Thin Layer Chromatography of Photosynthetic Pigments
Sample Preparation
Crushing the Plant Sample: Use a pestle and water to break the cell wall and membranes, releasing photosynthetic pigments from chloroplasts.
Chloroplast Membrane: Needs to be broken to release pigments.
Application to Chromatogram
Stationary Phase: Silica gel on plastic; handle with care to avoid contamination.
Silica Gel: Despite being called a gel, it's dried on plastic.
Pencil Line: Drawn with pencil to avoid dissolution by the solvent.
Pigment Application: Apply pigment mixture as a small dot on the line.
Allow it to dry between applications.
Typically repeated about 10 times for concentration.
Developing the Chromatogram
Mobile Phase: Solvent that dissolves pigments and moves them up the chromatogram.
Ensure solvent is below pencil line to prevent pigments from dissolving directly into solvent.
Smaller pigments travel further up due to less resistance in silica gel.
Calculating Rf Values
Distance Calculation:
Distance moved by pigment (compound) / Distance moved by solvent front.
Measure in millimeters for accuracy.
Comparison with Known Values: Match calculated Rf value with standard Rf values to identify pigments (e.g., Rf value of 0.42 might indicate Chlorophyll b).
Exam Preparation
Ability to estimate pigment identity from chromatogram.