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The separation of glutathione and glutamic acid using a simulated moving-bed adsorber system
[J. Ferment. Technol., Vol. 65, No. 1, 61-70. 1987]
The Separation of Glutathione and Glutamic Acid Using a Simulated Moving-Bed Adsorber System HARUHIK0 MAKI, HIDEKI FUKUDA, and HISASHI MORIKAWA Engineering ResearchLaboratory, KanegafuchiChemicalIndustry Co., Ltd., 1-8 Miyamae, Takasago, Hyogo 676, Japan
Glutathione (GSH) and glutamic acid (Glu) were continuously separated using a simulated moving-bed adsorber system. In this system, a specially-designed multi-port rotary valve was used to move the adsorbent particles, and its rotation speed and the liquid flow rate in the adsorber were controlled by a personal computer. Under the optimal conditions for the simulated moving-bed adsorber, both the purity and the yield coefficient of GSH in the raffinate stream at a steady state reached around 99%, and the concentration of GSH and the production of GSH for the amount of adsorbent used were greater by as much as ten times and eighteen times than for the conventional batch operation. The adsorption isotherms of GSH and Glu, which were non-linear and concentration-dependent, were well expressed using several parameters, and also the courses of GSH and Glu concentrations in transient changes to the steady-state condition could be predicted well by the intermittent moving-bed model.
L-Glutathione ( G S H ; y-glutamyl cysteinyl glycine) p r o d u c e d by yeast fermentation is a useful tripeptide as medicine for liver complaints, an antidote, etc. Industrially, G S H of a r o u n d 99% p u r i t y is usually required in the crystallization step. However, it is rather difficult to obtain such highly purified G S H , since the crude G S H extracted from yeast includes m a n y impurities such as salts, peptides, and a m i n o acids. A m o n g these impurities, glutamic acid (Glu) significantly limits the efficiency of recovery by crystallization. Consequently, m u c h effort has been directed towards the removal of these impurities. T h e most p o p u l a r a p p r o a c h has been the m e t h o d of isolation as a slightly soluble Cu or H g salt.l~ However, this m e t h o d has disadvantages for industry, such as low conversion in the Cu reaction with G S H , the production of harmful CuS and waste water, and a complicated operation. A l t h o u g h several c h r o m a t o g r a p h i c separations for G S H using various types of ion-exchange and thin-layer or p a p e r c h r o m a t o g r a p h s have been reported, *-5) they are not directly usable in an industrial
process since most of them were developed as a means of G S H analysis. Recently, there has been considerable interest in the use of a simulated moving-bed adsorber system originally developed b y Universal Oil Products Co. ( U O P ) , 6~ and this system has been successfully used in the industrial separation of various hydrocarbons. 7) A m o n g the advantages of a simulated moving-bed adsorber are: (11 reduction of the amounts of adsorbent and desorbent, (2) option of continuous operation, and (3) applicability to substances having similar adsorption isotherms. A theoretical treatment of a simulated moving-bed adsorber was done by H a s h i m o t o et al.s) for the continuous separation of a glucose/fructose mixture, and the validity of the m a t h e m a t i c a l models presented was experimentally confirmed. However, the behaviour of a G S H / Glu mixture is significantly different from that of a glucose/fructose mixture, since the former shows non-linear, concentrationdependent adsorption isotherms. T h e work presented in this paper is based on the separation of G S H / G l u using a simulated moving-bed adsorber system. For
MAKI, FUKUDA, and MORIKAwA
62
this purpose, the chromatographic conditions and the mathematical model for a G S H / G I u separation using a macroreticular ion-exchange resin were investigated first. The optimal operation of the simulated movingbed adsorber was then found experimentally, using a system controlled by a personal computer, in which the countercurrent movement of the adsorbent was caused by a specially-designed rotary valve. Thus, both the purity and the yield of G S H could be increased to around 99%, and the concentration of G S H in the recovered solution and the production of G S H for the amount of adsorbent were greatly improved. Mathematical
R-G++H +
(1)
_[R-G+I [H+I -- [G+] [R_H+ ]
(2)
where G +, H +, and R - are dissociated G S H or Glu, hydrogen ion, and fixed anion, respectively. Substitutions of p H and the activity coefficient of G + into Eq. (2) leads to: yCGqH where y, qH, Co, and qo represent the activity coefficient,9) the concentration of protonated fixed anion in the adsorbent, and the concentrations of GSH or Glu in the liquid phase and adsorbent, respectively. I n the ion-exchange reaction of G S H and Glu, an amino group ( - - N H s +) dissociates and reacts with the fixed anion. O n the assumption that thc dissociated carboxylic group ( - - C O 0 - ) of GSH and Glu bonds ionically with the amino group to some degree and this reduces the net plus charge, the activity coefficient based on the Henderson-Hasselbalch equation is used, with the parameter F instead of pKa for carboxylic groups, as follows: Yo :
1 -10fH--~i~- i- - -
(3) To find tile values of qH (for GSH and Glu) in the two-component system in Eq. (5), the following assumptions were made: 1. The effective capacity, Qoe, is introduced for each component, since neither GSH nor Glu reacts with all fixed anions of the ion exchange resin in which a high degree of cross-linkage is formed, 2. The value of Qo, for GSH, (0~0v,GSH), is smaller than that of Glu, (Qoe,Glu), since the molecular weight of the former is greater than that of the latter. 3. The amount of Glu adsorbed in the portion where two components can be adsorbed in equivalent to that in the remaining portion. Thus, the following equations can be derived for each qa. Q.Oe,GSH
and and K_
qo' 10~pH K : [{10PH_pK/i~ i-}cf~ {~-0F_-pi~_}_l }-x]-Co:qn
Model
Adsorption isotherms To find the optimal conditions for the operation of a simulated moving-bed adsorber, the adsorption isotherms of G S H and Glu onto the adsorbent are necessary. The ion-exchange reaction and the equilibrium constant between GSH or Glu and an ion-exchange resin (H + form) are given by G++R-H + ~
[.]. Ferment. Technol.,
qH,osa= Qoe,cSH- qosa -- qolu ~< Qo,,oi~-
qH,Olu = QOe,Glu -- qGSH-- qolu (7) Substituting the values of Qoe, K, and F for GSH or Glu into Eqs. (5)-(7), Co can he estimated from Eq. (5). Intermittent m o v i n g - b e d m o d e l s) In this study, the simulated moving-bed adsorber used was almost the same as the apparatus described by Hashimoto et al. s) Figure 1 shows a schematic representation of a simulated moving-bed adsorber divided into f o u r zones, each of which consists of four small columns connected to a rotary valve. The introduction (feed and desorbent) and withdrawal (extract and raffinate) points are intermittently shifted
-Desorbent (HCI solution)
*-Extract (Glu solution) Liquid flow \
"
-~ i-
(4)
Where p K a is the value for the amino group of GSH (8.75) or Glu(9.65). Substitution of Eq. (4) into Eq. (3) leads to:
- Feed (GSH.G~usolution)
-=- Raffir~te 03,58 solutior9
. . . . . . . .f_L I
1 ].(~F-pH
(6)
kRotary valve
Q Fig. I. Schematic representation ofsimulated movingbed adsorber.
