2017

Gel Filtration
Chromatography
Gebze Technical University
Turkey

Ebru AKHARMAN
142204026
 28.11.2017

GEL FILTRATION CHROMATOGRAPHY

Experiment 7
Ebru AKHARMAN, Gebze Technical University, Turkey

AIM:
Chromatography is the collective term for a set of separation techniques that operate based on the differential
partitioning of mixture components between a mobile and a stationary phase. The mobile phase (a liquid or a
gas) travels through the stationary phase (a liquid or a solid) in a defined direction. The distribution of
components between the two phases depends on adsorption, ionic interactions, diffusion, solubility or, in the
case of affinity chromatography, specific interactions. Depending on the experimental design, the separation in
a liquid mobile phase may be carried out via column or planar chromatography, on analytical or preparative
scales.

Chromatographic methods are important in the analytical and preparative separation of biological samples. Gel
filtration chromatography (size exclusion chromatography) is often the method of choice to purify
macromolecules, taking advantage of their different sizes and shapes. Ion exchange chromatography is also
useful for the separation of macromolecules, operating based on the various net charges on their surface, which
can be tuned via the pH of the medium. Biological specificity in enzyme-substrate, enzyme-inhibitor, receptor-
ligand, antigen-antibody (and other) interactions is utilised in affinity chromatography. In this method, one
interaction partner is immobilised on a solid surface (stationary phase) and can selectively bind its interacting
partner from a mixture in the mobile phase. The other components of the mixture can then be removed by
replacing the mobile phase (washing). The pure material is then eluted by applying a mobile phase that disrupts
the specific interaction. In this experiment, we will gel filtraion.

INTRODUCTION: and columns with exclusion limits ranging over

In a gel filtration chromatography column, the
stationary phase is composed of a porous matrix,
and the mobile phase is the buffer that flows in
between the matrix beads. The beads have a
defined pore size range, known as the fractionation
range. Molecules and complexes that are too large
to enter the pores stay in the mobile phase and
move through the column with the flow of the
buffer. Smaller molecules and complexes that are
able to move into the pores enter the stationary
phase and move through the gel filtration column
by a longer path through pores of the beads. Any
molecule or complex that is above the fractionation
range for a particular gel filtration chromatography
column will move through the column faster than
any molecule that can enter the stationary phase.
Therefore, any constituent in the sample that is
above the fractionation range will elute first (in the
void volume) before anything that is in the
fractionation range. The minimum size that will
remain in the mobile phase and not enter the
stationary phase is known as the exclusion limit.
Bio-Rad offers gel filtration chromatography media
three orders of magnitude, from 100 daltons to
100,000 daltons (100 kDa). Molecules and
complexes that can enter the stationary phase will
be fractionated according to their sizes. Smaller
molecules will migrate deep into the pores and will
be retarded more than larger molecules that do not
so easily enter the pores, and are thus eluted from
the column more quickly. This difference in pore
migration leads to fractionation of components by
size with the largest eluting first.
In gel filtration chromatography columns designed
for desalting, buffer exchange, and the removal of
small molecules such as nucleotides, the salts and
small compounds readily enter the pores, are
retarded, and migrate more slowly through the
column than the larger proteins or nucleic acids.
Therefore, the components of interest in the sample
are eluted in advance of salts, nucleotides, etc. DNA
cleanup kits using this mechanism often contain gel
filtration spin columns.
Resolution, here defined as the sharpness of the
boundaries between size fractions, is determined
by bead size and a number of other factors. Smaller

