Ion-exchange Chromatography - Biotechnology Engineering Video Lecture - Biotechnology Engineering (BT)

previous lecture we have gone through
the basics in chromatography also we
have discussed about the different
aspects of low pressure column
chromatography and high performance
liquid chromatography now in this
lecture we are going to discuss one of
the techniques which is widely used it
is ion exchange chromatography now as
you know many of the biological
molecules are charged molecules at a
particular pH so they carry certain
charge either positive or negative at a
particular pH like for example proteins
will carry positive charge below their P
I value and will carry negative charge
above their TI values and this
particular property of charge could be
utilized for their separation so I next
change chromatography involves
separation of macromolecules and other
molecules based on interaction between
charged solute molecules and oppositely
charged molecules covalently linked to a
chromatographic matrix now ion exchange
is the most frequently used
chromatographic technique for the
separation and purification of proteins
polypeptides nucleic acids and poly
nucleotides and other molecules which
are charged the reason for the success
of ion exchange chromatography is its
widespread applicability its high
resolving power its high capacity and
the simplicity and controllability of
the method before we go into details I
would like to show you on on your screen
in the very sim in very simple way the
mechanism and working of the ion
exchange chromatography so please focus
on your screen
and let's understand this whole
chromatography ion exchange
chromatography and then we'll go into
the details so what happens is that
first thing is you have a stationary
phase
now this stationary phase which is
particularly charged molecule or a
charged ionic charge which is
immobilized onto a support matrix say
bead now when you prepare your resin
then what happens is that your resin
will carry a particular charge it could
be a negative charge or it could be
positive charge and it is occluded with
a particular buffer so what is your
starting condition the starting
condition is if I say this is your bead
and this carries say particular charge
say positive charge then at the starting
point where you have equilibria to do
raising the positive charge will be will
be interacting with the counter ions so
if I say these are the counter ions
which are surrounding the raising the
charge on the resin so that is the
starting position now second thing will
be so what will happen how this whole
separation takes place that we will add
the protein or a biomolecule now when
you add a biomolecule biomolecule which
are oppositely charged that is here
resin is positively charged so
biomolecule which are negatively charged
will bind to this now they will replace
the counter ions and they can replace
the counter ions because they will have
high affinity for they will have high
affinity for the so biomolecules say
protein so protein is bound here and
protein is those protein molecule which
are carrying the negative charge will
bind in here so what you have is if I
show it here it is something like this
if it surface if this is positive charge
on resin then negative charge will be
binding and
might be of any molecule which are which
is present in here alright so once
protein is bound then you have these
protein molecules if there is a mixture
of protein molecules then they will bind
with different affinities depending on
the charge if they have more negative
charge they will bind with more affinity
if less then they will bind with less
affinity now what you have to do you
have to elute your protein or take out
your protein from the matrix and that is
to separate different analytes in a
mixture and this will be done on based
on the affinity is actually and here you
will put an element high ionic strength
element which will replace the protein
with those ionic species in the mobile
phase now what will happen depending on
as you will increase the concentration
of element or ionic strength like say
you can add NaCl and as you increase the
concentration of NaCl depending on their
affinities of proteins they will replace
it
so if protein has higher affinity then
you read need higher concentration of
NaCl so next step would be that is
illusion this was binding and next step
would be illusion where what you have
done you have eluted it to say NaCl or
some other element so what will happen
this positive charge will now it will be
replaced by say chloride ion and your
protein is you will get protein which is
eluted from now remember this protein
which will be like it's a mixture it
will come out at different strengths of
the salt alright once this is done so
you might get different peaks in here
which are there once it is done then
simply what you have to do regenerate so
again the simple buffer will be put in
an extra CL or extra element will be
washed off and fine you will re generate
the column which will be as the starting
position
and so this resin could then be reused
for another purification so this was a
very very simple way we I tried to
explain the mechanism of the ion
exchange chromatography
now let's little bit see how the working
of the chromatography is done so what is
done here is that you have a column and
there is a stationary phase in here
