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Selective separation of γ-linolenic acid ethyl ester using y-zeolite
[J. Ferment. Technol., Vol. 65, No. 5, 569-574. 1987]
Selective Separation of 7-Linolenic Acid Ethyl Ester Using Y-Zeolite MAKOTO ARAI, HIDEKI FUKUDA, a n d HISASHI MORIKAWA
Kanegafuehi ChemicalIndustry Co., Ltd., 1-8 M~amae, Takasago,Hyogo 676, Japan
To highly purify y-linolenic acid (GLA) from microbial lipids, various types of zeolites were investigated in a fixed-bed column system, where a two-step desorption operation mode was found to be superior in selective separation of GLA. By this system, GLA ethyl ester of 98 mol% purity was obtained from a mixture of various polysaturated and unsaturated fatty acid esters using cesium Y and methyl-, dimethyl- and ethylammonium Y zeolites.
C o n s i d e r a b l e interest has arisen from the p h a r m a c o l o g i c a l v i e w p o i n t in r e c e n t years in the p r o d u c t i o n o f y-linolenic a c i d ( G L A ; 6,9,12-oetadecatrienoie acid), since it is a n i m p o r t a n t i n t e r m e d i a t e p r o d u c t in the biosynthesis o f p r o s t a g l a n d i n s from linoleic acid. M u c h research has been d i r e c t e d t o w a r d s the d e v e l o p m e n t o f m i c r o b i a l p r o d u c t i o n o f GLA.x-6) R e c e n t l y , F u k u d a a n d M o r i k a w a 7) h a v e d e v e l o p e d a secretory f e r m e n t a t i o n process using nonionic surfactants a n d cells i m m o b i l i s e d in biomass s u p p o r t particles. T h i s g r e a t l y i m p r o v e d b o t h the p r o d u c t i v i t y a n d the yield of G L A , since m i c r o b i a l lipids i n c l u d i n g G L A were significantly secreted into the c u l t u r e b r o t h a n d / o r on t h e surface o f the cell wall. G e n e r a l l y , the use o f G L A in either p h a r m a c e u t i c a l or foodstuff a p p l i c a t i o n s requires a c o m p l i c a t e d purification process following t h e f e r m e n t a t i o n step, since microb i a l lipids c o n t a i n a large a m o u n t o f v a r i o u s fatty acids. T h e r e a r e several m e t h o d s for G L A purification in i n d u s t r i a l use: the u r e a a d d u c t , 8) the silver-complex s) a n d the supercritical fluid e x t r a c t i o n 9) m e t h o d s . H o w e v e r , the first involves several repetitions o f the o p e r a t i o n , t h e second involves the use o f very expensive silver n i t r a t e , a n d n e i t h e r r e a d i l y afforts high p u r i f i c a t i o n of G L A ,
while the t h i r d m e t h o d requires i n v e s t m e n t cost. T h e w o r k p r e s e n t e d in this p a p e r is based on the selective s e p a r a t i o n o f G L A using zeolite. By a system using c a t i o n - e x c h a n g e d Y-zeolite/fixed-bed c o l u m n , G L A ethyl ester o f 98 m o l % p u r i t y was s i m p l y o b t a i n e d in a single o p e r a t i o n . M a t e r i a l s and M e t h o d s A d s o r b e n t s and reagents The adsorbents used in this study were as follow: sodium A- and X-zeolites (Molecular Sieves 4A and 13X, Union Showa K.K., Japan); sodium Y-zeolite (TSZ-320 NAD, Toyo Soda Ind. Co. Ltd., Japan); potassium L-zeolite (TSZ-500 KOA, Toyo Soda Ind. Co. Ltd., Japan) ; sodium mordenite (TSZ-600 NAA, Toyo Soda Ind. Co. Ltd., Japan); Silicagel (Wakogel C-100, Wako Pure Chemical Ind., Ltd., Japan); Alumina (Aluminium Oxide, Nakarai Chemicals, Ltd., Japan). All of these adsorbents were dried at 160°C in an oven for 3 h before use. Sodium A-, X- and Y-zeolites and alumina were crushed and sized to 24 to 32 mesh. Commercial potassium L-zeolite (powder), sodium mordenite (powder) and silica gel (40-100 mesh) were used without sizing. The composition of the mixture of fatty acid ethyl esters (approx. 99%, Sigma Chemical Co., U.S.A.) used is shown in Table 1. The mixture was dissolved respectively in n-heptane and n-hexane (G.R. grades, Nakarai Chemicals, Ltd., Japan) in shake-flask and fixed-bed experiments. This mixture is very similar in composition to that obtained by microbial pro-
570
ARAI, FUKUDA, and MORIKAWA
[J. Ferment. Technol.,
Table 1. Composition of fatty acid ethyl ester mixture. Fatty acid Palmitic acid Palmitoleic acid Stearic acid Oleic acid Linoleic acid 7,-Linolenic acid
No. of carbon atoms
No. of double bonds
Percentage [tool o/o]
16 16 18 18 18 18
0 1 0 1 2 3
2.9 7.5 1.6 51.3 25.9 10. 8
