A two-dimensional nuclear magnetic resonance spectroscopy method for measuring tobacco cellulose content

Abstract

The invention discloses a two-dimensional nuclear magnetic resonance spectrum measurement method for tobacco cellulose content, wherein cellulose in the tobacco to be tested is converted into acetyl cellulose by acetylation reaction, Glu1 peak of acetyl cellulose is used as the target quantitative peak, benzene ring C peak of internal standard substance 1,3,5-trimethoxybenzene TMB is used as the internal standard reference peak, and two-dimensional heteronuclear single quantum coherence spectrum nuclear magnetic resonance measurement technology 2DHSQCNMR is used to realize the quantitative determination of cellulose in tobacco. The operation process of the invention is simple, the quantitative effect is good and the environment is friendly, and the practical requirements of the tobacco industry for fast, accurate and batch determination of tobacco cellulose content are met.

Description

Technical Field

[0001] The invention relates to a method for determining tobacco cellulose, and belongs to the research field of tobacco analytical chemistry. Background Art

[0002] Tobacco cellulose is an important type of cell wall macromolecular polymer. It has biological functions such as maintaining cell morphology and improving the lodging resistance of tobacco plants during tobacco growth. The chemical composition of tobacco is an important factor in determining the quality of tobacco leaves. Tobacco leaves with high cellulose content have good combustion properties and strong flame retention. However, the combustion of tobacco cellulose produces harmful substances such as carbon monoxide, polycyclic aromatic hydrocarbons, and tar, which endanger the health of consumers. The cellulose content has an important impact on the quality and safety of tobacco. Therefore, it is of great significance for analytical chemists to develop a rapid and non-degradable quantitative analysis method to determine the cellulose content.

[0003] At present, the tobacco industry basically uses chemical methods to determine the cellulose content in tobacco, that is, first use neutral detergent, acid detergent and 72% concentrated sulfuric acid to remove the soluble substances in the tobacco sample, then after the sample is dried and ashed, the neutral detergent fiber, acid detergent fiber and acid-washed lignin are obtained by weight method, and the cellulose, hemicellulose and lignin contents in the sample are obtained by calculation. Therefore, the chemical method for determining tobacco cellulose is cumbersome and time-consuming, and requires the use of a large amount of chemical reagents, which can easily cause environmental pollution and cannot meet the actual needs of the tobacco industry for rapid batch determination of tobacco cellulose content. Two-dimensional heteronuclear single quantum coherence spectroscopy nuclear magnetic resonance technology (2D HSQC NMR) is one of the most powerful tools for studying the structure and content of plant cell wall materials. This method combines 1 The signal detection sensitivity of HNMR experiments and 13CNMR spectra have the advantages of wide spectrum range and high resolution, and can maintain the initial state of natural complex macromolecules. The spectra can reveal the different functional group structures and bond compositions of cell wall macromolecules, and are widely used in the structural analysis of cellulose, hemicellulose, and lignin in plant samples (Green Chemistry, 2016, 18(3): 608-621). At the same time, obtaining quantitative information has also become one of the main requirements of 2D HSQC NMR technology (Analytical Chemistry, 2013, 85(6): 3213-3221). The commonly used quantitative technology is to optimize the 2D HSQC NMR pulse sequence to suppress the change of the target chemical bond coupling constant J and reduce the fluctuation of the HSQC signal (Journal of the American Chemical Society, 2003, 125(14): 4362–4367) to achieve the quantification of the target substance. Or select a suitable internal standard reference signal to reduce the error caused by T2 relaxation and resonance shift (Cellulose, 2020, 27 (14): 7929-7953) to improve the accuracy of measurement. In recent years, 2D NMR spectroscopy quantitative analysis methods have been applied to the detection of lactose components in milk (Journal of Agricultural and Food Chemistry, 2007, 55 (11), 4307-4311) and glucose and other components in tomato extracts (Metabolomics, 2015, 11 (5): 1231-1242), but usually, the target substances to be measured are mostly small molecule compounds. The 2D HSQC NMR quantitative analysis technology for plant macromolecules is still a hot spot in current technology development, but there are systematic errors in cellulose 2D HSQC NMR measurement (Journal of Pharmaceutical and Biomedical Analysis, 2015, 108, 78-85), and most of them are semi-quantitative analysis, which is greatly limited to the specific application of tobacco cellulose measurement. Summary of the invention

[0004] In order to overcome the defects of the prior art, the purpose of the present invention is to provide a 2D HSQC NMR quantitative analysis method for the content of tobacco cellulose macromolecules with simple process, good quantitative effect and environmental friendliness, so as to meet the practical requirements of the tobacco industry for rapid, accurate and batch determination of tobacco cellulose content.

