Precipitation - Downstream Processing Video Lecture - Chemical Engineering

today we are taking up a new topic in
the area of downstream precipitation
precipitation involves removal of solids
from a solution so you need to achieve
certain conditions so that the solids in
the solution comes out as a solid
material and then later on you may
purify the solids you may wash the
solids you may dry the solids and so on
actually so precipitation involves
removal of the solids so in many cases
the solid might not be 100% a pure if
you want 100 percent pure solid then you
may resort to something called a
slightly more complex process called a
crystallization so in precipitation we
can use this particular technique for
recovery of proteins we can use it for
even recovery of small molecules that
means the metabolites so its conversion
of soluble solutes into insoluble
solutes so we are here in precipitation
we are not bothered about the particle
size or the morphology but whereas in
crystallization you are more interested
in the morphology or the how the crystal
looks like the shape of the crystal the
size of the crystal the various planes
and so on whereas in precipitation we
are more interested in just removing the
solids from the solution so we convert
soluble solutes into insoluble solutes
so the solutes are small molecule the
solutes could be filled and proteins
once you do this you may resort to
something called as sedimentation or
settling or filtration or centrifugation
so we have looked at all these
separation processes before you know
early days so that's what precipitation
does so it's used in preliminary or the
early stages of downstream processing as
I said we are not bothered about the
purity of the solid product that's
formed so this process is resorted to in
the very early stages later on you may
do some washing to remove the and
purity Zoar toxins or other material
that's present in the solid so for
example if you take a globular protein
in aqueous solution it's got a
non-uniform distribution of positive and
negative charges it may have different
hydrophilic and hydrophobic regions
because some of the amino acids are
hydrophobics of them in acids are
hydrophilic so the solubility of these
proteins are affected by interactions
between various surfaces regions now you
have a hydrophobic process portions the
hydrophilic portions you have negative
charged portions you have positive
charged portions then you have the water
molecules surrounding it so ideally you
are disturbing these interactions so
that the proteins eliminate and then
they come out as a solid form and that's
what you are doing in a precipitation
okay if you look at proteins they
exhibit a negative charge on the surface
and attract positive ions to form stern
layer of counter ions close to the
protein we do proteins have charged if
you look at the peptide bond you all
know what's a peptide bond right and the
protein SAR made up of lot of peptides
so the nitrogen carbon bond here
okay it's called the peptide bond and
there is a keto bond C double bond o so
there is a resonance between this
particular configuration and this
particular configuration so when this
double bond moves down here this
nitrogen is going to acquire a positive
charge and this oxygen is going to
acquire a negative charge okay so this
type of resonance is going to give you
yeah negative charge of course it
depends on the pH if too much acidic
pairs are too much basic pH this
particular resonance is going to get
disturbed
under normal conditions so you are going
to have a negative charge so it's going
to attract positive ions around it and
that's called a stern layer okay now on
top of the stern layer
is counter ions because few are having
positive ions then you may have negative
ions around that and that's called ego
chop man layer of mobile contrarians
those contrarians are mobile because
they are much far away from the protein
so you have two protein and then you
have a stone layer and then you have the
chapman layer good shot man layer and
which are counter ions to this stern
layer we can show it pictorially like
this you know if you look at a protein
in an aqueous environment okay there are
hydrophobic patches and then you have o
minuses in the various places because of
those whoa - you are going to have maybe
a stern layer a primary layer and then
on top of that you are going to have the
counter ions that's the go chop man
layer that's the outer layer so if
protein is like this and there is
another protein like that so they are
not going to interact much and if you
disturb these layers and you make the
proteins agglomerate with each other
then it may form sufficient large sized
molecules which may then precipitate out
of your solution so there are different
ways by which you can disturb the these
various charge layers sometimes we use
pH sometimes we use counter ions
sometimes we use soils and so on
actually we will look at each one of
them in detail as we go along
okay now the stability of the protein
electrolyte Cloyd is due to the balance
between these attractive and repulsive
forces between these and so there is air
force which is preventing them to
aggregate now this thickness of this
double layer can be reduced by changing
solvent characteristics solvent solvent
characteristics could be the ionic
strength of the solvent the dielectric
constant of the Chara solvent or we can
change the protein surface
characteristics so by doing these we are
