FAKULTI TEKNOLOGI KEJURUTERAAN KIMIA DAN PROSES

Lab 04 SIZE EXCLUSION CHROMATOGRAPHY

BTP 3243

PROCESS BIOTECHNOLOGY TECHNIQUES

Lab objectives

By the end of this lab, students should be able to:

1. Separate a mixture of protein (hemoglobin and vitamin B12)

Student names Student ID Section Group

g NADIA WAHIDAH BINTI RAZAK

NUR ‘AMMARAH BINTI MOHD
TD18017
TD18021
01G
01G
4
4
GHAZALI
HASNI BINTI ABDUL RAHIM TD18024 01G 4
NUR NABILA ALYSHA BINTI TD18026 01G 4
ASMADI
AINA NAJWA BINTI MOHAMAD TD18032 01G 4
AZIZ
SYAZA ANIS BINTI SUBRAY TD18036 01G 4
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TABLE OF CONTENTS

No CONTENT PAGE
1 ABSTRACT 3
2 INTRODUCTION 4
3 MATERIALS & METHODOLOGY 5
4 RESULT 7
5 DISCUSSION 8
6 CONCLUSION 11
7 REFERENCES 12
8 APPENDIX 13

1. ABSTRACT

The purpose of conducted experiment, which is Size Exclusion Chromatography

(SEC) is to separate a mixture of protein which are hemoglobin and vitamin B12. In
this experiment, mobile phase liquid that called as buffer was used to dissolve with
biomolecules which are hemoglobin and vitamin B12. The mixture of biomolecules in
mobile phase liquid flow through the column bed since the column bed act as traps
that filter the small molecules to within the pores and allow the large molecules to
pass through it. Therefore, the large molecules will be collected in the first tube which
was tube 1. The additional buffer was filled inside the column bed after the sample
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has entered the bed. For the other tubes, 5 drops of buffer was collected in each tube
to allow the molecules with molecular weight below than 60,000 daltons penetrate the
pores of column bed. Since the hemoglobin has molecular weight of 65,000 daltons
whereas the vitamin B12 has 1,350 daltons of molecular weight, the hemoglobin will
be fractionated in early tubes while the vitamin B12 will be filtered in the latest tubes
through the pores. Thus, the color of the sample in early tubes will be brown color and
the other samples will be pink color. In conclusion, Size Exclusion Chromatography
(SEC) technique is an effective technique to separate the mixture of different size
molecules.

2. INTRODUCTION

Size exclusion chromatography (SEC), also known as gel filtration

chromatography is a technique used for a few purposes; mainly to separate protein
molecules according to its size and estimate the molecular weight of a protein. In
order to separate the molecules, a gel consisting of uniform-sized spherical beads is
filled into a column tube (Figure 1) and allowed to settle. The spherical beads have
pores of specific size that filter out molecules that are larger than the size of pores. A
protein sample is applied to the top of column. The solution will travel down the
column and the particles that are smaller than the size of pores will enter. The
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components in the sample will move down the column at rates that is affected by their
size. Larger molecules eluted faster compared to smaller molecules. The larger
molecules are eluted in the column’s void volume (interstitial volume) whilst the
small molecules diffused into the pores. The void volume shall take up one third of
the total column volume. The small molecules are claimed to travel throughout the
column volume with one third time is spent outside the bead whilst the other two third
time is spent inside the bead. Therefore, small molecules is said to elute in the total
column volume. Each molecules will be separated according to their size in order of
decreasing molecular weight. At the end of the column, the filtered solution is
collected for further assessment. The whole process is also referred as fractionation
step (Burgess, 2018).

Figure 1: A size exclusion column.

