Spectrophotometry and Thin Layer Chromatography using Chlorophyll Pigments PDF

Preview

Summary

Lab...

Description

Spectrophotometry and Thin Layer Chromatography using Chlorophyll Pigments Victoria Garofalo 0543619 BIOL 2910WA Due: February 12, 2015.

Abstract The pigments in coleus leaf species were studied to determine which pigments were present, the Rf values of each pigment, and the concentration of chlorophyll a in each leaf. ThinLayer Chromatography and spectrophotometry were used in this experiment. It was found that both of the leaves contained the same pigments, however they were contained in the leaf at different concentrations. The significance of these findings is that both the green and non-green leaf contain the same photosynthetic pigments, although their exterior pigments are greatly different. Introduction Chlorophyll is the main pigment involved in photosynthesis, but at least two other pigments, carotenoids and anthocyanins, play supporting roles in photosynthesis (Vermaas, 2007). Chlorophyll produces green colors in leaves, carotenoids produce yellow to orange and bright red colors, and anthocyanins produce violet and dark red to pink colors (Morgan et al., 2010). The chlorophylls, a and b, are the pigments of photosynthesis. They are produced in chloroplasts in the photosynthetic tissues of the leaf. Anthocyanins are water-soluble pigments produced via the flavonoid pathway in the cytoplasm of the colored plant cell. The objectives of this experiment are as follows; to calculate and compare the known Rf values of pigments in a coleus leaf to the Rf values calculated, to use spectrophotometry and TLC (Thin Layer Chromatography) to determine the molar concentration of chlorophyll a at a wavelength of 663nm, and to determine which pigments are present in both the red (non-green) and green coleus leaves. The technique of spectrophotometry is a common procedure for measuring leaf chlorophyll contents in plants. The spectrophotometer uses light beams emitted at wavelengths to record absorbance’s, which are later transformed by mathematical calculations to determine photosynthetic pigment concentrations. (Gonsiorkiewicz Rigon et al., 2013) Thin-layer chromatography is performed on a sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose (Speiser et al., 2015). It separates components of a mixture via their interaction between the mobile and stationary phase. This experiment uses Solvent B: mobile phase, 100mL petroleum ether, 11mL isopropyl alcohol, and 5 drops of water. From information regarding leaves and pigments, the hypothesis is made that; the green leaf sample will contains more chlorophyll a pigments than the non-green leaf sample. It should be found that the green leaf will contain the pigments; chlorophyll a and b. The non-green leaf will contain the pigments; chlorophyll a, b and anthocyanins.

Results Figure 1. Band Distances of Green B using eight samples.

Figure 1. The band distances of a Coleus Electric Lime green leaf. The bands are grey, unavailable, olive, bright green and yellow (from top to bottom) respectively. These bands are on Solvent B (refer to introduction for more information). This figure demonstrates Thin Layer Chromatography. By measuring the band lengths relative Rf values can be calculated (refer to Table 1.0).

Table 1. Band distances of Green B using a distance of 145mm in reference to Figure 1.0

Band Number

Colour

Band Distance (mm) 75 53 48

Grey Olive Bright green 5 Figure 2. Bands Yellow of Red B44 using eight samples. 1 2 3 4

Rf Values .52 .37 .33 .30

Table 2. Band distances of Red B using a distance of 137mm in reference with Figure 2.

Figure 2. The band distances of a Coleus Wizard Mix red leaf (with some green margins). The bands are grey, unavailable, olive, bright green and yellow (from bottom to top) respectively. These bands are on Solvent B (refer to introduction for more information). This figure demonstrates Thin Layer Chromatography. By measuring the band lengths, relative Rf values can be calculated (refer to Table 2.).

Graph for leaf Band Extract AnalysisRf of a Blank, Green, and a non-green sample. Samples Band 1. Results Colour Values Number Distance diluted 10x. (mm) 1 Grey 69 .50 2 3 Olive 47 .34 4 Bright 44 .32 Graph 2. Results greenfor pigment analysis of an olive pigment sample scraped off TLC plates. 5 Yellow 40 .29

2.5

Absorbance (Abs)

2 1.5 Green Non-Green

1 0.5 0 370

420

470

520

570

620

670

Wavelength (nm)

Graph 1. Green and non-green coleus leaves and their absorbance’s (abs). The green leaf displays a higher abs than the non-green leaf consistently. This displays that there are more pigments per leaf in the green leaf. Graph 2. The absorbance vs. wavelength of an olive pigment scraped off of a TLC plate. The curve of this graph resembles the curve of Graph 1. However, the 0.06

