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BIO 120 S-5L
EXERCISE 6. The Hill Reaction
Experiment Rationales
During the preparation of the experiment, solutions and glass wares were
pre-cooled to maximize the rates of reactions as centrifuge rotors and the blender
generate heat which denature enzymes involved. A hypertonic solution, 0.35M NaCl,
was mixed when grinding the leaves and suspending the chloroplasts to cause
plasmolysis, wherein the cells shrivel and become easier to lyze or break
After centrifugation, Pellet 1, which contained the heavier organelles and lipid
fragments, was discarded. Centrifugation speed was only at 1400 x g for 15 minutes
as chloroplasts are relatively lightweight and will sediment at this RCF.
During spectrophotometric measurements, the blank contained chloroplasts
to eliminate absorbance of chloroplasts in the mixture, as only DPIP absorbance is of
interest. The chloroplast suspension was diluted to 0.05 mg chl/mL to control the
levels of DPIP reduction. If the suspension is too concentrated, all DPIP will be
consumed too quickly.
Two different blanks were used as heated and unheated chloroplasts absorb
light differently. The wavelength used when determining the amount of chlorophyll
per mL of the extract was 652 nm because chlorophyll absorbs red light maximally.
Red light has a wavelength of 652 nm. All the other mixtures that contained DPIP
maximally absorb light at 605 nm, the wavelength of orange light.
Effect of Light on Hill Reaction and the Role of DPIP
In the presence of light, a process known as photo-phosphorylation occurs
wherein ATP is synthesized from ADP and Pi. The requirement of light entails that
photosynthesis is part of a photoinduced electron transport system which results to
the production of chemical energy to be supplied to the light-independent or dark
reactions in which CO2 and H2O are converted to carbohydrates.
2,6-dichlorophenolindophenol (DPIP), when added to the chloroplast solution,
substitutes NADP+ as the final electron acceptor in the photosynthetic electron
transport chain as it has a higher affinity for electrons (ie, higher reduction
DPIP is initially blue in its oxidized form and turns colorless in its reduced
form. During this reaction the nitrogen atom joining the two benzyl groups of the
compound is reduced and changes its double bond to a single bond, forcing several
carbon bonds in the entire left benzyl ring to change confirmation. This makes the

molecule reflect light differently and ultimately absorbs and reflects a different
range of wavelength, accounting for the change in color of the solution.

Figure 6.1. Spectrophotometric readings of three solutions consisting of heated

and unheated chloroplasts, phosphate buffer, NaCl, and DPIP
In the results of the experiment, the control setup had absorbance readings
that constantly fluctuated (although no extreme deviation is noted) as the control
contained heated chloroplasts. At high temperatures, enzymes in the chloroplasts
are mostly deactivated; hence the control exhibited random spectrophotometric
readings in the absence of photosynthetic activity.
The absorbance trend of the light setup is decreasing and eventually flat
lining. The extreme decrease in absorbance is attributed to the loss in color of the
solution caused by the light-dependent reaction reducing the DPIP dye indicator.
The flat lining of the graph indicates that all DPIP molecules have been used up by
the photosynthetic electron transport chain.
Lastly, the dark setup showed no increase or decrease in absorbance
readings and stayed constantly near 0. There is no change in the bluish color of the
solution. This is due to the absence of light energy supposedly powering the
transfer of electrons across the electron transport chain to finally reduce the DPIP
dye. This result confirms the requirement of light in the photosynthetic reactions
within the chloroplast.
Increasing absorbances may be attributed to impurities in the prepared
solution leading to the oxidation of DPIP or interference during measuring.
Other methods may be employed in the measurement of photosynthetic
activity. The manometric technique (which is based on direct measurement of the
pressure of CO2 or O2 in an isolated chamber with photosynthetic organisms), or
the electrochemical sensor method (which is based on the use of O2 and pH
electrochemical electrodes to measure the O2, CO2 or pH aqueous concentrations
of the analyzed sample) may be implied but requires more sophisticated

Giebek, P. E. (n. d.) Extraction of Chloroplasts from Plant Tissue and Their Use in
Demonstrating the Hill Reaction. Virginia Commonwealth University.
Richmond, Virginia, USA.
OCR (Oxford, Cambridge and RSA) pamphlet. 2014. The Hill Reaction Instructions
and answers for teachers. Retrieved on March 18, 2015 from