Construction of Large-Volume Monolithic Columns - American

over the entire monolith bed. It was shown that such a monolithic column exhibits flow-independent separation efficiency and dynamic binding capacity ...
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Anal. Chem. 2000, 72, 5693-5699

Construction of Large-Volume Monolithic Columns Alesˇ Podgornik, Milosˇ Barut, and Alesˇ S ˇ trancar

BIA Separations d.o.o., Teslova 30, SI-1000 Ljubljana, Slovenia Djuro Josic´

Octapharma Produktionsges, Oberlaaer Strasse 235, A-1100 Wien, Austria Tine Koloini*

Faculty of Chemistry and Chemical Technology, University of Ljubljana, Asˇ kercˇ eva 5, SI-1000 Ljubljana, Slovenia

Monolithic supports have become the subject of extensive study in the past years. Despite their advantageous features and many successful chromatographic applications in the analytical scale, only a very few examples of larger volume monoliths were described. In the case of GMA-EDMA monoliths, this can be attributed to the fact that due to the exothermic polymerization a pronounced temperature increase inside the monolith significantly affects the structure. The temperature increase depends on the thickness of the monolith, and consequently, there is an upper limit that allows the preparation of a unit with a uniform structure. In the present work, we have analyzed a heat release during the polymerization and have derived a mathematical model for the prediction of the maximal thickness of the monolithic annulus having a uniform structure. On the basis of the calculations, two annuluses of different diameters were polymerized and merged into a single monolithic unit with a volume of 80 mL. In addition, a special housing was designed to provide a uniform flow distribution in the radial direction over the entire monolith bed. It was shown that such a monolithic column exhibits flow-independent separation efficiency and dynamic binding capacity up to flow rates higher than 100 mL/min. The separation and loading times are in the range of a few minutes. The pressure drop on the column is linearly dependent on the flow rate and does not exceed 2.5 MPa at a flow rate of 250 mL/min. Monoliths are becoming very attractive stationary phases due to their advantageous hydrodynamic characteristics. The main difference in comparison to conventional particle beds is in their structure. Conventional particle-based supports consist of fewmicrometer-sized porous particles. Because the pores within the particles are closed, the liquid inside them is stagnant. Therefore, the molecules to be separated are transported to the active sites inside the close pores and back to the mobile phase mainly by diffusion. Since diffusion itself is a rather slow process, especially in the case of large molecules with a low mobility (low diffusion * Corresponding author: (e-mail) [email protected]; (tel) + 386 61 1760 441; (fax) + 386 61 1264 577. 10.1021/ac000680o CCC: $19.00 Published on Web 10/13/2000

© 2000 American Chemical Society

coefficient), it determines the overall separation time. Furthermore, the separation efficiency depends on the residence time of the sample inside the stationary phase and, therefore, on the linear velocity of the mobile phase through the separation medium.1 The monoliths, on the other hand, consist of a single piece of porous material. The pores are highly interconnected, forming a network of channels. Since the flow of the liquid within the channels is driven by the pressure difference, the molecules to be separated are transported to the active sites located on the surface of the channels by convection, increasing their mobility by several orders of magnitude. Because of that, it is possible to perform an efficient separation of large molecules within a very short time.2 Furthermore, the efficiency and the dynamic binding capacity are independent of linear velocity within the range of tested flow rates.3 Besides their interesting hydrodynamic characteristic, their preparation is another advantage. In contrast to particle preparation, where commonly particle size classification is required after polymerization is completed, monoliths are prepared with a bulk polymerization and their structure is defined already by monomer composition and polymerization temperature without further processing.4,5 There are several different monolithic supports described in the literature introduced in the late 1980s or in the early 1990s. They are basically synthesized from different chemical compounds to form acrylamide,6 silica,7 styrene,8 and methacrylate9 monoliths. Glycidyl methacrylate-based monoliths were introduced in 1990.9 They were polymerized from glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) in the presence of porogens and an initiator. The polymer is chemically and mechanically very stable and contains epoxy groups that can be further modified to prepare the separation units suitable for ion exchange, hydropho(1) van Deemter J.; Zuiderweg, F.; Klinkenberg, A. Chem. Eng. Sci. 1956, 5, 271. (2) Sˇ trancar, A.; Koselj, P.; Schwinn, H.; Josic´, Dj. Anal. Chem. 1996, 68, 3483. (3) Iberer, G.; Hahn, R.; Jungbauer, A. LC-GC 1999, 17, 998. (4) Sˇ vec, F.; Fre´chet, J. M. J. Anal. Chem. 1992, 64, 820. (5) Sˇ vec F.; Fre´chet, J. M. J. Ind. Eng. Chem. Res. 1999, 38, 34. (6) Hjerte´n, S.; Liao, J. L.; Zhang, R. J. Chromatogr. 1989, 473, 273. (7) Nakanishi, B. K.; Soga, N. J. Am. Ceram. Soc. 1991, 74, 2518. (8) Wang, Q. C.; Sˇ vec, F.; Fre´chet, J. M. J. Anal. Chem. 1993, 65, 2243. (9) Tennikova, T. B.; Belenkii, B. G.; Sˇ vec, F. J. Liq. Chromatogr. 1990, 13, 63.