3. MATERIALS AND METHODOLOGY

3.1. MATERIALS
- 12 units collection tubes
- 1 unit sizing chromatography column
- 1 unit column end-cap
- 1 unit pipette
- 1 unit lab marker
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- 1 unit test tube rack

- 1 vial protein mixture
- 1 bottle column buffer

3.2. METHODOLOGY

1. 12 collection tubes are placed in the test tube rack and labelled 10 of 12 collection
tubes with the sequence of 1 to 10. The remaining two test tubes are labelled as
‘waste’ and ‘column buffer’.
2. 4 ml of column buffer is pipetted into the ‘column buffer’ collection tube.
3. The cap is removed and the end of the Poly-Prep sizing column is snapped off and
all the buffer is drained into the ‘waste’ collection tube. The bottom of the column
is capped with the column end cap.
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4. The column is placed onto the collection tube 1. The placement is done lightly to
facilitate the flow of column and the protein sample is ready to be loaded onto the
column.
5. The end cap is removed from the column. The top of the column bed is observed
by looking over the column to ensure the ‘grainy’ appearance of the column beads
is visible. One drop of protein mix is pipetted onto the top of the column bed to
minimize the disturbance on the column bed.
6. The protein mix is allowed to enter the column bed. Then, 250µL of column
buffer is pipetted into the column and the buffer is let to run down the side of the
tube and onto the top of the bed. The drops are collected into the tube 1.
7. Another 250µL of column buffer is pipetted on top of the column once all the
liquid is drained from the column. The collection of drops are continued into the
tube 1.
8. 1 ml of column buffer is pipetted three times on top of the column after all the
liquid is drained from the column. The separation is not affected by the slight
disturbances to the column bed. The column is transferred to tube 2 and five drops
of buffer are collected into the tube 2.
9. The column is transferred onto the tube 3 and another five drops of buffer are
collected.
10. The collection of five drops of buffer is continued into the tube 4 until tube 9.
Once tube 10 is reached, ten drops of buffer are collected into it and the column is
capped after finishing the drops collection. The samples and column are stored in
the refrigerator and the results are sketched.

4. RESULTS

1. Examine & observe the fraction in tube.

The peak fractions vary significantly between the student groups; the peak may be
influenced by diffusion, column bed disturbance when the sample is prepared, etc.
The peak hemoglobin fraction can usually be contained in tubes3, 4, or 5. Tubes7, 8,
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or 9 should contain the peak vitamin B12 fraction.

2. Observe the intensity of color.

Both molecules are the heme or iron-containing group that give them their distinctive
red color. Iron is clearly red-think iron tablets or red, soil-containing iron.

3. Which tubes contains hemoglobin or vitamin B12.

The peak fractions vary significantly between the student groups; the peak may be
influenced by diffusion, column bed disturbance when the sample is prepared, etc.
The peak hemoglobin fraction can usually be contained in tubes3, 4, or 5. Tubes7, 8,
or 9 should contain the peak vitamin B12 fraction.

5. DISCUSSIONS

Size-exclusion chromatography which also can be called SEC is described as the

differential elution of the solutes from the bed of the porous chromatographic medium
due to the various degrees of steric exclusion of the solutes from the volume of pore due
to the variation molecular size of the solutes. Thus, in SEC, the solutes are strictly
elucidated by decreasing molecular size and the maximum volume available for
separation is equal to the total pore volume of the packing medium. The size exclusion
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chromatography which is an SEC kit is performed to teach basic gel filtering

chromatography techniques. This kit uses coloured haemoglobin and vitamin B12
molecules to describe the principles of the SEC.

The principles of size exclusion chromatography will be investigated in this

laboratory. Size exclusion chromatography is a very powerful technique for the physical
separation of molecules by size. A mixture of molecules is dissolved in a liquid which is a
mobile phase implemented to a chromatography column containing a solid support in
terms of microscopic porous spheres or "beads" which is a stationary phase. The mass of
the beads inside the column is often referred to as the column bed. The beads will act as
"traps" or "sieves" and to filter small molecules that are temporarily trapped inside the
pores. Larger molecules are passed around or "excluded" from the beads. It contains eight
columns prefilled with beads that differentiate or "subdivide" molecules that are
effectively below 60,000 daltons.

