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In this laboratory you will separate plant pigments using paper chromatography. You will also measure the rate of photosynthesis in isolated chloroplasts. The measurement technique involves the reduction of the dye, DPIP. The transfer of electrons during the light-dependent reactions of photosynthesis reduces DPIP, changing it from blue to colorless. (DPIP = 2,6-dichlorophenol-indophenol; Sigma Chem Co. at, Cat. No. D1878, approx $20 for 5 gm).

Before doing this laboratory you should understand:

After doing this laboratory you should be able to:

Lab-time will be subdivided into:

  1. A microscopic view of isolated chloroplasts.

  2. the measurement of photosynthesis using the DPIP-reduction technique.

  3. a branch of colorful biophysics to show you the wonders and potentials of looking a absorption spectra.

  4. a demonstration of paper chromatography.

(It is good to begin an afternoon lab with this part, because if started later, you will lose the brightness of the sun that you will need.)

0.1M Phosphate buffer: 174 g K2HP04 (dibasic) brought to 1 liter with distilled H20 and 136 g KH2P04 (monobasic) brought to 1 liter with distilled H20 (monobasic has a lower pH). Mix some monobasic with dibasic until the pH is 6.5 (try 685 mL of monobasic into 315 mL of dibasic). Since this solution is 1 M, 0.1 liter of the solution must be diluted with O.9 liter distilled H20 to prepare a 0.1 M solution.

Chloroplast suspensions: To prepare and prime the chloroplasts, incubate fresh spinach leaves under a light for a few hours. Do not allow the leaves to become hot. Pour 0.5-M cold sucrose into a blender so that it just covers the blender blades. This is probably 100 mL or 200 mL of solution. Pack fresh spinach leaves into the blender to a level one inch above the blades. Set up a beaker in ice with 2 layers of cheesecloth folded over a funnel. Blend spinach (about three short bursts - errrrrrr; errrrrr; errrrrrr!). Squeeze through cheesecloth (a cotton sock is better!) into a large funnel leading into a large beaker that is placed in an ice bucket.

  1. Before you go any further, look at the chloroplasts under a microscope; draw what you see. (Use a "wet mount:" Place a drop of the chloroplast suspension on a slide, use a cover-slip, and then start looking using the lowest magnification first. Obviously, look for green things!)

  2. DPIP Reactivity

    1. Obtain four test tubes, and place all four in a test tube rack. To each tube, add 6 mL of distilled H20, 2 mL of phosphate buffer, and 2 mL of DPIP to all four tubes.
    2. To tube #4 add an amount of cetavlon, which the instructor will tell you. (Cetavlon is a cationic detergent found in shampoos: NNNN-hexadecyl-trimethyl-ammonium bromide.)
    3. THEN-, LASTLY, add 4 drops of freshly swirled chloroplast suspension to tubes 2, 3 and 4. Immediately place tube 2 in a dark cabinet. The others immediately go out to bask in the sun (or sit in a greenhouse if it is raining. In summary, here is your tube set-up:

      Tube #Name
      1DPIP Control (Light)
      2Negative Control (Dark)
      3Positive Control (Light)
      4Cetavlon (Light)

    4. At timed intervals, measure the percent transmission of the tubes at 605 nm. Please don't forget to do a zero reading!

      The results should resemble those shown the accompanying graph.
      diamond = unboiled/dark
      square = unboiled/light
      triangle = boiled/light
      X = no chloroplasts

    5. ALTERNATIVE: For sake of shortening the time spent here, a qualitative technique may be used:

      Observe each tube and record the time needed for the tube to lose its blue color.

  3. Question: Would you expect chloroplasts to migrate through a semi-permeable (dialysis) membrane? Explain you answer.

  4. Questions: Would you expect chloroplasts to diffuse and migrate up a paper chromatogram? Explain your answer in light of the fact that your chromatography showed that the colored bands did move up the paper.


It was in the early 1940's that paper chromatography was invented by an observant person who saw application in how inks made rainbox hues when they "ran" on rain spattered paper. If you do not understand the principles behind "affinity" chromatography, you might find it helpful to look at an analogy.