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bead size generally yields higher resolution in a gel
filtration chromatography column. Compact
molecules diffuse through the stationary phase
faster than linear molecules.
Size exclusion, fractionation range, and elution rate
are affected by buffer composition, ionic strength,
and pH. For the fractionation of complex mixtures
of proteins, elution times and size exclusion limits
may need to be determined empirically.
ekil 1: Image of Pores
Gel filtration chromatography, a type of size
exclusion chromatography, can be used to either
fractionate molecules and complexes in a sample
into fractions with a particular size range, to
remove all molecules larger than a particular size
from the sample, or a combination of both
operations.
Gel filtration chromatography can be used to
separate compounds such as small molecules,
proteins, protein complexes, polysaccharides, and
nucleic acids when in aqueous solution. When an
organic solvent is used as the mobile phase, the
process is instead referred to as gel permeation
chromatography.
Gel filtration chromatography can also be used for:
Fractionation of molecules and complexes
within a predetermined size range
Size analysis and determination
Removal of large proteins and complexes
Buffer exchange
Desalting
Removal of small molecules such as
nucleotides, primers, dyes, and
contaminants
Assessment of sample purity
Separation of bound from unbound
radioisotopes.
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28.11.2017
Gel filtration chromatography media for all of the
above uses are available in prepacked gravity flow
columns, spin columns, low-pressure and medium-
pressure chromatography columns, and bottled
resins.
(I) Choosing the gel type
Several different gel filtration media are available,
which should be chosen according to the substance
to be separated. These media differ in the chemical
properties of the gel matrix, the pore size, the
particle size, as well as the physical and chemical
stability of the gel. The first developed and still
widely used gel matrix is made of crosslinked
dextran. Polymer beads made of dextran are known
by the trade name Sephadex.
ekil 2: Image of Beads
The pore size of the various Sephadex media is
controlled by the number of crosslinks. The most
popular ones are the entirely hydrophilic G-type
gels. Numbers accompanying the G-type mark refer
to the pore size and indicate the approximate
molecular mass of excluded molecules in kDa. For
example, Sephadex G-25 is used to separate
relatively small molecules in the molecular mass
range of 1000-5000 Da, including peptides.
Alternatively, it can also be used to desalt larger
proteins. To fractionate larger macromolecules up
to 200-300 , the G-150 or G-200 Sephadex gels
are to be used.
The mechanical properties of dextran gels having
large pores are unfavourable due to the low density
of crosslinks. These gels are easily compressible.
Therefore, more rigid gels made of synthetic
polymers are used to separate very large or
elongated molecules.

If the size difference between the compounds to be
separated is relatively large, e.g. during desalting of
a macromolecule, it is practical to choose a gel in
which the large-sized compound is eluted in the
excluded volume (Vo; thus Kav = 0), while the small
component elutes around Vt (thus = 1). In this
case, the fraction containing the macromolecules
appears sharply, with minimal band broadening
and dilution, in the shortest possible elution time.
In case of fractionating macromolecules and if the
molecular weight of the compound of interest is
known, the gel should be chosen so that the
component of interest will elute approximately at
the half of the entire fractionation range. For
example, if a 100- protein is to be isolated
from a protein mixture, the use of a gel that spans
the 10-250 fractionation range is
recommended.
(II) Choosing the particle size of the gel
Fine-sized beads fill the available space within the
chromatographic column more efficiently.
Therefore, the volume of the mobile phase will be
reduced. This will result in a similar reduction in
dilution and band broadening and, in turn, will
yield a better resolution. On the other hand, the
flow rate in a compact gel column is also reduced.
Therefore, larger pressure should be applied when
using super-fine beads. Indeed, special pumps are
needed below a particle size of 10 m. Naturally,
only rigid, non-compressible gel types can be used
in these cases. For most purposes, the Fine and
Medium type particle sizes (20-150 m) are
suitable. For preparative purposes and desalting,
where high flow rates are required and the
compounds of interest separate well even at a poor
resolution, Coarse type gels can be used too.
(III) Choosing the size of the column
During gel filtration, the distance between two
zones of separation increases proportionally to the
square root of the column length. Long columns
(> 100 cm) are used when a high resolution is
required, while shorter (< 50 cm) columns are
more practical when the aim is desalting or the
separation of compounds that can be eluted at
markedly different volumes. Columns with
diameters of around 1 cm are used for analytical
purposes. By increasing the diameter, the amount
of the applied sample, i.e. the capacity of the
column, can be increased.
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28.11.2017
(IV) Choosing the sample volume
A narrow start zone (relative to the column length)
is sought if maximal resolution is to be achieved,
e.g. for analytical purposes or in the case of
compounds whose separation is difficult. Therefore,
the sample volume in this case should be chosen to
be 1-5 % of the column volume. The resolution
cannot be further increased using smaller sample
volumes, while the dilution will be greater. The
sample volume can be increased to as much as 15-
20 % of the column volume if the compounds are
readily separable, especially when working on a
large scale.
ekil 3: Fraction Image
(V) Choosing the eluent
The composition of the eluent does not directly
influence the resolution of gel filtration. However,
all components that have an effect on the molecules
to be separated may influence the separation. The
pH, ionic strength or the presence of detergents
may influence the molecular state of the solutes.
For instance, changes in molecular shape or the
dissociation of multimeric proteins and enzyme-
inhibitor complexes will change their
chromatographic behaviour. In general, dilute
(0.01-0.1 M) buffers are used that do not influence
the structure of the compounds to be separated, but
restrict the unwanted adsorption interactions
between the gel matrix and the molecules of
interest. When the fractions containing the
separated compounds are to be later concentrated,
volatile buffers (e.g. ammonium bicarbonate) are
practical to use that easily disappear during
lyophilisation or film evaporation.

