A method for extracting and separating high-purity natural nicotine from tobacco

Abstract

This invention relates to a method for extracting and separating high-purity natural nicotine from tobacco. The invention utilizes high-speed countercurrent chromatography to prepare high-purity natural nicotine. By selecting a suitable solvent system, a system with an appropriate partition coefficient is obtained, significantly improving the separation and purification effect of nicotine. The process is simpler, more convenient to operate, results in no sample loss, is highly efficient and rapid, and allows for large-scale preparation with high purity. This invention also utilizes a eutectic solvent to extract natural nicotine from tobacco. This eutectic solvent is safe, low in toxicity, non-volatile, and reusable, making it a green and environmentally friendly solvent. The extraction method has high extraction efficiency, produces fewer impurities in the extract, and significantly reduces the amount of volatile organic solvents used, making it even more environmentally friendly.

Description

Technical Field

[0001] This invention relates to the field of tobacco chemistry, and more specifically to a method for extracting and separating high-purity natural nicotine from tobacco. Background Technology

[0002] Nicotine, also known as tobacco alkaloid, is a major alkaloid found in tobacco. It is a colorless to pale yellow, transparent, oily liquid and the main nitrogenous alkaloid component of tobacco. It exists in the plant as citrate or malate, and its content in tobacco leaves is 1-3%. Historically, it was used as an insecticide in agriculture. With the rapid development of the tobacco and pharmaceutical industries, the market demand for natural nicotine, especially high-purity nicotine, has increased daily, becoming one of the most sought-after products on the international market.

[0003] There are many extraction processes for nicotine, with classic methods including dry distillation, aqueous distillation, ion exchange extraction, aqueous organic solvent extraction, supercritical carbon dioxide extraction, and molecular distillation. Each method has its advantages and disadvantages, with aqueous organic solvent extraction being the most widely used. Several Chinese patent documents disclose methods for extracting nicotine from tobacco. For example, Chinese patent 89104689 discloses a method for producing nicotine sulfate. Chinese patent CN201980045405.8 also discloses the purification of nicotine. Chinese patent CN201410253273.7 discloses a method for separating and purifying nicotine using a supercritical-molecular distillation coupling technology. Chinese patent CN201611046202.5 discloses a method for purifying nicotine using a subcritical extraction-molecular distillation coupling technology. Chinese patent CN201910636858.X also discloses a combined process for large-scale continuous countercurrent extraction and supercritical carbon dioxide extraction of nicotine. Chinese patent CN202010614621.4 also discloses a method for purifying nicotine using a short-path molecular distillation apparatus and high-speed countercurrent chromatography.

[0004] High-speed countercurrent chromatography (HSCCC) is an advanced liquid-liquid partition chromatography technique. This chromatographic technique requires no solid support and separates substances based on the difference in their partition coefficients between two phases. Compared to traditional chromatographic techniques (such as thin-layer chromatography and high-performance liquid chromatography), it features no irreversible adsorption and high recovery rates, making it particularly suitable for the separation and purification of substances containing bioactive components. Chinese patent literature discloses several methods for extracting nicotine from tobacco. For example, Chinese patent CN102093461A discloses a method for purifying tea saponins using high-speed countercurrent chromatography. Chinese patent CN103086873A also discloses a method for separating and preparing high-purity DHA using high-speed countercurrent chromatography. Chinese patent CN101293872 also discloses a method for preparing high-purity hexamethasone using high-speed countercurrent chromatography. Chinese patent CN101519420 also discloses a method for preparing high-purity styraxin using high-speed countercurrent chromatography. Chinese patent CN1337397 also discloses a method for separating and purifying tanshinone using high-speed countercurrent chromatography. Chinese patent CN1445223 also discloses a method for separating and preparing EGCG monofatty acid esters using high-speed countercurrent chromatography.

