VI.

Discussion
Chromatography is used to separate
mixtures of substances into their components.
Chromatography is partially characterized by
the medium on which the separation occurs,
known as the stationary phase, which usually
include paper, thin plates coated with silica gel
or alumina, or columns packed with the same
substances. The mobile phase is the medium
that accompanies the analyzed substance as it
moves through the stationary phase.
Paper chromatography is a partition
type of chromatography wherein the stationary
phase is a very uniform absorbent paper, while
the mobile phase is a suitable liquid solvent or
mixture of solvents.
In this experiment, the mobile phase is
in the form of the organic solvent mixture of
butanol, glacial acetic acid, and distilled water.
The developing solvent was then poured into a
cylindrical transparent container, making sure
that the sets of the container did not get wet in
the process. The bottle was then sealed to
allow the solvent to saturate to ensure that it
will rise with the paper.
The stationary phase in this
experiment is the water that is tightly bound to
the cellulose fibers of the filter paper. Extra
care was taken in handling the filter paper so
as to avoid contamination that could possibly
lead to errors. Latex hand gloves were also
used in handling the filter paper. It is
paramount that no contact be made between
the body and the filter paper, as the human
body itself exudes amino acids of its own
found in sweat which might further add to
possible errors in the experiment.
To avoid mixing of amino acid
samples, a different capillary tube was used
for each of the amino acid solutions to avoid
mixing and contamination of the samples. In
specifically marked spots collinear to each
other about 2 cm to the edge of the paper,
each amino acid solution was individually
dropped. To prevent any overlapping, it is
important that the size of the final spot formed
by the drops on the paper be only 1-2mm in
diameter. When it comes to amino acid spots,
the area must be particularly 1-2mm in size as
excessively large spots result to bands that
tend to widen and overlap as they travel along
the surface of the filter paper. On the other
hand, miniature spots occur when the amount
of sample used is insufficient. In either case,
the results will make amino acids harder to

differentiate and detect. Afterwards, said spots

were dried using a handheld blow-dryer.
Once the paper has been sufficiently
dried, it is then rolled up neatly and stapled
and then lowered carefully into the cylindrical
container containing the solution, making sure
that the sides of the container do not touch the
rolled paper, as contact may affect the
vaporization of the solvent and the rate and
distance at with each of the amino acids would
be observed to travel.
The solvent mixture should be allowed
to saturate the chromatography chamber for
successful/effective capillary action and
separation of the components in the mixture.
When no saturation occurs, the solvent from
the system that is moving up the paper
evaporates out into the environment, stalling
the capillary action. The bands will then begin
to broaden as they continue to move up,
down, and sideways, while the solvent front
remains fixed. This spreading movement of
the bands is known as the Van Deemter
broadening, which must be avoided by
allowing the mobile phase to move faster.
The solvent was then left to rise for
about 30 minutes or until it has reached to
about 1 cm from the top edge of the paper.
The paper was the rolled open and laid flat on
a clean bond paper, where it was then dried
again. At the same time as it is being dried, a
small paint brush dipped in ninhydrin was
brushed very lightly onto the paper in the
direction of the solvent flow.
Ninhydrin was used as it is known to
react with amino acids resulting to a brownishviolet color. Specifically, Ninhydrin degrades
amino acids into aldehydes, ammonia, and
CO2 through a series of reactions; the net
result is ninhydrin in a partially reduced
form hydrindantin. Ninhydrin then condenses
with ammonia and hydrindantin to produce an
intensely blue or purple pigment, sometimes
called Ruhemann's purple:
Figure 1. Reaction of Ninhydrin with Amino
Acids

The particularly dark-colored areas

that appear on the paper indicate the distance
which the amino acid solution has traveled.
This distance is measured and recorded. The
paper was dried because the presence of heat
speeds up the reaction of ninhydrin with amino
acids.
The
abovementioned
measured
distances were then used in the calculations
for the Rf (retention or retardation factor) which
is given by dividing the distance travelled by
the amino acid with the distance travelled by
the developing solvent. Rf value described the
migration of a certain species through a
chromatographic medium. It is also indicative
of the amount of time a solute spends in the
stationary phase relative to the time it spends
in the mobile phase.

value because it is more soluble and has a

greater affinity for the stationary phase.
Aside from polarity, the differences in
molecular weight is another factor that affects
the Rf values. A heavier (greater molecular
weight) molecule will travel slower than a
relatively lighter one.
To further understand why different
amino acids have uniqure Rf values, it is
important to understand the structural features
of these molecules. Amino acids contains an
amino group, -NH3+ and a carboxylic acid
group, -COOH, with the generic amino acid
structure being:
Figure 2. Generic Structure of Amino Acids

The calculated Rf values of each of the

amino acids are 0.157 for Glycine, 0,139 for
Lysine, 0.652 for Leucine, 0452 for Tyrosine,
and 0.157 and 0.435 for the unknown. By
comparing the calculated Rf of the species in
the unknown, it was determined that the
species of amino acid present in the unknown
solvent mixture was Glycine and Tyrosine. For
reference, table 2 contains the theoretical R f
values of the amino acids.
Table 2. Theoretical Rf Values of Amino Acids
Amino Acids
Glycine
Lysine
Leucine
Tyrosine

Theoretical Rf Value
0.26
0.14
0.73
0.45

These theoretical Rf values of amino

acids are taken for a similar solvent setup as
that of the experiment, that is, 1-2mm diameter
spots of amino acid 2 cm from the bottom
edge of the paper and collinear to each other.
The observed differences of the Rf
values of amino acids is explained by how
dependent an amino acid is to the stationary
phase or mobile phase. Generally, more polar
amino acids are attracted to the more polar
water of the stationary phase, while the less
polar amino acid is attracted to the less polar
mobile phase (or developing solvent.) A truly
nonpolar analyte would not interact with polar
stationary phase. Thus, the nature and polarity
of the stationary phase affect the separation of
highly nonpolar analytes. Thus, the more polar
the amino acid, the less the distance it travels
across the filter paper and the lower the R f

Table 3. Amino Acid Structures and Trends

Amino
Structure
Relat
Rf
Acid
ive
Value
Polar
ity
Lysine

Glycine

Tyrosin
e

Increasin
Increasin
g
g

Leucin
e

As can be seen from table 3, the

different amino acids differ primarily on the
type of R-group attached to it. This is the basis
for the differences in their polarity and thus,
the differences in the Rf value. Lysine is
attached to a hydrocarbon and positive amine
(NH3+) R-group. The solvent in this case is
water, a protic solvent, meaning it readily
donates H+ that causes the NH 3+ to have a
positive charge. This positive amine is electron
deficient, which makes it easily attracted to the
electronegative oxygen in H2O. This causes
Lysine to be very polar, and thus have the
expected lowest Rf value of the group.

Tyrosine on the other hand has

aromatic and OH R-groups. The presence of
OH makes it slightly polar. Due to its relatively
weaker polarity, Tyrosine has a larger Rf value
than Lysine.
Leucine contains a Hydrocarbon Rgroup. This R-group is very stable and is not
easy to break. The strong hydrocarbon makes
it very non-polar therefore it has a higher
affinity for the non-polar mobile phase, which
means Leucine is expected to have the
highest Rf value of the group.