1358
I n d . Eng. C h e m . Res. 1989, 28, 1358-1364
Moving-Withdrawal Liquid Chromatography of Amino Acids Magdiel Agosto, Nien-Hwa Linda Wang,* and Phillip C. Wankat School of Chemical Engineering, Purdue University, West Lafayette, Indiana 47907
A new chromatographic technique called moving-withdrawal chromatography (MWC) was used to separate a four-component amino acid mixture. This technique can achieve significant improvements in large-scale separations with regard to product concentration, cycle time, and throughput, as comparisons between MWC and normal elution runs in this study have shown. T h e experimental conditions, such as bed lengths and stream switching times for these isocratic runs, were estimated with the Lapidus and Amundson linear dispersion model. Isocratic MWC experiments done in this study have demonstrated up to a 71.3% reduction in cycle time over normal elutions and a doubling of product concentrations for withdrawn species. Complementing these experiments were a set of step pH gradient MWC runs. These runs showed that the combination of pH gradients and MWC can lead to an 84% reduction in cycle time and a several fold increase in product concentration. The scale-up of chromatographic processes is an important area of interest in biotechnology research. Most of the efforts toward scale-up involving repetitive feedpulse processes aim to achieve high product concentrations and purities, short cycle times, and high throughput. By cycle time we mean the time from the start of one feed pulse to the beginning of the next, while throughput refers to the amount of feed that can be processed per hour. Many of these repetitive feed-pulse processes involve purification of complex mixtures through elution. Elution is often complicated when feed component affinities are far apart from each other. During the separation of such mixtures, large gaps often develop between component peaks, contributing to poor packing utilization and limiting when the next introduction of feed can be made. In scale-up, such underutilization could be quite costly. Ongoing design efforts have focused on alleviating these problems. One alternative is to apply the technique known as moving-withdrawal chromatography (Wankat, 19841, which is the subject of the present study. Moving-withdrawal chromatography (MWC) is a type of column-switching technique (Snyder and Kirkland, 1979; Wankat, 1986) that improves the efficiency in treating feed components having wide ranges in affinity. Its application involves the splitting of the normal column into shorter columns connected in series. During separation, a product can be withdrawn at these intermediate ports as soon as the product is sufficiently separated from other components. While this withdrawal is taking place, additional solvent is added to continue the separation downstream. This technique seeks to remove the slowest and fastest moving components as products earlier in the separation since it is these compounds that control the cycle time. This study applies moving-withdrawal chromatography (MWC) to the cation-exchange separation of a four-component amino acid mixture and provides experimental verification for the theory. The previous studies examined MWC through various mathematical models. For instance, Wankat (1984) used local equilibrium theory to show a 69.5% increase in throughput over normal liquid chromatography when separating a ternary mixture containing naphthalene, anthracene, and pyrene. For this same mixture, Miller and Wankat (1984) included the effects of an effective axial dispersion coefficient to show a 39% increase in throughput. Geldart et al. (1987) applied Rhee et al.’s (1970) nonlinear local equilibrium model (neglecting mass transfer) to the moving-withdrawal system to show that significant improvements can occur when applied to two cases: (1)the removal of slow-moving amino acids and 0888-5885/89/2628-1358$01.50/0
(2) the removal of both fast- and slow-moving components. Previous MWC studies were done isocratically, that is, at constant pH and ionic strength. However, because amino acid affinities have a strong pH dependency, we also studied the use of step pH gradients.
