Monolithic Polysaccharide Materials

F. Svec, T.B. Tennikova and Z. Deyl (Editors) Monolithic Materials

Journal of Chromatography Library, Vol. 67 2003 Published by Elsevier Science B.V.

Chapter 6

Monolithic Polysaccharide Materials Per-Erik G U S T A V S S O N and P e r - O l o f L A R S S O N

Department of Pure and Applied Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 O0 Lund, Sweden

CONTENTS

6.1 6.2

6.3 6.4 6.5

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Monoliths based on agarose . . . . . . . . . . . . . . . . . . . . . . . . 6.2.1 Properties o f agarose . . . . . . . . . . . . . . . . . . . . . . . . 6.2.2 Preparation o f agarose monoliths . . . . . . . . . . . . . . . . . 6.2.2.1 Principle o f preparation . . . . . . . . . . . . . . . . . 6.2.2.2 Experimental techniques . . . . . . . . . . . . . . . . 6.2.3 Factors influencing the properties o f monolithic agarose . . . . . 6.2.3.1 Diffusive pore size . . . . . . . . . . . . . . . . . . . 6.2.3.2 Flow-through pore volume (porosity) . . . . . . . . . 6.2.3.3 Flow-through pore size . . . . . . . . . . . . . . . . . 6.2.3.4 Mechanical strength . . . . . . . . . . . . . . . . . . 6.2.4 Characterization o f monolithic agarose . . . . . . . . . . . . . . 6.2.4.1 Flow-through pore volume . . . . . . . . . . . . . . . 6.2.4.2 Flow-through pore size, size distribution, and spatial distribution . . . . . . . . . . . . . . . . . . . . . . . 6.2.4.3 Chromatographic efficiency . . . . . . . . . . . . . . 6.2.5 Derivatization o f monolithic agarose . . . . . . . . . . . . . . . 6.2.6 Composite agarose monoliths . . . . . . . . . . . . . . . . . . . 6.2.7 Special formats o f monolithic agarose . . . . . . . . . . . . . . . 6.2.7.1 Membranes . . . . . . . . . . . . . . . . . . . . . . . 6.2.7.2 Monoliths in radial columns . . . . . . . . . . . . . . 6.2.7.3 Electrophoresis using the flow-through pores for internal cooling . . . . . . . . . . . . . . . . . . . . . . . . . Monoliths based on cellulose . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

122 122 123 123 123 124 127 127 128 128 129 130 130 130 132 133 134 136 136 137

139 139 140 141

122

Chapter 6

6.1 INTRODUCTION Continuous beds - monoliths - were developed for separations of biomolecules around 1990 and have since become accepted as an alternative format to the established beaded supports. They are manufactured from most well known base materials, such as silica, polystyrene, polymethacrylates, cellulose, agarose, and polyacrylamide. Characterising features of these continuous beds are that they consist of one single piece of material in which is distributed a continuous network of wide, flow carrying pores. Besides the flow carrying pores, also narrow diffusion pores may exist. The proportion of "diffusive" pores is very much related to the base material used. The advantages of the monolithic format compared to beaded supports are related to the production of the separation material as well as its performance. The preparation procedure is simplified because the monolith can be cast directly in the chromatography column avoiding the time consuming steps of sieving and packing usually associated with beaded supports. The chromatographic performance of monolithic supports is often remarkably good, which can be explained in terms of improved mass transport by convective flow in the flow-through pores. Furthermore, the percentage of active matrix in a continuous bed can be easily varied between 30 and 90 %, opening opportunities for high flow rates with very porous structures or high loading with denser structures. This chapter will focus on the preparation and characterisation of monoliths made of polysaccharide materials. The literature primarily deals with agarose [ 1], and cellulose [2-4]. The use of both stacks of cellulose membranes [2], and rolled cellulose sheets [3] for chromatographic applications is covered elsewhere in this book. The use of other types of polysaccharides, such as dextran, alginate, k-carragenan, and starch for the preparation of monoliths should certainly be possible but has so far not been pursued. 6.2 MONOLITHS BASED ON AGAROSE

Polysaccharide materials for chromatography have been used successfully for a long time and include for example supports made of agarose, cellulose, and crosslinked dextran. The success of these materials relies primarily on their hydrophilicity/protein compatibility and their regenerability, i.e. they can be repeatedly treated with strong alkali, e.g. 0.5 mol/L NaOH, a preferred sanitation agent in industry. Their drawbacks include a low mechanical strength, which restricts the choice of particle size and flow rates. However, the mechanical strength can be improved considerably by chemical crosslinking. Beaded polysaccharide materials are manufactured by twophase suspension processes [5].

Monolithic Polysaccharide Materials

CH2OH

123

H

O \ H

OH

O

H

Fig. 6.1. The repeating unit of agarose consisting of D-galactose and 3,6-anhydroL-galactose.

