Exploring Photosynthetic Pigments: Paper Chromatography, Solubility, and Divine Wisdom in Plant Creation

1. Objectives:1.1 Scientific Objectives:
   • Apply paper chromatography to separate individual photosynthetic pigments in plant tissue.
   • Determine the solubility of plant pigments in two different solvents.
   • Identify the optimal solvent ratio for pigment separation.
2. Introduction:2.1 Theory and Concept of Chromatography:

Chromatography, derived from Greek words Chroma (colour) and Graphe (write), is a technique for molecule separation based on differences in size, shape, mass, charge, solubility, and adsorption properties. It includes various types such as paper chromatography and thin layer chromatography, all operating on a stationary phase and a mobile phase principle.

The experiment focuses on chromatography of leaves to separate photosynthetic pigments. Pigments, molecules absorbing and reflecting light, influence leaf color and are primarily found in chloroplasts. Common pigments include chlorophyll a (dark green), chlorophyll b (yellowish-green), xanthophylls (yellow), carotenoids (orange), and anthocyanins (red/purple). Chlorophyll captures sunlight energy, while carotenoids and xanthophylls act as accessory pigments.

Paper chromatography is employed, separating pigments based on their migration rates. The stationary phase is porous paper, and the mobile phase is a liquid solvent.

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Separation occurs due to varying attractions between pigments and paper, with less soluble pigments moving slower. The result is a chromatogram depicting different pigment components.

The identification of components involves comparing an unknown mixture's chromatogram with known standards. The Rf value (distance traveled by compound divided by solvent distance) is used for quantitative analysis, enhancing the scientific rigor of chromatography. The experiment aims to understand pigment solubility, optimize separation conditions, and utilize Rf values for identification.

Hypotheses:

1. Anthocyanin is water-soluble and non-polar solvent-insoluble, while carotene, xanthophyll, and both chlorophyll a and b are water-insoluble and soluble in non-polar solvents.
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2. The more soluble the photosynthetic pigment, the greater its movement.
3. A higher rate of adsorption between pigment particles and chromatography paper results in greater movement above the paper.
4. Carotene is the most soluble pigment in a non-polar solvent, followed by xanthophyll, chlorophyll a, and finally chlorophyll b.

Variables:

• Manipulated variable: Chromatography solvent.
• Responding variable: Retention factor (Rf value).
• Constant variable: Types of leaves used.

Methodology:

3.1 Chemicals: 30mL propanone, 30mL water, 3 or 4 fresh spinach leaves.

3.2 Apparatus: Pair of scissors, a pencil, a ruler, pestle and mortar, 50mL beakers, chromatography paper, pin, glass tube, chromatography chamber.

3.3 Diagram:

[Diagram not provided]

3.4 Procedure:

Part A: Preparation of Pigment Extract:

1. Finely cut some spinach leaves and fill a mortar to about 2cm depth.
2. Mash the leaves using a pestle and mortar.
3. Pour 15ml of propanone into the mortar.
4. After 10 minutes, chlorophyll will dissolve in the propanone.
5. Remove the leaves from the mortar.
6. Repeat steps 1-5 using water as a solvent.

Part B: Preparation of Chromatography Chamber and Paper:

1. Use a thin strip of chromatography paper, ensuring it is not longer than the chromatography chamber.
2. Draw a pencil line 2cm from the bottom of the chromatography papers.
3. Use a fine glass tube to place liquid from leaf extracts onto the center of the line on two chromatography papers separately. Keep the spot small.
4. Allow the spot to dry and add another spot on top to build up a concentrated small spot.
5. Pin the paper to the hook just above its base.
6. Make a solvent with a 50:50 ratio of propanone and water.
7. Pour the solvent mix into the chromatography chamber.
8. Repeat step 1-6 with solvent ratios of propanone and water 100:0, 0:100, 70:30, and 30:70.
9. Hang the paper so that it dips in the solvent just below the pencil line.
10. Cover the chromatography chamber and avoid moving it during the process.
11. Leave the experiment until the solvent has soaked near the top, then remove the paper from the chamber.
12. Mark the solvent height on the paper with a pencil and let the chromatography dry.
13. Record and observe the obtained results in a table.

TABLE 1: DATA FOR EXTRACTION OF SPINACH LEAF USING SOLVENT RATIO OF PROPANONE:WATER, 100:0

TABLE 2: DATA FOR EXTRACTION OF SPINACH LEAF USING SOLVENT RATIO OF PROPANONE:WATER, 0:100.

TABLE 3: DATA FOR EXTRACTION OF SPINACH LEAF USING SOLVENT RATIO OF PROPANONE:WATER, 70:30.

TABLE 4: DATA FOR EXTRACTION OF SPINACH LEAF USING SOLVENT RATIO OF PROPANONE:WATER,30:70.

Adsorption refers to the attraction of atoms, molecules, or ions from a substance, whether it's a gas or dissolved solid and liquid, to the surface of another substance, known as an adsorbent. In contrast, absorption is the process in which a fluid is dissolved by a solid (absorbent) or a liquid. Paper chromatography utilizes capillary action to separate pigments from an initial concentrated solution. A solvent is placed at the paper's bottom, and as the solution travels upward, similar soluble pigments move with the solvent until weaker bonds break, resulting in their imprint at a specific height on the paper.