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bic interaction, reversed-phase, or affinity separations. The GMAEDMA monoliths described so far were mainly produced in two different geometries. In the form of the so-called disks (diameter > length), the maximal diameter described was 50 mm and the maximal length was 7 mm.10 Today, GMA-EDMA disks of 12-mm diameter and of 3-mm length are available on the market under the trademark CIM (Convective Interaction Media).11 An extensive review about the construction and application of these units was published by Josic´ and Sˇ trancar.12 Another geometrical shape extensively described in the literature is the so-called rod (length > diameter). They are usually prepared within the confines of conventional semipreparative stainless steel columns of 8-mm diameter.4 In the last few years, smaller rods in capillaries were also synthesized for the application in the capillary electrochromatography.13 Both shapes exhibit excellent hydrodynamic properties, typical for the monoliths, and were successfully used in a variety of different applications on an analytical scale: for separation and purification of proteins,2,10,14-23 DNA,24 and smaller molecules such as organic acids,25 hydroxybenzoates,26 and oligonucleotides and peptides26-28 as well as sensors incorporated in a FIA system.19,29-31 In both cases, however, the scale-up represents a big challenge. Scaling up of the disk-shaped GMA-EDMA monoliths can be achieved by further increasing the diameter. Unfortunately, the mechanical stability of such units and the difficulties with the uniform sample distribution are two main problems that limit such approach.32 As well, GMA-EDMA rods can be scaled in a radial (10) Josic´, Dj.; Reusch, J.; Loster, K.; Baum, O.; Reutter, W. J. Chromatogr. 1992, 590, 59. (11) Sˇ trancar, A.; Barut, M.; Podgornik, A.; Koselj, P.; Josic´, Dj.; Buchacher, A. LC-GC 1998, 11, 660. (12) Josic´, Dj.; Sˇ trancar, A. Ind. Eng. Chem. Res. 1999, 38, 333. (13) Sˇ vec, F.; Peters, E. C.; Sykora, D.; Yu, C.; Fre´chet, J. M. J. J. High Resolut. Chromatogr. 2000, 23, 3. (14) Abou-Rebyeh, H.; Ko ¨rber, F.; Schubert-Rehberg, K.; Reusch, J.; Josic´, Dj. J. Chromatogr. 1991, 566, 341. (15) Tennikova, T. B.; Sˇ vec, F. J. Chromatogr. 1993, 646, 279. (16) Josic´, Dj.; Lim, Y.-P.; Sˇtrancar, A.; Reutter, W. J. Chromatogr., B 1994, 662, 217. (17) Luksˇa, J.; Menart, V., Milicˇic´, S.; Kus, B.; Gaberc-Porekar, V.; Josic´, Dj. J. Chromatogr. 1994, 661, 161. (18) Kasper, C.; Meringova, L.; Freitag, R.; Tennikova, T. B. J. Chromatogr., A 1998, 798, 65. (19) Josic´, Dj.; Schwinn, H.; Sˇ trancar, A.; Podgornik, A.; Barut, M.; Lim, Y.-P.; Vodopivec, M. J. Chromatogr., A 1998, 803, 61. (20) Podgornik, H.; Podgornik, A.; Perdih, A. Anal. Biochem. 1999, 272, 43. (21) Platonova, G. A.; Pankova, G. A.; Il’lina, I. Ye.; Vlasov, G. P.; Tennikova, T. B. J. Chromatogr., A 1999, 852, 129. (22) Amatschek, K.; Necina, R.; Hahn, R.; Schallaun, E.; Schwinn, H.; Josic´, Dj.; Jungbauer, A. J. High Resolut. Chromatogr. 2000, 23, 47. (23) Tennikova, T. B.; Freitag, R. J. High Resolut. Chromatogr. 2000, 23, 27. (24) Giovannini, R.; Freitag, R.; Tennikova, T. B. Anal. Chem. 1998, 70, 3348. (25) Vodopivec, M.; Podgornik, A.; Berovic´, M.; Sˇ trancar, A. J. Chromatogr. Sci., in press. (26) Podgornik, A.; Barut, M.; Jancˇar, J.; Sˇ trancar, A., Tennikova, T. B. Anal. Chem. 1999, 71, 2986. (27) Sy´kora, D.; Sˇ vec, F.; Fre´chet, J. M. J. J. Chromatogr., A 1999, 852, 297. (28) Podgornik, A.; Barut, M.; Jane`ar, J.; Sˇ trancar, A. J. Chromatogr., A 1999, 848, 51. (29) Podgornik, A.; Vodopivec, M.; Podgornik, H.; Barut, M.; Sˇ trancar, A. In Stability and stabilization of biocatalysts, Progress in biotechnology; Ballesteros, A., Ed.; Elsevier: Amsterdam, 1998; Vol. 15, p 541. (30) Hagedorn, J.; Kasper, C.; Freitag, R.; Tennikova, T. J. Biotechnol. 1999, 66, 3. (31) Vodopivec, M.; Berovicˇ, M.; Jancˇar, J.; Podgornik, A.; Sˇ trancar, A. Anal. Chim. Acta 2000, 407, 105. (32) Sˇ trancar, A.; Barut, M.; Podgornik, A.; Koselj, P.; Schwinn, H.; Raspor, P.; Josic´. Dj. J. Chromatogr., A 1997, 760, 117.

