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Soap Lab

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Chromatography is a method of separating and analyzing different mixtures of chemicals. You make separation of closely related compounds happen by allowing a mixture to seep through different absorbents such as paper or gel so that each compound is absorbed into a separate often colored layer.
The objective of this preparative experiment is for students to become familiar with a technique known as Chromatography. This technique is used to take mixtures and separate small quantities of them into their component parts. There are many types of chromatographic techniques, but the ones that were used in this experiment were paper chromatography and thin layer chromatography. Paper chromatography was used to analyze food colors, where as thin layer chromatography was used to analyze amino acids. Both of these techniques have two phases, known as the stationary phase and moving phase. The stationary phase is the material used, through which the mobile phase flows. The mobile phase is usually known as the developing phase, and is a solvent or a mixture.
The compounds that were synthesized for paper chromatography were 1-propanol and 1-butanol. The compounds that were synthesized for thin layer chromatography were dimethoxyethane and absolute ethanol.
In the first experiment, paper chromatography was used to show the separation of food color components. This was done by using different food colorings and determining what colors were mixed in order to get red, green, blue, and yellow commercial food colors. In the second experiment, thin layer chromatography was used in order to separate three amino acids. These three included glycine, alanine, and phenylalanine. An unknown was also given, which consisted of one, two, or all three of the amino acids. Thin layer chromatography was used to determine what this unknown was.
Learning how to calculate the Rf value, known as a physical property, helped understand the mobility of the substance. To calculate Rf, the ratio between the distance traveled by the substance, and the distance traveled by the solvent is taken. Other skills learned included carefully using the chromatography jar and marking on the chromatogram without damaging it.

1. In a 50 ml graduated cylinder, 15 ml distilled water, 15 ml 1-propanol and 15 ml 1-butanol was mixed. The mixture was poured into a chromatography jar and twisted on, and the lid was tightened. The liquid was stirred and served as the development chamber.
2. 14 x 8 cm sheet of Whatman No. 1 paper was obtained. A pencil (not pen) was used to lightly draw a 14 cm long line about 2 cm up from the long edge of the sheet. Nine small marks were then measured along the line at 1.5 intervals.
3. Red 3, Red 40, Blue 1, Yellow 5, Yellow 6, Food Color Green, Food Color Blue, Food Color Red, and Food Color Yellow were spotted from left to right on the marks. The spots were made as small as possible (1-2 mm in diameter) so that they didn't tail and overlap after development.
4. After the spots dried, the paper was rolled into a cylinder and carefully stapled at the ends about 2 cm from the top and 1.5 cm from the bottom, making sure that the edges of the paper did not touch. The paper cylinder was held by the edges and placed into the development chamber making sure it did not touch the walls of the jar. The jar was sealed tightly.
5. The cylinder was removed after the solvent had ascended within 1 cm from the top of the paper, opened, and marked at the level the solvent reached with a pencil. The chromatogram was left to dry. A ruler was then used to mark the distance that each spot have traveled relative to the solvent front in millimeters. The Rf value was calculated.
The approved food dyes list was used to determine which particular dyes were used to formulate the food colors tested.

Thin layer Chromatography

1. In a 50 ml graduated cylinder, 2 mL Dimethoxyethane and 18 mL absolute ethanol were mixed and poured into the chromatography jar. The lid was tightened and the mixture was stirred well, serving as the development chamber.

2. A 10 cm x 5 cm sheet of Whatman silica gel flexible plate was obtained. A pencil (not pen) was used and a 5 cm long line about 2 cm up from the short edge of the sheet was lightly drawn. Four small marks at 1 cm intervals were measured and drawn on the short axis of the silica gel plate.
3. Glycine, Alanine, Phenylalanine, and the unknown amino acid were spotted from left to right. After spots were dried, the silica gel flexible plate was placed in the chromatography jar. After the solvent front moved within 1-2 cm from the top of the silica gel plate, the plate was removed and laid down on a paper towel. The location of the solvent front on the silica gel was marked with a pencil.
4. The chromatogram was placed in the drying oven for about 5 minutes, removed, and sprayed with ninhydrin solution under the hood.
The chromatogram was dried again and the amino acids were outlined. The composition of the unknown was determined.

5. Rf values were calculated for each

Substance Distance traveled by substance Distance traveled by solvent Rf Value
Red 3 45 mm 50 mm .9
Red 40 29 mm 50 mm .58
Blue 1 27 mm 50 mm .54
Yellow 5 15mm 50 mm .3
Yellow 6 26 mm 50 mm .52
Green Food Color 30 mm 50 mm .6
Blue Food Color 32 mm 50 mm .64
Red Food Color 28 mm 50mm .56
Yellow Food Color 15 mm 50 mm .3

Thin Layer:
Substance Distance traveled by substance Distance traveled by solvent Rf Value
Glycine 8 mm 70 mm .11
Alanine 15 mm 70 mm .21
Phenylalanine 30 mm 70 mm .43
Unknown 8 mm 70 mm .11

Rf value is the ratio between the distance traveled by the substance and the distance traveled by the solvent. The distance traveled by the substance depends on the absorption of material and how well it binds to the paper. In the paper chromatography experiment, Red 3 food coloring had the highest Rf Value. For the thin layer chromatography experiment, phenylalanine had the highest Rf Value while Glycine had the lowest. Once they were all calculated, the unknown was easy to determine because the distance traveled, the color and the Rf value were almost exactly the same. The unknown was found to be Glycine. Possible errors might have been not being precise when measuring the substance to make the mixture and not letting the paper soak in the mixture for the right amount of time.

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