I performed a TLC of plant pigments in my lab class with a solvent of petroleum ether/ acetone (7:3) and used a silica gel plate.
It is expected that the most polar pigments (chlorophyll) will be located near the bottom and the carotenoids near top.
In my experiment, it was the exact opposite - the green bands (belonging to chlorophyll) were located on the top and the yellow bands (belonging to the carotenoids) were on the bottom.
Can any one explain where I could have gone wrong in my experiment? Is a possibility that there was water in the solvent?
Any help would be greatly appreciated! Thanks!
The solvent ratio is about right for separating chlorophyll and plant pigments on a TLC plate. When you say the chlorophyll was at the top do you mean with the solvent front? If this is the case the mixture is too polar. The carotenes (orange to yellow band) should be at the solvent front with a cellulose band behind this (say at an Rf of 0.8). Carotenoids (rather than carotene) however are polar molecules and may be stuck to the base line (Rf=0) as an orange-brown band. I can't give you any reference as this is from experience. I would have used methyl ethyl ketone (MEK)/Pet Ether or cyclohexanone/Pet Ether mixtures but you are probably limited by the lab experiment.
The wikipedia reference below gives you the difference between carotene and carotenoid.
http://en.wikipedia.org/wiki/Carotene
Based on your experiment, it seems that the polarity of the pigments did not follow the expected pattern. This can be attributed to a few possibilities, and one of them is the presence of water in the solvent.
TLC (thin-layer chromatography) relies on the differential migration of compounds based on their polarity. In your case, the most polar pigments, such as chlorophyll, were expected to travel a shorter distance and be located near the bottom of the plate, while less polar pigments like carotenoids were expected to travel a longer distance and be located near the top.
To troubleshoot where you might have gone wrong, let's consider the presence of water in the solvent. Water can disrupt the separation of pigments because it is more polar than petroleum ether and acetone. If any water was present in the solvent mixture, it could have increased the overall polarity, leading to a reversal in the expected results.
Here are a few steps you can take to check if water was present in the solvent:
1. Check the purity of your solvents: Before performing the experiment, ensure that both petroleum ether and acetone are dry and free from any water contamination. Any water present in the solvents can affect the outcome.
2. Store solvents appropriately: Solvents like acetone are hygroscopic, meaning they can readily absorb water from the environment. Make sure to store them in tightly sealed bottles to minimize water absorption.
3. Drying the plate: Ensure that the TLC plate is thoroughly dried before spotting the pigments. Use an oven or a desiccator to remove any moisture and avoid introducing water to the system.
4. Investigate other possible factors: If ruling out water as a source of error, consider other factors that could have disrupted the expected results. This may include errors in spotting or using an incorrect ratio of solvent mixture.
By considering these steps and pinpointing the potential source of error, you can improve the accuracy and reliability of your TLC experiment.