CN105929092A - Method for measuring phenol compounds in tobacco - Google Patents

Abstract

The invention discloses a method for measuring phenol compounds in a tobacco. The method comprises the following steps: extracting the phenol compounds from the tobacco by using supercritical fluid, sectionalizing an extracting solution by using supercritical fluid chromatography to divide the extracting solution into a plurality of sections of fractions, and analyzing each section of fraction with gas chromatography-mass spectrometry. According to the method, the compounds between the separated sections are basically not superposed, so that the problem of limited peak capacity of GC-MS is solved. The result is satisfactory, and the quantity of the separated phenol compounds is much greater than that obtained by direct sample injection for analysis; a new method is provided for measurement of the phenol compounds in the tobacco.

Description

One assay method growing tobacco middle phenolic compound

Technical field

The invention belongs to technical field of analytical chemistry, be specifically related to a kind of supercritical fluid chromatography (SFC)-gas Phase chromatograph (GC)-mass spectrum (MS) combination measures the method for phenolic compound in Nicotiana tabacum L..

Background technology

Aldehydes matter is widely present in Nicotiana tabacum L., and and Nicotiana tabacum L. color and luster, quality and flue gas physiological strength etc. Aspect is closely related, is the compound that in tobacco industry, a class is important.Polyphenolic substance in Medicated cigarette is because boiling Point is higher, will not be directly entered in flue gas when burning, and major part all can be decomposed into simple phenolic compound In air inlet flue gas.But, the report major part about the simple phenolic compound in flue gas is negative, example As: respiratory mucosa is had strong impulse effect；Cause pungent, astringent taste etc., affect the mouthfeel of Medicated cigarette； The most also assist tumor promotion.Therefore, in Nicotiana tabacum L., the analysis detection of aldehydes matter also becomes domestic and international Nicotiana tabacum L. One of focus direction of industry research.

Conventional analysis method includes gas chromatography, GC-MS (GC/MS), liquid phase color Spectrometry (HPLC), ultra-performance liquid chromatography (UPLC), Liquid chromatography-tandem mass spectrometry combination method (LC/MS/MS) Deng.But phenolic compound is all little in the Nicotiana tabacum L. of these methods mensuration, become mainly due to chemistry in Nicotiana tabacum L. Point of a great variety, extremely complex, separating power beyond single column chromatographic, chemical compound lot cannot divide completely From, have impact on the reliability of retrieval result.Phenolic compound in Nicotiana tabacum L. to be realized accurately is analyzed, must Must look for another way.

Summary of the invention

The invention provides a kind of SFC-GC-MS and measure the method for phenolic compound in Medicated cigarette Nicotiana tabacum L., process temperature Spending low, in separation process, volatile ingredient will not lose；Each section of fraction GC-MS after SFC separates Analyzing, between each section, compound is the most overlapping, solves the problem that the peak capacity of GC-MS is limited simultaneously. Result is satisfactory, and isolated phenolic compound quantity is far more than direct injection analysis, for phenol in Nicotiana tabacum L. The mensuration of compounds provides new method, to evaluating bitting midges, promoting Less harmful cigarette Research and develop significant.

To achieve these goals, the technical solution used in the present invention is as follows:

One assay method growing tobacco middle phenolic compound, must be for analyzing sample with supercritical fluid extraction Nicotiana tabacum L.；Face with super Boundary's Fluid Chromatography first carries out drawing a section to for analysis sample, and each section of fraction after separation uses gas chromatography-mass spectrometry analysis again, specific as follows:

(A) supercritical fluid extraction: tobacco leaf is placed in supercritical fluid extraction equipment, after supercritical fluid extraction, Extract is concentrated, filters for analyzing sample；

(B) the supercritical fluid chromatography section of drawing: step A obtained enters into supercritical fluid chromatography (referred to as analysis sample SFC), transport pump flowing separate through supercritical fluid chromatography, cut continuously by retention time and be divided into 3-5 section fraction, every section Fraction is collected in gas chromatogram bottle automatically；

(C) gas chromatography-mass spectrometry analysis (referred to as GC-MS): each section of fraction step B obtained has respectively entered gas phase color Spectra system, Mass Spectrometer Method.

