Ind. Eng. Chem. Res. 1991,30, 1061-1067
1061
Continuous Separation of Proteins by Annular Chromatography Gene F.Bloomingburg, Jennifer S.Bauer,+and Giorgio C a r t a * Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia 22903-2442
Charles H.B y e r s Chemical Technology Division, Oak Ridge National Laboratory, f Oak Ridge, Tennessee 37831
T h e separation of protein mixtures by continuous annular chromatography (CAC) is studied in a preparative-scale apparatus. S-Sepharose, a strong-acid porous cation-exchange resin is used as the separation medium, and mixtures of albumin, hemoglobin, and cytochrome c are used as a model separation system. Equilibrium and mass-transfer parameters are developed for this system on the basis of fixed-bed chromatograph experiments. A mathematical model is then successfully used in conjunction with these parameters to simulate the performance of the CAC separations. The continuous separation performance of the annular apparatus is found to be essentially the same as the batchwise performance of an equivalent conventional chromatograph, making the unit attractive for preparative and process-scale applications where continuous throughput is desirable. Continuous annular chromatography (CAC) is a process concept that allows operation of chromatographic separations in a truly continuous, steady-state fashion. The process utilizes an annular sorbent bed, packed in the space between two concentric cylinders. When the system is operated as an isocratic chromatograph, the eluent is uniformly distributed at the top along the bed circumference, while the bed assembly is slowly rotated around its axis. The feed to be separated is continuously introduced a t the top of the bed at a point (or sector) that remains fixed in space. The chromatographic elution of the feed components coupled with the bed rotation causes the formation of individual helical component bands that extend to the bottom of the bed. Here the separated components are continuously recovered at different angular distances from the feed point. The early developmenta of the CAC technology have been reviewed by Carta and Byers (1989). Some significant advances have recently been made. Two improved operating modes, which are common in conventional chromatography, have been implemented: stepwise elution, which leads to gradient elution when multiple concentration steps are used, and displacement development. In addition some new applications of the technology have been investigated, including the separation of sugars (Howard et al., 1988; Byers et al., 1989, 1990), the separation of metal ion mixtures by stepwise elution (Carta et al. 1989),and the separation of mixtures of amino acids by displacement development (DeCarli et al., 1990). Both experimental and theoretical studies were carried out for these systems with CAC units ranging in scale from 10.2 to 45.1 cm in diameter and from 30 to 110 cm in depth. Various resins and sorbents were used with sizes varying from -30 to 400 pm in diameter. The general conclusion that can be drawn from these liquid chromatography studies is that, provided that hydrodynamic (axial) dispersion does not present a significant contribution to the broadening of chromatographic peaks, it is generally possible to specify conditions that permit continuous operation with the CAC while retaining the separation performance of an equivalent fixed-bed process. Thus, a quantitative evaluation of the relevant mass-
* Author to whom correspondence is to be addressed. 'Current address: Air Products and Chemicals, Inc., Allentown, PA 18195-1501. *Operatedby Martin Marietta Energy Systems, Inc., for the U.S. Department of Energy, under Contract No. DE-AC05840R21400.
