Investigation using thin layer of chromatography to separate photosynthesis pigment

The overall separation depends upon how strongly attracted the chemicals are to the mobile and          the stationary phases. This produces a chromatogram where different samples can be compared to find       the pigment group.

Thin layer chromatography is also like paper chromatography, but the stationary phase is a glass plate with a uniform layer of silica gel instead of paper. TlC chromatography has some advantages over paper chromatography such as the moving phase can move more quickly through the stationary phase, TLC also tend to produce more chromatograms by showing a bigger separation of the components which is then easier to analyse

Chromatography is used for separation. It can be applied in small scale for laboratory use or in large scale for industrial use. Chromatography can be used in the food industry to control the quality of the food and this is done by separating and analyzing the vitamins, preservatives, amino acids and proteins its also able to detect and separate containments such as aflatoxin. Chromatography can be used for a range of other purpose such as finding drug compounds in urine or other body fluids.

Aim
Paper and Tlc chromatography can be used to separate mixed chemicals and Rf value can then be calculated to find the pigments group

Equipment’s

• Pestle and mortar
• Leaves (Spinacia oleracea plant species)
• Pencil
• Ruler
• Goggles
• Propanone
• Chromatography solvent
• Filter Paper
• Funnel
• Test tube
• Micropipette
• Black polythene bag
• Chromatography tank in fume unit
• Thin layer silica gel chromatography plate
• Paper chromatography

Method

1. Firstly, place the leaves in the mortar then cover it with propanone, making sure to much isn't added
2. Then grind the leaves thoroughly with the pestle until a smooth pulp is obtained
3. Keep grinding the leaves until a dark green smooth paste is obtained, add more propanone if needed
4. Afterwards, fold the filter paper and place this in a funnel then pace the gunnel in a test tube
5. Pour the extracted green pigment into the funnel, collecting the filtered pigments in the test tube
6. Using a pencil and a ruler draw a line across a thin layer silica gel chromatography plate about 1cm from the end
7. Then, by using a micropipette transfer the pigments extracted to the TLC plate, creating a small spot in the center of the pencil line
8. Wait some few seconds to allow the spot to dry then add another spot. Repeat at least six more times, keeping the spot as small as possible
9. Transfer the TLC plate into the chromatography tank in the fume unit, making sure the solvent isn't touching the spot
10. Cover the tank with a black polythene bag whilst the chromatogram runs. Check every two minutes to check the solvent progress front
11. When the solvent front is roughly 2 mm from the top of the TLC plate remove the chromatogram and then mark the position of the solvent front in pencil and leave in the fume unit for solvent to evaporate
12. Mark the position of each colored pigment in pencil and take a photograph of the chromatogram as some colors fade rapidly
13. Measure the distance moved by the solvent front and the distance travelled by each pigment colors and record this in a table
14. Calculate the Rf value of each pigment and record the values in the table
15. Repeat the experiment using a paper chromatography
16. Finally using the information given, identify the pigments spots that’s been observed

Results and Calculations

Paper Chromatography

        Distance travelled by solvent

Therefore, Rf of Paper chromatography = 4.1

Rf of component A = 1.0/4.1= 0.24

Rf of component B = 2.0/4.1= 0.49

Rf of component C = 4.0/4.1= 0.98

TLC Chromatography

Rf = distance travelled by sample

        Distance travelled by solvent

Therefore, Rf of TLC chromatography = 5.1

Rf of component A = 0.6/5.1= 0.19

Rf of component B = 0.8/5.1= 0.23

Analysis

From the results I was able to observe that TLC chromatography gave me the most valid result close to the given Rf values as TLC chromatography was able to produce more chromatograms by showing a bigger separation of the components which is then easier to analyse.

Paper chromatography also gave me results as the first spot moved up by 3.5 for the Spinacia oleracea pigment and this gave a pigment of Pheophytin a, the second spot moved up by 3 giving the value of 3.8 for the Spinacia oleracea pigment and this also gave a pigment of Pheophytin a the final spot moved up by 2 giving the spot a distance of 4.0 for the Spinacia oleracea pigment and also gave a pigment of Pheophytin a. From the result for paper chromatography each pigments were the same and were high up the table meaning pheoythin a is very soluble

From the TLC chromatography result it gave a wider range of pigments compared to the paper chromatography. From the result table violaxanthin had the lowest Rf value whilst Pheophytin b had the highest Rf value this shows that Pheophytin b is the most soluble pigment with the specific solvent compared to violaxanthin which is the least soluble hence why it’s at the bottom

The speed at which a particular pigment moves will depend on its relative affinities for the two phases and it has no affinity for the water phase it will travel at a maximum speed and if the pigment has no affinity for the non-polar phase it will remain at the origin. The majority of the photosynthetic pigments such as chlorophyll b have some piece of the molecules have affinity for one solvent and portions with the affinity for the others and as a result these pigments will travel along with parts of them pulling in the stationary water phase and the greater the proportion of the pulling, the slower they move and as a result each pigments usually moves at specific rate, forming separate bands on the paper.

The proposed aim was correct, as the paper and tlc Chromatography did show that the solvent could be separated into various colors. There were range of colors that appeared during the experiment and the colors separated the way they did because of the differences in their molecular characteristics, specifically, their solubility in water and their rate of absorption by the paper and Tlc.

Evaluation

The experiment went well as results were obtained for both Tlc and paper chromatography but possible errors were include such as inaccurate measurements of the distances traveled by the solvent and mistakes when calculating the ratio traveled by the water and colors as the distance travelled wasn’t calculated from the center . Another error was when placing a dot on the paper and Tlc the dots were meant to be as tiny as possible but during our experiment the dots were a bit wide and this could affect our final result by making it inaccurate.

The sample should also be above the solvent level as if the solvent level covers the sample, this would mean the sample spot will be rinsed off into the solvent before it travels up the paper and TLC chromatography plate

The experiment could be improved in a number of ways such as using different type of plant as this will then give a range of result as for the paper chromatography the final result were more or less the same, also when calculating the Rf value we should calculate the distance from the center as this is more accurate also repeating the experiment is the biggest way to improve our result as there was no time to repeat the experiment so if we had more time we would make sure to reduce most of our error, also making more concentrated extracted would improve our result as the extract wasn’t as concentrated as it should’ve been during the experiment.

Risk Assessment

• Propanone: Highly flammable and can cause eyes and skin damage. To reduce the risk of this goggles should always be worn and ensure to keep away from naked skin.
• Chromatography Solvents: Highly flammable and harmful, lid should be kept on the chromatography tank as much as possible also avoid breathing vapor and goggles should always be worn during the experiment.

Reference