GEL FILTRATION
Rveyda AKN, Gebze Technical University, Turkey
Experiment 7
AIM
Gel filtration chromatography is a separation based on size. The following experiment has been
designed to give you experience in preparing and using gel filtration column.
INTRODUCTION
Gel filtration (also referred to as size exclusion particles, or mobile phase. Samples are eluted
chromatography, SEC) separates molecules isocratically so there is no need to use different
according to differences in size as they pass buffers during the separation. However, a wash
through a gel filtration medium packed in a step using the running buffer is usually
column. Unlike ion exchange or affinity included at the end of a separation to remove
chromatography, molecules do not bind to the molecules that may have been retained on the
chromatography medium so buffer column and to prepare the column for a new
composition does not directly affect resolution run.
(the degree of separation between peaks).
Proteins that are small enough can fit inside all
Consequently, a significant advantage of gel
the pores in the beads and are said to be
filtration is that conditions can be varied to suit
included. These small proteins have access to
the type of sample or the requirements for
the mobile phase inside the beads as well as
further purification, analysis or storage without
the mobile phase between beads and elute last
altering the separation.
in a gel filtration separation.
Gel filtration is well suited for biomolecules
Proteins that are too large to fit inside any of
that may be sensitive to changes in pH,
the pores are said to be excluded. They have
concentration of metal ions or co-factors and
access only to the mobile phase between the
harsh environmental conditions. Separations
beads and, therefore, elute first.
can be performed in the presence of essential
ions or cofactors, detergents, urea, guanidine Proteins of intermediate size are partially
hydrochloride, at high or low ionic strength, at included - meaning they can fit inside some but
37C or in the cold room according to the not all of the pores in the beads. These proteins
requirements of the experiment. Purified will then elute between the large ("excluded")
proteins can be collected in any chosen buffer. and small ("totally included") proteins.
To perform a separation, gel filtration medium Consider the separation of a mixture of
is packed into a column to form a packed bed. glutamate dehydrogenase (molecular weight
The medium is a porous matrix of spherical 290,000), lactate dehydrogenase (molecular
particles with chemical and physical stability weight 140,000), serum albumin (MW 67,000),
and inertness (lack of reactivity and adsorptive ovalbumin (MW 43,000), and cytochrome c
properties). The packed bed is equilibrated (MW 12,400) on a gel filtration column packed
with buffer which fills the pores of the matrix with Bio-Gel P-150 (fractionation range 15,000
and the space between the particles. The liquid - 150,000). When the protein mixture is applied
inside the pores, or stationary phase, is in to the column, glutamate dehydrogenase
equilibrium with the liquid outside the would elute first because it is above the upper
fractionation limit. Therefore it is totally
excluded from the inside of the porous
stationary phase and would elute with the void
volume (V0). Cytochrome c is below the lower
fractionation limit and would be completely
included, eluting last. The other proteins would
be partially included and elute in order of
decreasing molecular weight.
These separations can be described by this
equation;
Figure 1. (Construction of gel filtration)
where Vr is the retention volume of the protein,
V0 is the volume of mobile phase between the MATERALS AND METHODS
beads of the stationary phase inside the
M sodium phosphate buffer pH 7, G-25
column (sometimes called the void volume), Vi
is the volume of mobile phase inside the Sephadex, 0.1 ml diluted skim
porous beads (also called the included volume) milk(1/5), ependorfs, falcons, blue
and K is the partition coefficient (the extent to dextran
which the protein can penetrate the pores in 1. Before using a gel, it is necessary that dry
the stationary phase, with values ranging gel be allowed to swell in excess solvent. In
between 0 and 1). the case of G-25 Sephadex the maximum
time for proper swelling is 3 hours (room
In the mixture of proteins listed above, the temperature).
partition coefficient (K) for glutamate
2. Obtain a volume of suspension of G-25
dehydrogenase would be 0 (totally excluded),
Sephadex (in 0.01 M sodium phosphate
K = 1 for cytochrome c (totally included) and K
buffer, pH 7) that will contain about 3 g of
would be between 0 and 1 for the other
gel.
proteins, which are within the fractionation
3. Close the exit tube at the bottom of your
range for the column.
column and pour the suspension of
In practice, gel filtration can be used to Sephadex into column and attached
separate proteins by molecular weight at any powder funnel. Open the exit tube and
point in a purification of a protein. It can also allow the buffer to slowly drain from the
be used for buffer exchange - a protein column.