Wherein said term " draws section " and " segmentation " is synonym, each means the fraction in the range of the specific retention time of collection, And respectively enter subsequent analysis device with the fraction in the range of other retention time.

In technical solution of the present invention, the supercritical fluid extraction condition described in step A is as follows:

The extraction kettle temperature of supercritical fluid extraction is 30～65 DEG C, and extracting pressure is 200～400bar, static extracting 20～ 40min, dynamic extraction 30～60min, entrainer is dehydrated alcohol, flow velocity 0.01～0.2mL/min.

In technical solution of the present invention, the SFC condition described in step B is as follows:

Supercritical fluid chromatography uses 10mm × 150mm, the Silica 2-EP chromatographic column of 5 μm, and flowing is mass ratio mutually Being 50～the carbon dioxide/methanol of 90/10～50, flow rate of mobile phase is 20～30mL/min, each sample introduction 200～500 μ L.

In technical solution of the present invention, the GC-MS condition described in step C is as follows:

Capillary column DB-5MS (30m × 0.25mm × 0.25 μm)；Injector temperature is 250 DEG C；Carrier gas: high-pure helium is (pure Degree >=99.999%), flow velocity 1.0mL/min；Input mode: split sampling, sample size is 2 μ L, split ratio 30:1；Journey Sequence Elevated Temperature Conditions: initial temperature 50 DEG C, keeps 5min, and then 2 DEG C/min rises to 80 DEG C, then 3 DEG C/min rises to 230 DEG C, Keep 16min, last 12 DEG C/min to rise to 250 DEG C, keep 20min.

Mass Spectrometry Conditions: ionization mode: EI+；Ionizing voltage: 70ev；Sweep limits: 35-450amu；Ion source temperature: 230℃；Transmission line temperature: 260 DEG C.

Signals assigned: WILEY, NIST08 compose storehouse and retrieve.

Compared with prior art, it has the beneficial effect that the present invention

1, CO it is mainly mutually due to the flowing of SFC₂, comprise only a small amount of organic solvent (generally less than 10%) warp Sample after SFC is isolated and purified, CO₂Discharging from outlet, each section of fraction is not required to concentrate, and can be directly entered GC-MS Analyze.

2, for the sample of low content, each section of fraction separated through SFC can be allowed repeatedly to add up, run up to ideal Enter back into gas chromatographic analysis after amount of analysis, while realizing sample high-efficient purification, substantially increase analysis spirit Sensitivity

3, treatment temperature is low, and in separation process, volatile ingredient will not lose；After SFC separates each section Fraction GC-MS analyzes, and between each section, compound is the most overlapping, solves the peak capacity of GC-MS simultaneously Limited problem, it is possible to identify a greater variety of phenolic compound.

Accompanying drawing explanation

Fig. 1 is the SFC figure that a kind of SFC-GC-MS of the present invention measures tobacco sample in the method for phenolic compound in Nicotiana tabacum L..

Fig. 2 is that in a kind of SFC-GC-MS of present invention mensuration Nicotiana tabacum L., in the method for phenolic compound, tobacco sample faces without super Boundary's Fluid Chromatography segmentation directly analyzes gas chromatogram with GC-MS.

Fig. 3 is that a kind of SFC-GC-MS of the present invention measures in Nicotiana tabacum L. in the method for phenolic compound tobacco sample through supercritical First paragraph fraction (a), second segment fraction (b), the 3rd section of fraction (c), the 4th section of fraction (d) GC-MS after Fluid Chromatography segmentation Analyze gas chromatogram.

