1 IDENTIFICATION OF AMINO ACIDS IN UNKNOWN SAMPLES USING PAPER CHROMATOGRAPHY TECHNIQUE ASCENDING By: Alfath Agung Udayana (1013031004)

Chemistry Department of Education, Faculty of Mathematic and Sciences, Ganesha University of Education, Singaraja ABSTRACT Amino acids contained in a sample can be analyzed by using the technique of paper chromatography. Paper chromatography is one method of physical separation, in which the elements will be separated are distributed between two phases are the stationary phase and mobile phase. Determination of amino acids contained in the samples was done by comparing the Rf value of sample components with standard Rf, so that the Rf value equal or close to, then indentified as the same amino acid. Value of Rf of a compound on paper chromatography system depends on many variables, including solvent system, temperature, duration of elution and the type of paper. As the eluent used a mixture of n-butanes: glacial acetic acid: water and fenol solution. In this experiment, used the standard amino acid are leucine, systeine and glycine. The technique that use in this experiment is ascending technique. This experiment objectives is to separate the components of amino acids with paper chromatography technique and to identify the types of amino acids based in the value of Rf (retention factor) were compares with standard Rf. Based on the results of an experiment that has been done concluded that sample 4 contains the amino acid leucine. Keyword: amino acid, ascending technique, paper chromatography, eluent INTRODUCTION Chromatography comes from the word "chroma" and "graphein". In Greek, the word means both "color" and "write (Tika, 2010). Paper chromatography is one type of chromatographic mehod which has stationary phase and mobile phase that does not mix with each other. In the chromatographic process, the various components were separated by differential affinity of these components to the stationary phase (solid or liquid) carried by the mobile phase (gas or liquid). (Soebagio, et al; 2003). In this systems typically use a saturated solution of a nonpolar solvent (eg n-butanol) and polar solvents (eg water). This solvent mixture migrate throughout the paper, polar components adsorbed on the supporting media (cellulose) produced thousands of droplets are adsorbed on the support. The droplets are adsorbed stationary phase is by passed by the mobile phase in the form of nonpolar solvent so that the partition or separation of the sample mixture occurs. On paper chromatography (mixture of substances) dropped by a capillary tube at the paper, and then mixed solvent (eluent) migrate through the spots (spot) with an upward direction (ascending) or down (descending).

Picture 1. Paper Chromatography by Ascending position (Sumber: Ikiguci, 2008) Alfath Agung Udayana_1013031004

2 Implementation of separation by paper chromatography method is divided into three stages, namely the stage of spoting, stage of development, and the identification or the appearance of stains. At this stage of spoting, first prepared a certain size of filter paper. Following that is a line beginning at a distance of 2-3 cm with one end of the paper, and then spotted the solution samples using a micropipette or capillary tube at the beginning of the line was then dried. In practice this time, kind of a solution containing various amino acids will be tested by paper chromatography. In the identification phase or appearance of stains, if the stain was colored can be directly examined and determined its value Rf. The value of Rf stated degrees retention of a component in the stationary phase. The distance traveled by each compound from baseline relative to the distance the solvent/ eluent is defined as Rf can be formulated as follows.
Rf Distance from the sampleoutline Distance from the eluent outline

irradiation with UV light in a dark place. Stains or spots can also be specified location on paper using dye compounds, for example with ninhydrin (Tika, 2010). MATERIALS AND METHODS Experiment of identification of amino acids by chromatographic techniques was conducted at the Laboratory of Organic Chemistry Department of Chemistry, University of Education Singaraja Ganesha on March 19, 2013. The equipments used were beaker glass 100 mL (5 units), beaker glass 25 mL (1 unit), separation funnel 500 mL (1 unit), ring (1 unit), stative and clamp (1 pair), ruler 30 cm (1 unit), spatula (1 unit), glass road (1 unit), drop pippete (3 units), capillary pipe (3 units), chromatography room (1 unit), pinset (1 unit), scissor (1 unit), beaker glass 250 mL (2 units). The materials used were nbuthanol solution (100 mL), distilled water (500 mL), acetic acid glacial (25 mL), glicine solution (25 mL), sample solution 4 (25 mL), alcohol 50 mL Phenol solution50 mL Methods In the preparation of elution solution, 100 mL of n-butanol was added with 100 ml of distilled water and 24 mL of glacial acetic acid. The third solution was placed in a separating funnel and shaken. The two layers were formed and then separated. Process of chromatography using eluent phenol in the filter paper was prepared with the size of the container adapted to chromatography place. In the section about 1.5 cm from the bottom edge of the paper was marked with a pencil. Filter paper with a size of 15 cm x 25 cm spotted with a solution of tryptophan, leucine, tyrosine, methionine, glycine, a sample solution 4 using capillary pipette. Spot distance between each other is 1.5 cm. Things to note, that each droplet must
Alfath Agung Udayana_1013031004