[0005] All of the above patents have shortcomings. The main issue is the complexity of the operational steps, requiring multiple extraction and separation devices (including pressure equipment). While high-speed countercurrent chromatography (HSCGC) is used, it is limited to the final purification step; the preceding steps are too cumbersome and unsuitable for the rapid preparation of high-purity natural nicotine. The solvents used are conventional textbook designs (such as petroleum ether, ethyl acetate, methanol, and water), without specific experiments or optimizations for nicotine. The solvent system in Chinese patent CN202010614621.4 exhibits poor separation performance; before HSCGC, the crude nicotine needs to be distilled twice using a short-path molecular distillation apparatus.

[0006] This invention aims to overcome the numerous shortcomings of current nicotine production technologies by providing a novel method for the rapid preparation of high-purity natural nicotine. Compared with existing technologies, this invention offers the following advantages: the separation and preparation process is simple to operate, highly efficient, short in separation time, and yields a high product rate. Furthermore, it can be carried out continuously, and the purity of the prepared high-purity natural nicotine can reach over 99%, demonstrating significant advantages over traditional methods. Summary of the Invention

[0007] The purpose of this invention is to provide a method for extracting and separating high-purity natural nicotine from tobacco. The method employs high-speed countercurrent chromatography to separate and purify the tobacco extract, ultimately obtaining high-purity natural nicotine.

[0008] In one aspect, the present invention provides a method for extracting and separating natural nicotine, specifically comprising the following steps:

[0009] Prepare a mixture of n-hexane-chloroform-acetonitrile-n-butanol in a volume ratio of (9-11):(2-4):(6-8):(4-6). After separation, take the lower phase as the stationary phase and the upper phase as the mobile phase. Dissolve the tobacco extract in the mobile phase and separate it using high-speed countercurrent chromatography. Collect the target components to obtain the nicotine product.

[0010] In one embodiment, the method includes: accurately measuring a mixture of n-hexane-chloroform-acetonitrile-n-butanol, placing it in a separatory funnel to prepare a two-phase solvent system, using the lower phase as the stationary phase and the upper phase as the mobile phase; filling the column of a countercurrent chromatogram with the stationary phase, then pumping in the mobile phase until the two solvent phases reach dynamic equilibrium in the column; then dissolving and injecting the tobacco extract using the mobile phase as the solvent, separating it using high-speed countercurrent chromatography, monitoring under a UV detector, and collecting the target component to obtain the nicotine product.

[0011] Furthermore, the method also includes the preparation of a tobacco extract, which is selected from one of the following methods:

[0012] a. Extract tobacco raw material with petroleum ether, and then remove the petroleum ether to obtain a tobacco extract;

[0013] b. Extract tobacco raw material with an aqueous eutectic solvent, then extract the extract with an organic solvent, and remove the organic solvent from the organic solvent phase to obtain the tobacco extract.

[0014] Furthermore, the tobacco raw material can be tobacco stems, tobacco leaves, and tobacco dust, preferably tobacco waste used in industrial production, such as one or more of tobacco stems, tobacco leaf fragments, tobacco shreds, and powder. Its nicotine content is required to be greater than 1%.

[0015] Further, method a includes wrapping the tobacco raw material in filter paper, placing it in a Soxhlet extractor, and extracting it by reflux with petroleum ether.

[0016] Preferably, the petroleum ether is refluxed 6-10 times per hour for 6-10 hours.

[0017] Further, method b includes adding tobacco raw material to an aqueous eutectic solvent, ultrasonically extracting it at 40-100°C, centrifuging to collect the supernatant, extracting it with an organic solvent, removing the organic solvent from the organic solvent phase, and obtaining the tobacco extract.

[0018] Preferably, the eutectic solvent is a combination of choline chloride and glycerol in a molar ratio of 1:(2-3), with water added to a predetermined water content. The eutectic solvent is prepared by mixing choline chloride and glycerol in a specific ratio, heating and stirring at 80-100°C to form a clear and homogeneous system. The water content of the aqueous eutectic solvent is preferably 20-40 wt%, more preferably 25-35 wt%. Alternatively, the aqueous eutectic solvent can be prepared by mixing the eutectic solvent and water in a specific ratio, heating and stirring at 80-100°C, and then cooling.

