CN105021748A - Tea volatile aroma component analysis method - Google Patents
Tea volatile aroma component analysis method Download PDFInfo
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Abstract
The invention discloses a tea volatile aroma component analysis method based on the full-two-dimension gas chromatography-flying time mass-spectrometric technology. The method comprises the first step of tea volatile component preparing, the second step of full-two-dimension gas chromatography-flying time mass spectrometric analyzing and the third step of volatile component software qualitative analyzing. The method has the advantages that sensitivity is high and repeatability is good. Hundreds of volatile components can be analyzed at the same time. The method is suitable for basic studies related to tea characteristic aroma substances and tea classified studies.
Description
Technical field
The invention belongs to analytical chemistry field, relate to a kind of analytical approach of tealeaves aroma volatile, be specifically related to a kind of aroma volatile method in Simultaneous distillation-extraction and comprehensive two dimensional gas chromatography-flying time mass spectrum analysis tealeaves.
Background technology
The peculiar fragrance of tealeaves is the important element forming tealeaves local flavor, and as far back as the thirties in last century, foreign scholar just launches research to the fragrance component of black tea.Along with the progress of chromatogram and mass-spectrometric technique, at present, the tea aroma composition accumulation reported is more than 600 kinds.Complicated chemical component is the material base of the peculiar fragrance origin cause of formation of tealeaves, and for fragrance component of the same race, the smell under variable concentrations is also not quite similar.Therefore, if more fragrance component can by qualitative and quantitative analysis simultaneously, just can the origin cause of formation of further understanding fragrance and change, thus provide technical support for raising tea leaf quality.Traditional analytical approach based on one-dimensional gas chromatograph mass-spectrometric technique, cannot fragrance component that simultaneously determination and analysis is so many.It is large that comprehensive two dimensional gas chromatography-ionization time of flight has peak capacity, and resolution is high, the feature that analysis speed is separated with race soon, has the advantage that one dimension technology is incomparable in complex system compartment analysis.The content of most fragrance component is lower in addition, not easily direct-detection, needs enrichment to concentrate.The Simultaneous distillation-extraction combine with technique advantage of steam distillation and organic solvent extraction two kinds of methods, uses a small amount of sample just can obtain enough fragrance components, simultaneously easy and simple to handle, reproducible.
Summary of the invention
The problem to be solved in the present invention: in view of the problem that traditional one-dimensional technology peak capacity is little and resolution is low, the invention provides a kind of Simultaneous distillation-extraction in conjunction with analysis of aroma components method in the tealeaves of comprehensive two dimensional gas chromatography-flying time mass spectrum analysis, hundreds of active chemical can be detected simultaneously.
For achieving the above object, the technical solution used in the present invention.
(1) Simultaneous distillation-extraction method is used to extract volatile ingredient.5-20g Tea Samples powder, 0.1-0.3mg biphenyl (interior mark) and 200-400ml water are put into first flask, 50-70ml methylene chloride is put into second flask, connect SDE method, two flasks heat respectively, refluxing extraction 1-3h, then collects sample in second flask, adds anhydrous sodium sulfate and dewater, then be concentrated into 1-2ml revolving to steam in instrument, then store to be analyzed.
(2) comprehensive two dimensional gas chromatography-flight time mass spectrum is used to analyze obtained sample.
(3) for the raw data of comprehensive two dimensional gas chromatography-flying time mass spectrum analysis, by the chromatographic peak identification of Pegasus4D software and perk purity, then by the various aroma volatile of interior mark semi-quantitative analysis.Signal to noise ratio (S/N ratio) wherein for peak identification is set to 50.
In the present invention, comprehensive two dimensional gas chromatography-flight time mass spectrum is provided by LECO company; Wherein the first dimension chromatographic column is DB-5MS, and specification is 30m × 250 μm × 0.25 μm.Wherein the second dimension chromatographic column is RTX-200MS, and specification is 1.6m × 180 μm × 0.20 μm.
Be designated as biphenyl in using, before sample extraction, add 0.1-0.3mg in the first flask.
