Experiment 2: Separation of Amino Acids by Paper Chromatography
Group no. 2
Chem31.1, Section MEJ1
March 20, 2017
I. Abstract
Chromatography is the versatile separation technique based on the differences in affinities
of components of a mixture to a stationary phase and a mobile phase. In this experiment,
the liquid mobile phase is the solvent mixture of sec-butanol, glacial acetic acid and
distilled water; the liquid stationary phase is the water bound within the cellulose fibers of
the chromatography paper used. Retention Factor (R f), significant in performing
chromatography, is the ratio of the time the analyzed sample component resides in the
stationary phase relative to the time it resides in the mobile phase. This experiment aims
to identify two unknown amino acid samples through a comparison of calculated R f values,
attained through a type of chromatography known as paper chromatography of four other
amino acids: glycine, lysine, leucine and tyrosine. The computed R f values were 0.62, 0.22,
0.80, and 0.52, respectively. The identity of the unknown acid sample may prove to be
otherwise by respective theoretical R f values of the known acid solutions. Based on the
gathered data, the two unknown acid samples were identified to be as glycine and lysine
with Rf values of 0.20 and 0.61, respectively.
II. Keywords: paper chromatography, retention factor, solvent front, stationary phase,
mobile phase
III. Introduction Paper Chromatography uses a liquid
There are various separation solvent and paper as the mobile and stationary
techniques used in the laboratory, and is phase, respectively. It is commonly used to
used depending on the type of mixture separate dyes of an ink, and drugs from blood
and the chemical properties of the (Clark, 2016). This technique requires one
components of the mixture phase to be polar and the other is relatively
([Link], 2012). nonpolar. A normal phase chromatography
Chromatography is an example of a involves a polar stationary phase and a
separation method where the mixture [is relatively nonpolar mobile phase. On the other
passed] in a solution or suspension through a hand, a reversed phase chromatography has a
medium in which the components move at nonpolar stationary phase and a polar mobile
different rates ("chromatography," n.d.). phase. This is because [d]ifferent compounds
Chromatography has two phases: stationary in the sample mixture travel at different rates
and mobile. The stationary phase could be a due to differences in solubility in the solvent,
solid or a liquid adsorbed into an inert support, and due to differences in their attraction to the
while the mobile phase could be in a form of a fibers in the paper (Kumar, n.d.).
liquid or gas. Procedures in this method can be If the samples are colorless, they may
classified as adsorption, which is the be located by using detectors such as ninhydrin
distribution of the components of the sample for amino acids. The retention factor (Rf) is
between the liquid mobile phase and the solid calculated to determine the distance travelled
stationary phase ("Separation of amino acids by the sample relative to the solvent (Clark,
by paper chromatography," 2010, p. 5), while 2016). The formula is stated below:
the partition chromatography is between liquid
stationary phase and a liquid or gaseous mobile
phase ("Separation of amino acids by paper
The experiment aims to identify the Rf
chromatography," 2010, p. 5).
values of several amino acids, recognize the
stationary and mobile phases of paper
Chem31.1, Separation of Amino Acids by Paper Chromatography
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chromatography, and discover the factors that of bond paper. Ninhydrin solution was
affect the Rf values of the samples. sprayed lightly and evenly on the paper
along the direction of solvent flow and
IV. Methodology was constantly dried under a dryer until
Preparation of Solvent Mixture violet spots became visible. The edges of
This was performed in a hood. 24mL of the violet spots were traced and the
sec-butanol, 6mL glacial acetic acid and darkest spot in each of the spots were
10mL of distilled water were carefully marked. The distances were then
mixed in a bottle so as not to wet the measured in cm and the Rf values were
sides with the solvent. The bottle was calculated for each amino acid including
then covered and set aside. the unknown.
Preparation of the Chromatography Paper
A line was drawn 1 cm from the V. Results and Discussion
bottom in a 19 cm x 14 cm filter paper, a. The stationary phase in this
2 cm from the left edge of the filter chromatography experiment is the paper,
paper, light pencil cross marks were while the moving phase of the setup is
drawn 3 cm apart. With a clean capillary the solvent system (sec-butanol, glacial
tube, a spot of amino acid solution was acetic acid, and water). The setup is a
applied four times at the center of the normal phase chromatography since the
cross marks, creating a final spot of 1-2 moving phase is relatively nonpolar.
mm diameter. Prior to each application of The calculated Rf values are shown
an amino acid solution, the created spot in the table below. As evident from the
was dried with a dryer. Each amino acid data results in Table 1, only the
solution has its own distinct capillary tube experimental value Rf of glycine is far
and each spot on the chromatography from the theoretical one. The rest of the
paper was labeled respectively with a experimental values are relatively close
pencil marked X. The filter paper was to their respective theoretical values.
then rolled into a cylinder where the
edges were stapled about 1 mm apart.