duction of Mucor arabiguus IFO 67422) Preparation of cation-exchanged Y-zeolites A series of cation-exchanged Y-zeolites were prepared by the following procedure. About 5 g of a sodium Y-zeolite was dispersed in 100 ml of an aqueous solution of the desired cation chloride or nitrate (1.0 N), and this was shaken at 80°C for 24 h. The zeolite was then separated by filtration, washed with distilled water to completely remove the excess salts, and dried at 160°C for 90rain. A protonated Y-zeolite (HYzeolite) was prepared in a different way, by calcination of an ammonium Y-zeolite (NH4Y zeolite) obtained by bringing Y-zeolite into contact with an ammonium chloride solution. The degree of ion exchange was determined by quantitative analysis of sodium using flame spectrometry. Batch operation in shake flasks To investigate the selectivity of adsorption of GLA ethyl ester, shake-flask experiments were performed with various types of zeolites, silica gel and alumina. The mixture of fatty acid ester (500 rag) shown in Table 1 was dissolved in 30 ml of n-heptane in a 50-ml Erlenmeyer flask, then 1.0 g of adsorbent was added. Shaking was continued at 25°C until no change in composition of the solution was observed. Selective separation o f GLA in fLied-bed column system Figure 1 shows a schematic flow diagram of the fixed-bed column system used for selective separation of GLA. A mixture of around 2.5g of Y-zeolite and n-hexane was packed in a glass column of 1.0cm in inside diameter and 10cm in height (5). The mixture of fatty acid esters (i0 g) shown in Table 1 was dissolved in 100 ml of n-hexane, and this was fed from a storage bottle (1) to the column via a peristaltic pump (4). This feed was continued at a liquid hourly space velocity (LHSV) of about 1.0 until no change in composition of the effluent was observed. In the desorption steps, first, n-hexane was fed as a desorbent at the same feed rate as above until the concentration of GLA ethyl ester at the outlet became
® ® I 1 Fig. 1. A schematic diagram of the fixed-bed column system. 1. storage bottle (feed mixture); 2. storage bottle (desorbent); 3. valve; 4. peristaltic pump; 5. column; 6. adsorbent; 7. temperature controller; 8. water bath; 9. circulation pump; 10. peristaltic pump; 1 I. fraction collector. less than 0.1% of that in the feed mixture. Secondly, a complete desorbent operation was carried out with a mixture of n-hexane/ethanol ( 2 0 0 : 1 v/v). All samples were collected in an automatic fraction collector (11) via a peristaltic pump (10). Column temperature was maintained at 30°C by passing water through a jacket. Repeated regeneration of CsY-zeolite was investigated with drying at 160°C for 30 rain. A n a l y s i s o f f a t t y acid ethyl esters Fatty acid ethyl esters were quantitatively analyzed by a gas chromatograph (Shimadzu Seisakusho Ltd., Japan, Model GC-7A) fitted with a flame ionization detector, and the operating conditions were as follows: column, 3 . 0 m m × 3 . 1 m glass column packed with Unisole 3000 (Gasukuro Kogyo Inc., J a p a n ) ; column temperature, 210°C; temperature of injector and detector, 270°C; carrier gas, nitrogen (flow rate; 30ml]min). A digital integrator (Shimadzu Seisakusho Ltd., Japan, Type Chromatopac C-RIA) was used for quantitive analysis.
Results
and Discussion
Effects of zeollte type on GLA adsorption The results of GLA ethyl ester adsorption with various adsorbents in shake flasks a r e g i v e n i n T a b l e 2. I n t h i s T a b l e , the amounts of adsorbed GLA ethyl ester per u n i t a m o u n t o f a d s o r b e n t , q, a n d t h e e q u i l i b r i u m c o n s t a n t , S, a r e g i v e n r e s p e c t i v e l y b y the following equations.
q=(Qt-Qo)/W
(1)
Vol. 65, 1987]
571
Separation o f y - L i n o l e n i c Acid Ester T a b l e 2.
Adsorption o f y - l i n o l e n i c acid ethyl ester onto various adsorbents. SiOs/AlsOa
ratio
Largest pore[A]xa~ opening
1.06
2. 0 x0)
4. 0
5.60
2. 5 to)
7.4
17.3
5. 3 tl~
7.4
4. 90X 10-~
3. 30
6. 0 lt~
7.1
4. 10X 10-~
3. 43
9. 8 li)
7.0
Silica gel
8. 3 3 × 10-z
1.85
--
--
Alumina
4. 4 9 × 10-~
1.72
--
--
Adsorbent
q [mol/kg]
N a - A zeolite
9. 0 0 X 10- s
N a - X zeolite
1 . 2 8 × 10- t
N a - Y zeolite
1 . 5 4 × 10-1
K - L zeolite Na-Mordenite
s = [XoLA]A/[XoL,]U
(2)
where Qi, Q.e and W represent the moles of GLA ethyl ester at the initial and equilibrium values and the weight of adsorbent in a solution, respectively, and [XoL^]A and [XorA]u represent the adsorbed and unadsorbed mole fractions of GLA ethyl ester, respectively. It can be seen from Table 2 that GLA ethyl ester was selectively adsorbed by most adsorbents, and that the zeolites, except for sodium A type, showed higher selectivity than silica gel and alumina. O f the various zeolites, sodium X- and Y-zeolites (Faujasite structure type) were superior in terms of both q and S. Comparing sodium X- and Y-zeolites, the selectivity of Y was around three times that of X. Their SiOs/A1208 ratios are significantly different in spite of their having the same crystalline structure. Therefore, a high value of the ratio, Is) a relatively hydrophobic property, may enhance the GLA ester adsorption performance. Time courses of adsorption of various fatty acid ethyl esters onto sodium Y-zeolite are shown in Fig. 2. Figure 2 shows that each component was adsorbed without significant difference at the initial stage, but that a clear difference in the mole percentages of adsorbed fatty acid esters arose with time. It is worth noting that the percentages of C l s acid esters adsorbed increased with increasing number of double bonds in the acid, and this trend was also found with C16.