dissolved in a sodium acetate buffer, pH 4.8, 4. The eluant for this experiment is a 0.01 M
can be applied to a gel filtration column that sodium phosphate buffer, pH 7. Thus fill
has been equilibrated with tris buffer, pH 8.0. your reservoir with this buffer. Open the
Using the tris buffer, pH 8.0, as the mobile exit tube and start collecting 1 mL
phase, the protein moves into the tris mobile fractions. During this elution note how the
phase as it travels down the column, while the blue dextran behaves in your column.
much smaller sodium acetate buffer molecules 5. To determine the void volume of your
are totally included in the porous beads and column, you will need to determine the
travels much more slowly than the protein. elution volume for the blue dextran. If the
blue dextran is sufficiently concentrated,
this can be done visually by examining the
1 mL fractions you have collected. RESULT
Alternate methods of determining the
elution volume of the blue dextran make
use of the absorption bands that it has near
280 nm and near 625 nm. Measurement at
the first of these bands, however, calls for
an instrument that can be absorbances in
the low ultraviolet region. Accordingly, use
the cruder visual method to estimate the
elution volume for blue dextran and hence
the void volume for your column.
Assay for carbohydrate: Previously you
used the phenol-sulfuric acid method to
determine the concentration of an
unknown containing lactose (see Color Figure 2. (Sephadex pouring phase)
Reactions of Carbohydrates). You will now
use this method to determine the
distribution of carbohydrate among your
various fractions. To perform this assay,
remove 0.1 mL from each of your 1 mL
fractions, placing each of these aliquots in
a separate, clean test tube. To each tube
now add 0.9 mL water. To another tube
(tube 0) add 1 mL water. Now to each of
the 20 tubes add 1 mL %5 phenol solution
and mix. Then to each tube rapidly add 5
mL of concentrated sulfuric acid.
Figure 3. (Skim milk dropping phase)
Assay for protein: Use Bradford protein
assay as you did in Experiment 4 to
determine the protein concentration of
each of fractions. Use BSA as a standard
and draw standard curve as you did
previously. For each sample, apply three
different volumes: 10, 20 and 30 L (those
values in the region of the lineer range of
the standard curve can be taken into
account). Express these as g protein per
mL of eluate.
Figure 4. (Blue dextran solution dropping
phase)
STANDARTS FOR BSA METHOD
DNS
BSA 600
Concentration Avr- y = 404.98x + 18.339
(ng/ul) Rep 1 Rep 2 Abs. Avr. Blank R = 0.9902
500
2000 1,66 1,704 1,682 1,6704
Concentration
400
1500 1,335 1,298 1,3165 1,3049
1000 0,967 0,991 0,979 0,9674 300
750 0,694 0,654 0,674 0,6624
200
500 0,467 0,466 0,4665 0,4549
250 0,231 0,224 0,2275 0,2159 100
125 0,058 0,061 0,0595 0,0479
0 (Blank) 0,0105 0,0127 0,0116 0 0
0 0.5 1 1.5
Absorbance
BSA
2
1.8
y = 0.0009x + 0.0082 PROTEN CARBOHYDRATE
1.6 R = 0.9912 Absorbance1 Absorbance2 Abs. Avr. Avr-Blank Absorbance1 Absorbance2 Abs.Avr. Avr-Blank
1.4 Fraksiyon 1 0,019 0,021 0,02 -0,0105 0,034 0,031 0,0325 0,007
Fraksiyon 2 0,031 0,032 0,0315 0,001 0,034 0,048 0,041 0,0155
Absorbance
1.2 Fraksiyon 3 0,019 0,026 0,0225 -0,008 0,023 0,031 0,027 0,0015
1 Fraksiyon 4 0,031 0,046 0,0345 -0,004 0,031 0,019 0,025 -0,005
Fraksiyon 5 0,026 0,021 0,0235 -0,007 0,019 0,028 0,0235 -0,002
0.8 Fraksiyon 6 0,029 0,023 0.026 -0,0045 0,041 0,054 0,0475 0,022
0.6 Fraksiyon 7 0,089 0,094 0.0915 0,061 0,044 0,044 0,044 0,0185
Fraksiyon 8 0,065 0,045 0.055 0,0245 0,087 0,098 0,0925 0,067
