Gel-filtration chromatography Video Lecture - Biotechnology Engineering (BT)

previous lecture we have discussed about
ion exchange chromatography in ion
exchange chromatography the property of
charge which a particular analyte
carries on itself is utilized for
separation procedure in chromatography
in now in this lecture we are going to
discuss another important come to
graphic method that is gel permeation
chromatography now gel permeation
chromatography utilizes the molecular
size or shape of a molecule for
separation this is also called this has
different names and this is also called
gel filtration chromatography or gel
exclusion or we can say size exclusion
chromatography then molecular sieve
chromatography or even simply gel
chromatography now this technique is a
form of partition chromatography and it
is used for separations of molecules
like I said on the basis of the shape
and size now this technique exploits the
molecular si properties of a variety of
porous materials if you consider basic
principle of this techniques then
different kinds of molecules which
differ in sizes are partitioned between
solvent and stationary phase of a
defined porosity now the separation
process is carried out using a porous
gel material or gel matrix in bead form
and it is packed in a column surrounded
by the mobile phase or the solvent now
if a sample is applied which contain
mixtures of smaller larger and
intermediate sized molecules compared to
the pores of the stationary phase matrix
then separation will occur as one where
smaller molecules can enter the pores
and hence
slowly through the column this means
that they will spend more time on the
column because they can move in and out
of the beats more frequently than other
molecules so the smaller molecule will
rather spend more time on the column as
compared to other molecules now larger
molecules which are too large to enter
into the beats due to the pore size
limit they would be completely excluded
from the stationary phase and hence
elude first from the column and they
will come what we call in white volume
then there are molecules which are
intermediate in size and can enter the
stationary phase but it spends less time
it's a you can say that they spend
relatively less time than smallest
molecules and more time than the largest
molecules so depending on their
molecular size there will be a kind of
relative time spent in the gel or in the
column and accordingly they will be
purified so all the molecules which are
eluted they are eluted in order of their
decreasing size now let me explain this
now before we go into the details of
this method let me explain in a very
brief and this method to you on your
screen
alright so now first thing is that this
chromatography technique is based on
size and shape and then another thing is
since there is molecular size is the
basis of separation the column lands are
usually long so that the analyte needs
to be separated can be separated well as
they will they will travel a longer
distance that will allow them to be
separated to a certain extent that Peaks
do not overlap
second thing is that the column is
mostly narrow bore column or it has a
less it has a lower what we call
diameter of the column is less as
compared to other normal columns so that
there is no diffusion and run gel
filtration is run successfully so what
you have is now the beads which are
packed that's the most important part
here that is beats if we say see if
there is a column and like I said it
would be a long column and with narrow
bolt size or diameter and this column
will contain beads here now what is the
property of the bead like what is the
basis of separation here like we were
talking about so now these beads are
porous beads these are hollow beads
actually and these beads if I make it
here a single bead then these beads are
totally hollow inside and they have a
particular pore actually pore means this
is just a schematic to tell you these
are cross-linked polymers and they
present a gel-like structure and they
have a particular porosity because of
these cross-linking and porosity just
like to make you understand if this is
the pore size then what will happen a
molecule which is bigger than this pore
size if I say this is the molecule then
this cannot enter here but a molecule
which is smaller than pore size can
enter into the pores
this can go in into it help it can
access the inner volume of this bead
which is the trapped solvent or stripped
mobile phase but the larger molecule
cannot enter so what happens then that
larger molecules will not be able to
spend time it will only spend time
interstitial spaces or the space which
is surrounding this stationary support
material which which is which is in
which the stationary phases contain so
this will come very fast but the smaller
molecule will spend more time because if
you go back and forth as it travels
down the column it will move back and
forth into the stationary phase and then
mobile phase so if you compare different
sizes or molecules if I say there is one
size there's another size there is
another size there's a larger size then
all of these molecules according to
their molecular mass or molecular weight
they will spend relatively different
times smaller the longest time the
largest the least time and in between
they will be spending different times as
per their size so that's how the
