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Main Encyclopedia Of Bioprocess Technology Fermentation, Biocatalysis аnd Bioseparation
Encyclopedia Of Bioprocess Technology Fermentation, Biocatalysis аnd BioseparationFlickinger M.C.
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FlickingerM.C., EncyclopediaOfBioprocessTechnologyFermentation,Biocatalysis, AndBioseparationVol.1-5(JohnWileyA&Sons,NY,1999)/38223_a1.pdf 1 A ACTIVATED CARBON, DECOLORATION OF PHARMACEUTICAL PRODUCTS FREDERICK S. BAKER CHARLES E. MILLER ALBERT J. REPIK E. DONALD TOLLES Westvaco Corporation Charleston, South Carolina KEY WORDS Activated carbon Adsorption isotherms Adsorption of radionuclides BET surface area Emissions, motor vehicles Food, beverage, and cooking oil Pharmaceuticals Pore volume Powdered carbon regeneration Solvent recovery OUTLINE Introduction Physical and Chemical Properties Manufacture and Processing Thermal Activation Processes Chemical Activation Processes Forms of Activated Carbon Products Shipping and Storage Specifications Economic Aspects Analytical Test Procedures and Standards Health and Safety Liquid-Phase Applications Potable Water Treatment Groundwater Remediation Industrial and Municipal Wastewater Treatment Sweetener Decolorization Chemical Processing Food, Beverage, and Cooking Oil Pharmaceuticals Mining Miscellaneous Uses Gas-Phase Applications Solvent Recovery Gasoline Emission Control Adsorption of Radionuclides Control by Chemical Reaction Protection against Atmospheric Contaminants Process Stream Separations Gas Storage Catalysis Bibliography Additional Reading INTRODUCTION Activated carbon is a predominantly amorphous solid that has an extraordinarily large internal surface area and pore volume. These unique characteristics are responsible for its adsorptive properties, which are exploited in many dif- ferent liquid- and gas-phase applications. Activated carbon is an exceptionally versatile adsorbent because the size and distribution of the pores within the carbon matrix can be controlled to meet the needs of current and emerging markets (1). Engineering requirements of specific appli- cations are satisfied by producing activated carbons in the form of powders, granules, and shaped products. Through choice of precursor, method of activation, and control of processing conditions, the adsorptive properties of prod- ucts are tailored for applications as diverse as the purifi- cation of potable water and the control of emissions from bioproduct recovery processes. In 1900, two very significant processes in the develop- ment and manufacture of activated carbon products were patented (2). The first commercial products were produced in Europe under these patents: Eponite, from wood in 1909, and Norit, from peat in 1911. Activated carbon was first produced in the United States in 1913 by Westvaco Corp. under the name Filtchar, using a by-product of the papermaking process (3). Further milestones in develop- ment were reached as a result of World War I. In response to the need for protective gas masks, a hard, granular ac- tivated carbon was produced from coconut shell in 1915. Following the war, large-scale commercial use of activated carbon was extended to refining of beet sugar and corn syrup and to purification of municipal water supplies (4). The termination of the supply of coconut char from the Philippines and India during World War II forced the do- mestic development of granular activated carbon products from coal in 1940 (5). More recent innovations in the man- ufacture and use of activated carbon products have been driven by the need to recycle resources and to prevent en- vironmental pollution. PHYSICAL AND CHEMICAL PROPERTIES The structure of activated carbon is best described as a twisted network of defective carbon layer planes, cross- linked by aliphatic bridging groups (6). X-ray diffraction 2 ACTIVATED CARBON, DECOLORATION OF PHARMACEUTICAL PRODUCTS patterns of activated carbon reveal that it is nongraphitic, remaining amorphous because the randomly cross-linked network inhibits reordering of the structure even when heated to 3,000 �C (7). This property of activated carbon contributes to its most unique feature, namely, the highly developed and accessible internal pore structure. The sur- face area, dimensions, and distribution of the pores depend on the precursor and on the conditions of