Rf value of a compound on paper chromatography system depends on many variables, including solvent system, temperature, duration of elution, and paper type. Because it is influenced by many variables, the Rf of a compound that is known to be used as a standard or benchmark to determine Rf other compounds. For qualitative analysis, certain compounds that are known to be used in conjunction with the compound to be identified. Two different compounds in a given solvent system can have the same Rf value. Therefore, the analysis of the results obtained by paper chromatography techniques, must be justified by other methods.Compounds were analyzed by paper chromatography techniques can be determined location on the paper in various ways. If the tested compounds absorb UV light or fluoresce with UV rays, stains or spots can be detected by

3 be dried first with aerated spotted before the next drop. Spot should not exceed 0.4 cm. Paper kept clean and untouched as possible by finger. Furthermore, the paper is hung in space chromatographic elution for several hours in order to run. After elution solution runs 10 cm from the sample boundary, elution was stopped and removed from the filter paper chromatography. Solution boundary was marked with a pencil and paper filter is dried at a temperature of 100-105 C. The dried paper was sprayed with ninhydrin solution. Distance was measured by the distance eluent color formed. Process chromatography using the eluent, a mixture of n-butanol, distilled water, and glacial acetic acid begins by preparing the filter paper. The size and the procedure same as the previous procedure. Amino acid spot was visible. The distance was measured. Furthermore, it can be calculated the value of Rf. RESULT AND DISCUSSION In this experiment, carried out two experiments to identify the type of paper chromatography of amino acids based on its value comparison Rf values and Rf standards. This paper chromatography using an ascending technique (eluent moves from bottom to top). Filter paper used is ordinary filter paper. Standard solution used is systein, leucine, and glycine. The first step taken in this experiment was the preparation of the eluent solution. Eluent solution used is a mixture of n-butanol with acetic acid (CH3COOH) and glacial water, and a mixture of phenol and water. Mixture of n-butanol, water and acetic acid (CH3COOH) were made by glacial volume ratio 100 mL: 100 mL: 24 mL. During the preparation of the eluent solution, all three types of material was shaken in separating funnel. The third mixture is not completely mixed together. Water and acetic acid can be mixed perfectly remember the two mixture is a polar covalent compounds, as well as nbutanol and acetic acid can be mixed perfectly, both are both organic compounds. However, water and n-butanol mixed only partially so that when all three are mixed and shaken (to be perfectly distributed liquid) form two phases. It will form two layers. The bottom layer in the form of the solution is clear and colorless, while the upper layer in the form of a slightly turbid solution. The bottom layer is n-butanol is used as saturater solution in chromatography room, while the top layer of water is used as the eluent. In this case n-butanol acts as a mobile phase is nonpolar solvent, while the stationary phase is a polar solvent namely water.

(Picture 2. Making Eluent solution)

In making eluent, a solution acetic acid was added. The addition of acetic acid in the eluent is intended to distribute the two solvents which are not mixed together, where the n-butanol and water equally distributed in acetic acid so that the ratio of specific volume can be obtained mixture containing n-butanol, acetic acid, and water used for saturate the filter paper, the mixture migrate throughout the paper, where the polar component of the water will be adsorbed on the supporting media (paper). The molecules of water (as a polar phase) will be distributed on the surface of the paper. Where the interaction is very important in paper chromatography. Water acts as a stationary phase is possible because adsorbed on the surface of the
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4 paper and produces thousands of tiny drops. The droplets are adsorbed stationary phase is passed mobile phase is non-polar components in the eluent namely nbutanol, resulting in the partition or separation of the mixture can occur. It is given that the difference in the distribution of amino acids that make up the mobile phase and mixed with water (stationary phase). Before the process of identification by chromatographic techniques, the filter paper was prepared for phenol eluent of 5.5 cm x 19 cm, while the eluent n-butanol with glacial acetic acid of 6.5 cm x 28 cm. In the process of cutting paper, paper hand should not touch directly. This is because when using the hand, there will be contact between your hands with a paper that will be able to interfere with the chromatographic process, because our bodies sweat which is the main component of organic substances such as urea, oil. If the hand is used to pick up the paper, then the organic compounds will also migrate the non-polar phase (motion) that will affect the results of observations on the chromatographic process. So in this experiment used slippers hand so that the hand is not in direct contact with filter paper. Spotting of identification procedure followed by amino acid solution and samples on filter paper using a capillary tube. Spotting using capillary aims to obtain spot 0.4 cm diameter. If the diameter exceeds 0.4 cm, then the results are not perfect due to permeation mobile phase and stationary phase to be assertive, color detectable spread. Each spot solution to be tested, Totolan is dried. Furthermore spotting back another solution to be tested. This experiment uses two pieces of filter paper. The first filter paper filled with standard amino acids, namely tryptophan, leucine, tyrosine, methionine, and glycine. The second filter paper filled with standard amino acids, namely glycine, sample A, sample B, sample C and sample D. Using different paper aims to get stains of the same amount and does not exceed 0.4 cm in diameter. In addition, because the tool (space chromatography) are limited in the laboratory.