decreasing the solubility of the protein
so by doing this we are bringing two
proteins together and letting them
agglomerate reduce the solubility and
hence
the protein to precipitate if one is
interested to know more about these
various counter ions ions layers we can
look into this particular reference you
know downstream process technology new
horizon biotechnology by Krishna Prasad
prentice hall publication Oh protein
precipitation is based on two things one
is related to solvent one is related to
the solute solute means it could be
small molecule or it could be protein
and so on okay now solvent property pH
change so for example isoelectric
precipitation what we do we change the
pH so that the we reach the isoelectric
pH protein loses its charge so there
could be a precipitation let's call the
isoelectric precipitation the next one
could be change in ionic strength
that's called a salting out what do we
do we may add a salt which is highly
soluble when the salt gets dissolved
protein becomes less soluble it's coming
out let's call the salting out third
approach change in the dielectric
constant that means I can use an organic
solvent I add that organic solvent so
the dielectric constant of the solution
changes so what happened in the protein
precipitates out fourth approach change
in water availability I can add a non
ionic polymer so when I add a non ionic
polymer into the solution protein
precipitate reduces out so what we are
doing in this side the left hand side we
are changing the properties of the
solvent we are changing pH we are
changing and we changing the dielectric
constant we are changing the
availability of the water near the
protein so by doing all this we are
letting the protein precipitate out the
other side that's the right hand side we
can change the properties of the protein
so that it precipitate sources selective
interaction I can add materials or
polyelectrolytes or affinity reagents
so that the proteins get eliminated and
they participated it out let's call the
Selective interaction another approach
is selective denaturalization so I have
some unwanted protein so when I denature
the unwanted protein it precipitates out
how do i denature I can change the pH
when you change the pH as you all know
protein is active only in one selective
region of pH so if when I change the pH
protein may get denatured and then you
may allow the protein to precipitated or
thermal denaturation proteins are not
very stable at high temperatures so then
I increase the temperature then protein
gets denatured and then it may
precipitate or I can now add an organic
solvent by adding an organic solvent you
are allowing the protein to again get
denatured and it gets precipitated on
okay so this type of denaturalization
you saw - if you are not interested in a
particular protein okay we cannot use
this technique if you are interested in
that protein because the protein during
the precipitation is completely
denatured so if you want an active
protein then generally what we do we use
the solvent approach that means change
the property of the solvent or we can
even think about adding meat materials
or polyelectrolytes or affinity reagents
so that we can have a selective
interaction with the desired protein but
we never resort to this type of
linearization that means increasing
temperature or changing pH or adding a
toxic organic solvent which will
denature the protein so each of the
technique we resort to depending upon
the cost factors depending upon the
amount of protein your interest the
stability of the protein your interest
rate to this remove and what are the
other proteins present in the broth or
other metabolites present in the broth
and so on so there is nothing like an
Universal method but generally in
laboratory conditions what do we do we
use a salting out method it's very
simple we just add ammonium sulfate and
which is highly soluble
so the protein gets salting out okay one
of the advantages of protein
precipitation by doing this we are able
to reduce the volume of broth by a
factor of 10 to 50 times actually so
instead of processing the whole broth by
precipitating it out and then removing
the supernatant then we can process rest
of the impure protein and go about
purifying the protein and so on actually
so when you do protein purification we
are concentrating the desired product as
I originally said the protein after
precipitation is not going to be a
hundred percent pure you may have to go
further into purification stages to
really get it very pure but you are able
to get a concentrated product and now we
can do different downstream approaches
to purify it further and further okay
it's a less expensive technique very
very expensive very very less expensive
and it's a very robust and industrial
scale because ultimately we need to
consider scaling up we want to do it in
hundred liter thousand liter and so on
so when we do that sort of large scale
operations precipitation is a robust
technique which can be scaled up to any
level we can achieve rapid appraised
separation quickly precipitation happens
and then you go to filtration and then
you can stabilize the Lebel products
later actually so these are the
advantage of protein precipitation okay
for example if you take a immunoglobin
g-g-gee and albumin of about 99% purity
are obtained from human blood plasma in
in the steel scale by precipitating it
in five steps that means we do and
precipitation and then again you
precipitate like that you do it five
