Separation of Chlorophyll a, Chlorophyll B, and Beta Carotene by Paper Chromatography

There are two main types of chlorophyll, chlorophyll a which absorbs wavelengths of 430nm (blue) and 662 (red) and is the main photosynthetic pigment, and chlorophyll b, which doesn’t directly participate in the photosynthetic process, but is capable of donating its energy to chlorophyll a

The image above is our chromatogram, you can see at the top the orange bands of a carotene, next are the bands of xanthophylls which are yellow. The next light green bands directly bellow the yellow is chlorophyll a, the darker green bands are chlorophyll b. In order to test absorbance of the chlorophylls they were cut out of this chromatogram submerged into acetone and then used to get an absorption spectrum as seen below.

Lycopene and β-carotene are separated using thin layer chromatography (TLC). A mixture of tomato paste containing dissolved lycopene and β-carotene is first spotted on a TLC plate. The technique uses a stationary phase layer, on which the substances stick, and a mobile phase, which migrates upwards the plate and carries the components of the mixture with itself (Chemguide). Depending on their respective polarity, lycopene and β-carotene stick more or less to the stationary phase and so they migrate up the TLC plate at different rates (Chemguide). Thus, as the chromatography is completed, the two components will appear as distinct dots at different heights on the plate. Lycopene has a bright orange color, whereas β-carotene is light yellow.

Betalaines are the red pigments of beetroot. They are water-soluble and exist as internal salts in the sap vacuoles of plant cells. Betalaines are made up of red betacyanins and yellow betaxanthins. Betacyanines include about 90% of beetroot betalaines. The most important betacyanin is betain. This makes up 75-95% of the total colouring found in the beetroot. Jm

For part one of this experiment, I only experienced separation of colors with the green and brown M&M’s, along with the yellow food coloring. The green M&M separated into yellow and blue, with blue travelling farther up the paper. It is not surprising that green separated into blue and yellow because those are the primary colors that make up green. The brown M&M separated into red and orange, with the orange travelling farther up the paper. Finally, the yellow food coloring separated into yellow and red, with yellow travelling farther up the paper. This could be because it was such a concentrated, small amount of food coloring. The colors that didn’t travel very far up the paper, such as orange and brown, are probably less soluble than the others, like blue and green.

Answer: Once the chromatogram has been completed and is ready to be measured and calculated, on the plate that was used to perform the chromatogram you should see where the red and blue have completely separated. The red food coloring dye should be lower on the plate than the blue food coloring dye.

In the beginning of this experiment, our TA added water, salt, and 75/25 hexane/acetone to spinach leaves to a blender and blended the mixture to assume equal amounts for each group and to avoid erros if each student had to do the blending. The addition of the water to the mixture allowed the it to separate into a distinct organic layer after being run in a centrifuge, which was available to be collected at the top of the centrifuge. Salt reduces solubility, which will force the organic parts of the mixture (the desired pigments for example) to separate into the organic layer at the top. Lastly, 75/25 hexane/acetone is added because this is a moderately polar solvent and will useful for both the non-polar and polar pigments present within the spinach leaves. A mixed solution of hexanes and acetone must be used because acetone is very polar, while hexane in very non-polar, and the spinach leaves contain both non-polar and polar pigments in them that are important in the extraction and for further analysis. The mixture was placed in the centrifuge so the solids in the mixture (mostly cellulose) could be separated from the liquids into separate distinct layers for further extraction and testing. In the tube, the organic substances separated into the top layer, whereas the water layer remains at the bottom of the tube below the solid layer made up of mainly cellulose.

Abstract: The purpose of this lab is to separate and identify pigments and other molecules within plant cells by a process called chromatography. We will also be measuring the rate of photosynthesis in isolated chloroplasts. Beta carotene, the most abundant carotene in plants, is carried along near the solvent front because it is very soluble in the solvent being used and because it forms no hydrogen bonds with cellulose. Xanthophyll is found further from the solvent font because it is less soluble in the solvent and has been slowed down by hydrogen bonding to the cellulose. Chlorophylls contain oxygen and nitrogen and are bound more tightly to the paper than the other pigments.

This lab was conducted to explore the light energy, pigments and the rate of photosynthesis in magnolia leaves. In experiment one a magnolia leaf was used to see the separation of primary and accessory plant pigments using a process called paper chromatography. The importance of this process was to discover which pigment had the highest band along a piece of filter paper and identify various plant pigments in a magnolia leaf such as xanthophyll, chlorophyll a, chlorophyll b, and carotenoids that aid magnolia leaves during photosynthesis. Based on the conducted experiment, it can be concluded that chlorophyll a was the pigment that showed the highest band on the piece filter paper which means that chlorophyll a is the primary pigment in photosynthesis

The sixth lab I completed in Biology 101 taught me how autotrophs (self-feeders) and heterotrophs (other-feeders) make organic food molecules by using photosynthesis. Photosynthesis uses the energy from the sun and it is captured and stored in the chemical bonds of organic molecules. The sunlight consists of different wavelengths of light. In plant chloroplasts, they have different pigments that capture different wavelengths of light. Light capturing pigments in green plants are called chlorophylls and these absorb all the colors of light except green, which is mostly reflected. To separate molecules from each other according to their solubility in a particular solvent is done by the process of chromatography. This basically means that polar

When the beetroot sample is heated for a longer period of time, it is exposed to the vigorous and frequent vibrating molecules for a longer duration. This further disrupts the cell membranes making them more fragile, thus more pigment will leak out into the external solution. With more leakage of the betacyanin pigment, the % transmittance should be lower, indicating that the % of light that is able to pass through the beetroot solution should be less as duration heated increases.

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.

photosynthesis happens in two stages: light reaction and carbon fixation also known as calvin cycle.light reaction TAKES PLACE IN THYLAKOID USE light energy to produces atp and nadph whereas, calvin cycle takes place instroma uses energy derived from light dependent reaction to make GA3P from CO2 ( Bio166 lab execise manual, 2015). the purpose of this experiment was to separate plant

For lab 12, it is hypothesized that chlorophylls a and b are present in a plant leaf and contribute to the starch production in photosynthesis. Also, products of photosynthesis will be present in leaf tissue exposed to red and blue light wavelengths for several days, but a decreased presence in leaf tissue exposed to green and black light wavelengths. In lab 13, it is expected that since chlorophylls a and b are more polar and smaller molecules than the anthyocyanins and carotenoids, they will travel higher up the chromatography paper than the other pigments.

The next step was to place the strip of chromatography paper on a paper towel. Then dip a capillary tube into the plant pigment extract (spinach pigment extract) provided by the teacher. The tube will fill on its own. We applied the extract to the pencil line on the paper, blew the strip dry, and repeated it three to four times until the line on the paper is a dark