[0005] The present invention solves the technical problem by adopting the following technical solution:

[0006] A two-dimensional nuclear magnetic resonance spectroscopy method for measuring tobacco cellulose content is characterized in that the cellulose in the tobacco to be tested is converted into acetyl cellulose through an acetylation reaction, the Glu1 peak of acetyl cellulose is used as the target quantitative peak, the benzene ring C peak of the internal standard substance 1,3,5-trimethoxybenzene (TMB) is used as the internal standard reference peak, and the two-dimensional heteronuclear single quantum coherence spectrum nuclear magnetic resonance measurement technology (2D HSQC NMR) is used to achieve quantitative determination of cellulose in tobacco. Specifically, the following steps are included:

[0007] Step 1: Draw the standard working curve

[0008] A series of standard cellulose samples with a mass gradient were weighed and acetylated with acetic anhydride to generate acetyl cellulose. The obtained acetyl cellulose was dissolved in deuterated dimethyl sulfoxide / deuterated pyridine (DMSO-d) containing the internal standard substance 1,3,5-trimethoxybenzene (TMB). 6 / Pyridine-d 5 , volume ratio of 4:1) mixed solvent, using 2D HSQC NMR to obtain the spectrum of each acetyl cellulose sample;

[0009] Using MestReNova 14.0 software, the spectral peaks at δ C / δ H =99.1 / 4.75ppm of Glu1 target quantitative peak volume and TMB located at δ C / δ H =The ratio of the peak volume of the internal standard reference peak of 92.7 / 6.13ppm;

[0010] The absolute mass of cellulose in each standard cellulose sample is used as the abscissa, and the ratio of the peak volume of the target quantitative peak to the peak volume of the internal standard reference peak in the spectrum of each acetyl cellulose sample is used as the ordinate to obtain a standard working curve.

[0011] Step 2: Testing of cellulose in tobacco samples

[0012] Degreasing and ball-milling the tobacco sample powder to be tested to obtain a cellulose sample to be tested;

[0013] The cellulose sample to be tested is acetylated with acetic anhydride to generate acetyl cellulose; the obtained acetyl cellulose is dissolved in deuterated dimethyl sulfoxide / deuterated pyridine (DMSO-d 6 / Pyridine-d 5 , volume ratio of 4:1) in a mixed solvent, using 2D HSQC NMR to obtain the spectrum of acetyl cellulose of the tobacco sample to be tested;

[0014] Using MestReNova 14.0 software, the ratio of the target quantitative peak volume to the internal standard reference peak volume in the acetyl cellulose spectrum of the tobacco sample to be tested is obtained, and then the absolute mass of cellulose in the tobacco sample to be tested is obtained according to the standard working curve of step (1), and then the cellulose content M in the tobacco sample to be tested is obtained according to the absolute mass of cellulose in the tobacco sample to be tested and the mass of the sample to be tested. The calculation formula is shown in formula (1):

[0015]

[0016] Where M is the cellulose content in the tobacco sample to be tested (in %, mass fraction), m x is the absolute mass of cellulose in the tobacco sample to be tested (in mg), m 0 is the mass of the tobacco sample to be tested (in g), and W is the moisture content of the sample (%, mass fraction).

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] 1. The present invention utilizes two-dimensional heteronuclear single quantum coherence spectrum nuclear magnetic resonance measurement technology (2D HSQC NMR) to achieve quantitative determination of tobacco cellulose macromolecules. The process is simple, the quantitative effect is good, and it is environmentally friendly. It meets the actual requirements of the tobacco industry for rapid, accurate, and batch determination of tobacco cellulose content, is conducive to mastering and improving the quality of cut stems and tobacco flakes, and improving the quality of cigarettes, providing important support for the development and quality improvement of "heat-not-burn cigarettes".

[0019] 2. The present invention utilizes two-dimensional heteronuclear single quantum coherence spectroscopy nuclear magnetic resonance technology (2D HSQC NMR) to collect two-dimensional nuclear magnetic resonance signals. Compared with one-dimensional nuclear magnetic resonance spectrum signals, the spectrum has a wide range and high resolution; the standard working curve is used to realize the measurement of the absolute content of cellulose.

