Question

If you were provided with the leaves of a certain plant and asked to determine the absorption spectrum of the leaf pigments, how would you determine the same? Explain.

Answer 1

Absorption Spectrum - The curve representing the light absorbed at each wavelength by pigment is called absorption spectrum.

Procedure-

Sample preparation-

• 1. Prepare an extract of chlorophyll by weighing 0.3 grams of your leaf
  sample. Tear the leaf section into small pieces and place the tissue in a mortar
  for grinding.
  2. Add 10 mL of ethanol to the sample and grind with the pestle until only tiny
  specks of leaf tissue remain. Add an additional 20 mL of ethanol to the
  mortar.
  3. Filter this liquid into a beaker through a funnel lined with a coffee filter.
  4. Dispose of the filter in the designated trash bin.

Calibration- The extract must fall into a specified range to provide a reading with the spectrophotometer. A sample that is too concentrated will not provide a valid measurement. To assure the sample falls within a readable concentration range, follow the steps provided.

• 1. Set the spectrophotometer wavelength to 640 nm.
  2. Push the A/T/C button to select % transmittance. The digital readout will show
  %T.
  3. Label one of the 3 mL syringes ethanol. Label the second syringe leaf extract.
  4. Dispense 3 mL of ethanol into a clean glass cuvette. (Wipe off finger prints
  with a Kimwipe.) Important: This cuvette is a “blank” that will be used
  throughout this lab to calibrate the spectrophometer.
  5. Insert the blank in the cell holder and close the door.
  6. Press the 0 ABS/100%T key to set the blank to 100% transmittance.
  7. The blank provides a standard for reading. Remove the blank and proceed
  with testing the extract by following the same procedure.
  8. Dispense 3 mL of the extract into a clean glass cuvette. (Wipe off all
  fingerprints with a Kimwipe.)
  9. Insert the cuvette in the cell holder and close the door.
  10. A reading of 30%-40% transmittance is best. If %T is greater than 40%, you
  may skip the dilution step. If it shows less than 30% transmittance, dilution of
  the sample is required.
  11. To dilute the filtered extract, add 0.5 mL of ethanol (using the clean ethanol
  syringe). Tightly hold a finger over the cuvette and invert to mix. Re-test the percent transmittance. Continue with 0.5 mL increments of ethanol until the
  reading is greater than 30% transmittance.

Testing plant extract -

• 1.note the readings range from 400 nm – 700 nm (with increments of 20nm). This is the range for testing.
  2. Set the wavelength on the spectrophotometer to 400 nm.
  3. Set the mode to “absorbance” by pressing the A/T/C button until an A
  appears at the right of the digital display.
  4. Insert the blank (clean with a Kimwipe) in the cell holder and close the door.
  5. Press the 0 ABS/100%T key to set the blank to 0 absorbance.
  6. Remove the blank and insert the clean cuvette containing the chlorophyll
  extract.
  7. Record the digital readout for the absorbance in the data table.
  8. Continue to increase the wavelength by 20 nm each time and repeat steps
  4-7. Remember to use the blank for each new wavelength setting!!
  9. Graph the data for absorption spectrum.

Hope it is clear.