CHL793 TERM PAPER

Application of Membrane
Technology
For Protein
Purification

Submitted by
: Vinita Kumari
2011CH70188

Contents:
Page
no.

1.Introduction
3-4
2. Effect of operating parameters on the separation of
4-8
proteins in aqueous solutions by ultrafiltration
3. Membrane fouling in UF and MF during protein
separation
8-10
4. Modelling of constant flux based Protein Ultrafiltration
10-13
Notations:
14
References:

15

Introduction
Membranes are used for separation and purification purpose in
a wide range of industries including pharmaceutical ,food and
dairy industry ,other chemical industries for waste stream
treatment, water purification, defatting of skimmed milk and
whey streams, bioseparation of fermentation products,
deacidification of fruit juices etc. Membrane separation is
based on the principle of selective diffusion driven by
concentration (dialysis,osmosis) pressure
(microfiltration ,nanofiltration, reverse osmosis, gas seperation,
pervaporation) ,electric potential(electrodialysis) etc. There
are a variety of membranes based on the
material(ceramic,polymers,metals) from which they are made.
Organic membranes are made of polyethylene, polysulfone,
polyether sulfone, cellulose acetate etc and inorganic
membranes made of aluminium oxide, zirconium oxide etc.
There are liquid membranes also where thin layer of liquid film
serves as a membrane. Membrane separation processes do not
require additives and can be performed isothermally without
much energy consumption and capital investment as compared
to other thermal separation processes. The choice of
membrane depends on the objective of the application for
which it has to be used.
Most protein based products need purification before their use.
The requirements of protein purification are:

Concentration enrichment
Removal of specific impurities
Protein stability enhancement
Prevention of protein denaturation

Purification of protein is challenging because complexity of their

molecular structure, present in a very little amount in a
solution(dilluted) before separation and also inherently unstable
and subject to denaturation by heating, solvents and even
shearing. Therefore conventional separation techniques such as

distillation, solvent extraction absorption etc are of less use,

mostly chromatography and membrane separation processes are
used. Chromatography has some limitations-difficult to scale up,
expensive to operate, batch operation and detail knowledge of
solution system.
Although all membrane separation processes are used for protein
separation or purification, the most widely used are pressure
driven membrane separation processes ultrafiltration(UF),
microfiltration(MF)and nanofiltration(NF). MF membranes are
especially applicable for the separation of fine particles in the size
range of 0.110.0 m. While UF membranes having pore size in
the range of1100 nm are designed to provide high retention of
proteins and other macromolecules . The applications of UF are
limited to systems where the solutes to be separated have more
than 10-fold difference in molecular weight (MW) in comparison
with protein molecules. Molecular size becomes the sole criteria
for separation purposes in such cases. However, it is possible to
separate solutes having comparable molecular weight by
adequate manipulation of the parameters such as pH, ionic
strength, and transmembrane pressure (TMP).Ionic strength
should be kept low so that the thickness of diffused double layer
of charged solute is significant leading to high retentate while
uncharged solute permeating the membrane. Operating pH
should be near to isoelectric point(pI) of transmitted protein and
far from isoelectric point membrane of retained protein.
Buffer Condition

Recycle

Feed
TMP

Membrane unit

Permeate

NF (Nanofiltration) is useful for separation of peptides due to the

suitable cut-off of the NF membranes and due to the
electrochemical effects, which
plays an for
important
role in the case
Schematic
an ultrafiltration
of charged molecules. Negatively charged membranes have been
process
applied
to get cationic peptides (having antibacterial properties)
from cheese whey. A study on the desalting of peptide fractions
from whey protein hydrolysate using NF membranes has proved
the occurrence of specific rejection phenomena involving
negatively charged peptides by NF membranes. Variation in pH
and the ionic strength of the hydrolysate phase has proved the
charge effects on performance of peptides separation
Factors affecting the performance of an protein
separation/purifiacation are:

Ionic Strength of solution

pH of solution
transmembrane pressure
Stirring speed(if mixing required)
Membrane charge type(positive or negative)
Membrane material
Initial Protein Concentration

Last three factors cannot be modified and are varied prior to

conducting experiment.