S
lOO
Zso
~ 60 .....-ll
~ 4o
......
f...--,
~ 20 r
~,
~-.~.__
2
4 6 $ Shaking time [ h i
". . . . . . . .
e--
10
12
o 14
Fig. 2. Adsorption of various fatty acid ethyl esters onto NaY-zeolite. Symbols: 0 , y-linolenic acid;
O, linoleic acid;
A, oleic acid; ~, stearic acid; A, palmitoleic acid; 0 , palmitic acid.
Effects of ion-exchanged Y-zeolites in fixed-bed system The results of GLA ethyl ester adsorption using ion-exchanged Y-zeolites in the fixed-bed column system are listed in Table 3. It shows that the alkaline metal cations other than Li+ gave the highest amounts and mole percentages of GLA ethyl ester in desorbent, whereas the alkaline earth and transition metal cations gave significantly lower values. Among the alkaline metal cations, it should be noted that 9 8 m o l percentage of GLA ethyl ester was obtained with the Cs + cation. In general, adsorption of fatty acid ester onto zeolite is affected by surface acidity, electrostatic field and surface structure (ionic radius). It is well known that surface
ARAX,FUKUDA,and MOmKAWA
572
[J. Ferment. Technol.,
Table 3. Adsorption of y-linolenic acid ethyl ester onto various types of ion-exchanged Y-zeolite. Cation
Degree of exchange [%]
Amount of GLA* [mol/kg]
Purity of GLA** [mol%]
Electrostatic fieldt6,16)
radiust6, t6)
Alkaline metal
Li Na K Rb Cs
70 -81 64 65
0 8. 91X 10-2 6. 85X 10-2 6.76X 10-2 8. 14)<10-2
-71 83 81 98
2. 1 1.3 1.0 0. 8 0. 6
0. 6 0. 95 1.33 1.48 1.69
Alkaline earth metal
Mg Ca Sr Ba
71 76 71 70
0 6. 91 × 10-2 1.21X10 -2 4. 59X 10-2
-13 14 19
4. 9 3.8 3.2 2.8
0. 65 0. 99 1.13 1.35
Transition metal
Mn Co Cu Ag
73 73 66 92
0 0 1.21 × 10-2 1.67 × 10-2
--55 22
4. 8 4. 9 5.4 --
0. 80 0. 78 0. 69 --
Group
Ionic
w/A]
[A]
* Amount of GLA ethyl ester per unit weight of zeolite desorbed in the second step. ** Mole percentage of GLA ethyl ester recovered from the second step.
acidity of zeolite appears w h e n the s o d i u m ion in s o d i u m Y-zeolite is exchanged for such ions as Li+, Can+ a n d M g 2+.14) T a b l e 3, however, indicates t h a t the mole percentages of G L A ethyl ester o b t a i n e d with these cations were significantly lower t h a n those of N a + a n d K+, which have low surface acidity, t4) a n d t h a t there was no significant correlation b e t w e e n the electrostatic field a n d the selectivity of G L A ethyl ester. Thus, surface acidity does n o t seem directly to affect the selectivity of G L A ethyl ester. A l t h o u g h Taylort4) correlated the electrostatic field strength on Y-zeolite with the adsorbed a m o u n t of oleic acid, this m a y d u e to the difference b e t w e e n carboxylic acid a n d ester. T h e ionic radius v s . mole percentage of G L A ethyl ester shown i n T a b l e 3 is plotted i n Fig. 3, which shows that the selectivity increased with increasing ionic radius i n the alkaline a n d alkaline e a r t h m e t a l groups, with the exception of Li + a n d Mg2+. W i t h the t r a n s i t i o n m e t a l groups, however, the reverse correlation was observed. O n the other h a n d , there was no clear relationship b e t w e e n the ionic radius a n d the a m o u n t of G L A , as shown in T a b l e 3. I t is very
100 ~aG
Nay
~a ORb
~, 0 60 "6 ,s 4O u ~ 20
Ca Sr~a... ""
...0--0"
u~ 0
, C=
0
0.5 Ionic
'
1.0 radius [A]
1.5
2.0
Fig. 3. Ionic radius versus mole percentage of GLA ethyl ester. interesting that ionic radius affects G L A selectivity, a n d a further detailed investigation of this p h e n o m e n o n is required. T h e c o n c e n t r a t i o n s of various fatty acid ethyl esters are plotted against elution v o l u m e for the CsY-zeolite c o l u m n i n Fig. 4 (see T a b l e 3). T h e figure shows t h a t the ethyl esters of oleic, linoleic a n d other fatty acids
Vol. 65, 1987]
Separation of~,-Linolenic Acid Ester Adsorption Desorption .(solv. n-h@xarw) ¢ .'(,,
)."