transfer resistances of the separation system is crucial in evaluating the potential of CAC technology. The separation and purification of proteins is a potentially important application of the CAC technology. As with any new continuous, process-scale unit operation, implementation depends upon the establishment of appropriate evaluation and design procedures for predicting and optimizing the separation performance in a scaled-up configuration. Protein separation using the CAC is untried, and hence, this study has sought to establish scaling relationships. To this end, we have systematically studied the separation of model protein mixtures, using ion-exchange resins. The proteins studied include albumin, hemoglobin, and cytochrome c. The resin was S-Sepharose from Pharmacia (Piscataway, NJ), a strong-acid, porous cation exchanger. Currently it is generally not possible to predict solidliquid equilibria or mass-transport coefficients for proteins from first principles, and data for the system in question are at least in part lacking from the literature. An experimental determination of these coefficients was thus necessary. These parameters are most conveniently obtained by simple column chromatography. Hence, experimental work was carried out first with fixed beds and subsequently in a preparative-scale CAC unit, specifically designed for protein separations. A mathematical model was used to obtain equilibrium and mass-transfer parameters from the fixed-bed experiments. The applicability of these parameters was then tested by predicting the performance of continuous separations performed on the CAC unit. Experimental Section Fixed-Bed Apparatus. Chromatographic experiments were carried out in a conventional low-pressure liquid chromatography system to determine equilibrium and mass-transfer parameters. The apparatus comprised a 2.5 cm i.d. X 16.5 cm long glass column (Kontes). Eluent was supplied by a Chromaflex pump (Kontes), and feed samples were injected with a six-port Teflon Rheodyne injection valve. The effluent concentration was observed with a spectrophotometric monitor (Pharmacia, Model UV-2), capable of operating at two wavelengths simultaneously. Millivolt signals from the monitor were collected, stored, and subsequently reduced on a microcomputerbased data acquisition system. CAC Apparatus. The CAC unit used in this work was constructed of Plexiglas and PVC plastic and is com-
0S88-5885/91/2630-l061$02.50/00 1991 American Chemical Society
1062 Ind. Eng. Chem. Res., Vol. 30, No. 5, 1991 0.3
-
0
15
25
35
45
55
65
75
85
95
105
115
130
I
1
I
CNOc, = 200mM
Q, = 5
0
160
cm'/min
400
fi
---
I
I
-
EXPERIMENTAL MODEL
800
1200
1600
TIME (s) PARTICLE DIAMETER (um)
Figure 1. Particle size distribution for S-Sepharose resin. Mean = 89 pm; standard deviation = 25 pm.
mercially available through System Designs, Inc., Oak Ridge, TN. The annulus has an outer diameter of 12.7 cm with a width of 0.635 cm and is 45 cm deep. A geared electric drive attached to the bottom of the annular device rotates the bed assembly slowly about its vertical axis. The resin was slurry-packed in the annulus to a depth of 36 cm and was supported at the bottom by porous polyethylene plugs. A 6-cm layer of Dowex Monosphere (Dow Chemical Co., Midland, MI), a gel-type cation-exchange resin with a diameter of approximately 300 pm, was packed on top of the resin bed to provide adequate flow distribution and prevent convective mixing of feed and eluent. The resin was found to be completely inert to the proteins used in this work. The feed was introduced through a stationary stainless steel nozzle whose tip was located within the Monosphere layer, about 2.5 cm above the S-Sepharose resin. Feed and eluent were continuously supplied to the bed by Teflon positive displacement pumps (FMI). The effluent exited at the bottom through a set of 90 stainless steel tubes located at 4' intervals along the annulus. The product concentration profiles were determined at steady state by continuously withdrawing a steady stream from any one of the 90 effluent tubes. This stream passed through the spectrophotometric monitor, which in this manner received fluid from all circumferential locations during the course of a complete rotation of the apparatus. To minimize disturbances of the velocity profile within the resin bed, a peristaltic pump was used to meter the flow through the monitor at a rate equal to 1/90 of the total flow through the bed. Signals from the monitor were collected and digitized by a microcomputer-based data acquisition system. Separation experiments were done with mixtures of albumin and hemoglobin and albumin and cytochrome c. To determine the concentration of each component, the absorbance of the effluent stream was simultaneously recorded at two wavelengths: 254 and 405 nm. Albumin has negligible absorption at 405 nm, and thus, in a mixture the hemoglobin concentration can be determined directly, since at this wavelength it exhibits an absorption maximum. The albumin concentration was then determined from the 254-nm-absorbance (where both proteins absorb UV light) by subtracting the hemoglobin contribution. The procedure proved quite reliable and worked equally well for mixtures of albumin and cytochrome c. Materials. S-Sepharose (Pharmacia) resin consists of a cross-linked agarose porous network, functionalized with sulfonic acid groups. The nominal exchange capacity of the resin is 0.1W.25 mmol/mL, and the resin is chemically stable over a broad pH range (2-14). The resin is highly
Figure 2. Experimental and calculated concentration histories for the separation of an albumin-hemoglobin mixture in a fixed bed.
