DOI: https://doi.org/10.1038/nbt0789-686
BIOSEPARATIONS:
Design and Engineering of
Partitioning Systems
y the year 2000, th!:'.' wodd
bioch�mical market will
re;,.ch an estimated $4O- l 00
billion. 1 • Biop:roce3� engi
needng is a vital (but currently we:;;.k)
link hr:t.ween hb discove:des and the
fi.,lfilh:i::tcnt of thi.s comrne,·cialization
potential. T<) $trengthen the link, re
searchers are ei.p�9ring biochemically
based i;epatations, e!>peda11y for hiV,h
value-added chemicals. Biocherofcal
approacbes-focluding li<�uid-liquid
p;::i. rtiti.o:OJl)g-promise readily scalea
ble, econoroic;al separation of large
l:ii.9molec1.1k�, ai:; well a� an artswer to
industry's need for better st�dstical,
mec;hanical, ::i.nd thermodynamic
model� and measvrements
biose
paratiom.
One bioproi::es� in particular-two
pha8e aque•JU5 partitioning-has
great potential as an economical sepa
ration method for biqchemical prod
ucts. lt offers the poteri tial for strict
produ��t quality (On.trol, as well2 •
With many potential appli;::;ation, it)
the food and ph::1tmaceutical indus
tries foi: such things as enriching Ny
bean aw:l i;c:;,,n endo5perm prot.ein�
<1.nd harvesting IYso;,;vmes for use in
arr,ificial blood, partitioning serves
\.b.ree main roles:
• concentratim; dilute solutions of
biological substaiices of interest;
• purifying enr.ymes and other pro
tdns; a1,1.d
• ex.tractive bioconversion 3 •
Based r>n patdtioniri.g the compo
nents of an organic miic.t1.1te between
two hnmJscible (or partly mi�cibk)
solvents, this em:rgy-dfi6ei:it method
B
for
uses water a5 the �olvent in dealing who:re
with biological materials u .
M1 = molecular weioht of the partitioneo
In such a two-phase a.qu<oous sys
substance (i)
tem, incompatible polymers segro:-:
k = Boltzmann constant
gate in the water to form_ two phases.
T = absolute ternparature
While it doesn.'t work for some sm;i.ll
A = a constant related to tha two•phase
molec1.1 k� �uc.h as amino ;.a.cid� (which
syi,;tem's characteristics and ¢\her prop
erties of the partitioMd substance
distribute themselves evenly in the
rwo phases\, sud• a syst�m s1_1its Jarg�
This exponenti.al relation implies
biomolecules. These pa.rtition un
evenly to form variol.IS kinds of col that for large moleculi::s, a small
lokh 5 -7-.ind provide a ;::;onvenient change in J... �ignificantly influences
handle for separations (see figure 1). partitioning behavior.
In general, the partitiou codli.cient
for a soluble sub�tance is a function of
Calculating the
the foliowinfr
Partition Coefficient
• propenie� of the two phas�$;
A useful parameter for chataneriz,
• properties of the sarnple: and
ing the panition of component i of a
mixture in a two-phase matrix is the
• temperawte.
Note, however, that the coefficient
partition coeffidem K; = C)j/Cn,,
where C;t and C;b are the concentra ren)aim independent of the total vol
tions of the partitioned substance in ume of the system. Therefore, the
the rop and bottom ph;,_5es, respec partition coefficients for brge-,t:�I.;:
tively.
processes will be equal tci the values
According to Bronsted 8 the follow obtained in lab-scale experime.ms.
ing relationship exi5ts for the parti•
Calcubtlng the Partitiott Ratio
tion coefficient K;:
The parliLio11 ratio G1 i� !.he ratio
Ki "' exp (M; /./kT)
between the amount of sub�tance i in
�,..._..�----...........�..........��.......�'P"Wa.Ar.__.......,,__
ax
� Polar polym(!t
).fl+- Nonpol3r polymer
by:
M.Hossein Hatiri, Illinois Institute of
Technology, Chicago, IL USA;
James F. Ely, NIST, Department of Commerce,
Boulder, CO USA;
G.Ali.Mansoori*, University of Illiniois
at Chicago, Chicago, IL USA.
(*). Coresponding author email:
mansoori@uic.edu ��-�-....-�.....,...............-��
Figure 1 - Partition of mixture containing macromolecules A and B between two
polymer phases.
686
I
I
M.H. Hariri, J.F. Ely, G.A. Mansoori
Bioseparations: Design and Engineering of Partitioning Systems
Nature Biotechnology, Volume 7, Pages 686-688, July 1989
DOI: https://doi.org/10.1038/nbt0789-686
BIOSEPARATIONS:
Design and Engineering of
Partitioning Systems
the top s1nd bottom phases, s1nd is
written as
tling- rnt.e by adding a salt, a third
polymer, or an electric fit:ld.
