Structural Analysis of a Dipeptide
During this experiment we learned how to qualitatively determine the existence of amino acids in a solution and understand the basics of a new chromatographical technique.
Chromatography is a method of seperation. It is based on the flow rate of the mobile phase (which includes analyte) within the stationary phase. Therefore chromatographical methods can be modified by changing mobile and stationary phase and can also be categorized according to their types.
According to mobile phase, chromatography could be either liquid or gas.
According to stationary phase, chromatography could be adsorption, partition, gel exclusion, affinity, etc. Each of these methods have advantages of their own. For example; affinity chromatography has a specifically designed stationary phase for catching a type of molecule (usually proteins) while the mobile phase flows. So knowing which chromatographical technique to use while conducting an experiment is a very rewarding knowledge.
In paper chromatography, our stationary phase is a special filter paper and it is good for fast, quantitative observations. In this experiment, our filter paper was Whatman paper; a semi-dry paper composed of pure nitrocellulose and has a water flow rate of 130mm/30min.
Paper chromatography works by dropping the analyte solution on the paper and letting it absorb the chromatography solution. While the chromatography solution moves upward due to capillary movement and cohesion, it also drags the analyte solution along itself and seperating it during the process.
Our chromatographical solution in this experiment contains ammonium acetate (CH3COONH4) and acetonitrile (CH3CN). Ammonium acetate works as a buffer in this solution. As a volatile substance in low pressures, it is being used aqueously to replace cell buffers with non-volatile salts, in many chromatographical and/or spectrometrical techniques. Acetonitrile on the other hand is the actual solvent which is miscible with water and has medium-polarity.
The analyte solution which contains amino acids and water is colorless. After chromatography is done, we initially have no chance to observe the amino acids' migration. Hence we use ninhydrin, a molecule which reacts with free primary (NRH2) and secondary (NRR'H) amine groups and proteins through a series of reactions to yield aldehydes, ammonia (NH3) and CO2 by degrading amino acids. Then ninhydrin condenses with ammonia to form a purple-blue complex, which is sometimes called Ruhemann's purple as it reacts with free amine groups, the amino acid proline doesn't react with it and thus yields a yellow color which is highly characteristic.
Amino acids that we try to detect during this experiment are the monomers of macromolecules called proteins and they form proteins by forming peptide bonds between them (between their amine and carboxyl groups) and by weak intramolecular interactions. All amino acids have a central alpha carbon atom at the center which is surrounded by an hydrogen, an amine group, a carboxyl group and a functional group (R) which makes each of the 20 amino acid unique. According to their functional groups, amino acids can be polar, non-polar, acidic, basic, aromatic and cyclic. The only cyclic amino acid is proline which doesn't have a free amino group due to it's molecular structure. Although amino acids can be found as positively charged or negatively charged, in physiological pH they are generally found as zwitterion structure, which is formed by having both partial negative and positive charges on carboxyl and amine groups respectively.
The amino acids of interest in this experiment was glycine (which has H as a functional group), alanine (which has CH2-OH as a functional group) and proline (which has a cyclic structure bonded with amine group). We expect to see glycine and alanine residues as purple and proline as yellow.
After the technique is over and paper is rinsed with ninhydrin, from the colors of migration paths and lenghts we can obtain qualitative datas. One such data is the retention factor (Rf) which is calculated by dividing the migration distance of substance (often abbriviated as x) to migration distance of solvent (often abbriviated as y). The ratio between x and y is always between 0 and 1.
Retention factor is a calculation used most commonly in planar chromatographies like paper and thin-layer chromatography as they don't use detectors. Every substance used in these techniques will give a specific value for each mobile-stationary phase couple. So it has a distinctive spectra for substances as long as they are migrated in the same mobile-stationary phase couple. Although one can effect these values by other means too, like by changing the polarities of solvents. So it is important to use the right solvent and mobile-stationary phase couple to obtain the most distinctive spectra.
As our whatman paper is semi-dry and contains polar water, we assume the paper is polar and presume the chromatography solvent is non-polar in order to be able to push the analyte along the mobile phase and cause migration.
3. MATERIALS AND METHODS
* Erlenmeyer flask (x3)
* Glass stirrer
* Whatman paper
* Micropipette (20-200 µL)
* Pen and Ruler
· Chemicals and Organic Compounds
* Ammonium acetate
1. First we prepared 1% amino acid solutions by mixing 0,1 mg of each amino acid with 1 mL of water
2. Then we prepared chromatography solvent by mixing 300 mL acetonitrile with 200 mL 0,1M ammonium acetate.
3. After both of them was done, we dropped small drops of each aa solution on a side of Whatman paper. We dropped 40µL each of them to another spot and all of them to somewhere inside of the paper to make sure solvent will not mix with them.
4. After dropping the solutions, we stapled the paper to form a cylindrical shape and left the dropped area downwards.
5. We put that cylindrical paper into the flask with solvent (up to a finger below the drops) and as soon as we putted it, the paper began to absorb the solvent. We added solvent as needed.
6. After the paper absorbed enough solvent to become all wet, we took it and lefted it on heater for about half an hour to make solvent dry.
7. At this stage the paper had no signs of solution or solvent on it. We rinsed the paper with ninhydrin solution by applying lots of solution.
8. Again we left paper on heater for about 5 minutes this time.
9. After 5 minutes has passed, we began observing the migration paths as yellow or blue-purple.
10. We measured the average x distances and the whole migration path (y) with a ruler and recorded the results on papers.
4. CALCULATIONS AND OBSERVATIONS
This experiment was done by three different groups to compare the results.
Here's the calculations of Rf values of each amino acid for our group:
migration of analyte solutionmigration of solvent
Rf of Glycine:
Rf of Alanine:
Rf of Proline:
The Rf values of another group with seperate amino acid solutions.
Rf of alanine = 0,74
Rf of glycine = 0,6
Rf of proline = 0,8
This group was mixed the amino acids in solutions:
(amino acids' Rf values are written correspondingly)
Mobile phase = 10cm
Rf of ala + pro = 6,7 and 8,2
Rf of gly + ala = 5,6 and 6,7
Rf of gly + pro = 6 and 8,3
The drops were migrated along the mobile phase but they were expanded as they migrated. So the last spots we saw was obviously bigger than the initial drops which forced us to use an average center for measuring distance.
Also the proline was yellow while the glycine and alanine was blue-purple.
5. RESULTS AND DISCUSSIONS
Like we presumed, the colors of the amino acids with free amine groups was blue-purple unlike the cyclic amino acid proline which has no free amine group. The Rf values of each amino acid was:
Rf of Glycine < Rf of Alanine < Rf of Proline
And the distance they migrated were:
Xglycine < xalanine < xproline
Each of these orders has direct correspondance and therefore we can assume that the amino acids that have the higher Rf value should migrate longer in whatman paper and so we can seperate two different amino acids with greatly different Rf values from each other by this chromatographical method.
The polarity of the Whatman paper was assumed polar at the beginning of this experiment as opposed to chromatography solvent, a non-polar substance and this was what makes this method work.
So our solutions were repelled by the paper while they dissolved in the mobile phase and tried to flow and escape away from the paper as soon as possible. So the most non-polar amino acid was proline and after that alanine and glycine in decreasing order.
If we would use another substance as mobile phase other than chromatography solvent, the migration order wouldn't change but the distance would. So with different values we would have get the same order.
1. Yeditepe University Department of Chemical Engineering, “Lab Manual for Organic and Bioorganic Chemistry Courses”, 2008-09 Spring manual.