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Gel Filtration
Chromatography
Gebze Technical University
Turkey
Ebru AKHARMAN
142204026
28.11.2017
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.
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bead size generally yields higher resolution in a gel Gel filtration chromatography media for all of the
filtration chromatography column. Compact above uses are available in prepacked gravity flow
molecules diffuse through the stationary phase
faster than linear molecules. columns, spin columns, low-pressure and medium-
pressure chromatography columns, and bottled
Size exclusion, fractionation range, and elution rate resins.
are affected by buffer composition, ionic strength,
and pH. For the fractionation of complex mixtures (I) Choosing the gel type
of proteins, elution times and size exclusion limits
may need to be determined empirically. 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.
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If the size difference between the compounds to be (IV) Choosing the sample volume
separated is relatively large, e.g. during desalting of
a macromolecule, it is practical to choose a gel in A narrow start zone (relative to the column length)
which the large-sized compound is eluted in the is sought if maximal resolution is to be achieved,
excluded volume (Vo; thus Kav = 0), while the small e.g. for analytical purposes or in the case of
component elutes around Vt (thus = 1). In this compounds whose separation is difficult. Therefore,
case, the fraction containing the macromolecules the sample volume in this case should be chosen to
appears sharply, with minimal band broadening be 1-5 % of the column volume. The resolution
and dilution, in the shortest possible elution time. cannot be further increased using smaller sample
volumes, while the dilution will be greater. The
In case of fractionating macromolecules and if the sample volume can be increased to as much as 15-
molecular weight of the compound of interest is 20 % of the column volume if the compounds are
known, the gel should be chosen so that the readily separable, especially when working on a
component of interest will elute approximately at large scale.
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.
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The same considerations apply when the salt Determination of :
content of the gel filtration buffer should be
subsequently eliminated. 0.1 ml of blue dextran is slowly added into the
column containing the Sephadex. The exit tube
(VI) Choosing the flow rate of the eluent must be closed when in the insertion phase. After
the addition, the exit tube is opened and the entire
During gel filtration, increasing the flow rate will dye (in the form of 1.0 ml) is taken up in eppendorf
deteriorate the resolution, because it prevents the tubes. Eppendorf tube number gives us 0 value.
formation of equilibrium between the mobile and
the stationary phases. Generally, 5-10 mL/cm2x There are various formulas to determine the
hour is recommended as an optimal flow rate, but chromatographic gel volume.
in most cases, a several times excess of this will not
deteriorate the separation significantly. When = 0 + +
doing preparative work, or if the operation must be
performed quickly for some reason, the advantage 0
conferred by the higher flow rate may compensate
= ( for distribution )
for the deterioration of separation.
= + 0 = 0 ( for available )
To achieve a higher flow rate, of course, a larger 0
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
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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
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)
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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
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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
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
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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.
1+ 2 1+ 2 1+ 2 1+ 2
- -
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:
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.
1+ 2 1+ 2 1+ 2 1+ 2
- -
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.
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.
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