CN101487820A - Fast extraction and measurement method for plastid pigment in tobacco and tobacco products - Google Patents
Fast extraction and measurement method for plastid pigment in tobacco and tobacco products Download PDFInfo
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- CN101487820A CN101487820A CNA2008100491135A CN200810049113A CN101487820A CN 101487820 A CN101487820 A CN 101487820A CN A2008100491135 A CNA2008100491135 A CN A2008100491135A CN 200810049113 A CN200810049113 A CN 200810049113A CN 101487820 A CN101487820 A CN 101487820A
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Abstract
The invention relates to a method for rapidly extracting and measuring plastid pigment in tobacco and tobacco products, comprising the following steps: (1) a method of ultrasonic extraction is adopted to rapidly extract the plastid pigment in tobacco and tobacco products; (2) after ultrasonic extraction, the content of all the components in the extracted plastid pigment is measured by high performance liquid chromatography. The invention adopts the high performance liquid chromatography to rapidly measure the extracted plastid pigment, and the content of all the components in the plastid pigment can be rapidly and exactly measured, thus greatly improving the measuring accuracy. By adopting the extracting and measuring method, the working efficiency is greatly improved, and the cost is also reduced. Therefore, the invention has good social and economic values, and is easy to popularize and apply.
Description
Technical field
The present invention relates to one grow tobacco and tobacco product in the rapid extraction and the assay method of plamochromic pigment, relate in particular to a kind of by the method rapid extraction tobacco of ultrasonic extraction and the plamochromic pigment in the tobacco product, and measure the method for plamochromic pigment in the tobacco, belong to tobacco chemistry assay method field.
Background technology
The composition of plamochromic pigment is a lot of in tobacco and the tobacco product, mainly comprises chlorophyll a, chlorophyll b, bata-carotene, xenthophylls, neoxathin, violaxanthin etc., and these compositions are the principal ingredients that influence flue-cured tobacco flavouring essence quality and availability.If tobacco leaf pigment degraded in modulation, processing and ageing process is complete and proper, tabacco fragrance is increased, color and luster is vivid, can improve the quality and the availability of tobacco leaf.Set up the rapid extraction and the assay method of plamochromic pigment in the tobacco sample, will help further to determine pigment content and quality of tobacco relation, determine scientific and reasonable modulation and ageing condition and have great importance at the aspects such as the degree that becomes more meticulous that improve cigarette tobacco leaf formulation and be worth.Chlorophyll in the plamochromic pigment and carotenoid class pigment stability are poor, all require low temperature, lucifuge, quick during extraction and determination.And the extraction of existing plamochromic pigment concussion commonly used extraction etc., the concussion extraction extracts and needs the time longer, easily causes chlorophyll and carotenoids pigment oxidation in the plamochromic pigment to decompose; Measure normal spectrophotometric method, high performance thin layer chromatography scanning method and the liquid phase chromatography of adopting, accuracy is relatively poor during spectrophotometry, high performance thin layer chromatography scanning method and traditional liquid phase chromatography sample pre-treatments complexity, efficient is low, though adopt solid phase extraction can simplify sample pre-treatments, use cost is higher.
Summary of the invention
The object of the present invention is to provide one grow tobacco and tobacco product in plamochromic pigment rapid extraction and assay method, with realize fast, accurately, cost is low.
To achieve these goals, technical scheme of the present invention adopted one grow tobacco and tobacco product in plamochromic pigment rapid extraction and assay method, may further comprise the steps:
(2) adopt the method rapid extraction tobacco of ultrasonic extraction and the plamochromic pigment in the tobacco product;
(2) plamochromic pigment that extracts after the ultrasonic extraction adopts the content of its interior each composition of high effective liquid chromatography for measuring.
The ultrasonic extraction method concrete steps of described step (1) are: accurately take by weighing sample, use 90% acetone, include 0.1% antioxidant, at room temperature ultrasonic extraction 15-25min filters; Filter residue is used 90% acetone again, includes 0.1% antioxidant, wash 2～3 times, and be white to filter residue, merging filtrate, constant volume; Get extract, filter, filtrate is the plamochromic pigment solution of rapid extraction.
Described antioxidant is dibutyl hydroxy toluene.
The chromatographic column that the high performance liquid chromatography of step (2) adopts is anti-phase C18 liquid-phase chromatographic column, the octadecylsilane chemically bonded silica filling agent.