[0019] Preferably, the mass-to-volume ratio of the tobacco raw material to the aqueous eutectic solvent is 1:4 to 12 g / ml, and more preferably 1:5 to 8 g / ml.

[0020] Preferably, the temperature during ultrasound is 50–70°C. The ultrasound power is 200–600W, preferably 300–400W.

[0021] Preferably, the organic solvent is selected from one or more of ethyl acetate, diethyl ether, and petroleum ether.

[0022] Furthermore, after dissolving the tobacco extract in the upper phase, the pH of the solution is adjusted to 10–13, preferably 11.5–12.5. The purpose of adjusting the pH is to keep the nicotine in the tobacco extract in a free state, thereby improving the separation and purification effect.

[0023] Preferably, the pH value can be adjusted using an inorganic base, particularly an aqueous solution of an inorganic base. Examples of such inorganic bases include sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate.

[0024] Furthermore, the preferred volume ratio of the mixture of n-hexane-chloroform-acetonitrile-n-butanol is 10:3:7:5.

[0025] Furthermore, in the high-speed countercurrent chromatography method, the main unit rotation speed is 600-1000 rpm; the mobile phase flow rate is 1.0-3.0 mL / min, preferably 2.0 mL / min; and the temperature of the constant temperature circulator is 25℃.

[0026] Furthermore, the ultraviolet detection wavelength is 259 nm.

[0027] Beneficial effects

[0028] The key technologies of this invention are as follows:

[0029] 1. Choosing the correct solvent system. Selecting a suitable solvent system is crucial for preparing high-purity natural nicotine using high-speed countercurrent chromatography (HSCLC). Different solvent systems have different upper and lower phase ratios, and their viscosity, polarity, density, and other properties vary significantly. They exhibit different solubility and partitioning abilities for the same component, resulting in differences in partition coefficients and impacting the separation effect. Furthermore, after selecting the solvent system, it is sometimes necessary to optimize the settings of four instrument operating parameters (temperature, rotation speed, mobile phase flow rate, and injection volume). Currently, there is a lack of comprehensive theoretical framework for selecting the correct solvent system; it relies mainly on long-term experience and extensive experimental exploration. Experiments in this invention demonstrate that, using a suitable solvent system, high-purity natural nicotine can be separated and purified from tobacco raw materials using HSCLC. This process is simpler, easier to operate, results in no sample loss, is highly efficient and rapid, and allows for large-scale production of high-purity natural nicotine. It is an excellent method suitable for large-scale production of high-purity natural nicotine.

[0030] 2. Extraction of Nicotine from Tobacco Using Eutectic Solvents. This paper proposes for the first time the extraction of natural nicotine from tobacco using eutectic solvents. The hydrogen bond acceptor of the eutectic solvent is choline chloride, and the hydrogen bond donor is glycerol, both natural compounds that are safe and low in toxicity. The resulting eutectic solvent is non-volatile and reusable, making it a green and environmentally friendly solvent. The tobacco extract obtained using this eutectic solvent has a high nicotine content and few impurities. High-purity natural nicotine can be obtained using high-speed countercurrent chromatography with a high nicotine yield. This extraction method significantly reduces the amount of volatile organic solvents used, making it more environmentally friendly.

[0031] Instruction manual illustrations

[0032] Figure 1 This is the GC-MS total ion chromatogram of high-purity natural nicotine obtained in Example 2. Detailed Implementation

[0033] The following embodiments are provided to further illustrate the present invention, but they are not intended to limit the invention.

[0034] Preparation Example 1

[0035] Dry and pulverize tobacco leaf fragments to 70-90 mesh. Take 100g and place it in a filter paper tube. Place the tube in a Soxhlet extractor and add petroleum ether to 2 / 3 of the volume in the receiving flask. Heat to reflux and control the petroleum ether to reflux 6-8 times per hour for 8 hours to obtain tobacco petroleum ether extract. After removing and recovering the petroleum ether under reduced pressure, obtain tobacco petroleum ether extract for later use.