Column Design
Macromolecules and other biologi
where C; is the concentration of the c;al substances with differing paxtition
solute j and V is volume. Subscripts t coefficients can be separated either in
a11J l, d<:n<.>te top and bouom ph.i.ses. a sini,i;le-stagc (batch or continuous)
For efficient large-scale separation or ml�ltistage app«1'atus�,1o_
of componenr.s p and q of a mixture of
For industrial-scale partitioning,
biological macromolecules, par6tion multistage separati.ons rely on a liq"
tatios G and Gq m.ust have suitable uid-liquid partitioning column. In
values. The best separation occurs this arrangement, a pump feeds a
when
hcavy polymer/ha�� into the top of
the column an a lighter phase into
the bottom of the column. These im
miscible phases m.ove in opposite di
Another vitai pai·arneter, the tim<:: rections through alternatihg mbdng
required for phase separation, de and separation stages (see figure 2).
pends on both the viscosities and the
The mixing stages effect mass
density differences between the two transfer; settling stages allow for
phases, Ne:ar th� c:;ritic;al point, �t:l phase separation. To perform a sep,4tling time is long. due to the sm.all n.ation, the operator introduces;_; sam·
density difference. Far from the criti pk in one of two war: either by
cal point, settling time is also long, in feeding it alon15 with one of the two
this instance due to high phase-vi5cos polyrne, phases, or by injecting it
ity. The shortest setthng times occur directly into one of the mixing or
at intermediate polymer phase com �cttling stagc3.
positions, One can also enhance setAn alternative multistage system
LEGEND
i ,1:"� 13.ri�;
2
3
4
5
6
?
B
g
Stjrring .,�g�
SeltlinQ sla�e
Protein A pufiHcatiur1 unit
f'ro!llin B p1.nilic.1,ion vn11
Lower" i:;iolyr·ner �h�e: in
Low1'r ;:x:;,lym.u i:,i·i..1:.1-Q- out
l,JpPt, polyme:r ptla�" in
Upper polymer phase out
consi5ts <:>fa 5i'>.ries of stag.:=s 5epar::ited
by filter plates. Mixing ,md separat•
ing take place in the same stage. The
lighter ph<1.si'>. wt.er� from the bottom
of the column and move� upward
whJ.li:; the heavy phase is kept station·
ary. A.Jter allowing mixing and phase
�ep�.ra,�j,on to occur; the �y�t�m
pump1 � volume of the lighter pha�c::
mm the column from below. This
inflllx pushes an equal volume of
light ph;a.5<;, th,ovgh the fi).ter and into
the st=.a.ge above, where it mi.gr.ates \Jf)
thrqugh the stationary phase.
M11t.bematical Model�
of J>artitioni.n1r
Given that t�n o, more variables
affect a biological rnbstancc's parti
tion coefficierit ll , qctalitative attd
q1Jantitative understanding of thi5
process presents a formidable t,3.5k,
And although :a v:ast ;,mount of scat
tered data on protein partition coeffi
cients have been published l · 12 , these
data apply to 5ystems that are ther,
modynarnically unspecified and in
con�isLt:nL c1mong expei:iments.
Predictive models that illuminate
� Figure 2;, Multistage paroum.itng wilb
altiernating mixing and 21cpa£11ting
stages.
fi su .-e 3. Pha"e diag.a,:n_ r,f' .- mixt\U'€ •
of twQ J>(>lymets and wat.,r. Mixtures
having compositions repN>$e�ted l>v
points 11.b()ve the binuda.l curve: will
scpa.i:a� h:d.1J two phases; mixtures
repres=tcd by J>(lints below the bino•
Slngls-pha�8
!.!al cunr<" e,t.ist. a,, a single phase. A,
region
·--._-..:;
Tu/bid mi�\�r� L
s;riitcm with total composition F scpa
ratf!s into uppel." phase U and lowe:r
Folyme,· A, %
pha.!le .l., with thci ratio of the voh:ime�
of the two pha:oies 11.pproximiitell
equ,id to the ratio of the dl1i.bnc,:,5 F
and FU on the cc:>m).��i.�g !i!lt', Simi·
Two-phase
l,u:ly, a system 'With total ,;,om.
sition
f po
rey,�n
F' SCPaJ:ate9 h1to _phases f tu:id L: At
cnticiJ. point C, die two H<Juid pha:!<es
bec:r,me id<i!nticaL
li'igu.l."e 4, An aqlJrouil 21olution of ...
polydispm11e polymer1i1 A and ll.