In this experiment, propanone and water served as solvents to decolorize the leaf, with separation based on solubility differences. The polarity of molecules, characterized by one end being positively charged and the other negatively charged, played a crucial role. Water, being polar, dissolved polar molecules, while propanone, consisting of non-polar molecules, dissolved non-polar ones. Chromatography paper was employed to identify pigments present in the leaf, theoretically including chlorophyll a and b, xanthophyll, carotene, and anthocyanin. Carotene exhibited the highest pigment distance from the origin, confirming the presence of the five mentioned pigments.

Each pigment has distinct functions. Chlorophyll a absorbs light for photosynthesis, predominantly absorbing violet/blue and red light. Chlorophyll b, structurally similar but with a different absorption spectrum, absorbs more in the blue and orange-red ranges. Carotenoids act as accessory pigments, absorbing light wavelengths that chlorophyll cannot effectively absorb, appearing in various shades of yellow or yellow-oranges. Xanthophyll also serves as an accessory pigment working with chlorophyll a. Anthocyanin, while not participating in photosynthesis, provides color to flowers, leaves, and fruits, acting as a protective mechanism against excessive sunlight.

Analysis of the results from the extraction of spinach leaves using propanone: water (50:50) revealed that carotene had the highest pigment distance from the origin (7.2cm), followed by xanthophyll (6.4cm). The observations correlated with the expected colors of carotene (orange) and xanthophyll (yellow). Chlorophyll a (blue-green) had a higher distance than chlorophyll b (yellow-green), aligning with their expected colors. Chlorophyll b, being the most polar, had the lowest distance (4.9cm). Carotene's higher solubility was attributed to its non-polar molecular structure, while xanthophyll had intermediate solubility due to two polar groups. Chlorophyll a and b, with five and six polar groups, respectively, exhibited lower solubility.

Anthocyanin, extracted in propanone, showed no changes in pigment distances and solvent from the origin, indicating no Retention factor (Rf value). The absence of chlorophyll a and b, xanthophyll, and carotene extracts in water extraction was due to the hydrophobic nature of carotene and xanthophyll, rendering them insoluble in water but soluble in non-polar solvents.

The calculated Rf values demonstrated carotene's highest solubility (1.00), followed by xanthophyll (0.89), chlorophyll a (0.71), and chlorophyll b (0.68). Anthocyanin in water extraction showed changes, with an Rf value of 0.96, confirming its solubility in water.

Anthocyanin's solubility in water is attributed to its glycosides involving sugars, making it water-soluble with color variations based on pH. Anthocyanin's presence in spinach leaves contributes to their green color, turning purple in summer as anthocyanin pigments actively produce towards summer's end.

Precautions, including handling paper with dry hands, avoiding pen use for drawing lines, ensuring sufficient pigment transfer, and proper handling of propanone, were crucial. Immersing chromatography paper just below the pencil line further ensured accurate results.
In conclusion, the experiment successfully identified five types of pigments present in spinach leaves: xanthophyll, anthocyanin, carotene, chlorophyll a, and chlorophyll b. Each pigment produced distinct color spots on the chromatography paper, allowing for their identification. Chlorophyll a appeared dark-green, chlorophyll b was yellowish-green, xanthophylls were yellow, carotenoids were orange, and anthocyanin appeared pink, possibly due to the acidic nature of the water used.

The hypotheses were all accepted, with carotene exhibiting the highest Rf value, followed by xanthophylls, chlorophyll a, and chlorophyll b in propanone. This indicated that carotene is the most soluble pigment in non-polar solvent, with the furthest movement on the chromatography paper and the highest rate of adsorption between pigment particle and chromatography paper, followed by xanthophyll, chlorophyll a, and b. These four pigments were found to be soluble in non-polar solvent and insoluble in water. On the other hand, anthocyanins were soluble in water and insoluble in non-polar solvent.

The objectives of the experiment, including the application of paper chromatography to separate plant pigments, identification of color changes on chromatography paper, and determination of pigment solubility in water and propanone, were successfully achieved.

From an Islamic perspective, the experiment serves to explore the uniqueness of pigments in leaves, a creation by Allah (SWT). The various pigments, particularly chlorophyll, play a crucial role in sustaining the process of photosynthesis for the benefit of all living organisms. The Qur'an refers to chlorophyll as "Al-Khadir" (green substance) and emphasizes the wisdom of Allah in creating green factories (chloroplasts) within plants.

The experiment aligns with the Islamic belief in Allah as the best planner of all things. Plants, animals, and humans receive their sustenance from the food produced by plants in chloroplasts containing chlorophyll. The Qur'an draws attention to the creation of vegetation, grains, fruits, and other parts of plants through the process of photosynthesis.

Additionally, the experiment highlights the diversity and uniqueness of Allah's creation. Each pigment, such as anthocyanin, exhibits special characteristics, emphasizing the wisdom and creativity of the Creator. The Qur'an encourages believers to reflect on the signs in the universe, recognizing Allah's power over creation.

Overall, the experiment serves as a means to appreciate the diversity and significance of Allah's creation, acknowledging the wisdom behind the existence of different pigments in plants. It reinforces the Islamic concept of seeking knowledge and reflecting on the signs of Allah in the natural world.