RU2244916C1 - Method of determining chlorophyll in buckwheat plants - Google Patents

Abstract

FIELD: analytical methods in biology.

SUBSTANCE: method comprises disintegrating vegetable raw material, treating it with alcohol, and subsequent spectrometric measurement at two wavelengths: 665 and 649 nm, after which one calculates concentration of chlorophyll using following formulae: C_a = 13.7D₆₆₅ - 5.76D₆₄₉ and C_b = 25.80D₆₄₉ - 7.69D₆₆₅, where C_a and C_b are concentrations of chlorophyll a and b, D₆₆₅ and D₆₄₉ are measurable extinction coefficients, mg/L, at corresponding wavelengths. Treatment with alcohol consists in that disintegrated vegetable material is flooded with alcohol in containers, which are the tightly closed, placed in dark room, and 24 to 78 h later alcohol extract is subjected to spectrometric measurement.

EFFECT: simplified analytic procedure and reduced analysis time.

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Description

The invention relates to the field of plant biology and can be used in studies of plant physiology.

The main pigment of photosynthetic organs of all green plants is chlorophyll "a" - C ₅₅ H ₂₂ O ₅ Mg, usually accompanied by chlorophyll "b" and associated genetically. The main absorption maxima of chlorophyll "a" are located in the region of 665 nm, and for chlorophyll "in" is 649 nm.

A known method for determining the concentration of chlorophyll "a" and "b" by extinction values measured at two wavelengths, with further conversion to raw or dry matter (1). The method consists in the following: the vegetable sample is ground in a mortar with quartz sand or crushed glass, a small amount of chalk and alcohol. The alcoholic extract is sucked off through a glass filter, the residue is treated again with alcohol 2-3 times. Extracts combine. Next, a spectrometric measurement is performed at two wavelengths, followed by a calculation of the concentration of chlorophyll "a" and "b".

The disadvantage of this method is that it requires a large amount of time, limits the volume of research.

The objective of the proposed method is to reduce the time it takes and simplify the method.

To solve the problem in the known method for determining chlorophyll in plants, including grinding the raw material and treating it with alcohol, followed by spectrometric measurement at wavelengths of 665 and 649 nm, calculating the concentration of chlorophyll according to the formulas C _a = 13.7 D ₆₆₅ - 5.76 D ₆₄₉ ; With _b = 25.80D ₆₄₉ - 7.60D ₆₆₅ , according to the invention, the crushed vegetable raw materials are poured into containers with alcohol, which are tightly closed, placed in a dark place and after 24-48 hours subjected to spectrometric measurement to calculate the concentrations of chlorophyll.

Example: a freshly cut buckwheat plant of the Bogatyr cultivar in the flowering phase is divided into organs and their parts. The selected components are placed in a labeled box. First, leaves are separated, then petioles, branches. The main shoot is divided into two parts, since the upper part is almost similar to the branches in terms of chlorophyll content and mass, and the lower part is characterized by a large weight and low chlorophyll content. The combination of these parts leads to a decrease in the reliability of the analysis. Separation of the main shoot is carried out according to the fourth node, since lignification has time to spread to the flowering phase only up to this node. Then, using tweezers, they begin to separate the flowers from the main shoot and shoots of other orders. All selected organs and their parts are weighed. The flowers require the fastest execution of these works, since they are distinguished by the highest speed in the loss of water. After determining the wet weight proceed to the selection of samples. Flowers are taken with a mass of 100 mg and, without chopping, placed in a container. The leaves are cleaned of dirt with a damp soft tissue, the central veins are removed, and an average sample is prepared from which 100 mg samples are taken. The stems are cut in both horizontal and vertical directions, from their average sample, samples are taken weighing 300 mg. 300 mg are taken and fruits, which, due to the remontance of the studied culture, can be observed in the flowering phase. Fruits are placed in a container without chopping, otherwise their internal content goes into alcoholic extract, which increases its optical density. Boxes with plant material are placed in a drying cabinet with the subsequent determination of the dry weight of the material and the calculation of the percentage of dry matter. Samples are filled with 96% alcohol. To avoid the evaporation of alcohol, and accordingly the increase in concentration, the containers are tightly closed and placed in a dark place. After 24-48 hours, the containers are shaken and, pouring into the ditches, take measurements at two wavelengths. Using formulas: C _a = 13.7 D ₆₆₅ - 5.76 D ₆₄₉ ; C _b = 25.80 D ₆₄₉ - 7.60 D ₆₆₅ , where C _a , C _b are the concentration of chlorophyll "a" and " _b ", a D ₆₆₅ , D ₆₄₉ are the measured extinction coefficients in mg / l at the corresponding wavelength, calculated the concentration of chlorophyll, its further conversion to raw or dry matter.

