ABHISEKH UMRAO SINGH

MSc BIOTECHNOLOGY

7810595

FUNDAMENTALS OF BIOCHEMICAL ENGINEERING

 ƞƞ  
= ρ2RT +    4 () →1
 (ƞ)

This represents the equation of state which is mc millan mayer free energy.

Where,

T = temperature

V= volume of the solution

ρ 2= protein molar density

R= ideal gas constant

Nav= avogadro’s number

d2= protein diameter

η= fraction of the solution taken up the protein molecules

r= centre-to-centre separations between a pair of molecules.

1) Derive an analytical expression for the osmotic pressure and the protein
chemical potential.

Chemical potential is given by,

µ=µid+µex

We have the equation,

 ABHISEKH UMRAO SINGH

MSc BIOTECHNOLOGY

7810595
 
    "

 !
= 
"
×
!


  '
(
!   '
(
! "

= %& + +!,- ! 1 './
!
0(/)1/ + ρ2 %& × →2
 ! ()
)! ! 
()
)!  !

2 ƞƞ
Now solve the partial differentiation part
2ƞ (ƞ)

3 4ƞ − 3ƞ
3ƞ (1 − 7)
8 ú:;:́
We know that = ,
 

Using the above formula we get,

2 ƞƞ (ƞ) (=ƞ)>ƞƞ ?(ƞ)( )

2ƞ (ƞ)
=
(ƞ)@

Now simplifying this equation we get,

2 ƞƞ =ƞ
=
2ƞ (ƞ) (ƞ)A

Substituting this in eqn 2,

=ƞ 2ƞ
ρ2RT (ƞ)A ×2ƍ →3

2ƞ
Now solve2C ,

D A E F
GH
We know ƞ=
=

2ƞ D A FGH
= →4
2C =

Now substitute eqn 4 in eqn 3 we get,

=ƞ D A FGH
ρ2RT (ƞ)A × =
→5

Now solve the second term in eqn 2 i.e ,


   4 () →6

I
We know that w(r)= -ɛ( )J →7

 ABHISEKH UMRAO SINGH

MSc BIOTECHNOLOGY

7810595
Now substitute eqn 7 in 6,
 I
= −   4 (-ɛ( )J )dr

Simplifying the above eqn,

K
GHE ɛ@MNA

= →8
J

Substitute eqn 8 and 5 in eqn 2 we get,

O
2  ƞƞ =ƞ D A FGH S KGHE ɛ@MNA
P
= RT +RT A × +
2C (ƞ) (ƞ) = J

D A FGH S
Since we know ƞ= =
so substituting this in the second term of the above eqn,

2 
O K
ƞƞ =ƞ GHE ɛ@MNA
P
= RT +RT A ×ƞ+

→9
2C (ƞ) (ƞ) J

Now simplifying eqn 9 we get,

DKGHE
TƞUƞ VƞA NA
ɛ
ex
µ =RT(
(ƞ)A
)+ J
→10

total chemical potential is given by,

µ=µ i.d+µ e.x → 11

O
2 
2C
P
=µ e.x →12

µ i.d=RT lnρ2 →13

Now substitute 10 & 13 in eqn 11 we get,

DKGHE
TƞUƞ VƞA NA
ɛ
µ= RT lnƍ2+ RT( )+ →14
(ƞ)A J

eqn 14 depicts the protein chemical potential.

i) Osmotic pressure

The relation between osmotic pressure and macmillan mayer free energy is given by,
 ABHISEKH UMRAO SINGH

MSc BIOTECHNOLOGY

7810595
F=-ПV+µ 2N2 → 15

Considering excess conditions,

Fe.x=-Пe.xV+µ 2N2 →16

Divide eqn 16 by v,
WX.Z [ F
=-Пe.x+ →17
 

K
Since =ρ2 →18


Substitute 18 in 17 we get,
WX.Z

= -Пe.x+µ e.xρ2

WX.Z
Пe.x= µ e.xρ2- →19


Substitute 10 & 1 in 19 we get,

DɛK
ƞ ƞ GH NA
E
П =ρ2RT( (ƞ)A )+
e.x
J
→ 20

But we know that П=Пi.d+Пe.x →21

Пi.d=ρ2RT → 22

After Substituting 22 and 20 in 21 and simplifying we get \,

DɛK A E–
ƞ ƞA VƞV GH N
П=ρ2RT( (J)A
)+ → 23.
J

The equation 23 depicts the osmotic pressure derivation.

