Amino Acids Introduction This lab was done in order to detect the presence of amino acids. Amino acids are distinguished from other biomolecules because not only do they contain carbon, hydrogen and oxygen, but nitrogen as well. The Amine group (NH 2) is where the nitrogen is located. Amino acids contain two other groups, one is called the Carboxyl group (COOH) and the other is called the variable “R” group.
The “R” group is what makes amino acids different from one another. All of these groups are connected to what is called the “central” carbon. They also combine with one another in a line. These combinations are called polypeptides. The carboxyl group of one amino acid connects to the amine group of the other.
These connections or “bonds” are called peptide bonds (abbr C-N) and are the building blocks for proteins. Material and Methods Graduated cylinder (10 ml) (2) tes tubes (14) wax pencil Chromatography chambers test tube rack benedict reagent Plastic metric ruler test tube brush 2. 5% NaOH reagent 10 ul micro capillary tubes hair dryer Liquid soap SA TLC sheets chromatography solvent Procedures A. First we obtained a sheet of Ninhydrin thin layer chromatography (TLC).
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... separate and identify a mixture of amino acids by paper chromatography.MethodCut a piece of chromatography paper to about 25cms in length and ... means that it turns yellow. It is called an imino acid and belongs to the secondary amino group. Because of its unique colour it ... intervals.Using a micropipette, take the prepared albumen from the test tube and lightly dot a small amount on each pencil ...
The chromatography chambers where already set up for us with 50 ml of protein chromatography solvent at the base and 15 ml in the saturation pad.
We then drew a line across the bottom of the TLC sheet, about 2 cm above the edge. We split the line into 13 vertical segments 1 cm from the edges and 1. 5 cm in between, numbered and labeled them according to their samples. A line 10 cm above the first horizontal line was then drawn. 1. glutamic acid 6.
arginine 11. hydrolyzed casein 2. praline 7. glycine 12.
beef broth 3. tyrosine 8. urea 13. milk 4.
cysteine 9. biuret 5. alanine 10. casein Thirteen micro capillary tubes were obtained; each one was used to place drops of each sample onto its corresponding spot on the TLC sheet. About half of a tube was filled and emptied onto each spot, one drop at a time, and in 3 minute intervals. After all the spots had dried, we placed the TLC sheet into the chromatography chamber We allowed the TLC to sit in the chamber for about 1 and a half hours, which in that time, the solvent had traveled up to the 10 cm line we drew.
Immediately after taking the TLC sheet out, we drew another line to indicate the actual solvent front. The hair dryer was used next under the fume hood to speed up the drying of them TLC sheet. When the TLC sheet was completely dry, we circled each distinct spot and placed a dot in the middle of each spot, noting also the color of each one. The ruler was used to measure the distance from the sample origin to the center of the sample circle. We calculated Rf ration by measuring distance traveled from origin divided by distance from origin to solvent front. B.
biuret test While we were waiting for the TLC sheet to absorb the solvent in the chamber we labeled 13 clean test tubes with the wax pencil. Each one was labeled with a number corresponding to the samples. 1. 5 ml of each sample was put into the its own test tube. 1. glutamic acid 6.
arginine 11. hydrolyzed casein 2. praline 7. glycine 12. beef broth 3. tyrosine 8.
urea 13. milk 4. cysteine 9. biuret 5. alanine 10. casein We then added 1 ml of 2.
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5% NaOH solution, then 10 drops of benedict solution to each test tube. The test tubes stood in room temperature for about 5 minutes. During this time we observed the samples changing colors. We wrote down what color each sample had turned into. When all the tests were done we washed all the test tubes with soap and water. Results See attached pages Analysis For the TLC sheet test results were taken and interpreted.
We decided that each dot was a separate amino acid and the color and distance it traveled could be used to tell which amino acid it was. Some samples did not leave colored dots which lead us to believe that no amino acids were present in them. The biuret test showed us different color changes among the samples. Each color change details the amount of peptide bonds within the sample. A positive reaction is more intense with the greater number of bonds; colors range from light violet to a deep purple. Samples which lacked peptide bonds simply remained the blue color of the NaOH solution..
