Gel Filtration
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Gel filtration
•What is gel filtration and why use it?
•Some typical results
•Why and when to choose gel filtration
•Model of the mechanism
•How to get the expected results
•Unexpected results and what to do about them
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What is gel filtration?
Gel filtration is a technique of liquid
chromatography which separates
molecules according to their sizes.
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Gel filtration in the lab
•Simple to do
•Easy to understand
•Nothing to go wrong
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Why use gel filtration?
Some typical results
•Group separations: Desalting, Buffer exchange,
Removing reagents
•Purification of proteins and peptides: complex
samples, monomer/dimer
•Estimation size & size homogeneity
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Desalting proteins
Highly efficient desalting in less than 1 minute
10
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20
30
6
40
50 sec
Desalting proteins
Desalting in a simple column
Albumin
0
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NaCl
4
6
8
10
12
Elution volume (ml)
7
Column:
PD-10
Sample:
Buf fer:
HSA, 25 mg
NaCl 0.5M
Desalting the sample
Sample:
His tagged protein eluted from HisTrap
with 20 mM sodium phosphate, 0.5 M
sodium chloride, 0.5 M imidazole, pH 7.4
HiTrap Desalting 5 ml
20 mM sodium phosphate, 0.15 M sodium
chloride, pH 7.0
ÄKTAprime, 5 ml/min
Column:
Buffer:
System:
0
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1
min
8
Buffer exchange using HiPrep 26/10 Desalting column
Sample:
Column:
Buffer:
System:
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BSA dissolved in 50 mM piperazine,
0.5 M sodium chloride, pH 6.2
HiPrep 26/10 Desalting
20 mM sodium phosphate, 0.15 M
sodium chloride, pH 7.0
ÄKTAprime, 20 ml/min
Purification of recombinant IGF-1
AU280
Column: HiLoad 16/60 Superdex 75 prep grade
Sample: IGF-1, ZZ fusion protein and
uncleaved material
Buffer: 0.15 ammonium acetate, pH 6.0
Flow rate: 0.75 ml/min (22.5 cm/h)
0.1
0.05
IGF-1
1
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Time (h)
10
Purification of recombinant phosphatase
Sample:
Column:
Buffer:
System:
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4 ml concentrated eluate from a HIC run containing rPhosphatase
HiLoad 16/60 Superdex 75 prep grade
25 mM Tris-HCl, 0.3 M sodium chloride, 1 mM EDTA, 2 mM DTT, pH 7.4
ÄKTAprime, 0.5 ml/min (15 cm/h)
11
Enzyme purification - SDS-PAGE analysis
M 1 2 3 4 M
M
1
2
3
4
67k
43k
30k
12
- LMW markers
- Start material
- Pool from ion exchange
- Pool from HIC
- Pool from Superdex 75 pg
Separating dimer and oligomers from monomer
A
Monomer
280 nm
0.05
Column: Superdex 75 HR 10/30
Sample: A special preparation of
rhGH in distilled water
0.025
Dimer
Oligomer
VO
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20
13
VC
Time (min)
Characterisation: Size homogeneity
%B
mAU
100
1200
Mr 160 000
80
1000
60
800
mAU
mS/cm
11.84
600
40
60
10
mAU
mS/cm
11.94
60
10
400
20
200
0
0
0
50
100
HiTrap Chelating 5 ml
150 ml
8
8
6
6
40
40
4
4
2
2
20
0
20
0
0
5
10
15
20 ml
0
Superdex 200 HR 10/30
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5
10
15
20 ml
Estimating molecular size
Measure elution position
Calculate molecular size
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Why choose gel filtration?
Pros
Cons
Fastest
Very
for buffer exchange
Limited
gentle, high yields
Works
Poor
selectivity compared
with SDS-PAGE
in any buffer solution
Removes
dimers and
aggregates
Separates
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sample volume
by size
16
Use in group separations

Adjusting pH, buffer type, salt concentration during
sample preparation, e.g. before an assay.

Removing interfering small molecules, e.g. EDTA,
Gu.HCl

Removing small reagent molecules, e.g. fluorescent
labels, radioactive markers.

But not when the protein will precipitate
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Use in fractionation

Excellent during the polishing stage

Removes dimers and other aggregates

Transfers protein to buffer solution ready for the next
set of experiments

Not so suitable if the sample volume is large
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Use for size estimation