6.2.1 Properties of agarose Agarose is a natural polymer prepared from seaweed (red algae) and consists of the D-galactose and 3,6-anhydro-L-galactose repeating units shown in Fig. 6.1. Agarose can be dissolved in boiling water and a gel is formed after cooling this solution below 45~ as a result of extensive hydrogen-bonding between the agarose chains. The gelling temperature may vary due to monomer composition (methoxyl content) and concentration of the solution and may also be altered by chemical derivatization of the polymer such as hydroxyethyl derivatization or addition of destructuring salts [6]. The pore size of agarose gels depends on the agarose content. Beads with a 6 % agarose content are frequently used and have an average pore size of approximately 30 nm, whereas 4 and 2 % agarose beads have a pore size of 70 and 150 nm, respectively [7]. Agarose with even larger diffusion pores are used in gel electrophoresis to allow the passage of very large molecules such as DNA. Prominent examples of commercial beaded materials for chromatography include Sepharose FF, Superose (Amersham Biosciences), Ultrogel A (BioSepra/Sigma-Aldrich), Bio-gel A (BioRad Labs) and Thruput (Sterogene). 6.2.2 Preparation of agarose monoliths

6.2.2.1 Principle of preparation The concept of preparation of continuous agarose beds is explained in Fig. 6.2. Typically, an aqueous agarose solution (60~

is mixed with a water-immiscible

organic solvent containing a surface-active agent. The mixture is stirred vigorously for several minutes, forming a thick, white emulsion. The emulsion is poured into a mold such as a chromatography column. The emulsion actually consists of two continuous phases, an aqueous agarose phase and a water-immiscible organic phase. After a short time the mold is cooled and its content solidifies into a stiff rod. This rod consists of a

Referencesp. 141

Chapter 6

124

Aa

":-i~.......i~~;- ~iii . COOl Id]~ ~iamh >

,

>

ge~!g ~6~cPh)ase

/

Agarosediffusion pores(30nm) Flowpore(10~tm)

A

/ Continuous agarose

Fig. 6.2. Preparation of a continuous agarose bed suitable for chromatography. Experimental details can be found in ref. [1 ]. Note that the resulting agarose monolith has 10 ~tm wide flow-through pores as well as a system of much narrower 30 nm diffusive pores.

single piece of agarose gel, transected by a continuous flow pore system filled with the organic phase. Subsequently, the ends of the rod are trimmed, flow adapters are attached, and washing solutions are pumped through the column to remove the waterimmiscible organic phase. Figure 6.2 describes the preparation of a rodlike agarose monolith suitable for chromatography by allowing the a g a r o s e - organic phase emulsion to solidify in a tube. By using other molds for the solidification, a number of other useful monolith formats may be prepared, such as sheets, membranes, fibers, radial beds, and composites [1 ]. A few examples of these formats are shown in Fig. 6.3.

6.2.2.2 Experimental techniques Although the basic concept for the preparation of superporous agarose monoliths indicated in Fig. 6.2 is straightforward, a number of experimental details has to be considered. Five main steps are involved: preparation of agarose solution, preparation

Monolithic Polysaccharide Materials

125

Fig. 6.3. Different self-supported continuous agarose formats. From left to right: Monolithic agarose - hydroxyapatite composite, membranes, rod, rod derivatized with Cibacron blue, and fiber. (Reprinted with permission from ref. [ 1], copyright 1999 Elsevier Sciences B. V.).

of pore-forming organic phase, preparation of emulsion, casting of monolith, and washing. The first step is the preparation of aqueous agarose solution. Typically, agarose powder is suspended in water in a concentration of 4-8 %, and placed in a suitable container such as glass bottle or test tube, depending on the required amount of agarose solution. The suspension is conveniently heated in a microwave oven to 95-100~ and kept at that temperature for about 2 min. During heating the container must be occasionally shaken/stirred to keep the agarose powder well suspended and to level out non-uniform heating. The bottle/test tube must not be sealed during the heating since dangerous pressure may build up inside. Thus, a very loosely attached stopper should be used. The agarose solution is then placed in a water bath kept at 60~ until used (typically within one day). The major issue in the above procedure is to keep the agarose powder well suspended during the heating. The powder starts to hydrate and transform into a viscous solution at temperatures higher than about 70~

If the agarose is allowed to partially

settle before this temperature is reached, the bottom part of the container may contain a concentrated and extremely viscous agarose solution that is very difficult to mix/equilibrate with the rest of the solution. An alternative procedure that avoids this problem is to heat the agarose suspension in an oil bath under constant stirring using a propeller stirrer. Referencesp. 141