carbonization and activation. Pore sizes are classified (8) by the International Union of Pure and Applied Chemistry (IUPAC) as micro- pores (pore width �2 nm), mesopores (pore width 2–50 nm), and macropores (pore width �50 nm). The surface area of activated carbon is usually deter- mined by application of the Brunauer-Emmett-Teller (BET) model of physical adsorption (9,10) using nitrogen as the adsorptive (8). Typical commercial products have specific surface areas in the range 500–2,000 m2/g, but val- ues as high as 3,500–5,000 m2/g have been reported for some activated carbons (11,12). In general, however, the effective surface area of a microporous activated carbon is far smaller because the adsorption of nitrogen in micro- pores does not occur according to the process assumed in the BET model, which results in unrealistically high val- ues for surface area (10,13). Adsorption isotherms are usu- ally determined for the appropriate adsorptives to assess the effective surface area of a product in a specific appli- cation. Adsorption capacity and rate of adsorption depend on the internal surface area and distribution of pore size and shape but are also influenced by the surface chemistry of the activated carbon (14). The macroporosity of the car- bon is important for the transfer of adsorbate molecules to adsorption sites within the particle. Functional groups are formed during activation by in- teraction of free radicals on the carbon surface with atoms such as oxygen and nitrogen, both from within the precur- sor and from the atmosphere (15). The functional groups render the surface of activated carbon chemically reactive and influence its adsorptive properties (6). Activated car- bon is generally considered to exhibit a low affinity for wa- ter, which is an important property with respect to the ad- sorption of gases in the presence of moisture (16). However, the functional groups on the carbon surface can interact with water, rendering the carbon surface more hydrophilic (15). Surface oxidation, which is an inherent feature of ac- tivated carbon production, results in hydroxyl, carbonyl, and carboxylic groups that impart an amphoteric charac- ter to the carbon, so that it can be either acidic or basic. The electrokinetic properties of an activated carbon prod- uct are, therefore, important with respect to its use as a catalyst support (17). As well as influencing the adsorption of many molecules, surface oxide groups contribute to the reactivity of activated carbons toward certain solvents in solvent recovery applications (18). In addition to surface area, pore size distribution, and surface chemistry, other important properties of commer- cial activated carbon products include pore volume, parti- cle size distribution, apparent or bulk density, particle den- sity, abrasion resistance, hardness, and ash content. The range of these and other properties is illustrated in Table 1 together with specific values for selected commercial grades of powdered, granular, and shaped activated carbon products used in liquid- or gas-phase applications (19). MANUFACTURE AND PROCESSING Commercial activated carbon products are produced from organic materials that are rich in carbon, particularly coal, lignite, wood, nut shells, peat, pitches, and cokes. The choice of precursor is largely dependent on its availability, cost, and purity, but the manufacturing process and in- tended application of the product are also important con- siderations. Manufacturing processes fall into two catego- ries, thermal activation and chemical activation. The effective porosity of activated carbon produced by thermal activation is the result of gasification of the carbon at rela- tively high temperatures (20), but the porosity of chemi- cally activated products is generally created by chemical dehydration reactions occurring at significantly lower tem- peratures (1,21). Thermal Activation Processes Thermal activation occurs in two stages: thermal decom- position or carbonization of the precursor and controlled gasification or activation of the crude char. During carbon- ization, elements such as hydrogen and oxygen are elimi- nated from the precursor to produce a carbon skeleton pos- sessing a latent pore structure. During gasification, the char is exposed to an oxidizing atmosphere that greatly increases the pore volume and surface area of the product through