SEPARATION OF AMINO ACID MIXTURE BY PAPER CHROMATOGRAPHY PDF

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UNIVERSITI TEKNOLOGI MARA

FACULTY OF APPLIED SCIENCES BACHELOR OF SCIENCE (HONS.) APPLIED CHEMISTRY

CHEMICAL LABORATORY

COURSE EXPERIMENT NO EXPERTIMENT TITLE

: ANALYTICAL CHEMISTRY (CHM421) : EXPERIMENT 9 : SEPARATION OF AMINO ACID MIXTURE BY PAPER CHROMATOGRAPHY

NAME

: MIMI ATHIRAH BT R AZIMI

MATRIC NUM

: 2020897434

GROUP

: AS2451M3 LAB421

DATE OF SUMMISION

: 7 DECEMBER 2020

DATE OF EXPERIMEN

: 26 NOVEMBER 2020

LECTURER’S NAME

: DR NURULFAZLINA EDAYAH RASOL

TITTLE Separation of amino acid mixture by paper chromatography

ABSTRACT The main purpose of this experiment is to spot standard amino acid and the mixture, to separate the unknown mixture into individual components and to identify the component in the unknown mixture. This experiment also is using chromatography paper in order to do a technique of separation. 4 points are spots on the chromatography paper which is amino acid, alanine, leucine and methionine. Next, the chromatography paper is place in the chromatographic tank or beaker that contains a solvent mixture of (butanol : glacial : ethanoic acid with 12:3:5) as a ratio. The Rf for the first spot of pure amino acid mixture from the below (edge) of the filter paper is 0.25 , second spotted is 0.35 and the third one is 0.45. Hence , the Rf by the alanine is 0.23 which is lower than 0.25, leucine is 0.43 that is higher than 0.35 and methionine is 0.34cm which is also lower than 0.45.

OBJECTIVE 1. To Spot Standard Amino Acid and The Mixture 2. To Separate the Unknown Mixture into Individual Components 3. To Identify the Component in The Unknown Mixture.

INTRODUCTION Chromatography is a method by which a mixture is separated by distributing its components between two phases. It is also used to identity of a compound in a mixture when the Rf of the mixture is compared with the Rf of the known compound. A small amount of the mixture that need to analysed is spotted at the top of the line. It is then placed in the chromatographic beaker/tank to dissolved in the solvent. This is the mobile phase where the solvent will slowly rise up the chromatography. When the chromatography is taken out from the beaker/tank of the chromatographic and is dry, the spot is invisible hence we need to use the stain to determine the position of the spots. This equation will be use in this experiment to determine the Rf.

𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒

Rf = 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡

METHOD A line was drawn for 5 cm on a filter paper Whatman No.1 started from the end of the filter. Four points was mark on the line from the edge and leaving 3 cm space between them from one point to another. Then, amino acid mixture was dropped by using a capillary tube to obtained the diameter of 5mm. Another drop was put on the spot when the first drop has dried. The filter paper was placed in the chromatographic tank or beaker that contained a solvent mixture (Butanol: Glacial ethanoic acid: Water, 12:3:5). Then, the solvent was allowed to move upward through the filter paper until it reached about 5 cm from the top end. The filter paper has been sprayed with ninhydrin and placed in the oven at 100C for 5 minutes. The various spots on the filter paper was examined and compared. The distance from the original point to where it stops were measured. Lastly, the Rf for each of amino acid present were calculated

DATA AND RESULT

Distance travelled by amino acid mixture (cm) 1. 2.4 2. 3.4 3. 4.3 Distance travelled by pure (standard) amino acids (cm) 1. Alanine 2. Leucine 3. Methionine

= 2.2 = 4.1 = 3.3

CALCULATION 𝑅𝑓 =

𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡

Distance travelled by pure amino acids (cm) 2.4 = 0.25 9.6 3.4 = 0.35 𝑅𝑓 = 9.6 4.3 = 0.45 𝑅𝑓 = 9.6

𝑅𝑓 =

Distance travelled by pure (standard) amino acids (cm) 𝑅𝑓 = 𝑅𝑓 = 𝑅𝑓 =

2.2 9.6 4.1 9.6 3.3 9.6

= 0.23 (alanine) = 0.43 (leucine) = 0.34 (methionine)

DISCUSSION Based on this experiment, the mixture of amino acid was spotted on the filter paper. Then, the mixture of amino acid was compared with the standard solution by finding their Rf. The Rf for the first spot of pure amino acid mixture from the below (edge) of the filter paper is 0.25 , second spotted is 0.35 and the third one is 0.45. Hence , the Rf by the alanine is 0.23 which is lower than 0.25, leucine is 0.43 that is higher than 0.35 and methionine is 0.34cm which is also lower than 0.45. Some errors might occur in this experiment. One of it is when the solvent does not move up in a perfect line. Next , the filter paper is not handled from the edges to there was contaminating the results. Furthermore, the filter paper is kept in horizontal in the chromatography tank and Ninhydrin is sprayed outside the fume hood, so the smell might spread in the lab. The precautions that should be taken in this experiment is to make sure that the line on the filter paper is drawn using a pencil to avoid the ink to be in the solvent. Next, ensure the spots is not allowed to touch the solvent in the chromatography tank and make sure the filter paper is handled from the edges so as to prevent contaminating the results.

CONCLUSION As the conclusion, the main objective of this experiment which is to spot standard amino acid and the mixture, to separate the unknown mixture into individual components and to identify the component in the unknown mixture is successfully achieved. The Rf for the first spot of pure amino acid mixture from the below (edge) of the filter paper is 0.25 , second spotted is 0.35 and the third one is 0.45. Hence , the Rf by the alanine is 0.23 which is lower than 0.25, leucine is 0.43 that is higher than 0.35 and methionine is 0.34cm which is also lower than 0.45.

REFERENCES Palmer, C. (2020, September 01). What is Chromatography and How it Does it Work?. Retriened from https://www.coleparmer.com/tech-article/what-is-chromatography Harper College. (2020). Retention Factors. Retrieved December 03, 2020, from http://dept.harpercollege.edu/chemistry/chm/100/dgodambe/thedisk/chrom/wback3.htm Zumdahl, S. S., & Zumdahl, S.A. (2007). Chemistry. DC Health and Company, 800....