Molecules below 60,000 daltons will enter the bead and it will pass through the
column more slowly as the liquid flows through the column. The smaller the molecule,
the slower it moves through the column. Molecules that exceed 60,000 pass 15 of them
around the beads and are excluded from the column that is called the exemption limit of
the column. The liquid used to dissolve biomolecules for the mobile phase is called a
buffer. The mixture of biomolecules dissolved in the buffer is called the specimen. The
sample will be placed on the column bed, and the biomolecules within the buffer will
enter the top of the column bed, sieve through and around the porous beads. Then, it will
finally pass through a small opening at the bottom of the column. An extra buffer is
placed on the column bed after the sample has entered the bed to achieve that purpose.
The mobile phase which is liquid is collected as a drop in a series of collection tubes. A
set number of drops is collected in each tube. The larger molecules that pass through the
column quickly will end up in the early tubes or "fractions."

The tinier molecules that penetrate the pores of the stationary phase end up in the
subsequent fractions. The two biomolecules in your samples are haemoglobin and vitamin
B12. Haemoglobin, brown, has a molecular weight of 65,000 daltons. Vitamin B12 is
pink with a molecular weight of 1,350 daltons.

QUESTIONS
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1. Explain size exclusion chromatography (SEC) theory.

Size exclusion chromatography (SEC) is the filtration through a gel which separates
molecules based on their size. The gel is made up of spherical beads containing pores of
a particular distribution of size. When molecules of various sizes are included or
removed from the pores within the matrix, separation occurs.
Small molecules diffuse into the pores and, depending on their size, their movement
through the column is retarded, whereas large molecules do not reach the pores and are
eluted into the void volume of the column. Consequently, as they move through the
column, molecules differ based on their size and are eluted in order to minimize
molecular weight (MW).

A buffer is commonly called the solvent used to dissolve the biomolecules to make
the mobile phase. The sample is considered a mixture of biomolecules dissolved in the
buffer. The sample is placed on the column bed and the biomolecules enter the top of
the column bed within the buffer, filter through and around the porous beads, and
eventually pass through the bottom of the column through a small opening. An
additional buffer is placed on the column bed after the sample has reached the bed for
this method to be completed. The mobile phase liquid is collected, as drops, into
sequentially organized collection tubes. In each tube, a fixed number of drops is
normally collected. The bigger molecules that move through the column quickly will
end up in the early tubes or "fractions." In the later fractions, the smaller molecules that
enter the pores of the stationary phase terminate.

Operating conditions and selection of the gel depend on the application and the
resolution needed. Fractionation and desalting are two common forms of SEC
separations performed by (or buffer exchange).

2. Illustrates the separation of the hemoglobin and Vitamin B12.

The two molecules that are being separated in this lab experiment are haemoglobin
and vitamin B12. Haemoglobin has a molecular weight of 65,000 daltons, which is
brown, and is thus omitted from the column. Haemoglobin travels into the column more
rapidly and emerges in the early collection tubes, or fractions. The molecular weight of
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vitamin B12, which is pink, is 1,350 daltons and thus fractionated by the column. The
molecules of vitamin B12 enter the pores of the beads and become temporarily stuck. As
a result, they move through the column a lot more slowly and appear in the later fractions.
The following diagram shows the differential fractionation on a scale exclusion column of
big and small molecules.

Figure 2: Illustration of the differential fractionation of large and small

molecules on a size exclusion column.

3. Determine which biomolecules exit the column first.

Haemoglobin would be exit from the column first because it is larger than the pores of
the beads; thus it is excluded from the pores and would pass through the column quickly.
Vitamin B12 would be fractionated by the column—its molecular weight is much lower
than the exclusion limit, causing it to be filtered through the pores.

6. CONCLUSION

As a function of the SEC flow rate, the obvious size distributions obtained for starch
in the SEC have been studied. The data is provided as hydrodynamic radius functions as SEC
weight distributions, with the conversion of elution volume to hydrodynamic volume by
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uniform calibration together with the linear standards (pullulan) calibration whose Mark-
Houwink parameters are known. With decreasing flow rates, shear is reduced in an SEC
column. The data show that extensive shear splitting of the amylopectin chains occurs in
SEC, but there are limited effects on the smaller chains (the "amylose" region) for lower flow
rates. The splitting in a shear field of a strongly branched molecule in solution is seen to have
physical parallels to the irreversible shear break-up of droplets and flocks under shear, for
which detailed data and models are available. The use of a common concept in droplet break-
up, the dimensionless number that gives the maximum shear stability size, was found to be
useful for SEC of strongly branched polymers.

