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Photosynthesis Part II & III

Our investigations entailed determining the absorption spectrum of leaf pigments, the absorption spectrum of a yellow dye, and the pigments in leaves. Using a spectrophotometer for the analysis of absorption spectra, we found that the plant pigments absorbed all but the green wavelengths. Our paper chromatography method for separating leaf pigments failed.

Introduction

The purpose of the second and third parts of the photosynthesis lab was to determine the pigments in the leaves of two trees and was to determine the absorption of the light spectra by leaf pigments and a dye. The lab procedure hand out uses the misnomer chlorophyll for leaf pigments. Leaf pigments are tested because no process removed the pigments other than chlorophyll from the leaf solution. The determination of the pigments contained in leaves was investigated by paper chromatography. The procedure separates the pigments by their attraction to the solvent which, by capillary action, moves up a special piece of chromatography paper. The absorption of light was determined by placing a cuvette of leaf pigments in a spectrophotometer and varying the wavelength dial.

Our group hypothesized many things in this multi-part lab. For the paper chromatography section of the lab, we made the conjecture that red leaves would have less green pigments than green leaves. Similarly, green leaves should have significantly less red pigments. Our second hypothesis involved the spectrum of chlorophyll. We stated that chlorophyll would absorb all but green light. Furthermore, we were handed a yellow dye solution, we were asked to make a hypothesis about its absorption spectrum. We conjectured that the dye would absorb all but yellow light. These hypothesis were made on the basis that we see certain colors which bounce off leaves. Thus, the rest of the colors present in white light must be absorbed or somehow dissipated.

Pre-Lab Weirdness

These questions were quoted from our lab handout. Most of the answers are quoted from the lab report.

"WHAT YOU HYPOTHESIS CORRECT OR NOT? EXPLAIN!" First of all, we cannot begin to answer this question because no significant data was collected. Secondly, our hypothesis relies on the qualitative interpretation of the relative intensities of each of the separated pigments. The intensities of these pigments will fade with time so immediate appraisal of the chromatography paper is required.

"HOW CAN THE MACHINE BECOME DAMAGED DURING THIS LAB?" The color filter wheel can be moved outside the normal range and cause the mechanical linkage to break.

"WERE YOUR VALUES WITHIN THE ESTABLISHED RANGES FOR EACH COLOR? IF NOT, EXPLAIN." Our values for the qualitative color test differed a bit from the biology text's spectrum. One must remember that this test was qualitative, and thus no numbers were involved. Our collected spectral data reflects the spectrum on page 281 of the Menlo AP Chemistry Text.

"WHAT IS THE PURPOSE OF THE BLANK IN THIS LAB? (BE SPECIFIC) WHAT MUST YOU BE CAREFUL TO DO REGARDING THE HANDLING OF THE CUVETTE? WHY MUST YOU READJUST WITH THE BLANK AT EACH WAVELENGTH, RATHER THAN JUST ONCE?" We zeroed the spectrophotometer with an acetone blank after every change in wavelength. The zeroing step will eliminate any errors due to irregularities in the spectrophotometer filter or the light bulb's idiosyncrasies.

Procedure

The lab involved noxious fumes for both the chromatography and the spectrum sections. A fume hood was used to prevent exposure to the chromatography solution. Care was taken with the acetone solutions during the spectrum section of the lab.

The spectrophotometer used relies on a lamp, a filter, the specimen chamber, and a CdS photoresistive component tied to a digital readout. The instrument can measure percent transmittance and absorbance of a sample at a certain wavelength of light. Care was taken to avoid going past the spectrum limits of the spectrophotometer.

The spectrum section involved recording the qualitative color changes of the spectrophotometer. We used an empty cuvette with a scrap of white paper and visually determined the "colors." The second part of this section was to measure the absorbance of chlorophyll and the yellow dye. We zeroed the spectrophotometer with an acetone blank after every change in wavelength. The zeroing step will eliminate any errors due to irregularities in the spectrophotometer filter or the light bulb's idiosyncrasies. We measured the absorbance of chlorophyll and yellow dye at 25nm intervals over the range of the spectrophotometer.

The pigment section of our lab involved rubbing leaves onto chromatography paper. The pigments should then separate as the chromatography solution inches its way up the paper. The pigments should separate by their attraction to the solution versus their attraction to the paper and by their size. The Rf values are the relative distances from the pigment front to the front of the chromatography solution . A bar graph could easily be generated for each of the pigment's Rf values. Our hypothesis would also require a qualitative determination of which pigment was darker relative to the other sample. We would expect the larger chlorophyll molecules to travel less than the smaller carotenoids.