separation occurs because they are
moving down though you have a continuous
flow system and they are moving down at
different speeds or at different rates
actually which are eluted at different
rates so this is a very very simple
basic principle of column chromatography
of gel filtration chromatography now
these columns like I said are long and
they are narrow Bowl so there could be
for low pressure that will be coarse
beads or bigger beads and for
high-performance liquid chromatography
there could be finer beads which are
packed very nicely now this system
mostly can only cannot be run by gravity
feed so it is you require here if I have
certain things you require a pump here
which is a very important part of this
column so that it can give you a
constant flow rate so this is a
prerequisite that you should have a
pumping system to run the column if you
use certain like over you can put a
reservoir sort of over the column but
that will not give you a constant flow
rate so this is a very in a simple
method I tried to explain the principle
of this gel filtration now let's get
into the our discussion or detailed
discussion all right
so what we were talking about was what
we are talking about was that smaller
molecules can enter the pores hence they
spend more time in the column and larger
molecules are excluded so larger
molecules are excluded in void volume or
dead volume which is the total volume of
the column minus the volume occupied by
the beats so since it is not getting
inside the inside the beats it will be
excluded now here there is another
figure which explains it the same
principle if you can see here these are
this is a column where these are beats
actually and you have different sized
molecules there is a larger size
intermediate size in the smaller size
now if you can see them they are moving
at different rates larger moving out
very fast intermediate moving at a
relatively slower speed than the larger
ones and the smallest one will move at
the slowest speed and as the column runs
and as the as the time passes the first
to elute is the large one and you get a
separate fraction that's how you can see
very clearly the next one you can see
how the peak resolution can take place
if they are molecular sizes are further
apart far apart and they could be easily
separated in two different fractions and
could be utilized for different
applications as a purified product now
what I was trying to say why you need a
long column you need a long column as to
start a single entity in this when you
load these columns and as they move if
you have a smaller column then this
separation like here it's not really
separated quite a lot the separation
will be not complete and they will have
you can have say overlapping Peaks so as
long as the column is and narrow bore
column for avoid diffusions you will get
the better better separations could be
achieved in
filtration chromatography so this is
very in a very simple way this explains
the whole basis now if I have to say
like what are different terms and terms
in columns if you see here the shaded
part here this is called the void volume
it's the volume outside the support
matrix this is total volume like it is
taken both the volume occupied outside
the beads and inside the big beats
that's the total bed volume and then you
have volume which is inside the
stationary phase and that is called vs
or volume of the solvent held in the
beats so that's the kind of distribution
or you can say that's how the void
volume the total board were bed volume
and the volume occupied by a solvent
inside the stationary inside the support
beats is described now here elution
volume of a large totally excluded
molecule will be given by void volume VT
is physical volume and other like I said
mister you can divide or you can sorry
subtract the total wet volume by the
void volume and you can get the volume
of the beats
now elution volume is another term
called initial volume and that volume is
the volume between the point of
injection of the sample analyte at the
top of the column and the peak maximum
which is dependent on the fraction of
stationary phase available to the
analyte for diffusion so that will be
like elution volume is the volume which
is required for eluting the particular
and alike and that will be starting from
the point where you inject the sample
and the point where a peak appears or
that that analyte is eluted so if you
consider here for and these equations
here KD is of course
it's a distribution coefficient and kab
is proportion of ports available to the
molecule and both could be used so for a
totally excluded molecule since KD or KB
will be zero and so the elution volume
will equal the void volume that is for
completely excluded molecule totally
included molecule will be it's like
there is no fractionation happening and
illusion value will be almost equal to
the total volume of the column so and
intermediates could then be separated in
between they will have certain values in
between 0 and 1 so a total bed volume
can be measured by determining the
elution volume of a small molecule that
is which is not in the fractionation
range like salts actually or acetone
they could be utilized since they have
complete access they can easily move in
so they just move with the mobile phase
and they spent the most time and that
could be calculated
