Chromatographic Analysis of Spinach Pigments: Separation, Identification, and Quantification

Chromatography is a scientific discipline that investigates the separation of molecules based on differences in their structure or composition. This process involves dissolving a sample in a mobile phase (gas, liquid, or supercritical fluid) and passing it through an immobile, immiscible stationary phase. Components with varying solubilities in each phase experience different travel times through the stationary phase, resulting in the separation of sample components.

Various supports can be used for chromatographic separations, such as immobilized silica on glass plates, volatile gases, paper, or liquids with hydrophilic, insoluble molecules.

Column chromatography is a widely used technique in organic chemistry to isolate and purify chemicals from natural sources or reaction sequences.

For column chromatography, the progress of separation is often visually monitored when compounds are colored, as is the case with pigments in this experiment. However, when the compounds are colorless, various methods, including collecting labeled fractions and analyzing them through techniques like thin-layer chromatography, are employed to monitor the separation progress.

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The experiment specifically focuses on the column chromatography of spinach pigments, which can be classified into two main categories: chlorophylls and carotenoids. Chlorophylls, responsible for the green color in plants, absorb specific light wavelengths, converting them into chemical energy. Examples include chlorophyll a, chlorophyll b, pheophytin a, and pheophytin b. The structure of chlorophyll a is described, and chlorophyll b differs by replacing a methyl group with a –CHO group. Pheophytin a and b are similar to chlorophyll a and b, respectively, except for the replacement of the Mg2+ ion with 2H+ ions.

To separate and analyze pigments from spinach through column chromatography, focusing on the separation of carotenoids and chlorophylls.

Materials:

1. Spinach sample
2. Silica gel for column chromatography
3. Solvents: Hexane, Acetone, and Ethyl Acetate
4. Glass columns
5. Labeled tubes for collecting fractions
6. Thin-layer chromatography (TLC) plates
7. UV lamp for TLC analysis
8. Beta-carotene standard for TLC calibration

Procedure:

1. Preparation of Column: a. Pack a glass column with silica gel, ensuring a uniform and settled bed. b. Suspend the silica in hexane to remove air bubbles and allow it to settle. c. Add the spinach extract to the column.
2. Elution: a. Elute the column with a mixture of hexane, acetone, and ethyl acetate in increasing polarity. b. Collect fractions in labeled tubes.
3. Thin-Layer Chromatography (TLC) Analysis: a. Spot a small amount of each fraction on TLC plates. b. Develop the plates using a solvent system suitable for carotenoids and chlorophylls. c. Visualize under UV light.
4. Calculation of Rf values: a. Measure the distance traveled by each compound and the solvent front. b. Calculate Rf values using the formula: Rf=Distance traveled by solvent frontDistance traveled by compound​
5. Identification of Bands: a. Compare Rf values with beta-carotene standard. b. Assign bands to carotenoids (yellow) and chlorophylls (green).
6. Quantification:

1. a. Measure the area under the bands on the TLC plates. b. Use the area to estimate the relative amount of each pigment.
2. Discussion: a. Interpret the TLC results to identify and quantify pigments. b. Discuss the separation efficiency and the significance of Rf values.
3. Carotenoid Extraction Efficiency: a. Calculate the extraction efficiency of carotenoids from spinach. b. Use the formula: Extraction Efficiency=Amount of carotenoids extractedTotal amount of carotenoids in spinach×100Extraction Efficiency=Total amount of carotenoids in spinachAmount of carotenoids extracted​×100
4. Comparison with Seasonal Variation: a. Research the seasonal variation in carotenoid content in spinach. b. Discuss how the experiment results align with the natural variation.
5. Synthetic Carotene Comparison: a. Research and discuss the properties of synthetic carotene used for coloring butter and margarine. b. Compare the health and safety aspects of synthetic carotene with Butter Yellow.
6. Conclusion: Summarize the key findings of the experiment, including the separation efficiency and the identification of carotenoids and chlorophylls.

Calculation Example:

1. Rf Value Calculation:
   • Distance traveled by carotenoids = 2.6 cm
   • Distance traveled by solvent front = 4.0 cm
   • Rf=4.02.6​=0.65
2. Extraction Efficiency Calculation:
   • Amount of carotenoids extracted = Area under carotenoid band on TLCArea under carotenoid band on TLC
   • Total amount of carotenoids in spinach = Initial amount of carotenoids in the extractInitial amount of carotenoids in the extract
   • Extraction Efficiency=Area under carotenoid bandInitial amount of carotenoids×100Extraction Efficiency=Initial amount of carotenoidsArea under carotenoid band​×100

Note: Ensure proper safety measures are followed, including the use of appropriate protective equipment and disposal of chemicals according to laboratory guidelines.

Equipment Chromatography column Funnel Erlenmeyer flask (50 mL) * 6 Pipette (10 mL) * 4 Mortar and pestle Beaker (400 mL) Beaker (100 mL) Clamps, Ring Stand Suction bulb

Chemicals Silica Gel 40 Acetone (CH3COCH3) Petroleum ether Sodium chloride (NaCl) Calcium carbonate (CaCO3) Sodium sulfate (Na2SO4)

PROCEDURE

Column Preparation

1. Place glass wool at the column's bottom.
2. Pour 10 mL of a 7:3 petroleum ether and acetone solution into the column.
3. Weigh 20 g of silica gel 40 in a 50 mL Erlenmeyer flask, add petroleum ether acetone solution slowly while stirring with a glass rod.
4. Transfer the slurry into the column using a pipette. Place an Erlenmeyer flask under the column to drain excess solvent, ensuring silica doesn't dry in the column.
5. Reuse collected excess solvent for the next column.

Spinach Sample Preparation

1. Place spinach in a mortar; add 22 mL acetone, 3 mL petroleum ether, and a spatula tip of calcium carbonate (CaCO3).
2. Grind until leaves are finely broken; filter the pigment extract if necessary.
3. Put the filtrate in a separatory funnel; add 20 mL petroleum ether and 20 mL 10% NaCl solution.
4. Shake the funnel, drain the lower layer into a beaker.
5. Wash the upper layer twice with 5 mL distilled water.
6. Transfer the extract to a 50 mL Erlenmeyer flask; add a spatula tip of sodium sulfate (Na2SO4). Decant the solution into another flask. Evaporate excess solvent if needed.

Column Chromatography

1. Use a pipette to add 10 mL leaf extract to the column's top.
2. Open the column's bottom outlet; let the elute pass through slowly, allowing sample development.
3. Collect the eluent in a test tube when the yellow band reaches the column's bottom.
4. Change test tubes as the eluent color changes, achieving sample separation.
5. If necessary, evaporate excess solvent, seal sample tubes with parafilm, and store.