The results of determining the amount of chlorophyll in the organs of buckwheat plants of the Bogatyr cultivar in the flowering phase +20 days are presented in the table where the chlorophyll a and b contents and their amounts are given in mg per 1 g of dry matter.

Table Organs and parts of a plant Methods for determining chlorophyll Chlorophyll a Chlorophyll B Amount "a" + "b" 1 2 3 4 5 Flowers By a known method 5.79 0.52 6.31 According to the proposed method 5.81 0.51 6.32 Sheet By a known method 9.69 0.71 10.40 According to the proposed method 9.66 0.67 10.33 The main shoot is the upper part (from the 4th node) By a known method 5.68 0.48 6.16 According to the proposed method 5.70 0.50 6.20 The main shoot is the lower part (up to the 4th node) By a known method 2.12 0.09 2.21 According to the proposed method 2.87 0.14 3.01 Branches By a known method 5.70 0.60 6.30 According to the proposed method 5.71 0.62 6.33 Fruit By a known method 1.96 0.62 2,58 According to the proposed method 1.98 0.61 2.59 Petioles By a known method 4.48 0.80 5.28 According to the proposed method 4.48 0.81 5.29

Thus, comparing the amount of chlorophyll in the organs of plants, determined by the known method and by the proposed, we conclude that the proposed method has reliability and high accuracy. In addition, the proposed method is simple and affordable, and in the study of the coarsest parts of plants the most reliable.

When using the proposed method, the number of manipulations on the extraction of chlorophylls is reduced while maintaining the accuracy of this method, saving labor costs by reducing the operations of preparing crushed glass and grinding the plant material in a mortar. Also, when using the proposed method, the requirements for consumables are significantly reduced: chalk, crushed glass, quartz sand, a decrease in alcohol consumption is observed due to a reduction in possible losses (evaporation during processing of the filtrate, spraying during operation of the vacuum unit) and the need for filters in the vacuum unit is no longer necessary that leads to energy saving. In addition, conditions are created for a more productive distribution of working time. With careful filling of the cuvette, the extract does not require additional filtering, which also reduces the time spent on research.

Sources of information taken into account during the examination:

1. A.M. Grodzinsky, D.M. Grodzinsky. A quick reference to plant physiology. Kiev, 1973, Iz.: Naukova Dumka, pp 433-434 - prototype.

Claims (1)

A method for determining chlorophyll in buckwheat plants, including grinding the raw material and treating it with alcohol, followed by spectrometric measurement at two wavelengths of 665 and 649 nm and calculating the concentration of chlorophyll according to the formulas C _a = 13.7D ₆₆₅ -5.76D ₆₄₉ ; C _b = 25.80 D ₆₄₉ -7.60 D ₆₆₅ , where C _a and C _b are the concentrations of chlorophyll “a” and “b”, D ₆₆₅ and D ₆₄₉ are the measured extinction coefficients in mg / l at the corresponding wavelength, characterized in that the crushed plant raw materials are poured with alcohol in containers that are tightly closed, placed in a dark place, and after 24-48 hours the exposure is subjected to spectrometric measurement to calculate the concentration of chlorophyll.