elimination of volatile pyrolysis products and from carbon burn-off. Carbonization and activation of the char are generally carried out in direct-fired rotary kilns or multiple-hearth furnaces, but fluidized-bed reactors have also been used (22). Materials of construction, notably steel and refractories, are designed to withstand the high- temperature conditions (i.e., �1,000 �C) inherent in acti- vation processes. The thermal activation process is illus- trated in Figure 1 for the production of activated carbon from bituminous coal (23,24). Bituminous coal is pulverized and passed to a briquette press. Binders may be added at this stage before compres- sion of the coal into briquettes. The briquetted coal is then crushed and passed through a screen, from which the on- size material passes to an oxidizing kiln. Here, the coking properties of the coal particles are destroyed by oxidation at moderate temperatures in air. The oxidized coal is then devolatilized in a second rotary kiln at higher tempera- tures under steam. To comply with environmental pollu- tion regulations, the kiln off-gases containing dust and vol- atile matter pass through an incinerator before discharge to the atmosphere. The devolatilized coal particles are transported to a direct-fired multihearth furnace where they are activated by holding the temperature of the furnace at about 1,000 �C. Product quality is maintained by controlling coal feed rate and bed temperature. As before, dust particles in the furnace off-gas are combusted in an afterburner before dis- charge of the gas to the atmosphere. Finally, the granular product is screened to provide the desired particle size. A ACTIVATED CARBON, DECOLORATION OF PHARMACEUTICAL PRODUCTS 3 Table 1. Properties of Selected U.S. Activated Carbon Products Manufacturer Gas-phase carbons Liquid-phase carbons Property Precursor Product grade Product form Typical range Calgon coal BPL granular Norit peat B4 extruded Westvaco wood WV-A 1100 granular Calgon coal SGL granular Norit peat SA 3 powdered Westvaco wood SA-20 powdered Particle size, U.S. mesha,b �4 12 � 30 3.8 mm 10 � 25 8 � 30 60% �325 65–85% �325 Apparent density, g/cm3 0.2–0.6 �0.48 0.43 0.27 0.52 0.46 0.34–0.37 Particle density, g/cm3 0.4–0.9 0.80 0.50 0.80 Hardness number 50–100 �90 99 Abrasion number �75 Ash, wt % 1–20 �8 6 �10 6 3–5 BET surface area, N2 m2/g 500–2,500 1,050–1,150 1,100–1,200 1750 900–1,000 750 1,400–1,800 Total pore volume, cm3/g 0.5–2.5 0.8 0.9 1.2 0.85 2.2–2.5 CCl4 activity, wt% 35–125 �60 Butane working capacity, g/100 cm3 4–14 �11.0 Iodine number 500–1,200 �1,050 �900 800 �1,000 Decolorizing index Westvaco 15–25 �20 Molasses number Calgon 50–250 �200 Norit 300–1,500 440 Heat Capacity at 100 �C, J/(g K)c 0.84–1.3 1.05 1.05 Thermal conductivity, W/(m•K) 0.05–0.10 Note: Specific values shown are those cited in manufacturers' product literature (19). Typical ranges shown are based on values reported in the open literature. aUnless otherwise noted. bApproximate mm corresponding to cited meshes are (mesh:mm 4:4.76; 8:2.38; 10:2; 12:1.68; 25:0.72; 30:0.59; 325:0.04. cTo convert J to cal, divide by 4.184. �� Pulverizer Bituminous coal Briquette press Crusher Screen Fines Oxidizer kiln Devolatilizing kiln Off-gas Off-gas Off-gas Dust collector To atmosphere To atmosphere Steam Air Hot gas Multihearth furnace Screen Activated carbon Afterburner Incinerator Figure 1. Thermal activation of bituminous coal. typical yield of activated carbon is about 30–35% by weight based on the raw coal. The process for the thermal activation of other carbo- naceous materials is modified according to the precursor. For example, the production of activated carbon from co- conut shell does not require the stages involving briquet- ting, oxidation, and devolatilization. To obtain a high- activity product, however, it is important that the coconut shell is charred slowly prior to activation of the char. In some processes, the precursor or product is acid-washed to obtain a final product with a low ash content (23,25). Chemical Activation Processes In contrast to the thermal activation of coal, chemical ac- tivation is generally carried out commercially in a single 4 ACTIVATED CARBON, DECOLORATION OF PHARMACEUTICAL PRODUCTS Activation kiln Drying kiln Dust collector To atmosphere To atmosphere Screen Activated carbon Sawdust Mixer Acid make-up Concentrated acid