times and then you are able to get
almost 99% pure proteins from your human
blood plasma and this is done in a very
large scale industrial scale without any
problem if you want to do and any other
separation means it may be very very
expensive
so precipitation it's used for recovery
of protein the methods which we can
resort to for example salt induced
precipitation or addition of an organic
solvent in salt induced precipitation
one interesting thing is just like
salting or you can also have salting in
at very low concentration of the salt
solubility of the protein usually
increases slightly you know when you add
salt because of the changes in the ionic
strength sometimes the protein may
solubilize more but that is only in the
initial stages so you could have a
salting in but at high concentration
solubility of the protein drops
dramatically and that protein
precipitates out that's called a salting
out so you can have at low
concentrations of the salt little bit of
salting in and at high concentrations of
salt you may have salting or addition of
an organic solvent so if the medium
dielectric constant decreases with the
addition of an organic solvent that
means when I add a solvent and the
dielectric constant of the medium
decreases then the solubility of the
protein in that solvent also will
decrease so the protein will precipitate
out so you need to select solvents which
will decrease the dielectric constant of
the medium so you need to have some idea
about the dielectric constant of various
solvents that can be added of cause the
most important point you should you
don't want the solvent which you are
adding to denature the protein so you
need to keep that point also in mind
third approach changing the pH we all
know what's the isoelectric point okay
that's the pH at which the charge on the
protein is 0 that means protein will
have no net charge and there PA values
and electrostatic repulsion between the
protein are minimum at these conditions
ok so the solubility of the protein also
is minimum at its P I and this P I
values differ with different proteins
for different types of proteins you have
different types of PA values the next
approach we can do is addition of a non
ionic polymer or metal ions we will talk
about this also in detail so we can add
a polymer which is not ionic or we can
add a metal ion which will also change
the properties of the protein and will
allow the protein to precipitate out if
you look at the peptide bond once again
the NC double bond or the peptide bond
and because of the resonance you're
going to have a n plus and OH minus so
this is in a resonant stage so the
nitrogen that's the amino side and the
carboxyl here know they are going to
give you the charge at high pH that
means in alkaline conditions the
carboxyl groups are ionized you have a
negative charge
okay that's at high pH alkaline
condition and the aminos will have lost
their protons there will not be any
charge so at high pH generally will find
o minus at low pH with plenty of protons
low pH is acidic condition so you are
going to have plenty of protons so what
will happen the o- will disappear okay
the carboxyl will not get ionized but
the aminos are protonated so you'll have
n plus so there will be a net positive
charge on the protein so in acidic
condition you'll have a net positive
charge because the o- would have become
neutral and only the nitrogen will have
a positive charge at high ph in the
alkaline condition what will happen the
positive charge on the nitrogen would
have disappeared because you are in
alkaline condition but the o- will
remain so you will have a net negative
charge so pH will alter the charge on
the surface of the protein
so more negative ions are more effective
than less negative ions for salting out
okay so it's better to have more
negative ions then less negative ends
for salting out and similarly it is
better to have more positive ions than
less positive ions for salting out okay
we look at a problem on that so it's
better to have more negative ions than
less negative ions better to have more
positive ions than less positive ions
for salting out now solubility decreases
exponentially with the increasing
magnitude of charge so if the charge is
more and more then the solubility will
decrease in an exponential fashion what
are the other factors which affects the
solubility type of reactors I'm using
okay am I using your cylindrical reactor
or I am using a tubular reactor and
batch mode continuously mode rate of
mixing how fast the agitation is or how
slow is the agitation is what is the
length to the diameter of the reactor
and so on mode of addition of salt how
am i adding am i adding in different
Lots or am i adding in one Lord I'm
injecting in different places
temperature as you know solubility is a
function of temperature so what
temperature am i operating
am i operating at lower temperature or
am i operating at higher temperature so
all these factors affect the solubility
that means when I'm designing a
crystallizer I need to consider all
these aspects as well so when I am
designing a solid liquid separating
precipitation I need to consider all
these factors this is data taken from a
paper called advances in colloid and
interface science 2006 look at this the
various salts one can think about for
salting out proteins and various Sol's
one can think about for salting in
and you're also given the temperature
and pH conditions for example lysozyme i
can use sodium chloride 0 to 40 degrees