'Free' information

Gives an estimate of molecular size in any practically
any solution

Precision is not so good
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Nomenclature
• Gel filtration
(Porath, Flodin, 1959)
• Size exclusion chromatography
(Pedersen, 1962)
• Molecular sieve chromatography
(Hjertén, Mosbach, 1962)
• Gel permeation chromatography
(Moore, 1964)
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Separation mechanism
Qualitative model
• Gel structure
• Steric exclusion
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Gel structure
A good gel for
gel filtration
contains about
95% water
AGAROSE
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Gel structure
Agarose
Dextran
A hypothetical structure for Superdex
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Steric exclusion
Very large
molecules are
completely
excluded
Gel bead
Very small ones
get everywhere
Molecules are excluded from the gel bead to different extents according to their sizes.
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Steric exclusion leads to early elution
Molecules elute in order of
size.
The largest molecules come
first; the smallest ones come
last.
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Principles of gel filtration
• Terms used, defined
• Calibration curves
• Selectivity and efficiency
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Terms and explanations
Vo= Void volume
Vr = Elution volume within the separation range
of the gel
Vi = Inner pore volume = Vc - Vs - Vo
Vc = Total (geometric) volume of the column
Void volume Vo
Vr
Volume of the
gel matrix Vs
Vo
Vt
Pore volume Vi
Vc
1
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2
3
The void volume Vo
Concentration
Elution volume for very large molecules, Vo
Vo
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Volume
Vr
28
Vt and Vc
Concentration
Elution volume for very small molecules, Vt
Geometric volume of the gel bed, Vc
Vo
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Vr
Volume
Vt
Vc
29
The distribution coefficient, Kd
Vr Vo
Vr Vo
d  Vc Vo Vg  Vi
K
Kd is difficult to get because
Vi is difficult to measure
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The coefficient Kav
Vr Vo
av  Vc Vo
K
Kav is easy to get and
it is more useful in practice
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Kav for very large and very small molecules
For very large molecules eluting at Vo
Vr  Vo
Vr Vo
Vo Vo
av  Vc Vo  Vc Vo 
K
0
For very small molecules eluting at Vt
Vr  Vt
Vr Vo
Vr Vo
av  Vc Vo  Vr Vo 
K
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1
Concentration
Kav should always be in the range 0 to 1
Elution volume when Kav = 1
Vr
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Elution with Kav > 1
Adsorption has occurred
Volume
Vt
Elution with Kav < 0
Serious problem!
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The number of peaks is limited
Maximum number of peaks in gel filtration ca 15
Maximum number of peaks in RPC > 150
0
10
20 ml
but we are not
purifying peaks!
0
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200
34
400
ml
Resolution Rs
Rs = 0.6
Resolution = Peak separation
Av. peak width
Rs = 1
Vr 2  Vr 1
Rs 
W 1  W 2 
2
Rs = 4
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Resolution depends on
efficiency and selectivity
High efficiency
Efficiency is a measure of
peak width
Low efficiency
High selectivity
Low selectivity
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Selectivity is a measure of
peak separation
Resolution depends on
efficiency and selectivity
Low selectivity
high efficiency
High efficiency can compensate for
low selectivity.
low efficiency
High selectivity
high efficiency
low efficiency
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If selectivity is high, low efficiency
can be tolerated (if large peak
volume is acceptable).
Calibration curves
Plotting the elution volume versus
the logarithm of molecular mass
yields a calibration curve.
Vr
log Mr
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Selectivity curves
Plotting Kav versus the logarithm of
molecular mass yields the selectivity curve
of the matrix.
The steeper this curve, the greater the
difference in elution volume for two
molecules of different sizes.

Kav
Kav


log Mr

Kav
log Mr
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log Mr
Constructing a selectivity curve
Kav 1
1. Run standards and
determine the elution
volume for each.
2. Calculate Kav values.
0
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log (Mr)
40
3. Plot log (Mr) for each
standard against the
calculated Kav.
Exclusion limit
Kav 1
Exclusion limit
All molecules bigger
than this elute in the
void volume
0
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log (Mr)
41
Fractionation range
Kav 1
A selectivity curve is usually fairly
straight over the range Kav=0.1 to
Kav=0.7
0.7
0.1
0
log (Mr)
Fractionation range
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Separation ranges of modern supports
Kav
1: Superdex Peptide
2: Superdex 75
3: Superdex 200
4: Sephacryl S-100 HR
5: Sephacryl S-200 HR
6: Sephacryl S-300 HR
7: Sephacryl S-400 HR
1.0-
0.8-
0.6-
0.47
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-
10,000
3
-
1000
2 4 5
-
1
-
-
0.2-
100,000 1,000,000 10,000,000
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Results depend on selectivity
AU280
Sephacryl S-300
Increasing exclusion limit
Better for larger proteins
Sephacryl S-200
BSA
IgG
b-L
Cyt C
Cytidine
Best for these proteins
Sephacryl S-100
Better for smaller proteins
40
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80
120 ml
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Shape effects
A gel has different fractionation
ranges for
native, globular proteins and
denatured proteins in random coil
Kav 1
Native proteins
0.7
0.1
log (Mr)
Denatured proteins
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Shape effects
Kav 1
Linear polysaccharides
Globular proteins
log (Mr)
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Molecules with
different shapes have
different selectivity
curves.
Efficiency
Vr
N = 5.54 ( W
h
AU280
2
)
N = Number of theoretical plates
Wh = Peak width at
half peak height
Test: 1% solution of acetone (about
0.5% of column volume), 0.2 AUFS at
280 nm. Alternatively use 2 M NaCl and
conductivity monitor.
Vr
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Efficiency
Efficiency depends on:
Particle size of matrix
Particle size distribution of matrix
Packing quality of the column
Sample (volume and viscosity)
Flow rate
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Peak width
Band broadening effects
Large particles/large sample molecules
Small particles/small sample molecules
Flow rate
To increase efficiency: Lower the flow rate
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Peak width
Band broadening effects
Large molecules
Small molecules
Flow rate
To increase efficiency: Increase the flow rate
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Band broadening effects
Peak width
Uneven packing
Irregular particles
Good column packing
Uniform spherical particles
Flow rate
To increase efficiency: Use uniform beads in well packed columns
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Diffusion and the impact of flow rate
on peak width
Peak width
width
Large molecules Peak
peak width
Mass transfer
Eddy Diffusion
Diffusion
along column
Increasing flow rates lead to
broad peaks for large
molecules. Very low flow rates
are not suitable for very small
molecules as column diffusion
becomes too significant.
Human serum
albumin (Mr 68500)
Myoglobin (Mr 16900)
Tyrosine (Mr 181)
ml/min
Peak width
Small molecules
ml/min
ml/min
ml/min
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Peak width depends on particle size

