Chromatography is a physical method to separate a component out from a mixture of components. It is a method of separation in which the components to be separated are distributed between the stationary phase and the mobile phase.
Ion exchange chromatography is the most popular method in the purification of proteins and other charged molecules. It can be subdivided into cation exchange chromatography and anion exchange chromatography. In this experiment, we are doing the anion exchange chromatography. Anion exchange chromatography has a stationary phase that carries positive charge. Charged molecules in the liquid phase pass through the column until a binding site in the stationary phase appears. The molecule will not elute from the column until a solution varying pH or ionic strength is passed through it. Separation of component using ion exchange chromatography is highly selective.
Thin layer chromatography (TLC) is an adsorption chromatography in which the samples are separated based on the interaction between a thin layer of adsorbent and a selected solvent. It has a liquid mobile phase and a planar stationary phase. A small amount of the sample is spotted near the bottom of the TLC plate and it is placed in a shallow pool of solvent where the solvent are allowed to rise slowly up the TLC plate via capillary action. When the solvent move past the spot of sample, an equilibrium is established for each component of the mixture. The components differ in the solubility and strength of the adsorption to the adsorbent. This technique can be used to determine the number of different compounds present in a sample. The compound present in the sample can then be determined by calculating the Rf of the compound.
TLC can be subdivided into 1-D TLC and 2-D TLC.
Our Objective was:
1. To study separation method of amino acids through ion exchange method proceed by thin layer chromatography method.
2. To identify the component of amino acids mixture through Rf value and colour.
Procedure: anyone interested,plz follow this link
Results
Table 1: 2-D TLC
| Compound | Rf value | Colour |
| 1 | 1.5/2.9=0.52 | Yellow |
| 2 | 0.6/2.9=0.21 | Purple |
Table 2: 1-D TLC
| Compound | Rf | Color | Solution |
| Arginine | 0.8/2.8=0.29 | Purple | Alkaline Amino acid |
| Glutamic Acid | 1.7/2.8=0.61 | Yellow | Acidic Amino acid |
| Phenylalanine | 2.0/2.8=0.71 | Yellow- brown | Neutral Amino acid |
| A | 1.6/2.8=0.57 | Yellow | Acidic (pH 2) |
| B | 0.4/2.8=0.14 | Purple | Alkaline (pH 10 |
Discussion
pH of the three types of amino acids are:
Glutamic acid: pH 2 ,acidic
Arginine : pH 10 ,alkaline
Phenylalanine: lmost pH 7 ,neutral.
Ion exchanging chromatography can be classified into two types which are anion exchanging and cation exchanging chromatography. We are using anion exchanging chromatography, in which the resin is positively charged and is covalently bonded with negatively charge anions like SO3-or Cl--. Anion exchange chromatography targeting negatively charged analyte. The anion here will exchange with the negatively charged analyte ,resulting in the bonding of negatively charge analyte with resin and retained, while the anion together with other sample having neutral or positively charged samples will be eluted out. Later the negatively charged sample which is retained in resin stationary phase can be eluted by exchanging it with an anions.
Thin layer chromatography used a glass plate coated with silica gel as stationary phase. It uses the principle of adsorption. The component with more affinity towards the stationary phase travels slower, so travel through a shorter distance on the plate. On the other hand ,component with lesser affinity towards the stationary phase travels faster, and so travels a further distance on the plate. The distance travels by solvent front and samples are measured, and Rf value for each separated sample are calculated as below:
Rf of component A = dA /dS (where dA is the distance travels by sample A and dS is the distance travels by solvent front)
There are 2 types of thin layer chromatography namely 1D and 2D, 2D is actually a further step of 1D, in which the plate is turned 90 degrees anticlockwise or clockwise after 1D for better separation of spots, giving 2 dimensional effect.
The 2D TLC resulting in 2 spots (yellow and purple) ,proving that the mixture consisting of 2 samples of amino acids (AA+ and AA-) .The 2 samples are separated using the anion exchanging chromatography(AA- will be retained in stationary phase before been washed out with HCl) and their identity are determined by undergoing 1D TLC together with 3 known amino acids .Rf value of the samples in 1D and 2D TLC are calculated and shown above (please refer table a and 2). The identity of elute A and B are determined by comparing their Rf value of them with the Rf values of the 3 known amino acids , and their pH are tested also. Since Rf value of glutamic acid is closest to Rf value of sample A, and their colours on TLC are both yellow, sample A can be identified as glutamic acid. This can be further confirmed when both of their pH are acidic. On the other hand the Rf value of sample B is closest to that of arginine so it is known to be arginine .This is also further proven when both arginine and sample B possessing purple colour and basic pH.
Resin acts as a stationary phase in cation and anion exchanging chromatography, it can be negatively or positively charged and therefore can be covalently bonded with positively charge cation as well as negatively charge anions ,Cl--. Resin is used to remove heavy metal in some purification processes. Besides that ,resin is also largely used for therapeutic purposes and incense.
The precaution during the ion exchange chromatography is we have to ensure that resin is kept wet by the mobile phase and there is no cracking occur in the column .Besides that while using the capillary tube we must be careful and do not apply too much pressure on it, this is to prevent any breakage. In addition to that, we should read the mark of capillary tube upon eyes level to prevent any parallax error. While doing TLC we should 1st ensure that the plate is not bent or scratched. Also prevent any contamination to the plate like saliva ,these are to reduce any chance of mis-judgement during the analysis of result. Before we put the TLC plate with samples into the glass ware ensure that the glass ware is already pre-filled with the vapour of solvent. We should also ensure that proper solvent is used especially in 2D TLC. Cover also the glass ware containing TLC plate and solvent tightly. We should also make sure that the solvent moves through ¾ of the plate before we can stop the TLC. Do not include the 1st 0.5 cm into Rf calculation and ensure that the solvent does not exceed the drawn bottom line of the plate during the starting of thin layer chromatography.
Conclusion
1. Component A has yellow colour spot and Rf value of 1.6, which is almost the same as Glutamic Acid. Hence, it is Glutamic Acid.
2. Component B has purple colour spot and Rf value of 0.4, which is almost the same as Arginine. Hence, it is Arginine.
3. Eluate A is acidic. It contains AA- and H+.
4. Eluate B is alkaline. It contains AA+ and OH-.
References
1. Wikipedia.org.com.,my
2. Elearning.imu.edu.my
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