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direction. However, because of the difficulties in obtaining homogeneous monoliths (described later in detail), the largest working column reported was 12 mL with a diameter of 16 mm.33 The second logical scale-up of GMA-EDMA rods is by increasing their length. A column of volume and 15 mL and length of 300 mm was reported.34 The pressure drop of such a unit becomes a major problem, since it exceeds 10 MPa even at the flow rate of only 3.5 mL/min.34 One of the first attempts to overcome the abovementioned problems and to construct a large-scale GMA-EDMA separation unit was the introduction of a monolithic tube with a volume of 22 mL having the dimensions of 53 mm (height) × 23 mm (diameter) with a 1-mm hole in the middle. In contrast to the axial working mode of the monoliths described so far, the monolithic tube works on a principle similar to that of radial chromatography.35 Such a unit enabled an efficient separation and purification of clotting factor VIII from human plasma with a back pressure of only 0.8 MPa at a flow rate of 6 mL/min.32 Although this approach enables further scale-up, it still faces the problem of the production of large-scale GMA-EDMA monoliths with a uniform pore size distribution. To ensure the desired chromatographic characteristics of the monolith, its pore size distribution should be extremely well defined. Pore size distribution depends on, aside from the initial composition of monomers and porogens, mainly the polymerization temperature.36 The polymerization itself is a highly exothermic process.5 As a consequence, a temperature increase inside the monomer mixture during the polymerization cannot be avoided, resulting in an extremely inhomogeneous structure. Therefore, a diameter of 10-25 mm is considered to be the upper limit for such approach.5 All the monolithic (axial and radial) columns constructed so far fall within these values. One possible approach to overcome this problem was suggested by Peters et al.37 Control of the temperature increase during the polymerization was performed by slow addition of the monomer mixture, influencing in this way the polymerization rate. Although showing only moderate increase of the temperature during polymerization and a more uniform pore size distribution of the obtained monolith, no additional characterization was described. In this work, we introduce a different approach for the construction of large-scale monoliths and demonstrate their applicability for chromatographic separation and purification. THEORETICAL SECTION As already mentioned, an increase of the temperature inside the monolith during the polymerization of GMA-EDMA cannot be avoided. Furthermore, it was shown that even a slight change of the polymerization temperature shifts the pore size distribution considerably.36 In the case of the polymerization in a cylinder of 50 mm in diameter, an increase of almost 60 °C was observed, in this way substantially changing the monolithic structure.37 On the basis of the literature data, we derived a mathematical model based on the heat balances to predict the temperature increase for a particular monolith geometry. Since from all large-volume mono(33) Sˇ vec F.; Fre´chet, J. M. J. Biotechnol. Bioeng. 1995, 48, 476. (34) Sˇ vec F.; Fre´chet, J. M. J. J. Chromatogr., A 1995, 702, 89. (35) Saxena V.; Weil, A. E. BioChromatography 1987, 2, 90. (36) Sˇ vec F.; Fre´chet, J. M. J. Chem. Mater. 1995, 7, 707. (37) Peters E. C.; Sˇ vec, F.; Fre´chet, J. M. J. Chem. Mater. 1997, 9, 1898.

