Here’s a clear and classroom-ready method for performing Thin Layer Chromatography (TLC) using
Introduction
What Is Chromatography?
Chromatography is an analytical technique used to separate and identify components within a mixture. It relies on two phases: a stationary phase that stays fixed (solid or liquid supported on a solid) and a mobile phase (liquid or gas) that moves through or over it. Components partition between these phases based on differences in polarity, solubility, or affinity, causing them to travel at different rates and thus separate.
Principles of Thin Layer Chromatography (TLC)
Thin layer chromatography is a simple, rapid form of planar chromatography. A thin layer of adsorbent—usually silica gel or alumina—is coated onto a glass, plastic, or aluminum plate to serve as the stationary phase. A small spot of sample is applied near the plate’s base, which is then placed in a shallow pool of mobile phase (solvent). Capillary action draws the solvent upward, carrying sample components to different heights according to their interactions with the adsorbent.
Key features of TLC include:
- Visualization of separated spots using UV light or staining reagents
- Calculation of retention factor (Rf) to compare compound mobility
- Rapid screening of reaction progress and purity checks
Solvent Systems in Thin Layer Chromatography
Importance of Solvent Selection
Selecting the right solvent system is critical for achieving clear separation of components. The mobile phase must balance between dissolving sample spots and carrying them up the plate without all compounds moving together or hardly moving at all.
Polarity and Solvent Strength
Every solvent has a characteristic polarity index reflecting its ability to dissolve polar versus non-polar substances.
- Non-polar solvents (low polarity) favor moving non-polar analytes.
- Polar solvents (high polarity) will carry more polar compounds further up the plate.
By adjusting polarity, you tune how strongly each analyte interacts with the stationary phase (silica or alumina) versus the solvent front.
Common Solvents and Polarity Index
| Solvent | Polarity Index | Typical Use |
|---|---|---|
| Hexane | 0.1 | Non-polar; good for lipids |
| Petroleum ether | 0.1–0.2 | Similar to hexane |
| Toluene | 2.4 | Aromatics; moderate non-polar |
| Chloroform | 4.1 | Medium polarity |
| Ethyl acetate | 4.4 | Versatile; many general mixes |
| Acetone | 5.1 | Polar; moves polar dyes well |
| Methanol | 5.1 | Very polar; strong eluent |
| Water | 10.2 | Highest polarity; rarely alone |
Binary and Ternary Mixtures
- Binary systems combine two solvents (e.g., hexane : ethyl acetate) to fine-tune polarity.
- Ternary systems (e.g., butanol : acetic acid : water) introduce a third component, often an acid or base modifier, to adjust pH or selectivity.
- Start with a wide polarity gap and gradually increase the stronger solvent fraction until analytes have (Rf) values between 0.2 and 0.8.
Developing and Optimizing a Solvent System
- Choose a non-polar starting mix (e.g., 90 : 10 hexane : ethyl acetate).
- Spot your sample and run a test strip.
- Measure (R_f) values:
- (Rf = distance traveled by compound ÷ distance traveled by solvent front.
- If all spots remain near the baseline (Rf < 0.2), increase the polar solvent proportion by 5–10%.
- If spots run to the top (Rf > 0.8), decrease polar solvent or add a less polar cosolvent.
- Iterate until each component separates into distinct bands.
Example Solvent Systems
| Mixture | Ratio | Application |
|---|---|---|
| Hexane : Ethyl acetate | 7 : 3 | Non-polar lipids and esters |
| Hexane : Ethyl acetate | 4 : 6 | Moderate polarity dyes |
| Ethyl acetate : Methanol | 9 : 1 | Polar small molecules (e.g., amino acids) |
| Butanol : Acetic acid : Water | 4 : 1 : 1 | Plant pigments and food dyes |
Materials:
- Fresh plant petals (e.g. rose, hibiscus, marigold)
- Mortar and pestle or small blender
- Solvent for extraction Ethanol
- Mobile phase premixed 1-Butanol : Acetic Acid : Water (4 : 1 : 1)
- TLC plate (silica gel-coated)
- Capillary tubes
- TLC chamber (e.g. beaker with lid or jar with a lid)
- Pencil (not pen!)
- Ruler
- UV lamp (optional for visualization)
Method:
- Extract Pigments:
- Crush ~2 g of plant petals in a mortar with 5–10 mL of solvent.
- Filter or decant the extract to remove solid debris.
- Prepare TLC Plate:
- Use a pencil to draw a faint baseline ~1 cm from the bottom.
- Mark small dots evenly spaced along the line for spotting.
- Spot the Sample:
- Use a capillary tube or micropipette to apply a small drop of extract onto each marked spot.
- Allow to dry and reapply 2–3 times to concentrate the sample.
- Develop the Plate:
- Pour mobile phase into the TLC chamber to a depth of ~0.5 cm.
- Place the TLC plate upright in the chamber, ensuring the solvent doesn’t touch the spots.
- Cover the chamber and allow solvent to rise until ~1 cm from the top.
- Remove and Dry:
- Take out the plate and immediately mark the solvent front with pencil.
- Let the plate air dry.
- Visualize Spots:
- Observe pigment bands directly.
- Use UV light if spots are faint or colorless.
- Calculate Rf Values:
- Measure distance from baseline to center of each spot.
- Measure distance from baseline to solvent front.
- Use:
Rf = Distance moved by pigment ÷ Distance moved by solvent
Notes:
- Different petals yield different pigment profiles—great for comparative analysis.
- You can extend this into discussions on polarity, solubility, and plant biochemistry.
Safety & Waste Management:
- Safety Precautions for Solvents
- Wear nitrile gloves, safety goggles, and a lab coat at all times when handling solvents.
- Work in a fume hood or well-ventilated area to avoid inhaling vapors from 1-butanol and glacial acetic acid.
- Keep all sources of ignition (open flames, sparks, hot surfaces) away from 1-butanol, as it is highly flammable. Store it in a flammable-liquid cabinet when not in use.
- Handle glacial acetic acid with care: it is corrosive and can cause severe skin and eye burns. Always add acid to solvent slowly to minimize heat buildup.
- Rinse any skin or eye contact immediately with plenty of water and seek medical attention if irritation persists.
- Use spill trays when measuring and mixing solvents to contain accidental drips or spills. Clean up spills promptly with appropriate absorbent materials, then dispose of both waste and absorbents in a designated organic-waste container.
- Label all solvent containers clearly with chemical name, concentration or ratio, hazard pictograms, and date of preparation.
- After completing the experiment, decontaminate glassware with soap and water, and dispose of rinse water according to institutional guidelines.
Practical Science 