Rolled Stationary Phases: Dimensionally Structured Textile

tightly rolled in a cylindrical configuration, has a three-dimensional structure with sufficient hydrodynamic stability to withstand interstitial ...
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Ind. Eng. Chem. Res. 1999, 38, 865-872

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Rolled Stationary Phases: Dimensionally Structured Textile Adsorbents for Rapid Liquid Chromatography of Proteins Kent Hamaker,†,‡ Shiang-Lan Rau,§ Richard Hendrickson,† Jim Liu,§ Christine M. Ladisch,§ and Michael R. Ladisch*,†,‡ Laboratory of Renewable Resources Engineering, Department of Agricultural and Biological Engineering, and Textile SciencesDepartment of Consumer Sciences and Retailing, Purdue University, 1295 Potter Center, West Lafayette, Indiana 47907-1295

A woven textile fabric, consisting of 60% cotton/40% polyester, tightly rolled in a cylindrical configuration, has a three-dimensional structure with sufficient hydrodynamic stability to withstand interstitial eluent velocities of up to 300 cm/min when packed into standard liquid chromatography column assemblies. Demonstration of the pressure stability of the cotton/ polyester fabric was followed up with experiments in which the cotton (cellulose) portion was derivatized and the fabric evaluated for chromatography of proteins. When derivatized to give a (diethylamino)ethyl (DEAE) anion exchanger, a velocity independent plate height of 2 mm, a static capacity of 115 mg of bovine serum albumin/g of stationary phase, and a dynamic protein loading capacity which decreases only 25% over an 800% increase in mobile-phase velocity from 6.7 to 54 cm/min was achieved. The fibers that make up the stationary phase have a relatively nonporous structure which minimizes pore diffusional effects. A protein separation of Cytochrome C from β-lactoglobulin A is shown to be completed by ion-exchange chromatography in less than 10 min using an NaCl step gradient. Gradient chromatography of a hen egg white shows resolution of the proteins into two major components (lysozyme and ovalbumin) as well as two minor ones. A size exclusion separation of PEG 20 000 from glucose requires only 90 s. These characteristics, together with the ability of the cellulose-based stationary phase to withstand rapid flow rates, indicate that this type of stationary phase has potential for applications where chromatography using DEAE-cellulose particles has proven successful. Introduction The use of fibers as chromatographic stationary phases could expand the range of chemistries available for inventing new forms of packed columns for industrial bioseparations. Cellulosic, polyphenylene, and poly(ethylene terephthalate) fibers separate proteins. Acrylic fibers exhibit hydrophobic and ionic interactions with some types of dye molecules.1-3 Silica, polypropylene, polypeptide, carbonaceous, and other textile fibers represent additional scaffolds upon which biospecific ligands could be grafted or ion-exchange capacity derivatized. The surface chemistries of fibers used in the textile industry can be both hydrophilic (cellulose) and hydrophobic (polyester, aramid, and acrylic) (see Figure 1, parts a and b, for structures). Hydrophilic fibers with the appropriate porosities could prove useful as stationary phases for size exclusion chromatography. Derivatized forms of cellulose are viable for ion-exchange chromatography of proteins. Hydrophobic or reversed-phase interactions are possible with aromatic, vinyl copolymer, and carbonaceous fibers. These enable chromatographic separations to take place on the basis of interactions of the biomolecules with hydrophobic or ionic groups on the stationary phase * Address all correspondence to Michael R. Ladisch, LORRE, Purdue University, 1295 Potter Center, West Lafayette, IN 47907-1295. † Laboratory of Renewable Resources Engineering (LORRE). ‡ Department of Agricultural and Biological Engineering. § Textile SciencesDepartment of Consumer Sciences and Retailing.

Figure 1. Chemical structures of (a) cellulose (cotton), (b) poly(ethylene terephthalate) (polyester), and (c) DEAE cellulose.

when elution is carried out with an appropriate mobile phase. The potential of fibers and, in particular, particulate materials consisting of ion exchange celluloses has been recognized for over 40 years.4 The derivatization of fabric with (diethylamino)ethyl (DEAE) groups was reported in 1984.5 Derivatized rolled stationary-phase columns have been used for size exclusion chromatography.6 Oriented disks of fiber batting such that the fibers are perpendicular to flow have been used to estimate the pore size of bulk fabric.7,8 Extruded sheets

10.1021/ie970779u CCC: $18.00 © 1999 American Chemical Society Published on Web 02/02/1999