Figure 1. Schematic presentation of the polymerization of GMAEDMA annular monolith. The monomer mixture is placed between two stainless steel tubes with the radii r0 and r1 and closed at the top and bottom. Thermostated water on the inner and outer sides of the mold ensures constant temperature around the entire mould.

lithic GMA-EDMA columns described in the literature, the tube geometry columns exhibited the lowest pressure drop despite having the largest volume,32-34 we decided to apply this geometry. The goal was to define the maximal thickness of the monolith annulus that still exhibits a uniform pore size distribution. This problem is schematically presented in Figure 1. The polymerization mold consists of two thin stainless steel tubes of different diameters. Stainless steel was chosen because it is chemically inert and because of acceptable heat-transfer properties; therefore, a heat resistance through the mold walls can be neglected. The void between stainless steel tubes is filled with a monomer mixture, closed on both sides, and the whole mold is immersed in a thermostated water with a constant temperature during the entire polymerization process. Additionally, to avoid any temperature increase of thermostated water inside the smaller stainless steel tube, the thermostated water is pumped through it. Taking into account the geometry of the system, the following heat-transfer equation in cylindrical coordinates can be written

S ∂ ∂T - r) r λ ∂r ∂r

( )

(1)

with the boundary conditions

1. T ) T0; r ) r0 and 2. T ) T0; r ) r1 where S is heat released per unit volume (W/m3), λ is thermal conductivity of the monomer mixture (W/m‚K), T is absolute temperature (K), T0 is polymerization temperature (K), r is radius (m), r0 is inner radius of the annulus (m), and r1 outer radius of annulus (m). As seen from eq 1, the system is significantly simplified since it is assumed that the heat released per unit volume (S) is constant during the polymerization as well as uniformly released over the entire volume. Furthermore, it is assumed that the thermal

conductivity λ is constant and that the system is in thermal equilibrium. This is certainly not the case, since both S and λ are dependent on the polymerization rate and conversion, and consequently, the temperature profile inside the monomer mixture changes during the polymerization. However, since we are interested only in the maximal temperature reached during the polymerization, the time of interest is only around this point. Inside this time interval (a few minutes), there is insignificant temperature change (see Figure 2 in ref 37); therefore, the temperature profile inside the monomer mixture can be considered constant. Consequently, in this narrow time interval around the peak temperature, the system is assumed to be in the steady state. This simplification allows an analytical solution of the proposed model and requires calculation of only two unknown parameters: heat released per unit volume S and thermal conductivity of the monomer mixture λ. By integrating eq 1 taking into account the boundary conditions, the following solution is obtained:

