RU2805229C1 - Method for determining the content of rubber in the tissues of rubber plants - Google Patents

Abstract

FIELD: instrumental methods of physico-chemical analysis.

SUBSTANCE: method for determining the content of natural rubber in the tissues of rubber-bearing plants using the method of electron paramagnetic resonance (EPR). The method involves the preparation of a powdered sample from dried plant tissue with an average particle size of about 20 microns and subsequent analysis of the resulting sample using the spin probe EPR method using a stable nitroxyl radical 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) as a spin probe. The quantitative content of rubber in the sample is determined by the value of the integral intensity of the low-field component of the EPR spectrum using the linear dependence of the integral intensity of the specified component of the spectrum on the quantitative content of rubber in the sample. The technical result of the invention consists in the fact that a new method for determining the content of rubber in the tissues of rubber-bearing plants, based on the use of EPR spectroscopy of a spin probe, is proposed.

EFFECT: method involves the use of a commercially available nitroxyl radical TEMPO as a spin probe and allows you to quickly determine the content of rubber in plant tissues without separating it from rubber-containing biomass.

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Description

The method refers to instrumental methods of physicochemical analysis and is intended to determine the rubber content in the tissues of rubber plants. The method can be used to monitor the accumulation of the target product in plant tissues in technologies for the industrial cultivation of rubber plants, as well as when conducting scientific research on the cultivation of plants related to non-traditional sources of natural rubber.

Natural rubber (NR) is a strategically important raw material, which is a necessary component of rubber for the production of automobile and aircraft tires. The main source of rubber today is Hevea brasiliensis however, in recent decades, extensive research has been conducted to find alternative sources of this strategically important product. The guayule shrub (Parthenium argentatum) and close relatives of the dandelion - the root rubber plants kok-saghyz (Taraxacum kok-saghyz), Crimea-saghyz (Taraxacum hybernum), tau-saghyz (Scorzonera tau-saghyz) and some others are considered as such sources. However, for industrial production, wild forms of root rubber plants, in particular, kok-sagyz, which is a source of not only rubber, but also inulin, are unproductive due to their low growth rate, strong dependence on natural, climatic and agrotechnical growing conditions, the complexity of processing technologies and extraction of target substances. products. The key direction in solving the problems of increasing the productivity of Kok-Sagyz for rubber is genetic engineering research on genome modification and breeding work in order to obtain plants that can provide profitable industrial production. Technologies for the industrial cultivation of kok-saghyz in artificial conditions, in particular in aeroponic phytotrons, are being actively developed.

The widespread implementation of such studies creates a need for simple, reproducible and accurate methods for determining the rubber content in the tissues of rubber plants. Traditionally, a gravimetric method is used for this purpose, the use of which for the determination of resins and rubber in guayule is described, for example, in [L.T. Black, G. E. Hamerstrand, V.A. Rasnik "Gravimetric analysis for determining the resin and rubber content of guayule". Rubber Chem. Technol. 1983, 56, 2. p. 367-371]. The method includes drying rubber-bearing plant tissues, grinding them, sequential removal of water-soluble components and products soluble in polar organic solvents, extracting rubber by extraction with non-polar solvents, followed by drying the extract to a constant weight of the residue. Dried biomass before grinding can additionally be subjected to deep freezing, see, for example, [B.R. Kuluev, N.D. Minchenkov, G.R. Gumerov "Kok-saghyz (Taraxacum kok-saghyz Rodin): methods of rubber isolation and prospects for the use of biotechnological approaches." Biotechnology and plant breeding. 2019, 2 (2), p. 33-42]. Quantitative determination of rubber content by gravimetric method requires a significant amount of starting material for analysis, as well as a significant investment of time and reagents, which limits its use for conducting mass analyzes in order to monitor the accumulation of the target product during plant growth. However, the gravimetric method, which allows one to obtain absolute values of the quantitative content of rubber in plant tissues, remains indispensable as a comparison method for assessing the reliability of the results obtained by other methods.