Sbnded area i.ndicate-. :region'> in
which m;xrurei. will be i.omewbat tur
bid, dUt!: to wcom!;il• SiJh•hHity <.>f
PolymetA,%
certain mo1ecula.r welgbtt< of B i,,:, -A
��··'
and. vi11:e versa.
687
M.H. Hariri, J.F. Ely, G.A. Mansoori
Bioseparations: Design and Engineering of Partitioning Systems
Nature Biotechnology, Volume 7, Pages 686-688, July 1989
DOI: https://doi.org/10.1038/nbt0789-686
the imenelationships among system
variables should primarily predict the
p hase diagram of the polymers ( see
figun': � ) . But. �u<.:h modds are com
plicated by the polydisperslty o f the
pol y met:s and J:, y ti'.le effect of salt ions
in enhancing the partition of ,;:b,;,u;gcd
pt:oteins 1 3• Interactions between the
polymers and the proteins further
,;:omplkate m.mer!;;.
Q!;'). e (i'i!.1::1. , however , calculate phase
diagx.i.m$ of two-phase aqueous sys
tems from the equality relatiomhip
between the components ' chemical
potentials, To 1,.1n(;ierstand multicom
ponent interactions, the modeler sys
tematically rearrange$ them in(o bi
nary interactions fol:" calculation.
A diffic.:ulty jn predicting aqueous
chemical potentials stem$ from the
common assumption that po lymers in
two-phase systems are rnon
:uei;ms
•
' I e componen t s J 4 ' I �
is perse a s sing
PolymeJ"$ have a molecular w�igh t
distribution , so treating them as sin
gle components is not always satisfac
tory.
Ma.l'.l.roori and Ely's phase equilibri11rn model fo1· µo]ydJspene solu
tions 1 7, modified · and e'x.tended to
pol ydispem: polymer aqueo�s sol�
tions , take� the molecular weight dis
tribution into account. It leads to the
followi11g equations for chemical po
tentials of system5 �har-J.cterized as
p olydis p ¢rse polymer 1 /polydisper:se
polymer �/w;_i,ter ;
l"ll ,..
RT
.
ij "'
l n m 2 i + 6 m 2 1 + /j M2 1 (m1J1 +
m:iJ2)
l' w - ,.. ·w = _1_
-
RT
½
m � h + (fl m2
mw
rm1
L
+ m� + i m� f 1 +
2
'fvl� + a m1�1)(m1J1 + m:i.J2)]
where
In the equations above,
m1 = l: m 1 1, M1 = :r m11Mdm1
and mru's are the molalities of the i111
molecular - weight fraction of poly
mers 1 and 2. Mm's are the ratios of
molar volumes of the ith fraction of
polymeni l and 2 with respect to
w,1 1er (w) , r esp e ctivel y.
The chemical potentials in the
working conditions and refer<;r:u;;e
state: are denoted as µ, and µ,* , resp e c
tively. a , !3 ,8, and € are interaction
terms fol:" polymer-polymer and poly
mer - water systems .
Th e .rnolec11lar weight distribu tion
functions of the two polymers in the
twc, pha5es can be exorcssed as
F 1 ( I ,o·\ ,"1 1 ) and Fi ( l,0-2,"12)', where o- 1
and cr2 are variances and 'Il l an d 112
are mean molecular weights of the
two polymers , respectively. (] 2 1 f2 , rn 2 ,
and :M: 2 are defined similady to J 1 , f1 .
etc. )
UMng the " � uality of chemical p o·
tentials" algorithm fot continuous
mi:i.ture phase equilibtia 1 7 • 1 1' , we can
calculate equilibrium compositions of
the two phases.
If the biomolecule in t he tw<>·phase
�ystem is rnmidered as another poly
mer molecule , and assuming the
large biomolecule bears a net char ge
ZSMM • then:
µ BMM
- µpolymer + ! BMM F 4, phase
In this expression, IJ.polym�r is the
chemical potential of a polymer in the
.solution, F i5 the Faraday number,
and 4:>pha.t is the electrostatic poten
tial . The second term in this eq uation
contributes to partitioning when
there is an electrostatic potential dif
ference between the two pha5e� . A� a
result, the contribution o f macrom o
lecufa:r charge to it!! partitfon coeffi
cient is as follows :
Since each phase in cquilibtium
must be electricall y neutral, .:ilj) b(:
tween the two phases must 5atJ.5 fy the
electroneutrality condition5, which
vary for each two- phase partitioning
s y,tem. Ions present in the two
phases and, possibly, ioni.tadon of the
biomolecules affect the elec.:troneu
trality conditions of � two-phase sy$
tem.
ters between polymer$ and water and
between the po l ymers themselves us
ing �ooo.e bi!rn:t y poly1ner-w.i.ter wix
20
L u,i::: data ,
As5umin g adequ<1te R & D fundi ng
au(i plMnin g , partition-based biose
paration.i arc:· lik.dy 1.0 llml incrc:iilsi ng
u5c in biotechnology product de,·el
opme:nt, due to the system's biocom
patibilit y , am-enability to scale-up, and
favorab.1.e e<;;<>n.o�jc;;� com p ared wJ.tb
o0'.1¢r biq�e p ara�ion, �ec.:hni q 1,1es.