The moving phase of high performance liquid chromatography is made up of A and B in the step (2); Wherein A is that 80% acetonitrile and isopropyl alcohol are formed by concentration, and B is a ultrapure water.
Concentration is that 80% the acetonitrile and the volume ratio of isopropyl alcohol are 1:1 among the phase of the surging A.
Adopt linear gradient elution in the step (2); Flow velocity 0.5ml/min, sample size are 10 μ L, and column temperature is 40 ℃, and the detection wavelength is 450nm.
The present invention adopts the ultrasonic extraction method to carry out plamochromic pigment in rapid extraction tobacco and the tobacco product, shortened the pre-treatment time that plamochromic pigment is measured greatly, extracting only needed about 30 minutes, and prior art extraction plamochromic pigment often needs 4-5 hours, extracting method of the present invention has shortened extraction time greatly, has improved work efficiency.The present invention adopts high performance liquid chromatography that the plamochromic pigment that extracts is carried out fast measuring, can measure the content of each composition in the plamochromic pigment quickly and accurately, has improved the mensuration precision greatly.Adopt extraction of the present invention and assay method, improved work efficiency greatly, also reduced cost simultaneously, have favorable social and economic worth, be easy to apply.
Method of the present invention has been simplified the pre-treatment step of sample, adopt ultrasonic extraction method rapid extraction plamochromic pigment, plamochromic pigment in utilized high effective liquid chromatography for measuring tobacco and the tobacco product, obtain satisfied result, its easy control simple to operate, the measurement result precision is higher, for plamochromic pigment and cigarette quality relation in further research tobacco and the tobacco product provide feasible technical route.
Description of drawings
Fig. 1 is the high-efficient liquid phase chromatogram of plamochromic pigment among the present invention.
Embodiment
Embodiment 1
Extract cured tobacco leaf sample 500g according to the sample sampling standard, dry 4h under 40 ℃ temperature, pulverize, cross 40 mesh sieve, specimen preparation finishes; Accurately take by weighing preparation back sample 5.0000g (being accurate to 0.0001g), pour the 100ml triangular flask into, pour in the triangular flask ultrasonic extraction 20min under the room temperature into the acetone (including 0.1% dibutyl hydroxy toluene BHT) of 40ml 90%, filter, filter residue uses the acetone (including 0.1% dibutyl hydroxy toluene BHT) of 10ml 90% to wash again 2-3 times, and merging filtrate is settled to 100ml, get the 2mL extract, with 0.45 μ m filtering with microporous membrane, filtrate is carried out the HPLC analysis, and external standard method is quantitative; Wherein, chromatographic column is SymmetryC18 reverse-phase chromatographic column (3.9mm i.d. * 150mm, 4 μ m) (U.S. Waters company); Moving phase is: A, 80% acetonitrile: isopropyl alcohol=1:1, B, ultrapure water; Flow velocity: 0.5ml/min, online gradient elution (as table 1); Detect wavelength: 450nm, sample size: 10 μ l, column temperature: 20 ℃.
Among Fig. 1, the peak figure of 9.14min is a neoxathin, and the peak figure of 11.3min is a violaxanthin, and the peak figure of 17.5min is an xenthophylls, and the peak figure of 25.97min is a chlorophyll b, and the peak figure of 28.47min is a chlorophyll a, and the peak figure of 35.19min is a beta carotene.
The gradient condition of table 1 moving phase
Extract cigarette finished product sample 500g according to the sample sampling standard, dry 4h under 40 ℃ temperature, pulverize, cross 40 mesh sieve, specimen preparation finishes; Accurately take by weighing preparation back sample 5.0000g (being accurate to 0.0001g), pour the 100ml triangular flask into, pour in the triangular flask ultrasonic extraction 20min under the room temperature into the acetone (including 0.1% dibutyl hydroxy toluene BHT) of 40ml 90%, filter, filter residue uses the acetone (including 0.1% dibutyl hydroxy toluene BHT) of 10ml 90% to wash again 2～3 times, and merging filtrate is settled to 100ml, get the 2mL extract, with 0.45 μ m filtering with microporous membrane, filtrate is carried out the HPLC analysis, and external standard method is quantitative; Chromatographic column is SymmetryC18 reverse-phase chromatographic column (3.9mm i.d. * 150mm, 4 μ m) (U.S. Waters company); Moving phase is: A, 80% acetonitrile: isopropyl alcohol=1:1, B, ultrapure water; Flow velocity: 0.5ml/min, gradient elution; Detect wavelength: 450nm, sample size: 10 μ l, column temperature: 20 ℃.