In the present invention, comprehensive two dimensional gas chromatography-flight time mass spectrum condition: the first dimension column oven initial temperature: 60 DEG C; First dimension temperature programme: 60 DEG C keep 3min, are then warming up to 290 DEG C with 7 DEG C/min and keep 5min; Second dimension column oven initial temperature: 70 DEG C; Second dimension temperature programme: 70 DEG C keep 3min, and then 7 DEG C/min is warming up to 300 DEG C and keeps 5min; Constant voltage mode: inlet pressure, 200kPa; Injector temperature: 280 DEG C; Sample introduction pattern: shunting, split ratio 10:1; Solvent clipping time: 2min; Modulation period: 5s; Ion gun: EI, energy 70eV; Ion source temperature: 220 DEG C; Transmission line temperature; 270 DEG C; Karyoplasmic ratio scope: 33-600; Frequency acquisition: 50Hz; Detect voltage: 1.6kV.
The present invention reaches beneficial effect for prior art.
This method and other Measures compare, use comprehensive two dimensional gas chromatography-flight time mass spectrum to analysis of aroma components, can be separated simultaneously detect hundreds of compound, in conjunction with the high resolving power feature of flight time mass spectrum, by the examination of mass spectrum positive and negative similarity degree, improve qualitative accuracy.The method is checked by methodology, reproducible.For three class tea aroma constituent analyses, the content of multiple compounds in different tealeaves is found to there is significant difference.
Specific embodiment
Contribute to understanding the present invention by subordinate's embodiment, but do not limit content of the present invention.
Embodiment 1: method repeatability is investigated
For investigating the repeatability of holistic approach, experimental procedure is as follows
(1) Simultaneous distillation-extraction method is used to extract volatile ingredient.15g Tea Samples powder, 0.1mg biphenyl (interior mark) and 300ml water are put into the first flask, 60ml methylene chloride is put into the second flask, connect SDE method, two flasks heat respectively, refluxing extraction 2h, then collects sample in the second flask, adds 1g anhydrous sodium sulfate and dewaters, then be concentrated into 1ml revolving to steam in instrument, then store to be analyzed.Have 11 same samples to be extracted.
(2) comprehensive two dimensional gas chromatography-flight time mass spectrum is used to analyze 11 obtained samples.Comprehensive two dimensional gas chromatography-flight time mass spectrum is provided by LECO company.Comprehensive two dimensional gas chromatography-flight time mass spectrum condition: the first dimension column oven initial temperature: 60 DEG C; First dimension temperature programme: 60 DEG C keep 3min, are then warming up to 290 DEG C with 7 DEG C/min and keep 5min; Second dimension column oven initial temperature: 70 DEG C; Second dimension temperature programme: 70 DEG C keep 3min, and then 7 DEG C/min is warming up to 300 DEG C and keeps 5min; Constant voltage mode: inlet pressure, 200kPa; Injector temperature: 280 DEG C; Sample introduction pattern: shunting, split ratio 10:1; Solvent clipping time: 2min; Modulation period: 5s; Ion gun: EI, energy 70eV; Ion source temperature: 220 DEG C; Transmission line temperature; 270 DEG C; Karyoplasmic ratio scope: 33-600; Frequency acquisition: 50Hz; Detect voltage: 1.6kV.
(3) for the raw data of comprehensive two dimensional gas chromatography-flying time mass spectrum analysis, chromatographic peak identification and perk purity is carried out by Pegasus4D software, then by the various aroma volatile of interior mark semi-quantitative analysis.Signal to noise ratio (S/N ratio) wherein for peak identification is set to 50.
The parent mass peak area of each chromatographic peak is obtained with above-mentioned inventive method extraction and analysis sample.Corrected the parent mass peak area of each chromatographic peak by interior mark peak area, then calculate the retention time of each chromatographic peak and the relative standard deviation (RSD) of peak area.First investigate repeatability from overall angle, calculate the peak area distribution of the corresponding chromatographic peak under different peak area RSD interval, result is as shown in table 1.Then random selecting 8 kinds of fragrance components and interior mark investigate the repeatability of predetermined substance, and result is as shown in table 2.Result shows, and the peak area that peak area RSD value is less than the corresponding chromatographic peak of 20% accounts for 85.4% of total peak area.And select 8 kinds of its peak area of fragrance component RSD values are all less than 10%, its one dimension and two-dimentional retention time RSD value are all less than 1%.