,&ek,:e,:,�"-1'
1 . Humphrey, A. E. 19ij5. Critical needs in
biotechnology, Testimon y before the l.i.5 .
House of Representative� , S1.1b<::ommitti:<:
on Sci,::o�e. lt.e�arch, ;,;,d Technolog�.
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tioning of monoi;lisp•me/polydi�per� poly
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aq1,1eo11$ two-phase $�1em$ (A ri:emler Re
�a,ch Report). National Bureau of Stan
dar<;L� Techni�al l'iot<: 3. Alb(tt., >l()n, !'. A. 1 986. i•arthioning l)f' Cell
Po.rtide� and Ma�rom<>l=-...I;;;. ll�d �diti.,,1,
I . Wiley & Sons, New York.
4- Walter. H-, .8roolla. D- E,, and Fisher, D1985. Partitioninf in Aqueous Two-rhase
SYl'l<!':!1'1<, Ar.<1 d�ni� �<!':•� . Orhr1rlo, FL
5. Shaw, D. J 1 980. Introduction to CoHotd
a�<I S11rra�e Ch¢«)imy. :Std ewtion, Butter
worths, London , U . K.
6 . Voy11t5ky, S. ! 97.',. Colluid Chemi$try. MIR
Pltbl�h.tr;, �..fo;coiv, U.S.5. R. English trans
lation , 1 978.
7. J irgensons, B . and Straumanis, M. E, 1 954A Shori Tie-i<tbook of Colloid Chemi;try. J .
Wiley & Sons, New York.
8. Bro,1�1.ed , J N . 1 9� I . M9lecular Magnitude
and Phase mstribution. I. 'l • .l' hy�. C:hem.
Bodenstein-F�iband . p. 257.
9. Rometsch, R. 19M. Helv. Chim , ;\ct.J.
''(�6): ! 81l o. sd,elbel, I L c;. and Karr, A. E. 1 9/10. l ! itl.
En g. CJ:11:::m . ,U(6): 1 048.
1 1 . F,::rlur�, R. A. ·rt.c 1-'"nition of �i11gle com
pqi>•:mt .J.!lQ. binar}· �Q!llJ)Qf!�!H protein mix
t\.lres in polvethylt:,no, gly�ol-d�xtr�n " 'l ""
,,u,i two-phase systems. Natio11al Bu.-.::au of
Stw.�rds T�chnki-.1 Not,:,.
Ill. Walter, H., Brook$, I). E., and Fisher, D .
1 985. Plirtitior)ing h, 2quec;,us two• phas<t
5ystetn$- Academic l'resi, Odani:lo , FL
1 3. Johannon, (;_ 1 970. Partition of �alu and
th ,:,lr dfo�t., on partilion of proteins in a
de11tran-polyethylene glycol-water twc,
ph.-se Sy$W�- :Bio.:him. Siophys. Acta
!! ! :3B7,
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.8ioph)'s., Suppl. No, 1 ;39.
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aqueous two- phase systems. Presented at
1he AIChE annual meeting, New York,
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phase equilibri.- S0,5 7.
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ti<,n 9..,:,d ph= rule. Ch=- Ens- Comro
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ti� for biopnx11:ss ('ll.g:i r!¢¢tmg. Chem. En g.
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Blo.sep.:ratlc;,111 U$1ng piiirt.itioning of po) ydi.5...
P"r"' polymer, in aqu�ous twu-phue sys
tems. Pr�sented at AlChE meetintt, Hous
toi::1, "J'X, April !989.
Research Needs and Prospects
A recent N ational llureau of Stan•
dards workshop drew a dear consen
sus that more experimental data are
e5scntial to develop predictive parti
liouing modr:l� - CaJ:-e in choosing the
systems for 5tudy will en�urc: that the
number of measurements is. ke pt
low 19 . For example, using mol.alities
at the cntlO!l point of the solutiog, , For a free �of1Y of thi$ article (while. availabk),
one can calculate interaction parame- w·rite in .50? on Reader Ssroic11 Card
688