CN106226411A - A kind of SFC GC MS measures the method for ester type compound in Nicotiana tabacum L. - Google Patents

Abstract

The invention discloses a kind of supercritical fluid chromatography (SFC) gaschromatographic mass spectrometry (GC MS) and measure the method for ester type compound in Nicotiana tabacum L..This method is to use ester type compound in supercritical fluid extraction Nicotiana tabacum L., and extract supercritical fluid chromatography is divided into multistage fraction, and every section of fraction uses gaschromatographic mass spectrometric analysis respectively.It is the most overlapping that the present invention can realize compound between each section after separating, and solves the problem that the peak capacity of GC MS is limited.Result is satisfactory, and isolated ester type compound quantity, far more than direct injection analysis, provides new method for the mensuration of ester type compound in Nicotiana tabacum L..

Description

A method for the determination of ester compounds in tobacco by SFC-GC-MS

technical field

The invention belongs to the technical field of analytical chemistry, and in particular relates to a method for measuring ester compounds in tobacco by SFC-GC-MS.

Background technique

As people pay more and more attention to smoking and health issues, reducing the tar and harm of cigarettes has become an inevitable choice for the tobacco industry, and the research and development of low-tar cigarettes has also become an important topic for cigarette manufacturers. However, due to the development of tar reduction and harm reduction technology, the taste of tobacco products has been greatly reduced. Therefore, adding flavor, increasing flavor, and reducing the loss of cigarette smoke aroma have become an important direction of industry attention and research. An important prerequisite for adding aroma is to fully understand the composition and properties of aroma components in tobacco leaf raw materials. Therefore, it is of great significance to analyze and study the aroma components in tobacco.

Esters are a kind of spices commonly used in cigarette flavoring, which can not only give cigarettes a special taste, but also make cigarette smoke soft and delicate. At present, tobacco scientific and technological workers have also carried out a lot of research on the analysis of ester compounds. Commonly used analytical methods include gas chromatography, gas chromatography-mass spectrometry (GC/MS), high-performance liquid chromatography, liquid chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and the like. However, there are very few ester compounds in tobacco determined by these methods, mainly because the chemical components in tobacco are very complex and diverse, beyond the separation ability of single-column chromatography, many compounds cannot be completely separated, which affects the reliability of the retrieval results. Therefore, in order to achieve accurate analysis of ester compounds in tobacco, another method must be found.

Contents of the invention

The invention provides a method for measuring ester compounds in cigarette tobacco by SFC-GC-MS, the processing temperature is low, and the volatile components will not be lost during the separation process; the fractions separated by SFC are analyzed by GC-MS, There is basically no overlap between the compounds in each section, and at the same time, the problem of limited peak capacity of GC-MS is solved. The results were satisfactory, and the number of separated ester compounds was far more than that of direct injection analysis, which provided a new method for the determination of ester compounds in tobacco.

In order to achieve the above object, the technical scheme adopted in the present invention is as follows:

A method for measuring ester compounds in tobacco by SFC-GC-MS, comprising sample preparation and analysis, specifically the following steps:

(A) Sample extraction: the tobacco leaves are placed in a supercritical fluid extraction device, after supercritical fluid extraction, the extract is concentrated and filtered to obtain a sample for analysis;

(B) Supercritical fluid chromatography (abbreviated as SFC) segmentation: the extract obtained in step A enters the SFC separation system, the mobile phase is transported by the pump and separated by SFC, and is continuously cut into 3-5 fractions according to the retention time. Fractions are automatically collected into gas chromatography vials;

(C) Gas chromatography-mass spectrometry (abbreviated as GC-MS) analysis: the fractions obtained in step B were respectively sent to a gas chromatography system for detection by mass spectrometry.

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

The temperature of the extraction kettle for supercritical fluid extraction is 30-65°C, the extraction pressure is 200-400bar, the static extraction is 20-40min, the dynamic extraction is 30-60min, the entrainer is absolute ethanol, and the flow rate is 0.01-0.2mL/min.