Washing Water Acid recovery Off-gas treatment Off-gas Off-gas Figure 2. Chemical activation of wood. kiln. The precursor, usually wood, is impregnated with a chemical activation agent, typically phosphoric acid, and the blend is heated to a temperature of 450–700 �C (26). Chemical activation agents reduce the formation of tar and other by-products, thereby increasing carbon yield. The chemical activation process is illustrated in Figure 2 for the production of granular activated carbon from wood (23,27). Sawdust is impregnated with concentrated phosphoric acid and fed to a rotary kiln, where it is dried, carbonized, and activated at a moderate temperature. To comply with environmental pollution regulations, the kiln off-gases are treated before discharge to the atmosphere. The char is washed with water to remove the acid from the carbon, and the carbon is separated from the slurry. The filtrate is then passed to an acid-recovery unit. Some manufacturing plants do not recycle all the acid but use a part of it to manufacture fertilizer in an allied plant. If necessary, the pH of the activated carbon is adjusted, and the product is dried. The dry product is screened and classified into the size range required for specific granular carbon applica- tions. Carbon yields as high as 50% by weight of the wood precursor have been reported (26). Novel Manufacturing Processes. Different chemical ac- tivation processes have been used to produce carbons with enhanced adsorption characteristics. Activated carbons of exceptionally high surface area (�3,000 m2/g) have been produced by the chemical activation of carbonaceous ma- terials with potassium hydroxide (28,29). Activated car- bons are also produced commercially in the form of cloths (30), fibers (31), and foams (32) generally by chemical ac- tivation of the precursor with a Lewis acid such as alu- minum chloride, ferric chloride, or zinc chloride. Forms of Activated Carbon Products To meet the engineering requirements of specific applica- tions, activated carbons are produced and classified as granular, powdered, or shaped products. Granular acti- vated carbons are produced directly from granular precur- sors, such as sawdust and crushed and sized coconut char or coal. The granular product is screened and sized for spe- cific applications. Powdered activated carbons are obtained by grinding granular products. Shaped activated carbon products are generally produced as cylindrical pellets by extrusion of the precursor with a suitable binder before activation of the precursor. Shipping and Storage Activated carbon products are shipped in bags, drums, and boxes in weights ranging from about 10 to 35 kg. Contain- ers can be lined or covered with plastic and should be stored in a protected area both to prevent weather damage and to minimize contact with organic vapors that could reduce the adsorption performance of the product. Bulk quantities of activated carbon products are shipped in metal bins and bulk bags, typically 1–2 m3 in volume, and in railcars and tank trucks. Bulk carbon shipments are generally transferred by pneumatic conveyors and stored in tanks. However, in applications such as water treatment where water adsorption does not impact product perfor- mance, bulk carbon may be transferred and stored as a slurry in water. Specifications Activated carbon producers furnish product bulletins that list specifications, usually expressed as a maximum or minimum value, and typical properties for each grade pro- duced. Standards helpful in setting purchasing specifica- tions for granular and powdered activated carbon products have been published (33,34). ECONOMIC ASPECTS Excluding eastern European countries and China, where production figures have not been published, the world pro- ACTIVATED CARBON, DECOLORATION OF PHARMACEUTICAL PRODUCTS 5 Table 2. World Production Capacity, Estimated 1990 Country Capacity (103 t) United States 146 Western Europe 108 Japan 72 Pacific Rim, other 49 Total 375 Note: Excluding eastern Europe and China. duction capacity of activated carbon was estimated to be 375,000 metric tons in 1990 (35). The price of most prod- ucts was 0.70 to 5.50 $/kg, but some specialty carbons were more expensive (36, pp. 731.2001P, 731.2001Q). Forty per- cent of the production capacity was in the United States, 30% in western Europe, 20% in Japan, and 10% in other Pacific Rim countries (Table 2). Production capacity was almost equally split between powdered and nonpowdered activated carbon products. Powdered activated carbon, a less expensive form used in liquid-phase applications, is generally used once and then disposed of. In some cases, however, granular and shaped products are regenerated and reused (35). In 1990 produc- tion capacity for granular and shaped products was split, with about two-thirds for liquid-phase and one-third for gas-phase applications (37). Over the last decade, production capacity in the United States remained essentially unchanged, but minor fluctu- ations occurred in response to changes in environmental regulations (36pp.731.2000S–731.2000Y). A similar reac- tion was noted worldwide (35). The current demand for activated carbon is estimated at 93% of production capac- ity. The near-term growth in demand is projected to be ap- proximately 5.5%/year (37pp.4,5). In 1970 the U.S. Congress enacted the Clean Air Act, the Clean Water Act, and the Safe Drinking Water Act. Because activated carbon can often be used to help meet Environmental Protection Agency (EPA) regulations, the U.S. activated carbon industry reacted by increasing its production capacity. A proposed amendment to the Safe Drinking Water Act in 1979 required the use of granular activated carbon systems, but the amendment was not en- acted. In response to the projected increase in demand for activated carbon, production capacity remained high until the late 1980s, but when the anticipated need did not ma- terialize, some production facilities were shut down. Cur- rently, because of stricter EPA regulations implementing all three acts in 1990, the industry will increase production capacity by 25% during the next several years (35,38). The estimated production capacity of activated carbon in the United States is shown in Table 3 for seven manu- facturers (35pp.54–65). The principal producers are Cal- gon Carbon (37%), American-Norit (26%), Westvaco (19%), and Atochem (10%). Several other companies purchase ac- tivated carbon for resale but do not manufacture products. Western Europe has seven manufacturers of activated carbon. The two largest, Norit and Chemviron (a subsidi- ary of Calgon), account for 70% of western European pro- duction capacity, and Ceca accounts for 13% (35p.13), Japan is the third-largest producer of activated carbon, having 18 manufacturers, but 4 companies share over 50% of the total Japanese capacity (35p.25–32). Six Pacific Rim countries account for the balance of the world produc- tion capacity of activated carbon, 90% of which is in the Philippines and Sri Lanka (35p.13). As is the case with other businesses, regional markets for activated carbon products have become international, leading to consolida- tion of manufacturers. Calgon, Norit, Ceca, and Sutcliffe- Speakman are examples of multinational companies. Activated carbon is a recyclable material that can be regenerated. Thus the economics, especially the market growth of activated carbon, particularly granular and shaped products, is affected by regeneration and industry regeneration capacity. The decision to regenerate an acti- vated carbon product is dependent on the cost, size of the carbon system, type of adsorbate, and the environmental issue involved. Large carbon systems, such as those used in potable and wastewater treatment, generally require a high-temperature treatment, which is typically carried out in rotary or multihearth furnaces. During regeneration, carbon losses of 1 to 15% typically occur from the treat- ment and movement of the carbon (39). However, material loss is compensated for by the addition of new carbon to the adsorber system. In general, regeneration of spent car- bon is considerably less expensive than the purchase of new activated carbon. For example, fluidized-bed furnace regeneration of activated carbon used in a 94,600-m3/day water treatment system cost only 35% of new material (40). For this system, regeneration using either infrared or multihearth furnaces was estimated to be more expensive but still significantly less so than the cost of new carbon. Because powdered activated carbon is generally used in relatively small quantities, the spent carbon has often been disposed of in landfills. However, landfill disposal is becoming more restrictive environmentally and more costly. Thus large consumers of