Vol. 65, 1987]
Separation of Glutathione and Glutamic Acid
along with the direction of the liquid flow by the rotary valve. Thus, the positions of the raffinate, feed, extract, and desorbent streams are fixed at regular intervals. GSH and Glu are withdrawn respectively from a raffinate stream and an extract stream, since the former is weakly adsorbed compared with the latter. From the material balance in the adsorber, the concentration of component i in either the liquid or solid phase is given by
OCt . OCt
Oqt
uTi-~-~-~F + ai = 0
(8)
where u and ~ are the linear flow rates relative to the empty column and the void fraction of the bed, respectively. I n Eq. (8), the axial dispersion coefficient could be neglected in this study. T h e third term in Eq. (8) can be rewritten using the linear driving-force approximation as:x0, tl)
Oqt Ot : (KLa)i'(G -- Cl*)
(9)
where (KLa)t and Cl* are the overall volumetric mass transfer coefficient and the concentration equilibrium with qt for component i, respectively. T h e boundary conditions at the introduction points of desorbent and feed streams, and the withdrawal points of extract and raffinate streams in Eq. (8) are written respectively as follows: (10a)
uICiI0 = uIvCtivl
UIIICtIIIO=UIICilII+
t/fCif
(10b)
Ci,,0 =Gxt
(10c)
City0 =Ctlllt
(10d)
where subscripts 0 and 1 denote the inlet and outlet of the liquid stream in each zone, respectively, and Cif is the concentration of component i in the feed stream. The transient changes of GSH and Glu concentrations from t = 0 to t = T (interval of the valve rotation) are obtained by Eqs. (5)-(10) by using the method of finite differences.
63
were done in a constant-temperature room at 288K. T h e concentrations of G S H and Glu in the feed solution, the concentration of HC1 as desorbent, and the flow rate were 0 . 4 x 10-~--3.4× 10-B mol/l, 0 - 0.1 tool/l, and 2.5ml]min, respectively. T h e adsorption isotherms and the three parameters Qoe, K, and F for G S H or Glu were calculated by integration of the breakthrough curves and the method of least squares using the single-component system, respectively. T h e value of KLa was decided so that the calculated breakthrough curve by Eqs. (5)-(9) under the condition of Ci0=Cli would fit the experimental data. Chromatographic conditions for GSH/Glu separation Chromatographic conditions for GSH/ Glu separation were also arranged in the apparatus shown in Fig. 2 except that the column height was 100 cm. Two forms of ion-exchange resin (H + and Na + Amberlite IR200C) were used, and the concentrations of G S H and Glu in the feed solution, the amount of the feed solution, and the flow rate were set at 3.24× 10-~ mol/l, 3.40× 10-3 mol/l, 25 ml, and 2.5 ml/min, respectively. The concentrations of HCI (for the H + form ion-exchange resin) and N a O H (for the N a + form ion-exchange resin) as desorbents were investigated between 0--0.1 tool/l, and the concentration of HC1 or N a O H in the feed solution was adjusted to be the same as that of the desorbent. Simulated moving-bed adsorber system Experimental apparatus for simulated moving-bed adsorber Figure 3 is a schematic diagram of the simulated moving-bed adsorber system. Sixteen Pyrex glass columns were connected to a rotary valve, each column being 1.0 cm in inside diameter and 10 cm or 20 cm in length. T h e feed stream and the desorbent were
±
Constant-terrcecat ure
room
(288 K)
M a t e r i a l s and M e t h o d s A d s o r p t i o n i s o t h e r m s and overall v o l u m e t r i c m a s s transfer coefficient To decide the parameters of Qoe, K, and F included in Eqs. (5)-(7) and the value of KLa in Eq. (9), a simple batch operation was employed. Figure 2 shows a schematic flow diagram of the apparatus for measuring adsorption isotherms and the overall volumetric mass transfer coefficient. A Pyrex glass column, packed with the H + form of a macroreticular cation exchange resin (Rohm & Haas Co., Ltd., Amberlite IR200C, 350-590/~m), 20 c m in height and 1.05 cm in inside diameter is connected to a fraction collector. All experiments
Fig. 2. Schematic flow diagram of experimental apparatus for measuring adsorption isotherms and overall volumetric mass transfer coefficient. (1) feed or desorbent reservoir; (2) microtube p u m p ; (3) adsorption column; (4) fraction collector
64
MAKI, FUKUDA, and MORIKAWA
Liquid tlow
[J. Ferment. Technol.,
,
R
Fig. 3. Schematic flow diagram of experimental apparatus for simulated moving-bed adsorber. (1) adsorption column; (2) rotary valve; (3), (4) microtube p u m p ; (5), (8) fraction collector; (6), (9) plunger p u m p ; (7) feed reservoir; (10) desorbent reservoir; (11) personal computer supplied by plunger pumps (6) and (9), respectively. T h e raftinate stream was withdrawn into a fraction collector (5) by a microtube p u m p (4), and the extract stream was pushed into a fraction collector (8) by the internal pressure within the columns. The liquid flow in the adsorber was regulated by a microtube p u m p (3). Both the flow rate of p u m p (3) and the rotation of the rotary valve (2) were controlled by a personal computer (NEC, Type PC9801). A detailed diagram of the rotary valve used is shown in Fig. 4 (a). The rotary valve, made of Teflon, consists of two cylindrical sections, one fixed and the other rotating. The fixed section, 7.0 cm in diameter and 1.5 cm thick, has sixteen concyclic holes (in outside positions) connected to the sixteen columns, and another four holes (in inside positions) for raffinate, feed, extract and desorbent streams (see a - d in Fig. 4(a)). The rotating section, of the same diameter and thickness as the fixed section, has four grooves in concentric configuration, each of which connects inside and outside holes in the fixed section through one of four channels in the rotating section. H o w the feed stream exit is changed by a movement of the rotating section is schematically shown in Fig. 4(b). The feed stream exit at t = 0 , indicated as outside hole no. 1, is shifted to hole no. 2 after a certain period when t = T . Concerning the other three streams, the inlet or outlet positions are shifted in the same way. Thus, the specially-designed rotary valve is smoothly operated without any leakage of liquid. Criterion for good separation Considering the primary object of each zone (See Fig. 1), the conditions for good separation using a simulated moving-bed
adsorber can be found as follows. In zone l l I , for example, the amount of GSH carried by the transportation of adsorbent must be less than that carried by the liquid flow. This means that the direction of t5 15
Fixed section
Rotating section
(a)
"'
"
I
t=O
Im
I'
I=T
(b) Fig. 4. Schematic diagram of construction of rotary valve used in simulated moving-bed adsorber.
Vol. 65, 1987]
Separation of Glutathione and Glutamic Acid
65
Table 1. Experimental conditions for GSH/Glu separation with simulated moving-bed adsorber. Height of unit packed bed (cm) Desorbent Concentration of feed solution (moll/)
GSH Glu
8
16
0.05N HC1
0.05N HC1
1.62×10 -2 1.70X 10-3
3.24×10 -3 3. 40× 10-2
ui × A u~i × A um × A
14. 14 5. 65 8. 48
14. 14 6. 13 8. 95
utv × A Desorbent Extract Feed Raffinate
2.36 11.78 8. 48 2.83 6. 13
3.30 10. 84 8. 01 2. 83 5. 65
Interval of valve rotation (min)
10
20
Form of cation exchange resin
[H +]
[H +]
Flow rate (cmS/min)
the GSH stream is consistent with that of the liquid flow. O n the contrary, that of Glu must be reversed, since the primary object of this zone is selective adsorption of Glu. Thus, the following inequality must be held, using an average velocity of adsorbent flow (L/T): vm,clu < L / T < VIII,GSH
( 1 1)
where L, T, VIII,GSH~and viii,G1u represent the length of a unit column, the interval of valve rotation, and the average migration rates of the adsorption waves of GSH and Glu in zone III, respectively. Concerning the other zones, similar considerations lead to the following inequalities:
VhCSH>VI,C,lu>L/T VU,C,lu
for zone I
(12)
for zone II
(13)
for zone IV
(14)
To calculate the flow rate in each zone and the interval of valve rotation, the relationships between the linear flow rate and the average migration rates of the adsorption waves for GSH and Glu are necessary. Plotting the values of migration rate of the adsorption wave against the several linear flow rates in the singlecomponent system of GSH or Glu with HC1 concentration of 0.05 tool/l, the following equations were obtained. The value of the migration rate was estimated as the migration length in a certain time using Eqs. (5)-(9). vGsn=0.152 u
(15)
v61~ =0.088 u
(16)
Thus, the range for u can be set with a certain value of T (i.e. T ~ 6 0 0 or 1200see) using Eqs. (11)-(16). Simulating the distribution profiles of GSH and Glu concentrations in the adsorber using Eqs. (5)-(10) for several values of u, the optimal operating conditions were obtained as summarized in Table 1. Analysis The concentration of GSH was analyzed by the iodometric titration method, TM and the concentration of Glu was calculated by the difference between the value by the ninhydrin method 13,14) and the concentration of GSH.
