Extraction Of Pigments From Spinach

Have you ever really wondered how different variables can affect how plants go through photosynthesis? Well, in this experiment, the purpose was to see how various environmental conditions can affect the overall photosynthetic capacity of a specific plant. The factors, light, darkness, cold, and heat were applied to see how the different components would affect the photosynthesis on spinach plants. Each group was given a different factor to test. Out group was given the light factor. The hypothesis for this experiment is that when adding light as a factor, the light will affect the overall plant photosynthesis.

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

Experiment 55 consists of devising a separation and purification scheme for a three component mixture. The overall objective is to isolate in pure form two of the three compounds. This was done using extraction, solubility, crystallization and vacuum filtration. The experiment was carried out two times, both of which were successful.

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.

The cell membrane (Plasma membrane) functions to provide cell support, cell stability and control entry and exit of materials from the cell. This study was conducted to test the effects of environmental conditions such as the on beet root cell membrane (Beta vulgaris). Five trials using varied pH concentrations were tested and absorbance rates were monitored. The experimental results showed that the protein function decreased sequentially when the pH decreased. This allowed the betacyanin dye to leak out which created the color that was needed to determine the intensity and therefore the effect of the circumstances. This supported the hypothesis that the more acidic or basic the environmental condition around the beet cell, the more permeable the, membrane indicated by color intensity. Pigment leakage in the solution was analyzed by using a spectrophotometer.

The objective of this extraction experiment was to achieve a comprehensive understanding, as well as master the practice, of the technique of separating various individual components of a compound.

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

According to Oregon State University, there are three types of classes that plant pigments can fall under. They are, chlorophylls, carotenoids, and anthocyanins (Carol Savonen 2003). Often the pigments that fall under the category of chlorophyll look green to the eye. This is because they are absorbing all colors of the spectrum except for green. Next, the pigments that are known as carotenoids are going to look yellow or orange, due to the same reasoning. They are absorbing all colors but yellow and orange. Lastly, the anthocyanins will absorb blue and green, reflecting either purple or red (Volner Silva 2016). As the chromatography takes place and the solution is separated into its component substances, they will display the colors of the specific pigments present in them. For example, if Chlorophyll A is present in a leaf, the chromatography will show a blue-green color, while if xanthophylls, another type of plant pigment, are present, the chromatography will exhibit a yellow color. On the other hand, if the anthocyanin pigments are present in the leaf, the chromatography experiment should have resulted in red, blue, purple, or magenta colors (Joy Alkema and Spencer L. Seagerl 1982). Specific to this experiment, the green leaves should display more pigments under the chlorophyll category, while on the other hand, the darker leaves should display more pigments that fall

On a thin chromatography plate, five spots were placed ( as shown in table 2) and the plate was developed using chloroform/methanol. This was later visualized with dragendorff’s reagent under the UV light. All separated components were observed, identified and recorded.

This experiment demonstrates the effects of pH on the rate of photosynthesis by examining the behavior of leaf disks in different pH solutions under light. In this experiment, we used five different pH levels: pH 5, pH 6, pH 7, pH 8 and pH 9. These solutions were created using a combination of hydrochloric acid and sodium hydroxide. Spinancia olcerea or spinach, leaves were used in the experiment to examine the effects of pH on the rate of photosynthesis. The rate of photosynthesis was measured by counting the number of leaf disks that rose to the surface of the solution after each minute. In acidic solutions, the rate of photosynthesis increased while in basic solutions, the rate of photosynthesis decreased.

After wearing the gloves we obtained a chromatography vial from professor and label it with my and my peer initials. We dried up the chromatography vial in fume hood and added 1 ml of chromatography solvent to the vial. Then we took a chromatography strip and measure it 1.5 cm with ruler from one end of the strip and drew a line with pencil we cut two small pieces below the pencil line to form a pointed end. We applied spinach on the strip using quarter to rub the spinach leaf on the line that we drew on the strip and put it into the chromatography vial and placed that in fume hood. We observed as the solvent was moving up the chromatography strip by capillary action. When the solvent was reached approximately 1 cm from the top of the strip then we removed the cap from the vial. We took out the strip from the vial using forceps and marked up the location of the solvent front because it evaporates quickly. We measure out the distance as well as the pigment in order to find out the rf value. Moreover we compared rf values to the one in reference list in order to identify the

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.