TLC (Thin Layer Chromatography) is a very commonly used technique in synthetic chemistry for identifying compounds and determining their purity. It only requires small quantities of the compound and is much faster. Thin-layer chromatography consists of a stationary phase that is powerless on a glass or plastic plate, and an organic solvent. When the solvent front reaches the other edge of the stationary phase, the plate is removed from the solvent reservoir. Then the separated spots are visualized with ultraviolet light.
A common type of Liquid Chromatography is HPLC (High Performance Liquid Chromatography). High Performance Liquid Chromatographs (HPLC) uses a liquid mobile phase to separate the components of a mixture. These components are first dissolved in a solvent. Unlike Paper Chromatography the process is conducted at a high velocity and pressure drop.
Gas Chromatography is used in airports to detect bombs and is used is forensics in many different ways. It is used to analyze fibres on a person’s body and also analyze blood found at a crime scene. In gas chromatography helium is used to move a gaseous mixture through a column of absorbent material.
We used Paper Chromatography the method is outlined below.
- A spot of the substance (crushed leaves and ethanol) is left on the chromatography paper left to dry.
- Once dried another spot is added on top then the paper is suspended in a solvent which in this case was ethanol.
- As the solvent molecules move through the paper the components move up the paper.
- The substance eventually separates out into different spots.
Safety Precautions
All solutions should be disposed of in the liquid waste container. All solids should be disposed of in the solid waste container.
Chromatography works when the solvent rises up the chromatography paper. When the solvent reaches the "spot" it dissolves the mixture of coloured chemicals. This is when a solution is formed. The mixture of solutes dissolves in the solvent. The molecules of these different chemicals become all different sizes. During my experiment I observed this as I noticed that the big molecules moved slower than the small ones.
Results
The chromatogram can be analysed by measuring the distance travelled by the solvent front, and the distance from the origin to the centre of each spot. This is used to calculate the Rf (relative front) value for each spot:
Conclusion
Paper chromatography proved to be an accurate method of separating and observing the plant pigments, because the pigments dissolved in the solvent and moved upwards. The colours were observed and their movement distances were measured and recorded. The Rf value of each pigment was determined by dividing its movement by the migration of the solvent. It was shown that during my experiment four pigments were present in the original spot. Spot D was the most soluble in the solvent, while spot A was the least soluble. I also observed that some pigments move further up the chromatogram than others because the heavy pigments will not move fast and stay at the bottom, where as the lighter pigments remain at the top and move faster because they have a smaller relative molecular mass. From looking at my results I can see that my highest Rf value was 0.278 which indicates that it has the lowest molecular mass in the table.
Evaluation
Error Analysis
The chromatography paper touched the sides of the tube-test during the waiting time which caused the movement to go slightly to the side instead of straight to the top. The strip was also a little bent at the top which could have been a slight error in measuring the movement of the solvent front. However I feel that these errors were insignificant and so my results are reliable.
To correct these errors I could use some tweezers to slide the chromatography paper in the tube gently. For further study I would
Research