573
(soW. O.SvOl%eth=nol )
1sol
E
o
03 0.2 0.3 0.~; Etution volume [ I ) Fig. 4. Concentrations of various fatty acid ethyl esters versus elution volume for CsY-zeolite column. Symbols: O, 7-1inolenic acid; @, linoleic acid; z~, oleic acid; ~, stearic acid. (data not shown) adsorbed in the adsorption stage were almost all completely desorbed in the following first desorption step using n-hexane (elution volume of 0.1-0.3 l). But only about 300/o of the adsorbed G L A ethyl ester was desorbed in this step, while the rest was almost completely desorbed in the second step. T h e amounts of G L A ethyl ester desorbed per unit weight of zeolite in the first and the second steps were 3.45 × 10-~mol/kg and 8.14×10 -2 mol/kg, respectively. Thus, G L A of 98 m o l % purity was obtained by the recovery of desorbed fatty acid esters in the second step, and the yield coefficient of G L A ethyl ester based on the ratio of the amount recovered in the second step to that adsorbed in the first
stage was 7 0 % . T h e results of G L A ethyl ester adsorption on Y-zeolites ion-exchanged with a m m o n i u m and alkylammonium ions are shown in Table 4. T h e mole percentages of G L A obtained with methyl-, dimethyl- and ethyla m m o n i u m cations reached 98%, although the amounts of GLA were relatively low compared with that of CsY-zeolite. Also, the combination of a m m o n i u m and methylor ethyl group reduced the amount of G L A ethyl ester. T h e effects of ionic radius, surface acidity and electrostatic field were not investigated. However, the values of ionic radius of methyl-, dimethyl- and e t h y l a m m o n i u m seem to be greater than that of NH4 + ( 1 . 4 8 A l o ) . Therefore, the
Table 4. Adsorption of y-linolenic acid ethyl ester with protonated, ammonium and alkylammonium Y-zeolites. Cation
Degree of exchange [mol% ]
Amount of GLA in desorbent [mol/kg]
Mole percentage of GLA in desorbent [tool% ]
H
80
0
--
NH4 CHaNHa (CHs) =NHs C=HsNHs (Cd-Is)d~Hs
80 75 67 72 58
6. 45)< 10 -2
73 98 98 98 --
4. 48)< 10-~ 2. 63 × 10-~ 1.76 X 10-~ 0
574
ARAI, FtrgtmA, and MORXgAWA
10(
100
8<
8o~
06 %
20 "
2
zeolite/fixed-bed system are worth noting: It is highly advantageous to be able to purify G L A ethyl ester using only a single desorption step. Generally, several operations are required with the urea-adduct method, and this leads to decreased yield of G L A in the purification process. The cation-exchanged Y-zeolites used in this study can be prepared simply and are re-usable without change of G L A selectivity. Nomenclature
0
I
I
2 4 0 Number of regot~eration times
0 10
Fig. 5. Effect of number of regeneration times on GLA adsorption on CsY-zeolite. Symbols: ~ , amount of GLA; ©, mole percentage of GLA. high selectivity of G L A ethyl ester in these cases also m a y be due to the effect of ionic radius to some degree (see the monovalent cations in Fig. 3). Effects of regeneration
of CsY-zeolite
Figure 5 shows the adsorption of G L A ethyl ester against the n u m b e r of times CsY-zeolite was regenerated. T h e amount of G L A ethyl ester gradually decreased with increasing n u m b e r of regenerations, and after four times it remained almost constant at a value of around 50%. O n the other hand, the selectivity of G L A ethyl ester did not change during the eight repeated operations. Therefore, CsY-zeolite prepared in this study is superior in adsorption and stability, and this suggests that it is a suitable adsorbent for the purification of G L A ethyl ester in industrial operation. Conclusions
To attain a highly purified GLA, various types of zeolite were investigated, G L A ethyl ester of 98 m o l % was obtained from a fixed-bed column system using CsY-zeollte and methyl-, dimethyl- and ethylammonium Y zeolites. A two-step desorption operation using two different solvents was found to be superior in selective separation of GLA. Several general points concerning this
q
: adsorbed amount of G L A ethyl ester per unit amount of adsorbent, mol.kg -1 Q, : w e i g h t of G L A ethyl ester at equilibrium time, mol Qi : w e i g h t of G L A ethyl ester at initial time, mol S : equilibrium constant [XGLA]A : adsorbed mole fraction of G L A ethyl ester [X~LA]U : unadsorbed mole fraction of G L A ethyl ester W : weight of adsorbent, kg References
1) Shaw, R. : Biochim. Biophys. Acta, 98, 230 (1965). 2) Tryrell, D. : Can. J. Micro, 13, 755 (1967). 3) Mumma, R.O., Fergus, C.L., Sekura, R.D.: Lipids, 5, 100 (1970). 4) Deven, J.M., Manocha, M.S.: Can. J. Microbiol., 22, 443 (1976). 5) Hoffmann, B., Rehm, H.J.: Eur. J. Appl. Microbiol., 5, 189 (1978). 6) Suzuki, O., Yokochi, T., Yamashina, T.: Yukagaku, 30, 863 (1981). 7) Fukuda, H., Morikawa, H.: Appl. Microbiol. Biotecknol. (in press). 8) U.K. Patent No. 1240513 (1971) 9) Sako, T., Yokochi, T., Suzuki, O., Hakuta, T., Sato, M.: Bio Industry, 3, 750 (1986). 10) Ogawa, M.: Zeolite, 1, 7 (1984). 11) Nakano, M., Hironaka, T., Fujii, S., Sekizawa, K. : Toyosoda Kenkyu Hokoku, 29, 3 (1985). 12) Meier, W. M., Olson, D.H.: Zeoraito, (Hara, N., Takahashi, H.), Vol. 6, p. 6, Kodansha, Tokyo (1984). 13) Naraba, S., Hosonuma, S., Yashima, T.: J. Catal., 72, 16 (1981). 14) Taylor, D.R., Ungermann, C.B.: JAOCS, 61, 1372 (1984). 15) Ward, J.W.: J. Catal., 10, 34 (1968). 16) Ward, J . W . : J. Catal., 22, 237 (1971). (Received March 16, 1987)
Selective Separation of 7-Linolenic Acid Ethyl Ester Using Y-Zeolite MAKOTO ARAI, HIDEKI FUKUDA, a n d HISASHI MORIKAWA
Kanegafuehi ChemicalIndustry Co., Ltd., 1-8 M~amae, Takasago,Hyogo 676, Japan
To highly purify y-linolenic acid (GLA) from microbial lipids, various types of zeolites were investigated in a fixed-bed column system, where a two-step desorption operation mode was found to be superior in selective separation of GLA. By this system, GLA ethyl ester of 98 mol% purity was obtained from a mixture of various polysaturated and unsaturated fatty acid esters using cesium Y and methyl-, dimethyl- and ethylammonium Y zeolites.