These results, combined with this definition, indicate that it is possible to find SEC co
nditions where it is possible to reliably obtain size distributions of the smaller chains (the "am
ylose" region) essentially free of shear-scission objects. In summary, the applicability of
traditional size separation by SEC (GPC) to the study of the size distribution of starch using a
combination of experimental data and droplet-shear theory has been shown to be restricted to
the analysis of the smaller, less branched, amylose molecules due to the shear forces in the
system and their effect on amylopectin.

7. REFERENCES
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Bio-Rad Laboratories.Inc (2020). Size Exclusion Chromatography Instruction Manual

[PDF FILE]. Retrieved on 29 December 2020 from https://www.bio-
rad.com/webroot/web/pdf/lse/literature/4006103.pdf

Burgess, R. R. (2018). A brief practical review of size exclusion chromatography: Rules

of thumb, limitations, and troubleshooting. Protein Expression and Purification,
150, 81–85. https://doi.org/10.1016/j.pep.2018.05.007

Hagel, L., & Janson, J.-C. (1992). Chapter 6 Size-exclusion chromatography.

Fundamentals and Applications of Chromatography and Related Differential
Migration Methods - Fundamentals and Techniques, A267–A307. doi:
10.1016/s0301-4770(08)61567-5

8. APPENDIX
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Figure 1: A size exclusion column.

Figure 2: Illustration of the differential fractionation of large and small molecules on a size
exclusion column.

RUBRIC FOR LAB REPORT FORMAT AND ASSESSMENT

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Item Assessed Unacceptable Poor Average Good Score

(0) (1) (2) (3)

Abstract  No abstract  No highlight of the  Missing objective and/or  Objective 3

significant results and/or conclusion.  Summary of the methodology
methodology.  Summary of the results
 Conclusion

Introduction  No background  Some introductory  Introduction is nearly  Introduction complete and 3

information provided information, but still complete, missing some well written; provides all
or information is missing some major minor points. necessary background
incorrect. points. principles for the experiment.
Material and  No Material and  Methods copied directly  The whole sentences of  The whole sentences of 3
methods method explained from module event being methods are converted to methods are converted to past
referred to took place in past tense but not present the tense with creativity
the past. actual experimental block/diagram used.
 Some paste some procedure, only based on  Methods are also written
sentences are converted modules based on what actually
to past tense happened in experimental
procedure.
Results  Data sheet with  Results given but  Results given but a few not  Results shown for all the 6
stamp not provided significant amount not tally with the requirements of scopes of experiments
(x 2) with the report OR tally with the experiment
 Results not requirement/ scopes
compatible with
scopes
Discussion  Only mention the  Attempt to discuss but  Elucidation of result but  Elucidation and supported by 30
results without failed miserably contains some flaws proper references or logical
(x 10) meaningful explanations.
discussion
Conclusions  No conclusion  Conclusion missing the  Conclusions regarding major  Important/ significant results 3
sections OR important points OR points are drawn, but many are highlighted which also
 Conclusions totally  No recommendation are misstated, indicating a meets the scopes of
not reflecting the given to improve the lack of understanding OR experiments AND
scopes experiment.  Conclusion is too general.  Several recommendations have
Several recommendations been stated.
have been given but they are
too general and not
contributing to the
experiment’s improvement.
References  No references  Only refer to websites  Refer to websites and some  The whole references 3
related research articles accounted from books,
websites, articles and human
name (personnel
communication)
Appendixes  No appendixes which  Appendixes made on  Appendixes made from  Appendixes made from proper 3
it supposed to be copies of actual modules proper and tidy new paper paper sheet with original data
having paper sheet from own experiment

Grammar and  Unreadable and not  Numerous spelling and/or  Occasional spelling and/or  Correct use of words. 3
Spelling written in scientific grammar errors.. grammar errors.
way
Timeliness  Lab report  Lab report submitted  Lab report submitted 1 day to  Lab report submitted on time. 3
submitted more than more than 3 days late. 3 days late.
a weeks

Total Assessment Marks (60) 60