[0020] 3. The present invention adopts an acetylation method to derivatize the cellulose in the sample to be tested to generate acetyl cellulose, thereby achieving the purpose of 6 / Pyridine-d5, 4:1 v / v) mixed solvent.

[0021] 4. The present invention uses acetyl cellulose at δ C / δ H = 99.1 / 4.75ppm Glu1 peak as the target quantitative peak, the internal standard substance 1,3,5-trimethoxybenzene (TMB) is located at δ C / δ H = 92.7 / 6.13ppm benzene ring peak as the internal standard reference peak, effectively improving the impact of instrument signal fluctuations on the measurement results and reducing measurement errors.

[0022] 5. The test method established by the present invention reduces the waste of manpower and material resources, is environmentally friendly, and is suitable for batch testing of tobacco samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the classic 2D HSQC NMR spectrum of acetylcellulose obtained after acetylation of the microcrystalline cellulose standard sample in Example 1. Figure 1 The target quantitative peak of acetylcellulose Glu1 is located at δ C / δ H =99.1 / 4.75ppm, the internal standard substance 1,3,5-trimethoxybenzene (TMB) internal standard reference peak is located at δ C / δ H =92.7 / 6.13ppm, solvent DMSO-d 6 The central peak is located at δ C / δ H 39.52 / 2.50ppm.

[0024] Figure 2 to Figure 6 This is the 2D HSQC NMR spectrum of acetylcellulose obtained after acetylation of microcrystalline cellulose standard samples with different mass gradients (20.2 mg, 40.5 mg, 60.3 mg, 80.5 mg, and 100.1 mg, respectively) in Example 1.

[0025] Figure 7 This is the standard working curve diagram obtained in Example 1.

[0026] Figure 8 The tobacco sample to be tested in Example 1 (Yunyan 87B 2 F) 2D HSQC NMR spectrum after acetylation. DETAILED DESCRIPTION

[0027] The present invention will be further described below through specific implementation modes in conjunction with the accompanying drawings.

[0028] In the following embodiments of the present invention:

[0029] The pretreatment conditions of tobacco samples are as follows: (1) Degreasing: The tobacco samples to be tested were dried at 40°C for 2 hours and then mechanically crushed and passed through a 40-mesh sieve. 0.5 g of the sieved tobacco samples were subjected to Soxhlet extraction with 80 mL of toluene and 40 mL of ethanol for 6 hours to remove fat and dried for later use. (2) Ball milling: The degreased tobacco samples were placed in a 100 mL ball mill for 6 hours. The working environment of the ball mill was room temperature and the speed was set to 600 r / min. To prevent the temperature of the plant powder from rising excessively during the ball milling process, the ball mill was paused for 10 minutes after every 20 minutes of operation. (3) Derivatization: The ball milled samples were placed in a 10 mL reaction bottle, and 3.0 mL of a mixed solvent of dimethyl sulfoxide / 1-methylimidazole (the volume ratio of the two was 2:1) was slowly added. The sample powder was stirred to fully disperse in the solvent. Subsequently, 1.0 mL of pyridine-acetic anhydride (the volume ratio of the two was 1:1) was added to the reaction bottle at room temperature and protected from light for acetylation reaction. After 3 hours, the black solution after the acetylation reaction was completed was poured into 500 mL of distilled water with rapid stirring to precipitate acetyl cellulose. The acetyl cellulose was filtered, washed with distilled water and anhydrous ethanol, and vacuum dried for later use.

[0030] The preparation method of 2D HSQC NMR test samples is as follows: the acetyl cellulose obtained after acetylation of the sample to be tested is transferred to a 5 mm NMR quartz sample tube, and 500 μL of deuterated dimethyl sulfoxide / deuterated pyridine (DMSO-d 6 / Pyridine-d5, the volume ratio of the two is 4:1) in a mixed solvent, the content of the internal standard substance TMB in the solvent is 2.0% (m / v). Vortex oscillation for 10 minutes and ultrasonic for 20 minutes to completely dissolve the sample in the solvent.

[0031] The instrumental conditions for 2D HSQC NMR spectroscopy were as follows: All samples were tested on a Bruker 600 MHz superconducting NMR spectrometer equipped with a 5 mm Z-gradient field coil. 1 H / 13 C / 15 N triple resonance transverse cryogenic probe, at 298K, using Bruker standard pulse sequence. Specific parameters are as follows: F2( 1 The spectral width of the H dimension is 10ppm and the sampling points are 1024 (TD 1 ); F1( 13 The spectral width of the C dimension is 160ppm and the sampling points are 256 (TD 2 ); Scan number (NS) is 64; Scan delay (D 1 )1s. To obtain the NMR spectrum of acetylcellulose, DMSO-d 6 The solvent center peak (δ C / δH =39.52 / 2.50ppm) was used for chemical shift calibration, and the characteristic peaks in the HSQC spectrum were processed using MestReNova 14.0 software to obtain the target peak volume data.

