CN103235066B - Hanging drop liquid phase micro-extraction method for extracting aroma component in tobacco leaf - Google Patents
Hanging drop liquid phase micro-extraction method for extracting aroma component in tobacco leaf Download PDFInfo
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- CN103235066B CN103235066B CN201310130252.1A CN201310130252A CN103235066B CN 103235066 B CN103235066 B CN 103235066B CN 201310130252 A CN201310130252 A CN 201310130252A CN 103235066 B CN103235066 B CN 103235066B
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Abstract
The present invention discloses a hanging drop liquid phase micro-extraction method for extracting an aroma component in tobacco leaf, and belongs to the technical field of solution extraction. The method sequentially comprises the following steps: (1) a tobacco leaf sample pretreatment: weigh tobacco leaf powder, adding a mixed solution of methanol and water, extracting for 40 min, carrying out centrifugation for 8 min at a speed of 3000 rpm, and taking the supernatant to prepare a tobacco leaf sample solution; (2) hanging drop liquid phase micro-extraction: making a volume ratio of toluene to the tobacco leaf sample solution be 1:100-200, adjusting a rotation speed of a magnetic stirrer so as to stabilize the single drop toluene at the bottom of vortex, and extracting for 5-8 min; and (3) detection: adopting a micro sampler to suck the single drop toluene, and adopting a gas chromatograph-mass spectrometer to detect. The hanging drop liquid phase micro-extraction method has the following characteristics that: the apparatus is simple, the production cost is low, the extraction time is short, the enrichment factor of 80-90 can be achieved only by requiring 50-100 microliter of the organic solvent, and the low content aroma component in the tobacco leaf is easily detected.
Description
Technical field
The invention belongs to solvent extraction technique field, be specifically related to a kind of hanging drop liquid-phase micro extraction technique that extracts Changing Regulation of Aroma Materials.
Background technology
At present, distillation extraction method is widely used in the extraction of Changing Regulation of Aroma Materials simultaneously, and this method takes time and effort, and required organic solvent amount is large, and test sample amount is less.For realizing the preenrichment of sample, must concentrate the extract of large volume, can make like this object loss.For problems of the prior art, need a kind of novel method to solve.Prior art discloses a kind of liquid-phase micro extraction technique, its ultimate principle is that target analytes reaches partition equilibrium between sample and the extraction solvent of micro updating, thereby realize the micro-extraction of solute, at present liquid-phase micro extraction technique has application in fields such as some pesticide residues, middle medicine and nursing products, but Field of Tobacco to tobacco leaf in the mensuration of fragrance component have not been reported.The method (publication No. CN 102507806 A) of VFA in China's a kind of solid-phase micro-extracting device of publication and mensuration tobacco leaf, to adopt solid phase micro-extraction method to measure VFA in tobacco leaf, its assay method comprises and takes tobacco sample to headspace sample bottle, add interior mark compound and anhydrous sodium sulfate to mix, draw derivatization reagent in glass interpolation pipe simultaneously, interpolation pipe is put into head space bottle, sealed with head space bottle cap, carried out full-automatic headspace solid-phase microextraction.In recent years, the progressive direction of liquid-phase extraction method was updated by technical exactly, finally reached utilization organic extraction solvent, the effective enrichment of mass transfer velocity realize target thing from matrix faster still less.
Summary of the invention
For above shortcomings in prior art, the object of this invention is to provide a kind of simple in structure, easy and simple to handle, enrichment times is higher, extraction time is short, be suitable for the hanging drop liquid-phase microextraction method of fragrance component in the extraction tobacco leaf of a large amount of sample determinations.
For achieving the above object, the present invention realizes by following technical proposals: a kind of hanging drop liquid-phase microextraction method that extracts fragrance component in tobacco leaf, is characterized in that: it comprises the step of following order:
(1) tobacco sample pre-service: take tobacco leaf powder in round-bottomed flask, adding volume ratio is the methyl alcohol of 1:7 and the mixed solution of water, the mass ratio that makes mixed solution and tobacco leaf powder is 14～15:1, extract 40 min, after centrifugal 8 min of 3000 rpm, get its supernatant and make tobacco sample solution.
