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3 - Estimation of abscisic acid (ABA)
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.
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
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
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).
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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17.1 ULTRAVIOLET VISIBLE (UV–VIS)
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
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CHAPTER 17 Analytical techniques
Table 17.1 Relation Between Wavelength, Color, and Its Complementary Color
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.).
17.2 THIN LAYER CHROMATOGRAPHY (TLC)
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.