Results and Discussion Adsorption
isotherms
and
overall
volumetric mass transfer coefficient T h e d a t a for t h e a d s o r p t i o n i s o t h e r m s for G S H and Glu on the macroreticular cation e x c h a n g e r e s i n a r e p l o t t e d i n Fig. 5. The v a l u e s o f Q,o°, K, a n d F i n E q s . ( 5 ) - ( 7 ) w e r e f o u n d b y t h e m e t h o d o f l e a s t s q u a r e s so t h a t the estimated results by Eqs. (5)-(7) using set v a l u e s o f t h e p a r a m e t e r s m a y fit a l l t h e data in each figure. Thus, the values o b t a i n e d a r e s u m m a r i z e d i n T a b l e 2, a n d t h e r e s u l t s a r e s h o w n i n F i g . 5. I n s p e c t i o n o f Fig. 5 suggests that the mathematical model of Eqs. (5)-(7) with the parameters shown in Table 2 can give a good estimation in a wide r a n g e o f H C 1 c o n c e n t r a t i o n for b o t h G S H
MAKI,
a n d Glu, and also that a mixture of G S H and Glu can be separated chromatographically, since a significant difference appears in the adsorption isotherms between G S H and Glu. A gel-type cation exchange resin ( R o h m & H a a s Co., Ltd., Amberlite I R l l 8 ) was examined instead of a maeroreticular cation exchange resin, but the adsorption isotherms for G S H and Glu gave fairly similar profiles. Presumably, the degree of cross-linkage of the gel-type is relatively low c o m p a r e d with that of the macroreticular type, and this m a y cause the inferiority in selective adsorption 1.0
I
~--08 Z
I
I
I
I
I
I
(a) GSH
~
0.6 -
-
F
" 0.0
[J. Ferment. Technol.,
FUKUDA, a n d MORIKAWA
Table 2. Measured values of parameters in Eqs. (5)-(7). GSH
Glu
Qoe [moll/]
0. 44
1.13
K [--]
I. 05
0. 76
Y [--]
1.86
2. 07
between G S H and Glu Typical breakthrough curves for G S H and Glu are shown in Fig. 6. T h e concentrations of G S H and Glu in the feed solutions, the concentration of He1, a n d the flow rate were 1.62 × 10-= tool/l, 1.70× 10-s tool/l, 0.05 mol/ l, and 2 . 5 m l / m i n , respectively. Fitted to the experimental data by Eqs. (5)-(9), the values of KLa for G S H and Glu were calculated as 1.20rain -1 and 2 . 4 0 m i n -1, respectively. T h e calculated results are shown in the same figure. T h e values agree fairly well with the results of K a w a z o e et al.,Is) where they obtained values for sodium ion of 3 . 0 0 - 6 . 0 0 m i n - 1 with the equivalent Reynolds n u m b e r and coneen-
. . . . .q t
"-1"~"'Yg~=" . . . .
0.0
0.5
1.0
1.5
2.0
Z5
3.0
3.5
Concentration ( m o i l ! )
xl02
I
I
4.0
I
I
I
I
I
I
I
d
J
8
~ o . - ~ .
I
(~o--6-~ o-o-o~ - o X ~ O-o-wo-o-OO-o-O~ o,o
o/
I
(b) Glu
t
~o 2.( 1.6
I
I
(a) GSH
~1.2
1.0
t
0.4
o/
u
o oo Time
(rain)
.-.. -
/j"
/ oA
/
-
,,"
-/,'"
E 0 - 2 - ¢/ // / .~ A /
./ ./"
.I
/A
_.-°'" . ~ ..~"
_
2.4
-
~o 2,0 x
~./
..~.~
..-~.'" .,...~&"
v'""-
_
i
0.0, "~-~'-'-'~ - ''I I i I I I I O.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Concentration (mol I I ) xl02
I
I
I
Fig. 5. Effects of HCI concentration on adsorption isotherms for GSH and Glu. (©): no HCI; (A): 0.01 mol//; (O): 0.05 tool/l; (A) 0.1 mol/l. Each curve was obtained by Eqs. (5)-(7).
I
l
I
I
l
I
(b) GIu
oo/oOol;Oo°oo°OoOOOoO,
oi.6 E
g ~.z /0
~O.B
4.0
I
op
o/ °/°"
0n
o 0.0 0
Fig. 6.
30
60
90
120 150 180 210 3~0 270 300 330 Time (rain)
E x p e r i m e n t a l results of b r e a k t h r o u g h curves
for GSH and Glu compared with results calculated by Eqs. (5)-(9). The concentration ef He1 is 0.05 mol//.
Vol. 65; 1987]
Separation of Glutathione and Glutamic Acid i
03y 0,sL
0'0t g~ 00sl-
i
I,
I
I
I
I
I
67
I
l
I
I
I
'1)
I~ i
(c) 1
I! ',
,lo ,,
/0-%
,,,, ,
,
.
•
0 200 400 6000 200 400 6008000 25o Time
(rain)
Time (min)
qJ4
^
,I
^
4
o
1
O
zx . - ~ t x -
^"'~k,,,,~-"~
o
,4
^
I I
4~o 6oo 800 ~ooo ~2oo v-,oo-~6oo .time
(min)
Fig. 7. Effects of HC1 concentration on separability of GSH and Glu using [H +] form of cation exchange resin as adsorbent. The concentrations of HC1 in (a), (b) and (c) are 0.1 moll/, 0.05 tool//and 0.01 mol//, respectively. (O): GSH; (A) : Glu. Each curve was obtained by Eqs. (5)-(9). tration. O u r values were almost the same in a wide range o f flow rate from 2.0 to 15.0 ml/min, suggesting that the liquid-phase mass transfer resistance is negligible.
C h r o m a t o g r a p h i c c o n d i t i o n s for GSH/ Glu separation T y p i c a l courses o f the changes in G S H a n d Glu concentrations in effluents with HCI concentrations of 0.1mol//, 0.05mol//, a n d 0 . 0 1 m o l / l are plotted in Figs. 7(a), 7(b), and 7(c), respectively, along with the results calculated from Eqs. (5)-(9) with Cio=Cif. It seems f r o m Fig. 7(b) that a significant difference in migration rates of the adsorption waves between G S H and Glu was observed, and their adsorption waves were sharply separated. O n the contrary, with the concentration of 0.1 m o l l / t h e adsorption waves between G S H and Glu were fairly overlapped as shown in Fig. 7(a) and with 0.01 moll/ e x p a n d e d as seen in Fig. 7(c). Thus, in these cases, a large a m o u n t of ion-exchange resin, a long period for achieving a steady s t a t e , a n d removal o f m u c h C I - are required in a simulated moving-bed adsorber system. Figure 8 shows a typical course with a N a O H concentration of 0.005 mol/l. T h e migration rates of the adsorption waves between G S H and Glu were almost the same. A l t h o u g h we tried another investigation with N a O H concentrations of between 0 (pure water) and 0.1 mol/l, worse results than those
of Fig. 8 were obtained. This m a y be caused by the strong adsorption power of Na+ c o m p a r e d with H+; i.e. G S H a n d Glu were slightly adsorbed in Na+ form ionexchange resin. F r o m these results, a simulated moving-bed adsorber system was operated using a H+ form ion-exchange resin and a desorbent of 0.05 m o l / / H C 1 solution.
S i m u l a t e d m o v l n g - b e d a d s o r b e r system T y p i c a l courses of G S H and Glu I
I
I
I
I
\\
"xa
I
I
I
2.5 n
2.0--
v
I
i.5--
o~
E
1.0--
o
o
O.S-
0£0-
-..