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Figure 2. Representations of (a) fibers,12 (b) yarn,13 (c) woven fabric,13 and (d) fabric directions and orientation.22 Figures reprinted with permission.

of a porous polymeric material, embedded with derivatized particles and packed as stacked disks in a column, have been used as an ion exchanger for protein purification.9 Alternately, polymer rod continuous stationary phases represent a type of nonparticulate stationary phase in which the polymer is formed in situ10,11 and in some cases compacted by bed compression before being placed into service.10 Poor flow properties of fibers packed in chromatography columns has limited their application as adsorbents in fixed-bed systems. The layering of disks of fabric or membranes into a column have been used to obtain improved flow properties. Rolled stationary phases (RSP) made from fabrics enable a long bed depth to be attained while retaining good flow properties.1,6 The fibers in rolled stationary-phase columns are assembled into yarns, the yarns woven into fabric, and the fabric rolled and packed into a column in a manner which gives a mechanically stable bed, whose physical structure is in one piece, instead of individual particles.6 These columns differ from packed beds of fibers in that the rolled stationary phase positions the fabric in a three-dimensional structure which supports the fibers (Figure 2a)12 assembled into the yarns (Figure 2b),13 and the textile weave (Figure 2c)13 through contact between adjacent layers of fabric. Rapid chromatography is possible since this type of packed bed resists compaction by the hydrodynamic forces which characterize liquid chromatography systems, and therefore facilitates rapid chromatography on a process scale. Chromatography columns based on filaments,14,15 hollow fibers,16 polymer rods,10,11,17 and membranes18,19 represent other types of continuous stationary phases. The scale-up of fiber-containing columns suitable for preparative or process chromatography must consider the role of the column walls in supporting the stationary phase. Gel-type stationary phases packed in a column above a critical diameter are known to collapse and result in an excessive pressure drop so that flow is not possible.20 While capillary columns have been packed with fibrous cellulose acetate,14 a critical column diameter would rapidly be reached for this or other natural

and synthetic textile yarns, above which the walls would no longer support the bed, and the bed of fibers would collapse, resulting in a stopped flow. Process-scale chromatography not only seeks to achieve high mass transfer rates at low-pressure drops (i.e., a high ratio of mass transfer to momentum flow) but also requires volumes which would be 104-107 times greater than that of a glass capillary column, and therefore correspond to diameters measured in terms of centimeters to decimeters, rather than millimeters. Hence, other means are needed to support the fibers or filaments in a stationary packed bed. The challenge lies in the development of columns having larger total volumes, and therefore larger total adsorption capacity. This paper describes characteristics of fabric columns whose volumes are in the same range as columns packed with particulate stationary phases. The purpose of this paper is to present experimental data on protein separations and the velocity effect on pressure drop, plate height, and protein loading of a chromatography column packed with a DEAE-derivatized, cotton/polyester fabric. Materials and Methods Packing of Columns. This work is based on three continuous stationary phases: 100% cotton fabric, style no. 400M supplied by Testfabrics, Inc., P.O. Box 26, W. Pittston, PA 18643, a 60/40 blend of cotton (cellulose)/ polyester (PET, poly(ethylene terephthalate)), obtained from Cotton Inc., P.O. Box 30067, Raleigh, NC 17612, and a DEAE-derivatized form of the cotton/polyester fabric. Preparation and packing of the stationary phases into a liquid chromatography column are described elsewhere.6 Column bed dimensions were 10 mm i.d. × 150-200 mm in length, or 26 mm i.d. × 140 mm in length. The Sephadex G-50 (coarse), from Pharmacia Biotech (Piscataway, NJ), was equilibrated with deionized water and packed using standard procedures.21 Sephadex G-50 is not an ion exchanger and is used for size exclusion chromatography. Derivatization of Stationary Phase. The cotton/ polyester stationary phase was derivatized by a propri-