powdered carbon find that regeneration is an attractive alternative. Examples of re- generation systems for powdered activated carbon include the Zimpro/Passavant wet air oxidation process (41), the multihearth furnace as used in the DuPont PACT process (41pp.389–447,42), and the Shirco infrared furnace (40p.51,43). Other types of regenerators designed for specific ad- sorption systems may use solvents and chemicals to re- move susceptible adsorbates (44), steam or heated inert gas to recover volatile organic solvents (45), and biological systems in which organics adsorbed on the activated car- bon during water treatment are continuously degraded (46). ANALYTICAL TEST PROCEDURES AND STANDARDS Source references for frequently used test procedures for determining properties of activated carbon are shown in Table 4. A primary source is the Annual Book of American Society for Testing and Materials (ASTM) Standards (54). Other useful sources of standards and test procedures in- clude manufacturers of activated carbon products, the American Water Works Association (AWWA) (33,34), and the U.S. Department of Defense (54). 6 ACTIVATED CARBON, DECOLORATION OF PHARMACEUTICAL PRODUCTS Table 3. Production Capacity in the United States, Estimated 1990 Company Location Capacity (103 t) Acticarb Division, Royal Oak Enterprises Romeo, Fla. 6.8 American Norit Co. Marshall, Tex. 38.6 Barneby and Sutcliffe Columbus, Ohio 3.0 Calgon Carbon Corp. Catlettsburg, Ky. and Pittsburgh, Pa. 53.5 Ceca Division, Atochem NA Pryor, Okla. 15.0 Trans-Pacific Carbon Blue Lake, Calif. 2.3 Westvaco Corp. Covington, Va. 27.2 Total 146.4 Table 4. Source References for Activated Carbon Test Procedures and Standards Title of procedure or standard Source Standard Definitions of Terms Relating to Activated Carbon ASTM D2652 Apparent Density of Activated Carbon ASTM D2854 Particle Size Distribution of Granular Activated Carbon ASTM D2862 Total Ash Content of Activated Carbon ASTM D2866 Moisture in Activated Carbon ASTM D2867 Ignition Temperature of Granular Activated Carbon ASTM D3466 Carbon Tetrachloride Activity of Activated Carbon ASTM D3467 Ball-Pan Hardness of Activated Carbon ASTM D3802 Radioiodine Testing of Nuclear-Grade Gas-Phase Adsorbents ASTM D3803 pH of Activated Carbon ASTM D3838 Determination of Adsorptive Capacity of Carbon by Isotherm Technique ASTM D3860 Determining Operating Performance of Granular Activated Carbon ASTM D3922 Impregnated Activated Carbon Used to Remove Gaseous RadioIodines from Gas Streams ASTM D4069 Determination of Iodine Number of Activated Carbon ASTM D4607 Military Specification, Charcoal, Activated, Impregnated Ref. 47 Military Specification, Charcoal, Activated, Unimpregnated Ref. 47 AWWA Standard for Granular Activated Carbon Ref. 33 AWWA Standard for Powdered Activated Carbon Ref. 34 BET Surface Area by Nitrogen Adsorption Refs. 6, 8, 9, 48 Pore Volume by Nitrogen Adsorption or Mercury Penetration Refs. 10, 49–52 Particle Density Ref. 53 HEALTH AND SAFETY Activated carbon generally presents no particular health hazard as defined by NIOSH (55). However, it is a nuisance and mild irritant with respect to inhalation, skin contact, eye exposure, and ingestion. On the other hand, special consideration must be given to the handling of spent car- bon that may contain a concentration of toxic compounds. Activated carbon products used for decolorizing food products in liquid form must meet the requirements of the Food Chemical Codex as prepared by the Food and Nutri- tion Board of the National Research Council (56). According to the National Board of Fire Underwriters, activated carbons normally used for water treatment pose no dust explosion hazard and are not subject to sponta- neous combustion when confined to bags, drums, or stor- age bins (67). However, activated carbon burns when suf- ficient heat is applied; the ignition point varies between about 300 and 600 �C (58p.353). Dust-tight electrical systems should be used in areas where activated carbon is present, particularly powdered products (58pp.84,85). When partially wet activated car- bon comes into contact with unprotected metal, galvanic currents can be set up; these result in metal corrosion (59). Manufacturer material safety data sheets (MSDS) in- dicate that the oxygen concentration in bulk storage bins or other enclosed vessels can be reduced by wet activated carbon