The same considerations apply when the salt
content of the gel filtration buffer should be
subsequently eliminated.
(VI) Choosing the flow rate of the eluent
During gel filtration, increasing the flow rate will
deteriorate the resolution, because it prevents the
formation of equilibrium between the mobile and
the stationary phases. Generally, 5-10 mL/cm2x
hour is recommended as an optimal flow rate, but
in most cases, a several times excess of this will not
deteriorate the separation significantly. When
doing preparative work, or if the operation must be
performed quickly for some reason, the advantage
conferred by the higher flow rate may compensate
for the deterioration of separation.
To achieve a higher flow rate, of course, a larger
28.11.2017
Determination of :
0.1 ml of blue dextran is slowly added into the
column containing the Sephadex. The exit tube
must be closed when in the insertion phase. After
the addition, the exit tube is opened and the entire
dye (in the form of 1.0 ml) is taken up in eppendorf
tubes. Eppendorf tube number gives us 0 value.
There are various formulas to determine the
chromatographic gel volume.
= 0 + +
0
= ( for distribution )
= + 0 = 0 ( for available )
0

pressure must be applied. Therefore, in these cases,

the mechanical stability of the gel matrix must be
taken into consideration. Non-rigid gels may be
compressed at pressures higher than allowed,
which may lead to the complete clogging of the
chromatographic column.

MATERIAL AND METHODS:

Sephadex G 25
Chromatographic column( 0,9 x 15 cm )
ekil 4: Formula means
Blue Dextran Solution ( 0,1 ml )
Eppendorf Tubes
Separation of Protein and Carbohydrate
Components of Skim Milk:
Preparation of Gel Filtration Column:
Then, carefully 0,1 ml of diluted skim milk ( 1/5 ) is
Before using a gel, it is necessary that dry gel be
allowed to swell in excess solvent. In the case of G added in the chromatographic column. The solution
25 Sephadex the maximum time for proper swelling is added continuously to the chromatographic
is 3 hours ( room temperature ). Because of this, column. Otherwise, the beads are dried at the top of
you will be supplied with a suspension of swollen the column.
gel. It will be ready for chromatography.
The exit tube must be closed at this time. The exit
G 25 Sephadex ( in 0,01 M sodium phosphate
tube is then opened and the solution is pooled into
buffer, pH 7 ) that will contain about 3 g of gel is
added in the chromatographic column. When the 1 ml eppendorf tubes as 0 fraction. This process is
Sephadex gel is spilled, the exit column should be repeated twice for protein and carbohydrate.
closed and the solution should be poured with the
help of a funnel. Otherwise, the poured solution DNS assay for carbohydrates and BSA assay for
comes out directly. The solution is added proteins are maked and are measured absorance
continuously to the chromatographic column. and standard curve obtained. In this way, the
Otherwise, the beads are dried at the top of the
concentration of protein and carbohydrate is
column. Then the exit tube should be opened. The
buffer flows out of the exit tube in the solution and reached in the skim milk.
the gel becomes tight.