Embodiment 3
Get the new fresh tobacco leaf 3000g in field, freeze-drying under-45 ℃ temperature is pulverized, and crosses 40 mesh sieve, and specimen preparation finishes; Accurately take by weighing preparation back sample 1.0000g (being accurate to 0.0001g), pour the 100ml triangular flask into, pour in the triangular flask ultrasonic extraction 20min under the room temperature into the acetone (including 0.1% dibutyl hydroxy toluene BHT) of 40ml90%, filter, filter residue uses the acetone (including 0.1% dibutyl hydroxy toluene BHT) of 10ml90% to wash again 2～3 times, and merging filtrate is settled to 100ml, get the 2mL extract, with 0.45 μ m filtering with microporous membrane, filtrate is carried out the HPLC analysis, and external standard method is quantitative; Chromatographic column is SymmetryC18 reverse-phase chromatographic column (3.9mm i.d. * 150mm, 4 μ m) (U.S. Waters company); Moving phase is: A, 80% acetonitrile: isopropyl alcohol=1:1, B, ultrapure water; Flow velocity is 0.5ml/min, gradient elution; The detection wavelength is 450nm, and sample size is 10 μ l, and column temperature is 20 ℃.
It should be noted last that: above embodiment is only in order to explanation, and unrestricted technical scheme of the present invention, although the present invention is had been described in detail with reference to the foregoing description, those of ordinary skill in the art is to be understood that: still can make amendment or be equal to replacement the present invention, and not breaking away from any modification or partial replacement of the spirit and scope of the present invention, it all should be encompassed in the middle of the claim scope of the present invention.
Claims (7)
1, one grow tobacco and tobacco product in plamochromic pigment rapid extraction and assay method, it is characterized in that: may further comprise the steps:
(1) adopts the method rapid extraction tobacco of ultrasonic extraction and the plamochromic pigment in the tobacco product;
(2) plamochromic pigment that extracts after the ultrasonic extraction adopts the content of its interior each composition of high effective liquid chromatography for measuring.
2, plamochromic pigment rapid extraction and assay method in tobacco according to claim 1 and the tobacco product, it is characterized in that: the ultrasonic extraction method concrete steps of described step (1) are: accurately take by weighing sample, use 90% acetone, include 0.1% antioxidant, at room temperature ultrasonic extraction 15-25min filters; Filter residue is used 90% acetone again, includes 0.1% antioxidant, wash 2～3 times, and be white to filter residue, merging filtrate, constant volume; Get extract, filter, filtrate is the plamochromic pigment solution of rapid extraction.
3, plamochromic pigment rapid extraction and assay method in tobacco according to claim 2 and the tobacco product, it is characterized in that: described antioxidant is dibutyl hydroxy toluene.
4, according to plamochromic pigment rapid extraction and assay method in arbitrary described tobacco in the claim 1-3 and the tobacco product, it is characterized in that: the chromatographic column that the high performance liquid chromatography of step (2) adopts is anti-phase C18 liquid-phase chromatographic column, the octadecylsilane chemically bonded silica filling agent.
5, plamochromic pigment rapid extraction and assay method in tobacco according to claim 4 and the tobacco product is characterized in that: the moving phase of high performance liquid chromatography is made up of A and B in the step (2); Wherein A is that 80% acetonitrile and isopropyl alcohol are formed by concentration, and B is a ultrapure water.
6, plamochromic pigment rapid extraction and assay method in tobacco according to claim 5 and the tobacco product is characterized in that: concentration is that 80% the acetonitrile and the volume ratio of isopropyl alcohol are 1:1 among the phase of the surging A.
7, plamochromic pigment rapid extraction and assay method in tobacco according to claim 6 and the tobacco product is characterized in that: adopt linear gradient elution in the step (2); Flow velocity 0.5ml/min, sample size are 10 μ L, and column temperature is 40 ℃, and the detection wavelength is 450nm.