to a level that will not support life. Therefore, self- contained air packs should be used by personnel entering enclosed vessels where activated carbon is present (60). LIQUID-PHASE APPLICATIONS Activated carbons for use in liquid-phase applications dif- fer from gas-phase carbons primarily in pore size distri- bution. Liquid-phase carbons have significantly more pore volume in the macropore range, which permits liquids to diffuse more rapidly into the mesopores and micropores (61). The larger pores also promote greater adsorption of large molecules, either impurities or products, in many liquid-phase applications. Specific-grade choice is based on the isotherm (62,63) and, in some cases, bench or pilot- scale evaluations of candidate carbons. ACTIVATED CARBON, DECOLORATION OF PHARMACEUTICAL PRODUCTS 7 C on ce nt ra ti on Volume treated 0 C0C0 C1 C2 C3 C4 C0 C0 C0 C0 Contaminated influent stream Spent carbon Adsorption zone Fresh carbon Breakthrough curve Effluent stream Figure 4. Adsorption and breakthrough curve for fixed bed of granular or shaped activated carbon. Table 5. Liquid-Phase Activated Carbon Consumption (103 t) in the United States in 1987. Granular-shaped Powdered Total Potable water 4.5 13.6 18.1 Wastewater Industrial 6.4 6.6 13.0 Municipal 0.9 2.0 2.9 Sweetener decolorization 6.8 9.1 15.9 Chemical processing and misc. 4.1 2.3 6.4 Food, beverage, and oils 0.9 3.9 4.8 Pharmaceuticals 2.0 2.3 4.3 Mining 1.6 2.5 4.1 Groundwater 0.9 2.3 3.2 Household uses 1.4 0.9 2.3 Dry cleaning 0.7 0.4 1.1 Electroplating 0.2 0.4 0.6 Total 30.4 46.3 76.7 Stirred tank Separator Separator Stirred tank Contaminated liquor Slurry Spent carbon Slurry Partially spent carbon Partially purified liquor Purified liquor Fresh carbon Figure 3. Multistage countercurrent application of powdered ac- tivated carbon. Liquid-phase activated carbon can be applied either as a powder, granular, or shaped form. The average size of powdered carbon particles is 15–25 lm (62). Granular or shaped carbon particle size is usually 0.3–3.0 mm. A sig- nificant factor in choosing between powdered and nonpow- dered carbon is the degree of purification required in the adsorption application. Granular and shaped carbons are usually used in continuous flow through deep beds to re- move essentially all contaminants from the liquid being treated. Granular and shaped carbon systems are pre- ferred when a large carbon buffer is needed to withstand significant variations in adsorption conditions, such as in cases where large contaminant spikes may occur. A wider range of impurity removal can be attained by batch appli- cation of powdered carbon, and the powdered carbon dose per batch can be controlled to achieve the degree of puri- fication desired (61). Batch-stirred vessels are most often used in treating material with powdered activated carbon (64). The type of carbon, contact time, and amount of carbon vary with the desired degree of purification. The efficiency of activated carbon may be improved by applying continuous, counter- current carbon-liquid flow with multiple stages (Fig. 3). Carbon is separated from the liquid at each stage by set- tling or filtration. Filter aids such as diatomaceous earth are sometimes used to improve filtration. Granular and shaped carbons are generally used in con- tinuous systems where the liquid to be treated is passed through a fixed bed (41pp.8–19,64). Compounds are ad- sorbed by the carbon bed in the adsorption zone (Fig. 4). As carbon in the bed becomes saturated with adsorbates, the adsorption zone moves in the direction of flow, and breakthrough occurs when the leading edge of the adsorp- tion zone reaches the end of the column. Normally at least two columns in series are on line at any given time. When the first column becomes saturated, it is removed from ser- vice, and a column containing fresh carbon is added at the discharge end of the series. An alternative approach is the moving bed column (41pp.8–19). In this design the adsorp- tion zone is contained within a single column by passing liquid upward while continuously or intermittently with- drawing spent carbon at the bottom and adding fresh car- bon at the top. The total activated carbon consumption for liquid-phase applications in the United States in 1987 was estimated to be about 76,700 t, which accounted for nearly 80% of the total activated carbon use. The