[0036] Preparation Example 2

[0037] Choline chloride and glycerol were mixed in a molar ratio of 1:2 and heated and stirred at 90°C for 1 hour to form a clear and homogeneous natural eutectic solvent. 50% water was added, and stirring continued for 0.5 hours. After cooling, an aqueous eutectic solvent with a water content of approximately 33% was obtained. 600 ml of this solvent was taken, and 100 g of dried tobacco leaves, pulverized to 70–90 mesh, was added. The mixture was ultrasonically extracted at 60°C for 15 minutes at a power of 400 W. After cooling, the mixture was centrifuged, and the supernatant was extracted twice with an equal volume of ethyl acetate. The extracts were combined, and the ethyl acetate was removed and recovered under reduced pressure to obtain the tobacco eutectic solvent extract, which was then ready for use.

[0038] Example 1

[0039] Prepare a two-phase solvent system of n-hexane-chloroform-acetonitrile-n-butanol = 10:3:7:5 (v / v) in a separatory funnel, shake thoroughly, and let stand overnight. The lower phase is the stationary phase, and the upper phase is the mobile phase. Take 100 mg of the tobacco extract sample to be separated from Preparation Example 1, dissolve it completely in 10 mL of the lower phase solution, and adjust the pH to 12 with 30% NaOH solution.

[0040] The lower phase of the solvent system was pumped into the main unit and filled the separation solenoid. The circulating water bath was turned on and the temperature was set to 25℃. The main unit was powered on and rotated forward at 800 rpm. After the rotation speed stabilized, the mobile phase was pumped in at a flow rate of 2.0 mL / min. After the mobile phase flowed out of the column outlet and the baseline stabilized, the sample solution was injected through the injection ring. The effluent at the column outlet was continuously detected at a wavelength of 259 nm. Nicotine was separated and prepared with a purity of 99.32% and a yield of 88.45% (calculated based on nicotine in tobacco raw materials, the same below).

[0041] Example 2

[0042] Prepare a two-phase solvent system of n-hexane-chloroform-acetonitrile-n-butanol = 10:3:7:5 (v / v) in a separatory funnel, shake thoroughly, and let stand overnight. The lower phase is the stationary phase, and the upper phase is the mobile phase. Take 100 mg of the tobacco extract sample to be separated from Preparation Example 2, dissolve it completely in 10 mL of the lower phase solution, and adjust the pH to 12 with 30% NaOH solution.

[0043] The lower phase of the solvent system was pumped into the main unit and filled the separation solenoid. The circulating water bath was turned on and the temperature was set to 25°C. The main unit was powered on and rotated forward at 800 rpm. After the rotation speed stabilized, the mobile phase was pumped in at a flow rate of 2.0 mL / min. After the mobile phase flowed out of the column outlet and the baseline stabilized, the sample solution was injected through the injection ring. The effluent at the column outlet was continuously detected at a wavelength of 259 nm. Nicotine was separated and prepared with a purity of 99.85% and a yield of 92.57%.

[0044] Comparative Example 1

[0045] Prepare a two-phase solvent system of petroleum ether-ethyl acetate-methanol-water in a separatory funnel at a ratio of 4:4:5:5 (v / v), shake thoroughly, and let stand overnight. The lower phase is the stationary phase, and the upper phase is the mobile phase. Take 100 mg of the tobacco extract sample to be separated from Preparation Example 1, dissolve it completely in 10 mL of the lower phase solution, and adjust the pH to 12 with 30% NaOH solution.

[0046] The lower phase of the solvent system was pumped into the main unit and filled the separation solenoid. The circulating water bath was turned on and the temperature was set to 25°C. The main unit was powered on and rotated forward at 800 rpm. After the rotation speed stabilized, the mobile phase was pumped in at a flow rate of 2.0 mL / min. After the mobile phase flowed out of the column outlet and the baseline stabilized, the sample solution was injected through the injection ring. The effluent at the column outlet was continuously detected at a wavelength of 259 nm. Nicotine was separated and prepared with a purity of 97.13% and a yield of 85.46%.