[0032] Example 1

[0033] First, 10.2 mg of microcrystalline cellulose standard sample was weighed and acetylated with acetic anhydride to produce acetyl cellulose under the same conditions as the pretreatment conditions (3) of the tobacco sample. The derivatized acetyl cellulose was dissolved in deuterated dimethyl sulfoxide / deuterated pyridine (DMSO-d) containing the internal standard substance 1,3,5-trimethoxybenzene (TMB). 6 / Pyridine-d 5 The spectrum of acetyl cellulose sample was obtained by 2D HSQC NMR in a mixed solvent of 4:1 v / v. A typical 2D HSQC NMR spectrum of acetyl cellulose is shown in Figure 2. Figure 1 shown.

[0034] choose Figure 1 The chemical shift is located at δ C / δ H = 99.1 / 4.75ppm of acetylcellulose Glu1 peak as the target quantitative peak, chemical shift at δ C / δ H =92.7 / 6.13ppm 1,3,5-trimethoxybenzene (TMB) benzene ring carbon peak was used as internal standard reference peak. Figure 1 The center is located at δ C / δ H =39.52 / 2.50ppm DMSO-d 6 The chemical shift calibration was performed on the solvent center peak.

[0035] Next, 20.2 mg, 40.5 mg, 60.3 mg, 80.5 mg, and 100.1 mg of microcrystalline cellulose standard samples were accurately weighed and acetylated with acetic anhydride to generate acetyl cellulose. Each acetyl cellulose obtained by derivatization was dissolved in deuterated dimethyl sulfoxide / deuterated pyridine (DMSO-d) containing the internal standard substance 1,3,5-trimethoxybenzene (TMB). 6 / Pyridine-d 5 , 4:1 v / v) mixed solvent, the spectra of each acetyl cellulose sample were obtained by 2D HSQC NMR, as shown in Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 shown.

[0036] Select the wave in each spectrum at δ C / δ H =99.1 / 4.75ppm acetylcellulose Glu1 peak is the target quantitative peak, located at δ C / δ H = 92.7 / 6.13ppm TMB benzene ring carbon peak as the internal standard reference peak. Based on the ratio of the Glu1 target quantitative peak volume (A) of each acetylcellulose to the TMB internal standard reference peak volume (B), a cellulose standard working curve was established: I = 0.2127m + 2.4272 (R 2 =0.9951), such as Figure 7 The minimum detection limit of cellulose was calculated to be 0.74 mg / g and the minimum quantification limit was 2.45 mg / g at the signal-to-noise ratio of 3 times and 10 times.

[0037] Third, accurately weigh 0.5 g of flue-cured tobacco leaf sample powder (Yunyan 87B 2 F), after the above sample processing and data processing, according to the spectrum of the sample to be tested in δ C / δ H The peak volume (A) of the target quantitative peak of 99.1 / 4.75ppm acetylcellulose Glu1 is C / δ H = 92.7 / 6.13ppm Peak volume of TMB internal standard reference peak (B), see Figure 8 The ratio of the samples A and B to be tested is I x is 10.43, and then Figure 7 The working curve is represented by I x Calculate m x , so that the absolute mass m of the sample cellulose can be obtained x The water content W of the sample was 37.62 mg according to YC / T 31-1996 and was 12.00%.

[0038] Finally, the cellulose content in the tobacco sample to be tested can be calculated to be 8.55% by formula (1).

[0039] Example 2

[0040] Weigh 0.5g (accurate weighing to 0.0002g) (Yunyan 87B 2 F), five samples (sample numbers 1-5) were tested and the data were processed in Example 1 to obtain the average value, standard deviation and relative standard deviation of the five repeated tests of the cellulose content of the samples. The results are shown in Table 1.

[0041] Table 1. Mean, standard deviation and relative standard deviation of 5 repeated tests

[0042]

[0043] Example 3

[0044] Seven tobacco samples (sample numbers 1#--7#) were taken and each sample was divided into two parts. One part was tested according to the tobacco industry standard "Determination of Cellulose, Hemicellulose and Lignin in Tobacco and Tobacco Products Determination of Detergent Method", and the other part was tested according to the method steps of Example 1. The results are shown in Table 2.