Chapter 6

126

Yet, another way is to prepare a stable stock agarose suspension that can be heated without stirring. For this, two grams of agarose powder are added to 200 ml distilled water in a bottle and heated in a microwave oven to 95-100~

with occasional

shaking. The hot agarose solution is poured into 800 ml distilled water tempered to room temperature in a 1 L flask, forming a semiviscous agarose solution/suspension. Then, 58 g of agarose powder is added in portions with vigorous shaking. Finally, 100 mg of sodium azide is added this mixture to prevent microbial growth. This agarose suspension (6 % w/v) can be stored for long period of time and suitable portions withdrawn and conveniently heated without danger of settling of the agarose powder during the heating process. The second step, i.e. the preparation of the organic phase (flow pore-forming phase) is brought about by mixing a water-immiscible solvent with the surface-active agent. A number of combinations are possible. The preferred one, at least for smallscale preparations, is a mixture of cyclohexane and Tween 80. Typically, Tween 80 is added to cyclohexane and the mixture is thermostatted at 60~ in a water bath. The concentration of Tween 80 may be varied but is most often kept at 6 %. Since cyclohexane is a highly flammable liquid and used at a temperature close to its boiling point (boiling point for pure cyclohexane is 81 ~

boiling point for cyclohexane-water

azeotrope is 70~ work in a well-ventilated hood and use of explosion-proof electrical or pneumatic stirrers is recommended. The third step is the formation of emulsion. The organic phase is vigorously mixed before it is added to the agarose solution. The amount of organic phase used directly determines the flow-through pore volume in the final monolith. Typical volumetric ratio is 1/3 organic phase mixed with 2/3 agarose solution. The agarose/organic phase mixture is immediately emulsified at 60~ by stirring at 1000 rpm for 5 min with a large blade overhead stirrer. A viscous white emulsion is soon formed. A suitable stirrer should have a rather large blade that sweeps a large part of the vessel, since the emulsion is very viscous. This diminishes convective flow and material distribution in the vessel. Due to the high viscosity, the use of a smaller stirrer cannot be compensated for by a higher stirring speed, since this may lead to a heterogeneous emulsion. Fig. 6.4 shows suitable vessels for the preparation of 30 ml and 300 ml of emulsion. The emulsion, which contains two continuous phases, the agarose phase and the organic, flow pore-forming phase, is unstable and gradually changes its properties. Thus the time span between emulsion formation and casting must be timed to allow consistent results. The fourth step is the actual casting of the monolith. The agarose/organic phase emulsion is poured into a mold such as a glass tube pre-warmed to 60~ fitted with a rubber plug at the bottom end as shown in Fig. 6.2. After a short time, typically 1 min, during which time the emulsion matures, the system is cooled by transferring the glass

Monolithic Polysaccharide Materials

127

_

O

A

B

ollo z

--~ .f-r'-..

Fig. 6.4. Vessels suitable for formation of emulsion. Vessel with a stainless steel stirrer for preparing up to 30 ml of emulsion, constructed from a 50 ml disposable polystyrene plastic test tube (A). Thermostated glass reactor containing stainless steel baffles and stainless steel stirrer used for the preparation of 300 ml of emulsion with a total volume of 600 ml (B).

column to a water bath kept at 5~

The agarose solution solidifies when its tempera-

tures falls below about 45~ and a stiff superporous agarose monolith is obtained. In the fifth and final step, the agarose monolith is removed from the glass tube, trimmed to an appropriate length and then reinserted. The tube is fitted with flow adapters and the organic phase in this column is washed out typically with 5 volumes of water, 5 volumes of 50 % aqueous ethanol, and another 5 volumes of water.

6.2.3 Factors influencing the properties of monolithic agarose A number of factors affects the properties of monolithic agarose and some of them have already been briefly commented upon. 6.2.3.1 Diffusive pore size The agarose content in its solution determines the size of the diffusive pores. For example, 4 % agarose affords a monolith with an average pore size of about 70 nm, 6 % 30 nm and 8 % 20 nm. Admittedly, control of size of diffusive pores is usually less important. The standard material, 6 % agarose, contains pores that allow most proteins to diffuse rather unhindered. More importantly the agarose concentration greatly influences the mechanical strength of the monolith.

References p. 141

128

Chapter 6

6.2.3.2 Flow-through pore volume (porosity) The volume of the flow-through pores is determined by the volume of the waterimmiscible organic phase used in the emulsion. Therefore, it is usually easy to vary the percentage of these pores in the final monolith in the range 20-60 % and in some cases even broader.

