Experiment to identify an unknown mix of amino acids using paper chromatography

Experiment to Identify an Unknown Mix of Amino Acids Using Paper Chromatography

Aim

The aim of this experiment is to attempt to identify the amino acids present in the mixture (unknown solution provided) utilising the paper chromatography technique and a small range of amino acid solutions which may or may not be present in the mixture.

Introduction

Chromatography - (from the Greek “chroma” - colour; and “grafein” – to write) is the collective term for a family of laboratory techniques for the separation of mixtures. There are several different types of chromatography techniques including paper, thin layer, column, gas and liquid. It involves passing a mixture dissolved in a “mobile phase” through a “stationary phase”, which separates the analyte (the substance that is to be separated during chromatography) to be measured from other molecules in the mixture and allows it to be isolated.

In this experiment the chromatography technique employed was Paper Chromatography. Paper Chromatography is a technique which involves placing a small dot of sample solution onto a strip of chromatography paper. The paper is placed into a beaker containing a shallow layer of solvent and sealed with a watchglass cover. As the solvent rises through the paper it meets the sample mixture which starts to travel up the paper with the solvent. Different compounds in the sample mixture travel different distances according to how strongly they interact with the paper. This allows the calculation of an Rf value and can be compared to standard compounds to aid in the identification of an unknown substance. (www. en.wikipedia.org; Dec 2007)

Amino Acids have different mobilities because of their different pH values based on their structure. Some are also more polar than others, whilst some are completely non-polar (non-movers). Also, some are longer, more complex, while others are smaller. All these properties deal with mobility in chromatography.

The Retardation Factor referred to as the Rf value refers to the qualitative analysis performed by comparing (Rf) of the analyte components with the (Rf) of the known substances. The retardation factor is defined as the distance from the original sample spot that the component has moved divided by the distance that the mobile phase ...

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This practical experiment was conducted to identify what and how many, if any, of the amino acids used in this experiment were present in the unknown mix.

Method

It is important to wear gloves during the performance of this experiment as amino acids produced in the body, could through perspiration, soak into the chromatography paper and adversely affect the results obtained.

10 cm3of the ethanol; water; ammonia solvent at a ratio of 80:10:10, was poured into a 1000ml chromatography glass tank and covered with a watchglass cover to ensure a reasonably constant atmosphere within the tank throughout the experiment.
The chromatograph paper was prepared by marking in pencil, an origin line approximately one half inch from the bottom of the chromatograph paper. The paper was labelled “amino acids” 1- 6 (the mix). 1 = Leucine; 2 = Arginine; 3 = Aspartic Acid; 4 = Alanine; 5 = Lysine and 6 = Unknown Mix. With the use of capillary tubes, discrete spots of each solution were placed in their designated areas on the origin line of the chromatograph paper.
On drying, the chromatograph paper was folded to form a cylindrical shape and stapled at the top and bottom to hold it in place. This was then placed, with the origin line at the bottom, into the solvent in the chromatograph tank (carefully ensuring it did not touch the sides of the tank). A check was made to ensure that the solvent did not come above the origin line and the watchglass cover was placed back over the top of the chromatograph tank.
The experiment was left to run for one hour. After the hour elapsed the chromatograph paper was removed from the tank and a pencil line drawn across the top of the paper at the maximum height that the solvent had climbed the paper. This line is the “solvent front”. The chromatograph paper was left to dry which took 3-4 minutes.
The chromatograph paper was then taken to a fume cupboard where a “locating agent” (ninhydrin in acetone) was poured into a small “trough/bath” sufficient to cover the bottom. The chromatograph paper was then soaked in the locating agent.
The chromatograph paper was then placed in a small oven and heated at 105oC for two minutes. The amino acids formed purple spots which were then outlined in pencil and an “x” marked in the centre of each outline to indicate how far each had travelled from the origin line.

Results

Calculation of Rf (Retardation Factor)

Rf= the distance the amino acid moved from the origin/the distance of solvent from the origin expressed to two decimal places and a percentage

Rf Value of Leucine was 65mm/85mm = 0.76 or 76%
Rf Value of Arginine was 23mm/85mm = 0.27 or 27%
Rf Value of Aspartic Acid was 15mm/85mm = 0.18 or 18%
Rf Value of Alanine was 47mm/85mm = 0.55 or 55%
Rf Value of Lysine was 20mm/85mm = 0.24 or 24%
Rf Values of the mix were 65mm/85mm = 0.76 or 76% and 15mm/85mm = 0.18 or 18%

Table of Rf Values obtained for the range of amino acids tested and the unknown mix

(fig.1)

Conclusion

From the results obtained the mix was determined to be a combination of two amino acids – Aspartic Acid and Leucine.

This determination was made as a result of the mix displaying identical Rf values as Aspartic Acid (0.18 or 18%) and Leucine (0.76 or 76%) in their distances from the origin line.

Discussion

There were one or two potential errors in the procedure. Firstly the procedure/method indicated that the tank should have had 50cm3 of the solvent put in, but only 10cm3 was actually used. Presumably this change was made to allow for the origin line on the chromatograph paper utilised not to have been completely submerged from the outset. This allowed the solvent to travel up the paper past the origin line taking the amino acids with it.

Secondly, the procedure also indicated that the “run” should have been for a minimum of 1.5 hours whereas in actuality it was only left to run for 1 hour. This did not appear to affect the results though, as there was clear indication of what the unknown mix was.

References