647
Ind. Eng. Chem. Res. 1990,29, 647-651 Rajadhyaksha, R. A.; Chaudhari, D. D. Alkylation of Phenol by C9 and CI2 Olefins. Bull. Chem. SOC. Jpn. 1988, 61, 1379-1381. Reed, H. W. B. Alkylation. In Kirk-Othmer Encyclopedia of Chemical Technology; Grayson, M., Ed.; Wiley: New York, 1978. Roberts, R. M.; Khalaf, A. A. Rearrangements, Dealkylation, and Fragmentations of Arenes Induced by Friedel-Crafts Catalysts. In Friedel-Crafts Alkylation Chemistry: A Century of Discouery; Marcel-Dekker: New York, 1984. Rosenwald, R. H. Alkylphenols. In Kirk-Othmer Encyclopedia of Chemical Technology; Grayson, M., Ed.; Wiley: New York, 1978. Ryzhkov. 0. G.; Korenev, K. D.; Kazakov, E. I. The Interrelationship of the Structure and Properties of Mono- and Dialkylcatechols. Khim. Tuerd. Topl. 1975,9 (l), 129-133. Saunders, M.; Hagen, E. L.; Rosenfeld, J. Rearrangement Reactions of Secondary Carbonium Ions. Protonated Cyclopropane Inter-
mediates Formed from sec-Butyl Cation. J . Am. Chem. SOC. 1968, 90, 6882-6884.
Schmerling, L.; West, J. P. Isomerization Accompanying Alkylation of Benzene with 1-Chloro-3,3- and with 2-Chloro-2,3-Dimethylbutane. J . Am. Chem. SOC. 1954, 76, 1917-1921. Shrewsbury, D. D. The Infra-Red Spectra of Alkyl Phenols. Spectrochim. Acta 1960, 16, 1294-1311. Stanford Research Institute. Chemical Economics Handbook; Stanford Research Institute: Menlo Park, CA, Oct 1988. Varian Associates. High Resolution NMR Spectra Catalog; Varian: Palo Alto, CA, 1962; Vol. 1, Spectrum 315. Vogel, P. Carbocation Chemistry; Elsevier: New York, 1985. Received for review J u n e 13, 1989 Accepted November 30, 1989
Liquid Chromatographic Evaluation of Equilibrium and Kinetic Parameters of Large Molecule Amino Acids on Silica Gel Mohammad S. Uddin, Kus Hidajat, and Chi-Bun Ching* Department of Chemical Engineering, National University of Singapore, Kent Ridge, Singapore 051 1
Sorption and diffusion of four large-molecule amino acids (glutamine, methionine, phenylalanine, and tryptophan) in a uniform pore size silica gel adsorbent have been studied by the chromatographic method. All four species were adsorbed quite rapidly but with significant differences in both pore diffusivity and equilibrium constant. The diffusivities show a regular decrease with increasing molecular weight, but the variation in equilibrium constants and heats of sorption was less regular. Tortuosity factors calculated from the pore diffusivities are consistent with a molecular diffusion mechanism with some evidence of steric hindrance for the larger molecules. The potential of such adsorbents as a means of separating amino acids is briefly considered. Analysis and separation of amino acids are generally accomplished by techniques such as ion-exchange chromatograpy, reverse-phase HPLC, etc., which require special equipment and procedures and are difficult to scale up to a commercial operation. The possibility of carrying out such separations by selective adsorption is, in principle, attractive, provided that a suitably selective adsorbent can be found, since the design and scale-up of an adsorption system are relatively straightforward. To assess the viability of such a process, adsorption kinetic and equilibrium data are required, but there have been few reported studies of the behavior of amino acids on commercial adsorbents. Ching and Ruthven (1989) studied the adsorption of three of the lighter homologues (glycine, lysine, and alanine) on KX zeolite, and these studies were later extended to cysteine, threonine, and serine (Ching et al., 1989b) on the same adsorbent. The pore diameter of the X or Y zeolites (7.4 A) is, however, too small to admit larger amino acids so that phenylalanine, for example, was excluded. To separate the larger molecules therefore requires a larger pore adsorbent. One possibility is to use the recently developed monodisperse silica gel adsorbents which have a uniform pore size in the 20-40-A range. Such materials offer the possibility that the equilibrium selectivity may be enhanced by kinetic effects arising from sterically hindered diffusion. In the present study, we therefore investigated the sorption and diffusion of glutamine, methionine, phenylalanine, and tryptophan in a silica gel adsorbent. The results suggest that steric effects are of only minor importance, even for a relatively large molecule such as tryptophan. Differences in adsorption equilibria are sig-
* To whom
correspondence should be addressed.