filled in here and which is say up to
here there is a stationary phase now in
ion exchange column and this contains
resin which we can say ion exchange
resin here and it carries a particular
charge all right and like I said it is
in the form of beads actually it's like
these are beats which are which contain
the stationary phase in the support
matrix all right now what you will do is
there are two ways like through
peristaltic pump as we were discussing
in earlier lectures you can do it by a
peristaltic pump or you can manually
load the sample so if I have to manually
load the sample and I can load the
manually the sample with a micro pipette
so this micropipette I can load the
sample and this sample could be in ion
exchange you can have any volume of
sample as this is a displacement kind of
chromatography where the ions or ionic
species will bind to the oppositely
charged ion on the resin now even what
you can do is that you can put say in
the sample you can put the resin itself
and let it bind in a beaker so that is
also possible that in a beaker you can
bind it and then you can load it onto
the column that can also be done so what
happens is once you have prepared your a
scene and you have loaded your sample
your sample will get in and as sample
gets in what will
it will as it gets in and gets in
contact with resin the charges will be
saturated
so if resin is positively charged
negative charge biomolecule will bind to
it and slowly saturated and as these
beads which are in starting gets
saturated the biomolecule will move in
and it will bind to others so it depends
on the capacity of this stationary phase
as how many ionic stationary phase it
has once it is bound then what is done
that you have to wash it so that unbound
very weakly binding things could be so
you have a knob here which could be
opened and once it is and this could be
collected flow through could be
collected flow through will contain
biomolecules which have not bound to the
resin because they are of the same chart
and this could be then separated by
another chromatography ion exchange only
where it has oppositely charged
stationary phase now once you have done
then you will do washing so first is you
have prepared the column by equilibria
ting at with right buffer then second
thing is you are binding your analyte
that is oppositely charged and then you
will elute so after binding then
Ellucian has to be done that will be
done by certain ionic species which can
replace your analyte and as you increase
the concentration depending on the
affinities so what will happen if you
have different affinities as you will
ute like say I can use 50 milli molar
NaCl I can use 10 100 millimolar 200
milli molar and I can go up to 0.5 molar
of 1 molar NaCl and depending on the
affinities you will get the peaks here
which will be separated according to the
affinity so less affinity will come
first and higher affinity peak will come
later on now once you are
with this you can regenerate the column
as I have explained and again reused for
another purification step so this is how
the whole working of the column is done
so let's get into details actually this
was just to make you understand how this
whole thing works now let's get you in
the details of our discussion now theory
of ion-exchange says that separation in
ion exchange chromatography depends upon
the reversible adsorption of charged
solute molecules to immobilized ion
exchange groups of opposite charge
now most ion exchange experiments are
performed in five mill stages and these
steps are as I have explained also first
thing is the first stage is equilibrium
in which the ion exchanger is brought to
a starting state in terms of pH and
ionic strength so which allows the
binding of the desired solute molecule
so the exchanger groups are associated
at this time with exchangeable counter
ions now these are usually simple and
ions or cations it could be like h o x-
or CL minus or any plus so sodium
chloride or hydroxyl or X plus ions
could be the exchanger ions now second
stage is sample application and
adsorption in which solute molecules
carrying the appropriate charge will
displace the counter ions and bind
reversibly to the gel so unbound
substances can be washed out from the
exchanger bed using starting buffer just
called extensive washing now once that
is done in the third stage Ellucian will
take its place
so what is done the substances are
removed from the column by changing to
illusion conditions which are
unfavorable for ionic bonding of the
solute molecules or biomolecules now
this normally involves increasing the
ionic strength of the eluting buffer or
changing its pH the fourth and fifth
stages are the removal from the column
of substances not eluted under the
previous experimental
that you can use higher concentration of
alumina and then recalibration with the
starting buffer for the next
purification so these are the stages
which I also explained in a very simple
way that you have to go through when you
are performing column chromatography and
this also shown in Figure here which is
like first test where counter ions are
bound but in starting then you are these
molecules which needs to be separated or
bound to the column and one of those
will bind as per the charge then your
gradient times will be utilized like
NaCl or some other which will elute as
per the affinities and you have to maybe