T ) T0 +

[

]

ln(r/r1) 2 S (r12 - r2) + (r - r02) 4λ ln(r1/r0) 1

(2)

Since we are looking for the maximum temperature inside the monomer mixture, the radial position where the maximal temperature is reached can be calculated from the condition

dT/dr ) 0;

T ) Tmax

with the following result:

rmax )

x

r12 - r02

(3)

2 ln(r1/r0)

By inserting for the r value in eq 2 the expression for rmax defined in eq 3, the following expression for the maximal temperature inside the monomer mixture during the polymerization can be obtained:

Tmax ) T0 +

[

((

) )]

r12 - r02 1 - (r0/r1)2 S 2 r1 + ln -1 4λ 2 ln(r1/r0) 2 ln(r1/r0)

(4)

From the temperature measurements presented in ref 37 we were able to calculate the heat released per unit volume (S) as well as the thermal conductivity λ values for a particular GMA-EDMA mixture to be S ) 1.1754 MW/m3 and λ ) 7.35 W/m‚K (for a detailed calculation of λ and S see Appendix 1). Inserting the values for S and λ into eq 4, the relationship between the thickness of the annulus and the maximal temperature increase can be obtained (Figure 2). Knowing the maximal allowed temperature increase that still provides uniform pore size distribution, an annular monolith with the required inner diameter (r0) and the calculated thickness (r1 - r0) can be constructed. With the above-described approach one can construct a monolithic annulus of a required radius but limited thickness. However, since it is possible to construct the annuluses where Analytical Chemistry, Vol. 72, No. 22, November 15, 2000

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Figure 2. Effect of the annulus thickness on the maximal temperature increase during the polymerization of a GMA-EDMA monolith. Inner annulus radius is 10 mm; calculation is based on eq 4.

Figure 4. Schematic presentation of the housing for a large-volume annular GMA-EDMA monolith. Housing comprises inlet and outlet end fittings, two tightening nuts, and a body with distributor. Four O-rings (black circles) ensure proper sealing. Solid arrows indicate the direction of the mobile phase.

Figure 3. Construction of a large-volume GMA-EDMA monolithic unit of desired volume The monolithic unit (4) consists of three monolithic annuluses (1, 2, 3). Total thickness of unit 4 is a sum of the thicknesses of the monolithic annuluses 1, 2, and 3.

outer diameter of a smaller monolith is equal to the inner diameter of the larger one, a large-volume monolithic unit can be constructed by inserting two or several annuluses one into another as shown in Figure 3. In this way, a monolithic unit of required volume and uniform pore size distribution can be constructed. EXPERIMENTAL SECTION Polymerization and Modification. A monolithic unit consisting of two monolith annuluses was prepared. The preparation of a monomer mixture and the polymerization was performed by the procedure described elsewhere.38 The following reagents were used: glycidyl methacrylate and ethylene dimethacrylate both from Aldrich (Steinheim, Germany); cyclohexanol, dodecanol, and benzoyl peroxide from Fluka (Buchs, Switzerland). Polymerization of the outer monolithic cylinder was performed in an annularshaped stainless steel mold with the void inner diameter of 15 mm and outer diameter of 35 mm. The dimensions of the mould for inner monolithic annulus were 1.5 mm inner diameter and 15 (38) Sˇ vec F.; Bleha, T.; Tennikova, T. B.; Belenki, B. G. U.S. Patent 4 889 632, 1989.

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mm outer diameter. Both molds were placed in a termostated water bath in a vertical position; thus, all the shrinkage during polymerization occurred in an axial rather than in a radial direction.36 After the polymerization was completed, monoliths were removed from the molds and adjusted to a height of 84 mm to give a total volume of 80 mL. They were washed with ethanol to remove the porogens, and the smaller monolithic annulus was inserted into the larger one. The entire monolith was placed in a PTFE housing presented in Figure 4 and described in detail later. To introduce weak anion-exchange diethylaminoethyl (DEAE) groups on the monolith, 50 mL of pure diethylamine (Fluka) was pumped through the unit at a flow rate of 10-20 mL/min. The unit was closed on both sides and left overnight at room temperature. After the modification, the unit was washed with 5-10 column volumes of deionized water to remove the unreacted diethylamine. The content of diethylamine funcionalities was ∼2.5 mmol/g of dry monolith, determined from the mass difference before and after modification. Pore Size Distribution Measurement. Pore size distribution measurement was performed on mercury porosimetry PASCAL 440 (ThermoQuest Italia, Rodano, Italy) in the range of 14-20 000 nm. Samples of inner and outer monolith cylinder with a mass of ∼0.1 g were measured in the dry state. Pressure Drop Measurement. A pressure drop on the monolithic column at different flow rates was measured by a precision manometer (HBM, Darmstadt, Germany) connected to the system. At each flow rate, the value of the pressure of the system without and with the monolithic column was measured.