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etary method. The general reaction for preparing DEAE cellulose is given by Rousseau et al.;5 the chemical structure of DEAE cellulose is presented in Figure 1c. The nominal ion-exchange capacity was 0.09 mequiv/g of fabric or 0.15 mequiv/g of cellulose as calculated from elemental N analysis. Packing Density of Stationary Phases. The packing densities achieved for 100% cotton, cotton/polyester blend, and the DEAE-derivatized form, packed by the device described by Hamaker et al.,6 were 0.612 g/mL for cotton and 0.660 g/mL for both the cotton/polyester and DEAE-derivatized cotton/polyester. The packing densities for the cotton and cotton/polyester fabrics were determined by removing the rolled stationary phase from its column, measuring the oven dry weight of the material, and dividing it by the volume which the stationary phase occupied when packed in the column. Alternatively, column end fittings were removed and the entire column was placed in an oven for several days until the stationary phase was dried to a constant weight. The difference between the total weight and the tare weight of the column itself was then used to calculate the packing density. The packing densities of the underivatized and DEAEderivatized cotton/polyester stationary phases are from an average of four columns each. Standard deviations of the measurements were 0.06 and 0.03 g/mL for underivatized and DEAE cotton/polyester, respectively. The packing density of the cotton is based on a single column. The cotton and cotton/polyester stationary phases were packed so that flow of the mobile phase occurred over the fabric in a bias orientation. Figure 2d22 gives the orientation of a fabric which corresponds to the bias direction. The void fraction of the DEAEderivatized column ( ) 0.265) was much lower than the void fraction for the underivatized column ( ) 0.44), even though the packing densities (dry basis) were the same for both columns. This difference is associated with derivatization techniques, although we do not currently have an explanation for the difference. Measurement of Pressure Drop. The plate heights are in a range expected for preparative-scale chromatography, and the experimentally measured peak shape is consistent with other types of liquid chromatography columns. Flow rates of up to 100 mL/min, corresponding to an interstitial velocity of about 300 cm/min (or 18 000 cm/h) for the RSP column, were used to measure the pressure drop. The void fraction of the underivatized material was 0.44, as calculated from the elution volume of Dextran 2 000 000. The underivatized RSP column had a larger void fraction than the DEAE RSP column, but was similar to Sephadex G-50 used to obtain a pressure drop at the flow rate range of 0-30 mL/min. Both stationary phases were packed in a 10 mm i.d. column with a 17 cm high bed. Liquid Chromatography Instrumentation. The instrument used in evaluating the columns consisted of a programmable, single-head pump by ISCO (model no. 2350, Lincoln, NE), connected to a Rheodyne injector (model no. 7125, Cotati, CA) and either a Waters differential refractometer (model no. 401 or 403, Milford, MA) or an ISCO V4 UV-vis detector. The pump drew buffer from a 1.5 L reservoir and directed the flow through a pressure release (check) valve, through the injector, through the column, and finally to the detector. The signal from the detector was either recorded on a strip-chart recorder or digitally on a data acquisition

device from ComputerBoards, Inc. (Mansfield, MA). After the completion of runs, the column was flushed with 10 column volumes of 0.2 µm filtered 1 M NaCl solution. The column was then stored in 1 M NaCl until it was used again. High flow rate runs for both the 10 and 26 mm i.d. columns were carried out using a Novaprep preparative liquid chromatograph (R & S Technology, Wakefield, RI). Mobile Phase. An in-house source of 0.2 µm filtered, deionized water with a resistivity greater than 2 × 106 Ω‚cm was used in making up the mobile phase. Tris buffers at pH 8.0 were prepared using Trizma base (T1503) from Sigma Chemical (St. Louis, MO). The pH was adjusted to the desired level using HCl. Sodium chloride was added to the buffer to give a 1 M concentration. The NaCl suppressed binding of the protein to the DEAE anion exchanger. The phosphate buffer was made from mono- and dibasic sodium phosphate (Baxter Scientific Products, McGaw Park, IL). The run with the PEG 20 000/glucose was carried out using a mobile phase of deionized water. Molecular Probes and Proteins. The molecular probes used in this work were dextran 2 000 000, PEG 20 000 (P-2263), D(+)-glucose, and Dextran 66 300 (all from Sigma Chemical Co., St. Louis, MO). The proteins Cytochrome C and β-lactoglobulin A were from Sigma. The egg white separation was carried out using proteins from hen egg white. The protein elution profiles and loading were determined using bovine serum albumin (BSA fraction V powder, 98-99%, grade VII, from Sigma). The concentration used was 2 mg of BSA/mL in either 10 mM Tris-HCl (pH 8.4) or 20 mM phosphate (pH 6.0). Protein Loading. The amount of BSA which adsorbs on an ion exchanger, such as DEAE cellulose, gives a useful measure of its capacity and is referred to as protein loading. Static protein loading was measured using fabric cut into pieces that were approximately 0.25 cm2 in area. Approximately 120 mg of wet fabric was placed in a 13 × 100 mm glass test tube containing 5 mL of BSA solution at a concentration of 2 mg/mL in 8.7 mM Tris buffer at pH 8.4. The fabric pieces were kept in the solution for 6 h at ambient temperature. An aliquot of the supernate was then removed, appropriately diluted, and measured for protein content. The protein concentration was determined by measuring the change in UV absorbance at 280 nm and comparing against a BSA standard. Protein uptake by the fabric was calculated from the decrease in protein concentration in solution. The dry weight of the DEAE-cellulose fabric was determined by the following procedure. The fabric was first immersed in 5% NaCl solution for at least 1 h to remove the adsorbed protein from the stationary phase. The NaCl was rinsed from the fabric and the material was then dried in an oven at 105 °C overnight, cooled in a desiccator for 30 min, and weighed. Dynamic protein loadings were calculated from breakthrough profiles for 2 mg/mL of BSA in a 20 mM phosphate buffer at pH 6.0. Protein loadings achieved at interstitial velocities of 6.7-54 cm/min in a 26 mm i.d. × 140 mm long column of DEAE cotton/polyester were determined by calculating the point along the curves for which the area above the curve, up to the inlet concentration, and below the curve are equal. The amount of protein adsorbed, the protein loading, was