it could be increasing salt
concentration is used and then once this
is done it will be degenerated that's
how it's a very simple mechanism of ion
exchange chromatography it's a very
simple very versatile technique to be
you had now an anion exchanger consists
of an insoluble matrix to which charge
books have been covalently bound and
these charge groups are associated with
mobile counter the charge groups are
associated with mobile counter ions now
these counter ions can be reversibly
exchanged with other ions of the same
charge without altering the matrix the
matrix may be based on maybe inorganic
compounds synthetic resins
polysaccharides etcetera and the
commonly used method missus could be
like polystyrene cellulose agarose
dextran and so on now in ion exchange
chromatography there are two kinds of
ion exchangers one is called cation
exchanger and this depends on the charge
they carry so the cation exchanger and
anion exchanger now cation exchanger are
the ones which possess negative charge
as they are exchanging the cations today
by themselves possess negative charge
groups and will attract positively
charged cations these are also called
acidic kind exchangers because their
negative charge result from ionization
of acidic groups then there are anion
exchanger
and they purchase positively-charged
groups and will attract negative charged
anions these are also called basic ion
exchangers as their positive charges
result from the association of protons
with basic groups so if you see these
looks something like that is an ion
exchanger and there is a cation
exchanger so an ion exchanger will
exchange anion and cation exchanger will
exchange cations itself they're charged
accordingly this is positively charged
and cation exchanger is negatively
charged now there are a lot of examples
of commonly used ion exchangers as you
can see in this table there are strong
anion exchanger vk9 exchanger strong
cation exchanger and v cation exchanger
some examples are of a strong and ion
exchanger our trimethylamine Oh Ethel
which is also called time there is dial
2 hydroxypropyl these are strong and an
exchanger if you say we can dine
exchanger then amino ethyle and the dial
amino ethyle DEA these are weak weak and
ion exchangers then there are strong
cation exchangers like sulphur groups
are strong cation exchangers which is
salvo methyl or sulphur profile likewise
carboxy we can a cat ion exchangers
include carboxy group exchangers like
which contains carboxy group or carboxy
methyl groups so these are some examples
there could be many more of weak and
strong anion and cation exchangers now
when we say strong or weak it refers to
the extent of variation of ionization
with pH and not really the strength of
the binding strong ion exchangers are
completely ionized over a wide pH
whereas with VK an exchanger the degree
of dissociation and thus exchange
capacity varies much more markedly with
pH if you say some properties of what
are the properties of a strong or weak
and exchanges strong ion exchangers the
sample Lord
during capacity does not decrease that
high or low pH values they are due to
loss of charge from the ion exchanger so
they remain ionized at a wide range of
pH a very simple mechanism of
interaction exists between the ion
exchanger and the solute ion exchange
experiments are more controllable since
the charged characteristics of the media
do not change with changing pH and this
makes a strong exchanger ideal for
working with data derived from
electrophoresis titration curves
etcetera another important
characteristic is capacity now the
capacity of an ion exchanger is a
quantitative measure of its ability to
take up exchangeable counter ions and is
therefore of major importance now
capacity may be expressed as total ionic
capacity or available capacity or
dynamic capacity now total ionic
capacity is the number of charged
substituent groups per gram dry ion
exchanger per ml swollen gel total
capacity can be measured by titration
with a strong acid or base then there is
actual amount of protein which can be
bound to an ion exchanger under defined
experimental condition and that is
referred to as available capacity for
the gel now if the if the defined
capacity or defined conditions include
the flow rate at which the gel was
operated then the amount bound is
referred to as dynamic capacity for the
ion exchanger so when flow rate is
included it becomes dynamic capacity for
the ion exchanger available and dynamic
capacities will depend on many factors
and which includes say properties of the
protein now properties of the protein
will include say molecular size and
charge or pH relationships then
properties of the ion exchanger will
also affect this capacity like exclusion
limit of the matrix type and number of
the charge substituent's etcetera
then chosen experimental conditions like
pH ionic strength of the buffer nature
of the counter ion flow rate temperature
all these things will certainly all
these factors will certainly affect the
available and dynamic capacities of a
particular experiment a particular
blessing another important factor is
porosity of matrix now the porosity of
the matrix is an important feature
because the charge tubes are both inside
and outside the matrix and matrix is
support material so because of this the
matrix also acts as a molecular sieve
because matrix a flow has to be
maintained through the matrix so matrix
is not in itself or it's a mesh-like
structure and there is a porosity here
and when you immobilize designs they
will be all over the matrix they could
be totally exposed or they could be
somewhat inside now large molecules may
be unable to penetrate the pores it
might happen that certain ion and or
certain charged roofs may not be
approachable by the large molecules
because they will not be able to reach
while the small molecules will be able
to reach so capacity will be affected by
the porosity also so many times when you
are selecting the ion exchange matrix
you will also look for the porosity and
it's like when we say CMC 25 or some C
25 or 50 it refers to the porosity and
accordingly you can choose the matrix
for a particular size of the sample to
be purified mesh size is also important
which determines porosity like INEX
changes will come in a variety of mesh
size called a variety of size called
mesh size now finer mesh size means an
increased surface to volume ratio and
therefore increased capacity and
decreased time for exchange to occur for
a
volume of the exchanger on the other
hand finer mesh fine mesh means slow
flow rate which can increase diffusional
spreading so they're whole lot of
different mesh size are available and
accordingly one has to choose that mesh
size as we have explained before
mechanism
ion exchange is thought to be composed
of five distinct steps and this time
this is like a little different from the
procedure we have discussed earlier one
is the first step is diffusion of the
ion to the exchanger surface now this
occurs very quickly in homogeneous
solution as you put your sample the
diffusion occurs then diffusion line
through the matrix structure of the
exchanger to the exchange site actually
where it has to bind and this is
dependent upon the degree of cross
linking of the exchanger and the
concentration of the solution this
process controls the rate of whole ion
exchange process then exchange of files
at the exchanger site this is thought to
occurs instantaneously and to be an
equilibrium process so what happens this
happens like when you have a particular
ion a resin which is kind of interacting
with a counter ion and their analyte
comes or a protein comes then it is
replaced this will replace the counter
ion as in here and like in anion
exchanger similarly the analyte will
replace the counter ion as it has higher
affinity the more highly charged to
ionized molecules to be exchanged the
tighter it binds to the exchanger and
the less readily it is displaced by
otherwise so this depicts the the
affinity of a particular analyte for the
exchanger then diffusion of the exchange
iron through the exchanger to the
surface and the selective de saucé
desorption by the lumen and diffusion of
the molecules into the external element
so all these steps happen one by one and
this is how the the mechanism is unfolds
of the ion exchange chromatography now
the Selective desorption of the bound
line is achieved by it could be simply
pH or the concentration of a particular
ion that is ionic concentration could be
used so this was mechanism of ion
exchange chromatography now selection of
conditions for an exchange purification
depends on many factors a wide variety
of ion exchangers and adsorption
desorption conditions are available and
one has to choose according to what kind
of experiment they are doing what is
their analyte let's say a pH stability
what is the charges at particular pH all
these things has to be taken care and
likewise buffers and eleventh has to be
or has to be selected now the factors
involved in making each choice are
described here so if you say say choice
of matrix so we are discussing those
factors for choice of matrix for low and
medium pressure ion exchange you can use
say cellulose which could be fibrous
micro ground granular or beaded it could
you can use agarose dextran polyvinyl a
lot of other matrices are available for
high pressure on exiting you need strong
an exchanger or matrices like silica
based or other resins which have been
made for high pressure chromatography
these matrices mainly differ in their
particle size and pH range and those
could be selected accordingly a lot of
commercial resins are available for high
performance liquid chromatography
selection of functional group is another
important factor for example anionic or
cationic groups has to be selected
now if the material to be bound to the
column have a a single charge then
either positive or negative
so then choice is very clear now problem
comes in amphoteric materials like
proteins which might have both positive
and negative
charge so pH stability has to be
considered here if a protein is more
stable above its I selected point then
an anion exchanger should be chosen and
if it is stable below the isoelectric
point then a cation exchanger should be
chosen as the charge of the protein will
be accordingly be at its above or below
its p.i value for a weaker ionized
substances which requires very low or
high pH for ionization a strong ion
exchanger is used
however for certain other substances
Vica an exchanger are preferred because
strong exchangers are often capable of
distorting a molecule so much that the
molecule will might be nature so
accordingly one has to choose and see
the conditions if it's a very weakly