Theory Critical to the design of a MWC system is the accurate specification of column lengths and product withdrawal times. A model capable of predicting elution histories and profiles greatly aids in this specification and in the additional fine-tuning of the design by reducing the time and effort spent on preliminary trial runs. In this study, the amino acid feed used is dilute enough so that linear equilibrium can be assumed. For linear systems, the linear-dispersion model of Lapidus and Amundson (1952) can be applied. For sufficiently long columns, this solution, as reported by Lightfoot et al. (19621, reduces to the following asymptotic breakthrough expression: x = C / C F e e d = Y.(I + erf [PeZ1I2(V-P)/2(VP)1/2]) (1) where V = tuiAct is the elution volume, P = cAJ(1 + k ’ ) is a measure of bed capacity, and Pe, = uiz/(ED+ DM)is the Peclet number. In this study, feed pulses remained relatively small in comparison to the column volume. As a result, a differential-pulse solution was used. Such a solution can be developed from eq 1 by employing superposition (Lightfoot et al., 1962). The solution for a pulse is (2) Xpulse = x ( z , v ) - x ( z , v - VFeed)
A differential-pulse solution is obtained by taking the limit of the form XpuIse
as
VFeed
-
=
[
x(z,
v)- x ( z , v - VFeed) VFeed
0 to yield
Xdiff.pulse(Z,
I
VFeed
v) = VFeed ax(z, v)/av
which is
where
0 1989 American Chemical Society
(3)
(4)
Ind. Eng. Chem. Res., Vol. 28, No. 9, 1989 1359 Table I. Amino Acid Structures and Model Parameters fitted eff dispersion amino SeP coeff, factors cm*/min acid structure 1.1 asp NH,+CH(CH2C00-)C000.008 1.2 ala NH3+CH(CH&OO0.11 phe NH3+CH(CH2CBH6)COO0.75 5.0 lys NH3+(CH2)4CH(NH3+)C00- 4.90 0.3
Equilibrium Affinities. The magnitude of a solute’s affinity determines the solute’s characteristic retention time. Insight into this affinity or equilibrium behavior is often obtained from its chemical structure. It is the chemical structure of amino acids that helps provide a good basis to explain the elution orders observed. An amino acid’s structure can be represented by the formula NH,CHRCOOH. The molecule can exist as a zwitterion with the amino and carboxyl groups surrounding the a carbon being in either protonated or unprotonated forms. The R functional group along with the surrounding mobile-phase conditions determines the effective charge of the amino acid. This effective charge, in turn, affects how strongly an amino acid binds on to an ion exchanger. For instance, the structures of the four amino acids of interest, aspartic acid (asp), alanine (ala), phenylalanine (phe), and lysine (lys), are shown in Table I. This is the order they would elute during a cation-exchange separation using Bio-Rad AG50W-X8. The extra unprotonated carboxyl group contributes to a greater repulsion effect which causes asp to elute very quickly. Comparisons between ala and phe show that phe’s large aromatic ring would contribute a greater hydrophobicity than alanine’s smaller methyl group. As a result, phe has a longer retention time. Finally, lysine’s protonated amino group on the side chain provides an additional positive charge to make lys elute last. We now turn our attention to the underlying surrounding mobile-phase condition that influences the characteristic charge of an amino acid-specifically pH. In the pH range of interest, 1-7, the a-amino groups are essentially in their protonated form, NH3+, and are available to undergo ion exchange with a cation exchanger. However, in this range, the carboxyl group’s ionization is very sensitive to a change in pH. An increase in pH is able to shift the following chemical equilibrium toward a greater amount of unprotonated a-carboxyl groups (Yu et al., 1987): NH,+CHRCOO-
+ H+
-
NH,+CHRCOOH
In this form, an amino acid’s elution on a cation exchanger occurs much more rapidly. As a result, one can selectively speed up the elution of slower moving amino acids acids through pH gradients. I t was the intent of the gradient experiments to use this effect to produce additional improvements over isocratic MWC. Mass Transfer. While affinity accounts for the retention time of a component, a dispersion coefficient in the model helps to account for peak spreading due to mass transfer. In the original model (Lapidus and Amundson, 1952), the dispersion coefficient was used to describe axial dispersion. In this study, an effective dispersion coefficient was used to include the effects of both axial dispersion and internal mass transfer. This internal mass transfer includes film and intraparticle diffusion. This approach is valid for linear systems since variances add. Table I shows a summary of the fitted dispersion coefficients and separation factors against Na+ obtained for the four amino acids. Asp and ala parameters were fitted using data
INJECTIONVALVE
ASP,ALA.PHE,LYS FEED SOLUTION
/I PRODUCT
Figure 1. Single-column apparatus.
+7.
INJECTION VALVE
ASP,MPHE,LYS FEED SOLUTION
A
BUFFER
, I
E
, I1
LYs BUFFER
ASP.AL4.8 PHE
Figure 2. Moving withdrawal of lys. Valve switching times are all a t 16 min.
collected from a 26-cm column run. Phe parameters were fitted using data from a 15-cm column, while those for lys were obtained from a 4-cm column.