Absorbance (Abs)

0.05 0.04 0.03 Olive

0.02 0.01 0 370

420

470

520

570

Wavelength (nm)

absorbance values are much smaller than in Graph 1. Discussion

620

670

720

Coleus leaves are variegated in nature. Some have white patches and some have red patches. The white patches have no color pigments, the red patches have pigments called anthocyanins. Anthocyanins are not involved in photosynthesis, only chlorophylls and carotenoids are involved in photosynthesis (Vermaas, 2007). The actual Rf values of pigments are; Chlorophyll a - 0.59, Chlorophyll b - 0.42; yellowgreen Anthocyanins - between 0.32 and 0.62; red, pink, purple and blue colors Xanthophyll 1 0.28 (Galloway et al., 2015). Comparing these values with the calculated values in Tables 1. And 2., these values are relatively close to the actual values. This displays that the thin layer chromatography was successful. Referring to the graph 2. the Rf values calculated for a Green leaf are always higher than the red leaf. These values were calculated using the measurement of the band length of a solute over the distance of the solvent. Referring to Graph 1., it is shown that the absorbance of the pigment in a green coloured leaf is stronger and higher than it is in a non-green leaf. This aligns with the theory that green leaves contain more chlorophyll a pigment than a non-green leaf. The concentration of chlorophyll a in a green leaf is calculated to be 1.07 mg Chlorophyll A per gram of Leaf. While the concentration of chlorophyll a in a non-green leaf is calculated to be .162 mg Chlorophyll A per gram of leaf. The absorbance of a sample of olive pigment is shown in Graph 2. The highest absorbance recorded is .06. This aligns with the data shown in Graph 1.2 as the concentration of chlorophyll pigments would not be as high in a non-green pigment than a green pigment (Kobayashi et al., 1996). From Figures 1. And 2. It can be seen that both the green coleus and non-green (red) coleus leaves contain the same pigments. Both TLC sheets have the same bands; Grey, olive, bright green and yellow. This means that both coleus leaves have chlorophyll a and b, anthocyanin’s, and Xanthophyll. Red coleus leaves accumulated anthocyanin in the upper epidermal tissue, as well as green coleus types (Burger, 1996). This does not align with our hypothesis, however it is plausible that both would contain anthocyanin as leaves can always change colours in different seasons. From the Rf values, the band colours, and the absorbance’s read by the spectrophotometer, it can be determined that the green Coleus sp. Contains more chlorophyll a pigments than the non-green leaf sample. It is also shown through TLC that both the non-green coleus leaf and the green coleus leaf contain the same pigments. However, these pigments are contained at different molar concentrations per leaf.

References Vermaas W (2007) An introduction to photosynthesis and its applications. AZ ASU Center for Center for Bioenergy & Photosynthesis Tempe 28: 158-163. Morgan JG, Carter MEB (2010) Investigating Biology Laboratory Manual. San Francisco (CA): Pearson Benjamin Cummings, 7: 153-154.

Speiser A, Haberland S, Watanabe M. Wirtz M, Josef-Dietz K, Saito K, Hell R (2015) The significance of cysteine synthesis for acclimation to high light conditions. Front. Plant Sci 10: 3389. Kobayashi M, Wang Z, Yoza K, Umetsu M, Konami H, Mimuro M, Nozawa T (1996) Molecular structures and optical properties of aggregated forms of chlorophylls analyzed by means of magnetic circular dichroism. Spectrochimica Acta Part A: Molec and Biomolec Spectros Vol 52, Issue 5:585–598. Rigon J, Capuani S, Neto J, Beltrao N (2013) Indirect measurement of photosynthetic pigments in the leaves of Jatropha curcas. Semina: Ciências Agrárias, Londrina, vol 34:669-674 Burger J, Edwards G (1996) Short Communication Photosynthetic Efficiency, and Photodamage by UV and Visible Radiation, in Red versus Green Leaf Coleus Varieties. Dep of Botany, Washington St Un. Plant Cell Physiol. 37(3): 395-399 Galloway K, Bretz S, Novak M (2015) Paper Chromatography and UV–Vis Spectroscopy To Characterize Anthocyanins and Investigate Antioxidant Properties in the Organic Teaching Laboratory. J. Chem. Educ., 92 (1): 183–188...