porous, with a nominal exclusion limit of 4 X lo6 daltons. A commercial sample of the resin (Lot No. OD-06027) with a broad particle size distribution was used in this work. The particle size distribution was determined with a light-scattering particle size analyzer (HIAC/ROYCO, Model 4300) and is given in Figure 1. For these determinations the resin particles were heavily stained with methylene blue. Without staining, in fact, the difference in refractive index between the resin and the buffer solution was too slight to yield reliable measurements. The volume average diameter of these spherical particles was determined to be 89 pm in 10 mM dibasic sodium phosphate buffer. The resin, however, exhibited minimal volume change upon exposure to different pH and salt concentrations. The proteins used in this work were chosen to provide a range of molecular weight and isoelectric pH. Bovine serum albumin (BSA) has a molecular weight of -68 500 and a p l of 4.9. The material used in this study was obtained from Sigma Chemical as a powder with a purity of 98-t 5%. Bovine hemoglobin (Hb) has a molecular weight of -64500 and a pZ of 6.9. It was obtained from Sigma Chemical, in a doubly crystallized, dialyzed and lyophilized form. Finally, Sigma Chemical, type V-A (95-loo%), bovine heart cytochrome c (C-c) has a molecular weight of 13300 and a PIof 10.6. A 10 mM dibasic sodium phosphate buffer was used as the eluent in all chromatographic experiments. The pH was adjusted to 6.50 by the addition of acetic acid or sodium hydroxide. At this pH, albumin is negatively charged and is not significantly adsorbed by the resin (it can, however, still enter the resin true pores); hemoglobin has a small positive charge and is weakly adsorbed by the resin; and cytochrome c has a large positive charge and is strongly adsorbed. Blue dextran-2000 (MW = 2000000), obtained from Pharmacia, was used in some experiments to determine the interparticle porosity of resin beds. This material was found to be essentially completely excluded from the resin. All experiments were carried out at room temperature, 25 f 2 "C.
-
Results and Discussion Fixed-Bed Studies. Equilibrium and mass-transfer parameters can be obtained from analyses of experimental chromatographic peaks. The precision of such determination depends to a large extent upon the linearity of the equilibrium isotherms which should be established by means of experiments in which the feed concentration is varied. Concentrationprofiles for albumin and hemoglobin for a typical fixed-bed experiment are shown in Figure 2.
Ind. Eng. Chem. Res., Vol. 30, No. 5, 1991 1063 0.5 I
Table I. Effective Intraparticle Porosity solute" Dorositv NaN02 0.78 albumin 0.55 hemoglobin 0.67 cytochrome c 0.76
I
I
I
I
1
I
I
1.8
1.9
2.0
2.1
2.2
2.3
2.4
Y
" In 10 mM sodium phosphate buffer at pH 6.5.
zs)
-0
Equilibrium. The sorption equilibrium of these proteins on S-Sepharose depends on pH and salt concentration: the linear distribution coefficient, K , can be determined from the first moment, tR,of a chromatographic peak from the equation
-0.
1.7
log
F where t is the interparticle void fraction and tpl is the intraparticle porosity accessible by each solute. For a nonadsorbed solute, K = 0 and
Finally, for a solute that is so large as to be excluded from the resin, tpl = 0 and t
UtR/Z
'NaCl
m 3. Linear equilibrium distribution coefficient for hemoglobin
at pH 6.5 as a function of salt concentration. I
I
I
I
I
I
1
I
CYTOCHROME-C 10 mhj NAPHOS-pH6.5 S-SEPHAROSE
Y
-
0.5
(3)
Equations 1-3 can be obtained from a material balance (Sherwood et al., 1975; Ruthven, 1984). For a reasonably symmetrical peak the first moment becomes essentially equal to the peak maximum time, tma. A bed porosity t = 0.38 f 0.02 was determined from eq 3 from the retention time of blue dextran. The accessible intraparticle porosity t i was determined for different solutes from eq 2, and the results are given in Table I. Since sodium is present in excess in the buffer, and negative ions are excluded from the water-swollen resin matrix by the Donnan potential effect, sodium nitrite was used to determine the true resin porosity. This species should be able to penetrate most of the true pores, and a value of tpl = 0.78 was obtained. At pH 6.5, albumin should not be significantly adsorbed by the resin; Le., K = 0. Its retention time was in fact found to be nearly independent of salt concentration, and it actually increased very slightly at higher salt concentrations, likely as the result of small conformational changes of the resin. Equation 2 yielded an average value oft; of 0.55. To determine t i for hemoglobin and cytochrome c at the operating pH of 6.5, the salt concentration in the eluent was increased to about lo00 mM, to preclude any protein sorption on the resin. Above this salt concentration in the buffer no further change in the retention time could be detected, indicating the K was nearly zero for these conditions. As seen from Table I, the porosity accessible by cytochrome c is close to the "true total porosity" determined from the sodium nitrite experiment, indicating that a majority of the pores are sufficiently large to accommodate small proteins. The pore volume accessible by hemoglobin is considerably lower than the total porosity, however, and is comparable to that accessible by albumin, which has a similar molecular weight. These findings are somewhat in contrast to a porosity value of 0.94 used by Skidmore et al. (1990) for modeling the uptake of some proteins by S-Sepharose resin. This value was obtained by these authors from the chemical formulation of the resin, which consists of 6% agarose. It appears, nevertheless, that only a fraction of the total water content of the swollen resin beads is in the form of true
-0.5
2.2
I
I
I
I
2.3
2.4
2.5
2.6
2.7
2.8
2.9
log 'NoCI
Figure 4. Linear equilibrium distribution coefficient for cytochrome c at pH 6.5 as a function of salt concentration.
