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Paper Chromatography

Paper Chromatography

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Published by: Muslimah Anggun on Apr 16, 2013
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 By: Alfath Agung Udayana (1013031004)Chemistry Department of Education, Faculty of Mathematic and Sciences, GaneshaUniversity of Education, Singaraja
 Amino acids contained in a sample can be analyzed by using the technique of   paper chromatography. Paper chromatography is one method of physical separation, inwhich 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 R
value of sample components with standard R
 , sothat the R
value equal or close to, then indentified as the same amino acid. Value of R
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 inthis experiment is ascending technique. This experiment objectives is to separate thecomponents of amino acids with paper chromatography technique and to identify thetypes of amino acids based in the value of R
(retention factor) were compares with standard R
. Based on the results of an experiment that has been done concluded that  sample 4 contains the amino acid leucine.
: amino acid, ascending technique, paper chromatography, eluent
Picture 1. Paper Chromatography by
Chromatography comes from theword "chroma" and "graphein". In Greek,the word means both "color" and "write(Tika, 2010). Paper chromatography is onetype of chromatographic mehod which hasstationary phase and mobile phase thatdoes not mix with each other. In thechromatographic process, the variouscomponents were separated by differentialaffinity of these components to thestationary phase (solid or liquid) carried bythe mobile phase (gas or liquid).(Soebagio, et al; 2003).In this systems typically use asaturated solution of a nonpolar solvent(eg n-butanol) and polar solvents (egwater). This solvent mixture migratethroughout the paper, polar componentsadsorbed on the supporting media(cellulose) produced thousands of dropletsare adsorbed on the support. The dropletsare adsorbed stationary phase is by passed by the mobile phase in the form of non- polar solvent so that the partition or separation of the sample mixture occurs.On paper chromatography (mixtureof substances) dropped by a capillary tubeat the paper, and then mixed solvent(eluent) migrate through the spots (spot)with an upward direction (ascending) or down (descending).
(Sumber: Ikiguci, 2008)
Implementation of separation by paper chromatography method is dividedinto three stages, namely the stage of spoting, stage of development, and theidentification or the appearance of stains.At this stage of spoting, first prepared acertain size of filter paper. Following thatis a line beginning at a distance of 2-3 cmwith one end of the paper, and then spottedthe solution samples using a micropipetteor capillary tube at the beginning of theline was then dried. In practice this time,kind of a solution containing variousamino acids will be tested by papechromatography.In the identification phase or appearance of stains, if the stain wascolored can be directly examined anddetermined its value Rf. The value of Rf stated degrees retention of a component inthe stationary phase. The distance traveled by each compound from baseline relativeto the distance the solvent/ eluent isdefined as Rf can be formulated asfollows.
outlineeluent the fromce Dis outlin samplethe fromce Dis  R
Rf value of a compound on paper chromatography system depends on manyvariables, including solvent system,temperature, duration of elution, and paper type. Because it is influenced by manyvariables, the Rf of a compound that isknown to be used as a standard o benchmark to determine Rf other compounds. For qualitative analysis,certain compounds that are known to beused in conjunction with the compound to be identified. Two different compounds ina given solvent system can have the sameRf value. Therefore, the analysis of theresults obtained by paper chromatographytechniques, must be justified by othemethods.Compounds were analyzed by paper chromatography techniques can bedetermined location on the paper invarious ways. If the tested compoundsabsorb UV light or fluoresce with UVrays, stains or spots can be detected byirradiation with UV light in a dark place.Stains or spots can also be specifiedlocation on paper using dye compounds,for example with ninhydrin (Tika, 2010).
Experiment of identification of amino acids by chromatographictechniques was conducted at theLaboratory of Organic ChemistryDepartment of Chemistry, University of Education Singaraja Ganesha on March19, 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), glassroad (1 unit), drop pippete (3 units),capillary pipe (3 units), chromatographyroom (1 unit), pinset (1 unit), scissor (1unit), beaker glass 250 mL (2 units).The materials used were n- buthanol 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 solution50mLMethodsIn the preparation of elutionsolution, 100 mL of n-butanol was addedwith 100 ml of distilled water and 24 mLof glacial acetic acid. The third solutionwas placed in a separating funnel andshaken. The two layers were formed andthen separated.Process of chromatography usingeluent phenol in the filter paper was prepared with the size of the container adapted to chromatography place. In thesection about 1.5 cm from the bottom edgeof the paper was marked with a pencil.Filter paper with a size of 15 cm x 25 cmspotted with a solution of tryptophan,leucine, tyrosine, methionine, glycine, asample solution 4 using capillary pipette.Spot distance between each other is 1.5cm. Things to note, that each droplet must
(Picture 2. Making Eluent solution)
 be dried first with aerated spotted beforethe next drop. Spot should not exceed 0.4cm. Paper kept clean and untouched as possible by finger. Furthermore, the paper is hung in space chromatographic elutionfor several hours in order to run. After elution solution runs ± 10 cm from thesample boundary, elution was stopped andremoved from the filter papechromatography. Solution boundary wasmarked with a pencil and paper filter isdried at a temperature of 100-105 º C. Thedried paper was sprayed with ninhydrinsolution. Distance was measured by thedistance eluent color formed.Process chromatography using theeluent, a mixture of n-butanol, distilledwater, 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 distancewas measured. Furthermore, it can becalculated the value of Rf.
In this experiment, carried out twoexperiments to identify the type of paper chromatography of amino acids based onits value comparison Rf values and Rf standards. This paper chromatographyusing an ascending technique (eluentmoves from bottom to top). Filter papeused is ordinary filter paper. Standardsolution used is systein, leucine, andglycine.The first step taken in thisexperiment was the preparation of theeluent solution. Eluent solution used is amixture of n-butanol with acetic acid(CH
COOH) and glacial water, and amixture of phenol and water.Mixture of n-butanol, water andacetic acid (CH
COOH) were made byglacial volume ratio 100 mL: 100 mL: 24mL. During the preparation of the eluentsolution, all three types of material wasshaken in separating funnel. The thirdmixture 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 n- butanol and acetic acid can be mixed perfectly, both are both organiccompounds. However, water and n-butanolmixed only partially so that when all threeare mixed and shaken (to be perfectlydistributed liquid) form two phases.It will form two layers. The bottomlayer in the form of the solution is clear and colorless, while the upper layer in theform of a slightly turbid solution. The bottom layer is n-butanol is used assaturater solution in chromatographyroom, while the top layer of water is usedas the eluent. In this case n-butanol acts asa mobile phase is nonpolar solvent, whilethe stationary phase is a polar solventnamely water.In making eluent, a solution aceticacid was added. The addition of acetic acidin the eluent is intended to distribute thetwo solvents which are not mixed together,where the n-butanol and water equallydistributed in acetic acid so that the ratioof specific volume can be obtainedmixture 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 watewill 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 veryimportant in paper chromatography. Water acts as a stationary phase is possible because adsorbed on the surface of the

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