To determine which solvent system gives the best resolution of separation of the individual amino acids.
Key:
Ds represents the distance moved by the solvent up the chromatography paper
Da represents the distance moved by the individual amino acid up the chromatography paper
Rf represents the Rf value of the individual amino acids, that is, (Rf = Da / Ds)
The asterix, * indicates the uncertainty in the values calculated, which is, + 0.05cm.
Key:
Ds represents the distance moved by the solvent up the chromatography paper
Da represents the distance moved by the individual amino acid up the chromatography paper
Rf represents the Rf value of the individual amino acids, that is, (Rf = Da / Ds)
The asterix, * indicates the uncertainty in the values calculated, which is, + 0.05cm.
Bases of the evaluation:
To determine which solvent system gives the best resolution of separation of the individual amino acids, the following points would be taken into consideration:
- The spots of the individual amino acids either alone or in the mixture, must be visible. The solvent system, which gives the best clarity of the individual amino acids, would be the best.
- The distinct nature of the Rf values calculated for the individual amino acids alone. When I say distinct, this means that the spots should be at varying distances from the origin line, with the values for their distances being very different. Their values should not be too close and as such give a bad resolution.
- An equally distinct nature of the Rf values calculated ...
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- The spots of the individual amino acids either alone or in the mixture, must be visible. The solvent system, which gives the best clarity of the individual amino acids, would be the best.
- The distinct nature of the Rf values calculated for the individual amino acids alone. When I say distinct, this means that the spots should be at varying distances from the origin line, with the values for their distances being very different. Their values should not be too close and as such give a bad resolution.
- An equally distinct nature of the Rf values calculated for the individual amino acids in the mixture. This means, the separation of the individual amino acids in the “mixture” section should be visible in the first place, that is, it should be clear which spot corresponds to the each of the four amino acids in the mixture. For this to be possible, this would mean, the Rf values of the individual amino acids in the mixture should be equal or approximately equal to the Rf values of the amino acids alone.
Of course, since the experiments could not be carried to 100% efficiency, it is possible that not one of the solvent systems under investigation would satisfy all the criteria exactly. Therefore, the solvent system, which is able to satisfy most of these criteria, would be adjudged the best solvent system.
Observations:
After the experiments were carried out, the following observations were made:
- Light purple spots of the individual amino acids appeared on the chromatography paper at particular distances from the origin line. For some proportions of either solvent system, distinct spots of the individual amino acids appeared in the “mixture” section of the chromatography paper and for others, this would not the case.
- The proportion which gave the best resolution of separation of amino acids in the solvent system, methanol: chloroform: water, was the proportion, 8:1:1. This was adjudged the best for the following reasons:
- The spots of the amino acids obtained alone and in the mixture were the clearest in this proportion
- The Rf values calculated for the individual amino acids alone and in the mixture, were the most distinct compared to the results obtained for the other proportions. In addition, the spots obtained in the mixture section could easily be identified with the individual spots of each amino acids as their Rf were approximately equal.
- The proportion which gave the best resolution of separation of amino acids in the solvent system, methanol: ammonia: water, was the proportion, 8:1:1. This was adjudged the best for the following reasons:
- The clarity of the spots of the individual amino acids obtained alone in the proportion, 8:1:1, were relatively the best compared to the other proportions of the same solvent system. However, for all the proportions, including the best proportion, the separation of the individual amino acids in the mixture sections, were not visible at all. In relatively better situations, it was still very difficult to identify the individual amino acids in the mixture. As such, this criterion was not taken into consideration in determining the best proportion of this particular solvent system.
- The Rf values obtained for the four amino acids were the most distinct in the proportion 8:1:1 compared to the other proportions of the same solvent system.
- As the proportions of methanol in both solvents system mixtures were reduced and the proportions of ammonia or chloroform and water were increased, the spots of the individual amino acids were seen to move further up the solvent front, with the some of the spots reaching the top for the following proportions of solvent systems:
- 1:8:1
- 1:1:8
- 2:6:6
- 2:6:2
- 4:3:3
- 3:4:3
- 3:3:4
In these cases, the spots were observed to have moved relatively the same distance up the solvent front, and as such, their Rf values were very close and in some cases, the same.
Discussion:
Looking at the observations made above, there is no doubt that the solvent system methanol: chloroform: water is clearly a better solvent system than methanol: ammonia: water, regardless of the proportions used. For this investigation, initially, I had planned to use only the following proportions of the two solvent systems:
- 8:1:1
- 1:8:1
- 1:1:8
- 6:2:2
- 2:6:2
- 2:2:6
- 4:3:3
- 3:4:3
- 3:3:4
However, upon actually carrying out the experiments using these proportions, it is clear that the results obtained are not adequate and I still haven’t been able to come to a definite conclusion as to which proportion of the two solvent systems gives the best resolution. It would thus be necessary for me to carry out further experiments to thus come out with the desired conclusion.
From the results and observations obtained, it is clear that no proportions of the solvent system, methanol: ammonia: water, would give us the best resolution of the separation of the amino acids in it. As a result, in my subsequent experiments, I would not use this solvent system anymore. On the other hand, I would work around the methanol: chloroform: water, solvent system. Again, looking at the results obtained, it can be established that, as the proportion of methanol in the solvent, methanol: chloroform: water, decreased and that of the other components increased, the Rf values obtained for the individual amino acids alone and in the mixture, become less and less distinct. The best resolution for the separation of the amino acids obtained was the best in the proportion of the solvent system with methanol having the highest proportion and water and chloroform having the least proportion in the solvent, that is, 8:1:1. At this stage, this is still just a hypothesis and should not be taken as the conclusion.
I decided, therefore, to use a new set of proportions to test my hypothesis and come out with a conclusion. Majority of the proportions would apportion the greatest part of the solvent system, to methanol and the least parts to chloroform and water. Conversely, since this is just a hypothesis that I would be testing, I would still include proportions with methanol having a relatively smaller proportion of the solvent system compared to the other proportions used.
What would be done?
The procedure used previously, would be the same procedure used for these subsequent experiments. The proportions to be used would have a total ratio of 100. These proportions are:
- 90:5:5
- 85:10:5
- 85:5:10
- 80:10:10
- 70:15:15
- 60:20:20
- 50:25:25
Key:
Ds represents the distance moved by the solvent up the chromatography paper
Da represents the distance moved by the individual amino acid up the chromatography paper
Rf represents the Rf value of the individual amino acids, that is, (Rf = Da / Ds)
The asterix, * indicates the uncertainty in the values calculated, which is, + 0.05cm.
Observations: