If you plot absorbance versus concentration, the resulting graph yields a straight line. This is called an absorptions spectrum which illustrates the intensity of light absorbed to its relative wavelength. (Karp, 2010) The equation for the straight line can be used to determine the concentration of an unknown solution. (Karp, 2010) This is how the unknown concentration fast green is found in experiment number one. (Karp, 2010)
Materials and Methods
The protocol and materials used for the experiment were completed as written in the Department of Biology of the University of Waterloo Fall term 2010 Biology 130L Lab Manual, pages 34 to 37. No changes were made to the procedure.
Results
Dilution Sample Calculations
C1V1 = C2V2
C2 = (C1V1) / V2
C2 = (0.015mg/mL)(10mL)/(20mL)
C2 = 7.5 × 10-3
Table 1) Absorption Values of Stock Fast Green Solution (0.015 mg/mL) for Wavelengths 460 – 700 nm
Table 1 shows the results of the testing of absorption of the 0.015 mg/mL stock fast green solution using the spectrophotometer. The maximum absorbance or peak is observed at a wavelength of 625 nm.
Table 2) The Absorption Values of each Concentration of Fast Green at Wavelength 625nm
Table 2 shows the results of the testing absorptions of the different known concentrations of fast green solution and of the unknown # 207. The absorbances are all measured at the wavelength of 625nm.
Figure 1) Concentration Curve illustrating the Absorbance at Wavelength 625 nm
Figure 1 illustrates the results for the concentration curve of test tubes one through five of this experiment at a wavelength of 625 nm. This is done so the concentration of the unknown # 207 can be calculated. The equation of the line of best fit is y = 0.1694x – 0.1561. By analysing the graph the concentration of the unknown may be about 1.125 × 10-2mg/mL.
Figure 2) Drawing of Chromatogram of Chloroplast Pigments from Homogenized Spinach
The chromatogram illustrates the four chloroplast pigments which where separated from a chloroplast extract. The solvent, 90 petroleum ether: 10 acetone, dissolved the extract and allowed the pigments to migrate along the matrix. Carotene moved up the highest and it is the colour orange. Next is the yellow Xanthophylls, then the green chlorophyll A and finally the olive green chlorophyll B.
Table 3) Absorption Spectrum of Chlorophyll A and B
Table 3 shows the absorptions for chlorophyll A and B at different wavelengths. It is used to find the two peeks for each pigment. For chlorophyll A, the peaks are at wavelengths 430 nm and 665nm. For chlorophyll B, the peaks are at the wavelengths 455 nm and 640 nm.
Figure 3) Absorption Spectrums for Chlorophyll A and B
Figure 3 shows the peaks of both chlorophyll A and B. For chlorophyll A, the peaks are at wavelengths 430 nm and 665nm. For chlorophyll B, the peaks are at the wavelengths 455 nm and 640 nm.
Discussion
In experiment 1, a spectrophotometer took absorbance at specific wavelengths to make a graph illustrating the absorption of fast green solution. From this information, the maximum absorption was found and each of the remaining test tubes were measured at this wavelength to find their absorbencies. From these absorbencies, concentrations were calculated these were used to make a concentration curve. This concentration curve illustrated Beer’s Law which states that as concentration increases so does the absorption. (Vogelman and Evans, 2002)
However an error such as not properly handling the cuvettes or a reading mistake causes the graph to not be straight and linear. The curvature of Figure 1 does not disprove Beer’s Law but instead it shows that there is a source of error in the experiment. Because of this error the unknown concentration cannot be properly calculated. In other words, concentration and absorption are proportional. The absorbance of the unknown concentration can be used to find the concentration by drawing a line from the unknown absorbance and y axis to the point on the graph.
In experiment 2, chromatography paper was used to isolate the various pigments of chlorophyll of which include chlorophyll A, chlorophyll B, carotene and xanthophylls. (Karp, 2010) This resulted in the paper having many different colours and layers. These layers were very specific carotene (orange) at the top, xanthophylls below (yellow), and following this was chlorophyll A and B. (Karp, 2010) The solvent, 90 petroleum ether : 10 acetone, allowed the pigments to migrate up the matrix, chromatography paper. These pigments are compounds that appear coloured because they absorb only certain wavelength of light in the visible spectrum. Leaves on most plants are green because they contain chlorophyll. (Karp, 2010) Chlorophyll absorbs the red and blue light of the spectrum which leaves the colour green to be reflected to our eyes. (Karp, 2010) As seen in Figure 3, the chlorophyll A peaks at wavelengths 430 nm and 665nm and chlorophyll B peaks at the wavelengths 455 nm and 640 nm. Visible light ranges from 390nm to 760nm. (Petrucci, 2010) The colours which correspond with the peak wavelengths are blue which is between 450nm to 500nm and red which is between 650 nm to 700nm. (Petrucci, 2010) The chlorophyll absorbs the red and blue visible light photons which is used in photosynthesis. (Karp, 2010) Because the chlorophyll does not absorb the green light it is reflected. Since the human eye is able to detect wavelengths of light between 400 nm and 750nm, it is able to pick up the green wavelengths which have a wavelength of 500nm to 550nm. (Karp, 2010)
In conclusion, this lab experiment helped be become familiarized with spectroscopy and learn measure the concentration of an unknown solution and isolate the individual pigments of a chloroplast extract to measure the light absorption of chlorophyll A and B.
References
Department of Biology 2010 Introductory Cell Biology Laboratory Manual. University of Waterloo, Waterloo. pp. 32 – 37.
Jones, A. M., Reed, R., & Weyers, J. D. (2007). Spectroscopic techiques. Practical skills in biology (4th ed., pp. 366-371). Harlow, England: Prentice Hall.
Karp, G. (2010). Photosynthesis and the Chloroplast. Cell and molecular biology: concepts and experiments (6th ed., pp. 208 - 212). Hoboken, NJ: John Wiley.
Petrucci, R. H. (2010). Electrons in Atoms. General chemistry: principles and modern applications (10th ed., pp. 298 - 306). Toronto: Pearson Prentice Hall.
Vogelmann., & Evans. (2002). Profiles of light absorption and chlorophyll within spinach leaves from chlorophyll fluorescence. Plant, Cell & Environment, 25(10), 1313-1323. Retrieved November 1, 2010, from the Scholars Portal database.