Place a leaf over a piece of chromatography paper and roll the knerled edge of a coin over the leaf (using a ruler as a guide) so that the pigments of the leaf are driven into the chromatography paper 1.5 cm from the bottom. This will produce a straight line of pigment that can be chromatographed in a system such as:

The solvent of 1 part acetone and 6 to 9 parts of petroleum ether (or cigarette lighter fluid or naphtha or mineral spirits) should be used in a fume hood or outside (unless you want to get a headache or have the excitement of an exploded lab).

Discuss the pigments: what colors (wavelengths) do they absorb? Why are there more than one? (HINT) Also see THIS about why you usually don't see any red pigments on your chromatograms.


For an attached handout, see the description of how to use a small spectroscope for observing absorption spectra, which, in turn, give you a window onto how leaves can collect enough solar energy to rip electrons off water molecules to yield the reductive power needed to reduce carbon dioxide and make carbohydrates, as well as to make oxygen gas.

If you do not have access to a spectroscope, then you will have the opportunity for learning the same information PLUS gaining skill at using a spectrophotometer. The most common type of such machine today is called the Spec-20. There are four types of machines that are used to measure the amount of light that is either passed through or reflected from a test tube that holds a sample.

  • Colorimeter: This machine selects a wavelength of light by interposing a colored glass filter between a white light source and the sample.
  • Spectrophotometer: There are two types of these machines depending on how the desired wavelength is chosen. In both, a full spectrum (rainbow) of colors is made and then only a narrow band of those is chosen by interposing a slit between the rainbow and the sample tube. Rainbows can be made by either passing the white light through a prism or by reflecting the white light off of a diffraction grating. Spec-20's use the latter method.
  • Nephlometer: While the above machines measure the amount of light transmitted straight through the sample, a nephlometer measures the brightness at right angles. Nephlometers thus are preferred for observing fluorescence, and for measuring light that is reflected out of a cloudy suspension (nephlos = cloud).

Steps to preparing your sample for the Spec-20:

  1. Turn the Spec-20 "on" a few minutes before you will need to use it. Turn the left hand dial until it "clicks."

  2. The "BLANK" - Fill one cuvette half full with the sucrose solution that is used for isolating the chloroplasts from the leaves.

  3. The "EXPERIMENTAL" - Half-fill a tube with the chloroplast suspension.

  4. Wipe both tubes clean of spills and fingerprints. Hereonafter, hold the tubes by their top halves only.

  5. Without putting either tube in the Spec-20, and keeping the little hatch CLOSED, turn the left dial until the needle is exactly at the left end of the scales.

  6. Set the wavelength at 300 nm (deep blue)

  7. Now insert the BLANK and close the hatch. Adjust the right hand dial until the needle is exactly at the right end of the scales (100% or 0 absorption).

  8. Exchange the BLANK for the EXPTL tube. Read the LOWER scale. IF the reading is greater than 1.0, then dilute the sample four-fold (dump out 3/4 of the sample and add more sucrose solution up to halfway. Do this step again, and again until the reading gets within range of the machine (i.e.: 1.0 or less).

  9. Record your reading on a piece of graph paper.

  10. Here comes a hard part! Increase the wavelength from 300 to 340. AND, with the BLANK in the hatch, readjust the right hand dial so that the needle goes back to 0 absorption. If you do not recalibrate the machine after a wavelength change, you might just as well go home and take a zero for the lab. Now insert the EXPTL and record its reading.

  11. Continue increasing the wavelength, recalibrating the right hand side, recording the EXPTL and graphing it as you go UNTIL you get to about 750 nm (note that above 600 nm, you have to shift the lever at the lower left of the machine).

Question: Which color light has the more energy per photon - red or blue?

Question: Would you expect the auxilliary pigments to be nearer the blue end of the spectrum than the primary acceptor, or closer to the red end? (Hint: this is a tough one! Why not take a peek at a link called "photonics.")