Plants Pigment

Plant Pigments

Aims

The aim of the experiment is to determine the content of pigments (chlorophyll a and chlorophyll b) in spinach leaves and to test the pigments’ ability to absorb different wavelengths.

Method

Part 1: Estimation of the content of chlorophyll a and chlorophyll b in spinach leaves

Approximately 5g of spinach leaf was prepared for testing using a spectrophotometer (refer to practical notes for full method). Undiluted, 1:5 and 1:25 diluted supernatants of the spinach was placed in a plastic cuvette and the light absorbance at 663 and 645 nm was recorded. The concentrations of chlorophyll a and chlorophyll b were found and expressed in mg/g by using the equations:

A663 = 82.0 Ca + 9.3 Cb

A645 = 16.8 Ca + 45.6 Cb

Then, the effect of dilute acid on chlorophyll solution was tested and observed for any colour changes.  

Part 2: Thin layer chromatography of plant pigments

Thin layer chromatography (TLC) was used to test pigments in purple carrot and spinach (refer to practical notes for full method). Two TLC plates have been spotted using the aforementioned samples and placed in solvents (hexane: acetone) in ratios of 7:3 and 8:2.

The Rf is then calculated by using the distance from origin to component spot divided by the distance from origin to solvent front.

Results

Part 1: Estimation of the content of chlorophyll a and chlorophyll b in spinach leaves

Table 1 shows the absorbance reading for the spinach leaf under various dilution. The results shows that an increase in the dilution of the acetone in spinach decreases absorbance levels.

Table 1: Absorbance reading of spinach leaf under various dilution

All calculations can be found in the Appendix.

Table 2: Chlorophyll concentration (mg/g) in pigments

Dilute acid test on chlorophyll solution

Colour change: Green → Yellow

Part 2: Thin layer chromatography of plant pigments

All calculations for Table 3 and 4 can be found in the Appendix.

Table 3: Rf of Spinach and Purple Carrot in 7:3 solvent

In the 7:3 solvent the pigments identified for spinach leaf are: xanthophylls, chlorophyll b, pheophytin a, and carotene. The pigments identified for purple carrot are: xanthophylls, chlorophyll b and carotene. (Rf values are compared approximately to table X in the Appendix)

Table 4: Rf of Spinach and Purple Carrot in 8:2 solvent

In the 8:2 solvent the pigments identified for spinach leaf are: xanthophylls, chlorophyll b, pheophytin a, and carotene. The pigments identified for purple carrot are: xanthophylls, and carotene.  

[pic 1]

Fig. 1a:

[pic 2]

Fig. 1b:

Discussion

Reference

Appendix

Table