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Figure 3. Pressure drop for a 10 mm i.d. × 173 mm long underivatized rolled stationary-phase cotton/polyester column (O) and a 10 mm × 90 mm bed of Sephadex G-50 (- - -). The RSP mobile phase was deionized water; the mobile phase for the Sephadex column was 50 mM Tris with 500 mM NaCl at pH 8.

then calculated by dividing the total amount of protein removed from the solution by the dry weight of the fabric. The static equilibrium loading was measured in a Tris buffer at pH 8.4, while breakthrough profiles used to calculate dynamic loading were carried out using 20 mM phosphate buffer at pH 6.0. The analysis of Mosher et al.23 shows that the charge for BSA at pH 7.0 is -14 and at pH 6.5 the charge would be -10.5. Hence, the charge of BSA at pH 8.4 should be more negative and its adsorption of the DEAE cotton/polyester may be more favored than that at pH 6.0. The dynamic loading experiments at pH 6.0 were carried out with a protein which is less charged, and therefore less strongly retained. The decrease in retention (loading) with an increasing flow rate was therefore expected to be more apparent at pH 6.0 than pH 8.4 and easier to detect. Results and Discussion The evaluation of dimensionally structured textile adsorbents packed in chromatography columns was based on the pressure drop, plate height, separation of proteins, and adsorption capacity of bovine serum albumin. Pressure Drop. The structural rigidity of an underivatized rolled stationary phase (RSP) is demonstrated by the pressure drop curve in Figure 3. Stable operating pressures were achieved at flow rates up to 100 mL/min (18 000 cm/h) of deionized water through a column with an inside diameter of 10 mm. The pressure drop of the Sephadex column was 30 psi/cm or about 520 psi over the length of the bed. The particle Reynolds number corresponding to this flow rate was estimated to be about 8.7,6 and is still considered to be in the laminar regime (Rep < 10), although the decrease in the friction factor begins to level off in this range.24 A side-by-side comparison of a rolled stationary phase to a large particle size particulate stationary phase where both stationary phases had a characteristic dimension of 200-300 µm led to runs at high flow rates with Sephadex G-50 (coarse) even though G-50, a crosslinked Dextran gel, is not designed for high-pressure

Figure 4. Plate height of DEAE-cotton/polyester rolled stationary phase measured with NaCl and BSA. Column dimensions: 10 mm ID × 105 mm long. Mobile phase: 50 mM Tris, 500 mM NaCl, and pH 8. Sample size: 100 µL. Interstitial velocities correspond to volumetric flow rates of 0.5-10 mL/min.

operation. This material was packed in a 10 mm i.d. glass column to give an initial bed height of 90 mm. The interstitial void fraction prior to bed compaction was 0.39 based on Dextran 2 000 000. Plate Height. The plate heights of BSA for the DEAE-derivatized cotton/polyester rolled stationary phase (Figure 4) ranged from 1.5 to 1.65 mm. The plate height increases only 10% as interstitial velocities increase from 2 to 48 cm/min. The interstitial velocities are calculated based on the void fraction ( ) 0.265) measured using Dextran 2 000 000. Plate heights for BSA and NaCl with respect to the Sephadex were measured for flow rates up to 10 mL/ min (or 33 cm/min). As the flow rate was increased to 15 mL/min, there was an onset of vigorous mixing of the beads in the upper 10-15 mm of the column. When the flow rate was increased to 25 mL/min, compression of the bed height from 90 to 10 mm occurred in a matter of seconds together with a dramatic increase in pressure (Figure 3). While Sephadex G-50 is not intended for use at high flow rates, it was stable for a long enough time so that measurements of peak dispersion of injected solutes could be made. The BSA injections into the rolled stationary-phase column gave retention volumes that corresponded to void fractions from 0.268 at an eluent flow rate of 2.37 cm/min (0.5 mL/min) to 0.299 at 42.6 cm/min (10 mL/ min). BSA injected into the Sephadex column had velocities of 1.87-33.5 cm/min over the same range of flow rates and gave apparent void fractions ranging from 0.341 to 0.380. The increase in the apparent void fraction indicates that the bed volume explored by the BSA increases with an increasing flow rate. If the higher flow rates were to cause channeling, the apparent void fraction would not increase, and might decrease because of peak broadening from the solute bypassing part of the bed. The phenomenon of the increasing void fraction is consistent with the convective effects which help to explain velocity-independent plate heights for both stationary phases. Decoupling of Plate Height from Velocity. The dispersion or plate height of an injected solute band in