A modern alternative to the gravimetric method are methods of instrumental physicochemical analysis, the use of which in scientific research and industrial agricultural technologies has been actively developed in recent years.

It is known to use low-field NMR spectroscopy for the quantitative determination of rubber content in plant tissues [US 10859650 B2, publ. 12/08/2020]. The method involves transferring rubber from plant tissues into a solution and comparative analysis of the peak intensity of the NMR spectra of the test and standard samples with a predetermined rubber content. The complex procedure for preparing a sample for analysis and the relatively low sensitivity of the method lead to a high error, especially when analyzing samples with low rubber content.

To analyze the rubber content in kok-saghyz plants, the use of mass spectrometric methods based on the determination of rubber pyrolysis products, mainly isoprene and limonene, is described. [Y. Dong. T. Xiang, et al. "Rapid Quantitative Determination of Isoprene Monomer in Living Taraxacum kok-saghyz by Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry" China Petroleum Processing and Petrochemical Technology 2020, 22, 2, p. 30-36] and [Y. Dong, T. Guo, et al. "Determination of Natural Rubber Content in Taraxacum Kok-Saghyz by Pyrolysis Gas Chromatography-Mass Spectrometry" China Petroleum Processing and Petrochemical Technology. 2022, 22, 3, p. 43-48]. When interpreting the results, it should be borne in mind that similar products are obtained from the pyrolysis of low molecular weight rubbers contained in resins also present in the tissues of rubber plants. The method requires the use of complex equipment and painstaking preliminary preparation of samples and is not suitable for performing mass serial analyses.

As a method for determining the content of resins and rubber in plant tissues without lengthy preliminary sample preparation, it is proposed to use a portable version of IR spectroscopy in the near-infrared (NIR) range of 800-2400 nm. In the work [M. Taurines, L. Brancheriau et al. "Determination of natural rubber and resin content of guayule fresh biomass by near infrared spectroscopy." Industrial Crops and Products. 2019, 134, p. 177-184] the capabilities of the method are demonstrated by the example of analyzing the rubber content in the branches of the guayule bush. The method can be used to estimate the content of the target product both in individual plants and to estimate the average content of rubber and resins in plants across the entire field, based on a relatively small sample - about 70 measurements, for example, when studying the influence of natural and climatic conditions on accumulation of rubber in plant tissues or to determine the optimal harvest date. Using a portable NIR spectrometer, it is possible to determine the quantitative content of rubber directly in the field using raw or dried guayule branches, however, according to the authors, higher accuracy - up to ±0.3% is obtained when using powdered samples obtained by grinding dried ones for analysis rubber-containing plant tissues.

The present invention solves the problem of expanding the arsenal of technical means that make it possible, using modern instrumental methods of physicochemical analysis, to quickly and with high accuracy determine the quantitative content of rubber in the tissues of rubber-bearing plants without extracting it from rubber-containing biomaterial.

The problem is solved by the proposed method for determining the rubber content in the tissues of rubber-bearing plants, including the preparation of a powdered sample from dried plant tissue and subsequent analysis of the resulting sample using the instrumental method of physicochemical analysis, characterized in that the method of physicochemical analysis is the method of electron paramagnetic resonance (EPR) of a spin probe using the nitroxide radical 2,2,6,6-tetramethylpiperidin-1-oxyl (TΤΕΜΠΟ) as a spin probe, and the quantitative content of rubber in the sample is determined by the value of the integral intensity of the low-field component of the EPR spectrum ΤΕΜΠΟ using linear dependence of the integral intensity of the specified spectrum component on the amount of rubber in the sample.

The ΤΕΜΠΟ radical is an available commercial product, is characterized by stability at room temperature, has high volatility, allowing it to be introduced into the test substance from the gas phase; the molecule can easily be incorporated into the structure of the test object. The first of the three components recorded during field sweep is used as the low-field component of the EPR spectrum, since additional native signals are not superimposed on it.