pores which are freely accessible by proteins and other molecules. The linear equilibrium distribution coefficient, K, was determined from eq 1 for hemoglobin and cytochrome c for different salt concentrations. The results are given in Figures 3 and 4. The K values were found to be independent of the feed protein concentration over the range 0-2 g/L. However, the K values are strongly dependent upon the salt concentration and are well correlated by power-law functions K H =~ 4-73 X 103(cN&l)-''8 (4)
Kc-c = 1.58 x 1010(CN,c1)-3~8
(5)
where CNaClis the salt concentration in the buffer in mmol/L. Since the p l of cytochrome c is 10.6 and the experiments were performed at pH 6.5, the effective positive charge of this species is larger than that of hemoglobin, and hence the exponent in the cytochrome c equilibrium relationship (eq 5) has a greater negative value (Whitley et al., 1989). The equilibrium appeared to remain essentially linear down to about 60 mM NaCl for hemoglobin and to 100 mM NaCl for cytochrome c. This was confirmed independently by carrying out batch adsorption experiments at various salt concentrations. At lower salt concentrations, the equilibria becomes nonlinear and can be represented approximately by Langmuir isotherms (Bauer, 1990). Mass Transfer. Determination of mass-transfer parameters from chromatographic peaks can be made by fitting experimental results with appropriate mathematical
1064 Ind. Eng. Chem. Res., Vol. 30, No. 5, 1991
models. Generally speaking, linear chromatographic systems are quite insensitive to the exact nature of the mass-transfer mechanisms assumed in the model; i.e., an equally good fit can often be obtained by choosing axial dispersion, film resistance, intraparticle diffusion, or any combination of these as the controlling peak dispersion mechanism (Ruthven, 1984). In the case of proteins and porous resins such as S-Sepharose, however, the intraparticle diffusional resistance is generally dominant (Yamaxnoto et al., 1988). This was checked for our system by estimating axial dispersion and film mass-transfer coefficients from available correlations (Ruthven, 1984). Neither effect made a significant contribution to the broadening of chromatographic peaks when included in the overall mass-transfer computation. Carta and Bauer (1990) have developed an analytic solution for chromatographic operations carried out with adsorbent particles having an arbitrary particle size distribution. As was shown by these authors, for systems affected by large intraparticle diffusional resistance, use of the volume average particle diameter is not always accurate and an exact solution must be sought. The solution is given by the following series
-C --
Table 11. Free and Intraparticle Effective Diffusivities protein' D., cm2/s D,b cm2/s T albumin 5.4 X lo4 6.1 X lo-' 6.2 6.9 X lo-' 5.4 hemoglobin 8.5 X lo* cytochrome c 20.2 X lo* 11.4 X lo-' 4.3 "In 10 mM sodium phosphate buffer at pH 6.5. bValues from Zubay (1983).
Table 111. Base Conditions for CAC Operation parameter value units feed concentration" 1.0 gof BSA/L g of Hb/L feed flow rate 0.5 cm3/min eluent salt concentrationb 100 mM eluent flow rate 27 cm3/min rotation rate 250 deg/h bed length 36 em bed cross-sectional area 25 cm2 "In 10 mM sodium phosphate buffer at pH 6.5. *NaCl in 10
m M sodium phosphate buffer at pH 6.5.