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RESULTS:

PROTEN KARBONHDRAT
Absorbance1 Absorbance2 Absorbance1 Absorbance2
Fraksiyon 1 0,035 0,0310 0,021 0,029
Fraksiyon 2 0,051 0,0540 0,034 0,036
Fraksiyon 3 0,019 0,0310 0,051 0,051
Fraksiyon 4 0,013 0,0160 0,039 0,032
Fraksiyon 5 0,065 0,0630 0,074 0,035
Fraksiyon 6 0,058 0,0460 0,062 0,061
Fraksiyon 7 0,056 0,0780 0,041 0,046
Fraksiyon 8 0,064 0,0782 0,056 0,061
Fraksiyon 9 0,035 0,0480 0,145 0,134
Fraksiyon 10 0,145 0,1230 0,165 0,124
Fraksiyon 11 0,095 0,0780 0,125 0,135
Fraksiyon 12 0,235 0,2140 0,103 0,089
Fraksiyon 13 0,389 0,4120 0,345 0,333
Fraksiyon 14 0,213 0,2140 0,324 0,354
Fraksiyon 15 0,465 0,4320 0,642 0,632
Fraksiyon 16 0,632 0,6240 0,345 0,365
Fraksiyon 17 0,398 0,3660 0,785 1,020
Fraksiyon 18 0,312 0,2350 1,110 1,127
Fraksiyon 19 1,321 1,1230 0,965 0,974
Fraksiyon 20 0,954 1,0100 0,714 0,841
Blank (Tampon) 0,029 0,0360 0,037 0,041
Table 1: Absorbance of Carbohydrate and Protein

Avarage- Blank
BSA Concentration(ng/ul) REP 1 REP 2 Avarage AVG. concentration(ug/ml)
2000 1,6600 1,7040 1,6820 1,6704 2,000
1500 1,3350 1,2980 1,3165 1,3049 1,500
1000 0,9670 0,9910 0,9790 0,9674 1,000
750 0,6940 0,6540 0,6740 0,6624 0,750
500 0,4670 0,4660 0,4665 0,4549 0,500
250 0,2310 0,2240 0,2275 0,2159 0,250
125 0,0580 0,0610 0,0595 0,0479 0,125
0,000 0,0105 0,0127 0,0116 0,0000 0,000
Table 2: BSA Values For Protein

Lactose Concentration(ug / ml) REP 1 REP 2 Avarage Avarage - Blank AVG.

500 1,259 1,265 1,2620 1,2370
400 0,967 0,952 0,9595 0,9345
300 0,637 0,622 0,6295 0,6045
200 0,467 0,464 0,4655 0,4405
100 0,244 0,244 0,2440 0,2190
50 0,121 0,135 0,1280 0,1030
25 0,063 0,054 0,0585 0,0335
0 0,023 0,027 0,0250 0,0000
Table 3: DNS Values For Carbohydrate

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Standart Curve of DNS

1,4
1,237
1,2

1
0,9345
0,8

0,6
0,6045
0,4 0,4405

0,2 0,219
0,103
0,0335
0
0 100 200 300 400 500 600

y = 0,0024x - 0,0285 Standart Curve of DNS

R = 0,991
Dorusal (Standart Curve of DNS)
Graph 1: Standart Curve of DNS Graph

2
Standart Curve of BSA
1,8
1,6704
1,6

1,4

1,2 1,3049
0,9674
1

0,8

0,6 0,6624
0,4549
0,4
0,2159
0,2

0 0,0479
0 0,5 1 1,5 2 2,5

Absorbance of BSA

y = 0,8617x + 0,0058
R = 0,9929 Dorusal (Absorbance of BSA)