I -20 30 Time
I "°'l-o~t..,~,-T~ 40 5O 6O 70(rain)
I 80
Fig. 8. Experimental results of elution curves using the [Na+] form of cation exchange resin as adsorbent. The concentration of NaOH is 0.005 mol/l. Symbols are the same as in Fig. 7.
68
[J. Ferment. Technol.,
MAKI, FUKUDA, a n d MORIKAWA
,-, 2£
I
o x
I
I
i
I
I
!
I
I
~ 1,6 "5 E 1.2
.~'zx-S
zx a~ tx~2x-zx-~---zx-zx-°z~=-'Lx~'t~x'zx~-z~-tx-~--t~-t~ .... 7a- - :a-"
~x
,,
zx
zx
,,
o~
~ 0.~
E ,4~ i
u 0.~
s'
(a) Extract
200
400
600
I
I
I
2.0
800 1000 Time ( r a i n ) I
1200
I
I
1400
1600
1800
I
I
I
2000
%
"~1.6 --
.o.o.o.O-o °O.o.o~ o O.o.O.o o o o o °o-~-O.o.O O ° ° ° o-c~o.o.O.O-O.o° /O
~
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.~- 0.8
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o/°
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/ . . . . . . _k. . . . .
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(b) Raffinate ._A_Jd~A__&~x~_ ~,~._k~.,~_tt.E_~_^~ AJ_ __
~_~L~zx~.Jk~_
400
600
800 Time
1000
1200
1400
1600
1800
2000
(min)
Fig. 9. Courses of GSH and Glu concentrations in extract and raffinate streams using 16 cm of the unit packed bed. The concentration of HC1 is 0.05 mol//. Each curve was obtained by Eqs. (5)-(10). Symbols are the same as in Fig. 7. c o n c e n t r a t i o n s i n t r a n s i e n t c h a n g e s to t h e s t e a d y s t a t e u s i n g 16 c m a n d 8 c m o f t h e u n i t p a c k e d b e d i n h e i g h t a r e s h o w n i n Figs. 9 a n d 10, r e s p e c t i v e l y . Both the data and the calculated results using the intermittent m o v i n g - b e d m o d e l i n Figs. 9 a n d 10 w e r e a d o p t e d as t h e a v e r a g e v a l u e s for f r a c t i o n sizes o f 20 r a i n a n d 10 m i n . It can be seen f r o m t h e s e figures t h a t t h e c a l c u l a t e d c u r v e s for b o t h G S H a n d G l u i n t h e e x t r a c t a n d raffinate streams agree well with the data. The Glu concentration in the extract stream
and the GSH concentration in the raffinate stream in Fig. 9 reached steady-state values at about 800 min and 400 min, respectively, whereas those in Fig. 10 did so in a shorter period (about 300 min), since the amount of the ion exchange resin for the latter was half as much as that of the former. At the steady state in Fig. 9, the pH values of the raffinate and extract streams were respectively measured as 1.41 and 1.44, and in Fig. 10, they were 1.34 and 1.36. These values agreed well with the results calculated from
Table 3. Comparison of characteristics of simulated moving-bed adsorber system with conventional batch operation system in GSH]Glu separation. Simulated moving-bed adsorber
Conventional operation
Yield of GSH to obtain purity of 99% [%]
99
92
Concentration of refined GSH solution (mol//)
1.62 × 10-2
1.16 × 10-a
Productivity on the amount of adsorbent used (tool/l-adsorbent min)
4. 52 × 10.4
2.41 × 10-5
Vol. 65, 1987] 1.0
I
I
Separation of Glutathione and Glutamic Acid I
I
I
I
I
I
I
(a) Extract
I
I
°0.1 ~o0.1
-g ~0.~
.......... ~ - ~ X ~ - -~ . ~ l .~/~ ~ ~ ~
io.~
~ a~
0.(
50
100 ~0
I I 200 250
I
I
!
I L50 500
(rain)
Time
1.0
I 300 350 400
I
I
I
|
|
I
(b) Raffinate ~--°.0.8
~
0. 6
~0, ~o
~02: 0~ ~ r . ~ - - ; 0
50
100
. . . .~. . . . ~. . 150
Time
200
~a~a~2"~
250 300 350 400 450 500 (min)
Fig. 10. Courses of GSH and Glu concentrations in extract and raffinate streams using 8 cm of the unit packed bed. The concentration of HCI is 0.05 mol//. Each curve was obtained by Eqs. (5)-(10). Symbolsare the same as in Fig. 7. the Henderson-Hasselbalch equation. Both the purity and the yield coefficient of G S H in the raffinate stream at steady state in Fig. 9 reached around 99%. On the contrary, Fig. 10 shows only around 93% in both the purity and the yield coefficient of G S H , since the amount of adsorbent in each column was not sufficient for the complete separation of G S H and Glu. A comparison of the characteristics of the simulated moving-bed adsorber, using 16 cm of the unit packed bed in height, with those of the conventional chromatographic batch operation (see Fig. 7(b)) is summarized in Table 3. It is worth noting that the concentration of G S H and the productivity of G S H on the amount of adsorbent used by the simulated moving-bed adsorber were greater by as much as ten times and eighteen times compared with the conventional operation. Thus, the simulated moving-bed adsorber offers a significant improvement both in the
69
concentration and the productivity of GSH.
Concluding Remarks T h e continuous separation of G S H / G l u has been shown to be possible using a simulated moving-bed adsorber system, where the countercurrent movement of the adsorbent particles was operated by a specially-designed multi-port rotary valve. This compact valve was smoothly operated without leakage of liquid, and its rotation speed was controlled by a personal computer. Using the hydrogen form of a macroreticular cation exchange resin and diluted HC1 solution as desorbent, both the purity and the yield coefficient of G S H in the raffinate stream at steady state reached around 99%, and the concentration of G S H and the productivity of G S H on the amount of adsorbent used were greater by as much as ten times and eighteen times than with the conventional batch operation. The intermittent moving-bed model was adopted for the prediction of the courses of changes in G S H and Glu in the extract and raffinate streams. Although the adsorption isotherms of GSI-I and Glu were non-linear and concentration-dependent, they were well expressed using several parameters. Also, the calculated results using this model agreed well with the data in the range of the transient changes and steady state. Thus, from the economic point of view, the simulated moving-bed adsorber system offers a useful separation process for G S H / G l u in actual industrial production.
Nomenclature A C C* F K
KLa Qoo
: a r e a of the cross-section of the column, cm2 :concentration in the mobile phase, mol// :concentration equilibrium with q, tool// : parameter in equations (5), (6), - : equilibrium constant, - : overall volumetric mass transfer coefficient 1/min : effective capacity, mol//-bed
70
q Rt U
$
7 Suffix f i
MAgh FUKUDA, and MORIKAWA
: amount adsorbed, mol/l-bed : fixed a n i o n : time, rain : l i n e a r velocity r e l a t i v e to the e m p t y column, cm/min : a v e r a g e m i g r a t i o n rate o f the a d s o r p t i o n wave, c m / m i n : v o i d fraction, - : a c t i v i t y coefficient, - -
: feed s t r e a m : c o m p o n e n t in l i q u i d o r solid phase ( G S H or Glu) 1 : outlet 0 : inlet G : GSH or Glu H : hydrogen n ( I - I V ) : zone n u m b e r Acknowledgments
The authors are grateful to Professor K. Hashimoto and Assistant Professor K. Miura of the Department of Chemical Engineering, Kyoto University, for their helpful advice and encouragement during this study.
