In this experiment, pigments were isolated at first, and observed by using thin-layer chromatography. During extraction, petrol ether, acetone and calcium carbonate were added to spinach leaves and grinded together by using mortar and pestle. Because pigments only dissolve in organic solvent but some impurities such as flesh and fiber dissolve in inorganic solvent, aqueous sodium chloride and petrol ether were added to purify the extract. By using thin-layer chromatography, pigment fractions showed on the TLC silica gel plate. The solubility of different pigments in solvent was found out through calculating the Rf values of different pigment fractions. The results showed that yellow pigment has the smallest solubility strength in mobile phase; the olive green color has the biggest solubility strength. Golden pigment which should have the bigger solubility than olive green pigment, had the smaller absorption strength than olive green pigment in this experiment; the error will be discussed later.
The colored in plants are usually fall in two major categories: carotenoid and chlorophylls. β-carotene is golden colored pigment which is non-polar. Chlorophylls contain two types: chlorophyll a and chlorophyll b; chlorophyll a is blue green colored and chlorophyll b is yellow green colored. Different from β-carotene, chlorophyll contain several polar C-O, C-N bonds and magnesium chelated ion to an nitrogen atom thus they has strong polarity. There exist other small amount pigments such as lutein, xanthophylls and pheophytin. Xanthophylls look like chlorophylls but the magnesium ion is replaced by two hydrogen ions. Pigments isolation depends on different polarities of pigments. During using thin-layer chromatography to separate and purify the pigments, mobile phase and stationary phase are necessary. Mobile phase also called Developing solvent, which consists of several organic chemicals that can be changed. The components of mobile phase are chosen by experiment or from the textbooks; and different components mean that the polarity of solvent can be various.
1. Filter paper
2. TLC silica gel plate
3. Developing solvent (ether: acetone = 3:1)
6. Petrol ether
9. Calcium carbonate(CaCO3)
10. 10% Aqueous sodium chloride(NaCl)
11. Sodium sulfate(Na2SO4)
12. Spinach leaves
1. TLC chambers
2. Capillary tubes
3. UV lamps (UV 254nm, 325nm)
4. Mortar & pestle
5. Separating funnel 100 mL
6. Measuring cylinder 100 mL
7. Measuring cylinder 25 mL
8. Erlenmeyer round bottom flask 100mL
9. Rotary evaporator
First, some spinach leaves with dark green color were picked and put into a mortar; then some CaCO3 was added to the mortar. Pestle was used to grind the spinach leaves until it became paste. After that, measuring cylinder 25mL was used to measured 22mL acetone and 3mL petrol ether respectively, then petrol ether and acetone were mixed with spinach together after added to the mortar together. A piece of filter paper was placed in a funnel which was hold above a beaker. Follow by, paste of spinach leaves were poured though the funnel and waited for juice of spinach leaves dripped to the flask. Step2:
When the pigment extract was all filtered through filter paper, the extract is poured to a separating funnel; then 20mL of petrol ether and 20mL of 10% aqueous NaCl was measured by using measuring cylinder 25mL respectively and added to the separating funnel. The separating funnel was shaken 5-6 times carefully. The liquid in separating funnel was allowed to settle for several minutes until it divided to two layers. The lower layer was drain to a beaker through the separating funnel, and then was thrown away. After that, 5mL distilled water was used to wash the extract 3-4 times. Then the upper layer was poured to an...