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

Supercritical fluid chromatography adopts a 10mm×150mm, 5μm Silica 2-EP chromatographic column, the mobile phase is carbon dioxide/methanol, the mass ratio of the two is 50-90/10-50, and the flow rate of the mobile phase is 20-30mL/min. Inject 200-500 μL.

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

Capillary column DB-5MS (30m×0.25mm×0.25μm); inlet temperature is 250°C; carrier gas: high-purity helium (purity ≥ 99.999%), flow rate 1.0mL/min; sampling method: split injection, The injection volume is 2μL, the split ratio is 30:1; the temperature program conditions: the initial temperature is 50°C, keep for 5min, then rise to 80°C at 2°C/min, then rise to 230°C at 3°C/min, keep for 16min, and finally 12°C /min increased to 250°C and kept for 20min.

Mass spectrometry conditions: ionization mode: EI+; ionization voltage: 70ev; scanning range: 35-450amu; ion source temperature: 230°C;

Compared with the prior art, the present invention has the beneficial effects of:

1. Since the mobile phase of SFC is mainly CO ₂ , which only contains a small amount of organic solvent (generally less than 10%), the sample separated and purified by SFC, CO ₂ is discharged from the exhaust port, and the fractions of each section do not need to be concentrated, and can directly enter the GC -MS analysis.

2. For low-content samples, the fractions separated by SFC can be accumulated multiple times, and then enter the gas chromatographic analysis after accumulating to the ideal analysis amount, which greatly improves the analysis sensitivity while realizing efficient purification of samples

3. The processing temperature is low, and the volatile components will not be lost during the separation process; the fractions separated by SFC are analyzed by GC-MS, and the compounds in each section basically do not overlap, and at the same time, the limited peak capacity of GC-MS is solved problem, a wider variety of esters can be identified.

Description of drawings

Fig. 1 is the SFC graph of the tobacco sample in a method for measuring ester compounds in tobacco by SFC-GC-MS of the present invention.

Fig. 2 is a gas chromatogram of tobacco samples directly analyzed by GC-MS in a method for measuring ester compounds in tobacco by SFC-GC-MS of the present invention without supercritical fluid chromatography.

Fig. 3 is the first section fraction (a), the second section fraction (b), the third section after the tobacco sample is segmented by supercritical fluid chromatography in a kind of SFC-GC-MS method of the present invention measuring the ester compound in tobacco Fraction (c) and the fourth fraction (d) were analyzed by GC-MS gas chromatogram.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings, but the present invention is not limited in any way. Any transformation or replacement based on the requirements of the present invention belongs to the protection scope of the present invention.

Example 1

A kind of SFC-GC-MS measures the method for ester compound in tobacco, comprises the following steps:

(A) Sample extraction: the tobacco leaves were placed in a supercritical fluid extraction device, the temperature of the extraction kettle for supercritical fluid extraction was 55° C., the extraction pressure was 300 bar, the static extraction was 20 minutes, the dynamic extraction was 40 minutes, and the entrainer was absolute ethanol. The flow rate is 0.01mL/min, and the sample is obtained after supercritical fluid extraction;

(B) SFC segmentation: the extract obtained in step A enters the SFC separation system, and the supercritical fluid chromatography adopts a Silica 2-EP chromatographic column of 10mm × 150mm, 5 μm, and the mobile phase is carbon dioxide/methanol, and the mass ratio of the two is 90/10, the mobile phase flow rate was 30mL/min, and each injection was 500μL. Continuously cut into 4 fractions according to the retention time, and each fraction is automatically collected into a gas chromatographic bottle;

(C) GC-MS analysis: the fractions obtained in step C were respectively sent to a gas chromatography system for mass spectrometry detection. Capillary column DB-5MS (30m×0.25mm×0.25μm); inlet temperature is 250°C; carrier gas: high-purity helium (purity ≥ 99.999%), flow rate 1.0mL/min; sampling method: split injection, The injection volume is 2μL, the split ratio is 30:1; the temperature program conditions: the initial temperature is 50°C, keep for 5min, then rise to 80°C at 2°C/min, then rise to 230°C at 3°C/min, keep for 16min, and finally 12°C /min increased to 250°C and kept for 20min.