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| CNA2008100491135A CN101487820A (en) | 2008-01-18 | 2008-01-18 | Fast extraction and measurement method for plastid pigment in tobacco and tobacco products |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101876649A (en) * | 2010-07-02 | 2010-11-03 | 中国烟草总公司郑州烟草研究院 | Method for measuring residual maleic hydrazide in tobacco or tobacco product |
| CN102636592A (en) * | 2012-05-10 | 2012-08-15 | 重庆出入境检验检疫局检验检疫技术中心 | Method for simultaneous determination of a plurality of synthetic pigments in hot pot flavorings |
| CN103555422A (en) * | 2013-10-30 | 2014-02-05 | 河南亚临界生物技术有限公司 | Method for extracting plamochromic pigment in tobacco by use of subcritical extraction technology |
| CN106442788A (en) * | 2016-09-30 | 2017-02-22 | 中国烟草总公司郑州烟草研究院 | Method for judging quality of fresh tobacco leaf sample in tobacco metabonomics based on pigments |
| CN106501385A (en) * | 2016-09-19 | 2017-03-15 | 广西中烟工业有限责任公司 | A kind of supermolecule solvent extraction combines the method that Magnetic solid phases extract detection Plastid Pigment from Tobacco |
| CN106590018A (en) * | 2016-10-20 | 2017-04-26 | 东莞市东晟羊绒制品有限公司 | Tobacco natural plant dye, preparation method, and application thereof |
| CN108508105A (en) * | 2018-02-12 | 2018-09-07 | 云南中烟工业有限责任公司 | A method of measuring phytochrome in tobacco |
| CN110455951A (en) * | 2019-08-19 | 2019-11-15 | 中国农业科学院烟草研究所 | A kind of Tobacco Plastid Pigment analysis method |
| CN112858533A (en) * | 2021-03-23 | 2021-05-28 | 贵州中烟工业有限责任公司 | Method for measuring plastid pigment content in tobacco shreds |
| CN113030333A (en) * | 2021-03-23 | 2021-06-25 | 贵州中烟工业有限责任公司 | Method for measuring plastid pigment content in tobacco shreds |
-
2008
- 2008-01-18 CN CNA2008100491135A patent/CN101487820A/en active Pending
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101876649A (en) * | 2010-07-02 | 2010-11-03 | 中国烟草总公司郑州烟草研究院 | Method for measuring residual maleic hydrazide in tobacco or tobacco product |
| CN102636592A (en) * | 2012-05-10 | 2012-08-15 | 重庆出入境检验检疫局检验检疫技术中心 | Method for simultaneous determination of a plurality of synthetic pigments in hot pot flavorings |
| CN103555422A (en) * | 2013-10-30 | 2014-02-05 | 河南亚临界生物技术有限公司 | Method for extracting plamochromic pigment in tobacco by use of subcritical extraction technology |
| CN106501385B (en) * | 2016-09-19 | 2019-04-19 | 广西中烟工业有限责任公司 | A kind of method of supermolecule solvent extraction combination Magnetic solid phases extraction detection Plastid Pigment from Tobacco |
| CN106501385A (en) * | 2016-09-19 | 2017-03-15 | 广西中烟工业有限责任公司 | A kind of supermolecule solvent extraction combines the method that Magnetic solid phases extract detection Plastid Pigment from Tobacco |
| CN106442788A (en) * | 2016-09-30 | 2017-02-22 | 中国烟草总公司郑州烟草研究院 | Method for judging quality of fresh tobacco leaf sample in tobacco metabonomics based on pigments |
| CN106442788B (en) * | 2016-09-30 | 2018-10-19 | 中国烟草总公司郑州烟草研究院 | The method of discrimination of fresh tobacco leaves sample quality in a kind of tobacco metabolism group based on pigment |
| CN106590018A (en) * | 2016-10-20 | 2017-04-26 | 东莞市东晟羊绒制品有限公司 | Tobacco natural plant dye, preparation method, and application thereof |
| CN108508105A (en) * | 2018-02-12 | 2018-09-07 | 云南中烟工业有限责任公司 | A method of measuring phytochrome in tobacco |
| CN110455951A (en) * | 2019-08-19 | 2019-11-15 | 中国农业科学院烟草研究所 | A kind of Tobacco Plastid Pigment analysis method |
| CN110455951B (en) * | 2019-08-19 | 2023-10-10 | 中国农业科学院烟草研究所 | Tobacco plastid pigment analysis method |
| CN112858533A (en) * | 2021-03-23 | 2021-05-28 | 贵州中烟工业有限责任公司 | Method for measuring plastid pigment content in tobacco shreds |
| CN113030333A (en) * | 2021-03-23 | 2021-06-25 | 贵州中烟工业有限责任公司 | Method for measuring plastid pigment content in tobacco shreds |
| CN113030333B (en) * | 2021-03-23 | 2022-09-30 | 贵州中烟工业有限责任公司 | Method for measuring plastid pigment content in tobacco shreds |
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Application publication date: 20090722 |