Chlorophyll by Spectrophotometer

Title: Procedure for the determination of photosynthetic pigments (chlorophyll and Carotenoids)

 Reference(s):

Chlorophylls are estimated according to the procedure of Arnon (1949)

Carotenoids were determined following the method of Davis (1976)

Citation(s):

Arnon, D. I. 1994. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol., 24: 1-15.

Davis, B. H. 1976. Carotenoids, In: Chemistry and Biochemistry of Plant Pigments. T. W. Goodwin (ed.), (2nd Ed.) Academic Press Inc., London. Pp. 38-165.

Reagents: Acetone (80%)

Equipment: Cuvettes (one or two), pesto-mortar, pipette, spectrophotometer (double beam)

Principal: A spectrophotometer is employed to measure the amount of light absorbed by a sample. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector. The beam of light consists of a stream of photons. When a photon encounters an analyte molecule (cholorophyll), there is a chance the analyte will absorb the photon. This absorption reduces the number of photons in the beam of light, thereby reducing the intensity of the light beam that is received and measured at the detector in terms of OD.

 

Absorption maxima of chlorophylls:

In 90% acetone-water extract the peak absorption wavelengths can be read by spectrophotometer at:

  1. Chlorophyll a are 430 nm and 664 nm
  2. Chlorophyll b are 460 nm and 647 nm
  3. Chlorophyll c1 are 442 nm and 630 nm
  4. Chlorophyll c2 are 444 nm and 630 nm
  5. Chlorophyll d are 401 nm, 455 nm and 696 nm

Procedures:

The basic steps of chlorophyll measurements are

  1. Extraction
  2. Estimation
  3. Calculations

Step 1: Chlorophyll extraction:

Method 1:

  1. Take 0.1 g of fresh leaf and add 10 ml of 80% acetone
  2. Extract the chlorophyll by thoroughly grinding it in a pesto-mortar
  3. Filter the sample or centrifuge it at 10000 g
  4. Read the absorbance for photosynthetic pigments as soon as possible or chlorophyll starts disintegration

Method 2 (Less proffered)

  1. Weight 0.1 g of fresh leaf
  2. Finally chop it with a scissor
  3. Put it in a plastic bottle containing 10 ml of 80% acetone
  4. Place bottles in dark for 24 h
  5. Filter the sample or centrifuge it at 10000 g
  6. Read the absorbance (OD)

Step 2: Estimation

Warm-up: Plug in, turn on and allow for 30 minutes to  warm up

Adjust Zero: With no cuvette in the chamber the machine may be adjusted to read the absorbance without sample.

Select Wavelength: Select the desired wavelength of light at which absorbance will be determined

Blank adjust: Fill the B (blank) cuvette with the solvent used to dissolve specimen (mostly 80% acetone or distilled water ).  Polish to clean, Insert into the cuvette chamber. Close chamber cover and read blank.

Read specimen: Pour the sample into the S (specimen) cuvette, polish and insert into the chamber, and read specimen.

NOTE: This information pertains to single beam spectrophotometers. In most of the modern double beam digital spectrophotometers, however, you can set more than one wavelengths and insert blank and smaple cuvettes simultaneously.

Step 3: Calculation of photosynthetic pigments

Chlorophylls and carotenoids are estimated according to the following formula:

Chl. a (mg ml-1) = [12.7 (OD 663) – 2.69 (OD645)] × [V/1000] × W

Chl. b (mg ml-1) = [22.9 (OD 645) – 4.68 (OD663)] × [V/1000] × W

Carotenoids (g ml-1) = [Acar / Em100%] × 100

Acar = [(OD 480) + 0.114 (OD 663)-0.638 (OD645)]

Where,

V is the volume of the sample

W is the weight of fresh tissue taken for extraction

Em100% = 2500

Precautions:

During Extraction:

  1. After extraction, try to analyze samples as soon as possible. Extracted chlorophyll is sensitive to high light intensity. Unnecessary delay may cause the disintegration and reduced quantity of chlorophyll

Cuvettes:

  1. Should be handled with care so that they do not get scratched
  2. Should be stored separate from standard test tubes.
  3. Try not to touch them except at the top of the tube to prevent finger smudges which alter the reading
  4. A wipe should be available to polish them before insertion into the cuvette chamber

Instrument:

  1. It is very necessary to adjust zero, run a blank and properly adjust wavelengths

Estimation:

  1. Since the calculations are based on equations, the final concentration is sensitive to the fluctuations in (i) fresh wt taken (ii) accuracy in measuring the solvent (acetone)

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