Experimental Procedure The normal elution chromatography arrangement using a single column is shown in Figure 1. This separation conducted a t a bed length of 24 cm served as the basis for comparison of all MWC results. Moving-withdrawal chromatography runs were conducted at the same total bed length, flow rate, feed, and mobile-phase conditions as the single-column separation. These runs were made with two different arrangements. The first (Figure 2) divided the total bed length into two shorter columns connected in series. During the run, lysine was withdrawn between columns to achieve a shorter cycle time. In the second arrangement (Figure 31, the total bed length is segmented into three shorter columns. While lys is still removed between the first and second columns, phe is now removed between columns two and three. This earlier removal of the slower moving components, lys and phe, shortens the cycle time, thus increasing throughput, and it increases the lys and phe product concentrations. Further improvements in the moving withdrawal of lys and phe were obtained when step pH gradients were applied. In this experiment step, pH gradients were introduced into the first and second columns (see Figure 4) in order to sharpen product peaks and speed up the total separation. Details of the apparatus are given by Agosto (1988). The four-component amino acid feed mixture was made up of a 0.19 N sodium citrate buffer (pH 4.44) into which
1360 Ind. Eng. Chem. Res., Vol. 28, No. 9, 1989 1.00
INJECTIONVALVE ASP,AIA,PHE,LYS FEED SOLUTION
n m
b
+b-
0.90
rl
0.80
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180
240
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420
480
540
600
(mls.)
Figure 5. Single-column run ( L = 24 cm). Results of simulation using the Lapidus and Amundson model. Figure 3. Moving withdrawal of lys and phe. Valves 1-3 switch a t 16 min, while 4-6 switch a t 9 min.
+-
INJECTIONVALVE
ASP,AIA,PHE,LYS FEED SOLUTION
LYs
4
BUFFER
RECYCLE
PHE
+
0
RECYCLE
ASP B ALA
Figure 4. pH gradient moving withdrawal of lys and phe. Valves 1-4 switch a t 16 min, while 5-7 switch a t 9 min.
aspartic acid, alanine, phenylalanine, and lysine (from Chemical Dynamics Corp.) were added. The final feed concentrations for asp, ala, and phe were 0.0025 M each, whereas lys' feed concentration was 0.00125 M. The mobile phase was a 0.2 N sodium citrate buffer which was prepared at pH 4.44 and 8.5. The lower pH buffer was employed in isocratic separations while also acting as the initial mobile phase for step pH gradient runs. The cation exchanger used was a sulfonated polystyrene resin with 8% divinylbenzene cross-linkage (AG50W-X8). It was obtained in H+ form from Bio-Rad in 200-400-mesh size. Before use, the resin was converted to Na+ form following procedures described by Bio-Rad (1985). This conversion allowed it to conform more closely to the conditions of Spackman et al. (1958). Fractions were collected during cation-exchange runs, derivatized with o-phthalaldehyde (OPA), and analyzed by HPLC (Agosto, 1988; Jarrett et al., 1986). The equilibration of each column with the initial mobile phase (0.2 N sodium citrate buffer, pH 4.44) was done before carrying out any separation. Usually 4-5 bed volumes of the initial eluent were pumped through the system. The flow rate used for equilibration and for all separations was 2.0 mL/min. Feed injection was 1.0 mL.
