Organic Chemistry Lab 1: Dye Separation Techniques

Organic Chemistry Lab 1: Separation of Dyes & Spinach Pigments

• Lab Goal: Separate dyes (e.g., methylene blue, methyl orange) and spinach pigments using column and thin-layer chromatography.
• Chromatography Principle: Separates mixtures based on differing affinities for two immiscible phases (stationary and mobile).
• Stationary Phase: A stationary phase is a substance (e.g., silica gel, alumina) that remains fixed.
• Mobile Phase A mobile phase is a substance that moves through the stationary phase (e.g., solvent).
• Rf Values: Relative affinities for the mobile and stationary phases are expressed as Rf values (distance traveled by the solute / distance traveled by the solvent).

Safety Precautions

• Silica Gel Toxicity: Handle dry silica in a fume hood as it's toxic if inhaled.
• Methylene Chloride Toxicity: Evaporate this solvent in a fume hood as it's toxic.
• Reagent Handling: Always wear gloves and goggles when handling reagents.

Experiment 1 Goals & Data Collection

• Goal: Separate a dye mixture (e.g., methylene blue and methyl orange) via column chromatography.
• Method: Use different solvents to determine the effective separation, note results.
• Data to Collect: Note observations of dye separation and the time it takes.

Experiment 2 Goals & Data Collection

• Goal: Separate a mixture of dyes from spinach leaves using thin-layer chromatography (TLC).
• Additional Dyes: Bromocresol green, methyl red, basic blue 3, bromocresol purple, methyl blue.
• TLC Mobile Phase Compositions: Use different proportions of acetone and hexanes (e.g., 0:100, 20:80, 40:60, 60:40, 80:20, 100:0) .
• Rf Values for Spinach Pigments Analysis: Determine retention factors (Rf values) for each dye.

Partition Coefficient (K_c)

• Equilibrium: Solutes are in equilibrium between mobile and stationary phases.
• Kc Formula: Kc = [solute in stationary phase] / [solute in mobile phase]
• Kc Interpretation: Larger Kc values indicate a greater affinity for the stationary phase and vice-versa.

Chromatographic Separation

• Separation Basis: Solutes with larger Kc values move more slowly (stay in stationary phase longer).
• Separation Difficulty: Solutes with small Kc differences are harder to separate.
• Separation Efficiency: Longer travel distances (larger Rf differences) result in better separation.

Types of Chromatography

• Column Chromatography: Uses a packed solid stationary phase and a liquid mobile phase.
• Thin-Layer Chromatography (TLC): Uses a thin solid stationary phase coating a flat surface and a liquid mobile phase.

Eluting Power

• Solvent Selection: Different solvents have different eluting powers; use increasing polarities for better separation.
• Mobile Phase Mixtures: Mixed solvents can adjust eluting power.
• Mobile Phases: Mobile phases include water, methanol, ethanol, acetone, diethyl ether, chloroform, etc.

Silica Gel

• Stationary Phase: Silica gel is a common polar, solid, stationary phase composed of silicon dioxide (SiO2) and water.
• Applications: Also used as a desiccant (drying agent) and food preservative.
• Polarity: Polar molecules interact better with polar stationary phases.

Polarity & Kc Values

• Polarity: Polarity is the major factor affecting K values, measured by the dielectric constant (ε). Higher ε values = more polar.
• Interaction: Polar molecules interact better with polar stationary phases.

Relative Polarities of Functional Groups

• Elutropic Series: A series showing decreasing polarity of functional groups (e.g., carboxylic acid > alcohol > amine).
• Retention Time: Retention Time decreases with decreasing polarity of the compound.

Column Chromatography Set-up

• Components: Silica gel (stationary phase), solvent (mobile phase), column, and sand.
• Procedure: Compounds are loaded, solvent travels through, and components separate from each other.
• Separation Basis: Components with smaller Kc values travel faster (less interaction with the column stationary phase).

Thin-Layer Chromatography (TLC) Set-up

• Components: TLC plate, developing chamber, mobile phase, spotted samples.
• Procedure: Spotting of compounds, elution and development in chamber, visualization.
• Visualization: Visualize the separation of spots via staining or UV light.
• Rf Values Determination: Compare the distances traveled by spots to the solvent front to obtain RF values.

Spinach Pigments

• Identification: Use the course material to identify pigments (e.g., Chlorophyll a, Chlorophyll b, Xanthophylls, Carotenes) in spinach using color and Rf values.

This quiz focuses on the separation of dyes and spinach pigments through chromatography techniques such as column and thin-layer chromatography. Understand the principles of separation, including stationary and mobile phases, as well as safety precautions when handling toxic substances.