1) This lab contains of two major parts. The first part consists of the separation, isolation, and characterization of the pigments in spinach using an analytical technique known as chromatography in which various components of a solution can be separated. The second activity in this lab is the measurement of the rate of photosynthesis using spectrophotometry to measure the color change of various photosynthetic solutions in which DPIP (2,6-dichlorophenol-indohenol) has been substituted for the NADP carrier ion that is normally used in photosynthesis. As the DPIP is reduced, the color change from blue to colorless is easily distinguishable using a spectrophotometer.
2) Chromatography is the generic term for an analytical technique that has long been used to separate and identify the different components of a solution. In the basic procedure, the solution is first dissolved in an appropriate solvent. Once the solution has been fully dissolved, a thin sample to chromatography paper is placed so that a very small portion of it is immersed the solvent. As the Solvent is absorbed into the paper through capillary action the solute particles are pulled up as well. Because the solvent is designed to have very small attractive forces with the paper, the larger, more strongly attracted solute particles move up the paper with a slower speed related to particle size, and attractive forces. Because the forces and size on any particular molecule are quite different from other species, the ratio of its distance traveled to the distance the solvent traveled is unique and as such the particular molecules in a solution can be easily identified. There are also many different chromatography including thin layer chromatography, high and low pressure gas chromatography and various others. Although the means of obtaining the data and the procedures are considerably different, they all share the same basic principles.
3) The chromatography paper employed in paper chromatography is a special paper designed to minimize the attractive forces with the solvent used in the mobile phase of the chromatography. The paper acts as a wick �sucking� up the solvent by capillary action. As the solvent particles pass along the paper they are slowed significantly and are seen as highly concentrated bands of color, each representing a particular solute. After the paper is removed from the solution the solvent is evaporated and the colored bands remain stationary on the paper where is can be analyzed. This is known as a chromatograph.
4) Because of its specific properties we expect to see the carotene pigment to travel furthest, and therefore be closest to the solvent front. Because the molecules are attracted to the chromatography paper much less than the chlorophyll a and b molecules, and because it is much more soluble in the solvent, it can be expected to travel much further that the other pigments in the leaves.
5) In the middle of the chromatograph we would expect to see one of the remaining two primary pigments, either chlorophyll a or b. Because the chlorophyll b 7th carbon contains a ketone unit as opposed to chlorophyll a�s non-polar methyl group I would expect it to show slightly more attractive forces to the paper. As such, I would expect chlorophyll a to travel farther along the chromatography paper.
6) By the reasoning of the above question I would expect the chlorophyll b molecules to be nearer the bottom of the chromatography paper.
7) Chlorophyll a is responsible for absorbing the majority of the light used by most plants in photosynthesis. As photons of violet and red light strike the pigment it converts the energy into potential energy in the form of excited electrons. These electrons then follow a series of steps as the energy of their excited states is used to produce ATP�s for the organism to use.
8) The other two major pigments have roles that are roughly equivalent to that of chlorophyll a, except they absorb light at different wavelengths. Chlorophyll b is responsible for absorbing more centralized blue and orange pigments while the carotene absorbs light from the middle of the visible light spectrum, or the green colors.
9) The formula for calculating the reference front of a pigment is Rf = (Distance traveled by the substance)/(Distance traveled by the solvent)
10) As the frequency of electromagnetic radiation increases so does the energy contained by each photon by the formula Energy = hf where h is Planks constant. And since the wavelength is inversely proportional to the frequency of a particle, that is f (1/L), short electromagnetic waves contain more energy than long ones.
11) As light photons are absorbed by the pigments of a particular leaf the energy contained within those photons is used to energize low energy electrons into their excited states. These electrons are then transferred from pigment to pigment as they make their way to the reaction center of a thylakoid membrane where it is transferred to a carrier molecule such as NADP. From there, the electron is brought to the electron transport chain where the energy stored in its excited state is used to power the ATP synthase enzyme and ATP�s are created for use within the plant cell.
12) In its neutral state, the DPIP molecule is a blue color. As the molecule is reduced within the chloroplasts of a particular plant cell the molecule loses it color and becomes colorless. As such, a blue solution would indicate that there was no loss of color, and no DPIP was reduced. If there are no carrier ions being produces than that is an indication that no photosynthesis is taking place.
13) As photosynthesis takes place and the carrier ions in the system are reduced it should lose color because of the DPIP molecules absence of color is its reduced state.
14) The device used to measure the transmittance of light by a particular substance is known as a spectrophotometer.
15) As the color fades from our sample containing the DPIP the amount of light that is absorbed by the color in it will go down. This drop in absorbance also means an increase in the amount of light that is transmitted through the particular sample. Because a loss of blue color indicates the reduction of DPIP, which in turn indicates photosynthesis is taking place, an increase in transmittance shows that photosynthesis is occurring.
16) As is the case with any piece of scientific data, without a standard to compare newly found information to that data is quite meaningless. The purpose of tube 1 is to provide that standard. A solution which is exactly the same, except for the lack of DPIP, as the others is used to compare the data to. This control is used to ensure that any color change cause by the photosynthesis, and not the reduction of DPIP is accounted for. Cuvette 2 is used to create a control in which no photosynthesis is occurring (this is controlled by the lack of light caused by the foil covering. Cuvette 4 is used as a third control. In this vial the chloroplasts have been deactivated by boiling as should undergo no photosynthesis. Cuvette 3 is the vial in which photosynthesis is allowed to continue without interruption. This vial will be the vial from which actual photosynthesis data is collected.
17) Because the cuvettes must be positioned so close to the lamp to ensure that the rate of photosynthesis is sufficient to be measured within one class period it is important to reduce the amount of heat that is contacting the cuvettes. If the temperature of the solutions becomes too great, important enzymes in the process of photosynthesis could become inactive as their protein structures are denatured. The flask of water is put between the light source and the vials to ensure that the temperature of the vials does not reach these dangerous levels.
18) The cuvettes must be handled in a way that will minimize the amount of oils and dirt that will come in contact with the surface. It is very important to clean the cuvettes with a non-abrasive material before being inserted into the to ensure that the light can pass through the vials unobstructed. If any light is deflected or absorbed by something on the surface of the glass it could have great effects on the absorbance of the system.
19) We must prevent light from entering one of the cuvettes so that we can have one standard vial in which no photosynthesis took place. This vial will provide the control needed to compare the absorbance of the photosynthesis positive cuvette to.
20) I would expect the cuvette with the unboiled chloroplasts to have a lighter color that that of the boiled chloroplast. As the chloroplasts are heated, various protein structures including those of a great number of enzymes are destroyed thought the process of denaturing. As such, there should be no or very little photosynthesis taking place in the vial containing the boiled chloroplast. Because there is no photosynthesis taking place, there are no carrier ions being reduced and DPIP should retain its blue color.