pH of 310 or I can use the magnesium
chloride or I can use sodium acetate
similarly if you are I'm interested in
sorting out ribonuclease a I can use
some other salt and here you see I can
use polyethylene glycol of various
molecular weight PG 400 PG 20,000 and so
on for respite ating out or salting out
lysis I am big collector global Lin and
so on actually so the temperature
conditions also slightly varying pH
conditions are also slightly varying if
I'm interested in salting in that means
allowing more protein to solubilize that
also can be achieved I can use glycerol
and I can salt in lysozyme here and I
can use sodium chloride to salt in beta
lactone globin these are the conditions
25 degrees 4.6 and so on so this
particular table tells you what are the
various salts that can be used for
salting out or salting it ok for example
if you look at various proteins maximum
protein solubility is at a salt
concentration similar to that of the
cytoplasm that is about 0.15 to 0.25
molar for aquatic cells and point 3 to
point 6 molar for bacterial RNA
polymerase is more soluble at a point 3
molar KCl then at 0.25 0.35 so you need
a very optimum concentration of KCl to
enhance the solubility of this t7 RNA
polymerase
protein which are insoluble at very low
salt concentration may require point 2
to point 3 molar to be soluble but this
is more like a salting in type of
approach so you you see this particular
slide gives you a ballpark figure of the
concentration of the salt required to
achieve a the salting out or salting in
okay let's look at a very simple problem
so I have two proteins lysozyme
myoglobin both have different
isoelectric pH you all know what is I
selected pH that's the pH at which the
charge net charge is zero so lysozyme
the I selected pH is 11
myoglobin the isoelectric pH is 7 how to
precipitate myoglobin selectively using
ammonium chloride or ammonium sulfate so
which one is better
ammonium chloride or a Menem sulfate
ammonium sulfate is better because it
has got greater charge than chloride we
just saw a couple of slides back it's
better to have higher negative charge
then lower negative charge is better to
have higher positive charge than lower a
positive charge
okay so ammonium sulfate is better than
ammonium chloride ok what's the pH
condition which is which pH do you would
like to operate at neutral pH because it
is closer to the pi/4 myoglobin this is
the protein of our interest and not this
because the problem says how to
precipitate myoglobin selectively ok so
if I have a mixture of lysozyme and
myoglobin I would use ammonium sulfate
and I will work around pH of 7
understand so we need to look at the
charge on the salt and select
we need to look at the PA value for the
protein and select the operating pH of
course in sometimes the the salt cost
factors also may come into picture but
that's later but if you just consider
only the physical principles then this
is how you approach this problem why do
we generally use ammonium sulfate in our
labs we are told take ammonium sulfate
because of its high solubility it's
highly soluble in water about 3.6 molar
it's got a very high ionic strength
which is proportional to the square of
the charge on the ion right so ionic
strength of 1 molar ammonium sulfate is
three times that of one molar of sodium
chloride so it's always better to use
ammonium sulfate and then um sodium
chloride it's not 2 times but it is
three times because we also talked that
the it's not directly proportional to
the charge but it will be much more
exponentially so in most of the cases
that's why we resort to ammonium sulfate
as the reagent for salting out proteins
so if I want to increase the protein
solubility with increasing salt
concentration generally it happens at
very low salt concentration you know 1 0
to 0.5 molar very low concentration so
for salting out so what's the principle
there is something called common ion
effect you must have studied that long
time back so in common ion effects we
have lowering the solubility interaction
between hydrophobic regions of the
protein salt removes water from the
hydrated protein so it is exposing the
hydrophobic regions so the hydrophobic
regions interact and then the aggregate
hence the solubility goes down and hence
they come out as solubility so ions
referred to as layered tropic series
that is decreasing effectiveness in
causing the precipitation of proteins
cause I take anions citrate greater than
phosphate gaiter than tartrate greater
than sulfate greater than a fluoride I'd
date and so on okay if you take the
cations then thorium aluminum edge plus
barium two-plus right down to calcium
and magnesium so they come under the
electric series that is decreasing
effectiveness in causing the
precipitation of the protein so if you
look at the salting out solubility of a
protein as a function of ionic strength
so you may have a graph like this that
is a solubility with increasing ionic
strength
this could be for fibrinogen this may be
for hemoglobin
this could be for serum albumin this
could be for myoglobin so decreasing
molecular weight okay so if the
molecular weight is very very large then
it's much easier to precipitate out and
compared to very very simple molecular
weight so molecular weight also takes
place in a precipitation process
so basically salting out of protein is a
balance between salting in process due
to electrostatic effects and assaulting
out process d2 hydrophobic interaction
so both play a very important role the