Detailed description of the invention

The present invention is further illustrated below in conjunction with the accompanying drawings, but is any limitation as the present invention never in any form, based on this Any conversion that bright requirement is made or replacement, belong to protection scope of the present invention.

Embodiment 1

A kind of SFC-GC-MS measures the method for phenolic compound in Nicotiana tabacum L., comprises the following steps:

(A) supercritical fluid extraction: tobacco leaf is placed in supercritical fluid extraction equipment, the extraction of supercritical fluid extraction Still temperature is 55 DEG C, and extracting pressure is 300bar, static extracting 20min, dynamic extraction 40min, and entrainer is anhydrous second Alcohol, flow velocity 0.01mL/min, after supercritical fluid extraction, obtain sample；

(B) SFC segmentation: extract step A obtained enters into SFC piece-rate system, supercritical fluid chromatography uses 10mm × 150mm, the Silica 2-EP chromatographic column of 5 μm, flowing is the carbon dioxide/methanol of 90/10 for mass ratio mutually, flows Dynamic phase flow velocity is 30mL/min, each sample introduction 500 μ L.Cutting continuously by retention time and be divided into 4 sections of fractions, every section of fraction is certainly Move and collect in gas chromatogram bottle；

(C) GC-MS analyzes: each section of fraction step C obtained has respectively entered gas chromatography system, Mass Spectrometer Method.Capillary Tubing string DB-5MS (30m × 0.25mm × 0.25 μm)；Injector temperature is 250 DEG C；Carrier gas: high-pure helium (purity >=99.999%), flow velocity 1.0mL/min；Input mode: split sampling, sample size is 2 μ L, split ratio 30:1；Program Elevated Temperature Conditions: initial temperature 50 DEG C, keeps 5min, and then 2 DEG C/min rises to 80 DEG C, then 3 DEG C/min rises to 230 DEG C, Keep 16min, last 12 DEG C/min to rise to 250 DEG C, keep 20min.

Mass Spectrometry Conditions: ionization mode: EI+；Ionizing voltage: 70ev；Sweep limits: 35-450amu；Ion source temperature: 230℃；Transmission line temperature: 260 DEG C；Signals assigned: WILEY, NIST08 compose storehouse and retrieve.

For certain cured tobacco leaf, sample direct injected GC-MS is analyzed and has been identified 15 kinds of phenolic compounds altogether；And sample warp Analyze with GC-MS again after SFC segmentation and identified 27 kinds of phenolic compounds altogether.

Embodiment 2

A kind of SFC-GC-MS measures the method for phenolic compound in Nicotiana tabacum L., comprises the following steps:

(A) supercritical fluid extraction: tobacco leaf is placed in supercritical fluid extraction equipment, the extraction of supercritical fluid extraction Still temperature is 55 DEG C, and extracting pressure is 300bar, static extracting 30min, dynamic extraction 50min, and entrainer is anhydrous second Alcohol, flow velocity 0.01mL/min, after supercritical fluid extraction, obtain sample；

(B) SFC segmentation: extract step A obtained enters into SFC piece-rate system, supercritical fluid chromatography uses 10mm × 150mm, the Silica 2-EP chromatographic column of 5 μm, flowing is the carbon dioxide/methanol of 90/10 for mass ratio mutually, flows Dynamic phase flow velocity is 30mL/min, each sample introduction 300 μ L.Cutting continuously by retention time and be divided into 4 sections of fractions, every section of fraction is certainly Move and collect in gas chromatogram bottle；

(C) GC-MS analyzes: each section of fraction step C obtained has respectively entered gas chromatography system, Mass Spectrometer Method.Capillary Tubing string DB-5MS (30m × 0.25mm × 0.25 μm)；Injector temperature is 250 DEG C；Carrier gas: high-pure helium (purity >=99.999%), flow velocity 1.0mL/min；Input mode: split sampling, sample size is 2 μ L, split ratio 30:1；Program Elevated Temperature Conditions: initial temperature 50 DEG C, keeps 5min, and then 2 DEG C/min rises to 80 DEG C, then 3 DEG C/min rises to 230 DEG C, Keep 16min, last 12 DEG C/min to rise to 250 DEG C, keep 20min.