References
1) Voegtlin, C., Johnson, J . M . , Rosenthal, S.M.: J. Biol. Chem., 93, 435 (1931). 2) Purdie, J. W., Hanafi, D.E.: J. Chromatogr., 59, 184 (1971). 3) States, B., Segal, S.: Anal. Biochem., 27, 323 (1969). 4) Miyagawa, T.: Knrume Med. J., 177 (1971). 5) Blocklehurst, K.: Biochem. J., 133, 573 (1973) 6) Broughton, D.B.: U.S. Patent, 2985589 (1961). 7) Broughton, D.B., Neuzil, R.W., Pharis, J. M., Brearly, C.S.: Chem. Eng. Prog., 66, 70 (1970). 8) Hashimoto, K., Adachi, S., Noujima, H., Maruyama, H.: J. Chem. Eng. Japan, 16, 400 (1983). 9) Wevers, C . J . H . : Chem.Eng. Sci., 10, 171 (1959). 10) Glueckauf, E.: Trans.FaradaySoc., 51, 1540(1955). ll) Miura, K., Hashimoto, K.: J. Chem. Eng. Japan, 10, 490 (1977). 12) Woodward, G. E., Fly, E.G.: J. Biol. Chem, 97, 465 (1932). 13) Yemm, E.W., Cocking, E C.: Analyst., 80, 209 (1955). 14) Yemm, E.W., Cocking, E.C.: Biochem. J. (London), 58, 12 (1954). 15) Kawazoe, K., Takeuchi, Y.: Kagaku Kogaku, 31, 49 (1967). (Received June 26, 1986)
The Separation of Glutathione and Glutamic Acid Using a Simulated Moving-Bed Adsorber System HARUHIK0 MAKI, HIDEKI FUKUDA, and HISASHI MORIKAWA Engineering ResearchLaboratory, KanegafuchiChemicalIndustry Co., Ltd., 1-8 Miyamae, Takasago, Hyogo 676, Japan
Glutathione (GSH) and glutamic acid (Glu) were continuously separated using a simulated moving-bed adsorber system. In this system, a specially-designed multi-port rotary valve was used to move the adsorbent particles, and its rotation speed and the liquid flow rate in the adsorber were controlled by a personal computer. Under the optimal conditions for the simulated moving-bed adsorber, both the purity and the yield coefficient of GSH in the raffinate stream at a steady state reached around 99%, and the concentration of GSH and the production of GSH for the amount of adsorbent used were greater by as much as ten times and eighteen times than for the conventional batch operation. The adsorption isotherms of GSH and Glu, which were non-linear and concentration-dependent, were well expressed using several parameters, and also the courses of GSH and Glu concentrations in transient changes to the steady-state condition could be predicted well by the intermittent moving-bed model.
L-Glutathione ( G S H ; y-glutamyl cysteinyl glycine) p r o d u c e d by yeast fermentation is a useful tripeptide as medicine for liver complaints, an antidote, etc. Industrially, G S H of a r o u n d 99% p u r i t y is usually required in the crystallization step. However, it is rather difficult to obtain such highly purified G S H , since the crude G S H extracted from yeast includes m a n y impurities such as salts, peptides, and a m i n o acids. A m o n g these impurities, glutamic acid (Glu) significantly limits the efficiency of recovery by crystallization. Consequently, m u c h effort has been directed towards the removal of these impurities. T h e most p o p u l a r a p p r o a c h has been the m e t h o d of isolation as a slightly soluble Cu or H g salt.l~ However, this m e t h o d has disadvantages for industry, such as low conversion in the Cu reaction with G S H , the production of harmful CuS and waste water, and a complicated operation. A l t h o u g h several c h r o m a t o g r a p h i c separations for G S H using various types of ion-exchange and thin-layer or p a p e r c h r o m a t o g r a p h s have been reported, *-5) they are not directly usable in an industrial
process since most of them were developed as a means of G S H analysis. Recently, there has been considerable interest in the use of a simulated moving-bed adsorber system originally developed b y Universal Oil Products Co. ( U O P ) , 6~ and this system has been successfully used in the industrial separation of various hydrocarbons. 7) A m o n g the advantages of a simulated moving-bed adsorber are: (11 reduction of the amounts of adsorbent and desorbent, (2) option of continuous operation, and (3) applicability to substances having similar adsorption isotherms. A theoretical treatment of a simulated moving-bed adsorber was done by H a s h i m o t o et al.s) for the continuous separation of a glucose/fructose mixture, and the validity of the m a t h e m a t i c a l models presented was experimentally confirmed. However, the behaviour of a G S H / Glu mixture is significantly different from that of a glucose/fructose mixture, since the former shows non-linear, concentrationdependent adsorption isotherms. T h e work presented in this paper is based on the separation of G S H / G l u using a simulated moving-bed adsorber system. For
MAKI, FUKUDA, and MORIKAwA
62
this purpose, the chromatographic conditions and the mathematical model for a G S H / G I u separation using a macroreticular ion-exchange resin were investigated first. The optimal operation of the simulated movingbed adsorber was then found experimentally, using a system controlled by a personal computer, in which the countercurrent movement of the adsorbent was caused by a specially-designed rotary valve. Thus, both the purity and the yield of G S H could be increased to around 99%, and the concentration of G S H in the recovered solution and the production of G S H for the amount of adsorbent were greatly improved. Mathematical
R-G++H +
(1)
_[R-G+I [H+I -- [G+] [R_H+ ]
(2)
where G +, H +, and R - are dissociated G S H or Glu, hydrogen ion, and fixed anion, respectively. Substitutions of p H and the activity coefficient of G + into Eq. (2) leads to: yCGqH where y, qH, Co, and qo represent the activity coefficient,9) the concentration of protonated fixed anion in the adsorbent, and the concentrations of GSH or Glu in the liquid phase and adsorbent, respectively. I n the ion-exchange reaction of G S H and Glu, an amino group ( - - N H s +) dissociates and reacts with the fixed anion. O n the assumption that thc dissociated carboxylic group ( - - C O 0 - ) of GSH and Glu bonds ionically with the amino group to some degree and this reduces the net plus charge, the activity coefficient based on the Henderson-Hasselbalch equation is used, with the parameter F instead of pKa for carboxylic groups, as follows: Yo :
1 -10fH--~i~- i- - -
(3) To find tile values of qH (for GSH and Glu) in the two-component system in Eq. (5), the following assumptions were made: 1. The effective capacity, Qoe, is introduced for each component, since neither GSH nor Glu reacts with all fixed anions of the ion exchange resin in which a high degree of cross-linkage is formed, 2. The value of Qo, for GSH, (0~0v,GSH), is smaller than that of Glu, (Qoe,Glu), since the molecular weight of the former is greater than that of the latter. 3. The amount of Glu adsorbed in the portion where two components can be adsorbed in equivalent to that in the remaining portion. Thus, the following equations can be derived for each qa. Q.Oe,GSH
and and K_
qo' 10~pH K : [{10PH_pK/i~ i-}cf~ {~-0F_-pi~_}_l }-x]-Co:qn
Model
Adsorption isotherms To find the optimal conditions for the operation of a simulated moving-bed adsorber, the adsorption isotherms of G S H and Glu onto the adsorbent are necessary. The ion-exchange reaction and the equilibrium constant between GSH or Glu and an ion-exchange resin (H + form) are given by G++R-H + ~
[.]. Ferment. Technol.,
qH,osa= Qoe,cSH- qosa -- qolu ~< Qo,,oi~-
qH,Olu = QOe,Glu -- qGSH-- qolu (7) Substituting the values of Qoe, K, and F for GSH or Glu into Eqs. (5)-(7), Co can he estimated from Eq. (5). Intermittent m o v i n g - b e d m o d e l s) In this study, the simulated moving-bed adsorber used was almost the same as the apparatus described by Hashimoto et al. s) Figure 1 shows a schematic representation of a simulated moving-bed adsorber divided into f o u r zones, each of which consists of four small columns connected to a rotary valve. The introduction (feed and desorbent) and withdrawal (extract and raffinate) points are intermittently shifted
-Desorbent (HCI solution)
*-Extract (Glu solution) Liquid flow \
"
-~ i-
(4)
Where p K a is the value for the amino group of GSH (8.75) or Glu(9.65). Substitution of Eq. (4) into Eq. (3) leads to:
- Feed (GSH.G~usolution)
-=- Raffir~te 03,58 solutior9
. . . . . . . .f_L I
1 ].(~F-pH
(6)
kRotary valve
Q Fig. I. Schematic representation ofsimulated movingbed adsorber.