Results and Discussion A normal elution run (single-column separation) acts as the basis for comparing moving-withdrawal results. It is the standard upon which improvements need to be made. Experimental runs on the apparatus shown in Figure 1 were used to obtain estimates for the affinities and effective dispersion coefficients (Table I). The Lapidus and Amundson model was then used to predict the product concentrations for a 24-cm column (Figure 5). This plot shows some of the disadvantages associated with using normal elution techniques to separate feeds with a wide range of affinities. For instance, phe and lys have a large separation gap which causes poor bed utilization and a very high cycle time of 209.3 min, which at a flow rate of 2 mL/min translates into a cycle volume of 418.6 mL. This is the volume of eluent that must be passed through the column in order to sufficiently clear out the separated feed so that another feed pulse can be injected without interference. Because this cycle volume is so large, the throughput, defined as the volume of feed injected divided by cycle volume, turns out to be quite small, 0.0024. Another major disadvantage is the low product concentrations associated with phe and lys. These lower concentrations occur because of the extensive peak spreading exhibited by high-affinity solutes as they slowly elute from the column. Earlier removal of the slowest component, lys, can be done using the two column setup shown in Figure 2. The short first column sufficiently separates 1ys from the other three amino acids so that it can be withdrawn and collected as product. It was found that a 4-cm-long first column gives sufficient separation of lys and phe. The second column is then 20 cm long to give a total length of 24 cm. After the first three components pass on to the second column, lys is diverted through valve 1 and collected. Automatic valves 2 and 3 must also be switched simultaneously along with valve 1in order to allow fresh eluent to pass into the second column. Figure 6 shows the experimental and simulation results for both columns in Figure 2. There is no change in the results for asp, ala, and phe since they still travel through the full distance of 24 cm; however, there is quite a change for lys. It is collected much earlier now with its peak maximum at about 70 mL, an 84% reduction over a single-column run. Comparison of the two plots in Figure 6 shows that phe instead of lys determines the cycle time. The phe peak elutes completely at around 150 mL, while the last of the
Ind. Eng. Chem. Res., Vol. 28, No. 9, 1989 1361 IW
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lys peak elutes around 120 mL. Thus, the new cycle time based on phe is about 75 min, a 64% reduction over the single-column run. The throughput translates into 0.0067 or 2.77 times that of the single-column experiment. Shorter cycle times and a greater throughhput are not the only improvements. The lys product is also collected at about twice the concentration than in the single column run. Since the lys travels a much shorter distance, there is significantly less zone spreading. An earlier withdrawal of phe would decrease the cycle time and zone spreading even further, while allowing phe to be collected a t a higher product concentration. This was accomplished with the system shown in Figure 3. The first column remains at 4 cm, which is a sufficient bed length for a lys separation. If phe is allowed to travel another 11 cm (second column length), one finds that it is sufficiently separated from asp and ala to be withdrawn earlier. However, asp and ala still need to pass through 9 cm more of the bed (third column length) to obtain a good resolution. Figure 7 shows the results for this run. This time, the last of the phe peak elutes a t around 110 mL and has base peak spreading of about 90 mL, a 21.7% decrease over the two-column run. This decrease in base peak spreading translates into a comparable increase in phe product concentration. Because of phe's early removal, lys now dictates the cycle time at 60 min, a 71.33% decrease over the single-column run. With the cycle volume being 120 mL, the throughput is 0.008 33, about 1.25 times greater than the two-column run and about 3.47 times
200
(mls.)
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40
60
80
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Effluent Volume
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160
180
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(mls.)
Figure 7. Three-column moving withdrawal of lys and phe.
greater than the single-column run. Although the linear-disperson model works well for the above isocratic runs, an interesting question still arises: Is the observed spreading mainly due to axial dispersion or other mass-transfer effects? This question remains because effective dispersion coefficients were fitted from the data and were not estimated on the basis of axial dispersion alone. These fitted coefficients lump together the effects of axial dispersion, film mass transfer, and intraparticle diffusion. However, the Chung and Wen (1968) correlation gives us a method to estimate a true axial
1362 Ind. Eng. Chem. Res., Vol. 28, No. 9, 1989
dispersion coefficient for the system. This estimated effective dispersion coefficient (0.0699 cm2/min) was used in the linear dispersion model to generate effluent history curves whose peak spreading was due solely to axial dispersion. These curves were then compared to the experimental results to see to what extent mass transfer was actually present. These results (Agosto, 1988, Table I) demonstrated that film mass transfer and intraparticle diffusion contribute significantly to peak spreading for these runs. This suggests that any model used for a priori predictive purposes should take into consideration both of these mass-transfer effects. The above linear model with its adapted use of fitted dispersion coefficients is empirical in nature, allowing one to predict results a t varying column lengths if all other conditions are kept constant. This enables the quick estimation of column lengths and switching times for linear systems. Band spreading at withdrawal ports was minimal since intermediate values and fittings contained low dead volumes. An argument that supports negligible band spreading at withdrawal ports is that the same dispersion coefficients obtained from single-column runs could be used to fit moving-withdrawal data. Insight into the relative magnitudes of the fitted dispersion coefficients in Table I can be explained by the influence of separation factors on the overall mass transfer. This influence becomes more apparent if one examines the sum-of-resistances model stated as (Sherwood et al., 1975) - 1= - 1 + - 1 (7) KL
kL
p
1.80
8.4
2
1.80
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0.20
4.6
--------
0.0 0
4.0 12
24 36
48 60 72 84 96 108120
Effluent
Volume
(mls.)
