HBC1011 Biochemistry I

1. FACULTY OF INFORMATION SCIENCE & TECHNOLOGY
HBC 1011 BIOCHEMISTRY 1
TRIMESTER 1, 2017/2018
PRACTICAL 2: PURIFICATION AND ANALYSIS OF
BIOMOLECULES BY CHROMATOGRAPHY
MOHAMED REDA

2. 1
PART A: SEPARATION AND IDENTIFICATION TECHNIQUES
USING THIN LAYER CHROMATOGRAPHY
INTRODUCTION
Separation and identification of protein and other mixtures is very important step in
biochemistry especially when we are dealing with protein. There are a few ways to perform
this separation and identification. One of those is using chromatography methods. The
chromatography is for separation based on a few principles such as size, charge or affinity.
The components in chromatography are consisted of mobile phase and the stationary phase.
In this part of experiment, thin layer chromatography was the main concern. The short
form for thin layer chromatography is TLC. They consist of mobile phase and stationary
phase. The mobile phase is the sample being used or the sample that we would like to
separate. In this experiment dye mixture and amino acids mixture is being used. While the
stationary phase is based on type of chromatography used to perform the separation.
Therefore the stationary phased included in the experiment was the TLC sheet. The mobile
phase or the sample was applied on the stationary phase at the origin. The origin indicate
where the molecules started to travel while the solvent help facilitate the sample to move. The
sample with interact more with the solvent move faster. The sample which interacts more
with the stationary phase would move slower.
The length traveled by those samples is used to calculate the retardation factor, Rf.
The Rf is to calculate the ratio of the sample travel to the solvent travel. The higher the Rf the
geater the interaction between sample with the solvent and the less the interaction with the
stationary phase. Therefore a low Rf value mean the more the interaction between sample
with the stationary phase and less with solvent. The TLC method is normally not to separate
but to identify that there are how many molecule in the mixture by separation.
METHODOLOGY
Experiment I Separation of dyes
1. A TLC sheet was obtained without touching the coated surface since fingerprints
could leave significant quantities of protein which will affect the experiment. A light
pencil line about 1.5 cm was drawn from the bottom on both side of the sheet.
2. A small drop of dye mixture was gently spotted onto the pencil mark with applicator
side by side for 20 times using cut yellow tip and the sheet were allowed to dry. The
exact amount of sample applied was critical. Enough samples were a must so that the
developed spot could be detected, but overloading would lead to “tailing” and the lack
of resolution.
3. The sample was left for five minutes to let the spot dry.
4. Sufficient amount of solvent A was added to the bottom of the beaker until just
enough covering the beaker.

3. 2
5. The paper strip was placed in the beaker gently with sample spots facing down, the
beaker was covered with lid to minimize disturbance. The origin was made sure that it
was not submerged in the solvent. The solvent was allowed to travel up the sheet in
five to ten minutes, until the solvent reached almost the line of the pencil mark.
6. The TLC sheet was removed carefully using forceps and it was allowed to air dry.
The position of the solvent front and the sample spot when the solvent stopped
moving were marked.
7. 𝑅𝑓 values for each dye were calculated according to the following equation:
Rf = Distance traveled by sample spot
Distance traveled by solvent front
Rf values for each sample tested were recorded in the table.
8. The observation for TLC sheets was elaborated.
9. The chromatograms were kept for reference and were fully labeled.
Experiment II Separation of amino acids
1. A new TLC sheet was obtained without touching the coated surface since fingerprints
can leave significant quantities of protein and will affect the experiment. A light
pencil line about 1.5cm was drawn from the bottom of the sheet.
2. A tip or small drop of amino acid mixture was gently spotted onto the pencil mark
with applicator or cut yellow tip side by side for 20-40 times and the sheet was
allowed to dry.
3. The sample was left for five minutes to let the spot dry.
4. The amino acids mixture was separated in solvent B. Sufficient amount of solvent B
was added to the bottom of the beaker until just covering the beaker.
5. The paper strip was placed in the beaker gently with the sample spot facing down it
was covered with lid. The solvent was allowed travel up the sheet in 5 to 10 minutes,
until solvent reached the line of the pencil mark.
6. The TLC sheet was removed carefully by forceps and it was allowed to air dry. The
positions of the solvent front and the sample spots were marked.
7. The paper strip was taken from the tank and was heated over a hot plate for color
development. Amino acids would give purple or yellow colors when they react with
ninhydrin and the spots of Valine and Aspartic acid would show up dense colorations
of the paper. The spots were observed if there is any tailing. Extensive tailing means
that the chromatogram is overloaded and too much sample is applied.
8. The Rf values for these two amino acids on all the chromatograms were determined.
9. The observation for TLC sheets was elaborated. The chromatograms were kept for
reference and were fully labeled.