Vol. 65, 1987]
Separation of Glutathione and Glutamic Acid
along with the direction of the liquid flow by the rotary valve. Thus, the positions of the raffinate, feed, extract, and desorbent streams are fixed at regular intervals. GSH and Glu are withdrawn respectively from a raffinate stream and an extract stream, since the former is weakly adsorbed compared with the latter. From the material balance in the adsorber, the concentration of component i in either the liquid or solid phase is given by
OCt . OCt
Oqt
uTi-~-~-~F + ai = 0
(8)
where u and ~ are the linear flow rates relative to the empty column and the void fraction of the bed, respectively. I n Eq. (8), the axial dispersion coefficient could be neglected in this study. T h e third term in Eq. (8) can be rewritten using the linear driving-force approximation as:x0, tl)
Oqt Ot : (KLa)i'(G -- Cl*)
(9)
where (KLa)t and Cl* are the overall volumetric mass transfer coefficient and the concentration equilibrium with qt for component i, respectively. T h e boundary conditions at the introduction points of desorbent and feed streams, and the withdrawal points of extract and raffinate streams in Eq. (8) are written respectively as follows: (10a)
uICiI0 = uIvCtivl
UIIICtIIIO=UIICilII+
t/fCif
(10b)
Ci,,0 =Gxt
(10c)
City0 =Ctlllt
(10d)
where subscripts 0 and 1 denote the inlet and outlet of the liquid stream in each zone, respectively, and Cif is the concentration of component i in the feed stream. The transient changes of GSH and Glu concentrations from t = 0 to t = T (interval of the valve rotation) are obtained by Eqs. (5)-(10) by using the method of finite differences.
63
were done in a constant-temperature room at 288K. T h e concentrations of G S H and Glu in the feed solution, the concentration of HC1 as desorbent, and the flow rate were 0 . 4 x 10-~--3.4× 10-B mol/l, 0 - 0.1 tool/l, and 2.5ml]min, respectively. T h e adsorption isotherms and the three parameters Qoe, K, and F for G S H or Glu were calculated by integration of the breakthrough curves and the method of least squares using the single-component system, respectively. T h e value of KLa was decided so that the calculated breakthrough curve by Eqs. (5)-(9) under the condition of Ci0=Cli would fit the experimental data. Chromatographic conditions for GSH/Glu separation Chromatographic conditions for GSH/ Glu separation were also arranged in the apparatus shown in Fig. 2 except that the column height was 100 cm. Two forms of ion-exchange resin (H + and Na + Amberlite IR200C) were used, and the concentrations of G S H and Glu in the feed solution, the amount of the feed solution, and the flow rate were set at 3.24× 10-~ mol/l, 3.40× 10-3 mol/l, 25 ml, and 2.5 ml/min, respectively. The concentrations of HCI (for the H + form ion-exchange resin) and N a O H (for the N a + form ion-exchange resin) as desorbents were investigated between 0--0.1 tool/l, and the concentration of HC1 or N a O H in the feed solution was adjusted to be the same as that of the desorbent. Simulated moving-bed adsorber system Experimental apparatus for simulated moving-bed adsorber Figure 3 is a schematic diagram of the simulated moving-bed adsorber system. Sixteen Pyrex glass columns were connected to a rotary valve, each column being 1.0 cm in inside diameter and 10 cm or 20 cm in length. T h e feed stream and the desorbent were
±
Constant-terrcecat ure
room
(288 K)
M a t e r i a l s and M e t h o d s A d s o r p t i o n i s o t h e r m s and overall v o l u m e t r i c m a s s transfer coefficient To decide the parameters of Qoe, K, and F included in Eqs. (5)-(7) and the value of KLa in Eq. (9), a simple batch operation was employed. Figure 2 shows a schematic flow diagram of the apparatus for measuring adsorption isotherms and the overall volumetric mass transfer coefficient. A Pyrex glass column, packed with the H + form of a macroreticular cation exchange resin (Rohm & Haas Co., Ltd., Amberlite IR200C, 350-590/~m), 20 c m in height and 1.05 cm in inside diameter is connected to a fraction collector. All experiments
Fig. 2. Schematic flow diagram of experimental apparatus for measuring adsorption isotherms and overall volumetric mass transfer coefficient. (1) feed or desorbent reservoir; (2) microtube p u m p ; (3) adsorption column; (4) fraction collector
64
MAKI, FUKUDA, and MORIKAWA
Liquid tlow
[J. Ferment. Technol.,
,
R
Fig. 3. Schematic flow diagram of experimental apparatus for simulated moving-bed adsorber. (1) adsorption column; (2) rotary valve; (3), (4) microtube p u m p ; (5), (8) fraction collector; (6), (9) plunger p u m p ; (7) feed reservoir; (10) desorbent reservoir; (11) personal computer supplied by plunger pumps (6) and (9), respectively. T h e raftinate stream was withdrawn into a fraction collector (5) by a microtube p u m p (4), and the extract stream was pushed into a fraction collector (8) by the internal pressure within the columns. The liquid flow in the adsorber was regulated by a microtube p u m p (3). Both the flow rate of p u m p (3) and the rotation of the rotary valve (2) were controlled by a personal computer (NEC, Type PC9801). A detailed diagram of the rotary valve used is shown in Fig. 4 (a). The rotary valve, made of Teflon, consists of two cylindrical sections, one fixed and the other rotating. The fixed section, 7.0 cm in diameter and 1.5 cm thick, has sixteen concyclic holes (in outside positions) connected to the sixteen columns, and another four holes (in inside positions) for raffinate, feed, extract and desorbent streams (see a - d in Fig. 4(a)). The rotating section, of the same diameter and thickness as the fixed section, has four grooves in concentric configuration, each of which connects inside and outside holes in the fixed section through one of four channels in the rotating section. H o w the feed stream exit is changed by a movement of the rotating section is schematically shown in Fig. 4(b). The feed stream exit at t = 0 , indicated as outside hole no. 1, is shifted to hole no. 2 after a certain period when t = T . Concerning the other three streams, the inlet or outlet positions are shifted in the same way. Thus, the specially-designed rotary valve is smoothly operated without any leakage of liquid. Criterion for good separation Considering the primary object of each zone (See Fig. 1), the conditions for good separation using a simulated moving-bed
adsorber can be found as follows. In zone l l I , for example, the amount of GSH carried by the transportation of adsorbent must be less than that carried by the liquid flow. This means that the direction of t5 15
Fixed section
Rotating section
(a)
"'
"
I
t=O
Im
I'
I=T
(b) Fig. 4. Schematic diagram of construction of rotary valve used in simulated moving-bed adsorber.