0.4 Fraksiyon 9 0,112 0,098 0.105 0,0745 0,075 0,103 0,089 0,0635
0.2 Fraksiyon 10 0,089 0,061 0.075 0,0445 0,199 0,204 0,2015 0,176
Fraksiyon 11 0,214 0,197 0.2055 0,175 0,045 0,065 0,055 0,0295
0
Fraksiyon 12 0,124 0,123 0.1235 0,093 0,178 0,198 0,188 0,1625
0 500 1000 1500 2000 2500 Fraksiyon 13 0,214 0,231 0.2225 0,192 0,089 0,079 0,084 0,0585
Concentration Fraksiyon 14 0,099 0,1 0.0995 0,069 0,198 0,145 0,1715 0,146
Fraksiyon 15 0,356 0,325 0.3405 0,31 0,561 0,514 0,5375 0,512
Fraksiyon 16 0,454 0,564 0.509 0,4785 0,333 0,321 0,327 0,3015
Fraksiyon 17 0,398 0,378 0.388 0,3575 0,545 0,524 0,5345 0,509
Fraksiyon 18 0,387 0,389 0.388 0,3575 0,235 0,214 0,2245 0,199
Fraksiyon 19 0,854 0,88 0.867 0,8365 0,265 0,214 0,2395 0,214
STANDARTS FOR DNS METHOD Fraksiyon 20 0,931 0,965 0.948 0,9175 0,162 0,168 0,165 0,1395
Blank (Tampon) 0,032 0,029 0.0305 0 0,023 0,028 0,0255 0
Lactose
Concentration Avr- Table 1 (The absorbance of the fractions
(Ug/ml) Rep 1 Rep 2 Abs. Avr. Blank as a result of the experiment.)
500 1,259 1,265 1,262 1,237
Calculation:
400 0,967 0,952 0,9595 0,93455
300 0,637 0,622 0,6295 0,6045 For protein, absorbance is written instead of y.
200 0,467 0,464 0,4655 0,4405
y = 0,0009x + 0,0082
100 0,244 0,244 0,244 0,219
50 0,121 0,135 0,128 0,103 For carbohydrate, absorbance is written
25 0,063 0,054 0,0585 0,0335 instead of x.
0 (Blank) 0,023 0,027 0,025 0 y = 404,98x + 18,339
According to calculation of concentration;
It may originate from any error during the
Protein experiment. For instance, Sephadex can be
1200
frozen and the samples can not be obtained.
1000 Another important point is the comparison of
800 the concentrations of protein and
Concentration
carbohydrates. According to graphs, the
600
concentrations of protein fractions are higher
400 than fractions of carbohydrates. The building
200 blocks of carbohydrates are monosaccharides.
The building blocks of proteins are amino acids.
0
The amino acid molecules are more complex. It
1 2 3 4 5 6 7 8 9 1011121314151617181920
-200 also includes ring and radical groups.
Eluation Buffer (ml) Therefore, concentration of protein is higher
than that of carbohydrates.
REFERENCES
Carbohydrate
250 J. Reiland, Gel Filtration, Methods in
Enzymology, 22, 287 (1971).
200
https://people.rit.edu/pac8612/webionex/we
bsite/html/ione2fzt.html
Concentration
150
https://www.sigmaaldrich.com/life-
100 science/proteomics/protein-
chromatography/gel-filtration-
50 chromatography.html
Garrett, Reginald H.; Grisham, Charles M.
0
1 2 3 4 5 6 7 8 9 1011121314151617181920
(2013). Biochemistry (5th ed.). Belmont, CA:
Brooks/Cole, Cengage Learning. p. 108. ISBN
Eluation Buffer (ml)
9781133106296.
Paul-Dauphin, Stephanie; Morgan, Millan-
DISCUSSION Agorio; Herod, Kandiyoti (6 October 2007).
"Probing Size Exclusion Mechanisms of
A separation is performed according to the Complex Hydrocarbon Mixtures: The Effect of
molecular size in gel filtration chromatography. Altering Eluent Compositions". Energy & Fuels.
The most important thing we need to know in 6. 21 (6): 34843489. doi:10.1021/ef700410e.
gel filtration is to first separate large molecules Retrieved October 6, 2007.
from the column. On the other hand, by gel
filtration chromatography, of course, one
hundred percent pure substance can not be
obtained.
To interpret the experiment, first the graphics
of the standards are made and their equations
are found. Thus, we can calculate the
concentration of protein and carbohydrate
with the help of the equation. But Abs. Avr.-
Blank has a negative value for several fractions.
