in cigarette smoke and establish the detection method of holonomic system, initially set up the sample-pretreating method of chloroform extraction+n-hexane dissolution and washing, under ensureing the extraction ratio premise up to 77%, science removes impurity, gained sample treatment thing is after post processes, and impurity effect is decreased obviously, and target peak is not by impurity effect, can carrying out qualitative analysis, object reaches separation requirement。
Claims (6)
1. one kind is detected the sample liquid purifying treatment method of Squalene contained by cigarette smoke based on high performance liquid chromatography, it is characterised in that comprise the following steps:
S11. collect the solid granule phase substance of flue gas and extract: smoking cigarette, adopting filter disc to collect the solid granule phase substance of flue gas；
S12. extract: the solid granule phase substance cambridge filter of flue gas will be have collected and put in conical flask, and add extractant, mechanical shaking extraction under room temperature, obtain extracting solution；
S13. purifying: take step S12 gained extracting solution, the solution obtained after reprocessing, then through post, chloroform eluting, is collected eluent, rotary evaporated to dryness, with chromatograph acetonitrile constant volume, is crossed 0.22 μm of filter membrane and namely obtain analyte sample fluid；
Wherein, extractant described in step S12 is chloroform；
Described in step S13, reprocessing refers to step S12 gained extracting solution rotary evaporated to dryness, with n-hexane dissolution, then washs with normal hexane；
Crossing post described in step S13 is adopt N-propyl group ethylenediamine solid phase solid-phase extraction column。
2. high performance liquid chromatography detects the sample liquid purifying treatment method of Squalene contained by cigarette smoke according to claim 1, it is characterised in that smoking cigarette described in step S11 is according to GB/T16450 standard。
3. high performance liquid chromatography detects the sample liquid purifying treatment method of Squalene contained by cigarette smoke according to claim 1, it is characterised in that filter disc described in step S11 is the cambridge filter of Φ 92mm。
4. high performance liquid chromatography detects the sample liquid purifying treatment method of Squalene contained by cigarette smoke according to claim 1, it is characterised in that mechanical shaking extraction described in step S12 is with 130 turns/min vibration 20min with shaking table。
5. high performance liquid chromatography detects the sample liquid purifying treatment method of Squalene contained by cigarette smoke according to claim 1, it is characterised in that described in step S13, the number of times of washing is 3 times。
6. high performance liquid chromatography detects the sample liquid purifying treatment method of Squalene contained by cigarette smoke according to claim 1, it is characterized in that, the dress column method of N-propyl group ethylenediamine solid phase solid-phase extraction column described in step S13, for weighing 0.3gN-propyl group ethylenediamine solid-phase adsorbent dress post, adds 1.0g anhydrous sodium sulfate above it。
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