Vol. 65, 1987]
Separation of Glutathione and Glutamic Acid
65
Table 1. Experimental conditions for GSH/Glu separation with simulated moving-bed adsorber. Height of unit packed bed (cm) Desorbent Concentration of feed solution (moll/)
GSH Glu
8
16
0.05N HC1
0.05N HC1
1.62×10 -2 1.70X 10-3
3.24×10 -3 3. 40× 10-2
ui × A u~i × A um × A
14. 14 5. 65 8. 48
14. 14 6. 13 8. 95
utv × A Desorbent Extract Feed Raffinate
2.36 11.78 8. 48 2.83 6. 13
3.30 10. 84 8. 01 2. 83 5. 65
Interval of valve rotation (min)
10
20
Form of cation exchange resin
[H +]
[H +]
Flow rate (cmS/min)
the GSH stream is consistent with that of the liquid flow. O n the contrary, that of Glu must be reversed, since the primary object of this zone is selective adsorption of Glu. Thus, the following inequality must be held, using an average velocity of adsorbent flow (L/T): vm,clu < L / T < VIII,GSH
( 1 1)
where L, T, VIII,GSH~and viii,G1u represent the length of a unit column, the interval of valve rotation, and the average migration rates of the adsorption waves of GSH and Glu in zone III, respectively. Concerning the other zones, similar considerations lead to the following inequalities:
VhCSH>VI,C,lu>L/T VU,C,lu
for zone I
(12)
for zone II
(13)
for zone IV
(14)
To calculate the flow rate in each zone and the interval of valve rotation, the relationships between the linear flow rate and the average migration rates of the adsorption waves for GSH and Glu are necessary. Plotting the values of migration rate of the adsorption wave against the several linear flow rates in the singlecomponent system of GSH or Glu with HC1 concentration of 0.05 tool/l, the following equations were obtained. The value of the migration rate was estimated as the migration length in a certain time using Eqs. (5)-(9). vGsn=0.152 u
(15)
v61~ =0.088 u
(16)
Thus, the range for u can be set with a certain value of T (i.e. T ~ 6 0 0 or 1200see) using Eqs. (11)-(16). Simulating the distribution profiles of GSH and Glu concentrations in the adsorber using Eqs. (5)-(10) for several values of u, the optimal operating conditions were obtained as summarized in Table 1. Analysis The concentration of GSH was analyzed by the iodometric titration method, TM and the concentration of Glu was calculated by the difference between the value by the ninhydrin method 13,14) and the concentration of GSH.
Results and Discussion Adsorption
isotherms
and
overall
volumetric mass transfer coefficient T h e d a t a for t h e a d s o r p t i o n i s o t h e r m s for G S H and Glu on the macroreticular cation e x c h a n g e r e s i n a r e p l o t t e d i n Fig. 5. The v a l u e s o f Q,o°, K, a n d F i n E q s . ( 5 ) - ( 7 ) w e r e f o u n d b y t h e m e t h o d o f l e a s t s q u a r e s so t h a t the estimated results by Eqs. (5)-(7) using set v a l u e s o f t h e p a r a m e t e r s m a y fit a l l t h e data in each figure. Thus, the values o b t a i n e d a r e s u m m a r i z e d i n T a b l e 2, a n d t h e r e s u l t s a r e s h o w n i n F i g . 5. I n s p e c t i o n o f Fig. 5 suggests that the mathematical model of Eqs. (5)-(7) with the parameters shown in Table 2 can give a good estimation in a wide r a n g e o f H C 1 c o n c e n t r a t i o n for b o t h G S H
MAKI,
a n d Glu, and also that a mixture of G S H and Glu can be separated chromatographically, since a significant difference appears in the adsorption isotherms between G S H and Glu. A gel-type cation exchange resin ( R o h m & H a a s Co., Ltd., Amberlite I R l l 8 ) was examined instead of a maeroreticular cation exchange resin, but the adsorption isotherms for G S H and Glu gave fairly similar profiles. Presumably, the degree of cross-linkage of the gel-type is relatively low c o m p a r e d with that of the macroreticular type, and this m a y cause the inferiority in selective adsorption 1.0
I
~--08 Z
I
I
I
I
I
I
(a) GSH
~
0.6 -
-
F
" 0.0
[J. Ferment. Technol.,
FUKUDA, a n d MORIKAWA
Table 2. Measured values of parameters in Eqs. (5)-(7). GSH
Glu
Qoe [moll/]
0. 44
1.13
K [--]
I. 05
0. 76
Y [--]
1.86
2. 07
between G S H and Glu Typical breakthrough curves for G S H and Glu are shown in Fig. 6. T h e concentrations of G S H and Glu in the feed solutions, the concentration of He1, a n d the flow rate were 1.62 × 10-= tool/l, 1.70× 10-s tool/l, 0.05 mol/ l, and 2 . 5 m l / m i n , respectively. Fitted to the experimental data by Eqs. (5)-(9), the values of KLa for G S H and Glu were calculated as 1.20rain -1 and 2 . 4 0 m i n -1, respectively. T h e calculated results are shown in the same figure. T h e values agree fairly well with the results of K a w a z o e et al.,Is) where they obtained values for sodium ion of 3 . 0 0 - 6 . 0 0 m i n - 1 with the equivalent Reynolds n u m b e r and coneen-
. . . . .q t
"-1"~"'Yg~=" . . . .
0.0
0.5
1.0
1.5
2.0
Z5
3.0
3.5
Concentration ( m o i l ! )
xl02
I
I
4.0
I
I
I
I
I
I
I
d
J
8
~ o . - ~ .
I
(~o--6-~ o-o-o~ - o X ~ O-o-wo-o-OO-o-O~ o,o
o/
I
(b) Glu
t
~o 2.( 1.6
I
I
(a) GSH
~1.2
1.0
t
0.4
o/
u
o oo Time
(rain)
.-.. -
/j"
/ oA
/
-
,,"
-/,'"
E 0 - 2 - ¢/ // / .~ A /
./ ./"
.I
/A
_.-°'" . ~ ..~"
_
2.4
-
~o 2,0 x
~./
..~.~
..-~.'" .,...~&"
v'""-
_
i
0.0, "~-~'-'-'~ - ''I I i I I I I O.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Concentration (mol I I ) xl02
I
I
I
Fig. 5. Effects of HCI concentration on adsorption isotherms for GSH and Glu. (©): no HCI; (A): 0.01 mol//; (O): 0.05 tool/l; (A) 0.1 mol/l. Each curve was obtained by Eqs. (5)-(7).
I
l
I
I
l
I
(b) GIu
oo/oOol;Oo°oo°OoOOOoO,
oi.6 E
g ~.z /0
~O.B
4.0
I
op
o/ °/°"
0n
o 0.0 0
Fig. 6.
30
60
90
120 150 180 210 3~0 270 300 330 Time (rain)
E x p e r i m e n t a l results of b r e a k t h r o u g h curves
for GSH and Glu compared with results calculated by Eqs. (5)-(9). The concentration ef He1 is 0.05 mol//.
Vol. 65; 1987]
Separation of Glutathione and Glutamic Acid i
03y 0,sL
0'0t g~ 00sl-
i
I,
I
I
I
I
I
67
I
l
I
I
I
'1)
I~ i
(c) 1
I! ',
,lo ,,
/0-%
,,,, ,
,
.
•
0 200 400 6000 200 400 6008000 25o Time
(rain)
Time (min)
qJ4
^
,I
^
4
o
1
O
zx . - ~ t x -
^"'~k,,,,~-"~
o
,4
^
I I
4~o 6oo 800 ~ooo ~2oo v-,oo-~6oo .time
(min)
Fig. 7. Effects of HC1 concentration on separability of GSH and Glu using [H +] form of cation exchange resin as adsorbent. The concentrations of HC1 in (a), (b) and (c) are 0.1 moll/, 0.05 tool//and 0.01 mol//, respectively. (O): GSH; (A) : Glu. Each curve was obtained by Eqs. (5)-(9). tration. O u r values were almost the same in a wide range o f flow rate from 2.0 to 15.0 ml/min, suggesting that the liquid-phase mass transfer resistance is negligible.
C h r o m a t o g r a p h i c c o n d i t i o n s for GSH/ Glu separation T y p i c a l courses o f the changes in G S H a n d Glu concentrations in effluents with HCI concentrations of 0.1mol//, 0.05mol//, a n d 0 . 0 1 m o l / l are plotted in Figs. 7(a), 7(b), and 7(c), respectively, along with the results calculated from Eqs. (5)-(9) with Cio=Cif. It seems f r o m Fig. 7(b) that a significant difference in migration rates of the adsorption waves between G S H and Glu was observed, and their adsorption waves were sharply separated. O n the contrary, with the concentration of 0.1 m o l l / t h e adsorption waves between G S H and Glu were fairly overlapped as shown in Fig. 7(a) and with 0.01 moll/ e x p a n d e d as seen in Fig. 7(c). Thus, in these cases, a large a m o u n t of ion-exchange resin, a long period for achieving a steady s t a t e , a n d removal o f m u c h C I - are required in a simulated moving-bed adsorber system. Figure 8 shows a typical course with a N a O H concentration of 0.005 mol/l. T h e migration rates of the adsorption waves between G S H and Glu were almost the same. A l t h o u g h we tried another investigation with N a O H concentrations of between 0 (pure water) and 0.1 mol/l, worse results than those
of Fig. 8 were obtained. This m a y be caused by the strong adsorption power of Na+ c o m p a r e d with H+; i.e. G S H a n d Glu were slightly adsorbed in Na+ form ionexchange resin. F r o m these results, a simulated moving-bed adsorber system was operated using a H+ form ion-exchange resin and a desorbent of 0.05 m o l / / H C 1 solution.