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Figure 5. Plate height of Sephadex G-50 (coarse). Column dimensions of 10 mm × 90 mm. Mobile phase: 50 mM Tris, 500 mM NaCl, and pH 8. Sample size: 100 µL. Interstitial velocities correspond to volumetric flow rates of 0.5-10 mL/min.

Figure 6. Comparison of selectivity curves for 60%/40% DEAEcotton/polyester rolled stationary phase (RSP) to Sephadex G-50.

a chromatography column typically passes through a minimum and then increases significantly as the eluent flow rate increases. However, the plate height for NaCl increases slightly for the rolled stationary phase (Figure 4) and significantly for the Sephadex G-50 (Figure 5). The nearly constant peak dispersion for the NaCl in the rolled stationary-phase column is due to the relatively low fraction of small, dead-end pores in this stationary phase and its lower porosity compared to Sephadex G-50 (compare RSP and Sephadex curves in Figure 6). The rolled stationary phase has about a 6.5fold higher packing density (0.65 g/mL) than Sephadex (0.09-0.11 g/mL) and holds less eluent as a stagnant liquid (about 30% of its volume) compared to the Sephadex (about 90%). This corresponds to a lower Kav (Figure 6) where Kav is (Ve - Vo)/(Vt - Vo) and Ve represents elution volume, Vo interstitial void volume, and Vt empty column volume.21 The rolled stationary phase shows only a small increase in dispersion for NaCl, and the dispersion is nearly independent of the flow rate over the range of interstitial velocities shown

in Figure 4. This implies that the NaCl introduced into the column does not extensively explore pores in the cellulose and polyester since these pores are too small for the NaCl to penetrate, and their volume relative to the total column volume is also small. Nonetheless, rapid size exclusion separation of BSA from NaCl is achieved.6 Conversely, the porosity of the beads is large enough to facilitate diffusion of the NaCl into the bead, but is too small to allow significant penetration of BSA. Consequently, the velocity dependence of NaCl is more pronounced than that of BSA in the Sephadex G-50 (Figure 5) as well as more pronounced than that of NaCl with respect to the rolled stationary phase (compare Figure 4 to Figure 5). Velocity-independent plate heights were reported by Kelley and Billmeyer 25 in 1969 for hydrodynamic chromatography columns packed with nonporous, 115 µm glass beads which were run at reduced mobile-phase velocities of 10-10000. Hydrodynamic chromatography separates differently sized particles on the basis of the tendency of small molecules or particles to seek slower moving fluid streamlines near the surface of particles, compared to larger molecules which associate with faster streamlines. Consequently, the larger molecules elute first. Stegeman et al.26 showed that a flat plate height occurs in hydrodynamic chromatography columns packed with 1.5 µm diameter (dp), monodisperse, nonporous glass beads with an interparticle hydraulic radius of 0.32 × 10-4 cm. Plate heights were measured for molecular probes having molecular weights of 2200775000. Reduced velocities were approximately 5-160. The reduced velocity is given by u/(Dm/dp) where u is the interstitial velocity, Dm is the solute’s diffusivity, and dp is the characteristic size or diameter of the stationary phase. Ruthven27 suggested that pores in larger particles experience forced laminar flow (i.e., Poiseuille flow) when the pore radius is 10-4-10-5 cm. The flat plate heights of BSA for both the rolled stationary phase and Sephadex G-50 are consistent with the expected assymptotic behavior of a modified van Deemter equation as well as several other equations that anticipate flat plate heights (1):