Ellucian volume could be calculated by
utilizing a very small molecule if we
consider the efficiency of a column or a
gel filtration column if you could
recall we were talking about the
theoretical plates actually theoretical
plates more is the number of theoretical
plates where better is the resolution
better is the separation power of a
column so here efficiency is determined
by calculating number of theoretical
plates per meter and you can this
equation could be utilized for
calculating the the theoretical plates
where V is the elution volume V W is the
peak volume at half the height of the
peak and L is the column length so it's
like here half the height of the peak
and this is the initial volume so you
can calculate the theoretical plates
remember if you compare low pressure and
high-performance liquid chromatography
though the length of the
high-performance liquid chromatography
call
are not that long but still they contain
more number of theoretical plates
because the material which is packed
with the stationary phase or support
matrix is much finer material and so
they they can pack in many or more
material in a small area and there were
providing more number of theoretical
plates and enhancing the resolution or
the separation power of the column now
let us discuss all different aspects of
the gel filtration chromatography first
is gel permeation media now when you
take the media there should be certain
properties of this media that is a
column material one is that this should
be completely inert with respect to the
molecules being separated if the it is
not inert that nonspecific binding or
adsorption might be a big problem so the
medium or the column material should be
completely inert for better separation
there are a lot of different kinds of
medium or gel material which are
utilized if you could recall we have
discussed about this but as we go along
here in terms of gel filtration there
could be lot of different support media
or gels are available dextran dextran is
a homo polysaccharide of glucose
residues and it is prepared with various
degrees of cross-linking to control pore
size remember here it's receiving effect
which is or filtration effect it kind of
filtration which is taking place so pore
size is very important as one depend on
particular pore size of the gel material
the different kinds of molecules will be
separated so it is like cross-linking is
controlled for pore size it could be
obtained as dry beets or beets swelled
when water and it could be the beets
could be spelled when water is added we
will see how that is done now
this trade name is suffered ex mainly
used for separation of small peptides
and globular proteins which is small to
average molecular mass now when we say
we have to swell it and the swelling is
done like you take the dry beads which
are apply which are supplied by a
commercial commercially now those could
be put in the water or buffer initially
in the water and left for a certain
period of time say you can leave them
overnight or so and let them swell the
swelling could be observed by you can
look into the under the microscope and
you will observe the unsoiled and
swelled beads could be clearly observed
it could be enhanced by you can put this
beads within water in an oven at higher
temperature they will swell faster so
that could also be done then there is a
poly acrylamide these gels are prepared
by again cross linking with the
acrylamide with NN methylene bicycle
might the pore size is determined by the
degree of cross-linking and the
separation properties of this material
are mainly same as that of others like
dextrins and they are supplied as for
example by o GL P they are available in
wide range of pore size so you can
isolate or purified different range of
material or analytes then there is a
gross it's a linear polymer of
d-galactose and three six and hydrogen
galactose it forms a gel that is held
together by mostly hydrogen bonds and
the concentration of the material in the
gel term means the pore size so the
pores of the agro gel are much larger
than those of sephadex or bio gel pee
and it could be utilized for separation
of large globular proteins or long
linear molecules such as DNA dextran and
agarose beads are biodegradable so many
times you have to use antimicrobial
agents like sodium azide for storage
like another important part as we were
discussing was
large globular proteins and long and
Lydian molecules like I said this will
also affect like shape of the molecule
will also affect the separation remember
when we consider all these things we
ideally take spherical shape but maybe
two molecules of the same molecular mass
but differing in shape like say a
molecule which is spherical and a
molecule which is elongated elongated
molecule will certainly will not be able
to enter the beats as compared to the
same molecular mass is spherical
material so those things has to be has
to be taken into account while
purification so all when you are
standardizing the things most kit is
done for a spherical molecule so when
you are considering gel matrix like
different materials we have discussed
and there are certain factors which
needs to be considered while you are
choosing a gel matrix one is
fractionation range that is if all
proteins in a mixtures are to be
separated from relatively low molecular
weight solutes like say your molecule