Show that the method has good reproducibility from compartment analysis angle.
Table 1: the peak area distribution of each chromatographic peak in different peak area RSD value is interval
| RSD scope | 0-10％ | 10-20％ | 20-30％ | >30％ |
| The number percent of shared peak area | 27.1％ | 58.3％ | 7.3％ | 7.3％ |
Table 2: part fragrance component repeatability data in tealeaves
Embodiment 2: aroma volatile is qualitative
For aroma volatile in qualitative tealeaves, experimental procedure is as follows
(1) Simultaneous distillation-extraction method is used to extract volatile ingredient.15g Tea Samples powder, 0.1mg biphenyl (interior mark) and 300ml water are put into the first flask, 60ml methylene chloride is put into the second flask, connect SDE method, two flasks heat respectively, refluxing extraction 2h, then collects sample in the second flask, adds 1g anhydrous sodium sulfate and dewaters, then be concentrated into 1ml revolving to steam in instrument, then store to be analyzed.Have 1 quality control sample (being mixed with green tea, oolong tea and black tea) to be extracted.
(2) comprehensive two dimensional gas chromatography-flight time mass spectrum is used to analyze obtained sample.Comprehensive two dimensional gas chromatography-flight time mass spectrum is provided by LECO company.Comprehensive two dimensional gas chromatography-flight time mass spectrum condition: the first dimension column oven initial temperature: 60 DEG C; First dimension temperature programme: 60 DEG C keep 3min, are then warming up to 290 DEG C with 7 DEG C/min and keep 5min; Second dimension column oven initial temperature: 70 DEG C; Second dimension temperature programme: 70 DEG C keep 3min, and then 7 DEG C/min is warming up to 300 DEG C and keeps 5min; Constant voltage mode: inlet pressure, 200kPa; Injector temperature: 280 DEG C; Sample introduction pattern: shunting, split ratio 10:1; Solvent clipping time: 2min; Modulation period: 5s; Ion gun: EI, energy 70eV; Ion source temperature: 220 DEG C; Transmission line temperature; 270 DEG C; Karyoplasmic ratio scope: 33-600; Frequency acquisition: 50Hz; Detect voltage: 1.6kV.
(3) for the raw data of comprehensive two dimensional gas chromatography-flying time mass spectrum analysis, chromatographic peak identification and perk purity is carried out by Pegasus4D software, then by the various aroma volatile of interior mark semi-quantitative analysis.Signal to noise ratio (S/N ratio) wherein for peak identification is set to 50.
Quality control sample is analyzed by above-mentioned inventive method, and by the process of Pegasus4D software.When peak identification signal to noise ratio (S/N ratio) is set to 50, about 3000 chromatographic peaks are distinguished out.To these chromatographic peaks, NIST05 standard mass spectrum is utilized qualitative, as met following two indices: the positive and negative similarity of mass spectrum is greater than 800, and the absolute value of retention index deviation (calculate retention index-database retention index) is less than 20, then can compound representated by this chromatographic peak of preliminary judgement.Authenticating compound is as shown in table 3 below.450 kinds of effective volatile ingredients (having the peak of about 3000 identified, qualitative 450 kinds) can be detected by once analyzing, this is far better than the detectability of traditional one-dimensional gas chromatograph mass-spectrometric technique.For the sample extracted in above-mentioned experimental procedure 1, adopt one-dimensional gas chromatograph mass spectrophotometry.Analysis condition is with much the same in step 2, and unique difference is to remove the second dimension gas chromatographic column, is directly connected by one-dimensional gas chromatograph with mass spectrum.After analysis completes, processing mode is with step 3.When peak identification signal to noise ratio (S/N ratio) is set to 50, only have 550 peaks identified, far below the mass spectrographic analysis result of comprehensive two dimensional gas chromatography.