A difference between these two values at a defined flow rate was considered as a pressure drop on the monolithic unit. HPLC Equipment. Experiments were carried out on a preparative gradient HPLC system comprising two preparative pumps K-1800 allowing flow rates up to 1000 mL/min, an injection valve with a 1-mL SS sample loop, a preparative UV detector K-2500 set to 280 nm, a preparative mixing chamber, all connected by means of 1.5-mm-i.d. polyetheretherketone (PEEK) capillary tubes, and HPLC hardware/software (data acquisition and control station), all from Knauer (Berlin, Germany). Protein Separation. Myoglobin (Sigma, St. Louis, MO), conalbumin (Sigma), and soybean trypsin inhibitor (Fluka) were dissolved in a 20 mM Tris-HCl, pH 7.4 (binding buffer), in the following concentrations: 2, 6, and 8 mg/mL. The eluting buffer was a 20 mM Tris-HCl, pH 7.4, containing 1 M NaCl. A 1-mL aliquot of the protein mixture was injected for each analysis. Separation was performed using linear gradient elution (0-100% buffer B in 200 mL) and monitored at 280 nm. Different flow rates were applied. Dynamic Capacity Measurement. The bovine serum albumin (BSA, Fluka) was dissolved in a binding buffer (20 mM TrisHCl, pH 7.4) at a concentration of 10 mg/mL. The solution was pumped through the monolithic column at a defined flow rate, and the absorbency of the outlet at 280 nm was measured. The capacity of the monolithic column was calculated on 50% of the final absorbency value of the breakthrough curve. The monolithic column was regenerated with 240 mL of buffer A containing 2 M NaCl. RESULTS AND DISCUSSION Construction of a Large-Scale GMA-EDMA Monolithic Column. To verify our calculations, pore size distributions of the inner and outer cylinders were measured. In both cases, the monoliths have identical structure with the sharp maximum of pores around 1500 nm in diameter. Therefore, a uniform pore size distribution of the entire monolith was obtained. Once solving the problem with the preparation of a larger scale monolith, there is still a problem of making such a unit operational. This problem is associated with the fact that it is difficult to obtain a uniform sample distribution over the entire volume, which is essential for reproducible daily usage. One possible solution is a special housing presented in Figure 4. A mobile phase flows through the central bore of the inlet end fitting, and it is directed into a helical groove drilled inside the body serving as a distributor, which distributes the liquid over the entire outer surface of the monolith. The volume of the distributor is ∼3 mL; therefore, typical injected sample entirely enters into it before passing the monolith. Since the helical groove finishes in a dead end, the mobile phase is forced to penetrate into the monolithic annulus and it is collected in the central hole. The mobile phase exits through the bore of the outlet end fitting. Two tightening nuts fix the end fittings and the monolith inside the body with a distributor. The end fittings have on their outer side a connector for connecting the monolithic column to an HPLC system. Such a construction ensures a reproducible and uniform flow profile through the monolith. Characteristics of Large-Scale GMA-EDMA Monolithic Column. Since the constructed monolithic column is mainly intended for semipreparative or, in some cases, also preparative

Figure 5. Dependence of the pressure drop on the flow rate through the large-volume monolithic column. Mobile phase was distilled water.

