Organic Chemistry Laboratory 1
Column and Thin Layer Chromatography: The Separation of Spinach Pigments Russ Hoburg
The main goal in doing the column and thin layer chromatography was to separate spinach extract into its components based on polarity and then to analyze the components. The separation of the spinach extract was done using the column chromatography with the wet/slurry packing method. Alumina was inserted into the column to act as the stationary phase. After the components were separated and the solvents boiled out, the thin layer chromatography was performed to analyze the components of the spinach extract. This was done using a TLC plate and spotting each of the components on to the plate as well as spotting the standard spinach extract. A beaker was used with the developing solvent, filter paper, and TLC plate inside and a watch glass over top of the beaker to perform the thin layer chromatography. After the solvent front (30% ethyl acetate in hexane) reached about an inch from the top, the TLC plate was removed from the beaker and the developing spots were marked. After implementing the column and thin layer chromatography, the Rf value for component one was .95, for component two it was .28 and .61, and for the third component it was .41. The first component was the same as one of the components of the standard but the final two components were slightly off from the components of the standard likely due to human error.
Chromatography is the technique of separating components of a mixture based on its physical properties. The separation is done between two phases known as the stationary phase and the mobile phase and depends on if one component is more likely to adhere to the stationary phase more than another component. Moreover, the stationary phase is almost always more polar than the mobile phase [Padias pg. 163]. The rate at which these components move through the column depends on the partition coeffient. This is the distribution of the components between the two phases and shows the affinity of each component to either the mobile or the stationary phase. Therefore the components of a mixture will have different partitioning coeffient depending on polarity, solubility, hydrogen bonding, and among other interactions. If a component has a high partitioning coeffient than it has more affinity to the stationary phase and vice versa. Partitioning Coefficient [Kp(x)] = [x] Stationary phase
[x] Mobile phase
There are different types of Chromatography that are most commonly performed based on the types of phases. These are gas-liquid chromatography and solid-liquid chromatograph. Gas-liquid chromatography is just known as gas chromatography and has a liquid stationary phase and a gas mobile phase. The separation is mostly based on volatility of the components of a mixture and the polar interactions with the stationary phase [Padias pg. 179]. This is done in an extremely hot temperature controlled oven with a coiled column inside. The different columns used, all consist of an inert solid support that is coated with a non-volatile liquid serving as the stationary phase. An inert gas carrier is used to push the components through the column. A small syringe is used to transfer the mixture into the oven and a detector is used to detect the components of a mixture, since only a small concentration of the components is used. An attenuator can also be used to weaken the signal to the recorder so the peaks fit on the paper. There are two types of solid-liquid chromatography most often used in laboratory experiments. They are column chromatography and thin layer chromatography. Both kinds have a solid stationary phase and a liquid mobile phase. Moreover, both usually use silica gel or alumina as the stationary phase but is not limited to these. During the column chromatography the column is an actual pipet column and usually...