-
-0.3
-EXPERIMENTAL MODEL BASE CONDITIONS
ALBUMIN
i
\
ul v
CF
pZ
0.2
Q 0:
+ z W
0
t;
0.1
0
where ykj
[ ( + [Rj(-2 k ) l I 2 ] [Rj(-2r ak ) l I 2 ] [Rj(-r2ak ) l I 2 ] COS^ [ Rj (2k)lI2] [Rj(-2k)'I2] ra
= sinh Rj 2k)lI2] ra
sin
ra
Vkj
= sinh
-sin
hkj
=
-COS
ra
I
0
0
60
De 0 = 3(1 - €)De €pl (1- €pl)K'
+
In these equations, tF and t E are the feed and elution times, respectively, and De is the effective intraparticle diffusivity of the solute. The f j values are the volume fractions of particles with radius Rj and M is the number of fractions (Figure 1). Calculation of chromatographic profiles can be carried out typically with 5-50 additive terms in the series and is easily within the capabilities of a moderately equipped microcomputer. Only a few terms are usually required for systems with a large mass-transfer resistance, while more terms are needed for systems approaching local equilibrium conditions. The solution is not restricted to Gaussian peaks, which are obtained for infinitesimal feed pulses of a strongly retained component and low masstransfer resistance. It can be used to compute the periodic response of a chromatographic bed subjected to periodic pulses with arbitrary feed and elution periods. The only parameter not independently determined in eq 6 is the effective diffusivity, De, Its values were determined for the different solutes from a least-squares fit of experimental chromatographic peaks. Equation 6 provided an excellent fit as shown, for example, in Figure
180
240
Figure 5. Concentration profiles for the continuous chromatographic separation of an albumin-hemoglobin mixture a t base conditions (Table 111).
2, and the resulting effective diffusivities are given in Table 11. Tortuosity factors, 7, defined as T
a=
120
OFFSET FROM FEED (deg)
= eP1D/De
(7)
were also calculated and are given in Table I1 to illustrate the relationship of the effective diffusivities to the corresponding free diffusivity values. The latter were those reported by Zubay (1983). Consistent with their molecular weights, albumin and hemoglobin have similar effective diffusivities, while cytochrome c has a significantly higher value. The effective diffusivity values were essentially independent of salt and protein concentration at the relatively high salt concentrations used in the experiments. The effective diffusivities of albumin and cytochrome c reported in Table I1 are comparable with the values reported by Skidmore et al. (1990) of 8.0 X lo4 cm2/s for albumin and 4.7 X lo-' cm2/e for lysozyme obtained from batch uptake experiments for conditions of nonlinear equilibrium. The latter protein has a molecular weight of about 14500 and a free diffusivity close to that of cytochrome c ('&n and Gusek, 1990). The agreement with our results indicates that for this resin it is possible to obtain a reasonably accurate determination of diffusivity values from the chromatographic method and that these values may be useful for calculations for high-loading, nonlinear equilibrium conditions. CAC Studies. Typical operating conditions for the CAC are given in Table 111, and the concentration profiles
Ind. Eng. Chem. Res., Vol. 30, No. 5,1991 1065 1 .o
-
0.8
-
-
I
I
n
---
--
I
EXPERIMENTAL MODEL
-EXPERIMENTAL MODEL o = 170 deg/h
0.4 h
HEMOGLOBIN
1
\
-I
3 0.3
2 5 0.6 v
Z
P I< iE 0.2
F
a K
Z
z z
w
0.4
0 Z
0
0 0
0
0.1
0.2 I
0
n
0
180 270 OFFSET FROM FEED (deg)
90
360
Figure 6. Concentration profiles for the continuous chromatographic separation of a hemoglobin-cytochrome c mixture. Feed concentrations: Hb = 2 g/L, C-c = 2 g/L; C f i ~= 400 mM Q p = 1.0 cmg/min; Qe = 27 cmg/min; w = 270°/h.
