Daswani 6A (23)
Experiment 20
Paper Chromatography
Objective
To illustrate the use of paper chromatography for the separation and identification of amino acids.
Results
Special Questions
1.> For each amino acid, compare your two Rf values with each other and with the specimen results
Why do you think there is some variation?
Both Rf values are the same for aspartic acid but there is some variation for leusine & lysine. This may due to:
1.> The grease on our fingers contaminated on chromatographic paper will affect greatly on the amino acids that are less
polar.
2.> The measurement error of the distance travelled by solvent and/or amino acid.
3.> The shape of chromatographic paper is not exactly square-shaped, so that one side of the paper may be lifted and the
speed of the solvent travel in one side is faster.
4.> The different proportion of chemicals (∵measured by measuring cylinder) may be used between the researchers and us, and
the temperatures may also different, thus Rf values obtained are different.
2.> Why do Rf values change when a different solvent is used?
Each amino acid has different polarity and each solvent has different polarity, too. So, when a different solvent is used, amino
acids will travel at a different speed.
For example, if a more polar solvent is used, the more polar amino acid will travel at a faster speed
and has a larger Rf value.
On the contrary, if a less polar solvent is used, the non-polar amino acid will travel faster and the slowest one should be the
polar amino acid.
3.> Why is it so important to avoid touching the chromatographic paper with your fingers?
There are some secretion on our fingers - amino acids and grease.
Amino acids on our fingers will result in the unexpected colour zone appears on chromatographic paper after spraying
ninhydrin solution. This will make us confused and more difficult to obtain the appropriate Rf values.
Grease on our fingers will affect non-polar amino acids most, since they are more likely to dissolve in grease than more polar
developing solvent, they will move at a higher speed. Hence the Rf values are affected.
Moreover, there may have some chemicals on our fingers, e.g. ammonia, water, etc. These will affect the polarity of the
developing solvent and thus the Rf values.