C o n s i d e r a b l e interest has arisen from the p h a r m a c o l o g i c a l v i e w p o i n t in r e c e n t years in the p r o d u c t i o n o f y-linolenic a c i d ( G L A ; 6,9,12-oetadecatrienoie acid), since it is a n i m p o r t a n t i n t e r m e d i a t e p r o d u c t in the biosynthesis o f p r o s t a g l a n d i n s from linoleic acid. M u c h research has been d i r e c t e d t o w a r d s the d e v e l o p m e n t o f m i c r o b i a l p r o d u c t i o n o f GLA.x-6) R e c e n t l y , F u k u d a a n d M o r i k a w a 7) h a v e d e v e l o p e d a secretory f e r m e n t a t i o n process using nonionic surfactants a n d cells i m m o b i l i s e d in biomass s u p p o r t particles. T h i s g r e a t l y i m p r o v e d b o t h the p r o d u c t i v i t y a n d the yield of G L A , since m i c r o b i a l lipids i n c l u d i n g G L A were significantly secreted into the c u l t u r e b r o t h a n d / o r on t h e surface o f the cell wall. G e n e r a l l y , the use o f G L A in either p h a r m a c e u t i c a l or foodstuff a p p l i c a t i o n s requires a c o m p l i c a t e d purification process following t h e f e r m e n t a t i o n step, since microb i a l lipids c o n t a i n a large a m o u n t o f v a r i o u s fatty acids. T h e r e a r e several m e t h o d s for G L A purification in i n d u s t r i a l use: the u r e a a d d u c t , 8) the silver-complex s) a n d the supercritical fluid e x t r a c t i o n 9) m e t h o d s . H o w e v e r , the first involves several repetitions o f the o p e r a t i o n , t h e second involves the use o f very expensive silver n i t r a t e , a n d n e i t h e r r e a d i l y afforts high p u r i f i c a t i o n of G L A ,
while the t h i r d m e t h o d requires i n v e s t m e n t cost. T h e w o r k p r e s e n t e d in this p a p e r is based on the selective s e p a r a t i o n o f G L A using zeolite. By a system using c a t i o n - e x c h a n g e d Y-zeolite/fixed-bed c o l u m n , G L A ethyl ester o f 98 m o l % p u r i t y was s i m p l y o b t a i n e d in a single o p e r a t i o n . M a t e r i a l s and M e t h o d s A d s o r b e n t s and reagents The adsorbents used in this study were as follow: sodium A- and X-zeolites (Molecular Sieves 4A and 13X, Union Showa K.K., Japan); sodium Y-zeolite (TSZ-320 NAD, Toyo Soda Ind. Co. Ltd., Japan); potassium L-zeolite (TSZ-500 KOA, Toyo Soda Ind. Co. Ltd., Japan) ; sodium mordenite (TSZ-600 NAA, Toyo Soda Ind. Co. Ltd., Japan); Silicagel (Wakogel C-100, Wako Pure Chemical Ind., Ltd., Japan); Alumina (Aluminium Oxide, Nakarai Chemicals, Ltd., Japan). All of these adsorbents were dried at 160°C in an oven for 3 h before use. Sodium A-, X- and Y-zeolites and alumina were crushed and sized to 24 to 32 mesh. Commercial potassium L-zeolite (powder), sodium mordenite (powder) and silica gel (40-100 mesh) were used without sizing. The composition of the mixture of fatty acid ethyl esters (approx. 99%, Sigma Chemical Co., U.S.A.) used is shown in Table 1. The mixture was dissolved respectively in n-heptane and n-hexane (G.R. grades, Nakarai Chemicals, Ltd., Japan) in shake-flask and fixed-bed experiments. This mixture is very similar in composition to that obtained by microbial pro-
570
ARAI, FUKUDA, and MORIKAWA
[J. Ferment. Technol.,
Table 1. Composition of fatty acid ethyl ester mixture. Fatty acid Palmitic acid Palmitoleic acid Stearic acid Oleic acid Linoleic acid 7,-Linolenic acid
No. of carbon atoms
No. of double bonds
Percentage [tool o/o]
16 16 18 18 18 18
0 1 0 1 2 3
2.9 7.5 1.6 51.3 25.9 10. 8
duction of Mucor arabiguus IFO 67422) Preparation of cation-exchanged Y-zeolites A series of cation-exchanged Y-zeolites were prepared by the following procedure. About 5 g of a sodium Y-zeolite was dispersed in 100 ml of an aqueous solution of the desired cation chloride or nitrate (1.0 N), and this was shaken at 80°C for 24 h. The zeolite was then separated by filtration, washed with distilled water to completely remove the excess salts, and dried at 160°C for 90rain. A protonated Y-zeolite (HYzeolite) was prepared in a different way, by calcination of an ammonium Y-zeolite (NH4Y zeolite) obtained by bringing Y-zeolite into contact with an ammonium chloride solution. The degree of ion exchange was determined by quantitative analysis of sodium using flame spectrometry. Batch operation in shake flasks To investigate the selectivity of adsorption of GLA ethyl ester, shake-flask experiments were performed with various types of zeolites, silica gel and alumina. The mixture of fatty acid ester (500 rag) shown in Table 1 was