QualificationHigher National Diploma in Biomedical ScienceLevel5Module Name NumberBiochemistry (BIOM 502/1)Name of CandidateFathmath DheemaStudent Number516031822Title of PracticalSeparation of Amino acids by paper chromatographyDate of Practical8th May 2018Submission Date22nd May 2018 MARKS SCHEME LABORATORY REPORTIntroduction and Objectives/ 20Materials and Methodology/ 20Results/ 20Discussion and Conclusion/ 20Academic Writing/ 10Log Book / 10TOTAL / 100 Contents Introduction 4 Objectives5 Materials.. 5 Methodology.
. 6 Results7 Discussion 8, 9 Conclusion….
.. 10 Reference..
11, 12 Introduction This lab report is about Paper Chromatography and how it is used to identify an unknown sample of an amino acid by comparing it with known samples. The unknown sample is found using the Rf values calculated from the Chromatography paper. Ninhydrin was used which forms a colored compound with amino acids, hence assisting to navigate the spread of amino acid to the solvent front on the paper. History of Chromatography dates back to the 19th century, when the Russian Botanist, Mikhail.
S Tsvet discovered how to separate plant pigments by applying the physiochemical basis of separation. He described a technique in which a gas column was packed with surface assimilative material, and to the gas column, and to the column, plant pigment solution was added and washed with an organic solvent. The pigments separated into color bands on the column.
Other chemists innovated his idea and invented a technique called Partition Chromatography. Later Martin and his co-worker used a technique in which they used a filter paper as the stationary phase giving the Partition Chromatography an advancement giving rise to the now known Paper chromatography approach. (Giddings. C, 2016) Chromatography is a process that is used mainly to separate mixtures into their components. It works on the basis of a mobile and a stationary phase.
If a solute interacts more strongly towards the stationary phase their retention time will be longer, and vice-versa for mobile phase. There are many types of Chromatography techniques like Liquid Chromatography, Gas Chromatography, Reverse-phase Chromatography, etc. (Karger, 2014) In Paper Chromatography, the separation is based on liquid-liquid chromatography, because in Paper Chromatography, both mobile phase is a liquid and the stationary phase is amino acids suspended on the paper. For paper chromatography, various types of papers can be used. (Ahuja, 2003). In ascending method of paper chromatography, after applying the sample on the spots of a filter paper or chromatographic paper, it is immersed in a solvent. The solvent moves up by capillary action and the separation occurs. (Katoch, 2011) Objectives To use paper chromatography method to separate known amino acids To measure Rf values of amino acids To compare Rf values of known amino acids with an unknown sample.
Materials Chromatography paper Four samples of Amino acids Arginine Tyrosine Phenylalanine Unknown sample (A) of an Amino acid Two capillary tubes BAW solvent Access to a Fume cupboard Gloves Hairdryer Chromatography tank Drying Oven Ninhydrin in butanol Methodology Firstly, on a Chromatographic paper, a line of 1 cm was drawn above the bottom of the paper by using a pencil. Small marks were drawn along the line at 1.5 cm intervals.
Then a capillary tube was filled with the first sample of Amino acid. A small wet spot was made on the first mark made on the paper. After letting it dry, the same sample was applied to the spot for the second time. The same was done with the other three samples as well. After this, the Chromatography paper was placed inside a Chromatography tank which was kept inside the fume cupboard. The paper was kept inside it in such a way that the only part that touched the solvent was the bottom edge of the paper below the line drawn on it.
This set up was left for 1 hour. After keeping it for 1 hour, still inside the fume cupboard, the Chromatography paper was taken out of the tank. It was dried by using a hairdryer. Then with the Ninhydrin solution, the paper was splashed with it.
After doing that, it was dried again. The paper was removed out of the Fume cupboard and transferred to the drying oven. The paper was kept in the oven for 5-7 minutes at 100 degree Celsius. The distance was measured from the point of application to the solvent front and to the center of any spots revealed by the ninhydrin solution.
These values were recorded and the general color of the spots was also observed. Lastly, the Rf value was calculated, hence the composition of the unknown sample could be identified. Results Relative mobility ( Rf ) is calculated using the Following equation. Rf Distance of A moved through the paper Distance of the solvent moved SamplesDistance of the sample moved through the paper/ cmDistance of the solvent moved /cmRfUnknown (A)4.64.80.96Tyrosine2.
24.50.48Arginine1.94.70.40Phenylalanine4.85.
00.96 Table SEQ Table ARABIC 1. Results Discussion Amino acids are one of the most essential building blocks of a human body. There are 20 different amino acids in our body.
These join together to form proteins in our bodies. Their chemical properties are what dictates the biological action of a protein. (Baldwin, 2003) In this experiment, four samples of Amino acids were used. They are Ninhydrin is the hydrate of indane-123-trione. (2, 2-dihydroxyindane-1, 3-Dione) is the chemical formula for Ninhydrin. Ninhydrin reacted with the amino acids as shown in Figure 4. Different Amino acids have different R groups. When Ninhydrin and an Amino Acid react together they form a purple colored compound (Ruhemann purple), as was seen in this experiment.
(Miyoshi, 2013) A purple color was observed on the Chromatography Paper due to this reaction. The distant moved by the sample and the distance moved by the solvent was measured using the results obtained on the chromatography paper in Figure 5 by using a ruler. In this experiment, a pencil was used to draw a line on the Chromatography paper, because if the ink was used it would dissolve in the solvent. So the result would have been incorrect. Gloves were worn to prevent fingerprints to be formed on the paper because human skin also contains amino acids. (UKessays, 2013) The solvent front was not a straight line. Hence the solvent front varied for each spot. The first spot was for the unknown sample, then the second spot was Tyrosine, next Arginine and the last spot was Phenylalanine.
While applying the sample to paper using a capillary, each end was used for one amino acid to avoid contamination of samples with each other. In this experiment, the stationary phase was the paper and the mobile phase was the solvent, BAW. One half of the experiment was done inside a fume cupboard, due to the toxicity of Ninhydrin solution and it is also known carcinogenic. Conclusion The Rf value for Phenylalanine was 0.96. By measuring the solvent front and the distance the unknown sample traveled, it also had an Rf value of 0.96. Therefore it is confirmed that the unknown sample (A) was Phenylalanine.
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Online Available at https//chem.libretexts.org/titleCore/Organic_Chemistry/Aldehydes_and_Ketones/Reactivity_of_Aldehydes_26_Ketones/TollensE28099_Test Reusch, W., 2013. Proteins, Peptides and Amino Acids. Online Available at https//www2.chemistry.msu.
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phpcitethis DOI10.1016/B978-0-12-415806-1.00006-1 Accessed 20 May 2018. PAGE MERGEFORMAT 12 Figure 5. Results obtained from chromatography of the amino acids. Figure 3.
Structure of Phenylalanine Figure 2. Structure of Arginine (Baldwin, 2003) Figure SEQ Figure ARABIC 2 Structure of Tyrosine (Baldwin, 2003) Figure 4.The reaction of Ninhydrin with an Amino Acid. (Reusch, 2013) fimEv -ui/gSC_O h Q_qQ j7-az,fp_x/v Av ygi_ oZo.K UigDQwA54FOkkxY9Xn/eUx7sttxIrmO-d6lC .Ix [email protected]/95M/[email protected])[email protected](tN M9 @Swx w7 KurX.sN4g74l3Gj90FmwLeHOu4 akeXH085eVbgwOVJvedNaxw/7 gcFCY c.
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