COMMUNICATIONS TO THE EDITOR

Effect of pH on Solvent Flux during Stirred Ultrafiltration of Proteins

INTRODUCTION

Membrane ultrafiltration (UF) is a simple and effective process of concentration, fractionation, and separation of macromolecular solutions. Although separation is primarily
affected by the macrosolute size, v i s - h i s the membrane pore size distribution at a given
condition of stirring, macrosolute solution behavior as well as the solute-membrane interactions are of considerable importance in UF system performance. It is known that pH of
the solution significantly influences the solution conformations of protein molecules which
are dipolar. For example, the solubility of proteins is, in general, minimum at the isoelectric
point (IEP) and the agglomerating tendency is maximum. In addition, it has been reported
that protein adsorption on synthetic surfaces. and on surfaces of both cation exchange
and neutral polymer membranes3 increases as the IEP is approached. One would therefore
expect the solvent flux in protein ultrafiltration to be a strong function of solution pH.
Vilker, Smith, and Colton4 have observed that the UF flux of albumin solutions through a
total retention membrane in an unstirred batch cell increases at pH levels above the IEP.
We have therefore investigated the UF flux behavior of a number of different protein
solutions in a stirred batch cell with membranes of widely different permeabilities under
varying pH conditions. Fluxes were measured in two ways: 1) time-dependent initial transient flux achieving rapidly a steady state over a period of 0-15 mid-; and 2) steady-state
measurements over a period of 1 h., We report the results in this communication.

MATERIALS AND METHODS

Suspending Media

All the protein solutions of this study were prepared in appropriate buffer solutions made
from stock solutions of 0.1M citric acid and 0.2M disodium phosphate in double-distilled
water according to Table I.9
Solutes

The proteins investigated were bovine serum albumin (BSA, Cohn fraction V from V.P.
Chest Research Institute, New Delhi), ovalbumin (Wilson), hemoglobin (EM), and egg
albumin (Wilson). The last three solutes were supplied by Chempure Ltd., Bombay. Properties of these proteins relevant for this study are given in Table 11.

UF membranes
The membranes used were anisotropic synthetic membranes Diaflo PM30, XMIOOA, and
XM300 of Amicon Corp. (USA) with molecular weight cutoffs of 3 x lo4, 10 x lo4, and
30 x lo4, respectively. While PM30 is based on an aromatic polymer, the other two are
based on a substituted olefin polymer.

Biotechnology and Bioengineering, Vol. XXIII, Pp. 1873-1880 (1981)

0 1981 John Wiley & Sons, Inc.
CCC 0006-3592181/081873-08$01.OO

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BIOTECHNOLOGY AND BIOENGINEERING VOL. XXIII (1981)

TABLE I
McIlvain's Citric Acid-Phosphate Buffer"
PH
X

3.0
79.45

4.6
53.25

4.8
50.70

6.0
36.85

6.8
22.75

8.0
2.75

mL of O.IM citric acid (21 g C6H*07.H20/L)+ (100 0.2M disodium phosphate (35.6 g Na2HPO4.2H2O/L).

X)

mL of

Experiments were conducted in a batch system employing a magnetically stirred and

baffled 250-mL acrylic plastic U F cell over a pH range 3-8. A buffer solution reservoir was
always connected to the U F cell and maintained at the same pressure to replenish any
solvent lost from the cell during ultrafiltration conducted at a constant stirring speed of 800
rpm. Identical vortex-free stirring conditions thus were maintained in each experiment.
The initial time experiments were carried out with 0.5% protein solution for 15 min starting
with flux data at 0.5 min. The steady-state experiments were conducted with 0.25% protein
solutions for 1 h with flux data being presented at 6-min intervals except for the data at 3
min from start. The slope of the plot of the ultrafiltrate volume versus time of filtration was
utilized to calculate U F flux at any instant of time. An effective applied pressure of 1.38
X lo5 Pa (20 psig) was used in all experiments. The details of the experimental system and
procedure are available in a thesis by Swaminathan.' The initial time and steady-state
ultrafiltration procedures are described elsewhere.'.'
RESULTS
The results of initial time ultrafiltration studies of BSA and ovalbumin solutions through
PM30 membranes are shown in Figure I . In this, as well as in Figures 2 , 3, and 4, the
variation of filtrate volume with time has been shown and the steady-state ultrafiltration
rate achieved has been indicated by its value in each case. The steady-state ultrafiltration
of a 0.25% BSA solution through a PM30 membrane is given in Figure 2. Whereas these
results represent the behavior of an almost total rejection membrane for the two solutes
BSA and ovalbumin, we show in Figures 3 and 4 the steady-state ultrafiltration of several
solutes through membranes XMIOOA and XM300 which are quite open. Figure 3 indicates
the filtration rate of BSA and egg albumin solutions through XMIOOA membranes. The
ultrafiltration rates of a hemoglobin solution through an XM300 membrane are shown in
Figure 4. The pH of the solution was varied in each case from 3.0-8.0 with an intermediate
data point corresponding to the IEP of the protein.
It can be observed from these figures that the rate of ultrafiltration decreases with time
and reaches a steady value due to the dynamically forming polarized layer of proteins. It
is further evident that although concentration polarization takes place at all pH values
studied resulting in a substantially reduced steady-state flux. the extent of flux reduction
due to concentration polarization varies significantly with solution pH. One further notes
in each figure that the flux value at the IEP pH is almost always lower than those at other