4. 3
RESULT
Experiment 1
Distance from origin to the center of
spot
Rf value
Solvent front 8.20 cm -
Orange G 5.00 cm 0.610
Congo Red 3.40 cm 0.415
Light Green 8.10 cm 0.988
Experiment 2
Distance from origin to the center of
spot
Rf value
Solvent front 8.20 cm -
Valine 8.10 cm 0.988
Aspartic acid 7.00 cm 0.853
Thin Layer
Chromatography of dye
Thin Layer Chromatography
of amino acids

5. 4
Distance from origin to the centre of
spot
Rf value
Solvent front 9.0 cm -
Valine 4.8 cm 0.533
Aspartic acid 5.5 cm 0.611
Phenylalanine 5.3 cm 0.589
Proline 8.7 cm 0.40
DISCUSSION
Based on the two experiments that have been done in this first part, a few
observations have been obtained. The separation of dye is to give a basic understanding on
TLC, thin layer chromatography before we proceed into an actual objective which is to
separate the protein as an obvious indicator mixed dye has been used. So in the result we
would see a clearer separation of different colors of dyes. The separations of these colors are
because of the different in the charge of the dyes. The light green dye travel the longest
showing the least polar of the dye material. The second longest path was of orange-G
showing a greater polar the light green dye but still less polar than the congo red dye which
traveled the shortest.
In the second experiment mixture of amino acids has been used. In our sample we
used a mixture of aspartic acid with valine. Some difficulties we faced were the color of the
solution because the solution of amino acid mixture was colorless. Therefore when it was
applied on the TLC sheet it was hard to be seen and this might result in an undesired large
spot. A big spot would cause the result to be in accurate. The other circumstances was the
protein finger print from the holder as after we heat the sheet, some of the groups would see
the purplish color on abnormal part of the sheet ( the part was not the path traveled by the
mixture). From the result we could see that valine traveled longer than the aspartic acid. As
we know, that aspartic acid based on its name already showing that it is a polar molecule so
have more interaction with the stationary phase.
After the experiment was done, we were required to calculate the retardation factor, Rf.
This Rf. is to calculate the ration between the lengths traveled by the sample substance and the
lengths traveled by the solvent. The greater the ratio indicating the less interaction between
that particular sample with the stationary phase.

6. 5
QUESTIONS
1. Why do different compounds travel different distances on thin layer chromatography?
This depends on the characteristic of the compound the more soluble the compound in
the mixture it would travel faster. The other characteristic is the interaction between
compounds with the stationary phase. The more the interaction the slower it travels.
2. Name the stationary phase and mobile phase used in the experiment.
The stationary phase is the TLC sheet while the mobile phase is the substance either
dye or amino acid.
3. What is the use of an Rf value? What will be the range of an Rf value? You are given
Rf values for particle A and particle B, 0.2 and 0.8 respectively, what can you say
about the mobility of these particles?
Retardation factor is the ratio of the length travel by the substance with the length
travel by the solution in TLC. The range will be between 0 to 1. The mobility of
particle B is higher than particle A as it has higher Rf value.
4. Based on your experiment results, which has the highest mobility and which particle
is the least mobile?
In the experiment I, light green dye has highest mobility while congo red dye has least
mobility. In the experiment II, valine has highest mobility and aspartic acid has lower
mobility.
5. List the major sources of errors you can observe in this experiment.
The chromatography is to separate molecule mainly proteins based on their mobility.
The major sources of errors are the size of the spot made, and the time taken for
leaving the sheet in the solvent where the solvent front already reach the paper end.