ionized substance certainly strong ion
exchanger needs to be taken now the most
commonly used functionalities are weak
I know genic groups and these are in
common use these are weak exchangers
like die tuile amino tuile DEA commonly
known as this group is usually used in
anion exchanger to purify negatively
charged proteins while the carboxymethyl
is another commonly used a frequently
used cation exchanger for recovery of
positively charged groups so depending
on what kind of analyte you have you can
choose one of the exchanger either
cation or anion exchanger and most of
the time weak exchangers are utilized
now buffers used in ion exchange
chromatography again you have to be
careful in here for successful an
exchange chromatography now in ion
exchange chromatography buffers are
chosen in order to minimize pH
fluctuations and maximize the adsorbent
capacity now two important factors or
rules has to be taken care of one is
that is charged former buffers should
not interfere with the ion exchange
process and we'll discuss that and then
again another factor is temperature
which will affect the PK value of the
buffer and such that it's buffering
capacity will be different in
purification carried out in the cold
conditions for example Tris buffer will
have if you set the pH in say room
temperature and you carry it to the cold
room conditions then certainly a change
in buffer pH will take place and that
might hamper your protein purification
the commonly used buffers this is a
table gives you some commonly used
buffer with cation and anion exchange
chromatography for example with cation
exchange you can see acetic citric Mass
phosphate happies buffers are used with
Katan exchangers while with anion
exchanger you can say Elstad een Amida's
all Tritonal and I mean Tris and all
these buffers are used with an ion
exchanger so you have to worry about
that which kind of buffer which will not
interfere with the particular kind of
type of exchanger needs to be used now
selection of adsorption Andalucian pH is
an important factor and for protein
adsorption on the matrix a pH about one
pH unit above or below of the PI of the
target protein is chosen while for
protein Ellucian 0.5 of a pH unit from
its PII at an ionic strength of 0.5
molar nacl conditions are chosen and
these things needs to be standardized
these are not hard and fast rule here
when you do your experiment one has to
standardize that how much salt
concentration needs to be taken for
Ellucian or if it is done through pH
conditions how what changes in pH has to
be done now selection of adsorption
Andalucian ionic strength like i said
needs to be standardized needs to be
taken care of and one has to go through
the experiment to do that some
guidelines on the adsorption step the
highest honest strength which will allow
protein binding is used
while on illusion step the lowest
annexed rent is recommended so reason
for this are as follows so if you have a
buffer then you should use a buffer of
higher highest ionic strength when you
are binding the protein and when you are
in looting then you have to use the low
ionic strength buffer only thing you
have to take care that your protein
remains the stable in both conditions if
say at the low ionic spirit if it is not
stable then you have to avoid that now
if the ionic strength is too low on
absorption then the protein will bind
too tightly and effective Ellucian will
be made difficult so that's why one has
to use high in its ionic strength then
keeping the ionic strength as high as
possible on absorbs adsorption minimize
the binding of unwanted contaminants
that is very important that nonspecific
or unwanted contaminates will be avoided
if high ionic strength is utilized
conversely keeping the ionic strength as
low as possible or Lucian minimizes the
illusion of bound contaminants so you
can get a bit better
purification now the strategy outlined
above simplifies dilution step and the
salt concentration say of to 20 to 50
milli molar is usually used during the
absorption and say up to 0.5 molar NaCl
is used during Ellucian process this
graph here figure shows that you can use
something like safe this is your P I
value and your pH or a range of
stability for a protein is certain pH
range then that could be utilized for
that could be utilized and this could be
seen and particular pH of a stability
could be used for purification so the
net charge of a protein as a function of
pH and showing the pH range for
stability and for binding to cattle and
to anion exchanger you can say it will
bind accordingly as per its the pH
either it is pH is above or below so
this protein would be coronated draft on
an ion exchange
chromatography using a buffer adjustable
to pH 6.3 28.4 so accordingly it has to
be like if it is say an eye next if it's
an ionic protein is an ionic at
particular pH where it is stable then it
has to be extreme fide on an ion
exchanger and like here six point three
to eight point four is the right pH that
needs to be utilized
now choice of porosity and mass size
where we were talking about now small
molecules are best separated on matrices
with a small pore size whereas
macromolecule will need large pore size
as I was discussing many commercial
resins they tell that by certain
specifications that what is what could
be the mesh size and porosity and
accordingly if it's a macro molecule you
require to use a larger pore size if
