# Practical: Investigating Photosynthetic Pigments with Chromatography ## Introduction to Photosynthetic Pigments - Chloroplasts contain several different **photosynthetic pigments** within the thylakoids - These pigments absorb different wavelengths of light - There are **two main types** of pigments: **chlorophylls** and **carotenoids** - Accessory pigments surrounding the primary pigment absorb both similar and different wavelengths of light to chlorophyll - This expands the wavelength range that can be absorbed for use in photosynthesis

**Photosynthetic pigments** are molecules that absorb specific wavelengths of light energy to drive photosynthesis

### Chloroplast Pigment Types | Pigment Group | Name of Pigment | Colour of Pigment | |---------------|-----------------|-------------------| | Chlorophylls | Chlorophyll a | Blue-green | | Chlorophylls | Chlorophyll b | Yellow-green | | Carotenoids | β carotene | Orange | | Carotenoids | Xanthophyll | Yellow | **Chlorophylls** absorb wavelengths in the **blue-violet and red regions** of the light spectrum - They reflect green light, causing plants to appear green **Carotenoids** absorb wavelengths of light mainly in the **blue-violet region** of the spectrum

The pigments themselves have colour - this is different from the colours of light that they absorb

## Chromatography Overview - **Chromatography** is an experimental technique used to **separate mixtures** - Different components within the mixture travel through the material at different speeds due to their **size and charge** - This causes the different components to separate - A **retardation factor** ($R_f$) can be calculated for each component of the mixture ### Two Common Techniques **Paper chromatography:** - The mixture of pigments is passed through paper (cellulose) **Thin-layer chromatography (TLC):** - The mixture of pigments is passed through a thin layer of adsorbent (e.g. silica gel) - The mixture travels faster and separates more distinctly than with paper chromatography ## Apparatus - Leaf sample - Distilled water - Pestle and mortar - Filter paper - Capillary tube - Chromatography solvent - Acetone - Pencil - Ruler ## Method ### Preparation Steps **1. Draw a baseline** - Draw a straight line in pencil approximately 1 cm above the bottom of the filter paper - Do not use a pen as the ink will separate into pigments during the experiment and obscure the results **2. Extract the pigments** - Cut a section of leaf and place it in a mortar - It is important to choose a healthy leaf that has been in direct sunlight to ensure it contains many active photosystems - Add 20 drops of acetone and use the pestle to grind up the leaf sample and release the pigments

Acetone is an organic solvent - fats like lipid membranes dissolve in it, helping break down cell structures

**3. Apply the pigment** - Extract some of the pigment using a capillary tube and spot it onto the centre of the pencil line - Allow the spot to dry and reapply several times to concentrate the pigment ### Running the Chromatogram **4. Set up the chromatography** - Suspend the paper in the chromatography solvent so that the level of the solvent is **below the pencil line** - Leave the paper until the solvent has reached near the top of the paper - The mixture is dissolved in the **solvent** (called the mobile phase) and the dissolved mixture passes through a **static material** (called the stationary phase) **5. Mark the solvent front** - Remove the paper from the solvent and immediately draw a pencil line marking where the solvent moved up to - The pigment should have separated out with different spots on the paper at different heights above the pencil line

Work in a well-ventilated area as chromatography solvents are often flammable and produce harmful fumes

**6. Calculate $R_f$ values** $$R_f = \frac{\text{distance travelled by component (pigment)}}{\text{distance travelled by the solvent}}$$ - Always measure to the centre of each spot - Distances should be measured from the baseline to the centre of the pigment spot, and from the baseline to the solvent front ## Results and Interpretation - Chromatography can be used to **separate and identify** chloroplast pigments extracted from a leaf - Each pigment will have a unique $R_f$ value ### Understanding $R_f$ Values - The $R_f$ value demonstrates how far a dissolved pigment travels through the stationary phase - Molecules with a **higher affinity** to the stationary phase (such as large molecules) will travel **slower** and have a **smaller $R_f$ value** - Molecules that are **more soluble** in the mobile phase will travel **faster** and have a **larger $R_f$ value**

$R_f$ value = retardation factor, a ratio comparing how far a substance travels compared to the solvent

### Typical $R_f$ Value Patterns Although specific $R_f$ values depend on the solvent being used, in general: - **Carotenoids** have the highest $R_f$ values (usually close to 1) - **Chlorophyll a** has an $R_f$ value in the middle range (around 0.65) - **Chlorophyll b** has a much lower $R_f$ value (around 0.45) - **Xanthophyll** has an $R_f$ value between carotenoids and chlorophyll a (around 0.71) **Small $R_f$ values indicate:** - The pigment is less soluble in the mobile phase - The pigment has a larger molecular size - The pigment has a stronger affinity for the stationary phase The chart below shows typical $R_f$ values for the four main photosynthetic pigments, illustrating the separation pattern you would expect to observe:

### Expected Separation Pattern From top to bottom on the chromatography paper, you should observe: 1. **Orange band** - β carotene (highest $R_f$) 2. **Yellow band** - Xanthophyll 3. **Blue-green band** - Chlorophyll a 4. **Yellow-green band** - Chlorophyll b (lowest $R_f$) 5. **Origin spot** - Any remaining material that didn't travel ### Example Calculation If a pigment spot centre is 8.5 cm from the baseline and the solvent front is 10.0 cm from the baseline: $$R_f = \frac{8.5}{10.0} = 0.85$$ This $R_f$ value of 0.85 would suggest the pigment is likely **β carotene** or another carotenoid. ## Limitations **Not as specific as other techniques:** - Paper chromatography is sufficient to separate and distinguish different pigments and calculate their $R_f$ values - However, it doesn't provide data on the amount of each pigment present or the exact wavelengths they absorb - **Colorimetry** can be used to calculate these additional values **Subjective measurements:** - Determining the exact centre of a pigment spot can be difficult, especially if spots are irregular or overlapping - This introduces measurement uncertainty into $R_f$ value calculations **Environmental sensitivity:** - Temperature and humidity can affect how quickly the solvent moves up the paper - This can cause variation in $R_f$ values between experiments - Always run known standards alongside unknown samples for comparison **Pigment degradation:** - Some pigments, particularly chlorophylls, can degrade when exposed to light and oxygen during the procedure - Work quickly and in subdued light where possible

Run the chromatography in a sealed container to ensure the atmosphere is saturated with solvent vapour - this gives more consistent results

## Key Practical Skills This investigation develops several important practical skills: **Technique skills:** - Grinding plant material to extract compounds - Using capillary tubes for precise application - Setting up chromatography apparatus correctly - Taking accurate measurements with a ruler **Analysis skills:** - Calculating $R_f$ values - Identifying pigments based on colour and $R_f$ values - Comparing experimental results with known standards **Safety awareness:** - Handling organic solvents safely - Working in ventilated areas - Proper disposal of chemical waste ## Applications Beyond Photosynthesis While this practical focuses on photosynthetic pigments, chromatography principles apply to separating: - Amino acids in proteins - Sugars in food samples - Dyes in ink or food colouring - Drugs in pharmaceutical analysis Think of chromatography like a race where different molecules compete - lighter, more soluble molecules sprint ahead (like sprinters on a track), while heavier, less soluble molecules lag behind (like marathon runners carrying heavy packs). Each molecule finds its own finishing position based on its unique properties.