S i m u l a t e d m o v l n g - b e d a d s o r b e r system T y p i c a l courses of G S H and Glu I
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Fig. 8. Experimental results of elution curves using the [Na+] form of cation exchange resin as adsorbent. The concentration of NaOH is 0.005 mol/l. Symbols are the same as in Fig. 7.
68
[J. Ferment. Technol.,
MAKI, FUKUDA, a n d MORIKAWA
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Fig. 9. Courses of GSH and Glu concentrations in extract and raffinate streams using 16 cm of the unit packed bed. The concentration of HC1 is 0.05 mol//. Each curve was obtained by Eqs. (5)-(10). Symbols are the same as in Fig. 7. c o n c e n t r a t i o n s i n t r a n s i e n t c h a n g e s to t h e s t e a d y s t a t e u s i n g 16 c m a n d 8 c m o f t h e u n i t p a c k e d b e d i n h e i g h t a r e s h o w n i n Figs. 9 a n d 10, r e s p e c t i v e l y . Both the data and the calculated results using the intermittent m o v i n g - b e d m o d e l i n Figs. 9 a n d 10 w e r e a d o p t e d as t h e a v e r a g e v a l u e s for f r a c t i o n sizes o f 20 r a i n a n d 10 m i n . It can be seen f r o m t h e s e figures t h a t t h e c a l c u l a t e d c u r v e s for b o t h G S H a n d G l u i n t h e e x t r a c t a n d raffinate streams agree well with the data. The Glu concentration in the extract stream
and the GSH concentration in the raffinate stream in Fig. 9 reached steady-state values at about 800 min and 400 min, respectively, whereas those in Fig. 10 did so in a shorter period (about 300 min), since the amount of the ion exchange resin for the latter was half as much as that of the former. At the steady state in Fig. 9, the pH values of the raffinate and extract streams were respectively measured as 1.41 and 1.44, and in Fig. 10, they were 1.34 and 1.36. These values agreed well with the results calculated from
Table 3. Comparison of characteristics of simulated moving-bed adsorber system with conventional batch operation system in GSH]Glu separation. Simulated moving-bed adsorber
Conventional operation
Yield of GSH to obtain purity of 99% [%]
99
92
Concentration of refined GSH solution (mol//)
1.62 × 10-2
1.16 × 10-a
Productivity on the amount of adsorbent used (tool/l-adsorbent min)
4. 52 × 10.4
2.41 × 10-5
Vol. 65, 1987] 1.0
I
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Separation of Glutathione and Glutamic Acid I
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Fig. 10. Courses of GSH and Glu concentrations in extract and raffinate streams using 8 cm of the unit packed bed. The concentration of HCI is 0.05 mol//. Each curve was obtained by Eqs. (5)-(10). Symbolsare the same as in Fig. 7. the Henderson-Hasselbalch equation. Both the purity and the yield coefficient of G S H in the raffinate stream at steady state in Fig. 9 reached around 99%. On the contrary, Fig. 10 shows only around 93% in both the purity and the yield coefficient of G S H , since the amount of adsorbent in each column was not sufficient for the complete separation of G S H and Glu. A comparison of the characteristics of the simulated moving-bed adsorber, using 16 cm of the unit packed bed in height, with those of the conventional chromatographic batch operation (see Fig. 7(b)) is summarized in Table 3. It is worth noting that the concentration of G S H and the productivity of G S H on the amount of adsorbent used by the simulated moving-bed adsorber were greater by as much as ten times and eighteen times compared with the conventional operation. Thus, the simulated moving-bed adsorber offers a significant improvement both in the
69
concentration and the productivity of GSH.
Concluding Remarks T h e continuous separation of G S H / G l u has been shown to be possible using a simulated moving-bed adsorber system, where the countercurrent movement of the adsorbent particles was operated by a specially-designed multi-port rotary valve. This compact valve was smoothly operated without leakage of liquid, and its rotation speed was controlled by a personal computer. Using the hydrogen form of a macroreticular cation exchange resin and diluted HC1 solution as desorbent, both the purity and the yield coefficient of G S H in the raffinate stream at steady state reached around 99%, and the concentration of G S H and the productivity of G S H on the amount of adsorbent used were greater by as much as ten times and eighteen times than with the conventional batch operation. The intermittent moving-bed model was adopted for the prediction of the courses of changes in G S H and Glu in the extract and raffinate streams. Although the adsorption isotherms of GSI-I and Glu were non-linear and concentration-dependent, they were well expressed using several parameters. Also, the calculated results using this model agreed well with the data in the range of the transient changes and steady state. Thus, from the economic point of view, the simulated moving-bed adsorber system offers a useful separation process for G S H / G l u in actual industrial production.
Nomenclature A C C* F K
KLa Qoo
: a r e a of the cross-section of the column, cm2 :concentration in the mobile phase, mol// :concentration equilibrium with q, tool// : parameter in equations (5), (6), - : equilibrium constant, - : overall volumetric mass transfer coefficient 1/min : effective capacity, mol//-bed
70
q Rt U
$
7 Suffix f i
MAgh FUKUDA, and MORIKAWA
: amount adsorbed, mol/l-bed : fixed a n i o n : time, rain : l i n e a r velocity r e l a t i v e to the e m p t y column, cm/min : a v e r a g e m i g r a t i o n rate o f the a d s o r p t i o n wave, c m / m i n : v o i d fraction, - : a c t i v i t y coefficient, - -
: feed s t r e a m : c o m p o n e n t in l i q u i d o r solid phase ( G S H or Glu) 1 : outlet 0 : inlet G : GSH or Glu H : hydrogen n ( I - I V ) : zone n u m b e r Acknowledgments
The authors are grateful to Professor K. Hashimoto and Assistant Professor K. Miura of the Department of Chemical Engineering, Kyoto University, for their helpful advice and encouragement during this study.
References
1) Voegtlin, C., Johnson, J . M . , Rosenthal, S.M.: J. Biol. Chem., 93, 435 (1931). 2) Purdie, J. W., Hanafi, D.E.: J. Chromatogr., 59, 184 (1971). 3) States, B., Segal, S.: Anal. Biochem., 27, 323 (1969). 4) Miyagawa, T.: Knrume Med. J., 177 (1971). 5) Blocklehurst, K.: Biochem. J., 133, 573 (1973) 6) Broughton, D.B.: U.S. Patent, 2985589 (1961). 7) Broughton, D.B., Neuzil, R.W., Pharis, J. M., Brearly, C.S.: Chem. Eng. Prog., 66, 70 (1970). 8) Hashimoto, K., Adachi, S., Noujima, H., Maruyama, H.: J. Chem. Eng. Japan, 16, 400 (1983). 9) Wevers, C . J . H . : Chem.Eng. Sci., 10, 171 (1959). 10) Glueckauf, E.: Trans.FaradaySoc., 51, 1540(1955). ll) Miura, K., Hashimoto, K.: J. Chem. Eng. Japan, 10, 490 (1977). 12) Woodward, G. E., Fly, E.G.: J. Biol. Chem, 97, 465 (1932). 13) Yemm, E.W., Cocking, E C.: Analyst., 80, 209 (1955). 14) Yemm, E.W., Cocking, E.C.: Biochem. J. (London), 58, 12 (1954). 15) Kawazoe, K., Takeuchi, Y.: Kagaku Kogaku, 31, 49 (1967). (Received June 26, 1986)