Graph 2: Standart Curve of BSA Graph

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Absorbance1 Absorbance2 Avarage Avarage-Blank AVG.
Fraksiyon 1 0,035 0,0310 0,0330 0,0005
Fraksiyon 2 0,051 0,0540 0,0525 0,0200
Fraksiyon 3 0,019 0,0310 0,0250 -0,0075
Fraksiyon 4 0,013 0,0160 0,0145 -0,0180
Fraksiyon 5 0,065 0,0630 0,0640 0,0315
Fraksiyon 6 0,058 0,0460 0,0520 0,0195
Fraksiyon 7 0,056 0,0780 0,0670 0,0345
Fraksiyon 8 0,064 0,0782 0,0711 0,0386
Fraksiyon 9 0,035 0,0480 0,0415 0,0090
Fraksiyon 10 0,145 0,1230 0,1340 0,1015
Fraksiyon 11 0,095 0,0780 0,0865 0,0540
Fraksiyon 12 0,235 0,2140 0,2245 0,1920
Fraksiyon 13 0,389 0,4120 0,4005 0,3680
Fraksiyon 14 0,213 0,2140 0,2135 0,1810
Fraksiyon 15 0,465 0,4320 0,4485 0,4160
Fraksiyon 16 0,632 0,6240 0,6280 0,5955
Fraksiyon 17 0,398 0,3660 0,3820 0,3495
Fraksiyon 18 0,312 0,2350 0,2735 0,2410
Fraksiyon 19 1,321 1,1230 1,2220 1,1895
Fraksiyon 20 0,954 1,0100 0,9820 0,9495
Blank (Tampon) 0,029 0,0360 0,0325 0,0000
Table 4: Net Absorbance Values of Protein in Milk

Absorbance1 Absorbance2 Avarage Avarage-Blank AVG.

Fraksiyon 1 0,021 0,029 0,0250 -0,0140
Fraksiyon 2 0,034 0,036 0,0350 -0,0040
Fraksiyon 3 0,051 0,051 0,0510 0,0120
Fraksiyon 4 0,039 0,032 0,0355 -0,0035
Fraksiyon 5 0,074 0,035 0,0545 0,0155
Fraksiyon 6 0,062 0,061 0,0615 0,0225
Fraksiyon 7 0,041 0,046 0,0435 0,0045
Fraksiyon 8 0,056 0,061 0,0585 0,0195
Fraksiyon 9 0,145 0,134 0,1395 0,1005
Fraksiyon 10 0,165 0,124 0,1445 0,1055
Fraksiyon 11 0,125 0,135 0,1300 0,0910
Fraksiyon 12 0,103 0,089 0,0960 0,0570
Fraksiyon 13 0,345 0,333 0,3390 0,3000
Fraksiyon 14 0,324 0,354 0,3390 0,3000
Fraksiyon 15 0,642 0,632 0,6370 0,5980
Fraksiyon 16 0,345 0,365 0,3550 0,3160
Fraksiyon 17 0,785 1,020 0,9025 0,8635
Fraksiyon 18 1,110 1,127 1,1185 1,0795
Fraksiyon 19 0,965 0,974 0,9695 0,9305
Fraksiyon 20 0,714 0,841 0,7775 0,7385
Blank (Tampon) 0,037 0,041 0,0390 0,0000
Table 5: Net Absorbance Values of Carbohydrate in Milk

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1,2
Absorbance of Carbohydrate and Protein in Milk 1,4
1,1 1,3
1 1,2
1,1
0,9
1
Absorbance of Carbohydrate

0,8
0,9

Absorbance of Protein
0,7 0,8
0,6 0,7
0,5 0,6
0,4 0,5
0,3 0,4
0,3
0,2
0,2
0,1
0,1
0 0
-0,1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 -0,1
-0,2 -0,2
Fraction of Carbohydrate and Protein
Charbohydrate Absorbance - Fraction
Protein Absorbance - Fraction

Graph 3: Absorbance of Carbohydrate and Protein in Milk

Concentration of Carbohydrate and Protein in Milk

499,985 1,6

449,985 1,4
399,985
Concentration of Carbohydrate

1,2

Concentration of Protein
349,985
1
299,985
0,8
249,985
0,6
199,985
0,4
149,985

99,985 0,2

49,985 0

-0,015 -0,2
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Fraction of Carbohydrate and Protein
Concentration of Charbohydrate
Concentration of Protein

Graph 4: Concentration of Carbohydrate and Protein in Milk Graph

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CALCULATION:

For DNS Standart Curve Calculation: For BSA Standart Curve Calculation:

The average of REP1 and REP2 is calculated. The The average of REP1 and REP2 is calculated. The
obtanied averages are subracted from the average of obtanied averages are subracted from the average of
Blank. Blank.