dissolved in 30 ml of n-heptane in a 50-ml Erlenmeyer flask, then 1.0 g of adsorbent was added. Shaking was continued at 25°C until no change in composition of the solution was observed. Selective separation o f GLA in fLied-bed column system Figure 1 shows a schematic flow diagram of the fixed-bed column system used for selective separation of GLA. A mixture of around 2.5g of Y-zeolite and n-hexane was packed in a glass column of 1.0cm in inside diameter and 10cm in height (5). The mixture of fatty acid esters (i0 g) shown in Table 1 was dissolved in 100 ml of n-hexane, and this was fed from a storage bottle (1) to the column via a peristaltic pump (4). This feed was continued at a liquid hourly space velocity (LHSV) of about 1.0 until no change in composition of the effluent was observed. In the desorption steps, first, n-hexane was fed as a desorbent at the same feed rate as above until the concentration of GLA ethyl ester at the outlet became
® ® I 1 Fig. 1. A schematic diagram of the fixed-bed column system. 1. storage bottle (feed mixture); 2. storage bottle (desorbent); 3. valve; 4. peristaltic pump; 5. column; 6. adsorbent; 7. temperature controller; 8. water bath; 9. circulation pump; 10. peristaltic pump; 1 I. fraction collector. less than 0.1% of that in the feed mixture. Secondly, a complete desorbent operation was carried out with a mixture of n-hexane/ethanol ( 2 0 0 : 1 v/v). All samples were collected in an automatic fraction collector (11) via a peristaltic pump (10). Column temperature was maintained at 30°C by passing water through a jacket. Repeated regeneration of CsY-zeolite was investigated with drying at 160°C for 30 rain. A n a l y s i s o f f a t t y acid ethyl esters Fatty acid ethyl esters were quantitatively analyzed by a gas chromatograph (Shimadzu Seisakusho Ltd., Japan, Model GC-7A) fitted with a flame ionization detector, and the operating conditions were as follows: column, 3 . 0 m m × 3 . 1 m glass column packed with Unisole 3000 (Gasukuro Kogyo Inc., J a p a n ) ; column temperature, 210°C; temperature of injector and detector, 270°C; carrier gas, nitrogen (flow rate; 30ml]min). A digital integrator (Shimadzu Seisakusho Ltd., Japan, Type Chromatopac C-RIA) was used for quantitive analysis.
Results
and Discussion
Effects of zeollte type on GLA adsorption The results of GLA ethyl ester adsorption with various adsorbents in shake flasks a r e g i v e n i n T a b l e 2. I n t h i s T a b l e , the amounts of adsorbed GLA ethyl ester per u n i t a m o u n t o f a d s o r b e n t , q, a n d t h e e q u i l i b r i u m c o n s t a n t , S, a r e g i v e n r e s p e c t i v e l y b y the following equations.
q=(Qt-Qo)/W
(1)
Vol. 65, 1987]
571
Separation o f y - L i n o l e n i c Acid Ester T a b l e 2.
Adsorption o f y - l i n o l e n i c acid ethyl ester onto various adsorbents. SiOs/AlsOa
ratio
Largest pore[A]xa~ opening
1.06
2. 0 x0)
4. 0
5.60
2. 5 to)
7.4
17.3
5. 3 tl~
7.4
4. 90X 10-~
3. 30
6. 0 lt~
7.1
4. 10X 10-~
3. 43
9. 8 li)
7.0
Silica gel
8. 3 3 × 10-z
1.85
--
--
Alumina
4. 4 9 × 10-~
1.72
--
--
Adsorbent
q [mol/kg]
N a - A zeolite
9. 0 0 X 10- s
N a - X zeolite
1 . 2 8 × 10- t
N a - Y zeolite
1 . 5 4 × 10-1
K - L zeolite Na-Mordenite
s = [XoLA]A/[XoL,]U
(2)
where Qi, Q.e and W represent the moles of GLA ethyl ester at the initial and equilibrium values and the weight of adsorbent in a solution, respectively, and [XoL^]A and [XorA]u represent the adsorbed and unadsorbed mole fractions of GLA ethyl ester, respectively. It can be seen from Table 2 that GLA ethyl ester was selectively adsorbed by most adsorbents, and that the zeolites, except for sodium A type, showed higher selectivity than silica gel and alumina. O f the various zeolites, sodium X- and Y-zeolites (Faujasite structure type) were superior in terms of both q and S. Comparing sodium X- and Y-zeolites, the selectivity of Y was around three times that of X. Their SiOs/A1208 ratios are significantly different in spite of their having the same crystalline structure. Therefore, a high value of the ratio, Is) a relatively hydrophobic property, may enhance the GLA ester adsorption performance. Time courses of adsorption of various fatty acid ethyl esters onto sodium Y-zeolite are shown in Fig. 2. Figure 2 shows that each component was adsorbed without significant difference at the initial stage, but that a clear difference in the mole percentages of adsorbed fatty acid esters arose with time. It is worth noting that the percentages of C l s acid esters adsorbed increased with increasing number of double bonds in the acid, and this trend was also found with C16.