your proteins are large molecules and
you have to separate a smaller molecule
from the mixture from you have to
separate your protein from the smaller
molecules then you can have small
packings with small pores or gel
material and this is kind of a process
called desalting where proteins are
totally excluded we could you are using
the gel material which is of very small
pore size so your material does not or
your protein does not enter but a
smaller molecules will enter and you
will be able to easily purify this like
for example sephadex g 25 or bio gel p6
or p10 could be utilized for this
purpose for resolution of different
proteins which are more closely related
in size there is a range of size
actually then you have to take a matrix
where it's like molecules do do not get
excluded in wide volume or they are not
Nutan void volume and so there is a
fractionation range of the gel that is
of a particular pore size and it allows
all the analytes to enter unless you
want specifically that one of them is
excluded from the excluded and does not
fraction it actually then you have to
consider resolution for higher
resolution a smaller Beach should be
utilized they will give better
resolution because molecular diffusion
is slow is lower and there is less zone
broad broadening however smaller
particles gives higher a distance so you
have to use say high-performance
chromatography or very good pumps for
that purpose result and larger beads
allow faster flow rates and many times
they are used for large skip
applications so this is another factor
that what kind of resolution you are
seeking for and you can use either low
pressure chromatography or high
performance chromatography now another
factor very important factor is the
stability of the matrix matrix needs or
should be stable at different pH values
or in different buffer and organic
solvent conditions because you are going
to use different kinds of buffers or
solvents during the run and the matrix
needs to be physically and chemically
stable for these solvents and
necessarily proteins or any analyte
which needs to be purified should not be
affected by this particular problem so
this is another important part that is
stability now if you consider the
instrumentation of gel filtration is a
very simple very basic sort of thing as
we have already discussed earlier in for
low pressure chromatography and again
for high-performance liquid
chromatography in instrumentation part
the basic part is you require
a column which is like I said it's a
long column and narrow bore column you
require a detector so that you can go
through the peaks which are coming and
you can see the purification visually
then you require a fraction collector to
collect those fractions or different
Peaks separately then this like flow
rate has to be controlled and it is
mostly like I done you can have a
constant head reservoir but that's not
very suitable what you require a pumping
system it could be for low pressure to
be a peristaltic pump of high pressures
it could be like constant displacement
or other pumps could be utilized
now the system should be arranged with
the monitor connected as close as
possible to the column outlet and this
is to prevent the mixing or longitudinal
mixing in the tubing and similarly the
connecting tubing's and flow cells
should have narrow dimensions to prevent
mixing so that's a very important part
the column needs to be like when you are
purifying this the aim of the gel
filtration is to obtain greatest
possible resolution of the molecules of
interest and the resolution will be
improved by the distance between the
separated zones and their zone / zone
width so what you have is like I said
earlier longer the column better is the
resolution because you give more time
for the analytes to separate so that
when they travel through the mobile and
stationary phase the smaller columns
they will the peaks will overlap but as
the column length goes up then they will
have more time to separate from each
other
and separate peaks could be obtained
then columns if you say longer columns
it's up to 100 centimeter length columns
could be utilized the internal diameter
like I said it is very small like 1 or 1
point 2 5 centimeter or so and mostly
what is done is it is taken if you take
then the length should be 20 to 40
times longer than the then the wider so
that has to be remembered that you have
have you have to have a longer columns
with smaller diameter or internal
diameter of the column when you are
preparing the gels for column
chromatography what what is commercially
provided is either you will get dry
powders like say sephadex
or others and or you can get the swelled
or in certain in certain medium liquid
medium that particular matrix if you are
provided with dry powders then dry
powders has to be kept in a beaker or
container in the it has to be swelled in
the water and like I said you can swell
it overnight or you can spell in oven
which could be faster and you can check
for where the beats are spelled
uniformly by observing them under
microscope gels which are supplied pre
sweller like sefa krill and others what
you have to do is you require is that
you have to exchange them in the buffer
which is the proper element buffer
before used and then when you are again
storing them again you have to store
them in the and in particular solvent