(2) hanging drop liquid-phase micro-extraction: extraction flask is placed on magnetic stirring apparatus, the tobacco sample solution that adds magnetic stir bar and step (1) to make, open magnetic stirring apparatus, when tobacco sample solution forms whirlpool, draw toluene inject whirlpool place with microsyringe, the volume ratio of toluene and tobacco sample solution is 1:100～200, regulate the rotating speed of magnetic stir bar, make single toluene be stable at whirlpool bottom, cover tightly extraction flask bottle cap, extraction 5～8 min;
(3) detect: by single toluene sucking-off, put into the sample introduction bottle that 200 μ L interpolation pipes are housed with microscale sampler, auto injection, utilizes gas chromatograph-mass spectrometer (GCMS) to detect.
The rotating speed of the magnetic stir bar described in step of the present invention (2) hanging drop liquid-phase micro-extraction is 800-1100 rps.
The described condition that gas chromatograph-mass spectrometer (GCMS) detects of utilizing of step of the present invention (3) is: chromatographic condition: chromatographic column is DB-5MS (30 mm i.d. × 0.25, m × 0.25 μ m d.f.) Fused-silica capillary column, carrier gas He, flow velocity 1.0 mL/min, injector temperature is 280 ° of C, heating schedule is that 60 ° of C keep 1 min, rise to 135 ° of C with the speed of 5 ° of C/min, rise to 175 ° of C with 2 ° of C/min again, finally rise to 250 ° of C with the speed of 5 ° of C/min and keep 2 min, Splitless injecting samples, sample size is 1 μ L.Mass spectrum condition: solvent delay 5 min, transmission line temperature is 250 ° of C, and ion trap temperature is 220 ° of C, and cavity temperature is 40 ° of C, and the quality of scanning scope of counting is 50-350 u, adopts NIST2008 spectrogram library searching qualitative.
Beneficial effect of the present invention has:
(1) extraction time of the present invention is short, only needs 5～8 min, has saved the time, has reduced cost;
(2) apparatus of the present invention are simple, and production cost is low, only need the simple devices such as magnetic stirring apparatus, microsyringe, sampler;
(3) the present invention only needs the organic solvent of 50～100 microlitres, has reduced pollution, environmentally friendly;
(4) the present invention can reach the enrichment times of 80～99 times, is conducive to measure aroma substance that content is low as 3-acetylpyridine, o-methyl-benzene ethyl ketone, nicotine, neophytadiene etc., is applicable to the mensuration of a large amount of samples.
Accompanying drawing explanation
Fig. 1 is the present invention's hanging drop micro-extraction device of liquid phase structural representation used.
In figure: mono-extraction solvent of 1-magnetic stirring apparatus 2-magnetic stir bar 3-tobacco sample solution 4-.
Fig. 2 is the total ion current figure that in embodiment 1, actual sample is measured.
Fig. 3 has represented the impact of different volume of toluene on fragrance component extraction efficiency in tobacco leaf.
Fig. 4 has represented the impact of different extraction times on fragrance component extraction efficiency in tobacco leaf.
Embodiment
Below in conjunction with embodiment, the present invention is further described, but is not limited to embodiment.
Embodiment 1
(1) tobacco sample pre-service: take 1.00 g tobacco leaf powder in round-bottomed flask, adding volume ratio is the mixed solution of the first alcohol and water of 1:7, make mixed solution and the tobacco leaf powder mass ratio of first alcohol and water reach 14:1, extract 40 min, after centrifugal 8 min of 3000 rpm, get its supernatant and make tobacco sample solution;
(2) hanging drop liquid-phase micro-extraction: the extraction flask of 12mL is placed on magnetic stirring apparatus, add the magnetic stir bar of 15mm ' 6mm and the 10 mL tobacco sample solution that step (1) makes, open magnetic stirring apparatus, in the time that tobacco sample solution forms whirlpool, the toluene of drawing 100 μ L with the microsyringe of 100 μ L adds in whirlpool, and regulating the rotating speed of magnetic stir bar is 800 rps, makes single toluene be stable at whirlpool bottom, cover tightly bottle cap, extraction 8 min;
(3) detect: with microscale sampler bottom the whirlpool by single toluene sucking-off and put into the sample introduction bottle that 200 μ L interpolation pipes are housed, auto injection, utilizes gas chromatograph-mass spectrometer (GCMS) to detect, chromatogram and mass spectrographic testing conditions are:
Chromatographic condition: chromatographic column is DB-5MS (30 mm i.d. × 0.25, m × 0.25 μ m d.f.) Fused-silica capillary column, carrier gas He, flow velocity 1.0 mL/min, injector temperature is 280 ° of C, and heating schedule is that 60 ° of C keep 1 min, rises to 135 ° of C with the speed of 5 ° of C/min, rise to 175 ° of C with 2 ° of C/min again, finally rise to 250 ° of C with the speed of 5 ° of C/min and keep 2 min, Splitless injecting samples, sample size is 1 μ L.