Effect of operating parameters on the separation of

proteins in aqueous solutions by ultrafiltration:(BSA (Bovine
Serum Albumin))

Effects of TMP, initial concentration, and pH on the rejection of

protein:
R =1 Cp/Cf
Cp: protein concentration in permeate
Cf: protein concentration in feed
It has been found that rejection of protein increases with pH and
increasing TMP and initial concentration. At low TMP a maximum
rejection of protein is found at low pH, particular at pH lower than
pI(4.9 for BSA) value of the protein . This is because the positively
charged protein would adsorb onto the negatively charged
membrane at pH < pI. However, the influence of pH is negligible
at high TMP and high initial concentration

Rejectio
n

Conc

pressure

Rejectio
nm

pressur
e

pH

Effect of pH and TMP on the flux of protein solution:

It has been observed that flux increases sharply with TMP and
thenlevels off at lower initial protein concentration. Effect of
solution pH is more pronounced at low protein concentration. At
low pH(6 for BSA) positively charged molecules would absorb
easily on the negatively charged membrane and block the
membrane pores. However the repulsive force between
negatively charged protein and membrane reduce the fouling at
highr pH(>7).

Effect of pH and P on UF
flux

Effect of pH and TMP on the separation factor

=1-RBSA/1-RHb

RBSA=rejection of BSA molecules

RHb= rejection of Hb molecules
Higher separation factor is obtained at lower TMP and pH near to
the pI (isoelectric point) of Hb because of the self aggregation of
the neutral Hb molecules(pH7.1) and they are not adsorbed on
the surface of membrane by electrostatic attraction resulting in
reduced fouling.At this pH BSA molecules can easily pass through
the pores of membrane.At pH 6 or below serious fouling is
observed becaue of the preferential adsorption of positively
charged Hb molecules on surface of negaitively charged
membrane.At pH greater than 7.5 separation factor is less than 1
which indicates that amount of Hb passing through the membrane
is greater than the amount of BSA molecules( RHb> RBSA).It
occurs due to greater repulsion between BSA molecules and
membrane as compared tothat between Hb molecules and
membrane.

C0BSA=500pp
m

C0BSA=100pp
m

Membrane fouling in UF and MF during protein separation:

Membrane fouling refers to the irreversible alteration in

membrane properties, caused by specific interactions between
feed stream components and membrane.
Fouling of membrane during practical application for protein
separation results from its adsorption on membrane surface which
increases hydraulic resistance to flow, reduced filtration flux rate
an adverse effect on efficiency and economics of protein recovery
processes. Proteins are difficult foulants to deal with because they
readily adsorb onto membrane surfaces and pore walls. This leads
to the formation of a secondary barrier that decreases permeate
flux and solute selectivity is altered. Therefore, to minimize
fouling by making the membrane surface hydrophilic is a
challenge for achieving better membrane performance
Fouling can occur in following ways:
The formation of a gel layer as a result of concentration
polarization
Adsorption of specific species on the membrane surface and
inside the pore structure
Deposition and pore blockage after the formation of protein
aggregates caused by protein denaturation.
The techniques which are used to characterize membrane fouling
include measurements of the flux decline at constant pressure,
and the pressure increase during constant flow rate permeation.

Reducing Fouling:
Introduction of microsieves having well structured
morphology and controlled porosity,in place of conventional
MF membranes. It results in good separation behavior and
enhanced flow rate. It allows low pressure driven operation
and thus reduced operational cost. It helps in reducing
fouling because of the smooth surfaces and hindering the

trapping of proteins inside the pore network which normally

occurs in polymeric membranes.
Protein fouling during UF is mainly due to the formation of a
secondary (gel) layer on the upper surface of the membrane
which provides an additional resistance to both, solute and
solvent transport across the membrane. Hence surface
modification by increase in membrane surface hydrophilicity
can effectively minimize protein adsorption and prevent
membrane fouling. These methods include coating, surface
graft polymerization and chemical modification to reduce
the UF membrane fouling during protein separation .

Schematic diagram for mechanism of membrane fouling during protein

seaparation

Modelling of constant flux based Protein Ultrafiltration:

A simple mathematical model for increase in transmembrane
pressure during constant flux ultrafiltration considering the

contribution of concentration polarization, rapid initial fouling and

long term fouling has been discussed below.
In constant pressure ultrafiltration flux decreases continuously
and hence it may be concluded that osmotic pressure and rate of
fouling would decrease. The model assumes that a constant
pressure ultrafiltration process is made of large number of very
small constant flux steps. It has been assumed that initial flux in
constant pressure ultrafiltration would correspond to pure
water(or buffer) flux of a fresh membrane at the operating
pressure. Therefore the first constant flux step in the model
represent this. In all subsequent step osmotic pressure at that
instant and cumulative resistance increase due to membrane
fouling has been taken into account to calculate the permeate
flux. The protein used for the experiment is HAS(human serum
albumin) and the membrane is polyethersulfone(PES).
Concentration polarization builds up in a matter of minutes while
fouling may take place during the entire duration of an UF
process. In constant pressure UF, the effects of fouling and
concentration polarization are observed in the form of decline in
permeate flux with time.