say ethanol or others for long-term
storage so this is very important
now a packing is another important part
of running a column and here packing of
the gel filtration column is not that
easy if you compare it to the ion
exchange column and exchange columns are
smaller columns and you don't have to
worry about length and width so and the
diameter so much but here since these
are long columns and with a narrow ball
or a smaller internal diameter you have
to be very carefully packed the column
so that column is packed uniformly
without air bubbles without any gaps or
like say certain tracks so that run
could be proper so gels should consist
of a slurry when you are packing this of
approximately 75
percent settled gel and around 25%
supernatant liquid now suspensions will
be at constant done at constant
operating temperatures and like I said
earlier if you have different buffers
can change the pH and even there could
be certain other properties change due
to temperature so that has to be taken
care like for example if you sorta
suddenly take it into the cold room
conditions certain things could swell or
you know or could shrink load could
those could be our other problems so
suspensions should be at constant
operating temperature before and during
packing that's a very important so if
you want to do the experiment in cold
room then you can swell the gel in the
cold room
well the gel first and put it in the
cold room and then pack it now air
bubble formation and other things should
be prevented during column packing and
which can be monitored by using color
test sample such as like for example
blue dextran blue dextran is could also
be utilized for determining the void
volume as it will not enter the beads
and it is completely excluded from most
of the gel matrix now the packing is
mostly done with a pump like so you have
all the material is in a in a particular
container over the column it is packed
inside in merio flask and it could be
connected to a pump and it could be
packed properly manual packing might not
be a proper or good idea so it has to be
packed with a peristaltic pump or other
pump at a constant flow rate and it is
packed properly remember low pressure
beats
if you put too much of high pressure
they will flatten and then no separation
will take place so that has to be taken
care that they are packed properly they
are not subjected to very high pressure
which could be which will be like
totally damaging them and no proper
purification will take place then choice
of element for running the column is
important and when you are choosing an
element
it should be one which have a can where
your analyte could be stable and you can
add various cofactors or metals or other
things without any problem without any
interference so that is very important
if the sample is to be freeze-dried then
you can use volatile buffers let's say
you want to live flies the sample after
the purification then if you direct me
use the volatile buffers you really
don't have to exchange the buffer later
on let's say ammonium bicarbonate could
be utilized or water which is volatile
or other buffers could be utilized so
that's a very important part if you need
to live flies now one of the most
important part of this gel
chromatography is sample size or how
much amount needs to be loaded onto the
column the sample volume should be as
small as possible and must be lower than
the separation volume between components
in order to get higher resolution
typically it should be 1 to 5 percent of
the total wet volume so larger volume
will not give you a good separation I
will tell you why and so you have to
load the sample into in a higher
concentration but maybe what you call
smaller volume actually for group
separation where you are not so much
worried about separating many analytes
you can maybe use little larger volume
so when you sample when you have is so
gel filtration like is independent of
sample mass and so high concentration
could be utilized here without any
problem and you can get high resolution
however the concentration which can be
used is generally limited by the
viscosity if it's a too viscous or
sample then there could be a problem
relative to the xi so high viscosity
will results in zone stability and
irregular flow pattern so the protein
concentration of the sample applied to
the column could be around 10 to 20
milligram per ml
now let me first tell you why
the sample volume is very important in
here as come as compared to other
chromatographic techniques where larger
volume could be utilized in the column
so now let's understand this supposing
you have a column here and this is your
beats here in the column
I'm just trying this is just a schematic
this needs to be longer column now this
one you have your sample say if I have a
larger volume of sample why I am saying
why you require a small volume and you
have a different size things here all
right now what happens is that when you
have a larger volume of column this bead
here which is entering in the column or
this large size analyte which is
entering the column and this analyte
here is entering at the same time all
right
so they'll be separated accordingly but
what happens is these when they have
entered here in this beats this will
pass through interstitial space this
will go through this into the bead and
spend certain time but there will be a