Data


Wavelength     Leaf Pigments        Yellow Dye           
(nm)           (Absorption)         (Absorption)         

           405                 1.61                 1.31 

           425                 1.69                 1.37 

           450                 1.76                 1.42 

           475                 1.79                 0.89 

           500                 1.18                 0.12 

           525                 0.96                    0 

           550                 1.08                    0 

           575                 1.44                    0 

           600                 1.62                    0 

           625                 1.74                    0 

           650                 1.78                 0.01 

           675                 1.35                 0.01 

           700                  0.4                 0.01 




Wavelength     Color         
(nm)                         

440-470        Purple        

470-510        Blue          

510-570        Green         

570-600        Yellow        
               Orange        

600-660        Red           

660-700        Deep Red      



We made several observations about our cuvettes for the spectrum section of the lab. The leaf pigments cuvette was an opaque green while the yellow dye was transparent.

We found no appreciable data from our paper chromatography assay.

Results

[ Please See Attached Graphs ] The spectra absorbance graph has both the yellow dye and the chlorophyll on one scale. The Rf value graphs are not applicable.

Discussion

Our values for the qualitative color test differed a bit from the biology text's spectrum. One must remember that this test was qualitative, and thus no numbers were involved. Our collected spectral data reflects the spectrum on page 281 of the Menlo AP Chemistry Text.

The spectrophotometer used seems to have some variations and does not give 0.005 Absorbance precision. This can be noted in our Yellow dye sample which had very low readings. The readings on the order of 0.01 cannot be trusted because they are too small compared to the 0.005 Absorbance. Further errors were avoided by using the acetone blank which eliminated any variations because of the slightly varying opacity of the filter wheel.

The no data result from the paper chromatography section of the lab may have resulted from many causes. The whole afternoon class lacked any appreciable data which suggests some systematic error with the lab setup. Our group also experienced a delay between our leaf rubbing and actually doing the chromatography. This time lapse may have caused certain pigments to deteriorate. The solution can also be a source of this fiasco because the formulation of the solution was neither reviewed nor checked by us. It could have resulted in a solution is not soluble with certain pigments or a solution that destroys certain pigments.

Conclusion

Our hypothesis regarding the absorbance spectrum of leaf pigments was affirmed by the data collected. The green region (510-570 nm) on the absorbance graph has less absorbance. Thus the leaf pigments were absorbing all but the green section of the spectrum. The readings beyond 700nm are infrared and thus are not relevant.

Our hypothesis regarding the yellow dye is inconclusive. Although it does absorb the blue and violet regions of the spectrum (100-500 nm), the yellow does not absorbs any of the green, the orange or red areas of the spectrum. Perhaps the transparency of the sample should suggest a change in our hypothesis.

We do not have any data from the paper chromatography to conclude if our hypothesis regarding the relative concentrations of pigments in red and green leaves is correct or not.

Questions

  1. The question was answered above. This question is redundant as part of it was also answered in the Pre-Lab Weirdness section.
  2. No data was collected and we are unable to answer this question. This absence may be explained by one of several possible errors. We could have just not pressed hard enough to break up all the membranes in the leaf cells. The pigments could have significantly decayed because we had left the paper sitting around for a significant period of time. The solution could have been formulated incorrectly which would not move the pigments or destroy them.
  3. A spectrophotometer basically filters light from a white light source. The filter has many colors so that you can filter all but one wavelength of light. Then this filtered light tries to go through the sample. If any light does go through, it hits a photodetector which is hooked up to an electronic gizmo and spits out the number that we want.
  4. A = -log(T) where A is absorbance and T is transmittance written in decimal. This suggests that if we plotted transmittance, the graph would flip because T = e-A. Thus, in lay man's terms, everywhere where absorbance is zero, transmittance is one. Everywhere where absorbance is infinity, transmittance is zero.
  5. Our absorbance spectrum of the leaf samples seem to suggest that there are significant amounts of chlorophyll b. The "valley" is narrower for chlorophyll b and matches decently with our spectra absorbance. Carotenoids and chlorophyll a are probably present and should help smooth out the graph.

"WHAT FACTORS ARE INVOLVED IN THE SEPARATION OF THE PIGMENTS?" Factors such as the solubility of the pigment in the chromatography solution and the affinity of the pigment to the fibers of the paper affect how far the pigment travels up the paper. The size of the pigments also plays a role in how it can squeeze around the fibers of the paper.