0888-5885/9Q/2629-0647$02.50/0
nificant but probably not large enough for an economic separation process. Experimental Section The apparatus used in this study consists of a jacketed stainless steel column of internal diameter 1.1 cm and of height 10.0 cm. The column was fully packed with commercial silica gel supplied by Sigma Chemicals (S-4883). The gel particles, which were sieved to an average diameter of 509 pm, had the following physical properties (determined by nitrogen adsorption using the Quantasorb instrument manufactured by Quantachrome): mean pore diameter, 27 A; specific surface area, 680 m3/g; particle density, 1.09 g/cm3. The eluent (water) was degassed by using an ultrasonic bath and delivered to the column through a microporous filter by an HPLC pump. Injection of sample into the eluent was performed with a Rheodyne Type 7125 sampling valve fitted with a 200-pL loop. The output signal was detected by a refractive index detector (HewlettPackard Model 1037A). The signal from the RI detector was processed by a Hewlett-Packard Model 3497A data acquisition/control unit which performed the logging of the chromatographic response data and computation of the first moment and HETP values. The pulse samples for the experiments were 1 wt 70of the amino acids, glutamine, methionine, phenylalanine, and tryptophan (products of Sigma Chemicals). Starch and D,O solutions were used to estimate the bed voidage and axial dispersion contribution. Theoretical Section The chromatographic response curves were analyzed by the method of moments. For a column packed with silica 62 1990 American Chemical Society
648 Ind. Eng. Chem. Res., Vol. 29, No. 4, 1.990 Table I. S u m m a r y of Equilibrium a n d Kinetic P a r a m e t e r s sorbate (MW)
molec structure
F!II
glutamine (146)
HzNCCH&H$HCOOH
I
T,K
K
-AH, kcal/mol
293 310 328
0.83 0.78 0.75
0.59
293 310 3.28 293 310 328
1.41 1.12 0.93 1.87 1.36 1.12
2.26
293 310 328
0.99 0.86
106D cm!2/
E, kcal/mol
106D,, cm*/s
T
1.83 2.90 4.01
4.28
7.08 10.18 15.24
2.39 2.18 2.36
1.61 2.53 3.67 1.21 1.83 2.96 1.10 1.65 2.70
4.50
6.87 9.89 14.80 6.36 9.15 13.70 5.75 8.27 12.38
2.65 2.42 2.50 3.10 3.10 2.87 3.24 3.11 2.84
N HZ
methionine (149)
CH~SCH&HZCHCOOH I
NH2
ecH,,moH
phenylalanine (165)
NHZ
tryptophan (204)
1.22
cJH -Jfo$o;H H
2.78
1.94
gel, the first and second moments of the pulse response are related to the adsorption equilibrium constant ( K )and the macropore diffusional time constant ( D p / R p 2by ) the following equations (Ruthven, 1984): x m c tdt =
c1=
x m cdt U‘
-L
E
L12
HETP
-[
R. +
2DL u + 1 - € 3kf
&I[
-[€
€V
+ (1- € ) K ]
160
+
4.89
I
I
(1)
d’
i 1
4.61
eK]
l 7
P
t P
-2
(2)
40:
/
P
20
where u2 1
Jmc(t - ~
1 d) t /~J m c
dt
0
In a liquid-phase system, the contribution to the HETP arising from axial dispersion (2DL/u)is essentially independent of fluid velocity, especially in the low Reynolds number regime (Bischoff, 1960). It follows from eq 2 that a plot of HETP versus fluid velocity should be linear with the intercept corresponding to the axial dispersion (U>L/u) and the slope giving the overall mass-transfer resistance. At low Reynolds number, as in the present study, the external mass-transfer resistance may be estimated from Wakao and Funazkri’s (1978) correlation. If the term R , / ( 3 k f ) in eq 2 is much smaller than the term RY2/ (15t&,), the slope from the plot of HETP versus velocity will represent the mass-transfer resistance from macropore diffusion.
