RESULTS AND DISCUSSION

Separation processes are important in different

industries, especially in areas of chemistry and
biology. In some instances, unknown chemical
are to be separated in order to determine its
identity; on the other hand, proteins are also
separated in order to further analyze it. In fact,
there are already different separation techniques
available today depending on the phases
involved for separation, such as chromatography.
In chromatography, the separation is dependent
on the relative amount of components or solute
that will be separated. Generally, it is composed
of a mobile phase, and a stationary phase. The
mobile phase is composed of the mixture
samples that move through the solid phase and
depending on the affinity of the sample to the
nature of the solid phase, the time at which the
components pass over the solid take different
lengths.

beads, which is referred as the column bed.

These beads filter the mobile phase, which
contains the protein mixture, and allows the
larger molecules to pass through, while the
smaller molecules are directed towards these
beads and are temporarily trapped within its
pores. On the other hand, the mobile phase is
composed of a buffer and dissolved in here is the
main component, which is the mixture of
biomoleculesVitamin B12 and Hemoglobinthat
are to be separated.
Upon subjecting the protein mixture to SEC, it is
evident that hemoglobin, the brown-colored
solution, is eluting first (Refer to Figure 2).
Figure 2. The elution of the protein mixture upon subjecting to SEC

For
this
experiment,
size
exclusion
chromatography (SEC) was used in order to
separate the protein mixture (FIGURE 1).
Figure 1. Size Exclusion Chromatography Kit

Hemoglobin is an important component in red

blood cells (RBCs), since it serves as the carrier
of oxygen to the tissues of the body and it gives
the cells their red-brown color. This is because it
contains the heme, which is the iron-containing
group; and naturally, iron is red in color.
Hemoglobin is made up of four polypeptides,
which associate to form a large, globular protein
(Figure 3).
Figure 3. Hemoglobin Structure

It follows the principle of chromatography

discussed in the previous paragraph; however, it
is particular in separating the components
depending on the size. In SEC, the mobile phase
is in the form of microscopic porous spheres or

For the first and fifth test tube labeled A and E,

respectively, the solution was almost clear so we
had to put it at the end of the arranged tubes.
However, test tube A can be considered as the
tube with the lowest color intensity for the
hemoglobin elution, and test tube E for the
vitamin B12 elution. From here, a graph was
generated, which can be seen in Figure 5.
The beads that were filled in the SEC column
used can effectively fractionate molecules that
are below 60,000 Daltons. As the mobile phase
moves through the column, the component that is
below 60,000 Daltons enter the beads; therefore,
they elute slower as compared to the larger
molecules exceeding the said amount. Since
hemoglobin has a molecular weight of 65,000
Daltons, it did not enter the beads anymore;
rather, it simply elutes through it resulting to a
faster elution in accordance with the principle of
SEC.
On the other hand, Vitamin B12, which is also
called cobalamin, is a water-soluble vitamin that
has a key role in the normal functioning of the
brain and nervous system, and the formation of
red blood cells. In the protein mixture, it is the
component with dark pink color and has a
molecular weight of 1, 350 Dalton. Since its
molecular weight is much lower than 60,000
Daltons, it is expected that it will enter the beads;
thus, making it move at a much slower rate.
After performing SEC, the test tubes containing
that fractionated components were then arranged
according to their color intensity (Refer to Figure
4).
Figure 4. Test tubes with the fractionated proteins

Figure 5. Color Intensity vs. Test tube number

Color Intensity

Test tube number

From the graph, it is evident that the peak for

hemoglobin elution and vitamin B12 elution can
be found on test tubes 3 and 8, respectively
which simply indicates that these tubes contained
the most fractionated protein. As for test tube 5, it
can be seen that it has the lowest or light color
intensity; this can be caused by the transition
from the elution of hemoglobin to elution of
vitamin B12, thus, it possibly contains mostly of
the buffer used to dissolve the protein mixture.
Since the components are highly differed when it
comes to their molecular weight, the best way to
separate them is to use a chromatography where
the basis for the elution is size. Thus, size
exclusion chromatography is a very powerful
technique for the physical separation of the
proteins: Hemoglobin, and vitamin B12.