Mass spectrometry conditions: ionization mode: EI+; ionization voltage: 70ev; scanning range: 35-450amu; ion source temperature: 230°C;

For a flue-cured tobacco leaf, the sample was directly injected and analyzed by GC-MS without SFC segmentation, and a total of 15 ester compounds were identified; while the sample was analyzed by GC-MS after SFC segmentation, a total of 27 ester compounds were identified. an ester compound.

Example 2

A kind of SFC-GC-MS measures the method for ester compound in tobacco, comprises the following steps:

(A) Sample extraction: the tobacco leaves were placed in a supercritical fluid extraction device, the temperature of the extraction kettle for supercritical fluid extraction was 55° C., the extraction pressure was 300 bar, the static extraction was 30 minutes, the dynamic extraction was 50 minutes, and the entrainer was absolute ethanol. The flow rate is 0.01mL/min, and the sample is obtained after supercritical fluid extraction;

(B) SFC segmentation: the extract obtained in step A enters the SFC separation system, and the supercritical fluid chromatography adopts a Silica 2-EP chromatographic column of 10mm × 150mm, 5 μm, and the mobile phase is carbon dioxide/methanol, and the mass ratio of the two is 90/10, the mobile phase flow rate was 30mL/min, and each injection was 300μL. Continuously cut into 4 fractions according to the retention time, and each fraction is automatically collected into a gas chromatographic bottle;

(C) GC-MS analysis: the fractions obtained in step C were respectively sent to a gas chromatography system for mass spectrometry detection. Capillary column DB-5MS (30m×0.25mm×0.25μm); inlet temperature is 250°C; carrier gas: high-purity helium (purity ≥ 99.999%), flow rate 1.0mL/min; sampling method: split injection, The injection volume is 2μL, the split ratio is 30:1; the temperature program conditions: the initial temperature is 50°C, keep for 5min, then rise to 80°C at 2°C/min, then rise to 230°C at 3°C/min, keep for 16min, and finally 12°C /min increased to 250°C and kept for 20min.

Mass spectrometry conditions: ionization mode: EI+; ionization voltage: 70ev; scanning range: 35-450amu; ion source temperature: 230°C;

For another flue-cured tobacco leaf, the sample was directly injected and analyzed by GC-MS without SFC segmentation, and a total of 16 ester compounds were identified; while the sample was analyzed by GC-MS after SFC segmentation, a total of 16 ester compounds were identified. 28 phenolic compounds.

Example 3

Same as Example 1, under the same conditions parallel determination 7 times (same batch processing), mainly investigate retention time and total peak area value, test result shows that RSD is all less than 4.7%. At the same time, three kinds of compounds were selected to carry out the standard recovery experiment. The recovery rate of the traditional method was between 88-109%, and the recovery rate of this method was between 93-103%. The recovery rate of this method was obviously high.

Claims (4)

1. the method for ester type compound in supercritical fluid chromatography-gas chromatography-mass spectrometry Nicotiana tabacum L., it is characterised in that bag Include following steps:

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

(B) supercritical fluid chromatography segmentation: step A obtained enters in supercritical fluid chromatography, by pump for analysis sample Transport flow separates through supercritical fluid chromatography post, cuts continuously by retention time and is divided into 3-5 section fraction, and every section of fraction is automatic 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～ 40min, 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 is carbon dioxide/first mutually Alcohol, the two mass ratio is 50～90/10～50, and 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:

The capillary column DB-5MS of 30m × 0.25mm × 0.25 μm；Injector temperature is 250 DEG C；Carrier gas: purity >=99.999% High-pure helium, flow velocity 1.0mL/min；Input mode: split sampling, sample size is 2 μ L, split ratio 30:1；Temperature programming condition: 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, holding 16min, and last 12 DEG C/min rises to 250 DEG C, keeps 20min.

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