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3 - Estimation of abscisic acid (ABA)

3 - Estimation of abscisic acid (ABA)

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CHAPTER 16  Growth regulators

Preparation of reagents:

• 1% Acetic acid


1. Take leaf sample (2–5 g) in a flask and pour so much volume of acetone

containing 1% acetic acid that the sample dips into it completely and incubate it

overnight at 4°C.

2. Filter the extract through a filter paper (Whatman No. 4).

3. The extraction should be repeated twice or thrice (ie, repeat the above steps two

to three times; each time use the residue on the filter paper for extraction).

4. Evaporate acetone from the total volume of extract using a rotary evaporator at

40–50°C till some residue is left on the bottom.

5. Preserve the residue in a cool and dark place.

Sample preparation:

1. Take out the flask with the residue (sample) as mentioned earlier.

2. To it add 2-mL distilled water having 1% acetic acid (v/v) and sonic ate


3. Transfer the content of the flask into sample vials.

4. Make the volume in each sample vial up to 5 mL with distilled water containing

1% acetic acid (v/v). Store the sample vials for assay using HPLC.

5. Generally, the experimental conditions, for the analysis of ABA using HPLC,

are maintained as follows:

6. Column: reverse phase C18 column having particle size equal to 5 mrn.

Detector: UV detector

Wavelength: 265 nm

Flow rate: 1.5 mL min−1

Calibration curve: using 10-ppm solution of ABA in 95% ethyl alcohol.


A: 1% acetic acid in distilled water (HPLC grade) v/v.

B: 1% acetic acid in methyl alcohol (HPLC grade) v/v.

Both the solvents are filtered through Millipore nylon filter (13 mM) with the help of

vacuum pump solvent filter kit.


1. Filter the standard solution of ABA through nylon filter (0.45-nm pore size) and

inject 10 mL of this solution into HPLC.

2. Record the peak which generally appears after 30–50 s.

3. Inject 10 mL of the sample into the HPLC and record the peak area.

Calculation: The ABA content in the sample may be calculated using the equation

given here:

in 10 µL solution (ng) ABA content (ng)

Peak area ( cm 2 ) of sample × Amount of ABA


Peak area ( cm 2 ) of standard solution × Fresh weigth of the leaf sample (g)

16.4 Estimation of ethylene


Ethylene, a ripening hormone, is present in very small quantity in plants. It is estimated using a gas chromatograph. One may find the details regarding the estimation

procedure in the instrument manual; however, for the benefit of students at postgraduate level, the basic of chromatography along with the ideal operating conditions and

sample collection method are given here. It may be mentioned that in plant physiological studies, ethylene is generally assayed during different stages of ripening of

climacteric fruits such as mango (Reporter, 1987).

Sample Collection

1. Put mango fruits (known weight) in a cylinder or jar.

2. Seal the mouth with a gasket.

3. Incubate for at least 2–4 h at room temperature.

4. Withdraw the gas sample (ethylene) using a hypodermic syringe for assay. A

known volume of gas should be assayed.

Ideal operating conditions for gas chromatograph

Carrier gas: hydrogen/nitrogen mixture

Flow rate: 20–30 mL min−1

Gas for detector: hydrogen and air

Column: Porapak-Q 80/100 mesh packed

Oven/column temperature: 60°C

Injector temperature: 110°C

Detector temperature: 85°C

Detector (to be used): flame ionization detector (FID)

Retention time for ethylene: 1.3 min

1. The carrier gas from a cylinder is passed through flow regulator to an injection

port, where it picks up the sample for analysis.

2. The carrier gas + sample mixture then passes through the column in a

thermostatic oven where the components of the mixture are separated.

3. The area of the peak depends upon the amount of substance present, the detector

efficiency, and the degree of amplification used.

4. If the latter factor is hold constant, the recorded peak area is a direct measure of

the amount of substance present in the sample.

5. Prior to assay of sample, the instrument must be calibrated with a known

volume of standard ethylene gas.

6. For calculating the amount of ethylene produced per gram of sample per unit

time, refer to “Nitrogene” estimation.


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17 Analytical techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

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Analytical techniques




Introduction: According to electromagnetic theory of light, light travels in the form

of waves described by three attributes – wavelength, frequency, and energy. The visible portion of electromagnetic spectrum extends from 360 to 900 nm and the ultraviolet (UV) from 200 to 380 nm. The spectrophotometer uses a tungsten lamp

for measurements in the visible region (360–900 nm) and a deuterium lamp for the

ultraviolet region (200–380 nm) of the spectrum. A monochromator consisting of a

grating or a prism or a combination of both is used to obtain a narrow band of wavelengths continuously selectable through the exit slit of the monochromator. The beam

of light transmitted by the sample is detected with the help of a suitable detector

either a phototube or a photomultiplier tube (for greater sensitivity) and the optical

density displayed on a suitable analogue or a digital read out.

Light that includes all the rays in the wavelength range of visible rays is called

white light. When white light is irradiated on some substance and the substance

­absorbs the blue light, it appears yellow, which is the (additive) complementary color of blue. If blue monochromatic light is irradiated on this substance, the light is

­absorbed and the substance appears black, indicating that no color exists (Table 17.1).

Principle: Spectrophotometer measures the light absorbed by a sample solution at

a given wavelength. The absorption of light by molecules is governed by ­Lambert–

Beer law. When light with the intensity of I0 is irradiated on a certain substance and

the light with the intensity of I has transmitted, the following relational formula is

established, where K stands for proportional constant.

At this time, I/I0 is called transmittance (T), (I/I0) × 100 is percent transmittance (%T) and (I/T) = log (I0/I) is called absorbance (Abs). The absorbance (­optical

­density) of a solution is directly proportional to the concentration of the solute (Beer’s

law) and optical path length (Lambert’s law) through the solution.

Objective: Quantitative estimation of chemical substances was measured through

ultraviolet visible (UV–VIS) spectrophotometer (Fig. 17.1). The spectropho-tometer

consists of three different modes, viz., photometric, spectrum, and kinetic modes.

Spectro photometer measures the optical density values of different chemical

­substances (proteins, carbohydrates, amino acids, chlorophylls, etc.) of known wavelength through photometric mode. Spectrum mode scans the chemical substances of

Phenotyping Crop Plants for Physiological and Biochemical Traits. http://dx.doi.org/10.1016/B978-0-12-804073-7.00017-X

Copyright © 2016 BSP Books Pvt. Ltd. Published by Elsevier Inc. All rights reserved.



CHAPTER 17  Analytical techniques

Table 17.1  Relation Between Wavelength, Color, and Its Complementary Color

Sr. No

Wavelength (nm)


Complementary Color























Greenish blue

Bluish green


Yellow green




Purplish red

Yellow green




Purplish red



Greenish blue

Bluish green


FIGURE 17.1  UV–VIS Spectrophotometer.

unknown wavelength (scanning nano size chemical substances), whereas the kinetic

mode of spectrophotometer measures the enzyme activity of a substance (antioxidant

enzymes, nitrate reductase, nitrogenase, etc.).


Chromatography is highly useful in research laboratories to separate, identify, and

characterize unknown compounds.

Principle: Separation of closely related compounds in a mixture through equilibrium

distribution of the components between two immiscible phases, viz., the stationery

phase and the mobile phase.

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