2) Determine the spinodial(T vs ρ) and the critical point for a solution with
salt concentration equal to 1.0 M and the range of the potential n equal to
6.
Spinodal curve is used to find the stability of the solution. Basically it’s a limit; within this limit
any small fluctuation (infinitesimal) with respect to temperature, pressure and other parameters
will lead to spinodal decomposition. spinodal decomposition is the decomposition of the solution
When there is a change in the thermodynamic parameters such as temperature into multiple
 ABHISEKH UMRAO SINGH

MSc BIOTECHNOLOGY

7810595
changes. Above this curve the solution is exposed to fluctuations but it is metastable. Now we
have the lysozyme solution on which the salting out process is done and now ready for the phase
separations. We have to calculate all the thermodynamic parameters for the liquids which are
later used in phase separations. Now we have to find the spinodial and the critical temperature
for the lysozyme solution. Now in a phase separation, there are 2 phases one phase which is light
and is the top layer whereas the dense phase forms the bottom layer. So we are splitting it into
two phases ρ2L which represents the light phase (top layer) and ρ2D represents the dense phase
(bottom layer) which are the protein molar densities of the lysozyme solution. In mathcad, we
first derived the osmotic pressure and the chemical potential and we split it into dense and light
components. Since we have two phases which are light and dense there are two chemical
potentials (µ2L and µ2D). Now to check the equation we calculated the chemical potentials of
both light and dense as a function of molar density and temperature.

The figure above depicts the spinodal curve for light molar density lysozyme solution in
the protein phase diagram where chemical potential (as function of molar density and
temperature) is taken versus its molar density.
 ABHISEKH UMRAO SINGH

MSc BIOTECHNOLOGY

7810595

ρ2D

The figure above depicts the spinodal curve for dense molar density lysozyme solution in
the protein phase diagram where chemical potential (as function of molar density and
temperature) is taken versus its molar density.

From both graph the curve looks similar because they both are the same components but with
dense and light phases.any point above the curve will result in a single phase and below that
will result into two phases (spinodal decomposition).then we took the graph of osmotic
pressure versus the molar density (both light and dense) to get the spinodal curve.
 ABHISEKH UMRAO SINGH

MSc BIOTECHNOLOGY

7810595
The above graph depicts the spinodal where the osmotic pressure is plotted versus molar
density.
Now we have to find the critical point which is the extreme point of spinodal curve. In order
to find this we equate the osmotic pressure (both first derivative and second deriavative) to
zero.

d
0= Π( ρ2 , T)
dρ2

2
d
0 Π( ρ2, T)
2
dρ2

Using mathcad we get the following solution,

ρ2L=9.372
T= 328.264

Now plotting the graph temperature versus molar density of the protein we get the spinodal
curve of the lysozyme protein density.

The figure above depicts the spinodal curve and the critical point (the peak of the curve) of the
lysozyme solution where above the curve it will give a stable solution and if it is below it leads to
decomposition into two phases (slow crystallization). Also if its even below the co existence curve
spontaneously decompose.
 ABHISEKH UMRAO SINGH

MSc BIOTECHNOLOGY

7810595

3) calculate the liquid-liquid co-existence curve (T vs ρ) for solutions at salt

concentration equal to 0.5, 1.0 and 1.5M with n equal to 6.