Results and Discussion In calculation of the moments of the experimental response curves, a small correction was made to allow for the holdup and dispersion in the detector (volume 2.3 cm3). This correction was determined directly from pulse response measurement with the column removed from the system. The amino acid samples were of very low concentrations (1 w t %) and were further diluted in the column since the sample volume used was very small compared to the column volume. Therefore, system linearity with diffusivity independent of concentration can be assumed. A linear fit of the equilibrium data to eq 1,as shown later, confirms this assumption. Bed Voidage and Axial Dispersion. Starch is a high molecular weight carbohydrate, and the molecule is too large to penetrate the micropore of the silica gel adsorbent. i=
40
80
120
160
200
240
280
1 Wcm) E
Figure 1. Variation of first moment for unit bed height ( p / L ) with inverse of superficial liquid velocity ( l / t V ) for starch a t 293 K.
A plot of p / L versus l / t u should therefore yield directly the bed voidage ( E ) , as follows from eq 1with K = 0. Such a plot is shown in Figure 1, and the corresponding voidage is 0.51. In the other extreme limit, the D20 molecule is small enough to penetrate freely with very little mass-transfer resistance. Studies of dispersion in packed beds have shown that axial dispersion in a liquid system is always dominated by eddy mixing effects, with the result that DL is approximately proportional to fluid velocity and independent of the molecular diffusivity of the sorbate. Under conditions of axial dispersion control, the HETP is given by DL/u and should therefore be essentially constant and the same for all sorbates. The extent to which the experimental data conform to the expected trend may be judged from Figure 2. The figure shows some scatter in the experimental data. We believe that it does not show any significant effect of u on HETP. This has also been observed by Ching and Ruthven (1989) and Ching et al. (1989a). Equilibrium Data. The plots of the first moment as p / L against the reciprocal of the superficial velocity (l/tu) for all four amino acids were found to be linear and temperature dependent. Figure 3 shows a typical plot for phenylalanine. The adsorption equilibrium constants ( K ) , summarized in Table I, were derived from the slopes of the plots for the different sorbates according to eq 1. The differences in the K values between the four amino acids
Ind. Eng. Chem. Res., Vol. 29, No. 4, 1990 649 0.8
0.6
1 P
.
i
0
0.2
1-
i
i
j
1
-0.4
-0.6 3.0
0.000
0.008
3.2
3.1
3.4
3.3
0.024
0.016
1000/T ( l / K )
v (cm/s)
F i g u r e 2. H E T P versus interstitial liquid velocity ( V )for D20a t 293 K.
Figure 4. Temperature dependence of equilibrium constants: (0) glutamine, (0)tryptophan, ( 0 )methionine, (A)phenylalanine. 0.7
f
500
,Y
400
0.5 -
/
5
300
\
n
c
1
w
i a 200
/
03-
/
, ’
0,
,/p 4’
/
02-
, /
/f
100
01-
I
00 0
0.000 0
40
80
120
160
200
240
0.008
0.016
0.024
180
v (cm/s)
AC V
(s/cm)
Figure 3. Variation of first moment for unit bed height ( p / L )with inverse of superficial liquid velocity (l/cV)for phenylalanine: (0) 293 K, (A)310 K,(0) 328 K.
