COLUMN CHROMATOGRAPHY
(Gel filtration
chromatography)
(Ion exchange
chromatography)
(Affinity
chromatography)
Content:
Principle,
protocol for various types of column chromatography, pictures related to
chromatographic patterns.
Column
chromatography is one of many forms of chromatography. Others include paper,
thin-layer, gas, and HPLC. Most forms of chromatography use a 2-phase system to
separate substances on the basis of some physical-chemical property. One phase
is usually a stationary phase. The second phase is usually a mobile phase
(often a buffer in biochemistry) that carries the sample components along at
different rates of mobility. The separation is based on how well the stationary
phase retards the components versus how quickly the mobile phase moves them
along. Substances with different properties will thus elute (exit) from the
column at different times. Some common types of column chromatography used in
biochemistry are gel filtration, ion exchange, and affinity. You will have the
opportunity to use one or more of these during your projects. In this exercise,
you will use gel filtration chromatography.
(a) Gel Filtration (permeation)
Chromatography. Gel filtration uses a gel matrix as the stationary phase. The
matrix consists of very small porous beads. The large molecules of a sample
solution do not get “caught” in the pores of the gel and will travel through
the column more rapidly because they can go around the beads. They are said to
be “excluded” from the matrix. Smaller molecules that can enter the gel pores
must go through the beads, thus taking more time to reach the bottom of the
column. Medium-size molecules can enter larger pores, but not small ones. This
form is also referred to as “molecular sieve” chromatography, because the
components of a sample are separated according to their molecular size (and to
a certain extent, molecular shape). The gel matrices are commonly made of cross
linked polysaccharides or polyacrylamide, both of which can be made with varying
pore sizes. The information supplied by the manufacturer will state the size of
the beads, the approximate size of molecules that will be excluded, and the
range of molecular weight range that can be separated. By using gels of
different sizes and porosities, one can separate samples that have a large
variety of components.
A few useful definitions:
Bed volume (Vt) is the total volume inside the column.
Void volume (V0) is the volume of
solution not trapped in the beads.
Internal volume (Vi) is the volume of solution trapped in the beads.
Volume of the gel matrix (Vg): Vt = V0 + Vi + Vg. Elution volume (Ve) is
the volume necessary to elute a substance from the column.
(b) Ion Exchange Chromatography: In this type of chromatography,
the matrix is covalently linked to anions or cations. Solute ions of the
opposite charge in the mobile liquid phase are attracted to the resin by
electrostatic forces. There are 2 basic matrix types; anion exchangers bind
anions in solution and cation exchangers bind cations. As the sample components
go through the column, those with the appropriate charge bind and the others
are eluted. Proteins have many ionizable groups with different pK values, thus,
the charge on the protein will depend on the pH of the buffer used. Thus, one
must carefully choose the exchanger and pH of the buffer used for the mobile
phase. Once all unbound substances have passed through the column, the bound
molecules can be eluted by changing the buffer. One way is to increase the
ionic strength (either gradually using a gradient or all at once depending on
whether you wish to fractionate the bound components elute them all at once
respectively). The anions or cations in the salt will compete with the bound
molecules and cause them to dissociate from the matrix. The higher the charge
density on the bound molecules, the higher salt concentration will be required
to effectively remove them. Another option is to change the pH, and thus the
charge, of the proteins. Problem: You use ion exchange chromatography with DEAE
cellulose (an anion exchanger) to separate proteins with the following pI
values: 3.5, 5.2, 7.1, and 8.5. The proteins are loaded onto the column in a
low ionic strength buffer, pH = 7.0. The column is then washed and eluted with
a gradient of 0.05–0.50 M NaCl in the same buffer. What is the order of elution
of the proteins?