The technical result of the invention is that a new method, based on the use of spin probe EPR spectroscopy, is proposed for determining the rubber content in the tissues of rubber plants. The method involves the use of a commercially available nitroxyl radical ΤΕΜΠΟ as a spin probe and allows you to quickly determine the rubber content in plant tissues without isolating it from rubber-containing biomass.

The essence of the invention is illustrated by the following graphic illustrations.

In FIG. Figure 1 shows the ESR spectra of samples containing different amounts of rubber obtained from samples of the biomass of the root of the kok-saghyz plant grown under the conditions of an aeroponic phytotron. The low-field component of the spectrum is indicated by a dotted line. Sample weight 7 mg.

In FIG. Figure 2 shows the dependence of the integral intensity of the low-field component of the EPR spectrum ΤΕΜΠΟ on the amount of rubber in the sample in the concentration range from 0 to 17.5 wt. %.

The spin probe EPR method is widely used to study polymer systems [Md. A. Uddin, N. Yu, et al. "Recent progress in EPR study of spin labeled polymers and spin probed polymer systems" J. Polym. Sci., 2020, 58, p.1924-1948. DOI: 10.1002/pol.20200039]. The EPR spectrum of a nitroxyl radical provides information about chemical reactions, local molecular mobility, polarity, and molecular organization of the system under study. The present invention demonstrates the possibility of using the method for the quantitative determination of rubber content in the tissues of rubber plants.

The proposed method is based on the fact discovered by the authors of the selective sorption of the stable nitroxyl radical 2,2,6,6-tetramethylpiperidin-1-oxyl (TΤΕΜΠΟ) on rubber particles contained in plant biomass. It was shown that when the ΤΕΜΠΟ radical is introduced into a powder obtained by grinding dried rubber-containing plant tissue, ESR spectra are obtained similar to the spectra previously recorded for the ΤΕΜΠΟ radical in elastomers. The correlation times of rotation of the nitroxyl radical τ and the hyperfine interaction constant (HFC) a _N , obtained by the authors on samples of rubber-containing root biomass of the kok-saghyz plant, are: τ = 3.0 × 10 ^-10 s, a _N = 15.5 G ; values for natural rubber: τ=2.0×10 ^-10 s, a _N =15.4 Gs. [A.M. Wasserman, A.L. Kovarsky. “Spin labels and probes in the physical chemistry of polymers” Rep. ed. A.L. Buchachenko Academy of Sciences of the USSR, Institute of Chemistry. physics. - M. Nauka, 1986, 244 pp.].

It has been shown that the ΤΕΜΠΟ radical is localized exclusively in rubber formations of rubber-containing fabric. The fact of selective sorption of the ΤΕΜΠΟ radical on rubber particles is confirmed by the fact that after removing rubber from a biomass sample by extraction with a nonpolar solvent, the EPR spectra of ΤΕΜΠΟ are not recorded in the remaining material.

The degree of grinding of the dried biomaterial taken for analysis is of great importance. It has been shown that grinding dried tissue to a size of approximately 20 µm provides sample homogenization sufficient for rapid penetration of the ΤΕΜΠΟ radical into the rubber formations of the plant. The more rubber is contained in the sample, the greater the amount of radical penetrates into the sample from the gas phase, the greater the intensity of the ESR signal. As an illustration, FIG. Figure 1 compares the ESR spectra of samples of equal mass, containing different amounts of rubber, obtained from different samples of dried crushed root tissue of the kok-saghyz plant.