are small but outside the range of experimental uncertainty. Between glutamine, methionine, and phenylalanine, we see a regular increase in the equilibrium constant with molecular weight, as is to be expected. However, tryptophan, which has the highest molecular weight, has a K value lower than that of phenylalanine. The most obvious explanation is size exclusion from the smaller micropores. Heats of adsorption, calculated according to the van’t Hoff equation (K= KOexp(-AH/RT)) show mildly exothermic adsorption for all species (Figure 4). The difference in polarity may explain the lower K value that is observed for glutamine in comparison with methionine, which has a similar molecular weight. Kinetic Data. For significant mass-transfer resistance, it is expected that HETP should increase linearly with interstitial liquid velocity (eq 2). The gradient and intercept should yield the mass-transfer resistance and axial dispersion term, respectively. The external mass-transfer resistance (R,/3kf) in gel chromatography is generally small and can usually be
Figure 5. Variation of H E T P with interstitial liquid velocity (V) for glutamine: (0) 293 K, (A) 310 K,(0) 328 K.
neglected in comparison with the macropore diffusion resistance (RP/ Ching et al. (1989a) have shown that for silica gel, 5 lC tKD e external resistance is less than 10% of the macropore diffusion resistance. Assuming macropore diffusion to be controlling, the pore diffusivity may be estimated from the slope:
For systems with very rapid and strong adsorption, it is possible that the concentration profde through the particle becomes asymmetric, and this can lead to a significant additional contribution to the axial dispersion arising from direct transport through the solid. This effect particularly occurs with the gas-phase system as observed by Wakao and Funazkri (1978). However, in the present work, the axial dispersion contribution (2DL/u) is assumed to be constant for all the amino acids at the value 0.34 as derived from the D20 experiments. This was subtracted from the measured HETP for the amino acids, and the difference was plotted against the liquid velocity. Reasonably linear plots passing through the origin were obtained. A typical
650 Tnd. Eng. Chem. Res., Vol. 29, No. 4, 1990 Table 11. Tortuosity Factors for Diffusion in Liquid-Filled Pores -sorbate sorbate-solvent method breakthrough curves C6H,,-C6H6 Davison 525 5A moelcular sieve pellets C6H6-CCI4 C C ~ ~ - C ~ H G breakthrough curves Linde 5A molecular sieve pellets C6H6-C6H1'2 transient uptake NaCl-H,O porous Vycor glass
Si02-A1203
NaCl-H20 hydrocarbons
i,, A
€p
T*
-300
0.34
-500
0.33
5.3 -6.1 1.7 -2.3 4.5 2.9 3.1 3.5 2.3 3.2 1.6
25 92 234 476 185 32 57, 66
transient uptake transient uptake
0.23 0.54 0.58 0.60 0.7 0.44 0.6
ref Lee and Ruthven (1977) Lee and Ruthven (1977) Colton et al. (1975)
Satterfield et al. (1973) Prasher and Ma (1977)
I 240
1
0
I 210
I
t - 1 4 . 0 1 -
30
0
6ol
d
30 3.1
3.2
33
3 4
1000/T ( l / K )
Figure 6. Temperature dependence of pore diffusivities: (0) glutamine, ( 0 )methionine, (0) tryptopham, (A)phenylalanine.
plot for glutamine is shown in Figure 5, and the pore diffusivity data are included in Table I. This confirms the validity of the assumption in the present case. A similar conclusion was also made by Ching and Ruthven (1988, 1989). The HETP data points are found to be rather scattered, mainly because the second moment values are generally less consistent than the first moments. Significant differences in diffusivity values are observed, and they decreased regularly with increasing molecular weight. A similar trend was reported by Ching and Ruthven (1989) for smaller molecule amino acids on KX zeolite. The temperature dependence of the D, values for the four amino acids is shown on a Arrhenius plot (Figure 6) and used to estimate the diffusional activation energy. The activation energy does not vary significantly with molecular size; only a slight increase is observed for the higher molecular weight species. For comparison, the free liquid diffusivities of the amino acids are estimated by using the Wilke and Chang (1955) equation. The values are shown in Table I. The effective pore diffusivities are found to be lower than the free diffusivities by a factor of 4-5. The pore and free diffusion coefficients are used to calculate the tortuosity factor of the gel particles from the equation T
= tpDm/Dp
is calculated from the first moment data obtained for p / L versus reciprocal of superficial fluid velocity (Figure 7) is E + (1 - c)cp, since for D20there is no significant adsorption relative to water. This gives E = 0.62. The calcuyated tortuosity values are shown in Table I. For comparison purposes, tortuosity factors for diffusion t
&O. The slope of the plot of
/
'0
40
SO
120
1
160
200
240
280
(s/cm)
Figure 7. Variation of first moment for unit bed height ( p / L ) with inverse of superficial liquid velocity (l/cV) for DzOat 293 K.