it's a it's a smaller molecule then a
smaller pore size could be utilized
selecting a mesh size is difficult and
determined empirically as a small mesh
size improves resolution but decreases
flow rate so accordingly one has to
choose the conditions alright so this
was like all different factors we have
discussed regarding when you are
attempting to do ion exchange
chromatography and what are the factors
you have to take care you have to
accordingly design your ion exchange
chromatography experiment now if we see
the simple procedure as I was explaining
earlier in a very simple way the
procedure in ion exchange separation is
quite straightforward
the first step to be done is you have to
select the matrix once you have selected
the matrix the pretreatment of matrix
has to be done now remember matrix will
be available either in say powder form
or as in some solvent say 30 percent
ethanol
so the matrix has to be in both ways
matrix has to be swollen it has to be
put in proper buffer which needs to be
calibrated or you can start with water
and then you can put it in buffer so
what will it be done like as you put the
matrix in the buffer or suitable buffer
of water mostly you can start with water
but then you have to exchange it to the
suitable buffer in a be weaker in a
beaker so once you do that then certain
fine
fines which are really not cannot be
used and they will be unnecessarily
increasing the volume of the matrix can
be removed by decanting then you will
allow dope in ion exchange swelling is
not so important but you will allow it
to exchange with the buffer and then it
will wash it and eek liberate it to the
proper buffer and then if say it has to
be converted to a correct counter ion
that will be done so matrix P turret
treatment is the first stage of ion
exchange separation once you have done
that then matrix has to be loaded on to
the column you can also load your sample
before loading it onto the column and
there are various methods of adsorption
so different adsorption methods could be
one could be batch adsorption now batch
adsorption is carried out in free
solutions but it has a lower efficiency
like I said you can do it in a beaker it
is ideally suited for initial treatment
of large volumes of sample say if you
have a litters of sample and what you
can do is suitable amount of matrix
could be directly put into them or
preliterate matrix could be directly
putted put into the this large volume of
the sample and you can gently stir the
sample and the protein or the analyte
will bind to the to the stationary phase
now this does not suffer from problems
of bat swelling and shrinkage now once
it is done then Ellucian can be carried
out either in batch mode or ion exchange
slurry may be packed
to a column and then eluted lecture in a
batch mode say once you have bound the
the analyte to the stationary phase the
sample could be decanted and then
elution buffer could be first you can
wash and then elution buffer could be
put in the beaker and batch solution
like so you can directly use point five
molar NaCl and let it be there which is
little gentle stirring and then whole
analyte will be looted into that buffer
like say 0.5 molar NaCl and that could
be decanted and could be used for
further use now further applications or
you can so this is batch mode or what
you can do is rather than eluting in
batch mode you can pack whole thing in
the column and then elute accordingly in
column adsorption rather than better
option you can also do it in column
adsorption so in batch adsorption of
protein is purified by adsorption of
contaminants which allows the desired
protein to pass through the column
without binding now low concentration of
protein results in here in column
binding rather than batch mode you can
also do it by Ellucian method now here
an ilusion method
it could be static ion exchange or
dynamic ion exchange my static ion
exchange protein is fully a jerk to the
bed and completely looted by
displacement into mobile phase using a
small volume of strong element like I
said you can take point 5 molar of 1
molar NaCl and you can elute the protein
now this is useful for concentration of
protein from a large volume of sample
because what you are doing is you are in
looting though it's a strong reagent
but you are in looting it in smaller
volume so what you get is much
concentrated solution of the protein in
dynamic ion exchange separation of
protein is achieved by their relative
speed of migration through the column so
what is done all the sample components
will migrate but they will be separate
depending on their relative equilibrium
distribution or this
tribution coefficient we can say or we
can say relative affinities to the
stationary phase so what will happen as
you increase the ionic concentration the
molecules with different display
affinities will be lifted and as I
showed you earlier
the they will be eluted in different
fractions and those fractions can be
collected in fraction collector and they
could be a jar and absorb served on a
spectrophotometer or if the if your
Renato graph contains you be monitored
then they could be observed in form of
different Peaks there are three types of
Aleutian conditions which could be
utilized it could be isocratic elution
in isocratic elution sample volume
should be between 1% and 5% of the red
volume and the long column is used with
