Column and Thin Layer Chromatography: the Separation of Spinach Pigments

The spinach plants were hole punched, mixed with 0.2% NaOH and dish detergent, placed in a syringe with the solution to have the oxygen

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.

16) Heat some of the hexane to boiling and place the solid to be crystallized into an Erlenmeyer flask.

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.

To tube 2 and tube 3 a boiling chip is added. The two tubes are boiled to remove any residual ether. Next, the tubes are cooled to room temperature and placed into an ice bath to allow for crystallization. The solution is then removed from the solid in each tube and discarded. To tube 2 and 3 ~0.5 ml of H2O is added for recrystallization, the tubes

The purpose of this experiment is to determine the specific pigments that are found in each of the chosen plant leaves, as well as, discover the intermolecular forces present in each leaf. This is accomplished by using a technique called chromatography, which splits a mixture or solution into its different parts based on the mixtures ability to dissolve in a chosen solvent. Chromatography works by placing a strip of chromatography paper, that has a small amount of mixture on it, into a cup of the specific solvent chosen. In this experiment, one hundred percent acetone was chosen to be the solvent. Once the small amount of the mixture is aligned with the solvent, the mixture will begin to separate and rise by capillary action based on the solubility,

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.

     In liquid chromatography, the separator is called the column and consists in most cases of a tube filled with porous material called the stationary phase. A liquid, called the mobile phase, flows through the tube between the particles of stationary phase material. A liquid sample is taken from a mixture to be analyzed and introduced to a part of the system that is at elevated pressure. The sample is then transported to a separator by the flow in the system.

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

Exploring Thin Layer Chromatography Thin layer chromatography (TLC) is used to to identify organic compounds with common over-the-counter medicine; and the relative strength of the intermolecular forces and how they affect the compound’s polarity within the medications tested. TLC is the physical separation of a mixture into its individual components by distributing the components between a stationary phase, or silica gel; and a mobile phase, or movement of sample up the TLC plate to the solvent front. The mobile phase is considered to be non-polar, the non-polar components of the sample will travel farther up the TLC plate than the polar components that will stay near the stationary phase.

Different components move up the strip at different rates based on their polarity, size, and solubility. The ratio of the distance moved by a pigment to the distance moved by the solvent, its retention factor, is characteristic for each pigment. Thin layer chromatography allows the separation of molecules based on their affinity for the mobile phase, which is usually non-polar hydrophobic. The Rf value for light-grown barley (0.974) is greater than the Rf value for dark-grown barley (0.692) (Table 1).

The separation of the spinach is caused by each compound’s solubility and its adsorption to the silica gel. Each compound is dissolved in the solutions to a different extent, and they travel with it as it moves up the TLC strip. The compounds will be attracted to the OH groups on the silica’s surface if they are polar, causing more polar molecules to move more slowly up the strip. The compounds are separated when they all move at different speeds up the TLC.

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

AIM : Thin-Layer Chromatography can show many different characteristics of a mixture. It is recognized for isolation , separation ,identification, and anaylsis of the mixture’s components. The purpose of this experiment is to separate carbohydrates into its pure components such as mixtures of monosacrides by TLC. TLC is used to identify sugars in normal and pancreatic disease urine, the procedure is easy and reproducible .