Table 3, identifies tealeaves aroma volatile
Embodiment 3:
For finding at green tea, discrepant fragrance component between oolong tea and black tea, experimental procedure is as follows
(1) Simultaneous distillation-extraction method is used to extract volatile ingredient.15g Tea Samples powder, 0.1mg biphenyl (interior mark) and 300ml water are put into the first flask, 60ml methylene chloride is put into the second flask, connect SDE method, two flasks heat respectively, refluxing extraction 2h, then collects sample in the second flask, adds 1g anhydrous sodium sulfate and dewaters, then be concentrated into 1ml revolving to steam in instrument, then store to be analyzed.Have 12 green tea, 12 oolong tea and 9 black tea samples to be extracted.
(2) comprehensive two dimensional gas chromatography-flight time mass spectrum is used to analyze obtained all 33 samples.Comprehensive two dimensional gas chromatography-flight time mass spectrum is provided by LECO company.Comprehensive two dimensional gas chromatography-flight time mass spectrum condition: the first dimension column oven initial temperature: 60 DEG C; First dimension temperature programme: 60 DEG C keep 3min, are then warming up to 290 DEG C with 7 DEG C/min and keep 5min; Second dimension column oven initial temperature: 70 DEG C; Second dimension temperature programme: 70 DEG C keep 3min, and then 7 DEG C/min is warming up to 300 DEG C and keeps 5min; Constant voltage mode: inlet pressure, 200kPa; Injector temperature: 280 DEG C; Sample introduction pattern: shunting, split ratio 10:1; Solvent clipping time: 2min; Modulation period: 5s; Ion gun: EI, energy 70eV; Ion source temperature: 220 DEG C; Transmission line temperature; 270 DEG C; Karyoplasmic ratio scope: 33-600; Frequency acquisition: 50Hz; Detect voltage: 1.6kV.
(3) for the raw data of comprehensive two dimensional gas chromatography-flying time mass spectrum analysis, chromatographic peak identification and perk purity is carried out by Pegasus4D software, then by the various aroma volatile of interior mark semi-quantitative analysis.Signal to noise ratio (S/N ratio) wherein for peak identification is set to 50.Then statistical method is utilized to find the discrepant compound of content in three class tealeaves samples.
Adopt said method, to green tea, oolong tea and black tea fragrance component are analyzed, each 12,12 and 9 examples of three class tealeaves samples, totally 33 examples.By the peak area of each for gained chromatographic peak after interior mark peak area corrects, filter out compound in 74 altogether by statistical method, as shown in table 4, at least there is significant difference in them, namely p value is less than 0.05 in two class tealeaves.
Table 4, three class tealeaves samples find the fragrance component of difference
The advantages such as the method has highly sensitive, reproducible, can analyze hundreds of volatile ingredient simultaneously, the fundamental research that the material being applicable to tealeaves characteristic perfume is correlated with and Classification of Tea research.
Claims (5)
1. an aroma volatile analytical approach in tealeaves, is characterized in that: comprise following steps:
(1) Simultaneous distillation-extraction method is used to extract volatile ingredient: 5-20g Tea Samples powder and 200-400ml water are put into first flask, 50-70ml methylene chloride is put into second flask, connect SDE method, two flasks heat respectively, refluxing extraction 1-3h, then collects sample in second flask, adds anhydrous sodium sulfate and dewater, then be concentrated into 1-2ml revolving to steam in instrument, then store to be analyzed;
(2) comprehensive two dimensional gas chromatography-flight time mass spectrum is used to analyze obtained sample;
(3) for the raw data of comprehensive two dimensional gas chromatography-flying time mass spectrum analysis, chromatographic peak identification and perk purity is carried out by Pegasus4D software, then by the various aroma volatile of interior mark semi-quantitative analysis.
2. method according to claim 1, is characterized in that: wherein the first dimension chromatographic column is DB-5MS, and specification is 30m × 250 μm × 0.25 μm.
