Chromatography Lab Used to Examine Spinach and Red Cabbage

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 purpose of this experiment is to determine the maximum absorbance of fast green, and the chlorophylls, also in the case of fast green create a concentration curve to determine an unknown substance. Each test will use the spectrophotometer.

Set up work station by gathering weighing dish and graduated cylinder and One Quart Zip-Lock® bag.

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

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.

1. Paper Chromatography is a method used for the separation of colors which are also referred to as colored chemicals/substances or pigments. This method is used for experiments, to identify coloring agents and to separate out a compound into its various components.

The argument in the scenario was being made between Abby and Estelle over the pigments in the multicolored coleus plants. Abby was certain that the green section contained chlorophyll, whereas, Estelle claimed that both the green and purple parts of the coleus plants consisted of chlorophyll. A paper chromatography test was conducted to clearly separate the mixture of the pigments in which the mixture was dropped onto a slip of paper and the end was then placed into a solvent.

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

Chromatography is a separation technique in which the mixture to be separated is dissolved in a solvent and the resulting solution, often called the mobile phase, is then passed through or over another material, the stationary phase. The separation of the original mixture depends on how strongly each component is attracted to the stationary phase. Substances that are attracted strongly to the stationary phase will be retarded and not move alone with the mobile phase. Weakly attracted substances will move more rapidly with the mobile phase.

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.

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

This was an investigation into the effects of different wavelengths of light on methylene blue and carmine red on the absorbance value on a spectrophotometer. A spectrophotometer is used to measure light intensity by emitting a single light source through a cuvette of coloured solution. The particles in the solution, which are coloured, absorb the light depending on how concentrated it is and this produces an electronic reading from the photometer which is the absorbance value. The maximum absorption was found for both solutions and was used to calculate the molar extinction coefficient of methylene blue. An unknown concentration of methylene blue was calculated by using

The purpose of this lab is to determine which pigments in a plant support or effect photosynthesis, based on starch production, which wavelengths of light are involved in photosynthesis, and identify plant pigments found in a plant leaf by means of paper chromatography.

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