purification and separations, high flow rates are expected to be used. To examine the suitability of the 80-mL unit for high flow rates, the pressure drop was measured. The results are presented in Figure 5. It can be seen that even at the flow rate of 250 mL/ min the pressure drop was below 2.5 MPa, which is much lower than in the case of the 15-mL monolithic rod.34 As shown also for GMA-EDMA monolithic rods and disks,4,11 the relation is linear, demonstrating no deformation of the monolith even at the highest flow rate applied. The possibility of running a monolithic column at high flow rates indicates that separations and purification can be performed in a very short time. This is demonstrated by the separation of the protein mixture containing myoglobin, conalbumin, and soybean trypsin inhibitor under the conditions of a linear salt gradient. At a flow rate of 200 mL/min, a good separation within 1 min was achieved (see Figure 6). Although the constructed monolithic column works in a radial direction mode, there is a significant difference in the geometry when compared to radial columns containing conventional particle media. In the case of the porous particles, the column efficiency is highly dependent on the linear velocity according to the Van Deemter equation.1 Since in the case of radial chromatography the mobile-phase flux increases from the outer surface toward the inner surface, the linear velocity also increases linearly with a diameter decrease. To diminish this effect, radial columns of large diameter and small thickness are used. In the case of monoliths, however, the characteristics were demonstrated to be flow-independent.3,11,28,39 Therefore, it can be expected that the difference between the inner and outer diameters can be much larger. In fact, in the case of the presented monolithic column, the outer diameter is 35 mm while the inner diameter is only 1.5 mm. Because of that, the linear velocity increases more then 23 times from the outer to the inner surface. To verify the flow independence, a gradient separation of proteins at different flow rates was performed. Each time, a gradient was adjusted to be constant when normalized to elution volume. In this way, the normalization of the chromatograms, although performed at (39) Mihelicˇ, I.; Koloini, T.; Podgornik, A.; Sˇ trancar, A. J. High Resolut. Chromatogr. 2000, 23, 39.

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Figure 6. Gradient separation of a protein mixture on an 80-mL monolithic column. Conditions: mobile phase (buffer A) 20 mM TrisHCl buffer, pH 7.4; (buffer B) 20 mM Tris-HCl buffer + 1 M NaCl, pH 7.4; flow rate, 200 mL/min; gradient, 0-100% buffer B in 60 s; sample, 2 mg/mL myoglobin (peak 1), 6 mg/mL conalbumin (peak 2), and 8 mg/mL soybean trypsin inhibitor (peak 3) dissolved in buffer A; injection volume, 1000 µL; detection, UV at 280 nm.

Figure 7. Effect of the flow rate on the separation efficiency. Separation of a protein mixture at six different flow rates (40, 80, 120, 160, 200, and 240 mL/min) normalized to the elution volume. Conditions: mobile phase, (buffer A) 20 mM Tris-HCl buffer, pH 7.4; (buffer B) 20 mM Tris-HCl buffer + 1 M NaCl, pH 7.4; flow rate, 200 mL/min; gradient, 0-100% buffer B in 200 mL; sample, 2 mg/mL myoglobin (peak 1), 6 mg/mL conalbumin (peak 2), and 8 mg/mL soybean trypsin inhibitor (peak 3) dissolved in buffer A; injection volume, 1000 µL; detection, UV at 280 nm.

different flow rates, clearly demonstrates no changes in the separation efficiency. The normalized chromatograms are shown in Figure 7. As can be seen, all chromatograms overlap even at the highest applied flow rate of 250 mL/min. In this case, the separation was completed in 50 s. Therefore, although at the highest flow rate the linear velocity on the inner surface of the monolith was 4000 cm/h, the column separation efficiency remained practically the same. Furthermore, the dynamic binding capacity of such a unit is of utmost importance. Again, the question of the flow rate 5698 Analytical Chemistry, Vol. 72, No. 22, November 15, 2000

Figure 8. Effect of the flow rate on the dynamic binding capacity. Conditions: flow rate, 50, 100, and 150 mL/min; sample, 10 mg/mL BSA in a 20 mM Tris-HCl buffer, pH 7.4; detection, UV at 280 nm.