H)A+

B vchrom

+

DCvchrom D + Cvchrom

(1)

where H ) plate height; vchrom ) chromatographic velocity (cm/s); A ) Eddy diffusion contribution to the plate height, cm; B ) coefficient of axial dispersion (cm2/ s); C ) coefficient of linear velocity (s); D ) coefficient representing the convective contribution to plate height, cm. Note that the definition of velocity in eq 1 is given as cm/s since the associated coefficients (A, B, C, and D) as reported by Yang et al.1 are in terms of seconds (rather than minutes). At high velocities, eq 1 simplifies to

H)A+D

(2)

that is, a constant plate height. Porosity. PEG probes having a molecular weight larger than 3350 are excluded from this type of DEAEcotton/polyester stationary phase,6 resulting in a rapid separation of PEG 20 000 from glucose (Figure 7). The chromatogram in Figure 7 also demonstrates that a relatively large sample volume (0.5 mL) of these two components can be separated in 90 s. Similar results

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Figure 7. Size exclusion separation of the molecular probes PEG 20 000 and glucose for rolled stationary phase consisting of 60%/ 40% cotton/polyester derivatized with DEAE. Column dimensions of 10 mm i.d. × 105 mm long; 8.3 mL volume. Sample size of 500 µL, PEG 20 000, and glucose dissolved in deionized water at 5 mg/ mL. Flow rate 5 mL/min.

have been obtained for protein/salt separations as described elsewhere.6,28 Interstitial Void Fractions. The plate heights29 of BSA for Sephadex G-50 (Figure 5) ranged from 1.31 to 1.6l mm at interstitial velocities of 2-33 cm/min (for  ) 0.392 measured using Dextran 2 000 000) and correspond to reduced plate heights of 6.6-8.1 for a particle diameter of 200 µm. The corresponding interstitial velocities for the Sephadex G-50 are smaller than those for the rolled stationary phase at equivalent volumetric flow rates since the void fraction of Dextran 2 000 000 and BSA in the Sephadex G-50 column is larger than that for the DEAE-derivatized rolled stationary phase (RSP), although the protein elutes at a volume that is close to the interstitial volume in both cases. Interstitial velocities based on Dextran 2 000 000 were used in order to compare plate heights on an internally consistent basis. Separation of Large from Small Molecules. The literature (see review by Hamaker and Ladisch29), together with the results in Figures 5 and 6, are consistent with a simple explanation for the separation of large from small molecules based on size. Nonporous particles, in monodisperse, agglomerated, aggregated, or woven forms, when packed in fixed beds (i.e., chromatography columns) have channels between them. In a situation analogous to a liquid distribution plate, sufficiently high interstitial flow rates cause a pressure drop across the channels. The pressure drop promotes even distribution of flow across all the channels as long as their characteristic hydraulic width exceeds about 10-5 cm. As a consequence, stagnant pockets of fluid are minimized. Since all the fluid in the packed bed is moving, plate heights approach a constant value, and for nonadsorbed solutes, the column takes on the operating characteristics of hydrodynamic chromatography. Therefore, PEG 20 000 and glucose (Figure 7) or BSA and NaCl 6 are rapidly separated in as little as 25 s. Exclusion regions are associated with the stagnant fluid in the laminar sublayer next to the particles’ or fibers’ surfaces,26 and the convective contribution to axial dispersion would be expected to decrease with an increasing solute size as proposed by Guttman and DiMarzio.30 This characteristic would be expected for Sephadex G-50 and other particulate stationary phases when the solute is too large to penetrate the internal pores of the stationary phase, as well as for the rolled stationary phase. Hence, flat plate height behavior is observed for BSA with respect to Sephadex G-50, and

Figure 8. A step gradient of 100 mM NaCl followed by 500 mM NaCl elutes Cytochrome C and β-lactoglobulin A, respectively, from a 60%/40% DEAE-cotton/polyester column. 10 mm × 10.6 cm column; 0.2 mL sample volume.