7. 6
PRACTICAL 2(B) BIOMOLECULE SEPARATION;SIZE EXCLUSION
CHROMATOGRAPHY(SEC)
INTRODUCTION
The separation or purification of biological molecule is very important in doing
researches in the field of biochemistry. Chromatography is commonly used for separation or
purifying biological molecules, like proteins. Normally chromatography will separate the
molecules from other molecule in a mixture based on a few characteristic such as affinity,
size and charge.
One of a common chromatography is size exclusion chromatography. Based on the
name it is surely the separation of molecules using the principle of differences in size. As we
all known every type of molecule would have different size based on the composition of
different element in different portion. Based on the two components of chromatography
which are stationary phase and mobile phase, we can identify what is stationary phase for size
exclusion chromatography. In size exclusion chromatography (SEC), microscopic beads
which contain tiny holes packed into a column is considered the stationary phase while the
mixture of the hemoglobin with vitamin B12 is the mobile phase.
When the sample, the mobile phase was applied into the column different molecules
would traveled differently. A smaller molecule would pass through the bead and travel longer
while a bigger molecule does not need to travel through those beads as its size does not fit the
size of the path in the beads. So, it has traveled shorter path.
METHODOLOGY
Extraction of hemoglobin
1. To extract hemoglobin, the skin of a fingertip was pierced using an auto-lancet and a
micropipette with a sterile tip was used to collect 50 µl of blood.
2. The 50 µl of blood was mixed with 200 µl of an isotonic solution (0.100 M NaCl) in a
1.5 ml microcentrifuge tube and was centrifuged for 2 minutes at 6500 RPM
(approximately) to pellet the red blood cells.
3. The blood serum (supernatant) was removed.
4. The pellet of red blood cells was re-suspended in 250 µl of a hypotonic solution
(0.065 M KCl) and was vortex vigorously to lyse all cells.
5. The lysed red blood cell solution was centrifuged at 13,000 RPM (approximately) for
5 minutes to remove any undissolved materials (such as membranes).
Protein Separation
1. The 12 collection tubes were placed in a test tube rack. Ten collection tubes were
labeled sequentially from 1 to 10. The last two tubes were labeled with “waste” and
“column buffer”.

8. 7
2. Each group was given 1 ml pipette with indicators.
3. Three milliliters of Column Buffer was pipetted into the tube labeled “column buffer”.
4. The seal of both ends of the chromatography column was removed. The entire buffer
was drained into the “waste” collection tube.
5. The column was gently placed onto collection tube 1 without jamming the column
tightly into the collection tubes; otherwise the column would not flow.
6. The end seal was removed from the column. The top of the column bead was
observed; all of the buffer should have drained from the column. About 250 µl
undiluted biomolecule mix was carefully loaded onto the top of the column bead. The
pipette was inserted into the column and the drop was loaded just above the top of the
column so that it minimally disturbs the column bead. The column was kept hydrated
all the time.
7. The protein mix was allowed to enter the column bead. This is best observed by
looking directly over the column. The 250 µl of column buffer was carefully loaded to
the top of the column. This was best done by inserting the pipette tip into the column
so that it rests just above the column bead. The buffer was carefully let to run down
the side of the tube and onto the top of the bead. Drops were collected into tube 1.
8. When all liquid had been just drained from the column, another 250 µl of column
buffer was added to the top of the column. The buffer was added as before, by placing
the pipette just above the top of the column and let the buffer running down the side
of the tube. Drops were again collected into tube 1.
9. When all liquid have been drained from the column, 3 ml of column buffer was added
to the top of the column. This was done by adding 1 ml from the pipette three times.
The column was transferred to tube 2 and the drops that enter into each tube were
counted. Five drops of buffer were collected into tube 2.
10. When 5 drops had been collected into tube 2, the column was transferred onto tube 3.
Five drops of buffer were collected into each collection tube. When 5 drops had been
collected into a tube, it was lifted off and was transferred to the next tube.
11. The collection of 5 drops was continued into each tube. When tube 10 was reached, a
final 10 drops was collected. The column was capped when finished collecting drops.
The samples and column were stored according to the tutors’ instructions.
12. In the results, the observations on the colors separation in the column (which color
runs faster), colors changes of every tube were listed down in a table.