S
lOO
Zso
~ 60 .....-ll
~ 4o
......
f...--,
~ 20 r
~,
~-.~.__
2
4 6 $ Shaking time [ h i
". . . . . . . .
e--
10
12
o 14
Fig. 2. Adsorption of various fatty acid ethyl esters onto NaY-zeolite. Symbols: 0 , y-linolenic acid;
O, linoleic acid;
A, oleic acid; ~, stearic acid; A, palmitoleic acid; 0 , palmitic acid.
Effects of ion-exchanged Y-zeolites in fixed-bed system The results of GLA ethyl ester adsorption using ion-exchanged Y-zeolites in the fixed-bed column system are listed in Table 3. It shows that the alkaline metal cations other than Li+ gave the highest amounts and mole percentages of GLA ethyl ester in desorbent, whereas the alkaline earth and transition metal cations gave significantly lower values. Among the alkaline metal cations, it should be noted that 9 8 m o l percentage of GLA ethyl ester was obtained with the Cs + cation. In general, adsorption of fatty acid ester onto zeolite is affected by surface acidity, electrostatic field and surface structure (ionic radius). It is well known that surface
ARAX,FUKUDA,and MOmKAWA
572
[J. Ferment. Technol.,
Table 3. Adsorption of y-linolenic acid ethyl ester onto various types of ion-exchanged Y-zeolite. Cation
Degree of exchange [%]
Amount of GLA* [mol/kg]
Purity of GLA** [mol%]
Electrostatic fieldt6,16)
radiust6, t6)
Alkaline metal
Li Na K Rb Cs
70 -81 64 65
0 8. 91X 10-2 6. 85X 10-2 6.76X 10-2 8. 14)<10-2
-71 83 81 98
2. 1 1.3 1.0 0. 8 0. 6
0. 6 0. 95 1.33 1.48 1.69
Alkaline earth metal
Mg Ca Sr Ba
71 76 71 70
0 6. 91 × 10-2 1.21X10 -2 4. 59X 10-2
-13 14 19
4. 9 3.8 3.2 2.8
0. 65 0. 99 1.13 1.35
Transition metal
Mn Co Cu Ag
73 73 66 92
0 0 1.21 × 10-2 1.67 × 10-2
--55 22
4. 8 4. 9 5.4 --
0. 80 0. 78 0. 69 --
Group
Ionic
w/A]
[A]
* Amount of GLA ethyl ester per unit weight of zeolite desorbed in the second step. ** Mole percentage of GLA ethyl ester recovered from the second step.
acidity of zeolite appears w h e n the s o d i u m ion in s o d i u m Y-zeolite is exchanged for such ions as Li+, Can+ a n d M g 2+.14) T a b l e 3, however, indicates t h a t the mole percentages of G L A ethyl ester o b t a i n e d with these cations were significantly lower t h a n those of N a + a n d K+, which have low surface acidity, t4) a n d t h a t there was no significant correlation b e t w e e n the electrostatic field a n d the selectivity of G L A ethyl ester. Thus, surface acidity does n o t seem directly to affect the selectivity of G L A ethyl ester. A l t h o u g h Taylort4) correlated the electrostatic field strength on Y-zeolite with the adsorbed a m o u n t of oleic acid, this m a y d u e to the difference b e t w e e n carboxylic acid a n d ester. T h e ionic radius v s . mole percentage of G L A ethyl ester shown i n T a b l e 3 is plotted i n Fig. 3, which shows that the selectivity increased with increasing ionic radius i n the alkaline a n d alkaline e a r t h m e t a l groups, with the exception of Li + a n d Mg2+. W i t h the t r a n s i t i o n m e t a l groups, however, the reverse correlation was observed. O n the other h a n d , there was no clear relationship b e t w e e n the ionic radius a n d the a m o u n t of G L A , as shown in T a b l e 3. I t is very
100 ~aG
Nay
~a ORb
~, 0 60 "6 ,s 4O u ~ 20
Ca Sr~a... ""
...0--0"
u~ 0
, C=
0
0.5 Ionic
'
1.0 radius [A]
1.5
2.0
Fig. 3. Ionic radius versus mole percentage of GLA ethyl ester. interesting that ionic radius affects G L A selectivity, a n d a further detailed investigation of this p h e n o m e n o n is required. T h e c o n c e n t r a t i o n s of various fatty acid ethyl esters are plotted against elution v o l u m e for the CsY-zeolite c o l u m n i n Fig. 4 (see T a b l e 3). T h e figure shows t h a t the ethyl esters of oleic, linoleic a n d other fatty acids
Vol. 65, 1987]
Separation of~,-Linolenic Acid Ester Adsorption Desorption .(solv. n-h@xarw) ¢ .'(,,
)."