Figure 8. pH gradient moving withdrawal versus isocratic run (lys, 4 cm). pH gradient: solid line represents connection of experimental data points. Isocratic: solid line represents results of the Lapidus and Amundson model.
---pH
rl
2 00
grad
(experimental)
i s o c r a t i c (model)
X v
mkR
When the local slope of the isotherm, m, is large, then the tendency is for the effective intraparticle mass-transfer resistance to be small. Thus, the overall mass-transfer coefficient is large and band spreading or the fitted dispersion coefficient small. This, to a degree, helps explain comparisons between fitted coefficients for asp, ala, and lys in Table I. Phe represents the exception because of its aromatic R functional group. Like all weak electrolytes, phe coexists in its charged and neutral states. A t pH 4.4, phe is mostly in its neutral state. Physical adsorption of phe's neutral state is likely to occur because of the similarity between its hydrocarbon R group and the aromatic backbone of the polystyrene-divinylbenzene resin. This physical adsorption of neutral phe is favored by Van der Waals forces and dipole-dipole interactions and can be as important as ion exchange at low phe concentrations (Liu, 1985; Helfferich, 1962). The possible presence of physical adsorption along with ion exchange may explain why phe has such a large fitted dispersion coefficient. Zone spreading had a substantial effect in reducing product concentration for the isocratic runs. This spreading was still quite significant in moving withdrawal despite the obvious improvements. In order to further reduce this spreading, step pH gradients were tried since pH alters the affinities of the slower moving components. For instance, introducing a step change in pH from 4.44 to 8.5 into the first and second beds of the three-column moving-withdrawal arrangement shown in Figure 4 helped to speed up and focus the lys and phe peaks. This increase in pH allowed the last of the lys peaks to exit a t about 66 mL, making the cycle time 33 min (see Figures 8 and 9). This new cycle time is a 45% reduction over isocratic moving withdrawal and an 84.2% reduction over the isocratic single-column runs. The use of a step pH gradient also reduced the peak width by more than half. This type of focusing translates into a 2.4-fold increase in lys product concentration. Throughput, which is based on cycle vol-
o
E
24
36
Effluent
48
80
72
Volume
84
96
108
120
(mls.)
Figure 9. Moving withdrawal (pH gradient versus isocratic).
ume (66 mL), was 0.015 15 for the step pH gradient run or 6.34times the results for an isocratic single-column run and about 1.8 times that of an isocratic moving-withdrawal run. The effect of the step pH gradient on phe had somewhat mixed results. This was mainly due to inflexible switching times. Even though phe was diverted from the system as early as 9 min into the run, the step pH gradient could not be introduced until lys was ready to be removed a t 16 min (compare lys and phe curves in Figures 8 and 9). Any earlier switching of valves 1-3 would split the phe between the first and second columns, and some of the phe would exit in the lys product. To avoid this, switching was done at 16 min, which caused part of the phe peak to elute under a pH 4.44 environment while the rest eluted under a pH of 8.5 (see Figure 9). This occurs because the pH wave introduced into the second column at 16 min does not have a chance to completely overtake the phe peak before phe starts to exit. Another method to employ step pH gradients and avoid phe switching problems is to conduct the moving-withdrawal separation with two columns. The first column length and the lys withdrawal time would remain the same as well as for the pH gradient introduction time. However, the second column would be longer, allowing the pH wave to surpass phe before phe begins to elute. In this ar-
Ind. Eng. Chem. Res., Vol. 28, No. 9, 1989 1363 rangement, the second column must be sized so that the higher pH wave does not catch up to asp and ala. This will allow phe product concentrations to be higher than before. Each moving-withdrawal design must cater to and is dictated by the various distributions of affinities in the feed mixture. When the feed mixture is comprised of components having a wide range of affinities, the problem becomes one of removing the key components that will shorten cycle times and maximize column utilizations. The general strategy may appear like this: 1. Establish the total column length needed to separate the two feed components closest in affinity. 2. By use of this total length as a basis, proceed to divide the column into a series of shorter ones. 3. These shorter column lengths should be based on which components are to be withdrawn. They could be the slowest moving or the fastest moving components that contributed t o the longer cycle times. They can even comprise an intermediate cut of components that would otherwise leave large segments of packing underutilized (Geldart et al., 1987). 4. Good models are ideal for arriving at the proper short column lengths and product switching times once accurate equilibrium parameters and dispersion coefficients are established. They are useful in predicting effluent histories for any given length and in allowing one to estimate the proper switching times for product withdrawal. When a good model is lacking and the system is linear, one could always revert to two rules of thumb in order to estimate proper lengths and switching times. They are (a) retention time is directly proportional to the length of the bed when all other conditions are held constant, and (b) peak width is directly proportional to the square root of the column length. When the isocratic moving-withdrawal arrangement is established, one can look into the use of gradients to see whether improvements like the ones shown for the case of lys and phe are possible. Another question that should not be ignored is whether gradients applied to the normal single-column arrangement will get the separation done rapidly enough so that there is no need for the added complexity of moving withdrawal.
