Ha Nguyen

Size-exclusion Chromatography and Ion Exchange Chromatography

Purpose:

In two experiments, the main purpose is to separate a mixture containing blue dextran,
cytochrome c, and DNP-glycine by using size-exclusion chromatography and ion exchange
chromatography. Part a of this experiment is size-exclusion chromatography and part b is ion-exchange
chromatography. Size-exclusion chromatography (SEC) is used for separating and quantifying protein
mixtures based on the difference in molecular mass. In ion-exchange chromatography, the method
relies upon the fact that different biomolecules will have differential electrostatic affinities for a charged
solid support due to differences in their overall charged states. For part B of the experiment, after the
separation, based upon the results of the experiment, the goal is also to establish whether the resin
used in the column corresponded to DEAE-sepharose or CM-sepharose.

Materials and Method:

Reference:

Part A: Size-exclusion chromatography.

The procedure followed is found on chapter 5 (page 152) of the lab test: “Fundamental
Laboratory Approaches for Biochemistry and Biotechnology”.

Part B: Ion-exchange chromatography.

This lab is part B of the chromatography separation technique. All instructions were followed as
the protocol handout available on: Blackboard, in BioChemistry Lab II section, under Course Document
tab, file name “Ion Exchange Chromatography”.

Materials:

The majority of materials were used for both size-exclusion chromatography and ion-exchange
chromatography experiment. However, buffer B with potassium acetate and sodium chloride was not
used for size-exclusion chromatography and pH 7.0 of potassium acetate was used for SEC.

1. 5ml glass column (pipet) provided by the TAs.

2. Ion exchange Resin slurry obtained from the TAs for Ion-exchange chromatography.
3. Sephadex G-75 slurry obtained from TAs for size-exclusion chromatography.
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4. 0.1M potassium acetate, pH 6.0 (Buffer A, 200ml) diluted from 0.5M potassium acetate
(premade from lab 08/29/2017).
5. 0.1M potassium acetate, 1M NaCl, pH 6.0 (Buffer B, 50ml) was made from the first day of the
experiment.
6. Glass wool
7. Mixture of blue dextran, cytochrome c and DNP-glycine provided by the TAs.
8. Variable pipettor and pipet tips (200microliters).
9. 4-5mm inner diameter tygon tubing (~3” long piece)
10. Tubing clamps.
11. Parafilm (Pechiney Plastic Packaging).
12. Disposable plastic Pasteur pipets & rubber bulb.
13. Eppendorf tubes (2mL).
14. Ring stand and clamp (Precision company; Burette Holder; Pat. No. Des. 188,563).

Modification:

Part A:

There was modification to the materials in the experiment comparing to the materials in the
textbook. In the text book, the materials are sodium phosphate elution buffer and a sample mixture of
blue dextran, cytochrome c, and potassium chromate.

There was no modification to the procedure in the textbook as the experiment was carried out.

Part B:

There was no modification to the procedure and the materials in the experiment.

Results:

Part A: After the mixture was purified by size-exclusion chromatography, a picture with the
measured volume for each Eppendorf tubes was taken by our group. Based on the color from each
collected tube, the mixture was purified completely. Because of the difference in color, the identity of
each biomolecule from the mixture can be determine. The first solution eluted from the column was
blue dextran, following with cytochrome c, and the final one was DNP-glycine.
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Figure 1: image of three compounds after separation by size-exclusion chromatography.

From the text book questions, a set values of volume were measured and calculated. Table one
is the collected data for both techniques that we performed in two experiments.

Vo Vt Vi Vg
Size-exclusion 1.75 mL 10.0 mL 0.15 mL 6.35 mL
chromatography
Ion-exchange 0.70 mL 5.70 mL 0.60 mL 3.70 mL
chromatography
Table 1: values of different volume measured during the experiment.

The total volume (Vt) of a column packed with a gel that has been swelled by solvent is given by
Vt = Vg + Vi+ Vo, where Vg is the volume occupied by the solid matrix of gel, Vi is the volume of solvent
held in the pores or interstices and Vo is the free volume outside the gel particles.

Part B: Ion-exchange chromatography

After the mixture was purified and separated by ion exchange chromatography, a photo with
the recorded volume of each biomolecule was taken by our group. Based on the color of each
biomolecule, it can be illustrated that three compounds- blue dextran, cytochrome c, and DNP-glycine,
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from the mixture were separated from one another. The identities of each compound can be determine
based on the difference in physical properties.