It should be noted that the degree of grinding of the initial rubber-containing material is also essential for obtaining correct results using the traditional gravimetric method, which is something that the authors of publications describing the weight determination of rubber in plant tissues usually do not pay attention to. The spin probe method allows you to control the completeness of natural rubber extraction from the plant. For example, it has been found that when rubber is extracted from a powder with a particle size of approximately 150 μm, a noticeable amount of rubber remains in the sample. Additional dispersion of the material ensures the penetration of the radical into rubber formations, which in large particles can be blocked by inulin and/or other components of plant tissues that are impermeable to the radical.

The proposed method for determining the rubber content in the tissues of rubber-bearing plants is based on the existence of a linear relationship between the value of the integral intensity of the low-field component of the ESR spectrum ΤΕΜΠΟ and the quantitative content of rubber in the sample. The low-field component of the EPR spectrum is indicated by a dotted line in Fig. 1. Integral intensity a is calculated using the formula:

where I is the intensity of the low-field component of the EPR spectrum ΤΕΜΠΟ, ΔН is the width of the low-field component of the EPR spectrum ΤΕΜΠΟ.

The integral intensity is proportional to the amount of radical present in the rubber formations and, accordingly, to the amount of rubber in the sample. As an illustration, FIG. Figure 2 shows the calibration linear dependence obtained by the authors of the integral intensity of the low-field component of the EPR spectrum ΤΕΜΠΟ on the quantitative content of rubber in the standard samples. The amount of rubber in the samples from which the reference samples used to construct the calibration curve were prepared was determined by the gravimetric method. The linear relationship between the value of the integral intensity of the low-field component of the EPR spectrum ΤΕΜΠΟ and the quantitative content of rubber in the sample makes it possible to determine the amount of rubber in the sample of the test sample. Since a zero NR content in a sample corresponds to a zero signal intensity of the low-field component of the EPR spectrum ΤΕΜΠΟ (the calibration graph passes through 0), to construct it it is sufficient to use one or two reference samples, the rubber concentration in which is preferably close to the expected rubber concentration in the test sample. Moreover, in order to obtain correct results in each experiment, the saturation of the test and reference samples with the ΤΕΜΠΟ radical is carried out simultaneously under the same conditions; the EPR spectra of all samples in the series must also be recorded under the same conditions.

Samples for analysis are prepared as follows.

The rubber-containing biomaterial is dried at 60°C in an oven for 15-16 hours, after which it is crushed to obtain particles measuring approximately 20 microns. The particle size is controlled by sifting them through a sieve.

Samples of the resulting powdered samples are taken into ampoules for EPR analysis. The optimal sample amount, determined by the technical characteristics of the spectrometer, is 7 mg. The ΤΕΜΠΟ radical is introduced into samples from the gas phase at room temperature. To do this, ampoules with samples are placed in a desiccator, which contains approximately 10 mg of the ΤΕΜΠΟ radical. It has been experimentally shown that with the selected quantities of components, the exposure time, which ensures obtaining the most accurate and well-reproducible results, is about 10 minutes, while the concentration of the ΤΕΜΠΟ radical in the samples does not exceed 1 × 10 ^-5 M. Increasing the exposure time leads to exceeding the optimal concentration and interaction of radicals with each other, which can cause distortion of the spectrum shape and introduce an error in determining the signal intensity. After introducing the radical, the sample ampoules are sealed.

An EPR spectrometer Bruker EMX (Germany) was used for measurements. When recording spectra, the microwave power in the resonator was 2 mV, the modulation amplitude was 0.5 G.

The rubber content in the analyzed sample is determined from the linear relationship between the integral intensity of the low-field component of the EPR spectrum ΤΕΜΠΟ and the amount of rubber in the sample obtained using a reference sample with a known NR content determined by an independent method. The error in determining the integral intensity of the low-field component of the spectrum from three consecutive measurements for a sample containing 5.0% NR (0.35 mg in 7 mg of sample) is 7.5%, which corresponds to an error in the absolute amount of rubber equal to ±0.03 mg.

The possibility of using the proposed method is demonstrated by examples of determining the rubber content in the roots of kok-saghyz plants grown under different conditions and having different concentrations of NR.