TABLE I1
Properties of Solutes
Solute

BSA

Molecular weight
lsoelectric pH

69,000
4.8

Hemoglobin Ovalbumin
67,000
6.8

45,000
4.6

Egg albumin
44,000
4.6

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COMMUNICATIONS TO THE EDITOR

50

45

40

35

I,

30

:
3

-l

t
2

25

;
3

>

>

w
I-

a 4
a

20 w
IQ

LL

3
-LL

LL

15

10

I 0

2.0

3-0

4 . 0

T I M E , MINUTEE

5 0

6.0

7 0

Fig. 1. Initial time ultrafiltration of BSA and ovalbumin through PM 30 membrane as

a function of pH. Ce = 0.05%, pressure = 20 psig. pH 3.0: (-)BSA, (A) ovalbumin; pH 8.0:
(o)BSA, ( 7 ) ovalbumin; pH 6.0: (-I)BSA,)(. ovalbumin; pH 4.8:(o)BSA, ( 0 ) ovalbumin.
pH values. This behavior becomes clearer when the steady-state UF flux for both steadystate experiments and initial time experiments are plotted against pH for each case. As
Figures 5(a) and 5(b) show, the solvent flux reaches a minimum at the isoelectric pH in
each case and increases at pH values on both sides of the isoelectric point.
DISCUSSION AND CONCLUSIONS

Although such results have not been previously reported in literature, related behavior
has been observed in several investigations. In a study on UF of starch factory effluents

BIOTECHNOLOGY AND BIOENGINEERING VOL. XXlIl(1981)

1876

180

160

140

120

z
_I

100

0
>

w
I-

-'i8C
LL

6C

4C

2c

__

12

-18
T I M E , MINUTES

I
30

I
36

I
42

Fig. 2. Steady-state ultrafiltration results for BSA through PM 30 membrane as afunction

of pH. CB = 0.25%. pressure = 20 psig. pH: (T3.0: (43.0; (0)4.8.
through noncellulosic membranes, Fane and Fell" had observed a minimum in flux at pH
4-5 with a maximum at pH 3. They had also pointed out that the minimum in flux that
occurred at the IEP pH was probably due to agglomeration and lowered diffusivities. Friedli
et aI.l3 have reported a sharp reduction in the rate of filtration of dilute albumin solution
as the IEP was reached. ForbesI4 had also observed similar events with proteinaceous
solutions. Chemical treatment of cheese whey to reduce fouling of U F membranes was
studied by Lee and Mersonls and Hayes, Dunkerley, and Muller," wherein acidification
of whey was found to increase the ultrafiltration rates. This was presumably due to pH-

I877

COMMUNICATIONS TO THE EDITOR

360

180

3 20

160

280

140

24c

w2 200

100

3
J

0
>

0
>

180

80 w
4

IX

IJ

I20

60

80

40

40

20

0
0

I2

I8

LL

24

TIME, MINUTES

30

36

42

48

Fig. 3. Steady-state ultrafiltration results for BSA and egg albumin through XMIOOA
membrane as a function of pH. CB = 0.25%. pressure = 20 psig. pH BSA: ( ~ ) 8 . 0(a)3.0:
;
;
(*)4.6 (isoelectric pH).
(0)4.8 (isoelectric pH). pH egg albumin: ( ~ ) 3 . 0(~)8.0;

induced changes in the state of P-lactoglobulin which constitutes about 50% of the whey
protein. The marked effect of pH on the association-dissociation behavior of P-lactoglobulin
is well k n o ~ n . Lee
~ ~and
~ Merson had also shown, from electron micrograph studies of
the fouling deposits on the membrane, that the form of the deposits is strongly dependent
upon pH. Addition of NaOH destabilized the protein, causing thick deposits of a granular
matrix to form a nonporous fouling layer. On the other hand, acidification stabilized the
proteins and only light deposits were found. Thus it would appear that ultrafiltration rates
will be high if the proteins are maintained in a dispersed state by controlling the pH. This
will not only reduce the amount of deposit on the membrane surface due to polarization but
it will also reduce the protein adsorption on membrane s ~ r f a c e sIn
. ~ addition, the deposits
will be much more porous.
The concentration profile measurements of Vilker, Smith, and Colton4 with an unstirred
batch UF cell provide direct evidence in this connection. For BSA ultrafiltration in 0.15%
saline through a total retention membrane, the membrane surface concentration reached the
highest value of 40.2 g % at a pH of 4.5 compared to 31.5 g % at pH 5.4 and 24.9% at pH