(c) Affinity Chromatography:
Affinity chromatography utilizes the specific interaction between one kind of
solute molecule and a second molecule that is immobilized on a stationary
phase. For example, the immobilized molecule may be an antibody to some
specific protein. When solute containing a mixture of proteins is passed by
this molecule, only the specific protein reacts to this antibody, binding it to
the stationary phase. This protein is later eluted by changing the ionic
strength or pH. Alternatively, an excess of the molecule immobilized on the
stationary phase may be used. For example, if the molecule you wish to purify
binds glucose, it can be separated from molecules that don’t by using a glucose
affinity column (the matrix contains immobilized glucose molecules). Only
glucose-binding molecules will bind to this matrix. The bound molecules can be
eluted by adding glucose to the elution buffer. This will compete with the
matrix-bound glucose for the binding sites on the protein and the proteins (now
bound to free glucose) will dissociate from the matrix and elute from the
column. This method is gentler, but can only be used in some cases. This
elution method is only feasible when the immobilized molecule is small, readily
available, and cheap, as is the case with glucose.
Exercise for Gel Filtration
Chromatography
Determine the “bed volume” of the glass column by filing the column with water and measuring with a graduate cylinder.
Preparation of the Gel
1. You will use Sephadex G-100 for this experiment. The gel has a fractionation range for proteins of 4000–150,000 daltons. Sephadex is supplied as a dry powder and must be hydrated before use. The amount of water absorbed and the time required depends on the type of gel. Sephadex G-100 takes 3 days at room temperature or 3 hours in a boiling water bath. One gram of dry powder will make about 15–20 mL of gel. Weigh out the powder and add a large amount of water. Gentle stirring may be used, but vigorous stirring will break the beads.
2. When the gel is ready, decant the water. Some of the very fine
particles will also be decanted. This is not a problem. In fact, it is good to
remove the “fines” as they will pass through bottom support screen of the
column or clog the column and slow the flow. Replace the water with phosphate
buffered saline (PBS) and stir to equilibrate the gel with the buffer. Allow to
settle and decant again.
3. Degas with a gentle vacuum just before use.
Packing the Column
4. Close the outlet of the column. Stir the gel to create slurry and
carefully fill the column without creating areas of different densities. The
most even packing will be achieved if you pour all the necessary slurry into
the column at once. If necessary, stir the settling gel to prevent layers of
gel from forming. Open the outlet and add buffer as the gel packs. Do not let
the buffer drop below the top of the gel bed! If it is necessary to add more
Sephadex, stir the top of the gel bed before adding more slurry.
5. If layers or air bubbles are still present in the column, invert the
column and allow it resettle, doing this as many times as is necessary to
obtain a well-packed column.
6. Connect the column to the peristaltic pump and equilibrate the column
by eluting 1 bed volume of PBS buffer at a flow rate of 1 mL/min. Collect the
eluent in a graduated cylinder.
7. Determine the void volume and check the packing. Blue Dextran is a
large polysaccharide (average molar mass is about 2 million daltons). It is
excluded from the beads and will be eluted in the void volume. Add Blue Dextran
solution to the top of the column and let it run into the gel. Immediately
start collecting the eluent in a graduated cylinder. Gently put more buffers
over the gel and run the peristaltic pump. Measure the amount of PBS eluted
during the time it takes the Blue Dextran fraction to run the length of the
column. This volume is the void volume. If your column was evenly packed, the
Blue Dextran should run as a horizontal well-defined band through the column.
8. Prepare your protein mixture to 1 mg/mL concentration and add it
carefully to the top of the column like you did for the Blue Dextran. For the
best resolution, the sample volume should not exceed 1%–2% of the column
volume. You will run the following substances: hemoglobin, myoglobin,
cytochrome c, and vitamin B12. Vitamin B12 has a molar mass of 1355 D and
should be completely included in the Sephadex beads. All of these substances
are colored various shades of red or brown, so you should see them as they make
their way down the column and in the collected fractions. Run the column at a
rate of 0.5 mL/minute. Rates that are too fast will decrease resolution and
compress the gel. Start collecting 1-mL fractions and start the chart recorder
as soon as you add the sample. The eluent passes through an absorbance detector
(280 nm) and will detect the proteins as they elute.
9. Note the elution volume of each substance. A plot of log molar mass
versus elution volume should be linear over the useful fractionation range (for
roughly spherical proteins).
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| COLUMN CHROMATOGRAPHY - Affinity chromatography |
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