the void fraction explored by BSA increases as the volume associated with stagnant pockets of fluids decreases. The porosity of the Sephadex G-50 is large enough, however, to allow NaCl to explore the significant volume inside the particle. Hence, peak broadening occurs, and the plate height increases with an increasing interstitial velocity (Figure 5). Even though Sephadex G-50 also separates large from small molecules, the mechanism is probably different from the mechanism that facilitates the same separation in RSP columns. Adsorption followed by elution using a step gradient of NaCl gives a clean separation, as shown in Figure 8 for Cytochrome C (MW ) 12 400, pI ) 10) and β-lactoglobulin A (MW ) 18 600, pI ) 5.1). The loading of the protein was carried out in 20 mM Tris-HCl buffer at pH 8.0, at a concentration of 5 mg/mL for each protein and a flow rate of 8 mL/min. There was no elution of protein until a step gradient of 100 mM NaCl was introduced at 8 mL/min, which caused Cytochrome C to elute. This was followed by a 500 mM NaCl step gradient which caused the β-lactoglobulin A to elute. A subsequent wash of the column with 1 M NaCl gave no further elution of protein. Another illustration of the separation capability of the rolled stationary-phase column is shown for hen egg white lysozyme and ovalbumin (Figure 9) using a multistep, salt gradient. The major peaks are lysozyme (peak 1, MW ) 14 600, pI ) 10.7 to 11.3) which elutes first and ovalbumin (peak 3, MW ) 45 000, pI ) 4.6) which elutes at 150 mM NaCl. The two minor peaks represent conalbumin (peak 2, MW ) 80 000, pI ) 5.8 to 6) and ovamucoid (peak 4, MW ) 28 000, pI ) 3.9-4.3). Peaks 2 and 4 eluted at 50 mM and 300 NaCl step gradients, respectively. Protein Loading Capacity. The adsorption of BSA onto DEAE cellulose was measured as a function of time in test tubes, and as a function of flow rate for the rolled stationary phase packed into a 26 mm × 140 mm column. The DEAE-cotton/polyester fabric gives a protein loading of at least 115 mg of BSA/g of fabric for an initial BSA protein concentration of 2 mg/mL. Since the derivatization procedure functionalizes only the cellulose portion of the cotton/polyester fabric, the capacity calculated per unit weight of cellulose gives a loading of 196 mg of BSA/g of cellulose ()115 mg of BSA/g of fabric ‚ 1 g of fabric/0.6 g of cotton ‚ 1 g of cotton/0.98 g of cellulose).

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Figure 9. Separation of hen egg white lysozyme over 60%/40% DEAE-cotton/polyester stationary phase. Conditions: column dimensions of 10 mm i.d. × 102 mm long. Step gradients of NaCl in 50 mM Tris-HCl buffer, pH 7.5: 0-8 min/0 M NaCl; 8.1-13 min/50 mM NaCl; 13.1-20 min/150 mM NaCl; 20.1-25 min/300 mM NaCl at a flow rate of 3 mL/min. Sample size of 500 µL of 50 mL of egg white dissolved in 200 mL of buffer (courtesy of Dr. Prabha Painuly, R & S Technology).

Figure 11. Protein breakthrough profiles for 60%/40% DEAE cotton/polyester packed as a 26 mm i.d. × 140 mm long bed. BSA was dissolved at a concentration of 2 mg/mL in a 20 mM phosphate buffer at pH 6.0. Note that the buffer pH is lower than the pH used for batch studies shown in Figure 10. CF denotes concentration of protein in the feed and C the concentration in the effluent.

Figure 10. Comparison of batch equilibrium loading in the presence and absence of agitation for 60%/40% DEAE cotton/ polyester. Conditions: initial BSA concentration of 2 mg/mL in 8.9 mM Tris-HCl buffer at pH 8.4 and ambient temperature.

The static loading runs showed that the BSA attained its equilibrium loading more quickly if the stationaryphase/protein solution is agitated (Figure 10). Fabric samples which were left to sit in the glass test tube without agitation exhibited a slower uptake of protein than those in the test tubes which were gently agitated by shaking in a shaker bath. While the extent of agitation is small (i.e., gentle shaking in a water bath), it is sufficient to bring the depleted protein concentration near the surface of the textile back to the bulkphase concentration much more quickly than the diffusional processes or Brownian motion associated with a quiescent fluid. A small amount of fluid motion can have a significant effect on mass transfer. The protein loadings obtained at the average residence times of 15 s (at 54 cm/min) to 2 min (at 6.7 cm/min) are similar to the static loading at contact times close to zero for the intermittently agitated test tubes (Figures 10-12). An exact comparison is not possible since the agitated batch experiment gives a steep slope at short times. The static loading at short times (Figure 10) and dynamic loadings at interstitial velocities of 6.7-53.9 cm/min (Figure 12) are about 4.6-6 times lower than the maximum static loading (Figure 10) for the same starting concentration of protein. The increase in interstitial velocity, ue, resulted in an earlier breakthrough and a 27% decrease in loading. However, this is small relative to the 800% increase in interstitial velocity from 6.7 to 53.9 cm/min

Figure 12. Capacity of 60%/40% DEAE-cotton/polyester rolled stationary phase for BSA protein as a function of interstitial velocity. Void fraction of 0.28 as measured used dextran 66 300. Packing density of 0.66 mg/mL.