in liquid-filled pores extracted from various references are included in Table 11. These tortuosity values are calculated as T* = D,/D,, where E, has not been included. On a similar basis, the toruosity values obtained in the present study would be in the range 3.5-5.2, which are consistent and agree well with the reported values. This agreement provides strong evidence that the surface diffusion makes no significant contribution to the solute transport through the liquid-filled pores (Lee and Ruthven, 1977). Satterfield et al. (1973), Prasher and Ma (1977), and Colton et al. (1975) observed that the ratio of the effective pore diffusivity to bulk diffusivity decreases with an increase in the ratio of the critical molecular diameter to pore diameter, h (=rs/Pp). The h values for glutamine, methionine, phenylalanine, and tryptophan are calculated to be 0.231, 0.256,0.267,and 0.306, respectively. The critical molecular diameters of the amino acids are estimated from the bond lengths and bond angles. The corresponding values of D / D m are 0.269, 0.246, 0.205, and 0.200, respectively. Tfh shows that D,/D, decreases with an increase of A, and it follows that log (DJD,) is approximately inversely proportional to h as observed by Satterfield et al. (1973) and others. Conclusions Application of the liquid chromatographic technique using commercial silica gel to study adsorption equilibrium and diffusional kinetic parameters of large amino acid molecules has been demonstrated. The results show that all the four amino acids, glutamine, methionine, phenyl-
I n d . E n g . Chem. Res. 1990, 29, 651-659
alanine, and tryptophan, are adsorbed quite rapidly, and there are significant differences in equilibrium constants and pore diffusivities. Pore diffusivity decreases with molecular size. The polarity of the functional group and the type of side chain or ring, such as aliphatic or aromatic, are shown to have some influence on the adsorption equilibrium. Tortuosity factors for the gel particles calculated from the pore diffusivity data show that the kinetic data are quite consistent with the assumption that the transport rate is determined primarily by molecular diffusion.
Acknowledgment We thank Prof. D. M. Ruthven for his valuable comments and suggestions and Yong Yian Lee for doing the experiments. Nomenclature c = fluid-phase concentration, mol/cm3 D, = molecular or free diffusivity, cmz/s DL = axial dispersion coefficient, cm2/s D, = pore diffusivity, cmz/s E = diffusional activation energy, kcal/mol HETP = height equivalent to a theoretical plate, cm AH = heat of adsorption, kcal/mol kf = external film mass-transfer coefficient, cm/s K = dimensionless equilibrium distribution constant KO = constant in the van't Hoff equation L = bed height, cm f , = average pore radius, A r, = critical solute molecular radius, A R = gas law constant, kJ/(mol K) R = gel particle radius, cm ,rP= temperature, K t = time, s u = interstitial liquid velocity, cm/s
65 1
e, = porosity of gel particle p = mean of response curve first moment, s u2 = variance of response curve second moment, s2
= tortuosity factor of gel particle Registry No. H-Gln-OH, 56-85-9; H-Met-OH,63-68-3; HPhe-OH, 63-91-2; H-Trp-OH,73-22-3. T