120 1ao OFFSET FROM FEED (deg)
240
60
0
Figure 7. Concentration profiles for the continuous chromatographic separation of an albumin-hemoglobin mixture at a rotation rate of 170°/h. I
for an albumin-hemoglobin experiment are shown in Figure 5. The solid lines are model calculations which are based on the equilibrium and mass-transfer parameters determined from the fixed-bed studies. Equation 6 can be used directly for these calculations. As reviewed by Carta and Byers (1989),several authors have recognized that, in the absence of axial dispersion, equations describing the transient behavior of conventional (fixed-bed) chromatographic operations can be used to describe the steady-state performance of the CAC, simply by making the transformation 8 = wt
(8) Here 11) is the angular position in the CAC unit, w its rotation rate, and t the time of the corresponding fixed-bed operation. In eq 6, t is replaced by O/w and the feed time tF by QF 360° tF = (9)
w where Q F and Q E are the feed and eluent flow rates, respectively. From Figure 5 it appears that the steady separation performance of the CAC is quite close to predictions using parameters determined from fixed-bed experiments. This indicates that no significant channeling or other nonidealities take place in the CAC. Minor material balance differences between the theoretical and experimental results in Figure 5 and subsequent CAC runs are generally within the error of the experimental procedures. Concentration profiles obtained for the separation of a mixture of hemoglobin and cytochrome c are shown in Figure 6 in comparison with model predictions. Blue dextran was also present in the feed mixture used in this experiment and eluted in the column dead volume. Model predictions, based entirely on the parameters determined from the fixed-bed studies, are seen to be again in excellent agreement with the experimental results. The effecte of reducing the rotation rate from 250 to 170°/h on the albumin-hemoglobin separation are shown in Figure 7, where the experimental chromatogram is compared with model predictions. The peaks are sharper since they elute closer to the feed point, but the separation is somewhat reduced. A greater throughput is however possible as these experimental conditions would allow the Q F + QE
0
200
A
HEMOGLOBIN ALBUMIN RESOLUTION MODEL
2.0
h
P
U
150
1.5
v
m W
z
v)
0 c
g n
Pc
100
1.0 2
0
z
Y
9 n 50
3.5
0
100 200 300 400 ROTATION RATE (deg/h)
500
Figure 8. Experimental and calculated effects of rotation rate on peak position and resolution for the continuous chromatographic separation of an albumin-hemoglobin mixture at base conditions (Table 111).
simultaneous introduction of up to four feeds, while only three feed points would be possible at the higher rotation rate used in Figure 5. The experimental and theoretical peak positions and resolution obtained in a series of runs at different rotation rates are shown in Figure 8. The resolution between albumin and hemoglobin was defined as (10) where and OZ are the peak positions for the two components and W,and W 2are the baseline widths of the two chromatographic peaks. Base-line widths were obtained by extending the tangents at half of the peak height to both experimental and calculated peaks. Peak position and resolution are in reasonable agreement with model predictions. The resolution remains approximately constant for the experimentalrange of rotation rates. At lower rotation rates, however, the resolution is reduced. This is readily explained by eq 9, which shows that reducing w corresponds to an increased loading of feed to the chromatographic bed.
1066 Ind. Eng. Chem. Res., Vol. 30, No. 5, 1991
0.2
I
--
-EXPERIMENTAL -MODEL Q, = 5 1 c m Y m i n
-
n
_1
v
v
z
0.8 -
-
0.6
-
-
6 0.4 0
-
-
-
-
Z
0 +
p
,Y
w
I-
Z
-
h
1
-2 a
-- -EXPERIMENTAL MODEL
-
1.0
-
0.1 -
-
W
0 Z
Q
+ z
0 0
0 0
0.2 0
I
0
270
180
90
0
360
OFFSET FROM FEED (deg)
Figure 9. Concentration profiles for the continuous chromatographic separation of an albumin-hemoglobin mixture at an eluent flow rate of 51 cm3/min. 300
I
I\
I
I
I
-
n (5,
2. 200
I
1
I
- 1.0 MODEL
-
0.8
0.8
I
I
I
A -
-
I
EXPERIMENTAL MODEL
-
E
i 3
0
0
-I
0
a
-
100
0.4
- 0.2 01 0
I
I
I
I
I
I
10
20
30
40
50
60
-
ffl
0.6
i
;zl
m
0 k m
9 a
1.0 I
1.2
Z
Y
Figure 11. Concentration profiles for the continuous chromatographic separation of an albumin-hemoglobin mixture at a feed flow rate of 3.9 cm3/min.
J O 70
ELUENT FLOW RATE (cm3/min)
Figure 10. Experimental and calculated effects of eluent flow rate on peak position and resolution for the continuous chromatographic separation of an albumin-hemoglobin mixture at base conditions (Table 111).