Figure two is the image of the ion exchange chromatography of our containing mixture of three
compounds. Each volume of each compound was also measured after the purification. Table two is
included for further discussion and explanation.

Figure 2: image of three compounds after separation by ion exchange chromatography.

Molecule Color Mass (daltons) Charged Properties

Blue Dextran Blue >500,000 Nonionic
Cytochrome c Red 12,400 Ionic protein, pI = 10.7
(net cation pH< 10.7)
DNP-glycine Yellow 241 Anion above pH 3.0

Table 2: Compounds in colored solution applied to Resin (provided on Blackboard, Biochem Lab II,
Course Document tab, file name “Ion Exchange Chromatography”).

Based on the table one with the physical properties of each biomolecule and the observed
results, it can be concluded that each compound was successfully separated from the mixture. The
light blue color solution was first collected from the column, which is corresponding to molecule blue
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dextran. The second compound was obtained is DNP-glycine with a yellow color. Finally, a light orange
solution was eluted lastly corresponds to cytochrome c.

Discussion:

Based on figure one and two from the result section, it can be illustrated that both technique
had completely separated and purified the mixture. The presence of different color in the solution is
crucial evidence to prove the success of the experiment.

For size-exclusion chromatography, the separation of mixture is based on the molecular size
(more correctly, their hydrodynamic volume) of the components. Separation is achieved by the
differential exclusion or inclusion of solutes as they pass through stationary phase consisting of
heterosporous (pores of different sizes) cross linked polymeric gels or beads. From table two, the
expected result is that blue dextran would be the first molecule to be eluted because it has the
heaviest mass. Following is the elution of cytochrome c and final is DNP-glycine. The observed result
was consistent with the expected one.

Question 5-6 from the textbook: “Were the colored molecules completely separated during your
chromatography run? Explain.

Yes, the three colored molecules were completely separated. Each molecule was eluted at
different rate because of its molecular weight and size. Blue dextran with the heaviest mass was eluted
first from the column, then cytochrome c and DNP-glycine.

For ion-exchange chromatography, charged substances are separated by column

chromatography with resins that carry charged ionic groups. In the experiment, blue dextran does not
contain any charge hence it was the first molecule to be eluted. Because of the different charge on
cytochrome c and DNP-glycine, each molecule was eluted at different rate. Since the resin could be
positively charged or negatively charge, the flow of these two molecules can be considered to establish
whether the resin used in the column corresponded to DEAE-sepharose or CM-sepharose. If the resin
used was CM-sepharose with negatively charge group, the expected results would be the elution of
DNP-glycine, then cytochrome c. If the resin was DEAE-sepharose, the opposite result would be
expected. However, based on the observed result, DNP-glycine with the yellow color was eluted first,
which illustrates that the resin used was CM-sepharose. Because cytochrome c has a positive charge, it
was bind to the resin and did not move along the column when buffer A was being added. In order to
separate cytochrome c, buffer B containing salt solution was used to bread the electric interaction
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between cytochrome c and the beads in the resin. Increasing the salt concentration results in the
shielding of the charges on the protein’s surface and effective binding to an exchanger is inhibited.

In conclusion, from two experiments, two techniques were demonstrated to show different way
to separate protein based on the differences in charge and molecular mass. Overall, both experiments
were a success because each molecule was separated from the mixture. The elution pattern reflects a
major difference in these two techniques. In SEC, the elution pattern was blue dextran, cytochrome c,
and DNP-glycine. With ion-exchange chromatography, the pattern was blue dextran, DNP-glycine, and
cytochrome c. In both techniques, the observed pattern in size-exclusion chromatography was
consistent with the expected. However, the amount of molecule was the different between the two
techniques. In SEC, 6.70 mL of DNP-glycine was collected while only 2.0mL of DNP-glycine was eluted in
ion-exchange. The intensity in the color was also varied between these two techniques. Particularly,
with ion-exchange chromatography, the color in each molecule is more intense compared to those in
SEC. The difference in the color intensity could have resulted from loosely packed column in SEC. In our
group SEC experiment, the column was not as tightly packed as the column in ion-exchange. During SEC,
the molecule moved faster and this could explain for the color intensity. If both experiments were
carried out, it would be better to pack the column carefully with no air bubble and good amount of
cotton wool. In two techniques, the color of cytochrome c was not as red as expected from its given
physical property. This could due to the concentration of cytochrome presented in the mixture. If
another experiment was carried out, increasing the concentration of cytochrome c in the mixture would
give a better color intensity.
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