Paper Chromatography Lab

From this graph and chart we can see that the higher the concentration the higher the absorbance, all the different concentrations were tested at the same wavelength (625nm). Also we can determine our unknown substances concentration by using the absorbance we got for it. The red dot on the graph followed by the line towards the horizontal axis indicates that the concentration of fast green was 34% or 5.1x10-3.

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.

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.

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

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.

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.

The procedures for experiment A, B, and C all start the same. The first step is to put on goggles and get the data collection device set properly. The labquest needs to be plugged into the colorimeter accurately so that a click is heard when putting it in. The labquest needs to be reading digitally and the colorimeter needs to be set to 635 nm. Then shake the chloroplast solution and take a clean cuvette and fill it with 3 mL of distilled water, 3 drops of the chloroplast solution, and cap it. This is used as a blank to calibrate your labquest. Double check that the labquest is reading absorbance, this assures that the colorimeter is plugged into the labquest accurately. Insert the blank into the colorimeter and hit the calibration button. Take out the blank and empty it. The labquest is now set to experiment with. Make sure that the heat bank is set in front of the lamp and that the lamp is on. The cuvette must be placed on the opposite side of the heat bank in the path of light in the box so that no other light can interfere with the experiment.

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

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

It is possible to separate these pigments from each other using a technique called paper chromatography. In this process, plant tissue extract is applied to a piece of chromatography paper. “A solvent is allowed to travel up the paper, and if the pigment is soluble in the solvent, it will be carried along with it.” (Benya, 2009) Different pigments have different affinities for the solvents or polarity and will travel at different rates. Chlorophyll, anthyocyanins, and carotenoids are typically non-polar.

Throughout the experiment, Allura Red was continually mixed with distilled water to form many different concentration solutions. The colorimeter was then used to determine the absorbance of each solution. Red light is around 650 nanometers on the visible light spectrum, but the colorimeter was set at 470 nanometers. This was done because Allura Red has the greatest absorbance when exposed to a light that is blue in color, which is why 470 nanometers was used.

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

First, we collected spinach leaves and cut out 60 disks using cork borer and then placed them in a syringe. Then, we added sodium bicarbonate into the syringe leaving about a third of the syringe empty and replace the plunger to the syringe. We aspirated the leaves by pulling the plunger down

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.

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.