3. method according to claim 1, is characterized in that: wherein the second dimension chromatographic column is RTX-200MS, and specification is 1.6m × 180 μm × 0.20 μm.
4. method according to claim 1, is characterized in that: be designated as biphenyl in use, adds 0.1-0.3mg before sample extraction in the first flask.
5. method according to claim 1, is characterized in that: comprehensive two dimensional gas chromatography-flight time mass spectrum condition: the first dimension column oven initial temperature: 60 DEG C; First dimension temperature programme: 60 DEG C keep 3min, are then warming up to 290 DEG C with 7 DEG C/min and keep 5min; Second dimension column oven initial temperature: 70 DEG C; Second dimension temperature programme: 70 DEG C keep 3min, and then 7 DEG C/min is warming up to 300 DEG C and keeps 5min; Constant voltage mode: inlet pressure, 200kPa; Injector temperature: 280 DEG C; Sample introduction pattern: shunting, split ratio 10:1; Solvent clipping time: 2min; Modulation period: 5s; Ion gun: EI, energy 70eV; Ion source temperature: 220 DEG C; Transmission line temperature; 270 DEG C; Karyoplasmic ratio scope: 33-600; Frequency acquisition: 50Hz; Detect voltage: 1.6kV.
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Cited By (6)
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| CN105755097A (en) * | 2015-12-30 | 2016-07-13 | 中国人民解放军第二军医大学 | Method for comprehensively screening anti-leukemia active constituents from natural indigo and method for determining anti-leukemia active constituent targets |
| CN106818893A (en) * | 2017-01-17 | 2017-06-13 | 长江大学 | To the volatile extract and its bactericidal composition in section Chinese wax with bactericidal activity |
| CN107764917A (en) * | 2017-09-29 | 2018-03-06 | 云南中烟工业有限责任公司 | The assay method of crucial volatile ingredient in a kind of quick-fried pearl of cigarette |
| CN108088922A (en) * | 2017-12-12 | 2018-05-29 | 湖南省检验检疫科学技术研究院 | Fragrance component database in essence rapid screening database and tealeaves in essence high flux examination detection method, tealeaves in tealeaves |
| CN108645932A (en) * | 2018-05-16 | 2018-10-12 | 陕西科技大学 | A kind of method of violated addition essence and flavoring agent in rapid screening dilute cream |
| CN111879867A (en) * | 2020-06-29 | 2020-11-03 | 普洱市质量技术监督综合检测中心 | Method for detecting 20 volatile components in tea |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105755097A (en) * | 2015-12-30 | 2016-07-13 | 中国人民解放军第二军医大学 | Method for comprehensively screening anti-leukemia active constituents from natural indigo and method for determining anti-leukemia active constituent targets |
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| CN106818893A (en) * | 2017-01-17 | 2017-06-13 | 长江大学 | To the volatile extract and its bactericidal composition in section Chinese wax with bactericidal activity |
| CN106818893B (en) * | 2017-01-17 | 2019-09-10 | 长江大学 | To volatile extract and its bactericidal composition with bactericidal activity in section Chinese wax |
| CN107764917A (en) * | 2017-09-29 | 2018-03-06 | 云南中烟工业有限责任公司 | The assay method of crucial volatile ingredient in a kind of quick-fried pearl of cigarette |
| CN108088922A (en) * | 2017-12-12 | 2018-05-29 | 湖南省检验检疫科学技术研究院 | Fragrance component database in essence rapid screening database and tealeaves in essence high flux examination detection method, tealeaves in tealeaves |
| CN108645932A (en) * | 2018-05-16 | 2018-10-12 | 陕西科技大学 | A kind of method of violated addition essence and flavoring agent in rapid screening dilute cream |
| CN108645932B (en) * | 2018-05-16 | 2020-09-01 | 陕西科技大学 | Method for rapidly screening prohibited essence and spice added in cream |
| CN111879867A (en) * | 2020-06-29 | 2020-11-03 | 普洱市质量技术监督综合检测中心 | Method for detecting 20 volatile components in tea |
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Application publication date: 20151104 |