electrostatic forces hydrophobic
interactions and at very high
concentration of the salt protein just
precipitates out so you may have a salt
concentration is increased electrostatic
forces increasing as salt concentration
C's you may have hydrophobic effect so
both may be combining together to give
you a salting out so initially
solubility will increase and then
solubility will decrease that is why at
very very low concentration 0.3 molar or
around that any region the addition of
salt will allow the protein to
solubilize
so that is why you will hide generally
have a graph like this you know it may
be very very close to your to the
left-hand side but still you will have
little bit of salting in taking place
depending upon the type of salt which we
are using so if you take a another
method that is precipitation by the
addition of organic solvents so we can
consider organic solvents like ethanol
acetone and so on so by adding these
solvents we are changing the dielectric
constant by changing the dielectric
constant the protein solubility goes
down as I mentioned very early lower the
dielectric constant lower will be the
solubility hence the protein insulin
disability out so what are you doing
when you are adding this type of organic
solvents reduction in water activity and
solvating power of water for a charged
hydrophobic protein molecule so it is
described this way log s solubility
equal to K by d square plus log s nod D
is the dielectric constant of reagent
and water K is some constant related to
the dielectric constant of original
acquiesce s naught is the extrapolated
solubility so if I keep increasing D I
will be decreasing the solubility so
involvement of electrostatic forces
dipolar interactions in protein
aggregation is supported by the fact
that precipitation occurs at lower
organic solvent concentration that is at
the isoelectric points of the protein so
the mechanism of aggregation is similar
to that tau in ice electric
precipitation so hydrophobic
interactions play a minor role in
protein precipitation because of their
solubilizing effect here now the pair at
equilibrium the chemical potential of
the solid precipitate and the solute
which is in the solution have to be
equal right mu
to precipitate equal to MU in solution
now mu in solution can be given like
that mu naught plus RT lon C R is your
gas constant T is your temperature mu
naught is a standard chemical potential
and C is your concentration at the
concentration is very very low we can
call it this equation as RT C freedom by
Randolph's equation okay so the chemical
potential of this solute in the solid
phase is equal to the chemical potential
of the solute in the dissolved phase so
this is at equilibrium so if you have
solute concentration and mu that's the
chemical potential when organic solvent
is added in small quantity to an aqueous
solution chemical potential of the
solute in the mixed medium changes okay
and varies with the solute concentration
okay so you have the aqueous medium and
then you have the mixed well vent as the
solute concentration increases but the
chemical potential of the precipitate is
always constant because it is a solid
material okay so if you look at the
chemical potential diagram this is how
it will look like so for the aqueous
medium this is the chemical potential
graph as a function of solute
concentration when you are adding a
solvent the chemical potential in the
mixed medium of the solute in the mixed
medium changes and that also will have
this type of shape okay whereas for the
solid precipitate chemical potential
will remain constant that is why it is
parallel to the x axis so in when you
are using AA organic solvent to
precipitate and the mole reciprocation
depends on the molecular size of the
protein so if the protein is larger then
I will require lower concentration of
solvent to precipitate out so if the
precipitate if the protein is smaller
then I will require more quantity of the
solvent so its
ideal for very large proteins no you
just have to add little solvent and the
protein will precipitate out so what are
the advantages quite a lot the variation
in dielectric constant pH temperature
ionic strength and putting concentration
provides a refined method for protein
flaxen ation so that's main advantage of
this type of technique actually another
method by which I can achieve protein
precipitation is adding ionic poly
electrolyte so it's almost like
flocculating agent what does a
flocculating agent do suppose you have
suspended solids they are not settling
down because they are very light they
are not agglomerating because there are
net charge which are repelling each
other so we add something called a poly
electrolyte which will disturb the
charges or neutralize the charges so
what will happen those floating flop
flocculants flocculating they will come
together and they form flocks and when
they form flocks the weight will be
higher then they may settle down okay
that's the process called a flocculation
so same principle is resorted to in in
this particular approach where you are
adding an equality lights so
electrostatic forces are responsible for
protein precipitation so when you add an
equality lights
you are disturbing these and allowing
the proteins to come to each other and
then settle down so we will require very
low concentration of polyelectrolyte
very small point zero five to 10.1
weight percent and low price sometimes
we can use very cheap polyelectrolytes
to achieve this type of separation there