Picture 3. Chromatography process in Eluent Phenol

The next filter paper was saturated with vapor chromatographic eluent in chromatography room. After saturation is reached, then starting with the elution. Saturation above goal is to accelerate the process of permeation eluent on filter paper. In this experiment, use the principle of ascending, where filter paper hung from the top of the room so immersed in the solvent and moving his way up by capillarity power. Immersion or installation of filter paper is not soaked spot standard amino acid and sample, so amino acid samples that have different solubility in eluents so the speed of movement in the paper also different (differential migration). When the eluent was approaching the boundary line elution process was stopped. After elution process is complete, the filter paper was taken and dried in an oven in order to evaporate the water molecules. After dry filter paper sprayed with ninhydrin solution colorless. Spraying with ninhydrin aims to reveal the amino acid stains. Spot that looks is purple. Purple color indicates the occurrence of a reaction between amino groups of free amino acids with ninhydrin. Purple color can be used to determine the amino acid quantitatively by measuring absorbance at 570 nm. Under the right conditions, the intensity of the color
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5 produced can be used to measure the concentration of amino acids. After sprayed with ninhydrin, the filter paper was dried in an oven again. The results of this drying causes the purple color on the filter paper more apparent. In this experiment, there are 4 samples analyzed by comparing the value of its Rf with standard solution, namely tryptophan, leucine, tyrosine, methionine, and glycine. In the second paper chromatography using phenol eluent and water. After the chromatography process is complete, chromatograms are treated the same as the first filter paper, by drying in the oven and when dry sprayed with ninhydrin solution. Then dried again in the oven. After arise color, then measured the distance of each eluent and amino acids contained in the paper.Based on the experimental results, the data obtained as follows. Based on the observation result, it was gotten the data of distribution distance of ech solution as follows. Table 1. Distribution data of standard solution and sample solution as well as the Rf value with phenol as eluent Distance (cm) Solution Elue Rf Note Component nt Glycine 10 5.5 0.55 Systeine Leusine Sample 4 10 10 10 9.6 10 9.9 0.96 1.0 0.99 Leuci ne Table 2. Distribution data of standard solution and sample solution with mixture of n-buthanol + aquades + acetic acid glacial as eluent. Solution Glycine Systeine Leusine Sample 4 Distance (cm) Eluen Compon t ent 10 5.5 10 10 10 9.6 10 9.9 Rf 0.55 0.96 1.0 0.99 Note Leuci ne

According to the second experiment, the Rf value of sample 4 in the mixture of n-buthanol + aquades + acetic acid glacial as eluent same as the solution leusine namely the Rf value of sample 4 and leucine is 0.9. CONCLUSION Based on comparation between the Rf value of sample solution and amino acid solution, the sample solution 4 is Leucine because the Rf value of sample 4 in the phenol as eluent close to the solution leusine namely the Rf value of sample 4 is 0.99 and the Rf value of leucine is 1.0. And according to the second experiment, the Rf value of sample 4 in the mixture of n-buthanol + aquades + acetic acid glacial as eluent same as the solution leusine namely the Rf value of sample 4 and leucine is 0.9. ACKNOWLEDGMENT Thank for the advice and guidance of Drs. I Nyoman Tika, M.Si as the lecture of biochemistry experiment. Thank also to Mr. I Ketut Lasia as laboran staff that help to prepare the materials and equipments for the experiment. And also for my partner, Putu Prema Swari that already help to conduct the experiment

Based on the data, the Rf value of sample 4 in the phenol as eluent close to the solution leusine namely the Rf value of sample 4 is 0.99 and the Rf value of leucine is 1.0.

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6 REFERENCES Anwar, Chairil, dkk. 1996. Pengantar Praktikum Kimia Organik. Yogyakarta : Dedikbud Fessenden & Fessenden. 1982. Kimia Organik Edisi Ketiga Jilid 2. Terjemahan Pujaatmaka, Aloysius Hadyana Organik Chemistry, Third Edition. Jakarta: Erlangga Soebagio, Endang Budiasih, Sodiq Ibnu, Hayuni Retno Widarti, Munzil. 2003. Common Textbook (Edisi Revisi) Kimia Analitik II. Malang: Universitas Negeri Malang Tika, I Nyoman. 2010. Buku Penuntun Praktikum Biokimia. Singaraja: Universitas Pendidikan Ganesha Wiratma, I Gusti Lanang, I Nyoman Selamat, I Dewa Ketut Sastrawidana. 2003. Dasar-dasar Pemisahan Analitik. Singaraja: IKIP Negeri Singaraja.

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