Discussion Questions
1. What is chromatography? Describe two uses in society. [3 Marks]
Chromatography is a process whereby the components of a solution or mixture are separated into individually identifiable substances based upon their physical characteristics. These characteristics can include molecule size, hydrophobicity, and more. All forms of chromatography involve a stationary phase and a mobile phase. One use of chromatography in society is its use in the field of drug testing. By having a urine sample undergo chromatography the presence of illegal substances can be identified as they separate from the other components of the urine. This is useful in the fields of athletics and criminal justice. A second use of chromatography is the analysis of water. Through the use of chromatography pollutants and other additives to water sources can be isolated and identified. This process allows for municipal water to be quality controlled for the general populous.
2. List three chromatographic techniques (besides paper chromatography), and briefly state how they work. [3 Marks]
One chromatographic technique is "Thin Layer Chromatography". Thin layer chromatography (TLC) uses a thin layer of silica gel spread on an inert piece of glass or metal as its stationary component. Once the sample is deposited on the gel a solvent is added and the different components of the sample migrate up the gel at different rates. The different components separate and can be analyzed based on their migration distance. A second chromatographic technique is gas chromatography. Gas chromatography uses a high boiling liquid as its stationary phase and an inert gas such as helium as its mobile phase. A vapourized sample is placed into a tube containing the gas and liquid. The gas forces the sample to interact with the liquid along the walls of the tube and separate. As the separated substances leave the tube they pass through a sensor which records the identity and quantity of each substance that composed the original sample. This information is represented on a graph. A third chromatographic technique is gel filtration chromatography. A bed of porous gel beads acts as the stationary phase while liquid solvents act as the mobile phase. Using different solvents the sample is passed through the bed of beads. Smaller molecules permeate the porous gel beads and move through the bed slower while the larger molecules pass through with less resistance. This separates the different substances based on molecular shape and size.
3. What is Rf? Show the calculation of the green fragment from your lab results. [2 Marks]
Rf is a short form used to represent Retention Factor. Retention factor is the distance a substance will travel along the stationary phase when in the presence of a specific solvent. This value is constant for each substance given that all other factors remain constant.
4. List and briefly explain two factors that would determine your chromatographic profile (theory of paper chromatography separation). [2 Marks]
One factor that helps to determine the chromatographic profile of the sample is the polarity of the molecule. The solvent that was used in the lab was at a 9:1 polar to non-polar ratio. As the solvent was more polar the polar components of the sample were more readily dissolved. This resulted in an increased ability to migrate up the filter paper. Another factor that helps to determine the chromatographic profile of the sample is the variety of chromatography paper which is used. Different varieties of paper have different characteristics which affect a substance's ability to travel through it. Paper with more dense characteristics limit the movement of substances whereas less dense paper is conducive to the migration of the various samples.
5. List three factors that would improve your chromatogram (sources of error). [3 Marks]
One source of variability in the lab was the lack of a predetermined incubation period. As each group exposed the ethanol and spinach mixture to heat for different amounts of time a different amount of the green substance was extracted from the spinach. These varying concentrations in the initial sample could affect the accuracy of the final product between the different groups. Another source of variability within the lab was the number of samples deposited on the chromatography paper. The number of drops being placed on the paper from the capillary tube was not specified. As a result each group could have a different concentration of the sample being tested. This variability in concentration could affect the final results. A third factor which added to the variability found in the lab was the lack of a predetermined value for the amount of ethanol used to extract the chloroplast pigments from the spinach. Each group used a different amount of ethanol based on an approximation and as a result the concentration in each sample varied. All in all the slight variation between each group's interpretation of the vague instructions affected the accuracy of the final result. By changing the concentration of the substance being tested the rate of migration was affected and the possibility for difference in Rf values was increased. Note that Rf values are only constant when all other variables remain the same but due to the variation between groups a uniform Rf value may not be found.
6. List two proposals for future experiments. [2 Marks]
A future experiment which could generate additional information pertaining to the chloroplast pigments in spinach leaves involves the use of gas chromatography. Through the use of gas chromatography the exact values of each component of the sample could be analyzed. By identifying exact molecules and the concentrations in which they are present more in depth analysis of chloroplast pigments in spinach could be undertaken. A second lab proposal would be the comparison of varying vegetables. By having a variety of different lettuces, and other green plants undergo chromatography comparisons could be drawn between samples. The possibility for identifying a component present in all or many of the samples arises and comparative studies could be done expanding on the results. By analysing the different pigments present in different samples information can be inferred about environmental effects on the presence of different pigments.
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