Spinach Paper Chromatography

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.

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.

The electromagnetic spectrum is the range of wavelengths over which electromagnetic radiation extends (Merriam-Webster Dictionary). The visible part of the spectrum is light and we can see colors from blue to red. On the left side of the spectrum is blue where the wavelength is shorter. On the right side of the spectrum is red where the wavelength is much longer than the blue end. These wavelengths are called the visible spectrum and an example of this is a rainbow. For a light wave to be absorbed by an object, the single frequency light wave must come in contact with the object. Although light colors reflect part of the visible light, black absorbs all energy and wavelengths.

Scientists use an instrument called a spectrometer to quantitatively determine the amount of light absorbed by a solution. The primary inner parts of a typical spectrometer are described below. The spectrometer has a light source that emits white light containing a vast mixture of different wavelengths of electromagnetic radiation. The wavelength of interest is then selected using a monochromator (“mono” meaning one and “chromate” meaning color) and an additional exit slit. The separation of white light into different colors (wavelengths) is known as diffraction. The selected light then reaches the sample and depending on how the light interacts with the chemical compound of interest, some of the light is absorbed and some passes straight through. By comparing the amount of light entering the sample (P0) with the amount of light reaching the detector (P), the spectrometer is able to tell how much light is absorbed by the sample.

My lab partners and I performed an experiment that involved placing spinach disks into separate cups of distilled water (dH2O) and 0.2% sodium bicarbonate (NaHCO3) solution to examine photosynthesis in leaf tissue (Department of EEB, 2015). Discovering that the spinach disks quickly floated to the top of the 0.2% NaHCO3 solution and not in dH2O, we wondered if varied concentrations of carbonation would affect the rate of photosynthesis (PS). We tested this by halving the 0.2% NaHCO3 solution (using equal parts dH2O and 0.2% NaHCO3 solution to make 0.1% NaHCO3 solution). I hypothesize that if the spinach disks are placed in the 0.1% NaHCO3 solution, then they will have a slower PS compared to the disks placed in 0.2% NaHCO3. CO2

Which one of the statements concerning valence bond (VB) and molecular orbital (MO) bond theories is correct?

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.

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

Oparina/Miller hypothesis theorized was because of absence of free oxygen, chemicals, and so on could shape inorganic mixes for emerged on this planet.

Lab 8: Investigation 5 Lab question: How do you separate molecules that are attracted to one another? Procedure: Place a small amount of solvent in separate beakers. Then place the mixture on chromatography paper and put the paper in each beaker. Controls:

Pigments extracted from different greens have different polarities and may be different colors. Mixed pigments can be separated using chromatography paper. Chromatography paper is able to separate mixed pigments due to their polarity and solubility. Pigments of chlorophyll a, chlorophyll b and beta carotene will be separated on chromatography paper because each has its own polarity and solubility, which results in different distance traveled up the paper. Beta carotene is non-polar so it travels the highest distance, followed by chlorophyll a. Chlorophyll b is the most polar; therefore, it travels the shortest distance. The separated pigments on the chromatography paper can be eluted in acetone and absorbance spectrum is

The concepts of pigments in metabolism are applicable to this condition and are important in societal understanding. Pigments can be found in leaves, medicine, and food. Pigments contain chlorophyll a and b depending on the type of plant, where chorin wavelengths of both colors of blue and yellow are shown in the white light. This color gives the plant a green pigment. Plants reflecting green pigments are when the bouncing through the accessory pigments, going to the antenna complex, and then going to the reaction center occurs on a leaf. This bouncing of color of the leaf happens during the process of photosynthesis and absorbing all other colors in ROYGBIV. Anthocyanins comes from Greek origins meaning anthos =flower and kyanos=blue. Anthocyanin pigmentation is evident in foods mainly in fruits and vegetables, resulting in the blues, greens, purples, and red flavonoid pigments

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.

Chromatography Investigation Chromatography is a highly regarded technique used to separate the components of a mixture. It is based on the principle that each component possesses a unique affinity for a stationary phase and a mobile phase. The components that are more inclined to enter the mobile phase will migrate further on the chromatogram and distinguish themselves from the other components. The type of solvent used in chromatography is known to directly affect the separation of the mixture. In this experiment, thin-layer and column chromatography will be utilized to separate the numerous chlorophyll and carotenoid pigments of a spinach extract.

Gel-Filtration Chromatography is a commonly used method used in order purify a protein from a mixture, by means of separations. Different biomolecules differ in size, or their molecular weight. In the gel matrix inside the chromatography column, there are gel beads which are porous to allow certain sized molecules to enter. The molecules that are able to enter the pores of the gel, are held in stationary phase and will elute from the column later on, these are usually smaller, to medium sized molecules. Larger molecules that are not able to fit in the pores will elute out of the column first, they are involved in mobile phase where they just go straight through the column without interacting with the gel beads. Smaller molecules will have a higher elution volume, while the larger molecules will have a lower elution volume. The volume to elute the protein is inversely proportional to the molecules size.