[0047] Comparative Example 2

[0048] Prepare a two-phase solvent system of petroleum ether-ethyl acetate-methanol-water in a separatory funnel at a ratio of 4:4:5:5 (v / v), shake thoroughly, and let stand overnight. The lower phase is the stationary phase, and the upper phase is the mobile phase. Take 100 mg of the tobacco extract sample to be separated from Preparation Example 2, dissolve it completely in 10 mL of the lower phase solution, and adjust the pH to 12 with 30% NaOH solution.

[0049] The lower phase of the solvent system was pumped into the main unit and filled the separation solenoid. The circulating water bath was turned on and the temperature was set to 25°C. The main unit was powered on and rotated forward at 800 rpm. After the rotation speed stabilized, the mobile phase was pumped in at a flow rate of 2.0 mL / min. After the mobile phase flowed out of the column outlet and the baseline stabilized, the sample solution was injected through the injection ring. The effluent at the column outlet was continuously detected at a wavelength of 259 nm. Nicotine was separated and prepared with a purity of 98.56% and a yield of 88.79%.

[0050] Example 3

[0051] The eutectic solvent used in Preparation Example 2 was reused to extract tobacco leaves in the same manner as in Preparation Example 2, and purified by high-speed countercurrent chromatography as in Example 2. The purity and yield of nicotine after five reuses are as follows:

[0052]

[0053]

[0054] As can be seen from the above embodiments, the present invention utilizes a two-phase system of n-hexane-chloroform-acetonitrile-n-butanol for high-speed countercurrent chromatography separation, which can effectively purify tobacco extracts, thereby obtaining high-purity natural nicotine products. Compared with the existing petroleum ether-ethyl acetate-methanol-water system, the separation effect is greatly improved. In addition, the eutectic solvent extraction method of the present invention can effectively extract nicotine from tobacco raw materials, reduce impurities in tobacco extracts, further improve the effect of high-speed countercurrent chromatography separation, and obtain natural nicotine with higher yield and better purity.

[0055] Obviously, the above description of the embodiments is only for the purpose of helping to understand the method and core idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for extracting and separating natural nicotine, comprising the following steps: Prepare a mixture of n-hexane-chloroform-acetonitrile-n-butanol in a volume ratio of 10:3:7:

5. After separation, use the lower phase as the stationary phase and the upper phase as the mobile phase. Dissolve the tobacco extract in the lower phase and separate it using high-speed countercurrent chromatography. Collect the target components to obtain the nicotine product. The method further includes the preparation of tobacco extract: extracting tobacco raw material with an aqueous eutectic solvent, then extracting the extract with an organic solvent, and obtaining tobacco extract after removing the organic solvent phase; the eutectic solvent is a combination of choline chloride and glycerol in a molar ratio of 1:(2-3), with water added to a specified water content of 25-35 wt%; the mass-volume ratio of the tobacco raw material to the aqueous eutectic solvent is 1:5-8 g / ml.

2. The method according to claim 1, characterized in that, The method includes: accurately measuring a mixture of n-hexane-chloroform-acetonitrile-n-butanol, placing it in a separatory funnel to prepare a two-phase solvent system, using the lower phase as the stationary phase and the upper phase as the mobile phase; filling the countercurrent chromatography column with the stationary phase, then pumping in the mobile phase until the two solvent phases reach dynamic equilibrium in the column; then dissolving and injecting the tobacco extract using the lower phase as the solvent, separating it using high-speed countercurrent chromatography, monitoring under a UV detector, and collecting the target components to obtain the nicotine product.

3. The method according to claim 1, characterized in that, The preparation of the tobacco extract includes adding tobacco raw material to an aqueous eutectic solvent, ultrasonically extracting at 40-100℃, centrifuging to collect the supernatant, extracting with an organic solvent, removing the organic solvent from the organic solvent phase, and obtaining the tobacco extract.

4. The method according to claim 1, characterized in that, The organic solvent is selected from one or more of ethyl acetate, diethyl ether, and petroleum ether.

5. The method according to claim 1, characterized in that, After dissolving the tobacco extract in the lower phase, the pH of the solution was adjusted to 11.5–12.5.

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