Superdex Peptide 13-15 µm

Superdex 30 prep grade 24-44 µm
AU214
0.06
AU214
0.14
0.12
0.05
0.1
0.04
0.08
0.03
0.06
0.02
0.04
0.01
0.02
0
0
0
10
20
30
40
50
Retention time (min)
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10
20
30
Retention time (min)
53
40
50
Superdex and Sephadex
Sephadex G-200
Smaller beads can give both
better resolution and
shorter separation times
600 min
Superdex 200 prep grade
Bed length:
Sample:
Sample size:
Flow rate:
80 min
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60 cm
Mouse monoclonal cell supernatant IgG1
1.2 ml (1 % Vc)
0.2 ml/min (6 cm/h), Sephadex G-200
1.6 ml/min (50 cm/h), Superdex 200 prep grade
Resolution depends on column length
Increasing column length increases resolution
1 x Superdex® Peptide HR 10/30
2 x Superdex® Peptide HR 10/30
AU214
0.08
0.09
0.08
0.07
0.07
0.06
0.06
0.05
0.05
0.04
0.04
0.03
0.03
0.02
0.02
0.01
0.01
0
0
0
5
10
15
20
25
10
20
30
Retention time (min)
Retention time (min)
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55
40
50
Resolution depends on sample volume
Column: Superdex Peptide HR 10/30
A214
0.1
AU214
0.1
25 µl
0.08
200 µl
0.08
0.06
0.06
0.04
0.04
0.02
0.02
0
0
0
25µl
5
10
15
20
25
0
5
Retention volume (ml)
10
15
Retention volume (ml)
AU214
0.08
400 µl
0.06
0.04
0.02
0
0
5
10
15
Retention volume (ml)
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20
25
400 µl
20
25
200 µl
Resolution depends on sample viscosity
Relative viscosity: 1.0
Relative viscosity: 4.2
Samples which are much
more viscous than the eluent
give very poor results
Relative viscosity: 11.8
even for a simple group
separation.
Haemoglobin and NaCl, viscosity changed by adding dextran
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Recommended gels
Desalting and other group separations

Sephadex G-25 Fine or Superfine grades
Fractionation





Use a pre-packed column!
Superdex or Superdex prep grade
Native proteins: Superdex 200
Recombinant proteins: Superdex 75
Peptides: Superdex Peptide or Superdex 30
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Column size
Desalting and other group separations


Volume four times the expected sample volume
Length is not so important
Fractionation


Volume ca 20-200 times the expected sample volume
Length 30-100 cm
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Column packing
A well-packed column is ESSENTIAL
• Choose a properly designed column
• Prepare the gel slurry carefully
• Pack the column at the temperature at which it will be
•
•
•
•
used
Add all the slurry in one smooth movement
Pack at constant pressure or constant flow rate
Stabilise and equilibrate the packed bed
Check the efficiency; re-pack the column if necessary
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Preparing and applying the sample
Preparation
•
•
•
•
Check the viscosity and remove nucleic acids and
polysaccharides if necessay
Filter or spin to remove particles
Volume for desalting: up to 25% column volume
Volume for fractionation: 0.5-5% column volume
Application
•
•
Through an adapter at the top of the column or
Layering under the eluent
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Increasing resolution
•
•
•
•
•
•
•
Check the column efficiency
Clean and/or re-pack
Check that the separation is in the fractionation range
Reduce the sample volume
Reduce the flow rate
Change to a gel with smaller beads
Connect two columns in series
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Trouble shooting
Resolution and recovery
See the Notes view for ideas which may be
of use in answering questions on this topic
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Trouble shooting
Elution earlier than expected
See the Notes view for ideas which may be
of use in answering questions on this topic
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Trouble shooting
Elution later than expected
See the Notes view for ideas which may be
of use in answering questions on this topic
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