6. 2.3.3 Flow-through pore size The flow-through pore size is controlled by both input of kinetic energy in the system during the preparation of the emulsion and concentration of the surface-active agent. A higher stirring speed gives narrower pores. Obviously, also the shape of the stirrer and baffles in the vessel affect the energy transfer in the system and the pore size. The effect of the surface-active agent is less transparent. We found that within certain limits, a higher percentage of surface-active agent affords monoliths with larger pores. Another factor of critical importance for the pore size is the time span between the emulsion formation and the agarose gelling. The bi-continuous phase system formed during the emulsification process is inherently unstable. As soon as the stirring is switched off, the bi-continuous phase structure starts to degrade. The kinetics for this degradation is dependent on many factors. Initially this is manifested in an increase in the size of the flow-through pores and decrease in their number. Delayed cooling/solidification of an unstirred emulsion enables the preparation of monoliths with larger pores. The size of the pores is obviously a very important factor controlling the flow properties of the monolith. This is an important issue considering that agarose gels have a limited mechanical strength. The flow resistance in a monolith is according to the Kozeny-Carman equation inversely proportional to the square of the pore diameter [8]. Thus, it is prudent to keep the pore size under control. As a rule of thumb, it is difficult to use monoliths with pore sizes smaller than 5 lam made of non-stabilized agarose with a bed length exceeding a few centimeters if reasonable flow rates are attempted. The literature describes agarose monoliths with pores often as large as 50 ~tm [1,9,10]. Monoliths with small pores are also desirable from another point of view. At a fixed total volume of the flow-through pores determined by the volume of organic phase used for the preparation, a decrease in the pore size increases the number of flow-through pores and, consequently, diminishes the distance between them. A shorter distance between the flow-through pores is desirable since it provides shorter diffusion path, resulting in faster mass transport,, which is beneficial for improved

Monolithic Polysaccharide Materials

129

chromatographic performance of the monolith. The choice of pore size is usually a compromise between both mechanical and mass transport requirements. The fact that a longer period of time between formation of the emulsion and gelling increases the flow-through pore size imposes restrictions on the size of agarose monoliths. The emulsion is cooled from the outside and consequently the outer parts gel first and the inner parts later. This generates larger pores in the center of the resulting monolith. This effect is potentially very serious since it may lead to an uneven flow distribution with a considerably faster flow through the center of the monolith. Typically, monoliths wider than 3 cm prepared using standard techniques exhibit an appreciable distortion of the flow profile and are unsuitable for chromatography. However, a smart solution to this problem is to use the monoliths not in the traditional axial but rather in the radial direction. In this approach the asymmetric pore size distribution does not lead to uneven flow profiles. In fact, the slightly larger pores near the center of such radial-flow columns are beneficial at high flow rates. Due to the many factors influencing flow-through pore size, it is difficult to predict the resulting pore properties with a certain type of equipment run under a certain set of conditions. However, after a few test runs with concomitant checking of the pore size in a microscope as described later, suitable conditions can readily be established.

6.2.3.4 Mechanical strength As touched upon above, the mechanical strength of monolithic agarose may become a limiting factor for applications at high flow rates using deep beds. Once the pressure drop across the monolithic bed is too high as a result of a high flow rate, the structure compresses gradually. This compression is fully reversible once the flow rate is decreased again. Since the compression of the bed decreases primarily the size of the flow-through pores, an additional increase in the pressure drop can be observed leading to further compression of the bed. The pore structure completely collapses after exceeding a certain flow rate and the flow stops in much the same way as observed for a bed packed with agarose beads. Several design features may be used to improve the mechanical strength. For example, higher agarose concentration and the use of chemical crosslinking of the agarose chains may help significantly. Even without a specific crosslinking, considerable improvement of mechanical strength is usually achieved upon chemical derivatization introducing ligands of various kinds such as ion exchange and affinity. The mechanical strength is also improved if the total volume of flow-through pores is kept low. A high percentage of these pores afford a monolith with a "spongy" feel. However, a decrease in the pore volume by necessity also limits the pore cross-section

Referencesp. 141

130

Chapter 6

area, which in turn leads to a higher flow resistance. This in fact counteracts the positive effects of lowering the pore volume. 6.2.4 Characterization of monolithic agarose

Characterization of properties of a new monolith is important to reproducibly achieve the desired function. Some of these characterization methods are standard techniques of determining the chromatographic performance, while others are specifically tuned to reveal the basic properties of the monolith, such as pore volume, pore size and its distribution, spatial distribution of pores, presence of dead end pores, etc.

6. 2.4.1 Flow-through pore volume The flow-through pore volume can be determined using size-exclusion experiments with molecules that do not penetrate the agarose matrix such as 0.5 ~tm latex particles or Blue Dextran (Mw 2,000,000). The elution volume for these molecules is a direct measure of the flow-through pore volume of the continuous agarose bed. Practical tests [ 1,11 ], have shown that the measured flow pore volume usually agrees very well with the designed pore volume represented by the volume of added organic phase in the emulsion.

6. 2.4.2 Flow-through pore size, size distribution, and spatial distribution Fundamental properties of the monolith such as the pore size, and spatial distribution of the pores can be rapidly determined by observing thin slices of the monolith under a microscope. The technique is very useful when developing new recipes. Thus, a few milliliters of an emulsion under preparation are withdrawn and solidified in a test tube or on a glass surface. The piece of gel is sliced by a microtome or a razor blade. It is not necessary to obtain extremely thin slices. The slices are then inspected and photographed using a microscope. A useful technique to improve the contrast between the flow-through pores and the agarose matrix is to allow the monolith surface to dry slightly. This partial drying leads to a preferential loss of water from the pores, which make them considerably more visible when illuminated from above and at an angle with no cover glass attached. Another simple trick to improve the contrast is a brief washing of the slices with ethanol. An example of this observation is given in Fig. 6.5. The pores are the dark areas, typically 50 lam large. The light areas constitute the agarose phase containing diffusive pores with a size of 30 nm that are not visible. A complementary method to improve the visibility is filling the pores with a suspension containing colored latex particles that do not enter the agarose phase. A

