INTRODUCTION
Proteins play an essential role in living cells. The basic forming units of those proteins are the amino acids which are made up of similar composition i.e. amine group, carboxyl group and a variable R-group. The laboratory tests to identify these amino acids work on identifying the variable R-groups that distinguishes each amino acid. It is very important to note that all amino acids with alpha-amino group turn purple ranging from pink to deep violet. Proline is the notable exception (only amino acids that turns bright yellow). Proline is the only one with its unique color. Other amino acids has color ranges hence identification with a single test might not be conclusive. The need for combination of various tests to identify the unknown is essential.
This laboratory report is based on the two experiments that were conducted with different samples to show the characteristics of amino acid. The two tests are the Ninhydrin test and Biuret test. The Ninhydrin tests which is combined with linear chromatography experiments is based on the different separating characteristics of the R-groups to identify the amino acids and the color changes that will occur with the heating of the samples. The chromatography separates and identifies the respective R-groups. It has two major phases which are the stationary and mobile phase. It is based on the use of ascending chromatography which allows the movement of the solvent up the filter paper. The action is a form of capillary movement that is dependent on the Rf of the amino acids. The color produced by the sample is also an added characteristics needed in the identification process.
Biuret tests on the other hand perform its identification process by identifying the presence of peptide bonds in the sample. The numbers of peptide bond corresponds to the presence of certain form of bonds. Identification of the amino acids will be done by comparing the colors and Rf of the unknown samples to that of the known samples.
MATERIALS AND METHODS
The required materials are the;
Graduated cylinder (10ml), test tubes (14), test tube racks, test tube brush, wax pencil, Benedict reagent, 2.5% NaOH reagent, plastic metric ruler and liquid soap.
Others are; 10ul microcapillary tubes, SA TLC sheets, hair dryer, infrared heat lamp and chromatography solvent.
Procedures
Ninhydrin Thin Layer Chromatography experiments.
1 sheet of TLC filter paper was used and this was handled with care by ensuring that fingerprints and body oils do not stain or contaminate the paper thereby interfering with the results. A solvent saturation pad is made to wet with 15ml of protein chromatography solvent. The important precaution here is that the solvent was handled with care because of the noxious and carcinogenic characteristics of the solvent. A line is then drawn on the TLC sheet after the orientation has been determined. The required lines are drawn. 13 clean 10um microcapillary tubes were obtained and those tubes were handled in the middle and not the tip to prevent contamination. The first microcappilary tube was loaded with the first sample. The tube was filled to the half. The solution at the first interval on the sheet was identified. The small amount of the solution was ensured to be out at a time and the entire content of the microcapillary tube are on the TLC sheet. This procedure was repeated for the other solutions to ensure that they all maintain their proper spots on the TLC sheet. The sheet was allowed to dry for several times as each of those spots are being placed on the sheet.
Another important precautionary factor considered was to allow the solvent level be below that of the sample spots to prevent the sample from dissolving in the solvent. The whole process for the travelling of the solvent was about 1hour 35mins and before this procedure, I actually checked the movement intermittently. Immediately as the TLC sheet was removed, a line was drawn across the new upper level of the solvent (solvent front) and the sheet was then allowed to dry. It was after the TLC sheet got dried itself that the gently heating with hair dryer was done. This heating now resulted in the formation of colored spots. The colored spots on the chromatogram were circled, distance travelled measured (distance from each sample origin to the dot in the center of each spot), and the Rf values was then calculated.
Biuret test
This second test was done using 14 clean test tubes. A graduated cylinder was used to add 1.5ml. The major consideration to prevent contamination was that after each addition involving a sample, the cylinder was rinsed properly with tap water. 1ml of 2.5% NaOH solution was then added to the test tubes, followed by 10 drops of Benedict solution to each of the tubes. The tubes were then allowed to stand undisturbed for 5 minutes and thus color changes starts. The changes were noted per sample and this was evaluated against the control. The results of the biuret test were combined with that of ninhydrin-TLC to differentiate between the samples having amino acids contents and those that contain peptide chains.
RESULTS
The results from the experiments are provided below
Ninhydrin-TLC experiment result
Amino Acids
Color
Glutamic acid
Red
Proline
Yellow
Tyrosine
Violet
Cysteine
Purple
Alanine
Red
Arginine
Red
Glycine
Purple
Urea
Non
Biuret
Non
Casein
Purple
Hydrolyzed casein
Purple
Beef broth
Purple
Water
Non
Biuret test experiment results
Glutamic acid
Blue
Proline
Blue
Tyrosine
Blue
Cysteine
Black
Alanine
Blue
Arginine
Blue
Glycine
Blue
Urea
Blue
Biuret
Violet
Casein
Purple
Hydrolyzed casein
Purple
Beef Broth
Dark purple
Water
Blue
DISCUSSION
These laboratory experiments focusing on the amino acid has highlighted some of the major physical and chemical characteristics of amino acids and proteins. The physical outcome of the chemical interactions was highlighted by the experiments while the chemical reactions resulting in different movement actions were also highlighted by the experiment.
The ninhydrin-Thin layer chromatography part show two different features of an amino acid. The filter paper help identified some amino acid based on their movement distance and Rf calculations from the filter paper. The control for the experiment which is water is not being tested for in the case of ninhydrin-TLC experiment because of the limitation of the paper that was used. The Rf calculations for those samples that are different from the known amino acids, their Rf is higher when compared to that of the known amino acids.
The colored spot on the TLC sheet after gentle heating is as a result of reactions between amino acids and ninhydrn. Their color is more of purple which is similar to that of cystein and glycine. The implication of this is that those samples might contain more cystein and glycine as their amino acid subunits. It can be concluded that those samples are made up of amino acids with α-amino groups.
The test also shows that some of the solutions are made up of single amino acids while some contain several amino acids and some non. The solution coming out with purple coloration could be regarded as those with several amino acids while those with other colors as red or violet are with single amino acids.
In case of the biuret tests, the control for the experiment is the water and it helped serve as a guide towards establishing the validity of the color changes that are being noticed with other samples. The results also show that there are some of the solutions with few peptide bonds, some neither and some many peptide bonds. Majority of the solutions are those with no C-N bonds. These include; glutamic acid, proline, tyrosine, alanine, arginine, glycine, urea and water. Those with many of the bonds are casein, hydrolyzed casein and beef broth. Those with few are biuret.
REFERENCES
Van Thiel, Linda. Experiments In Biology From Chemistry to Sex. Dubuque: Kendall/Hunt, 1985.