Conclusions This study has shown that MWC can increase throughput, shorten cycle times, and increase product concentrations of feeds which contain solutes with a wide range of affinities. Cation-exchange separations of asp, ala, phe, and lys were done with normal elution and with MWC. Isocratic MWC experiments were done with a two-column moving withdrawal of lys and a three-column moving withdrawal of phe and lys. Comparisons between results showed the clear advantage of the early removal of the slowest moving components. Throughput for the two-column moving-withdrawal experiment was 2.77 times greater than for normal elution. This throughput increase is linked to the 64% decrease in cycle time that the earlier lys removal allowed. Also, the product concentration for lys averaged about twice that of normal elution. In three-column MWC, lys removal remained the same, and no further increase in lys product concentration was expected. However, the earlier removal of phe increased phe concentration to twice as high as before, and the cycle time was reduced by 71.33% compared to normal elution. Application of step pH gradients to MWC columns 1 and 2 allowed the cycle time to be shortened to 33 min, a 45% reduction over isocratic moving withdrawal and an 84.2% reduction over the isocratic single-column results.
This combination of pH gradient focusing and moving withdrawal gave the best results in terms of cycle time, throughput, and product concentration. The model developed by Lapidus and Amundson (1952) and later modified by Lightfoot et al. (1962) predicted the isocratic experimental results quite satisfactorily when an effective dispersion coefficient found from experiments was used. For linear isocratic systems, this model was used to decide column lengths and switching times for MWC. The results shown in this study for the separation of amino acids have general implications for designing other large-scale chromatography units. For instance, this study showed that for the given system and conditions a vast improvement in the overall large-scale separation scheme can be achieved by combining two approaches to the separation. One approach was to physically and operationally alter the normal elution setup by using in its place a column switching method like moving withdrawal. The other approach was to make use of a solute’s known chemistry to alter its affinity through the use of gradients. The combination of both of these approaches, as shown in this study, will generally offer the best alternative toward reaching the most efficient large-scale design.
Acknowledgment This research has been supported by a Purdue University Fellowship and two NSF grants, CBT-8604906 and ECE-8613167.
Nomenclature A , = cross-sectional area, cm2 C = concentration of an amino acid, M Cfeed = concentration of amino acid in feed, M CRT = wet resin capacity, mequiv/mL or N CT = total ionic concentration in the mobile phase, N DSM = pore diffusion coefficient, cm2/min ED + DM = effective axial dispersion coefficient, cm2/min k ’ = relative retention = [(l- c)/c]aA,Na(CRT/CT),for linear monovalent ion exchange k L = liquid-phase mass-transfer coefficient, cm/min k R = resin-phase mass-transfer coefficient, cm/min KL = overall mass-transfer coefficient based on liquid phase, cm/min L = length of a column, cm m = local slope of an ion-exchange equilibrium curve Pe, = uiz/(ED + DM), Peclet number t = time, min ui = interstitial velocity, cm/min V = cuiAct,elution volume, mL V = cA&(1 + k ’ ) , bed capacity at saturation, mL x = C/Cfed, dimensionless concentration in the mobile phase z = axial distance along column, cm Greek Letters a A , N a = separation factor of amino c = interparticle bed porosity
acid A against Na+
= defined in eq 6 = bed density, g/cm3 Registry No. Aspartic acid, 56-84-8; alanine, 56-41-7; phenylalanine, 63-91-2; lysine, 56-87-1. K
pB
Literature Cited Agosto, M. Moving-Withdrawal Liquid Chromatography of Amino Acids. MS.ChE. Dissertation, Purdue University, West Lafayette, IN, 1988. Bio-Rad. Ion Exchange Manual. Richmond, CA, 1985. Chung, S. F.; Wen, C. L. Longitudinal Dispersion of Liquid Flowing Fixed and Fluidized Beds. AIChE J. 1968, 14, 857. Geldart, R. W.; Wang, N.-H. L.; Wankat, P. C. Non-linear Analysis of Multicomponent Moving-Withdrawal and Moving-port Chro-