Monolithic Polysaccharide Materials

131

Fig. 6.5. Optical micrograph of a thin gel slice of continuous agarose bed. The contrast between the flow pores and the agarose has been enhanced by partial drying of the surface.

powerful technique, although not tested yet, would be to use fluorescent dyed latex particles and study the slice of a monolith with confocal microscopy. Related techniques are known to provide valuable structural information with superporous agarose beads [ 12-14]. We have also described a more elaborate way of determining pore size as well as the three-dimensional structure of the flow-through pore network [15]. In this technique, a replica of the pore structure is made and studied by scanning electron microscopy (SEM). Figure 6.6 shows the micrograph. A solution of ethylene dimethacrylate in toluene containing a free-radical initiator (azobisisobutyronitrile) was pumped through a continuous agarose bed column. The column inlet and outlet were then closed and polymerization was initiated using ultraviolet light (366 nm). After the polymerization was completed, the continuous bed was removed from the tube and the agarose phase was melted away in a boiling water bath. The obtained replica was then studied by SEM. The structure presented in Fig. 6.6 shows a fairly evenly distributed network of flow-through pores with diameters of between 25 and 75 ~tm. In some other monoliths the network was clearly less randomly distributed. Instead, the flow-through pores had a preferred orientation. Similar observations had been made under the light microscope, although not with such a clarity. A study of a set of gels prepared under different conditions demonstrated that the non-random pore distribution was due to mechanical stratification of the emulsion before the solidification of the agarose occurred. By careful treatment of the emulsion, the stratified pore system could be avoided. On the other hand, a stratified pore system could possibly be advantageous in some applications.

References p. 141

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Chapter 6

Fig. 6.6. SEM micrograph of pore structure replica of an agarose monolith. (Reprinted with permission from ref. [ 15], copyright 1996 Elsevier Sciences B. V.).

6. 2. 4.3 Chromatographic efficiency The chromatographic efficiency of the continuous agarose beds can be determined by size-exclusion experiments. Pulse injection experiments with both a low-molecular-weight tracer such as sodium azide and medium-sized protein e.g. bovine serum albumin are routinely used in our laboratory to characterize continuous agarose beds. These results can be compared with the chromatographic efficiency of columns packed with standard agarose beads of different particle sizes. These comparisons reveal that the chromatographic efficiency of the continuous agarose beds is equal to the chromatographic efficiency of columns packed with standard agarose beads, having a diameter roughly equal to the distance between the flow-through pores of the continuous agarose matrix as determined by microscopy. Fig. 6.7 shows the HETP values obtained by pulse injections of sodium azide in a radial flow column as a function of the flow velocity. Also shown are theoretical HETP curves in packed columns for standard agarose beads with a particle size similar to the diffusion distance between the pores in the continuous radial column. The observed HETP-minimum is reasonably close to twice the distance between the flow-through pores, which could be expected for a well-behaved column. Due to the transparency of continuous agarose beds, a convenient quick visual test of the homogeneity of the beds can be made by injecting colored latex samples to check that no gross pore distribution inhomogeneity exists. Similarly, by injecting a

Monolithic Polysaccharide Materials

133

1.0

~-~'~"

0.8

0.4

~I

...............

0

0.4 0.8 1.2 1.6 2.0 Flow velocity (cm/min)

Fig. 6.7. Effect of flow velocity on efficiency of the monolithic agarose column expressed as HETP (curve 1). Column: 65 ml radial flow agarose monolith. Injection: 2 ml pulses of sodium azide solution (2 mg/ml). The dashed curves represent theoretical curves for 220 ~tm (curve 2) and 160 pm (curve 3) homogeneous agarose beads. (Reprinted with permission from ref. [ 11], copyright 2001 Elsevier Sciences B. V.).

colored low-molecular-weight sample an overall flow profile check can be obtained. By these experiments a quick check can be made to determine possible reasons for poorly performing beds. 6.2.5 Derivatization of monolithic agarose

Continuous agarose can be provided with functional chemistries equal to those known for agarose in bead form [ 16]. However, the protocols developed for agarose beads must be transformed to in situ activation/coupling conditions, which require pumping of reagent solutions through the continuous agarose bed column. This is usually successful. Difficulties may include the fact that derivatization often leads to slight shrinkage of the agarose gel. An obvious remedy is to cast the original gel slightly oversized. For small gel beds an axial compression of the gel with an aid of flow adaptors typically affords good result. Another potential difficulty may be associated with in situ derivatization using very reactive reagents. In such cases, preferential derivatization of the inlet portion of the continuous bed may result, while the outlet portion is poorly converted. This can be avoided by lowering the reaction rate by e.g. reacting at a low temperature and circulation of the reagents through the bed as