Mass Spectrometry Conditions: ionization mode: EI+；Ionizing voltage: 70ev；Sweep limits: 35-450amu；Ion source temperature: 230℃；Transmission line temperature: 260 DEG C；Signals assigned: WILEY, NIST08 compose storehouse and retrieve.

For another cured tobacco leaf, the sample after supercritical fluid extraction processes without the SFC section of drawing, and analyzes and has identified 16 altogether Plant phenolic compound；And sample is analyzed with GC-MS after SFC segmentation again and has been identified 29 kinds of phenolic compounds altogether.

Embodiment 3

Same as in Example 1, parallel assay 7 times (same to batch processed) under the same conditions, main investigation retention time and Zong Feng Area value, result of the test shows that RSD is respectively less than 4.3%.Have chosen wherein 3 kinds of compounds simultaneously and carry out recovery of standard addition in fact Testing, the response rate of traditional method is between 87-110%, and the response rate of this method is between 92-104%, and the response rate of this method is bright Aobvious height.

Claims (4)

1. an assay method growing tobacco middle phenolic compound, it is characterised in that comprise the following steps: use supercritical fluid extraction cigarette Careless must supplying analyzes sample；First carrying out drawing a section to for analysis sample with supercritical fluid chromatography, each section of fraction after separation uses gas phase again Spectrometry, specific as follows:

(A) supercritical fluid extraction: tobacco leaf is placed in supercritical fluid extraction equipment, after supercritical fluid extraction, Extract is concentrated, filters for analyzing sample；

(B) the supercritical fluid chromatography section of drawing: step A obtained enters into supercritical fluid chromatography, by pump for analysis sample Transport flow separates through supercritical fluid chromatography, cuts continuously by retention time and is divided into 3-5 section fraction, and every section of fraction is received automatically Collect in gas chromatogram bottle；

(C) gas chromatography-mass spectrometry analysis: each section of fraction step B obtained has respectively entered gas chromatography system, mass spectrum is examined Survey.

Method the most according to claim 1, it is characterised in that the supercritical fluid extraction condition described in step A is as follows:

The extraction kettle temperature of supercritical fluid extraction is 30～65 DEG C, and extracting pressure is 200～400bar, static extracting 20～40 Min, dynamic extraction 30～60min, entrainer is dehydrated alcohol, flow velocity 0.01～0.2mL/min.

Method the most according to claim 1, it is characterised in that the supercritical fluid chromatography condition in step B is as follows:

Supercritical fluid chromatography uses 10mm × 150mm, the Silica 2-EP chromatographic column of 5 μm, and flowing for mass ratio is mutually 50～the carbon dioxide/methanol of 90/10～50, flow rate of mobile phase is 20～30mL/min, each sample introduction 200～500 μ L.

Method the most according to claim 1, it is characterised in that the gas chromatography-mass spectrum condition in step C is as follows:

Capillary column DB-5MS (30m × 0.25mm × 0.25 μm)；Injector temperature is 250 DEG C；Carrier gas: high-pure helium is (pure Degree >=99.999%), flow velocity 1.0mL/min；Input mode: split sampling, sample size is 2 μ L, split ratio 30:1；Program Elevated Temperature Conditions: initial temperature 50 DEG C, keeps 5min, and then 2 DEG C/min rises to 80 DEG C, then 3 DEG C/min rises to 230 DEG C, Keep 16min, last 12 DEG C/min to rise to 250 DEG C, keep 20min.

Mass Spectrometry Conditions: ionization mode: EI+；Ionizing voltage: 70ev；Sweep limits: 35-450amu；Ion source temperature: 230℃；Transmission line temperature: 260 DEG C.