independence is set forth. We performed the experiments by loading bovine serum albumin on the monolithic column at three different flow rates. After each loading, the elution was performed with 2 M sodium chloride in binding buffer. The results are shown in Figure 8. From overlapping of the normalized breakthrough curves, it can be concluded that the capacity was not affected by increased flow rates. The total capacity of the unit was 1.4 g of protein, which gives a capacity of 17.5 mg/mL of support, somewhat less than the values reported for commercially available GMA-EDMA monoliths,11,39 indicating that further increase of specific activity is possible. It should be emphasized, however, that the loading step in the case of a flow rate of 150 mL/min was performed in less than 2 min. Approximately 3 min was required for the elution and washing steps. Therefore, at least 12 loading and eluting steps can be performed within 1 h, gaining more than 15 g of a pure protein. CONCLUSIONS The approach described in this article allows the construction of large-volume monolithic columns with a uniform pore size distribution. Because of the modular approach, the volume of the monolithic unit can be freely selected. The appropriately designed housing allows a uniform sample distribution over the entire monolith volume. Since the characteristics of the monoliths are flow independent, such a unit can efficiently be used for the separation and purification of proteins and other large molecules at elevated flow rates, but still moderate pressure drops. Consequently, such monolithic columns can be a method of choice when high productivity in protein purification is required. ACKNOWLEDGMENT Support of this research through Project No. L2-1552-0158-99 from the Ministry of Science and Technology of Republic of Slovenia as well as through Eureka project “EU 1766 FAST1” is gratefully acknowledged. The authors thank J. Jancˇar for his technical assistance. APPENDIX 1 With the same arguments described in the text, a calculation of S and λ is based on the assumption of a steady-state condition

inside the time interval within which the maximal temperature inside the monomer mixture is reached. Since the polymerization was performed in a cylindrically shaped mold (in contrast to the mold described in the text, which had a shape of the annulus), eq 1 can again be applied but with different boundary conditions:

T ) T2; ∂T/∂r ) 0;

πr221S )

2π1λw(T2 - T3) ln(r3/r2)

(A4)

From eq A4. the heat released per unit volume S can be calculated:

S)

r ) r2

2λw(T2 - T3) ln(r3/r2)r22

(A5)

r ) 0 (center of the cylinder)

where S is heat released per unit volume (W/m3), λ is thermal conductivity of a monomer mixture (W/m‚K), T is temperature (K), T2 is temperature on the inner wall (K), r is radius (m), and r2 is inner wall radius (m). The solution is given by equation

T ) T2 + (S/4λ)(r22 - r2)

(A1)

The heat flux through the mold wall is described by equation

Q)

2π1λw(T2 - T3) ln(r3/r2)

(A2)

Inserting eq A5 into eq A1, the thermal conductivity λ can be calculated:

[1 - (r/r2)2] T2 - T3 λ ) λw 2 ln(r3/r2) T - T2

(A6)

Since the polymerization was performed in the glass tube37 the thermal conductivity λw was assumed to be 1 W/m‚K.40 The tube wall thickness was 2.35 mm.41 The maximal measured temperature during the polymerization in the middle of the monomer mixture was 113 °C, on the half of radius 108 °C and on the inner side of the wall 88 °C.37 The polymerization was performed at 55 °C in a cylinder mold with a radius of 25 mm.37 Inserting these values into eqs A5 and A6, the following values are obtained:

S ) 1.1754 MW/m3 where Q is heat flux (W), λw is thermal conductivity of the wall (W/m‚K), l is cylinder height (m), T3 is outer wall temperature ) polymerization temperature (K) (it is assumed that the water thermal resistance can be neglected), and r3 is outer wall radius (m). The heat flux during the polymerization (again assuming steady-state conditions) is given by

Q ) πr221S

In equilibrium, the two fluxes must be equal:

(A3)

and

λ ) 7.35 W/m‚K Received for review June 14, 2000. Accepted September 7, 2000. AC000680O (40) Koloini, T. Heat and Mass Transfer; FKKT University Press: Ljubljana, 1999; p 253. (41) Sˇ vec, F. Personal communication, 1997.

Analytical Chemistry, Vol. 72, No. 22, November 15, 2000

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