time gap between these two entering and
these two entering actually so what will
happen yes
this one has traveled but it will travel
further but this one will since this a
like addition to the void volume if
suppose this is excluded then is
addition to the void volume and so the
there could be a problem when you have
many analytes of peak overlaps or peak
broadening because peak broadening wilt
and you'll not get you will not get a
sharp peak or this sort of peak rather
you will get a spread peak and what is
the problem is because these will be
entering at different times so the
purification will not be proper so it is
very important that sample is applied in
a very small volume so that all of them
all analytes almost enter the gel at the
same time
and then they separate according to
their retention capacity or retention
time sex retention volumes as per their
molecular size so that's a very
important factor actually and which
needs to be looked into so this is
another part which is important now when
you are applying sample application here
sample application as we have discussed
in ion exchange chromatography here also
you have to take lot of clear when you
are applying the sample so care should
be taken when applying the sample to the
surface of the bed and one is the bed
should not be disturbed if you are
disturbing the bed then again you have
to wait so that it settles down and it
might settle down
not so uniformly now when samples needs
to be applied first is you can remove
the or drain the bed surface on the top
without disturbing the bed surface and
then slowly very carefully load put your
sample on top of the bed then you can
layer the sample under the element if it
is viscous little bit like say high
concentration proteins will not unless
you mix very harshly they'll not mix and
even leak could be layered in the
element like I have told you in ion
exchange chromatography technique also
or you can use the loop injector or
three-way valve could be utilized for
this purpose as we have discussed in
high-performance liquid chromatography
so there could be different ways so if
you are applying it like manually then
the above plunger needs to be again
mobile phase needs to be put in after
the after the sample has entered the bed
or the stationary support material and
then you can layer the mobile phase and
close the column and connect it to the
pump so the maximum resolution is
obtained like I said using long columns
using slow flow rates and using
concentrated but less volume of the
samples after optimum flow rate could be
like 2 ml per centimeter square per hour
but it could go very high for
sefa krill like 30 ml per centimeter
square per hour but remember for
low-pressure chromatography if you use
higher flow rates then beats could be
flattened because of much higher
pressure so those things has to be taken
care of once you have utilized the
column your analytes are done you can
recreate the column for next run or
otherwise gels may be cleaned up you can
treat it with say 0.2 molar sodium
hydroxide or certain other non ionic
detergents clean it up and then you can
store it in the presence of
antimicrobial agents like sodium azide
which could be put in a very very small
amount or say 20 percent ethanol can
also be utilized so this way you can
easily store your material now this was
the basis and Mac you can say mechanism
and working of the gel filtration
chromatography gel filtration
chromatography has lot of advantages a
lot of application now before like so
when you are utilizing the this method
it's the best method for separating
molecules which are differing in
molecular weight or molecular size so
here like temperature pH ionic strength
buffer composition etcetera will not be
making a difference like in exchange you
have to have a particular buffer
condition of pH condition because they
will be charged accordingly here only
you have to worry about whether your
analyte or proteins stable so that's the
important part and separation can be
carried out under any conditions mostly
it's the isocratic flow like in it could
be in the presence of ions or cofactors
detergents and other particular
conditions could be utilized there is a
very little adsorption because these are
inert columns material it's a lot less
zonal spreading than other techniques
elution volume is related to the
molecular weight like I said
so this could be utilized for for say
determining molecular mass if you
consider the applications of gel
filtration chromatography there are many
applications of gel filtration
chromatography and let me list you some
of them one is like we were discussing
purification purification is the most
important application of gel filtration
chromatography now it is like mean
application and you can have separate
them on the basis of this shape or size
proteins enzymes hormones antibodies
nucleic acids polysaccharides and many
more could be separated on or purified
on this chromatographic column in
general gel filtration is considered a
low resolution chromatography and it is
it is not the first step in the
chromatographic procedure when you are
purifying an analyte rather it is a
final step but in certain cases it
depends from case to case you can use it
before also but mostly it is the final
polishing step registry relative
molecular mass determination could be
performed on gel filtration column so