Buffer filtration
through fresh

TM
P

Buffer filtration through

fouled membrane

Concentration
polarization
and rapid
fouling

Long term linear fouling

(slope=Jv)

Time

Approach for predicting flux decline in constant pressure ultrafiltration.

Equations governing the model:

Increase in TMP with time explained by modified form of osmotic
pressure resistance model:
P = + Jv(R0m + Rm* + t)
Rf changes with time due to deposition and adsorption of
foulant and expressed as:
Rf = Rm+ t (=m/Jv)
The mass balance equation for protein(foulant) for the
concentration polarization layer assuming that diffusion and
convection within this layer takes place perpendicular to
membrane surface:

C
C
2C
+ Jv
=D
t
y
y2

Permeate flux can(Jv) be expressed using the osmotic pressure

model:
Jv=(P-)/Rm
Jv,i =( P i1)/Rmi1 + ((Rm* t)/tR) + ((mi1 *t)/Jv,i1)
t<tR
Jv,i = (P i1)/Rmi1 + ((mi1* t)/Jv,i1)
t>tR
Initial condition:C(y,0)=Cb at t=0
Boundary Conditions:
For y=0
For y=

C(0,t)=Cb ,

JvC(,t)=D

C ( ,t )
y

Detailed scheme for predicting permeate flux decline including working

equations.

Notations:
C: foulant(protein) concentration (kg/m3)
Cb:foulant bulk concentration (kg/m3)
Cw:foulant wall concentration (kg/m3)
D:diffusion coefficient of foulant (m2/s)
Jv:volumetric permeate flux (m/s)
:water flux (m/s)
k::mass transfer coefficient (m/s)
m:slope of the linear portion of TMPtime profile in constant flux
ultrafiltration (kPa/s)
P:transmembrane pressure (kPa)
Rf: fouling resistance (kPa s/m)
Rm :total membrane resistance (kPa s/m)
R0m :membrane hydraulic resistance (kPa s/m)
Rm*:initial rapid fouling constant (kPa s/m)
t:time (s)
t:small time increment (s)
tR:duration of initial rapid fouling phase (s)
y:distance from the edge of concentration polarization layer
toward membrane (m)
:fouling rate constant (kPa/m)
:boundary layer thickness (m)

:osmotic pressure (kPa)

References:
[1]Almcija et al., 2007 M.C. Almcija, R. Ibez, A. Guadix, E.M. Guadix Effect of pH
on the fractionation of whey proteins with a ceramic ultrafiltration
membrane,Journal of Membrane Science, 288 (2007), pp. 2835
[2] R.W. Baker,Membrane technology and applications,Wiley, Chichester (2004)
[3]M.C. Almcija, R. Ibez, A. Guadix, E.M. Guadix,Effect of pH on the fractionation
of whey proteins with a ceramic ultrafiltration membrane,Journal of Membrane
Science, 288 (2007), pp. 2835
[4]M.Y. Teng, S.H. Lin, C.Y. Wu, R.S. Juang Factors affecting selective rejection of
proteins within a binary mixture during cross-flow ultrafiltration,J. Membr. Sci., 281
(2006), pp. 103110
[5]W.R. Bowen, D.T. Hughes,Properties of microfiltration membranes. Part 2.
Adsorption of bovine serum albumin at aluminum oxide membranes,J. Membr. Sci.,
51 (1990), p. 189
[6] R.F. Boyd, A.L. Zydney,Analysis of protein fouling during ultrafiltration using a
two-layer membrane model,Biotechnol. Bioeng., 59 (1998), p. 451
[7]R. Ghosh. Study of membrane fouling by BSA using pulsed injection techniqueJ.
Membr. Sci., 195 (2002), p. 115
[8]E. Matthiasson The role of macromolecular adsorption in fouling of ultrafiltration
membranesJ. Membr. Sci., 16 (1983), p. 23