Example 1. Determination of rubber content in the roots of a kok-saghyz plant grown in an aeroponic phytotron for two months.

The plant root weighing 100 g is dried for 16 hours at a temperature of 60°C in an oven and ground in a ball mill for 10 minutes to obtain a powdered sample with an average particle size of approximately 20 μm.

Gravimetric determination.

A sample weighing 0.3 g is extracted with chloroform in a Soxhlet apparatus for 3 hours at a temperature of 60°C. Rubber is precipitated from the extract with a threefold excess of ethyl alcohol, washed with water, acetonitrile and ethyl alcohol and dried at a temperature of 60°C for 24 hours until constant weight. The amount of rubber in the test sample based on the results of three measurements is 42 ± 3 mg (14.0 ± 1% based on dry weight).

Determination by the proposed method.

To determine the amount of rubber in the test sample, a reference sample is used, the rubber content of which, according to the results of gravimetric analysis, is 17.5%. In ampoules for EPR analysis, 7 mg of reference and test samples are weighed, then the ampoules are placed for 10 minutes in a desiccator containing approximately 10 mg of the ΤΕΜΠΟ radical (Sigma-Aldrich), after which the ampoules are sealed and the EPR spectra are recorded. The values of intensity I and width ΔH of the low-field component of the spectrum are determined and, using formula (1), the values a (arb. units) of the integral intensity of the low-field components of the EPR spectra ΤΕΜΠΟ of the reference and test samples are calculated. Based on the value of a for the reference sample, a calibration linear dependence passing through 0 is constructed, from which the quantitative content of rubber in the test sample is then determined. The obtained value of 1.026 mg (14.6 wt. %) coincides, within the experimental error, with the result obtained by the gravimetric method.

Example 2. Determination of rubber content in the roots of the kok-saghyz plant grown in vitro for two months.

The plant root weighing 30 g is dried for 16 hours at a temperature of 60°C in an oven and ground in a ball mill for 10 minutes to obtain a powdered sample with an average particle size of approximately 20 μm.

Gravimetric determination.

A sample weighing 2 g is extracted with chloroform in a Soxhlet apparatus for 3 hours at a temperature of 60°C. Rubber is planted from the extract with a threefold excess of ethyl alcohol, washed with water, acetonitrile and ethyl alcohol and dried at a temperature of 60°C for 24 hours to constant weight. The amount of rubber in the tested sample based on the results of three measurements is 53.4 ± 3.0 mg (2.67 ± 0.15% based on dry weight).

Determination by the proposed method.

To determine the amount of rubber in the test sample, reference samples are used, the rubber content of which, according to the results of gravimetric analysis, is 1.3% and 2.8%. Preparation of samples for analysis, EPR analysis and processing of results are carried out similarly to example 1. The obtained value of 0.195 mg (2.78%) of NA coincides, within the experimental error, with the result obtained by the gravimetric method.

The proposed method is easy to implement, does not require a large amount of starting material, and can be used both in scientific research and for monitoring the accumulation of rubber in plant tissues during their industrial cultivation in soil or in artificial phytotron conditions.

Claims (2)

1. A method for determining the rubber content in the tissues of rubber-bearing plants, including the preparation of a powdered sample from dried plant tissue and subsequent analysis of the resulting sample using an instrumental method of physicochemical analysis, characterized in that the average particle size in the powdered sample is about 20 microns, and As this method, the spin probe electron paramagnetic resonance method is used using the nitroxide radical 2,2,6,6-tetramethylpiperidin-1-oxyl as a spin probe, and the quantitative content of rubber in the sample is determined by the value of the integral intensity of the low-field component of the EPR spectrum using linear dependence of the integral intensity of the specified spectrum component on the quantitative content of rubber in the sample. 2. The method according to claim 1, characterized in that the specified nitroxyl radical is introduced into the test sample from the gas phase at room temperature.

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