Concentration profiles for an eluent flow rate of 51 cm3/min are shown in Figure 9, and a summary of the effects of varying the eluent flow rate is given in Figure 10. The peaks emerge closer to the feed point as QE is increased, while the resolution between albumin and hemoglobin is reduced. T h e agreement between model and experiment is again quite good, indicating that hydrodynamic effects do not play a significant role in determining the dispersion of the concentration profiles. The effects of increasing the feed flow rate from 1.0 to 3.9 cm3/min are shown in Figure 11. Note that, in this case, the chromatographic peaks approach the corresponding feed concentrations and tend to be flat at the top. Equation 6 predicts this behavior quite well, indicating the ability of the system to work in a nearly ideal manner at high feed loadings. Obviously, increasing the feed loading by a factor of 4 over the base conditions, reduces resolution. This is illustrated in Figure 12, which gives a summary of a series of experiments carried out at different feed flow rates. At low feed flow rates, the theoretical predictions approach a horizontal asymptote. For these conditions the feed loading is essentially infinitesimal and the resolution is independent of the feed flow rate. At higher feed flow rates, the separation deteriorates in a nearly linear manner. Increased loading of the CAC translates to greatly increased product throughput and less dilution. So, it may be advantageous to absorb a product-recycle penalty to
m
&
0.4
-
t
o’2 0 ‘ 0
I
I
I
I
I
1
2
3
4
5
FEED F L O W RATE (cm3/min)
Figure 12. Experimental and calculated effects of feed flow rate on resolution for the continuous chromatographic separation of an albumin-hemoglobin mixture at base conditions (Table 111).
gain these advantages. Obviously, factors associated with specific separations, such as economics, product characteristics, and regulatory issues, would enter into the optimization of the process operating conditions. The protocol presented here allows prediction of the performance factors of the CAC.
Concluding Remarks The separation of protein mixtures by continuous annular chromatography has been demonstrated by using a preparative-scale CAC unit. The separation performance was adequately described by using a particle diffusion model, whose equilibrium and mass-transfer parameters were obtained from simple fixed-bed experiments. The CAC unit behaved in a nearly ideal manner, indicating that hydrodynamic and extracolumnar nonidealities are virtually absent or have no effect on the separation. The experiments were, however, limited to conditions for which the system exhibits linear equilibrium and a modest protein loading on the resin. At low salt concentration, the equilibrium becomes nonlinear and a numerical solution of the partial differential equations that describe the chromatographicpropagation of the solutes may be needed for an accurate prediction of performance. However, our diffusivity values, determined from the chromatographic response of a fixed bed for linear equilibrium conditions,
Ind. Eng. Chem. Res. 1991,30, 1067-1075
are quite close to literature values recently obtained for similar proteins on the same resin from batch uptake experiments for nonlinear equilibrium conditions. This appears to indicate that our diffusion parameters may be suitable for describing the behavior of the process even for high-loading, nonlinear equilibrium conditions, which are likely t o be found in some processing applications. In conclusion, the CAC experiments which were performed under isocratic chromatography conditions (single eluent) show the applicability of the technology to protein separations. Previous work (Byers et al., 1989; DeCarli et al., 1990), however, clearly indicates the feasibility of implementing step elution and displacement techniques for the continuous separation of proteins a t high throughput. These types of operations are currently under study in our laboratories.