Liquid –liquid co-existence curve is defined as the condition where the 2 distinct liquid phases
may co-exist. In this condition the chemical potential of a particular solution component is equal
in that distinct phase of the solution in which it exists. In order to calculate the Liquid –liquid co-
existence we defined the chemical potential and osmotic pressure of both light and dense
components in mathcad. Then we adjusted the salt concentration(ρ3) to 0.5,1.0 and 1.5. after
adjusting we input values of T(200),ρ2L(0.5) and ρ2D(20). The critical point of ρ2 was found to
be 9.372 so we adjusted ρ2L<9.4 and ρ2D>9.4 so that in lies in the range of critical point.then
we equated the chemical potential and osmotic pressure of both light and dense fluids to each
other.

μ2L( ρ2L, T)= μ2D( ρ2D, T)

ΠL( ρ2L, T) ΠD( ρ2D, T)

From the above equation we found various values ρCO as a function of T with find command in
mathcad to give us various values of temperature and the corresponding ρ2L and ρ2D which was
written in a excel sheet. Plotting the graph give us Liquid –liquid co-existence curve for ρ3=0.5,
1.0 and 1.5 with n=6 as constant.

350
300 ρ3=0.5
250
200
temperature

150 Light fluid

100 Dense fluid

50
0
0 10 20 30
ρ2
 ABHISEKH UMRAO SINGH

MSc BIOTECHNOLOGY

7810595

350
300
temperature

250
200
150 Light fluid
100 Dense fluid
50
0
0 5 10 15 20 25 30
ρ2

400
350 ρ3=1.5
300
250
temperature

200
150 Light fluid
100 dense fluid
50
0
0 10 20 30
ρ2

The above three graphs shows different Liquid –liquid co-existence

existence curve for different ρ3 but
constant n=6.the critical temperature rises as the concentration of the salt is increased. The
solution has less components of protein and more concentration of the salt as the salt
concentration increases and afterter the critical point the curve slides down because of less
temperature at more and more salt concentration. The light fluid may end at critical temp and
dense fluid starts at the critical temp and slide down with changing molar density(ρ2).
density(
 ABHISEKH UMRAO SINGH

MSc BIOTECHNOLOGY

7810595
4) Determine the effect of changing n on the liquid-liquid co-existence
curve for a solution with salt concentration equal to 1.0 M. What are the
implications of this result for the shape of the protein phase diagram?

For this problem, in mathcad we have previously defined chemical potential, osmotic pressure
and also the critical point for both light and dense. Now we change n from values 1 to 9 so
totally 8 cases keeping everything constant and salt concentration to 1.0 M. we feeded the values
into an excel and plotted the graphs.

1000
n=1(light)
temperature

800
600 n=1(dense)
400 n=2(light)
200 n=2(dense)
0
n=4(light)
-200 0 5 10 15 20 25 30
-400 n=4(dense)
-600 n=5(light)
-800 n=5(dense)
-1000
ρ2

350
300 n=6(light)
temperature

250 n=6(dense)
200 n=7(light)
150 n=7(dense)
100 n=8(light)
50 n=8(dense)
0 n=9(light)
0 5 10 15 20 25 30 n=9(dense)
ρ2
 ABHISEKH UMRAO SINGH

MSc BIOTECHNOLOGY

7810595

critical temperature vs n
1500

1000

500

0
0 5 10 15
-500 critical
temperature
-1000

-1500

From the temperature vs. ρ22 graph

graph,, it implies that as the n which is the variable of proteins
molecules that surrounds the protein increases the temperature also decreases. At n=4, the
temperature (becomes positive) decreases as n increases. This is due to more protein molecules
that harbour the protein. at n=3 the whole function becomes infinite due the term of the chemical
potential equation (n-3).liquid
3).liquid liquid equilibrium exists where there is a equilibrium ium for two
disordered phases. One dilute with the protein (which which forms liquid after salting out) and the other
concentrated with a protein (which forms crystals) which is disordered phases. During salting out
process, first we take little amount of the sasalt
lt to be added to the protein solution. As the salt
concentration increases the attractive forces between the proteins also increases and it goes
below the spinodal curve, there is a slow crystallization but as the salt is increased further it
buries into the spinodal giving a liquid
liquid- liquid phase which the solution decomposes into 2
liquids. From the critical temperature vs. n graph as the n increases the critical temperature
decreases. This results in less area which is below the curve where the protein ccrystallizes
rystallizes and
increased are above the curve which represents stable protein. So as more the n increases the
critical temperature decreases and the liquid
liquid-liquid
liquid separation will be ineffective.

