Column Chromatography of Plant Pigments

Background

Column chromatography is a separation technique used to purify mixtures by exploiting differences in the affinity of components for a stationary phase and a mobile phase. In organic chemistry, it is commonly used to isolate and analyze compounds based on their polarity and molecular structure.

• Stationary phase: Typically silica gel or alumina packed in a column.

• Mobile phase (Eluent): An organic solvent or mixture of solvents that moves through the column, carrying the sample components.

• Components are separated based on their interactions with the stationary phase and their solubility in the mobile phase.

Example: In this experiment, pigments from Capsicum frutescens (red pepper, locally known as siling labuyo) are separated using silica gel and solvents of increasing polarity.

Dielectric Constants and Solvent Polarity

The dielectric constant of a solvent is a measure of its polarity. More polar solvents (higher dielectric constant) can move polar components faster through the column. The dielectric constant of a mixture is calculated as the sum of the products of the dielectric constant of each solvent and its corresponding concentration.

• Formula:

Table 1. Dielectric constants of common solvents:

Experimental Procedure

Extraction of Plant Pigment

Plant pigments are extracted from red pepper using organic solvents and separated based on their polarity.

1. Remove seeds from 2 pcs of red pepper (siling labuyo), chop, and grind with sand.

2. Mix 0.5 g of chopped peppers with 10 mL dichloromethane (DCM), stir thoroughly.

3. Filter the mixture, discard the solid residue.

4. Add 10 mL saturated sodium chloride solution to the extract, mix to form an emulsion.

5. Allow layers to separate; use a Pasteur pipette to collect the lower DCM layer.

6. Add a pinch of anhydrous sodium sulfate to dry the DCM layer; collect the resulting solution as the pigment extract.

Preparation of the Column

A micro-scale column is prepared using a Pasteur pipette packed with silica gel.

1. Insert a small piece of cotton to plug the bottom of the column.

2. Pack silica gel into the column, avoiding air bubbles and over-packing.

3. Add 1:1 hexane/dichloromethane solution until all silica gel is wet; tap to eliminate air bubbles.

4. Allow solvent to drain until the level reaches the top of the silica gel.

Figure 1: Set-up for micro-scale column chromatography (Pasteur pipette, clamp, column plug, silica gel, pigment extract).

Separation of the Pigments

Pigment extract is loaded onto the column and eluted with solvents of increasing polarity.

1. Add 0.5 mL of pigment extract dropwise onto the column.

2. Elute with hexane/dichloromethane (1:1) and collect colored bands in test tubes.

3. Change eluent to dichloromethane/methanol (1:1) when the previous eluent becomes colorless; collect new colored bands.

4. Continue elution until all bands are collected and eluents are colorless.

5. Discard silica gel and remaining eluents appropriately.

Table 2. Eluting agents for pigment separation:

Treatment of Results

• Identify the possible compounds in each colored band (e.g., chlorophylls, carotenoids).

• Use references to support identification.

• Calculate the dielectric constant of each eluent using Table 1 values.

• Discuss how polarity of the compounds affected their separation.

Guide Questions

1. What is the stationary phase and what is/are the mobile phase(s) in this experiment?

2. What compounds would elute out first and last? Relate this to the polarity of the solvents and the pigments.

3. What is the significance of using a dried-out column (solvent level below top of silica) before introducing the elution and isolation of pigments?

Key Concepts and Definitions

• Stationary phase: The solid material (silica gel) that adsorbs sample components.

• Mobile phase: The solvent or mixture of solvents that carries the sample through the column.

• Elution: The process of washing out components from the column using the mobile phase.

• Polarity: A property that affects how strongly compounds interact with the stationary phase and how quickly they move with the mobile phase.

• Dielectric constant: A measure of solvent polarity; higher values indicate more polar solvents.

Applications and Examples

• Chlorophylls: Green pigments, more polar, elute later with more polar solvents.

• Carotenoids: Yellow/orange pigments, less polar, elute earlier with less polar solvents.

• Capsaicin and capsorubin: Red pigments found in mature red peppers, elute depending on their polarity.

Additional info: The experiment demonstrates the principle of chromatographic separation based on polarity, and the use of dielectric constants to select appropriate solvents for effective separation of plant pigments.