(Figure 11). If forced-convective flow in the interyarn and interfiber void spaces is the dominant mechanism for transporting protein to the ion-exchange sites, then the adsorption capacity should approach a constant value which reflects adsorption kinetics rather than diffusional resistances. Conclusion Dimensionally structured textile adsorbents consisting of rolled stationary phases of a DEAE-derivatized cotton/polyester fabric blend are mechanically stable at high flow rates. This enables the operation of the cellulose-based stationary phase at high flow rates and also measurement of plate height, separations, and protein adsorption at high flow rates to take place. Plate heights and protein loadings showed only a small dependence on the eluent velocity. A size exclusion effect could explain the elution of high-molecular-weight species followed by low-molecular-weight components. However, the increase in the apparent void fraction at a constant bed height with increasing flow rates suggests that hydrodynamic chromatography is another possible mechanism. The static capacity of 115 mg of BSA/g of

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stationary phase (198 mg/g of cellulose) shows that textile adsorbents can attain capacities which are in the same range as particulate materials. Separation of proteins by ion exchange in 10-20 min, and by size exclusion in less than 90 s, demonstrates that this type of stationary phase has the potential for preparativescale, rapid liquid chromatography of proteins. Textile-based rolled stationary phases offer chromatographers a new approach for studying established chemistries of textile fibers which have yet to be tested for purification of protein molecules, and which could result in new types of chromatographic stationary phases. Research is now needed to further improve the operational characteristics of these materials and expand the derivatization chemistries of fibers for use in chromatographic separations. Elucidation of fundamental mechanisms which govern separation characteristics of continuous stationary phases, ranging from membranes and polymer rods to dimensionally structured textile adsorbents, will help to identify process applications for which this class of stationary phases could provide unique capabilities. Acknowledgment The material in this work was supported by R & S Technology (Wakefield, RI), National Science Foundation Grants 8907304 and BCS 8912150, The National Aeronautics and Space Administration, Grant NAGW2329 (NSCORT), and the Purdue University Agricultural Experiment Station. We thank Dr. Joe Weil, Craig Keim, and Chenghong Li of Purdue University, and Dr. Paul Schauer and Robert Ritacco of R & S Technology for helpful comments and review of this manuscript. We thank Dr. Prahba Painuly of R & S Technology for the chromatogram used in Figure 9. Literature Cited (1) Yang, Y.; Velayudhan, A.; Ladisch, C. M.; Ladisch, M. R. Protein Chromatography Using a Continuous Stationary Phase. J. Chromatogr. 1992, 598, 169-180. (2) Yang, Y.; Velayudhan, A.; Ladisch, C. M.; Ladisch, M. R. Liquid Chromatography Using Cellulosic Continuous Stationary Phases. In Advances Biochemical Engineering/Biotechnology; Tsao, G. T., Feichter, A., Eds.; Springer-Verlag: Berlin, 1993; pp 123-146. (3) Yang, Y.; Ladisch, C. M. Hydrophobic Interaction and Its Effect on Cationic Dyeing of Acrylic Fabric. Textile Res. J. 1993, 63, 283-289. (4) Peterson, E. A.; Sober, H. A. Chromatography of Proteins. I. Cellulose Ion-Exchange Adsorbents, J. Am. Chem. Soc. 1956, 76, 751. (5) Rousseau, R. W.; Ferrell, J. K.; Reardon, R. F. Synthesis of Diethylaminoethyl Cellulose on Cotton Fabric. Ind. Eng. Chem. Prod. Res. Dev. 1984, 23, 250. (6) Hamaker, K. H.; Liu, J.; Ladisch, C. M.; Ladisch, M. R. Transport Properties of Rolled, Continuous Stationary Phase Columns. Biotechnol. Prog. 1998, 14, 21. (7) Bertoniere, N. R. Accessible Internal Volume Determination in Cotton. In Modern Textile Characterization Methods; Raheed, M., Ed.; Marcel Dekker: New York, 1996; pp 265-290. (8) Bertoniere, N. R.; King, W. D. Effect of Scouring/Bleaching, Caustic Mercerization, and Liquid Ammonia Treatment on the Pore Structure of Cotton Textile Fibers. Textile Res. J. 1989, 59, 114-121.

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Received for review November 6, 1997 Revised manuscript received September 22, 1998 Accepted November 2, 1998 IE970779U