5) What happens if the range of w(r) becomes very large (i.e. small n)?
Why does this happen?

We know that w(r) is the attractive forces between the protein molecules where r is the
center-to-center
center separations between a pair of proteins. As the w(r) range increases the
attractive forces between a pair of protein increases which results in precipitation. As
 ABHISEKH UMRAO SINGH

MSc BIOTECHNOLOGY

7810595
previously explained we know that as n reduces the critical temp increases which results
in liquid- liquid separations. Increasing salt solution moves the protein into crystal-liquid
phase where the concentrated protein becomes crystal and the dilute ones becomes liquid.
But at this point we have a large activation barrier in this region which prevents the
protein crystallization and as more salt concentration is increased the protein decomposes
into liquids and the separation is ineffective. And above the co-existence curve the
protein becomes stable. So having small n will make large liquid- liquid phase and higher
critical temperature so that the protein crystallizes in the crystal-liquid phase and
overcomes the activation barrier.

MATHCAD

T := 200.. 350

R := 8.314
23
Nav := 6.203⋅ 10

π := 3.14

ρ3 := 1.0

R
Kb :=
Nav

ε := Kb( 750 + 120ρ3)

−9
d := 3.5⋅ 10
2

−9
d := 3.5⋅ 10
2

n := 6

η :=
( 2)3⋅ Nav
π⋅ d

6 *

 2π ⋅ ε ⋅ ( Nav ) 2 d 3 ( ρ2) 2 

 ( ρ2η) 2 − ( ρ2η) 3 + ( ρ2⋅ η) +
Π( ρ2, T) := ρ2R⋅ T⋅ 
1 
+
 ( 2) 
   
 ( 1 − ρ2⋅ η)
3

( 3 − n) 
 ABHISEKH UMRAO SINGH

MSc BIOTECHNOLOGY

7810595

ηL :=
( 2)3⋅ Nav
π⋅ d

 8( ηL⋅ ρ2L) − 9⋅ ( ηL⋅ ρ2L) 2 + 3 ( ηL⋅ ρ2L) 3   4π Nav ρ2L⋅ ε ⋅ ( d2)  
2 3
µ2L( ρ2L, T) := T⋅ R⋅ ln( ρ2L) + T⋅ R⋅   + 
 3   ( 3 − n) 
 [ 1 − ( ηL⋅ ρ2L) ] 

ηD :=
( 2)3⋅ Nav
π⋅ d

 8( ηD⋅ ρ2D) − 9⋅ ( ηD⋅ ρ2D) 2 + 3 ( ηD⋅ ρ2D) 3   4π Nav ρ2D⋅ ε ⋅ (d 2)  
2 3
  +
µ2D( ρ2D, T) := ( T⋅ R) ⋅ ln( ρ2D) + T⋅ R⋅ 
 
  
3 ( 3 − n)
 [ 1 − ( ηD⋅ ρ2D) ]

µ2L( 1 , 200) = −615.298

µ2L( 2 , 200) = −67.689

ρ2L := 0.1, 0.2.. 60

µ2L( ρ2L, 250) =

-4.843·103
-3.46·103
-2.674·103
-2.133·103
-1.726·103
-1.404·103
-1.14·103
-919.031
-730.557
-567.776
-425.755
-300.852
-190.304
-91.959
-4.112
...
 ABHISEKH UMRAO SINGH

MSc BIOTECHNOLOGY

7810595
µ2D( 1 , 200) = −615.298

µ2D( 2 , 200) = −67.689

ρ 2D := 0.1 , 0.2 .. 60

µ2D( ρ2D, 250) =

-4.843·103
-3.46·103
-2.674·103
-2.133·103
-1.726·103
-1.404·103
-1.14·103
-919.031
-730.557
-567.776
-425.755
-300.852
-190.304
-91.959
-4.112
...

