This Chromatography lab was done to identify pigments in different colored leaves and to evaluate the relationship between the pigments and photosynthesis. Spinach leaves and Zebrina leaves are used in this experiment. Mobile Phase: solvent into which sample is dissolved and helps sample travel through stationary phase. When these pigments were obtained they were used to measure the wavelengths by way of spectrophotometer of each and the total of all the pigments wavelength. I believe the spinach leaf in distilled water will have a higher pigment of chlorophyll a and the red leaf lettuce will have a higher pigment of anthocyanin. Remarkably, only two solvents were observed to separate the pigments, pure hexane; and 30% acetone, 70% hexane. This causes most plant leaves to appear green most of the time.
Because Rf values are consistent across consistent conditions, the value can be used to identify a substance by all scientists. Data Analysis and Determination of an Unknown Concentration of Chlorophyll Derived Solution This section of the lab will address some of the ways that analytical data are presented and evaluated. Take care to cut the strips so that they contain no more than one horizontal band of pigment. For some species you may need to add more than the suggested 10mL of acetone. A retention factor, also abbreviated as an Rf value, is the measurement of the distance that the sample had traveled. This different technique, known as paper chromatography, separates mixtures in a liquid into individual components. With a bit of luck five pigments can be identified: chlorophyll a blue-green , chlorophyll b yellow-green , xanthophylls yellow , carotene orange and phaeophytin grey, it is a breakdown product of chlorophyll.
By using chromatography we can manipulate these to move at different speeds through the system, thus separating them. Rf value also determines the solubility of a solute which in this lab were the photosynthetic pigments in a particular solvent which is the mixture of carbon tetrachloride and a scoop of anhydrous sodium sulfate. Background: A leaf is filled with many pigments. The beet leaf contained more pigments in the leaf compared to the spinach leaf. The chromatography paper usually contains pure cellulose, which is a linear polymer of D-glucose with β-1,4 linkages and many polar side groups.
Most reactions of organic compounds require extraction at some stage of product purification. Apply the extract with the edge of the applicator, not the wide face. These brilliant colors can be separated another way. Because different pigments absorb light of different wavelengths, by comparing the occurrence of different pigments we can deduce the plants' habitat; l We can do further experiment to determine the absorption spectrum of each pigment. The reason for this is that if the solvent and the samples mixed this would cause unreliable and faulty results. The mobile phase refers to any solvent known as the developing solvent that is partially miscible mixable with water.
Carotenoids also help aid the chlorophylls by also absorbing light. During those 15 minutes, the solvent will meet with the colored dot, and will continue to travel, separating color by color. In this way they become separated from one another and can be identified by their different colours and positions. This is used to isolate and identify the different lipids causing the different pigmentations. In the other development, the solvent consisted of 30% acetone and 70% ethanol, and without separation the R f was measured at 0. Make sure that the strip is in a perfectly vertical position.
A photosynthetic pigment accessory pigment;chloroplast pigment;antenna pigment is a pigment that is present in chloroplasts or photosynthetic bacteria and capture the light energy necessary for photosynthesis. Properties to be considered: Calcium Carbonate Naphthalene Solubility in water slightly insoluble Solubility in ethanol insoluble partially Melting point 825 degrees C 80. The components of a mixture are not chemically combined. In paper chromatography, paper marked with an unknown, such as plant extract, is placed in a beaker covered with a foil containing a specified solvents. In the hood wear gloves Prepare 25mL of chromatography solvent by first adding 25mL of Petroleum ether into your chromatography jar followed by 250µL of n-propanol.
Let it sit for about 15 minutes to let the solvent rise upon the paper. Generally, there is a yellow pigment band migrating with the solvent front , followed by 2 green bands, and finally 0—2 yellow bands. In this laboratory, we have observed how the particular affinities and solubilities of the various photosynthetic pigments carotenes, xanthophylls, chlorophyll a, chlorophyll b from the chloroplast of three different plants Zea mays,Hibiscus rosa-sinensis, Codiaeum variegatum cause them to migrate up a piece of chromatography paper at different rates. Set the Spectronic20+® spectrophotometer to the wavelength that gave maximal absorption for chlorophyll derivative in Part A and zero the instrument using water as the blank. Photosynthetic pigment is located in the chloroplast of the leaf. Chlorophyll b passes its exited electrons to chlorophyll a, which passes its excited electrons to other molecules, which store the energy as chemical potential energy.
These pigments are divided into two categories primary chlorophyll and accessory carotenoids pigments. United States of America: Lansing Community College. The molecules do this by absorbing specific wavelenghts of light rays red and blue-violet light which excite electrons contained within the double bonds of the porphyrin ring of the chlorophyll molecule. Take out the chromatography strip when the solvent front comes close to the top. Capillary action makes water draw up the paper. The technique is based on the fact that each substance in a mixture has a specific affinity for a solid surface and a specific solubility in different solvents.