Literature Cited Bischoff, K. B. Notes on the Diffusion Type Model for Longitudinal Mixing in Flow. Chem. Eng. Sci. 1960, 12, 69-70. Ching, C. B.; Ruthven, D. M. A Liquid Phase Chromatographic Study of Sorption and Diffusion of Glucose and Fructose in NaX and KX Zeolite Crystals. Zeolite 1988, 8 (Jan), 68-73. Ching, C. B.; Ruthven, D. M. Sorption and Diffusion of Some Amino Acids in KZ Zeolite Crystals. Chem. Eng. J . 1989, 40, Bl-B5. Ching, C. B.; Hidajat, K.; Rathor, M. N. Chromatographic Evaluation of Sorption and Diffusion Characteristics of Glucose, Maltose and Maltotriose in Silica Gels. J. Chromatogr. 1989a, 463, 261-270. Ching, C. B.; Hidajat, K.; Uddin, M. S. Evaluation of Equilibrium and Kinetic Parameters of Smaller Molecular Size Amino Acids on KX Zeolite Crystals via Liquid Chromatographic Techniques. Sep. Sci. Technol. 1989b,24 (7, 81, 581-597. Colton, C. K.; Satterfield, C. N.; Lai, C.-J. Diffusion and Partitioning of Macromolecules within Finely Porous Glass. AIChE J . 1975, 21 (2), 289-298. Lee, L.-K.; Ruthven, D. M. An Experimental Method for the Determination of Macropore Diffusivities for Liquids in Molecular Sieve Pellets. Ind. Eng. Chem. Fundam. 1977, 16 (2), 290-294. Prasher, B. D.; Ma, Y. H. Liquid Diffusion in Microporous Alumina Pellets. AIChE J. 1977, 23 (3), 303-311. Ruthven, D. M. Principles of Adsorption and Adsorption Processes; John Wiley: New York, 1984. Satterfield, C. N.; Colton, C. K.; Pitcher, W. H. Restricted Diffusion in Liquids within Fine Pores. AIChE J . 1973, 19 (3), 628-635. Wakao, N.; Funazkri, T. Effect of Fluid Dispersion Coefficients on Particle-to-fluid Mass Transfer Coefficients in Packed Beds. Chem. Eng. Sci. 1978, 33, 1375-1384. Wilke, C. R.; Chang, P. C. Correlation of Diffusion Coefficients in Dilute Solutions. AIChE J. 1955, 1, 264-270.
Greek Letters t = voidage of packed bed
Received for review June 2, 1989 Accepted November 29, 1989
Synthesis of L-Phenylalanine Methyl Ester Stanley B. Mirviss,* Samun K. Dahod, and Mark W. Empie Stauffer Chemical Company, Eastern Research Center, Dobbs Ferry, New York 10522
The synthesis of L-phenylalanine, a synthetic sweetener raw material, comprises a six-step sequence using hydantoin and benzaldehyde as raw materials. These react to form 5-benzalhydantoin, which is hydrogenated to 5-benzylhydantoin at atmospheric pressure with Raney nickel using triethylamine as a promoter in alkaline media. The benzylhydantoin is hydrolyzed to D,L-phenylalanine with dilute caustic after acidification. The D,L-phenylalanine is esterified with methanol in the presence of hydrogen chloride, and the methyl D,L-eSter hydrochloride is then neutralized with sodium bicarbonate in the presence of toluene. The toluene solution of the free base D,L-ester is now resolved by stereoselective hydrolysis of the L-ester with chymotrypsin enzyme in the aqueous phase. The chymotrypsin enzyme can be used immobilized or as is. The L-phenylalanine precipitates from the aqueous phase. The D-eSter in toluene is racemized for recycle with a resin made from salicylaldehyde. Economical synthesis of L-phenylalanine methyl ester is important for the manufacturing of the synthetic sweetener, Aspartame. Many schemes have been developed for this synthesis, and some have been practiced on a commercial scale. Generally, L-phenylalanine is made first and then esterified to the methyl ester. The methods of commercial production of L-phenylalaninehave included fermentation (Hwang et al., 1985; Finkelman and Yang, *Present address: 90 Surrey Rd, Stamford, CT 06903. 0888-5885/9012629-0651$02.50/0
1985), enzymatic addition of ammonia to cinnamic acid (Chibata et al., 1978; Schruber, 1985), and enzymatic reductive amination of phenylpyruvic acid with ammonia or another amino acid (Matsunaga and Kitamura, 1987; Asano et al., 1986). The literature is replete with fully synthetic approaches and combinations of organic synthesis with biochemical approaches. A large majority of the myriad of processes were evaluated for economics, simplicity, raw material availability, and equipment costs. High-pressure processes were considered unsuitable. The 1990 American Chemical Society
