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Separation of amino acids by ascending paper Chromatography
Introduction: Chromatography is a method of separating and analyzing mixtures of chemicals. The paper chromatography is used for the testing of purity of compounds and identifying substances and a very useful method as it is quick and very small quantities of materials used It is used to separate the mixture of substances into their components. They all have a stationary phase and a mobile phase. The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates. In this chromatography the stationary phase is a very uniform absorbent paper. The mobile phase is a suitable liquid solvent or mixture of solvents.

Principle: In this experiment a small spot with amino acid onto a strip paper is made out. The bottom of the strip will then be placed in a wide-mouth jar of mixture of solvent, 40% Et-OH (ethanol) and 60% H2O and the solvent then shook up into the paper. The solvent is the mobile phase and the paper is the stationary phase. The attraction of the solvent to the paper is larger than the attraction of the water too itself hence the mixture of solvent moves up to the paper. The amino acid which also be attracted to the paper to itself and to the water differently and thus a different component will move a different distance depending upon the strength of attraction to each of these  objects we can calculate retention factor, Rf of the sample Rf =(distance traveled by the sample component)/(distance       traveled by the solvent)

Materials and instrument: Apparatus:

1.	Ruler 2.	Pencil, (two) 3.	Capillary tube 4.	A wide moth jar 5.	10-15 identically sized strips of paper.

Solvent and sample: 1.Mixture of ET-OH (ethanol) and water (H2O) 2.Amino acids.

Locating reagent: 1.Ninhydrine solution

Procedure: 1.The paper strips are cut about one by four (4) inches in area. 2.Then drawn a line across it horizontally to one cm (1 cm) from the bottom of one of the paper strips by using a ruler and pencil. This is the original line. 3. Then a small amount of solvent is pour into the jar (there be barely enough for the paper strips to hang inside of the jar and just touch the solvent) 4.Then place a small dot (.) of the sample onto the line by using one of the capillary tubes.

5.Then the paper is tapped with a pencil and hung it into the jar of solvent so that the bottom edge of the paper which is just barely touching. 6.Then it to be keep for some time for rising up onto the strip. 7.Then the strip needed to be remove from the jar and a mark is to be made to how far the solvent rise with a pencil. 8.Then the paper strip is to be dry in normal sunlight and dip in locating reagent (Ninhydrine solution). 9.Then the strip is to be expos UV light and then dry and indicate the spot. 10.Then the distance of the component and solvent travel from the starting point is to be measure and calculate the  Rf  value for each component. 11.Repeat the experiment for each type of component

Experimental data: Determination of distance: 1.Experiment no. 2.Samples (A,B,C,mixture) 3.Distance travelled by the sample,d1 (cm) 4.Distance travelled by the solvent,d2 cm Calculation:

We know, Retention factor, Rf =( distance traveled by the sample      component)/( distance traveled by the solvent)

Determination of Rf value of each amino acid sample: Exp no.	Samples 	(d_1 cm)/(d_2 cm)	  Rf 01	A 02	B 03	C 04	D(mixture)

Result: 1.The Rf value for the sample, A = 2.The Rf value for the sample, B = 3.The Rf value for the sample, C=  4.The Rf  value for the mixed sample, D = By comparing the above Rf value the component which present in the mixture is=

Discussion:

Related questions: 1.What is the retention factor Rf ? 2.What is the significance of the retention factor? 3.What types of factors that affect the Rf value in paper Chromatography? 4.Why do different compounds travel different distances on the piece of paper? 5.What is chromatography used for?