9. 8
RESULT
Tube 1 until tube 10 respectively
Tube 1 2 3 4 5 6 7 8 9 10
Color Colorless Colorless
Slightly
red
Red Red
Slightly
red
Slightly
red
Slightly
yellow
Slightly
yellow
Yellow
DISCUSSION
In this second part of the lab experiment, we did the size exclusion chromatography. From
the name itself we could know that this type of chromatography is for separation of
molecules based on the size of sample. In the experiment the solution used was the mixture of
hemoglobin and vitamin B12.
After collecting the eluent, we could make and observation that the tube 2 which is the first
tube to collect the sample contain colorless solution. This shows that the desired samples
(hemoglobin or vitamin B12) have not yet been eluted. Tube 3 starts showing reddish
solution which indicates that some of hemoglobin started to be eluted off the column. The
solution in tube 4 and tube 5 was in red color showing hemoglobin content. In the sixth and
the seventh tube the color was only slightly reddish indicating the hemoglobin is finishing
eluted.
In the 8th tube onward, the solution inside the becoming more and more yellowish indicates
the vitamin B12 content. So we can conclude that the hemoglobin elute first then followed by
the vitamin B12. The difference of time taken for the two molecules to be eluted showing that
these two molecules have different size. The order of the solution to exit the column help us
in knowing which molecule is bigger and which molecule is smaller. Hemoglobin which has
been eluted first is bigger in size compared to vitamin B12 which eluted last.

10. 9
QUESTIONS
1. Briefly explain how the chromatography in this practical functions.
In this experiment we used size exclusion chromatography technique. This technique
separates molecules based on the size, a bigger size molecule moves faster and the
smaller moves slower. This type of chromatography provides a longer path for
smaller molecules to travel.
2. If a size exclusion chromatography is said to have an exclusion limit of 40,000
daltons, would hemoglobin (64,000 daltons) be fractionated or excluded from the
column and why? Would vitamin B12 (1,350 daltons) be fractionated or excluded
from the column? Why?
Since the hemoglobin is 64,000 daltons and the exclusion limit is 40,000 daltons, the
hemoglobin would surely being excluded. Meanwhile for vitamin B12, as it is 1,350
daltons which is lower/smaller than 40,000 daltons it will be fractionated and move
out later than the hemoglobin.
3. Why did you need to add more buffer after the protein mixture was loaded onto the
column (at step 7 and 8 of the protocol)?
The reason to add the column buffer is to make sure that all the protein will be eluted
out and none of them would remain or stuck inside the column.
4. Which molecule exited the column first? Would this molecule be the larger or smaller
of the two?
The hemoglobin exited the column first followed by the vitamin B12. The first
molecule exited is larger than the next; hemoglobin is larger than vitamin B12.
5. If the following mix of molecules were purified using size exclusion chromatography
with exclusion limit of 80,000, what would be the order in which the molecules pass
through the opening in the bottom of the column? Mixture containing: hemoglobin,
64,000 daltons; myoglobin, 17,000 daltons; myosin, 180,000 daltons.
a. First molecule to appear: Myosin
b. Second molecule to appear: Hemoglobin
c. Third molecule to appear: Myoglobin

11. 10
CONCLUSION
From the experiment, we have experienced a few way of separating molecules mainly
proteins. Some of protein separations we have done are thin layer chromatography and size
exclusion chromatography. The separation was depending on a few basic principles such as
size, charge, affinity and weight. This experiment give us a better understanding on
chromatography and help in making decision on which type of chromatography to be used in
the future molecule separation.
REFERENCES
Kumar, P. (2016). Top 12 Types of Chromatographic Techniques | Biochemistry. Retrieved
August 3, 2017, from BiologyDIscussion:
http://www.biologydiscussion.com/biochemistry/chromatography-techniques/top-12-
types-of-chromatographic-techniques-biochemistry/12730