References p. 141

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Chapter 6

TABLE 6.1 OVERVIEW OF SURFACE CHEMISTRIES USED WITH SUPERPOROUS AGAROSE MONOLITHS

Activation chemistry

Ligand

Bed dimensions Application (length x diameter, mm)

Ref

Chlorotriazine

Cibacron Blue Various + various textile dyes

Affinity [ 11] chromatography

CNBr

NAD+-derivative

60 x 16

Affinity [ 1] chromatography

Glycidol/periodate- Lactase oxidation

60 x 25 (radial column)

Bioreactor

CNBr

Acetylcholine esterase

15 x 5

Biosensor [ 10] (electrochemical)

Tresylchloride

Glucose oxidase

15 x 5

Biosensor

CNBr

Antibody

15 x 5

Analyte trapping [9]

[11 ]

[9]

fast as possible to ensure that all parts of the bed are in contact with the same concentration of the active reagent. Table 6.1 gives an overview of chemistries used. Coupling of antibodies to a continuous agarose bed using the cyanogen bromide activation is an example of application [9]. The method was adapted from the activation procedure run at room temperature developed by March et al. [ 17], and modified for in situ conditions. The protocol was designed for a continuous agarose bed with dimensions of 15 x 5 mm but can be scaled to suit any bed dimension by following ordinary scale-up/down rules such as by maintaining constant both contact time and agarose/reagent ratio.

6.2.6 Composite agarose monoliths In composites, two or more materials are combined to afford special advantages to the resulting product. An early example of this approach was the Ultrogel AcA media developed for size-exclusion chromatography of biomolecules [18]. Here, the good size-exclusion selectivity of the soft polyacrylamide gel could be exploited by incorporating the gel in the more mechanically stable agarose matrix. Another example is the Streamline adsorbents developed for expanded bed chromatography [19]. The

Monolithic Polysaccharide Materials

13 5

TABLE 6.2 AGAROSE MONOLITH COMPOSITES

Composite component Application in addition to agarose monolith

Main advantages

Dimensions Ref (length x diameter, mm)

Yeast cells

Catalytic reactor

Column usage possible

5 • 16, 10 • 10

[20]

Hydroxyapatite

Chromatography Avoids column clogging

14 x 16

[1]

Ion-exchange beads

Chromatography Application of dirty feed stock possible

5 • 16

[20]

1-2 ~tm graphite powder

Chromatography

Cheap reversed-phase adsorbent

5 x 16

[20]

Reticulated vitreous carbon

Electrochemical biosensor

Increased surface area/ Improved mechanical stability

15 • 5

[10]

Streamline adsorbents consist of quartz particles incorporated into agarose beads to increase their density. By addition of a filling material to the agarose solution before making the agarose/organic phase emulsion, a range of composite agarose monoliths for different applications can be prepared (Table 6.2). The properties of the filling material such as its particle size and hydrophobicity determine the maximum percentage of the agarose phase that can be substituted with the filler. Mostly, up to 50 % of the agarose phase can be replaced while maintaining the stability of the agarose monolith. The preparation method is exemplified by the incorporation of hydroxyapatite particles in an agarose monolith [ 1]. A different way of making composite agarose monoliths is to cast the agarose/organic phase emulsion into a larger structure such as reticulated vitreous carbon (RVC). This preparation method was used for the preparation of a biosensor where RVC, apart from improving the mechanical stability of the bed, also provided an electrically conductive network through the bed [ 10]. This monolithic composite was then subjected to a cyanogen bromide activation followed by coupling of acetylcholinesterase. By using the electrode arrangement shown in Fig. 6.8, this composite monolith was used for the on-line determination of the pesticide paraoxon.

Referencesp. 141

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Chapter 6

Counter electrode

Composite material (working electrode)

IIII IF3 p Reference electrode (SCE)

RVC (black) + superporous agarose with immobilised acetylcholinesterase

From sample injection valve

Working electrode

Fig. 6.8. Device for FIA determination of paraoxon that includes the continuous agarose/ reticulated vitreous carbon composite. Pore size 25 ~tm.

6.2.7 Special formats of monolithic agarose

6.2. 7.1 Membranes Monolithic agarose can also be manufactured in thick membrane/disc format. This format makes it easy to attain separations characterized by high flow velocities and short residence times at minimal pressure drops. Monolithic agarose membranes have potential as a cheap adsorbent for analysis in various chromatographic modes. A typical simple membrane-casting device that we assembled is shown in Fig. 6.9. It consists of two thick glass plates separated by a U-shaped gasket made of 5 mm thick silicon rubber. The rubber serves both as a seal and spacer, determining the thickness of the final membrane. The assembly is kept together by strong paper clamps and is pre-warmed to 60~ in a water bath. The agarose/organic phase emulsion is subsequently filled in the mold. After a pre-determined time the mold is transferred to a cold water bath to solidify the agarose. The casting device is then dismantled and circular pieces of the milky white membrane are punched from the sheet by a sharp steel puncher with the appropriate diameter. The membrane discs can be inserted in