are no waste disposal problem also no
because you add the polyelectrolyte
protein precipitates then you don't need
to
some other some other chemical because
the quantity you're adding is very less
for example we can add alginate or
pecked eight or carboxymethyl cellulose
carrageenan all these are very good
polyelectrolytes and they are called
acidic polysaccharides they can help in
the precipitation we can also add
anionic polyelectrolytes such as poly
acrylic acid
or poly meta acrylic acid we can even
add cationic polyelectrolytes such as
poly ethylene amine polystyrene based a
quaternary ammonium salt so all these
are cationic material and poly acrylic
acid or poly methacrylic acid or anionic
material or we can add a acidic neutral
material like alginate or carboxymethyl
cellulose and so on and all these are
electrolytes which will help in the
flocculation of the protein and allow it
to settle down and the quantity required
here may be very very less when compared
to salting in where you may have to add
large amount of ammonium sulfate you can
also do this precipitation by using
metal ions for example you can use
polyvalent metal ions which will help in
the aggregation and settling down of the
protein molecules for example if I use
divalent manganese iron cobalt nickel
copper zinc cadmium they will bind
strongly to the carboxylic acid you have
a OH - right and sometimes they will
also add bind to the nitrogen groups and
amine and heterocyclic groups divalent
calcium barium magnesium and lead bind
to carboxylic acid but not significantly
to the nitrogenous ligand
mono valent silver mercury and lead
binds to sulfide rail compound or groups
okay so we can consider various
violent that means divalent of
monovalent metals which will selectively
go and bind to the o - or the n plus
regions and then it will resort to the
precipitation of the proteins what are
the advantages they have greater power
in precipitating even dilute protein
solutions can be processed using this
metal ion precipitation so can be easily
removed by chelating agents or cation
exchange resin okay so once the
materials go and bind it to these places
then I can use your ion exchange column
and the protein gets removed calcium
barium zinc salts used to modulate
ethanol precipitation of human plasma
protein this is a good example of where
metals are used for removing a human
plasma protein selectively denaturation
of unwanted protein so if you are not
interested in a certain protein you want
it to be removed it's not getting
removed because of the ionic charges and
electrostatic charges what do you do you
denature the protein so once it is
denatured it will just precipitate out
so this is good if you are not
interested in the product whereas
whatever I talked before you are
interested in the particular protein so
for example I have some proteins which
are stable at extreme pH and temperature
there are impurity proteins which are
very sensitive to pH and temperature
then I can remove those impurity protein
by changing the temperature of pH there
whereas the protein of my interest which
is very stable
will remain unaltered so that is called
selective denaturation
so denaturation of protein is due to
destruction of the tertiary quaternary
structure resulting in the formation of
random coiled a polypeptide chain so it
is no more a protein it is a polypeptide
chain so what happens these chains
aggregate once they aggregate they are
less soluble so they easily come out or
easily precipitate out of this solution
so how do I denature I can use higher
temperature so as I increase temperature
the activity of the protein will go down
it will form it will follow an arena is
relation so the once the activity goes
down it becomes like a polypeptide and
it will aggregate it will precipitate
out I can use extreme pH as you all know
proteins have stable only at a range of
pH when I change the pH beyond this
protein will get denatured addition of
organic solvents at high temperature
range at 25 to 30 so I can add solvents
which will denature my protein so there
are certain solvents which may remove
the water of water surrounding these
proteins it means it may disturb the
coil structure the tertiary or the
quaternary structure thereby the protein
gets denature so all these techniques
can be resorted to but you have to be
very careful in using these techniques
but because they may affect the protein
of your interest also for example let us
look at a simple problem
they brought 18 litres it contains a
desired protein at twenty twelve point
eight grams per liter as well as another
protein this protein I am NOT interested
but it is present as one point eight
grams per liter now calculate the
ammonium sulfate concentration required
to require recover 98% of the desired
protein okay that is listen this protein
if the precipitation constants beta
and k of the desired protein at 0.93 3
and 1.1 respectively and that of the
contaminated protein or 0.88 and point
nine five respectively what will be the
purity of the desired protein at 98%
recovery
okay now total amount of the desired
protein in the broth 80 liters into
twelve point eight correct okay 1024 I'm
interested in recurring ninety-eight
percent that means 98 by 100 into 1024
that gives you one thousand three point
five so what is the amount of protein
remaining in the solution 1024 - one
thousand three point five that gives you
20 point five grams so this much amount
of protein is left behind correct