3
Π ( 1 , 200) = 1.356 × 10
3
Π ( 2 , 200) = 2.112 × 10

ρ 2 := 0.1 , 0.2 .. 60
 ABHISEKH UMRAO SINGH

MSc BIOTECHNOLOGY

7810595
Π( ρ2, 250) =
204.984
404.253
597.832
785.744
968.016
1.145·103
1.316·103
1.481·103
1.641·103
1.796·103
1.945·103
2.088·103
2.226·103
2.359·103
2.486·103
...

ρ2 := 1

T := 250

Given

d
0 Π( ρ2, T)
dρ2

2
d
0 Π( ρ2, T)
2
dρ2

 9.372 
Find ( ρ2, T) =  
 328.264

Given

d
0 Π( ρ2, T)
dρ2

T( ρ2) := Find ( T)

ρ2 := 0.1 , 0.2 .. 40
 ABHISEKH UMRAO SINGH

MSc BIOTECHNOLOGY

7810595

ηL :=
( 2)3⋅ Nav
π⋅ d

6 *

 [ ( ρ2L) ( ηL) ] 2 − [ ( ρ2L) ( ηL) ] 3 + [ ( ρ2L) ⋅ ( ηL) ] + 1   2π ⋅ ε ⋅ ( Nav ) ( d 2) ( ρ2L)  
2 3 2
  + 
ΠL( ρ2L, T) := ( ρ2L)R ⋅ T⋅ 
 
  
3 (3 − n)
 [ 1 − [ ( ρ2L) ⋅ ( ηL) ] ]

 8( ηL ⋅ ρ 2L) − 9⋅ ( ηL ⋅ ρ2L) 2 + 3 ( ηL⋅ ρ2L) 3   4π Nav ρ 2L⋅ ε ⋅ ( d 2)  

2 3
µ2L( ρ 2L, T ) := T ⋅ R ⋅ ln( ρ2L) + T ⋅ R ⋅  +
 
 3   (3 − n) 
 [ 1 − ( ηL ⋅ ρ2L) ] 

ηD :=
π⋅ d( 2)3⋅ Nav
6 *

 [ ( ρ2D) ( ηD ) ] 2 − [ ( ρ2D) ( ηD ) ] 3 + [ ( ρ2D) ⋅ ( ηD ) ] + 1   2π ⋅ ε ⋅ ( Nav ) ( d 2) ( ρ2D)  

2 3 2

ΠD( ρ2D, T) := ( ρ2D)R ⋅ T⋅  +

 
 3   ( 3 − n) 
 [ 1 − [ ( ρ2D) ⋅ ( ηD ) ] ] 

 8( ηD ⋅ ρ2D) − 9⋅ ( ηD ⋅ ρ2D) 2 + 3 ( ηD ⋅ ρ2D) 3   4π Nav ρ2D⋅ ε ⋅ ( d 2)  

2 3
µ2D ( ρ2D, T) := ( T⋅ R ) ⋅ ln( ρ2D) + T⋅ R ⋅  +
 
 3   (3 − n) 
 [ 1 − ( ηD ⋅ ρ2D) ] 

T := 200

ρ 2L := 0.5

ρ 2D := 20

Given

µ2L( ρ2L, T) µ2D( ρ2D, T)

ΠL( ρ2L, T) ΠD( ρ2D, T)

ρ2L < 9.2

 ABHISEKH UMRAO SINGH

MSc BIOTECHNOLOGY

7810595

ρ2D > 9.4

ρCO( T) := Find ( ρ2L, ρ2D)

 3.615 
ρCO( 300) =  
 17.135 