1878

BIOTECHNOLOGY AND BIOENGINEERING VOL. XXIII (1981)

---

- _

240

I
12

16

30

24

T I M E , MINUTES

36

42

Fig. 4. Steady-state ultrafiltration results for hemoglobin through XM300 membrane as

a function of pH. CB = 0.25%, pressure = 20 psig. pH: (0)3.0; ( ~ ) 8 . 0(0)6.7.
:

7.4. Although the osmotic pressure at a given concentratlon is lowered as pH approaches

the IEP, a higher wall concentration will ensure a greater reduction in flux. Further, the
diffusion coefficients are lowered significantly when the IEP is approached leading to a
lower flux. In addition, an increase in diffusion potential as the solution goes away from
IEP will increase U F flux significantly as shown recently by Dileo, Smith, and Colton'' for
an unstirred batch ultrafiltration of a BSA solution. Thus we conclude that the results of
these two investigation^^.'^ on unstirred batch U F lend support to our observations in stirred

COMMUNICATIONS TO THE EDITOR

1879

(a)

20

XMIOOA]
16

ISOELECTRIC

0
Y

EGG ALBUMIN
X M IOOA

0
2

PH

( b)

OVALBUMIN

I
8

PH
Fig. 5 .
pressure

Effect of pH on ultrafiltration flux. (a) Steady-state experiments: Cg = 0.2576,

20 psig. (b) Initial time experiments: Cs = 0.0576, pressure = 20 psig.

batch UF of protein solutions. Other conditions remaining constant, the stirred ultrafiltration
flux of a protein solution will be a minimum at the isoelectric pH and will increase as the
pH changes on both sides of the IEP.

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BIOTECHNOLOGY AND BIOENGINEERING VOL. XXlIl(198l)

References

I . A. D. McLaren, J. Phys. Chern., 58, 129 (1954).

2. E . I. F. Pearce and B. G. Bibby, Arch. Oral B i d . , 11, 329 (1966).
3. W. J . Dillman and J. F. Miller, J. Colloid Interface Sci., 44,221 (1973).
4. V. L. Vilker, K. A. Smith, and C. K. Colton, paper No. 102e presented at the 68th
Annual AIChE Meeting at Los Angeles, November 16-20. 1975.
5. T . Swaminathan, Ph.D. thesis. 1.1.T.. Kanpur, India, 1978.
6. T. Swaminathan, M. Chaudhuri, and K. K. Sirkar, paper No. 8b presented at the
86th National Meeting of AlCHE, Houston, Texas, April 2, 1979.
7. T. Swaminathan, M. Chaudhuri, and K. K. Sirkar, J. App!. Polym. Sci., 24, 1581
(1979).
8. T. Swaminathan, M. Chaudhuri, and K. K. Sirkar, J. Colloid Interface Sci., 76(2),
573 (1980).
9. H . A. Sober, Handbook of Biochemistry (CRC, Cleveland, OH, 1968).
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Melin, and H. L. Taylor, J . Am. Chem. Soc., 68, 459 (1946).
11. J . S. Fruton and S. Simmonds, General Biochemistry (Asia Publishing House, New
Delhi, 1960), p. 102.
12. A. G. Fane and C. J. D. Fell, AlChE Symp. Ser. 73(163), 198 (1977).
13. H . Friedli, E. Fournier, T. Volk, and P. Kistler, Vox Sang., 31, 283 (1976).
14. F. Forbes, Chem. Eng. (London),257, 29 (1972).
15. D. N. Lee and R. L. Merson, J. Food Sci.,41, 778 (1976).
16. J. F. Hayes. J. A. Dunkerley. and L. L. Muller, Aust. J. Dairy Technol., 29, 132
( 1974).
17. D. N. Lee and R. L . Merson, J. Dairy Sci., 58, 1423 (1975).
18. H. A. McKenzie, Milk Protein I1 (Academic, New York, 1971).
19. A. J. Dileo, K. A. Smith, and C. K. Colton, paper No. 126a presented at the 71st
Annual AIChE Meeting at Miami, November 16, 1978.
T. SWAMINATHAN*
M. CHAUDHURI
K. K. SIRKAR
Indian Institute of Technology
Kanpur-208016, U.P., India
Accepted for Publication March 12, 1981

* Present address: National Environmental Engineering Research Institute, Nagpur440020, India.

r Correspondence should be sent to: Department of Chemistry and Chemical Engineering,
Stevens Institute of Technology, Hoboken, NJ 07030.