573
(soW. O.SvOl%eth=nol )
1sol
E
o
03 0.2 0.3 0.~; Etution volume [ I ) Fig. 4. Concentrations of various fatty acid ethyl esters versus elution volume for CsY-zeolite column. Symbols: O, 7-1inolenic acid; @, linoleic acid; z~, oleic acid; ~, stearic acid. (data not shown) adsorbed in the adsorption stage were almost all completely desorbed in the following first desorption step using n-hexane (elution volume of 0.1-0.3 l). But only about 300/o of the adsorbed G L A ethyl ester was desorbed in this step, while the rest was almost completely desorbed in the second step. T h e amounts of G L A ethyl ester desorbed per unit weight of zeolite in the first and the second steps were 3.45 × 10-~mol/kg and 8.14×10 -2 mol/kg, respectively. Thus, G L A of 98 m o l % purity was obtained by the recovery of desorbed fatty acid esters in the second step, and the yield coefficient of G L A ethyl ester based on the ratio of the amount recovered in the second step to that adsorbed in the first
stage was 7 0 % . T h e results of G L A ethyl ester adsorption on Y-zeolites ion-exchanged with a m m o n i u m and alkylammonium ions are shown in Table 4. T h e mole percentages of G L A obtained with methyl-, dimethyl- and ethyla m m o n i u m cations reached 98%, although the amounts of GLA were relatively low compared with that of CsY-zeolite. Also, the combination of a m m o n i u m and methylor ethyl group reduced the amount of G L A ethyl ester. T h e effects of ionic radius, surface acidity and electrostatic field were not investigated. However, the values of ionic radius of methyl-, dimethyl- and e t h y l a m m o n i u m seem to be greater than that of NH4 + ( 1 . 4 8 A l o ) . Therefore, the
Table 4. Adsorption of y-linolenic acid ethyl ester with protonated, ammonium and alkylammonium Y-zeolites. Cation
Degree of exchange [mol% ]
Amount of GLA in desorbent [mol/kg]
Mole percentage of GLA in desorbent [tool% ]
H
80
0
--
NH4 CHaNHa (CHs) =NHs C=HsNHs (Cd-Is)d~Hs
80 75 67 72 58
6. 45)< 10 -2
73 98 98 98 --
4. 48)< 10-~ 2. 63 × 10-~ 1.76 X 10-~ 0
574
ARAI, FtrgtmA, and MORXgAWA
10(
100
8<
8o~
06 %
20 "
2
zeolite/fixed-bed system are worth noting: It is highly advantageous to be able to purify G L A ethyl ester using only a single desorption step. Generally, several operations are required with the urea-adduct method, and this leads to decreased yield of G L A in the purification process. The cation-exchanged Y-zeolites used in this study can be prepared simply and are re-usable without change of G L A selectivity. Nomenclature
0
I
I
2 4 0 Number of regot~eration times
0 10
Fig. 5. Effect of number of regeneration times on GLA adsorption on CsY-zeolite. Symbols: ~ , amount of GLA; ©, mole percentage of GLA. high selectivity of G L A ethyl ester in these cases also m a y be due to the effect of ionic radius to some degree (see the monovalent cations in Fig. 3). Effects of regeneration
of CsY-zeolite
Figure 5 shows the adsorption of G L A ethyl ester against the n u m b e r of times CsY-zeolite was regenerated. T h e amount of G L A ethyl ester gradually decreased with increasing n u m b e r of regenerations, and after four times it remained almost constant at a value of around 50%. O n the other hand, the selectivity of G L A ethyl ester did not change during the eight repeated operations. Therefore, CsY-zeolite prepared in this study is superior in adsorption and stability, and this suggests that it is a suitable adsorbent for the purification of G L A ethyl ester in industrial operation. Conclusions
To attain a highly purified GLA, various types of zeolite were investigated, G L A ethyl ester of 98 m o l % was obtained from a fixed-bed column system using CsY-zeollte and methyl-, dimethyl- and ethylammonium Y zeolites. A two-step desorption operation using two different solvents was found to be superior in selective separation of GLA. Several general points concerning this
q
: adsorbed amount of G L A ethyl ester per unit amount of adsorbent, mol.kg -1 Q, : w e i g h t of G L A ethyl ester at equilibrium time, mol Qi : w e i g h t of G L A ethyl ester at initial time, mol S : equilibrium constant [XGLA]A : adsorbed mole fraction of G L A ethyl ester [X~LA]U : unadsorbed mole fraction of G L A ethyl ester W : weight of adsorbent, kg References
1) Shaw, R. : Biochim. Biophys. Acta, 98, 230 (1965). 2) Tryrell, D. : Can. J. Micro, 13, 755 (1967). 3) Mumma, R.O., Fergus, C.L., Sekura, R.D.: Lipids, 5, 100 (1970). 4) Deven, J.M., Manocha, M.S.: Can. J. Microbiol., 22, 443 (1976). 5) Hoffmann, B., Rehm, H.J.: Eur. J. Appl. Microbiol., 5, 189 (1978). 6) Suzuki, O., Yokochi, T., Yamashina, T.: Yukagaku, 30, 863 (1981). 7) Fukuda, H., Morikawa, H.: Appl. Microbiol. Biotecknol. (in press). 8) U.K. Patent No. 1240513 (1971) 9) Sako, T., Yokochi, T., Suzuki, O., Hakuta, T., Sato, M.: Bio Industry, 3, 750 (1986). 10) Ogawa, M.: Zeolite, 1, 7 (1984). 11) Nakano, M., Hironaka, T., Fujii, S., Sekizawa, K. : Toyosoda Kenkyu Hokoku, 29, 3 (1985). 12) Meier, W. M., Olson, D.H.: Zeoraito, (Hara, N., Takahashi, H.), Vol. 6, p. 6, Kodansha, Tokyo (1984). 13) Naraba, S., Hosonuma, S., Yashima, T.: J. Catal., 72, 16 (1981). 14) Taylor, D.R., Ungermann, C.B.: JAOCS, 61, 1372 (1984). 15) Ward, J.W.: J. Catal., 10, 34 (1968). 16) Ward, J . W . : J. Catal., 22, 237 (1971). (Received March 16, 1987)