Mass spectrum condition: solvent delay 5 min, transmission line temperature is 250 ° of C, and ion trap temperature is 220 ° of C, and cavity temperature is 40 ° of C, and the quality of scanning scope of counting is 50-350 u, adopts NIST2008 spectrogram library searching qualitative.
Adopt the method for the present embodiment to carry out 5 replicate determinations to same sample, the linear equation simultaneously obtaining by standard addition method is measured the recovery, and recovery experimental result is in table 1, and enrichment times is in table 2.
Table 1
In the present embodiment, methyl alcohol and water mixed liquid and tobacco leaf powder mass ratio reach 15:1, and the volume of toluene is 70 μ L, and extraction time is 5min, rotating speed 1100 rps of magnetic stir bar, and all the other are with embodiment 1.Gas chromatograph-mass spectrometer (GCMS) testing result is in table 3.
Table 3
Embodiment 3
In the present embodiment, methyl alcohol and water mixed liquid and tobacco leaf powder mass ratio reach 14.6:1, and the volume of toluene is 50 μ L, and extraction time is 6min, rotating speed 1000 rps of magnetic stir bar, and all the other are with embodiment 1.Gas chromatograph-mass spectrometer (GCMS) testing result is in table 4.
Table 4
Claims (1)
1. a hanging drop liquid-phase microextraction method that extracts fragrance component in tobacco leaf, is characterized in that: it comprises the step of following order:
(1) tobacco sample pre-service: take tobacco leaf powder in round-bottomed flask, adding volume ratio is the methyl alcohol of 1:7 and the mixed solution of water, the mass ratio that makes mixed solution and tobacco leaf powder is 14～15:1, extract 40 min, after centrifugal 8 min of 3000 rpm, get its supernatant and make tobacco sample solution;
(2) hanging drop liquid-phase micro-extraction: extraction flask is placed on magnetic stirring apparatus, the 10 mL tobacco sample solution that add magnetic stir bar and step (1) to make, open magnetic stirring apparatus, when tobacco sample solution forms whirlpool, draw 50～100 μ L toluene with microsyringe and inject whirlpool place, the volume ratio of toluene and tobacco sample solution is 1:100～200, regulating the rotating speed of magnetic stir bar is 800-1100 rps, make single toluene be stable at whirlpool bottom, cover tightly extraction flask bottle cap, extraction 5～8 min;
(3) detect: with microscale sampler by single toluene sucking-off, put into the sample introduction bottle that 200 μ L interpolation pipes are housed, auto injection, utilize gas chromatograph-mass spectrometer (GCMS) to detect, testing conditions is: chromatographic condition: chromatographic column is DB-5MS 30 mm i.d. × 0.25, m × 0.25 μ m d.f. Fused-silica capillary columns, carrier gas He, flow velocity 1.0 mL/min, injector temperature is 280 ° of C, heating schedule is that 60 ° of C keep 1 min, rise to 135 ° of C with the speed of 5 ° of C/min, rise to 175 ° of C with 2 ° of C/min again, finally rise to 250 ° of C with the speed of 5 ° of C/min and keep 2 min, Splitless injecting samples, sample size is 1 μ L, mass spectrum condition: solvent delay 5 min, transmission line temperature is 250 ° of C, and ion trap temperature is 220 ° of C, and cavity temperature is 40 ° of C, and the quality of scanning scope of counting is 50-350 u, adopts NIST2008 spectrogram library searching qualitative.
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| CN105004826A (en) * | 2015-07-10 | 2015-10-28 | 云南中烟工业有限责任公司 | Method for accurately characterizing and evaluating green fragrance characteristics of cigarette fragrance |
| CN104991024A (en) * | 2015-07-10 | 2015-10-21 | 云南中烟工业有限责任公司 | Method for exactly characterizing and evaluating wood aroma characteristics in cigarette aroma |
| CN106501420B (en) * | 2016-09-30 | 2018-07-03 | 中国农业科学院烟草研究所 | One grows tobacco the extraction purification of middle neophytadiene and detection method and its application |
| GB201707758D0 (en) | 2017-05-15 | 2017-06-28 | British American Tobacco Investments Ltd | Ground tobacco composition |
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