Table1. Experimental and Theoretical R f
Chromatography Setup Values of Amino Acid Standards.
In the hood, the paper was Theoretical
carefully lowered into the bottle Amino Acid Experiment Rf Value
containing the developing solvent with Standards al Rf Value (Steane,
the sample edge down. It was made
n.d.)
certain that the paper does not touch the
sides of the bottle and that the line amino Glycine 0.62 0.26
acid solution spots were not immersed in Lysine 0.22 0.14
the solvent. The bottle was tightly closed Leucine 0.80 0.73
and the set-up was left untouched for Tyrosine 0.52 0.45
about two hours until the solvent front Unknown Lysine - 0.61 Lysine -
was only about 1 cm from the top edge of amino acid 0.73
the paper. The paper was then carefully mixture Glycine - Glycine -
removed and let stand on a Petri dish 0.20 0.26
cover.
The solvent front was immediately
marked with a pencil and the paper let The theoretical value of leucine has
dry for a few minutes with a dryer. The the largest value because it is the most
staples were removed and the filter paper nonpolar among the given amino acids.
was laid front side down on a clean sheet
Chem31.1, Separation of Amino Acids by Paper Chromatography
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This is followed by tyrosine, then glycine, the solution ("Separation of amino acids
and the last one is lysine. Since the by paper chromatography," n.d.).
solvent is relatively nonpolar, the most Common errors in chromatography
nonpolar amino acid would have the involve applying too much sample that
highest affinity with respect to mobile may make the samples travel horizontally
phase. Therefore, the solvent would carry and mix with the others. This is the case
the amino acid father than the other for the glycine in the experiment done by
samples. The unknown amino acid the researchers. Solvent mixtures should
mixture is determined by the two solvent also be covered to prevent solvent
fronts in the chomatograph. The evaporation and reaction to the
calculated Rf values are compared to the environment. The chromatography paper
experimental values that helped the should always be handled with gloves as
researchers identify the components of to avoid contamination and prevent
the mixture. transferring the amino acids of the
researcher [through sweat or oil] to the
experiment setup.
VI. Conclusion and Recommendations
The Rf value varies in every amino
acid in the experiment. These Rf values
are affected by the pH of the solvent,
affinity, and the pH level of the solvent
involved in the experiment.
It is recommended that the
spotting must be carefully performed to
prevent the common error of tailing. Dry
the spot as fast as possible to prevent
diffusion of amino acids to different
Figure 1. Structures of the amino acids directions. All materials to be used in the
used in the experiment. experiment, especially the filter paper,
should be cleaned and uncontaminated
Various factors affect the Rf value as these impurity factors may lead to
of a solute. One of which, as mentioned in inaccuracy and failure of the experiment.
the previous paragraph, is affinity. Higher It is also recommended to have relatively
affinity with respect to the stationary larger or longer gloves and lab coat for it
phase would have a lower R f value, and is hazardous for the skin to be in contact
higher affinity with respect to moving with the ninhydrin solution as this may
phase would have a higher R f value. cause undesirable effects.
Polarity is also a factor, since polar
substances mostly interact with the paper VII. References
that is relatively polar and therefore [Link]. (2012). Separation of
travels a shorter distance, compared to mixtures using different
the nonpolar molecules that interact with techniques. Retrieved March 19,
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level also affects the R f value because all [Link]
amino acids, even those with neutral side sub=73&brch=2&sim=96&cnt=1
chains, contain an acidic COOH group Caceres, C., & Velilia, A. (2014, May
and a basic -NH2 group. The most 1). Experiment 2 [Prezi slides].
prevalent ionic form of an amino acid in Retrieved from
solution therefore depends on the pH of
Chem31.1, Separation of Amino Acids by Paper Chromatography
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[Link] [Link]
ll6djy/experiment-2/?webgl=0 of-experiment-2-oral-report/
chromatography. (n.d.). In Oxford Separation of amino acids by paper
dictionaries. Retrieved from chromatography. (2010).
[Link] In Laboratory manual in organic
efinition/chromatography chemistry (pp. 5-7). Manila,
Clark, J. (2016, July). Paper Philippines: University of the
chromatography. Philippines Manila.
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