Extraction of Chlorophyll from Spinach Leaves
2-15-11 Purpose: The purpose of this experiment was to take spinach leaves and extract the chlorophyll and carotenoid pigments by using acetone as the solvent. The chlorophyll and carotenoid pigments were extracted by using column chromography and alumina was used as the solvent. Solvents of different polarities were used, starting with the least polar, to extract the certain components from the leaves. They were then analyzed by using thin- layer chromatography. Procedure: The first part of the experiment dealt with breaking down the spinach leaves in a mortar and pestle.
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Acetone was added to this to help with the breakdown of the spinach leaves. Once the spinach leaves were broke down enough that you could see the particles clearly, they were all put into a centrifuge tube. It was sometimes necessary to add a little bit more acetone if it evaporated. Next, 2 mL of hexane was added to the centrifuge tube. The tube then had 2 mL of water added to it. This was shaken up and then centrifuged for a couple of minutes so that the layers would separate and to get rid of the emulsion, which was the green layer.
Once the centrifuging process was completed, the bottom layer was removed using a pipet. This layer was the hexane layer. A column was assembled for the hexane layer to travel through. This column was a pipet with a little bit of cotton down at the bottom and . 5g of sodium sulfate towards the top. The sodium sulfate was necessary because it allows for the hexane to dry, or remove and water from the solution. The column was clamped down in a vertical position and a test tube was placed under it which was labeled E.
The hexane was transferred to the column using a pipet. Once it was completely drained, . 5mL of hexane was added to the column so that it was known that all of the pigments were extracted from the sodium sulfate. Tube E was then put in a warm water bath with air flowing onto it to evaporate it. The residue that was left after the evaporation process was dissolved into . 5mL of hexane. This was then stoppered and put aside to use later in the experiment. The next portion of the lab was the process of the column chromatography.
The column was set up with the cotton in the bottom and 1. 25g of alumina on top. The four different polar solutions that could be used in this process were hexane, 70% hexane- 30% acetone, acetone, and 80% acetone- 20% methanol. The hexane was the least polar solution, and the 80% acetone- 20% methanol was the most polar solution. Next, the column was placed in a vertical position and test tube 1 was placed under it. About half of the pigment solution was added to the column after it had been rinsed with hexane into tube number 1.
The pigment solution would go in tube number 2. The first polar solution was added to the column as soon as the pigment had gone down far enough that it reached the top of the alumina. When the hexane was added, it was noted that a yellow band started to separate from the top of the column. This meant that the least polar solution was able to react to separate this particular band. This yellow band was drained into tube number 2 and once it was gone, tube number 3 was placed under the column.
The next most polar solution was added to the column in order to try to separate a green band from the column. The next polar solution was 70% hexane- 30% acetone. When this was added to the column, and green band formed and started to move down the column. However, this band was not collected into tube number 3. This band was collected in tube number 4, but, you did not switch to tube number 4 until the green band was at the bottom of the column. After the green band was collected, tube number 5 was placed under the column to collect whatever was left in the column.
This solution was not useful to the experiment. Tubes numbered 2 and 4, along with tube E were all evaporated. These were all stoppered and were to be used in the TLC analysis. In the TLC analysis, the first step was to draw a fine pencil line across the bottom of the silica gel plate. This was important because when the spotting process took place, you needed to know where the original spot was placed. Three micropipets were used to spot each of the solutions.
Tube number 2, which was the yellow solution, was spotted then 2 new pipets were used to spot both tube number 4 and tube E. But before they were spotted, each of the tubes had 70% hexane- 30% acetone added by a couple of drops. The TLC plate was placed into a chamber, which consisted of a beaker, aluminum foil cover, and the solution of 70% hexane- 30% acetone in the bottom. It was important that the solution in the bottom of the beaker was not higher than the pencil line on the TLC plate because you wanted to know for sure where the spots would go on the plate.
If the solution level was too high, then the spots would be very random on the TLC plate and you would not get accurate results. Results: Once the spots were found, they were circled with pencil so that it was known where they were when it dried, and what color they were. The “E” solution ended up having the most spots because it was the pigment fragments. The Rf values could be calculated for all of the spots by taking the distance traveled by the spot and dividing it by the total distance traveled by the solvent front.
The calculations are as shown: Rf= distance traveled by spot (cm)distance traveled by solvent front (cm) Tube E: 25. 1= . 39 (green) 4. 25. 1=. 82(yellow) . 55. 1= . 098 (yellow) 1. 55. 1= . 29 (yellow) 15. 1= . 196 (green) Tube 2 (yellow solution): 45. 1= . 78 (yellow) Tube 4 (green solution): 25. 1= . 39 (green) 1. 35. 1=. 254 (yellow) By looking at the table on page 142 of the text book, and through the Rf values that were calculated, it was possible to determine what each spot was in tubes numbered 2 and 4.
Through looking at my TLC plate, I concluded that the yellow spot that I found 4cm away from the origin was most likely to be carotene because this was the component with the highest Rf value. Unfortunately, this was the only spot that showed up on my TLC plate for solution number 2. As for solution number 4, I found two distinct spots which were located 2cm away from the origin and 1. 3cm away from the origin. I thought that tube number 4 contained both Chlorophyll b, Xanthophylls, and pheophytin.
The reasoning for this is that Chlorophhyll b has a relatively low Rf value, and it is also the color green, which is what showed up on the plate. I thought Xanthophylls was also in tube number 4 because it has the lowest Rf value and can be 3 yellow spots. It looks like there might have been little yellow spots on my plate. Also, Pheophytin a may have been in tube 4 because it shows up as a slightly gray color which I found on my plate also. Part 3 Questions: 1. Chlorophylls are a bigger particle so they would not move as fast on the column chromatography.
They also have lower Rf values for the same reason, they are bigger and denser so it would take them longer to travel up the TLC plate, so carotenes which are smaller can travel farther and therefore have the higher value. 3. You would be increasing the polarity of the developing solvent, so it would be most likely that the Rf values would decrease because the pigments would dissolve into the solvent. 4. No, each band did not result in a single component because it was almost impossible to cleanly separate out the bands so that you only got what was needed into the test tubes that were analyzed.