For Example; For Example;

1+ 2 1+ 2 1+ 2 1+ 2
- -
2 2 2 2

1,259 +1,265 0,023 +0,027 1,6600 +1,7040 0,0105 +0,0127

= 1, 2370 - = 1, 6704
2 2 2 2

This process is repeated for all values, and the This process is repeated for all values, and the
following equations are obtained. following equations are obtained.

Equation 1: Equation 2:

y = 0,0024x - 0,0285 y = 0,8617x + 0,0058

R = 0,991 R = 0,9929

For Carbohydrate Concentration Calculation: For Protein Concentration Calculation:

The average of REP1 and REP2 is calculated. The The average of REP1 and REP2 is calculated. The
obtanied averages are subracted from the average of obtanied averages are subracted from the average of
Blank. Blank.

For Example; For Example;

1+ 2 1+ 2 1+ 2 1+ 2
- -
2 2 2 2

0,021 +0,029 0,037 +0,041 0,035 + 0,031 0,029 + 0,036

- = - 0, 0140 ( Net Absorbance ) - = 0, 0005 ( Net Absorbance )
2 2 2 2

Then, Equation 1 is used to find concentration of Then, Equation 2 is used to find concentration of
every fraction of carbohydrate in skim milk. every fraction of protein in skim milk.

y = 0,0024x - 0,0285 ( y is absobance ) y = 0,8617x + 0,0058 ( y is absorbance )

-0, 0140 = 0, 0024x 0, 0285 0, 0005 = 0, 8617x + 0, 0058

0, 0285 0, 0140 = 0, 0024x ( x is concentration) 0, 0005 0, 0058 = 0, 8617 ( x is concentration )

X = 6, 0417 g / ml X= - 0, 0062 g / ml

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DISCUSSION: Dry Total
TYPE Substances Fat Protein Kasein Lactose Mineral
The nutritional value of milk is due to the COW 12,6 3,7 3,4 2,8 4,7 0,7
presence of lactose, proteins, lipids and SHEEP 18,8 7,5 5,6 4,6 4,6 1
inorganic elements. Bioactive compounds in GOAT 13,2 4,5 3,6 3 4,3 0,8
MANDATE 17,5 7,5 4,3 3,6 4,8 0,8
milk perform many functions other than
CAMEL 13,4 4,5 3,6 2,7 4,5 0,8
nutritional, e.g., immune system, hormones and
Table 6: Compounds Rates of Milk
related compounds, antibacterial agents,
enzymes, enzyme inhibitors and cryptic When we look at the real component rates of the
peptides. Biologically active milk compounds in milk, we see that the carbohydrate values are high.
the form of immunoglobulins, antibacterial The experiment we made is reasonable according
peptides, antimicrobial proteins, to this datas.
oligosaccharides and lipids protect neonates
and adults against pathogens and illnesses. Some points on the charts are showing negative
How many of these compounds are found in results. At these points we can say that the
the milk? For this experiment, skim milk was concentration in the fraction is low. Also negative
results may be due to personal errors. Incorrect
used. In this way the amount of protein and
calculations may have been made.
carbohydrate can be easily calculated. Gel
filtration chromatography separates proteins
and carbohydrate solely on the basis of
molecular size.
RESOURCES:
Separation is achieved using a porous matrix to
http://www.bio-rad.com/featured/en/gel-
which the molecules, for steric reasons, have filtration-chromatography.html
different degrees of access--i.e., smaller
molecules have greater access and larger http://elte.prompt.hu/sites/default/files/tananyag
molecules are excluded from the matrix. Hence, ok/IntroductionToPracticalBiochemistry/ch06.htm
proteins are eluted from the gel filtration l
column in decreasing order of size. This unit
describes the experimental theory behind gel http://www.mikeblaber.org/oldwine/bch5425/lec
filtration and contains many useful tables t31/lect31.html
listing the properties and characteristics of
currently available matrices. Depending on the
content of the milk, the minerals of skim milk
in the gel filtration column will enter the pores
of the beads and will occur fraction the
carbohydrates and proteins according to their
molecular weights.

Once we have made the necessary calculations,

we will examine the resulting graphs. It is seen
that the absorbance and concentration of
carbohydrate is higher than that of protein.
Based on these results, we can say that the
carbohydrates in the skim milk are higher than
the protein.

If we look at actual nutritional values of the

skim milk.

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