what happens is like I said illusion
volumes of globular proteins are a
characteristic value and they could be
determined by the relative molecular
mass so if you have a if you run
standards certain standard proteins
which you know the molecular mass and
you know the aleutian volumes then you
have a standard graph which could be
plotted and then an unknown sample could
be run and through the calibration curve
the proteins of similar shape or like a
spherical proteins ideally their
molecular weight could be determined so
that's a very good technique to
determine the molecular mass only thing
is that all the standard conditions
which have been maintained for running
the standards needs to be maintained for
running the unknown sample so this is
how you can you can plot log of
molecular weight and the
Ellucian volume there will be a plot and
from this plot you can determine the
unknown sample you can concentrate
certain sample here say you have a
purified sample or other so what is done
is solution of say high molecular weight
substances can be concentrated by simply
adding dry sephadex g25 now as earlier I
told you these are not for purification
but kind of desalting so what will
happen when you put the dry powder it
will swell and absorb lot of water and
so low molecular substances will be in
the gel but high molecular substances
will be excluded and they will remain in
solution so after certain period of time
you can at a very low speed you can
centrifuge them and you can get the
solution or the solvent which contains
your high molecular weight analyte and
it is concentrated because lot of water
has been absorbed by the material this
could be utilized this it is frequently
utilized for desalting so why by use of
sephadex g 25 solutions
high molecular mass compounds may be
desalted where i know you can piece well
the gel in this case so the high
molecular mass substances will just move
in the void volume whereas the salts
will be distributed and will be in the
stationary phase so this could be
another important part it is very fast
and more efficient method than dialysis
which is a little cumbersome there are
lot of applications of this particular
method you can remove like for example
removal of phenol from nucleic acid
preparations that could be utilized it
could be utilized for removal of
ammonium sulfate from protein
preparations it could be utilized for
removal of salts from samples eluted
from ion exchange like for example an
exchange will contain lot of salt and to
take it to the next level
you need to remove the salt in certain
cases you can do protein binding Studies
here protein binding studies like you
can be used the gel filtration
chromatography to study the reversible
binding of a ligand to its to a macro
molecule and there are a lot of
combinations like say receptor hormone
or say Proteus or any beater likewise it
could be utilized antibody antigen and
these could be like when you will have a
separate ligand and protein they will
have a different illusion volumes but
when they combine and if they are
interacting with each other then elution
volume will be different because they
will be excluded they will be alluding
earlier Esper increase in the molecular
mass so there is a procedure and you can
like go through that and even experiment
could be repeated as a series of ligand
concentration and you can calculate
binding constants can also be calculated
so it's a very useful technique you can
also determine quaternary and tertiary
structure here so what is done when you
have purified proteins and if you run
through a gel filtration column through
the elution volume or through the
retention time their hydrodynamic radius
of typical protein will play a role and
so there will be change in illusion
volumes and so two forms could be
separated as well as you will have an
indication there is certain change in
the quaternary or tertiary structure of
a particular protein so that is another
way many times in polymer synthesis it
could be utilized to measure the size
and poly dispersity of a synthesized
polymer so that is the ability to be
able to find the distribution of of the
size of the polymer molecule and if a
standard so for known size are run
previously then a calibration curve can
be created to determine the size of
polymer molecules of interest in the
solvent so these are different
applications of gel filtration
chromatography
technique so as you see here in this
this is a very useful technique and it
is has many many applications it is on
the basis of size or shape
like I said ideally we talk about
spherical size but if it's a elongated
shape then certainly say molecular mass
a protein will be excluded faster than a
spherical protein so this is a very
simple very nice technique could be done
in low pressure chromatography or
high-performance liquid chromatography
with different types of columns with
more number of theoretical plates as
their finer particles effect and it is
like one of the techniques where you
don't have to worry about certain
factors physical properties except for
size here like pH or other things only
thing to worry about is stability of the
protein in particular but for condition
so this completes the gel filtration
chromatography in the next lecture we
are going to discuss about another
important technique which is affinity
chromatography thank you