Formal Report on Exp 5

7. Tape the strip to a pencil and rest the pencil on top of the jar so that the strip hangs into the jar. The goal is to have the end of the chromatography strip just touching the surface of the solvent solution, with the colored dots above the surface of the liquid. Make sure that the colored spots do not come in direct contact with the liquid in the bottom of the glass.

The solvent mixture we decided to use for our TLC was a 3:1 ratio of ethanol to ethyl acetate. Originally when testing plates, we found that ethanol was the solvent that created the most clear and vibrant image, while ethyl acetate made an image, in a much smaller amount of time, but this image was very light and unclear. At first we decided to use only ethanol for the final TLC, but after waiting 20 minutes and having the plate being nowhere near finishing, we decided to add some ethyl acetate to speed up the process. When testing, we added ethyl acetate 1 mL at a time until we found a combination of speed and clarity we were happy with. This is how we settled on the mixture of 75% ethanol and 25% ethyl acetate.

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.

The instructor's manual also parallels the laboratory manual, exercise by exercise, providing labels for unlabeled diagrams and answers to questions that appear in the laboratory reports. For some exercises, special instructional suggestions that propose alternative procedures, laboratory equipment, or laboratory techniques are provided.

The spots applied to the TLC plate must be above the level of the developing solvent or else the spots (compounds) will simply dissolve in the solvent and there will not be any spots left.

Abstract. Thin Layer Chromatography is a simple procedure that allows you to determine how many and what kind of compounds are in a mixture. By understanding the properties of the TLC system and how it combines with the functional groups of amino acids the retardation factor (Rf) values can be calculated and compared to specific hydropathy values of amino acids. Thin layer chromatography is used to separate and recognize a certain compound. The silica gel that is located on the outside of the TLC sheet functions as the stationary phase and the solvent mixture functions as the mobile phase.

Two TLC plates were prepared with pencil. Plate one was marked with five marks. The marks were placed 1 cm apart in this order 0 seconds, 15 seconds, 30 seconds, Fluorenone, and Fluorenol. A second TLC plates was then marked with four marks. The marks were 1 cm apart and placed as followed 60 seconds, 120 seconds, fluorenone, and fluorenol. A 5.0 mL conical vial was obtained and weighted. 0.20 g of fluorenone and 4.0 mL of methanol was added to the conical vial. We then placed a magnetic stir bar into the vial. The vial was then clamped in place on a magnetic stir plate. The mixture was stirred until all the solid had dissolved. At the time the 0 second sample was taken with a micropipet and the TLC number one was spotted. Approximately

Objective: The purpose of the experiment is to extract pigments from spinach leaves and separate them by column and thin layer chromatography, determining Rf values for the pigments.

Thin-layer chromatography (TLC) is a chromatography technique used to separate the components of a mixture. It can be used to monitor the progress of a reaction, determine the purity of a substance, and identify compounds present in a given mixture. TLC is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminum oxide, or cellulose. This layer of adsorbent is known as the stationary phase. After the sample has been applied on the plate, a solvent or solvent mixture is drawn up the plate via capillary action (known as the mobile phase).

By dissolving in a (easily evaporated) solvent the sample produces a very dilute solution (around 1%) that can be analysed. The stationary phase is the powdered silica gel or alumuina that has been coated onto a plastic sheet. By using a micropipette, spotting transfers a small amount of the dilute solution to one end of a TLC plate. By rapidly evaporating, the spotting solvent leaves behind a small spot on the material.

Development is placing the bottom of the TLC plate into a shallow pool of a development solvent, which then travels up the plate through capillary action. As the solvent travels up the plate, it moves over the original spot. A contest is set up between the silica gel plate or alumina plate and the development solvent for the spotted material. The very polar silica gel or alumina tries to hold the spot in its original place and the solvent tries to move the spot along with it as it travels up the plate. The outcome of the movement depends on the balance among three polarities which is of the plate, the development solvent and the spot material. If the development solvent is polar enough, the spot will move some distance away from its original

A development chamber was also set up with filter paper and 70% hexane -30% acetone as the solvent. This solvent was also used to dissolve the dried pigments, which were then spotted onto the TLC plate. The TLC plate was then placed into the development chamber and removed once the solvent traveled near the top. Image 1 shows the TLC plate after this experiment. Each of the pigments could be identified for each spot on the TLC plate. The top pigments that are yellow-orange are the carotenes; they are indicated under the extract and yellow band spots. In decreasing order from top to bottom, the spots are carotenes, pheophytin a, pheophytin b, chlorophyll a, chlorophyll b, and xanthophylls. Xanthophylls are yellow spots and are the last three spots towards the bottom of the plate. For the green band, there were no yellow spots and the green spots have the most color; this most likely proven that the green band are the chlorophyll pigments. However, almost all of the colors on the plate were not as intense as they should be; the pigments were spotted as small dots and thus, less concentration of each pigment interacted with the solvent. While performing thin-layer chromatography though, a possible error might have occurred, which was adding a little too much solvent in the development chamber. When the TLC plate was placed into the chamber, the solvent was above the line where the pigment spots were located. Nevertheless, all of the pigments in the carotenoids and chlorophylls were distinguishable on the plate. Data Table 1 shows the Rf value for each pigment spot, including the

The silica gel on the TLC plate is infused with a fluorescent substance that glows under ultraviolet (UV) light. The fluorescence will interfere with a spot and appear as a dark spot on a glowing background.

Chromatography, which was originally discovered and developed by Mikhail Semenovich Tswett in the early 1900s, is a laboratory technique used to separate and distinguish between components of a mixture. The various components of a sample mixture are known as analytes. Chromatography was originally used to separate the various molecules that make up plant pigmentation. Because these components, carotenes, chlorophyll, and xanthophylls, are different colors, the separation of them is responsible for the name of this laboratory technique. Throughout the early to mid-1900s, different styles of chromatography were developed and allowed this technique to be applicable to a wide variety of analyses and separation processes. Chromatography can be applied to analytical process, which aim to examine the presence and relative concentrations of various analytes within a sample. The goal of preparative chromatography is to separate analytes within a mixture for isolation and purification purposes. Chromatography was originally developed by Tswett for the purpose of isolation of particular compounds (10). Moreover, more recent advancements in chromatography are facilitating the separation and distinction of analytes that are extremely comparable to one another and increasing the resolution of the technique.

In the Thin Layer Chromatography (TLC), the eluates were applied on a 5 cm x 8 cm per coated TLC plate by spotting 10 times. The member performing the application allowed each spot to dry before applying the next and made sure the spots are small as possible. Meanwhile, another member prepared the developing chamber by placing an approximate amount of the solvent system, DCM-hexane (1:1). The inner wall of the chamber with filter paper was lined, covered with a watch glass, and was then allowed to