137

Monolithic Polysaccharide Materials

Space filled with agarose emulsion Cla Glass plate 3 x 130 x 130

Silicon rubber; thickness 5 mm

Fig. 6.9. Casting device for agarose membranes. (Reprinted with permission from ref. [1], copyright 1999 Elsevier Sciences B. V.).

membrane holders and the organic phase removed by pumping water, 50 % ethanol, and water through the discs. Another apparatus has also been constructed for continuous casting of monolithic agarose membranes. This device is schematically shown in Fig. 6.10. A hot agarose/organic phase emulsion is introduced in the container located at the top of the apparatus, where a turbine keeps the emulsion agitated. The emulsion is then drawn into the casting channel of the device by two endless Teflon sheets. The Teflon sheets are in contact with a thermostatted aluminum structure, which at the top is kept at 60~

and at the lower part at 10~

by aid of circulating water. When the agarose

emulsion enters the zone of low temperature, it gels. At the bottom of the apparatus a continuous agarose membrane is delivered at a rate of 10 cm/min, which is determined by the speed of the Teflon sheets. 6.2. 7.2 Monoliths in radial columns

The preparation of rods with a diameter exceeding 3 cm imposes problems due to the temperature gradient formed upon cooling the emulsion. As a result, the central part of the rod solidifies later than the outer parts, affording larger pores in the center. The resulting uneven flow profile leads to a decrease in chromatographic efficiency of References p. 141

Chapter 6

138

Continuous membrane casting

Stirred, gel-forming -:.i:i:i.":!i.!~emuIsion

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the monolithic agarose. To circumvent this problem, large diameter rods should be operated in the radial direction. In this implementation, the asymmetric pore size distribution does not lead to uneven flow profiles. Figure 6.11 shows two types of radial column constructed for the application of monolithic agarose [ 11 ]. The radial column of Fig. 6.1 l a is constructed from two octagonal glass plates with a central hole functioning as end-pieces and a standard chromatography solvent filter from sintered stainless steel which acts as an outlet flow distributor. This simplified con-

Fig. 6.11. Radial columns for larger scale applications of monolithic agarose.

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139

struct was used submerged in a beaker with a centripetal (inward) flow accomplished by connecting a pump or a vacuum to the center outlet. The radial column of Fig. 6.1 lb was designed to work in both centrifugal and centripetal flow directions. This column consisted of Plexiglas cylinder, Plexiglas top and bottom plate, and a stainless steel flow distributor. The self-supported structure of the monolithic agarose greatly simplifies the packing procedure of the radial columns. A radial bed can be removed from the radial column B and inserted in the column A, and vice versa, ready for a new chromatographic run in a few minutes. The column in Fig. 6.1 l a also demonstrates another advantage of the self-supported structure of monolithic agarose. It enables a simple low cost construction of separation devices, which cannot be achieved with particlebased adsorbents. The flow properties of the radial beds are also excellent, allowing flow rates of over 100 ml/min to be used at a pressure drop less than 0.1 MPa.

6. 2. 7. 3 Electrophoresis using the flow-through pores for internal cooling A special format of monolithic agarose was briefly investigated for use in electrophoresis [ 1]. The idea was to use the flow-through pores to dissipate the Joule heat created during electrophoresis, and make it possible to use larger blocks of agarose for preparative electrophoretic separations. The heat normally produced in a larger gel block would lead to a severe temperature build-up with resulting loss of resolution or even breakdown of the gel structure. While this is not a problem for thin gels, it had a significant impact on the development of a large-scale use of this high-resolution technique [21 ]. The conceptual idea as well as one of the prototype designs is shown in Fig. 6.12. Initial tests showed indeed that this idea is viable since a substantial lowering of the internal gel temperature was observed. However, problems with protein recovery were observed, probably due to protein absorption at the agarose - organic solvent interface. 6.3 M O N O L I T H S BASED ON C E L L U L O S E

Cellulose found in the cell wall of plants consists of glucose molecules linked by 1,4 13-glucosidic bonds. Three different types of cellulose are currently available for chromatography, fibrous, microgranular, and regenerated. Fibrous cellulose consists of a highly inhomogeneous structure with essentially non-porous microcrystalline regions intertwined with less dense amorphous regions. This structure results in a material with little porosity. By acid treatment of cellulose followed by chemical cross-linking, microgranular cellulose have been developed featuring increased porosity and mechanical stability. Regenerated cellulose is prepared by first dissolving