now ionic strength of ammonium sulfate
solution is the molar concentration now
the corn equation that is an equation
called Cohen equation which relates the
solubility and the ionic strength in
this fashion log s is equal to beta
minus K into I beta is a constant K is a
constant s is the solubility and I is
the ionic strength now I put twenty
point five that is the amount of protein
that's left behind in the solution I am
recovering 98% so two percent is left
behind okay I is the ionic strength of
ammonium sulfate which I'm adding that's
about three molar concentration
okay so ammonium sulfate three the K is
1.1 and the AMA concentration of
ammonium sulfate so I can calculate so
ammonium sulfate concentration required
to precipitate 98% of the desired
protein will be two point four three
molar
no total amount of contaminated protein
in the broth it is how much one point
eight grams per liter and I'm starting
with 80 so 18 to 1.8 is 144 gram at two
point four three molar ammonium sulfate
concentration is 32 point three six
grams and the contaminated protein also
will precipitate out okay so the product
desired protein when it precipitates out
that means desired protein of so much
thousand three point five grams is
precipitating out I will also have about
32 point three six grams of the
contaminant protein also precipitating
out so as I originally said protein
precipitation is not going to give you a
very very pure solid product but it is a
very good method to the sort to at the
early stages of downstream whereas
crystallization can give you a very pure
solid product so this particular problem
tells you something there is a equation
called Cohen's equation which gives you
some idea between the solubility and the
ionic strength this is used for salting
out technique and these are constants
the beta and the K so log s is directly
related to the ionic strength so log s
and ionic strength are linearly related
and if once I know the beta and the K if
I if I know the ionic strength I can
calculate what is the constant amount of
protein is left behind after
precipitation or if I know what is the
amount of protein left behind after
precipitation I can calculate the ionic
strength okay so I can do both this way
or that way so if I have the details for
beta and K for various proteins I can
calculate how much concentration of the
salt needed to precipitate out 98 or 99
percent of the protein that is present
originally
so the total amount of precipitate of
both the proteins so I need to add the
initial protein amount the second
protein amount to get the total amount
and the percentage purity of the desired
protein in the precipitate obviously
it's having certain impurity 32.3 six
grams the desired protein is thousand
three point five grams so that will give
you the percentage purity it's quite a
simple problem but this particular
relationship is necessary and these
values like beta and k are also
necessary for you to calculate the
concentration of this ammonium sulfate
required actually okay so this problem
gives you a nice idea about the
concentration of salt that is required
to precipitate one protein when I say
one protein if there is another protein
you cannot guarantee that the second
protein will not precipitate out because
the second protein also will be here
losing some of its solubility when the
first protein loses its solubility and
when that happens the second protein
also will be partially precipitating out
and hence you will always have a little
bit of impurity in the solid that's
coming out as a precipitate now one can
do air Reese all Yubel is asian and
removal of the unwanted protein and so
on so that is further downstream but
when you do salting out and you cannot
expect a hundred percent pure protein if
you have a mixture of proteins present
in your original product but if you have
only one protein then you will be ending
up in one single solid protein that is
sure so the precipitation as you can see
is a quite a simple method we can use it
in the early stages of downstream we can
change the properties of the solution we
can change the properties of the protein
how do you change the properties of the
solution
we can do
things like changing the isoelectric pH
we can change the dielectric constant we
can change the ionic strength so so many
different approaches by doing this we
are doing the precipitation or we can
change the the the attractive forces
between the protein by adding metal ions
or we can add poly electrolytes so this
disturbs the electrostatic forces and
those type of methods are also very good
in precipitating out your protein the
another method where if you are not
interested in the protein but you want
to remove it as an impurity we can
denature the protein so how do you
denature the protein drastically
changing its pH or drastically changing
the temperature or adding a solvent so
it will denature the protein once the
protein becomes denatured loses its
tertiary structures and it becomes a
polypeptide it will deliberate and then
just come out as a precipitate so all
these methods can be used for protein
precipitation and one important point
you need to keep in mind is during
precipitation you don't want the protein
lose its activity that means you don't
want the protein to lose its
three-dimensional structure the folded
structure which is the most important
parameter which maintains its activity
so that particular point needs to be
kept in mind when one is designing a
protein precipitation technique