diameter to length ratio of around 1 is
to 20 so the starting buffer is used
throughout the separation its isocratic
elution where you are not changing the
buffer as such is a one single buffer
which will be looting it and could be
concentrated buffer or high amount of
element might be utilized then there is
a stepwise solution now in stepwise
Ellucian a sequential discontinuous
change in pH or salt concentration will
be done like for example say you can
change PS sequentially like you can
first start with say pH if I have to
loot I can start with say 7 then I could
come down to 6 5 4 likewise or I can go
up in that order or like my salt
concentration could be increased like
for example I can start with 100
millimolar salt concentration then I can
go to 150 200 250 and up 2.5 molar nacl
now column volume is determined by the
exchanger capacity and the sample volume
here so the sample should adsorb to 5 to
10 percent of the total bed capacity and
the column length is usually shorter so
here it is a stepwise Ellucian and
different fractions will contain
different Peaks and those analytes
having different affinities will elute
accordingly more affinity one will elute
later on
and less affinity analyte will later
first will elute first you can use
gradient elution also rather than
stepwise Ellucian so it is a continuous
gradient rather than step in the
gradient so the composition of element
that is pH or ionic concentration is
changed continuously
and sample contents should be 5 to 10
percent of the bed capacity ideally and
column length is usually 20 to 40
centimeter with a diameter to length
ratio of not more than 1.5 one is to 5
the volume of the buffer required is
determined empirically and in two
steeper gradient resolution will be lost
while two shallower gradient resulting
unnecessary dilution as long and long
separation times so accordingly one has
to choose the condition the total volume
of sample should be about five times the
bed volume these things are you know not
huddle fast and one has to really go
through standardizing these values so in
continuous gradient could be made by two
pumps and a gradient mixture could be
used where you can say start with zero
molar NaCl buffer and say one molar NaCl
buffer and accordingly you can have a
gradient which could be too steep or you
can choose the gradient it could be
simple linear gradient otherwise now if
you see here the chromatographic
purification has been compared for a
steep gradient and not somewhere so
steep gradient and you can see that here
these two peaks are for one is here only
first peak is very clear other Peaks are
not very clear but there here you can
see two peaks are separating and other
Peaks are visible so that is a very
important that separation of four peaks
by ion exchange chromatography is taken
here but in steep higher steep gradient
only one peak is very clear other Peaks
are not so clear but instead lower
gradient of peak 2 & 3 also becomes
clear and existence of 4th peak becomes
of
so these are like again as you go along
doing these things general guidelines
are very clear but one has to
standardize its own conditions to get
best results finally once you have
separated your biomolecules on column by
combination of say cation or anion
exchange chromatography chromatography
then the column could be reused many
times and so it has to be regenerated
and if you are not doing right away the
next purification you can store at also
regeneration involves removal of tightly
bound contaminants and conversion of the
support to the required contour line
form ready for equilibrium and protein
adsorption removal of tightly bound
protein is achieved say using 2 molar
nacl and then followed by an alkaline
wash for storage antimicrobial agents
are used as to avoid microbial growth
and it could be high organic solvents
are recommended or sodium azide could
also be used like antimicrobial agents
like sodium azide can be used in certain
conditions where antimicrobial agents
cannot be used then say 30 percent
ethanol or other organic solvents could
be used for this purpose so this
completes our ion exchange
chromatography section and as you have
as we have gone through this particular
section I think you have that you have
you are able to understand that how
column ion exchange chromatography is
performed it's a very straightforward
chromatography but depending on the type
of biological moly molecules to be
separated dependent you have to worry
about lot of different factors and which
include say conditions of the experiment
the stability of your analytes the mesh
size or porosity of the column and the
versity of the column standard
conditions for running the column kind
of buffer you have to use kind of ill
you elements you have to use all the all
these things has to be taken care of
general guidelines are very clear as
it's a very straightforward if it's a
your analyte is positively charged you
have to use negatively charged cation
now negatively charged the column if
your and vice versa so but certainly one
needs to standardize standardize all the
conditions and then now once for a
particular analyte you have a
standardized the condition that could be
followed in your lab and purification
can be performed in the next lecture we
are going to discuss about another
important chromatographic technique and
that is exclusion chromatography thank
you