Referencesp. 141

Chapter 6

140

Heptane coolant Superporous E/ectrode ~ /agaros%

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Fig. 6.12. Monolithic agarose for electrophoresis with internal cooling. The left part of the figure shows the concept of the internal cooling: A water-immiscible solvent is pumped through the electrophoretic bed carrying away the Joule heat formed during electrophoresis. The right panel shows a prototype design, where the superporous separation gel is surrounded by a layer of normal homogeneous agarose to prevent the escape of the heptane coolant pumped through the superporous separation gel

natural cellulose in a solvent, followed by a solidification step, leading, for example to beads with a high porosity suitable for separation of biomolecules. Cellulose has numerous hydroxyl groups available for coupling chemistries typical of all polysaccharide-based materials [ 16,22]. Cellulose membranes were used for chromatographic separations of biomolecules [23,24]. They are available in different formats such as layered stacks [2,25,26], and rolled layers [3,27]. These formats are described in detail elsewhere in this book. Cellulose-based monoliths have been commercialized by Sepragen (San Leandro, CA) under the trade name SepraSorb [28]. These products are made of regenerated cellulose and fabricated in sheets. The monoliths have a continuous pore structure, with a pore diameter of 50-300 ~tm. The monoliths are available for ion-exchange chromatography, derivatized with both weak and strong ion-exchange functionalities. The large flow-through pore size of these monolith enables operation at high flow rates exceeding 100 ml/min at a very low back pressure. These monoliths can be used for capture steps with crude particle-containing feed streams without prior clarification. 6.4 ACKNOWLEDGEMENT This work was supported by the Swedish Centre for BioSeparation (CBioSep).

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6.5 REFERENCES 1 2 3 4

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

P.-E. Gustavsson, P.-O. Larsson, J. Chromatogr. A, 832 (1999) 29. J.E. Kochan, Y.-J. Wu, M.R. Etzel, Ind. Eng. Chem. Res., 35 (1996) 1150. J.F. Kennedy, M. Paterson, Polym. Int., 32 (1993) 71. R. Noel, A. Sanderson, L. Spark, in: J.F. Kennedy, G.O. Phillips, P.A. Williams (Eds.) Cellulosics: Materials for Selective Separations and Other Technologies, Horwood, New York, 1993, p. 17-24. R. Arshady, J. Chromatogr., 586 (1991) 181. A.S. Medin, Studies on Structure and Properties of Agarose, Doctoral thesis, Department of Biochemistry, Uppsala University, Sweden, 1995. P. Serwer, S.J. Hayes, Anal. Biochem., 158 (1986) 72. G. Sofer, L. Hagel, Handbook of Process Chromatography, Academic Press, San Diego, 1997, p. 263. M.P. Nandakumar, E. P~lsson, P.-E. Gustavsson, P.-O. Larsson, B. Mattiasson, Bioseparation, 9 (2000) 193. M. Khayyami, M.T. P6rez Pita, N. Pena Garcia, G. Johansson, B. Danielsson, P.-O. Larsson, Talanta 45 (1998) 557. P.-E. Gustavsson, P.-O. Larsson, J. Chromatogr. A, 925 (2001) 69. P.-E. Gustavsson, A. Axelsson, P.-O. Larsson, J. Chromatogr. A, 795 (1998) 199. E. P~lsson, A.-L. Smeds, A. Petersson, P.-O. Larsson, J. Chromatogr. A, 840 (1999) 39. Anders Ljungl6f, Amersham Biosciences, personal communication. P.-E. Gustavsson, P.-O. Larsson, J. Chromatogr. A, 734 (1996) 231. G.T. Hermanson, A.K. Mallia, P.K. Smith, Immobilized Affinity Ligand Techniques, Academic Press, London, 1992. S.C.March, I. Parikh, P. Cuatrecasas, Anal. Biochem., 60 (1974) 149. J. Uriel, J. Berges, E. Boschetti, R. Tixier, C. R. Acad. Sc. (Paris) S6rie D, 273 (1971) 2358. Expanded Bed Adsorption, Principles and Methods, Amersham Pharmacia Biotech, ISBN 91-630-5519-8. P.-E. Gustavsson, P.-O. Larsson, WO 00/12618, 2000. W. Thormann, in: J.-C. Janson, L. Ryd6n (Eds.), Protein Purification, Principles, High-Resolution Methods and Applications, Wiley, New York, 1998, pp. 651-678. R. Arshady, J. Chromatogr., 586 (1991) 199. D.K. Roper, E.N. Lightfoot, J. Chromatogr. A, 702 (1995) 3. C. Charcosset, J. Chem. Technol. Biotechnol., 71 (1998) 95. X. Santarelli, F. Domergue, G. Clofent-Sanchez, M. Dabadie, R. Grissely, C. Cassagne, J. Chromatogr. B, 706 (1998) 13. D. Zhou, H. Zou, J. Ni, L. Yang, L. Jia, Q. Zhang, Y. Zhang, Anal. Chem., 71 (1999) 115. K. Hamaker, S.-L. Rau, R. Hendrickson, J. Liu, C.M. Ladisch, M.R. Ladisch, Ind. Eng. Chem. Res., 38 (1999) 865. http://www.sepragen.com