1067
Greek Letters t = bed void fraction
tpl = accessible intraparticle porosity 19 = angular coordinate: offset from feed, rad G i = peak position of component i, rad w = rotation rate, rad/s Literature Cited Bauer, J. S. Separation of Proteins by Ion Exchange Chromatography. M.S. Thesis, University of Virginia, Charlottesville, VA, 1990. Byers, C. H.; Sisson, W. G.; DeCarli, 11, J. P.; Carta, G. Pilot-Scale Studies of Sugar Separations by Continuous Chromatography. Appl. Biochem. Biotechnol. 1989,20,635-654. Byers, C. H.; Sisson, W. G.; DeCarli, 11, J. P.; Carta, G. Sugar Separations on a Pilot Scale by Continuous Annular Chromatography. Biotechnol. Prog. 1990,6, 13-20. Carta, G.;Byers, C. H. Novel Applications of Continuous Annular
Chromatography. In New Directions in Sorption Technology; Keller, G . E., Yang, R. T., Eds. Butterworth Stoneham, MA,
Acknowledgment
1989;pp 167-185. Carta. G.: Bauer. J. S. Analvtic Solution for ChromatonraDhv with Nonuniform Sorbent Paiticles. AIChE J. 1990,36,-14?-i50. Carta, G.; DeCarli, 11, J. P.; Byers, C. H. Separation of metals by ContinuousAnnular Chromatography with Step Elution. Chem. Eng. Commun. 1989,79,207-227. DeCarli, 11, J. P.; Carta, G.; Byers, C. H. DisplacementSeparations by Continuous Annular Chromatography. AIChE J. 1990, 36, 1220-1228. Howard, A. J.; Carta G.; Byers, C. H. Separation of Sugars by Continuous Annular Chromatography. Ind. Eng. Chem. Res. 1988,27, 1873-1882. Ruthven, D. M. Principles of Adsorption and Adsorption Processes; Wiley: New York, 1984;Chapter 8, pp 235-250. Sherwood, T. K.; Pigford, R. L.; Wilke, C. R. Mass Transfer; McGraw-Hill: New York, 1975;Chapter 10,pp 548-592. Skidmore, G.L.;Horstmann, B. J.; Chase, H. A. Modelling SingleComponent Protein Adsorption to the Cation Exchanger S-Sepharose FF. J. Chromatogr. 1990,498,113-128. Tyn, M. T.;Gusek, T. W. Prediction of Diffusion Coefficients of Proteins. Biotechnol. Bioeng. 1990,35,327-338. Whitley, R. D.; Wachter, R.; Liu, F.; Wang, N.-H. L. Ion-Exchange Equilibria of Lysozyme, Myoglobin and Bovine Serum Albumin. J. Chromatogr. 1989,465,137-156. Yamamoto, S.; Nakanishi, K.; Matsuno, R. Zon Exchange Chromatography of Proteins; Marcel Dekker: New York, 1988. Zubay, G . L.Biochemistry; Addison-Wesley: Reading, MA, 1983; Chapter 2,p 59.
This research was sponsored, in part, by the Office of Industrial Programs, US.Department of Energy, under Contract No. DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc., and by the Virginia Center for Innovative Technology. Nomenclature C = liquid-phase solute concentration, g/L C F = feed solute concentration, g/L D = free diffusivity, cm2/s De = intraparticle effective diffusivity, cmz/s = volume fraction of particles with radius Rj = equilibrium distribution coefficient M = number of fractions Q F = feed flow rate, cm3/s Q E = eluent flow rate, cm3/s R = resolution, defined by eq 10 R j = radius of particle fraction j , cm t = time, s t,, = peak maximum time, s t R = first moment of a chromatographic peak or retention time, s tF = feed time, s tE = elution time, s u = superficial velocity, cm/s W i= base-line width for component i, rad z = bed axial position, cm 2 = bed length, cm
it
Received for review July 2, 1990 Revised manuscript received October 25, 1990 Accepted November 7, 1990
Mass Transfer in a Thermally Regenerable Ion-Exchange Resin by Continuous Cycling Tah-Ben Hsu* and Robert L. Pigford' Department of Chemical Engineering, University of Delaware, Newark, Delaware 19711
The equilibrium properties and the mass-transfer rates in an amphoteric, thermally regenerable resin, Amberlite XD-2, were studied. The equilibrium resin capacity for sodium chloride adsorption is strongly influenced by temperature, solute concentration, and pH of the aqueous solution. The kinetic study was carried out with repeated temperature cycling, either sinusoidal or square wave. A surface resistance model represents the rate data better than another model based on pore diffusion. Introduction The cycling zone adsorption (CZA) process (Pigford et al., 1969, 1970) utilizes the forced cycling of a thermody-
* Address correspondence to this author at ARC0 Chemical Company, 3801 W. Chester Pike, Newtown Square, PA 19073. Deceased.
0888-5885/91/2630-1067$02.50/0
namic variable, such as temperature, in a fured-bed reactor, packed with a porous solid adsorbent, e.g., activated carbon, silica gel, or ion-exchange resins. Cyclic variation of the thermodynamic variable changes the equilibrium between fluid and solid phases; hence a solute is forced to move between the phases. If the feed flow to the bed has a constant composition, the effluent composition varies 0 1991 American Chemical Society
