Investigation of Photosynthetic Pigments in Spinach
Michael Yacoub BIOL&211 Lori Hayes
Purpose: To demonstrate the existence of the pigments of photosynthesis in spinach by separating them using chromatography, then measure them using spectrometry. There are multiple pigments in photosynthesis, the varying pigments allow the plant to absorb light at various wavelengths which allows the plant to derive energy from photons from most, if not all, light available in that region it grows in.
In this lab there were a variety of pigments that were looked at, they included, chlorophylls, carotenes, nd xanthophylls. Chlorophylls are any of the few green pigments associated with certain bacterial membranes that are responsible for trapping light for energy in photosynthesis. Carotenes can be red, orange, or yellow lipid pigments and is often found as an accessory pigment in photosynthesis. Xanthophylls are yellow or orange pigment also usually found as an accessory pigment in photosynthesis.
Although, these are not quite clear in the drawing shown below because they were hard to find in the provided samples, one can find these various pigments are found within the hloroplasts and more specifically within the thylakoid membrane within photosystems one and two. When testing to see what wavelengths are involved in the process of photosynthesis an action spectra was created; this is a graph that graphs the biological processes versus light wavelengths. An absorption spectrum however, is a graph of light absorption versus wavelength of light and allows one to see how much light is absorbed at each wavelength.
This can also help show which pigments are present in the plant. In this lab an absorption spectra was created in order to elp identify the varying pigments present in the spinach leaf. Spectrophotometry at a specified wavelength. This experiment was testing the hypothesis that the wavelength shown through the pigment in solution will affect the amount of light absorbed by the solution. In specific, it was predicted that the darker the solution the greater the absorbance.
Furthermore, various pigments will have higher absorbance values at specific wavelengths than other pigments at other wavelengths. There are two independent variables; the wavelengths of light shown through the pigments in olution, a quantitative variable, and the pigments in solution, a qualitative variable. There is only one dependent variable however and it is the absorbency measured, also a quantitative variable.
Experiment: The procedure in the Biology 211: Majors Cellular Laboratory Manual was used as reference in the experiment.
The experiment began with obtaining a piece of chromatography paper cut with 12 cm x 14 cm dimensions. Then a line 1. 5 cm from the bottom was made by rolling a penny over a spinach leaf, producing a 14 cm long green spinach line. Then, the chromatography paper was rolled into a cylinder and stapled to hold together; the line made by the spinach leaf was on the outside. Meanwhile, 20 mL of separation solvent was placed in a 600 mL beaker and the cylindrical chromatography paper was placed in it without having the solvent touch the green line and covered with plastic wrap.
This set up was left alone to sit, vented by lifting up a corner of the plastic wrap, resealed and allowed to sit until the solvent line was approximately 1 cm from the end of the paper. Once the solvent line was bout 1 cm from the end of the paper it was removed from the solvent and allowed to dry. Then each section of pigment was cut as without including any other pigment. The instructor pooled all of the classes’ pigments and placed them in 80% acetone in separate beakers and sealed until all the color was eluted from the paper.
One pigment in solution was given to individual lab groups and one group was given a solution that included all the pigments (pigment 5). Each group used spectrophotometry to measure the absorbance of each pigment starting with a avelength of 400 nm all the way to a wavelength of 710 nm with 10 nm increments; the 80% acetone was used as a blank and the machine was blanked every time the wavelength was changed. The absorbencies were recorded, the data was pooled and absorption spectrum was created. Our group was analyzing pigment E.
Figure 1 and Table 2 provide the collected chromatography data, the qualitative data was collected here. Table 1, Figure 2, and Figure 3 provide the spectrometry data. The quantitative data was collected here.
Shows conclusive evidence of peaks for pigment absorbance. Also the absorbance from the spinach leaf was included. Chromatography of Spinach Pigment # Pigment Color Distance Moved (mm) Rf Value Dark Green 11 0. 169 2 Light Green 28 0. 431 3 Light Yellow 0 0. 769 4 Dark Yellow 65 1. 00
Table: Shows different color of pigments as well as relative polarities in relation to the separation solvent. Figure : Shows the absorbance of Just the spinach leaf. Provides a comparison to pigments.
Figure: Shows the relative peaks of absorbance for each pigment given the varying wavelength.
Discussion: The hypothesis was proven to be correct, it was clear that different pigments were in the spinach leaf and each of those pigments was absorbing more light at specific wavelengths while having very low to no absorbance at other wavelengths- this can e seen in Figure 3, as well as Table 1. This is what helps the plant make use of as much light as possible in photosynthesis and not let anything go to waste. Pigments A, B, and C have two peaks while pigments D and E have only one peak in about the exact same spot.
Something one can notice about the absorbance of the spinach leaf is that there is a range of wavelengths where there is very low absorbance. This definitely supported the proposed hypothesis because different pigments did have extremes, clearly showing that different pigments were better suited for varying wavelengths. The absorbances were so low at points because the pigments, Chlorophyll A, Chlorophyll B, Xanthophyll, and Carotene absorb the most light at different optimal wavelengths, so the absorbance would be low at times because it at a wavelength where the pigment would absorb the most light.
Furthermore, this supports the hypothesis more simply because, in fact, the wavelength did affect the absorbance. Lastly, the darker solutions in fact did have higher absorbency peaks. This makes sense because Figure 2 models the spectra of Pigment A,B, and C in Figure 3. These pigments were the darker color which means they were most bundant in the spinach leaf. Therefore since these graphs model each other it would conclude that these were the major pigments.
This also goes to define the concentrations of the pigments found in spinach. Pigment B since it closely modeled the spectra for Just spinach found in Figure 2, must be the major pigment component in spinach due to the fact that spinach is a combination of all the pigments that were isolated. Error that could be found in this lab probably is from misusage of the Spec-20. Mistakes such as not completely “zeroing” the machine occurred; this could esult in errors in the absorbance data. This could be fixed by getting better lab equipment.
References: Biology 211 Majors Cellular Laboratory
Manual Self Assessment:
1. I chose to use scatter-line plots. I chose this because I felt like it easily showed the peaks that I kept talking about. Also it showed the relation between the spinach and pigment absorption spectra well.
2. They are excellent. They have titles, the axes are labeled, and that have citations.
3. Excellent. I feel like I learned all the objectives for the lab, and I was able to effectively display the information.