Plant Pigments and Photosynthesis Lab Report

Plant Pigments and Photosynthesis Lab Report Background: (Part A)Paper chromatography is a useful technique for separating and identifying pigments and other molecules from cell extracts that contain a complex mixture of molecules. As solvent moves up the paper, it carries along any substances dissolved in it. The more soluble, the further it travels and vice versa. Beta carotene is the most abundant carotene in plants and is carried along near the solvent front since it is very soluble and forms no hydrogen bonds with cellulose. Xanthophyll contains oxygen and is found further from the solvent front since it is less soluble in the solvent and is slowed down by hydrogen bonding to cellulose. Chlorophyll a is primary photosynthetic pigment in plants. Chlorophyll a, Chlorophyll b, and carotenoids capture light energy and transfer it to chlorophyll a at the reaction center. (Part B) Light is part of a continuum of radiation r adiation or energy waves. Shorter wavelengths of energy have greater amounts of energy. Wavelengths of light within the visible spectrum of light power photosynthesis. photosynthesis. Light is absorbed by leaf pigments, electrons within each photosystem are boosted to a higher energy level and this energy level is used to produce ATP and reduce NADP to NADPH. ATP and NADPH are then used to incorporate CO2 into organic molecules called carbon fixation. In place of the electron accepter, NADP, the compound DPIP will be substituted. It changes chloroplasts from blue to colorless. Purpose: (Part A) The lab groups were to separate pigments and calculate Rf values using plant pigment chromatography, describe a technique to determine the photosynthetic rate, compare photosynthetic rates at different light intensities using controlled experiments, as well as explain why rate of photosynthesis varies under different environmental conditions. (Part B) The lab groups were to use chloroplasts extracted from spinach leaves and and incubated with DPIP and use use the dye-reduction technique. technique. When the DPIP is reduced and becomes colorless, the resultant increase in light transmittance is measured over a period of time using a spectrophotometer. Hypothesis: (Part A) If pigments are separated, then RF values can be determined. (Part B) If we compare unboiled chloroplasts to boiled, then unboiled should have higher Ps rates since it is undamaged. Materials: (Part A) Various leaves without a thick cuticle ● Ruler ● Chromatography solvent ● ● Graduated cylinder Coin ● Pencil ● ● Tape ● Chromatography paper strips (Part B) Chloroplasts extracted from spinach leaves Incubated DPIP ● Spectrophotometer ● ● Light Procedure: ●

(Part A) ●

Obtain 30cm strip of chromatography paper, cut into V-shape

Titles you can't find anywhere else

Try Scribd FREE for 30 days to access over 125 million titles without ads or interruptions! Start Free Trial Cancel Anytime.



●

● ● ●

Measure 2cm vertically from bottom of each strip and draw a horizontal line across paper strip using a pencil Using a quarter extract pigments from leaf cells Place chromatography paper in graduated cylinder Place chromatography chamber in fume hood and allow chromatograms to develop (Part B)

Turn spectrophotometer to warm up the instrument and set wavelength to 605 nm Setup an incubation area Keep two beakers with chloroplasts on ice ● Label cuvettes 1-5 respectively ● ● Add 1ml phosphate buffer to each cuvette Bring spectrophotometer to zero ● Obtain unboiled chloroplast suspension suspension and transfer three drops to cuvette 2. Insert in ● spectrophotometer sample holder. Obtain unboiled chloroplast suspension suspension and transfer three drops to cuvette 3. Insert in ● spectrophotometer sample holder. Obtain boiled chloroplast suspension and transfer three drops to cuvette 4. Insert in ● spectrophotometer sample holder. Cover and mix contents of cuvette 5. Insert in spectrophotometer sample holder. ● Data & Observations: During this lab, several observations were recorded. Firstly, the spinach leaf was green, which in effect turn pigment on the paper green. Also, carotene, found in carrots, is an orange pigment. Besides this, the cuvettes with chloroplasts were blue and soon after turned clear after light hit the chloroplasts. Lastly, some chloroplasts had shown transmittance of chloroplasts solution when there should not have been any. ● ●

(Part A) Distance Moved by Pigment Band Band number Distance (mm) 1 22 2 34 3 42 4 44

Band Color Yellow-Green Bright Green Yellow-Orange Yellow

(Part B) Transmittance of Chloroplast Solution (%) Time(min) Unboiled/Dark Unboiled/Light 0min 41% 43% 5min 43% 55% 10min 44% 61% 15min 43 64% Analysis: (Part B) Transmittance through DPIP graph

Boiled/Light 51% 52% 53% 54%

Rf value .5 .773 .955 1

No Chloroplasts 57% 57% 56% 55%



lower readings since the chloroplasts settle at the bottom. Also, not covering the cuvettes would cause light to reach the chloroplasts and activate it. Another error may have been that the paper was too thin for solvent to travel across the paper. It may also have been too thick for the solvent to travel across a cross the paper. Faulty DPIP may also have caused an issue if it had not been working properly, giving poor data results. Lastly, the cuvette may not have been boiled completely causing transmittance in the chloroplast. Discussion: The hypothesis in this lab for part A was “If pigments are separated, then Rf values can be determined” was accepted. Band number 1 yielded an Rf value of .5 with a distance of 22mm. Band number 2 traveled 34 mm with an Rf value of .773. Band number 3 traveled 42 mm with an Rf value of . 955. Lastly, band number 4 had a distance of 44mm with an Rf value of 1. In part B the hypothesis was “If we compare unboiled chloroplasts to boiled, then unboiled should have higher Ps rates since it is undamaged” which was accepted, since Unboiled/Light had the greatest transmittance over the fifteen minute period, ending with 64%. Meanwhile, Boiled/Light yielded an average of about 53%--lower than the unboiled/light test. Thus, both hypotheses were accepted and proven correct. Discussion Questions: Part A: 1. The solubility, size of particles, and their attractiveness to the paper are all involved in the separation of pigments. 2. Hydrogen bonding affects how pigments travel up the chromatography paper based off of solvent solubility. 3. No, the different solubility of pigments would change Rf values, such as chlorophyll b is only soluble to fat solutions. The plant with the most accessory pigments was the spinach. Their role in photosynthesis is to absorb specific wavelengths of light which power photosynthesis. 4. The most polar pigment was the chlorophyll a/b and the least polar was the carotene pigment.

The Chlorophyll a and b pigments would be expected to travel the furthest across the chromatogram due to solvent solubility. 6. If the primary pigment color of the leaf was purple the results would be similar since retinal acts very similar to chlorophyll. The complex of retinal and membrane proteins is known as bacteriorhodopsin, bacteriorhodopsin, which generates electrons which establish a proton gradient that powers an ADP-ATP pump, generating ATP from sunlight without chlorophyll. (Part B) 1. The purpose of DPIP in the experiment was to accept electrons. 2. DPIP replaced NADP in this experiment. 3. The source of the electrons was the chloroplast. 5.



7.

In the dark cuvette, there was no light energy available, so no flow of electrons and no photolysis of water, while in the light cuvette these processes were allowed to continue.

Plant Pigments and Photosynthesis Lab Report

Recommend Documents