[0045] Table 2 Comparison of the results of the determination of cellulose in tobacco samples by the method of the present invention and the results of the determination by the standard method

[0046]

[0047]

[0048] Therefore, the method for determining the tobacco cellulose content provided by the present invention is a simple, rapid, accurate and environmentally friendly quantitative analysis method, which provides an effective technical method and means for the tobacco industry to determine the tobacco cellulose content in batches.

Claims

1. A two-dimensional nuclear magnetic resonance spectroscopy method for measuring tobacco cellulose content, Features: The cellulose in the tobacco to be tested is converted into acetyl cellulose through acetylation reaction, and the Glu1 peak of acetyl cellulose is used as the target quantitative peak, and the benzene ring C peak of the internal standard substance 1,3,5-trimethoxybenzene TMB is used as the internal standard reference peak. The quantitative determination of cellulose in tobacco is achieved by using the two-dimensional heteronuclear single quantum coherence spectrum nuclear magnetic resonance measurement technology 2D HSQC NMR; The method for converting cellulose in the tobacco to be tested into acetyl cellulose through acetylation reaction is as follows: degreasing and ball-milling the tobacco sample powder to be tested to obtain a cellulose sample to be tested; placing the cellulose sample to be tested in a 10 mL reaction bottle, slowly adding 3.0 mL of a mixed solvent of dimethyl sulfoxide and 1-methylimidazole in a volume ratio of 2:1, and stirring to fully disperse the sample powder in the solvent; then, adding 1.0 mL of a mixed solution of pyridine and acetic anhydride in a volume ratio of 1:1 to the reaction bottle at room temperature and in the dark, and performing an acetylation reaction; after 3 hours, pouring the black solution after the acetylation reaction into 500 mL of rapidly stirred distilled water for precipitation to precipitate acetyl cellulose; The acetyl cellulose was filtered, washed with distilled water and anhydrous ethanol, and vacuum dried for later use.

2. The method for measuring tobacco cellulose content by two-dimensional nuclear magnetic resonance spectroscopy according to claim 1, It is characterized in that The following steps are involved: Step 1: Draw the standard working curve A series of standard cellulose samples with a mass gradient are weighed and acetylated with acetic anhydride to generate acetyl cellulose; the obtained acetyl cellulose is dissolved in a deuterated dimethyl sulfoxide / deuterated pyridine mixed solvent containing an internal standard substance 1,3,5-trimethoxybenzene (TMB), and the spectrum of each acetyl cellulose sample is obtained by 2D HSQC NMR; The MestReNova 14.0 software was used to obtain the δ C / δ H = 99.1 / 4.75 ppm of the Glu1 target quantitation peak volume and TMB at δ C / δ H = 92.7 / 6.13 ppm ratio of the internal standard reference peak volume; The absolute mass of cellulose in each standard cellulose sample is used as the abscissa, and the ratio of the peak volume of the target quantitative peak to the peak volume of the internal standard reference peak in the spectrum of each acetyl cellulose sample is used as the ordinate to obtain a standard working curve; Step 2: Testing of cellulose in tobacco samples The cellulose in the tobacco to be tested is converted into acetyl cellulose by acetylation reaction; the obtained acetyl cellulose is dissolved in a deuterated dimethyl sulfoxide / deuterated pyridine mixed solvent containing an internal standard substance 1,3,5-trimethoxybenzene (TMB), and the spectrum of the acetyl cellulose of the tobacco sample to be tested is obtained by 2D HSQCNMR; Using MestReNova 14.0 software, the ratio of the target quantitative peak volume to the internal standard reference peak volume in the acetyl cellulose spectrum of the tobacco sample to be tested is obtained, and then the absolute mass of cellulose in the tobacco sample to be tested is obtained according to the standard working curve of step (1), and then the cellulose content in the tobacco sample to be tested is obtained according to the absolute mass of cellulose in the tobacco sample to be tested and the mass of the tobacco sample to be tested.

3. The method for measuring tobacco cellulose content by two-dimensional nuclear magnetic resonance spectroscopy according to claim 2, Features: The volume ratio of deuterated dimethyl sulfoxide to deuterated pyridine in the deuterated dimethyl sulfoxide / deuterated pyridine mixed solvent used in step 1 and step 2 is 4:1.

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