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I n d . Eng. C h e m . Res. 1989, 28, 1364-1369
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Received for review December 28, 1988 Accepted May 29, 1989
Selective Extraction of Oleic, Linoleic, and Linolenic Acid Methyl Esters from Their Mixture with Supercritical Carbon Dioxide-Entrainer Systems and a Correlation of the Extraction Efficiency with a Solubility Parameter Yutaka Ikushima,* Norio Saito, and Tomio Goto Government Industrial Research Institute, Tohoku, Nigatake 4-chome, Miyagino-ku, Sendai 983, Japan
Oleic, linoleic, and linolenic acid methyl esters were able to be well extracted and separated from their mixture by supercritical carbon dioxide (SC-C02)with an entrainer. We used several entrainers, some paraffins, and some esters, and these were alternately added to the C02. The SC-C02 with a single entrainer flowed over the mixture and then through a separation chamber packed with AgN03-doped silica gel. The use of the chamber and the entrainer made it possible t o effectively extract and separate the desired component from the mixture. The amount of each substance extracted was estimated by a model including the solubility parameter. The affinity of the substance for the mobile (SC-C02 or SC-C02and an entrainer) and stationary (AgN0,-doped silica gel) phases was regarded as the ratio of the activity coefficient of the substance in the two phases. An approximately linear relationship exists between the variation in the ratio of the activity coefficient and the amount of substances extracted. In the fields of food and pharmacy, supercritical carbon dioxide (SC-C02) extraction is noted to be a useful method for extracting valuable materials such as docosahexenoic acid (DHA), eicosapentenoic acid (EPA), linoleic acid, linolenic acid, and others contained in natural resources (Yamaguchi and Murakami, 1986; Sako et al., 1986). The utility of these acids is described elsewhere (Lossonczy, 1978; Hirao et al., 1980; Sanders and Younger, 1981). However, it is difficult to extract selectively a specific component from the mixture of such higher fatty acids because these are similar in chemical and physical properties. We (Ikushima et al., 1988) previously reported that the selective extraction of stearic (clw) and linolenic (c18-3) acid methyl esters from a equimolar mixture of stearic, oleic (CIB-J, linoleic (CIB-J,and linolenic acid methyl esters becomes feasible by means of a chamber packed with both AgN0,-doped silica gel and ethyl acetate as the entrainer. (In the subscripts, the first and second figures refer to the number of carbon atoms and the degree of unsaturation in the molecule, respectively.) When flowing out the extractor and then passing through the AgN0,-doped silica gel chamber, fatty acid methyl esters having a higher deOSSS-5885/89/2628-1364$01.50/0
gree of unsaturation, such as C18-2and Clgg methyl esters, were found to be held in the chamber, probably because these two might form some adducts with AgNO,. It was possible to isolate C18-3 methyl ester in adequate purity from the chamber by the addition of entrainer to the COz. However, c18-1 and c18-2 acid methyl esters could not be separated well from the mixture. The property of the solvent for the extraction can be easily changed according to an entrainer added to the COz (Ikushima et al., 1988), and the amounts of Clgl and Clg2 methyl esters extracted through the chamber might be adjustable with several entrainers, which are alternately added to the COz. The first objective of this work is to present a method to extract and isolate a desired component in high purity from a mixture of oleic (Clgl), linoleic (Clg2), and linolenic acid methyl esters by the alternate addition of suitable entrainers to COz for a given extraction system in which AgN0,-doped silica gel is used. The gradual variations in the solvent polarity may occur by means of the alternate addition of entrainers. The second objective is to apply chromatographic analysis to the estimation of the amount of each fatty acid methyl ester extracted through the chamber. The amount 6 1989 American Chemical Society