consumption by application is summarized in Table 5 (36pp.731.2000V–731.2001L) Potable Water Treatment Treatment of drinking water accounts for about 24% of the total activated carbon used in liquid-phase applications (36pp.731.2000V–731.2001L). Rivers, lakes, and ground- water from wells, the most common drinking water sources, are often contaminated with bacteria, viruses, natural vegetation decay products, halogenated materials, 8 ACTIVATED CARBON, DECOLORATION OF PHARMACEUTICAL PRODUCTS and volatile organic compounds. Normal water disinfection and filtration treatment steps remove or destroy the bulk of these materials (65). However, treatment by activated carbon is an important additional step in many plants to remove toxic and other organic materials (66–68) for safety and palatability. Groundwater Remediation Concern over contaminated groundwater sources in- creased in the 1980s, and in 1984 an Office of Groundwater Protection was created by the EPA (36pp.731.2000V– 731.2001L). This led to an increase in activated carbon con- sumption in 1987 for groundwater treatment to about 4% of the total liquid-phase usage, and further growth is ex- pected in the 1990s. There are two ways to apply carbon in groundwater cleanup. One is the conventional method of applying powdered, granular, or shaped carbon to adsorb contaminants directly from the water. The other method utilizes air stripping to transfer the volatile compounds from water to air. The compounds are then recovered by passing the contaminated air through a bed of carbon (69,70). Industrial and Municipal Wastewater Treatment Wastewater treatment consumes about 21% of the total U.S. liquid-phase activated carbon (36pp.731.2000V– 731.2001L), and governmental regulations are expected to increase demand over the next several years. Wastewater may contain suspended solids, hazardous microorganisms, and toxic organic and inorganic contaminants that must be removed or destroyed before discharge to the environ- ment. In tertiary treatment systems, powdered, granular, or shaped carbon can be used to remove residual toxic and other organic compounds after the primary filtration and secondary biological treatment (71). Powdered carbon is also used in the PACT process by direct addition of the carbon to the secondary biological step (41pp.389–447). Sweetener Decolorization About 21% of the liquid-phase activated carbon is used for purification of sugar (qv) and corn syrup (36pp.731.2000V– 731.2001L). White sucrose sugar is made from raw juice squeezed from sugarcane or sugar beets. The clarified li- quor is decolorized using activated carbon or ion exchange resins (58pp.87–125,274–292). High-fructose corn sweet- eners (HFCS) are produced by hydrolysis of corn starch and are then treated with activated carbon to remove un- desirable taste and odor compounds and to improve stor- age life. The demand for HFCS rose sharply in the 1980s primarily because of the switch by soft drink producers away from sucrose (83). Chemical Processing Activated carbon consumption in a variety of chemical processing applications is about 8% of the total (36pp.731.2000V–731.2001L). The activated carbon re- moves impurities to achieve high quality. For example, organic contaminants are removed from solution in the production of alum, soda ash, and potassium hydroxide (58pp.87–125,274–292). Other applications include the manufacture of dyestuffs, glycols, amines, organic acids, urea, hydrochloric acid, and phosphoric acid (35pp.92– 135). Food, Beverage, and Cooking Oil Approximately 6% of the liquid-phase activated carbon is used in food, beverage, and cooking oil production (36pp.731.2000V–731.2000L). Before being incorporated into edible products, vegetable oils and animal fats are re- fined to remove particulates, inorganics, and organic con- taminants. Activated carbon is one of several agents used in food purification processes. In the production of alcoholic beverages, activated carbon removes haze-causing com- pounds from beer, taste and odor from vodka, and fusel oil from whiskey (58pp.87–125,274–292). The feed water for soft drink production is often treated with carbon to cap- ture undesirable taste and odor compounds and to remove free chlorine remaining from disinfection treatment. Caf- feine is removed from coffee beans by extraction with or- ganic solvents, water, or supercritical carbon dioxide prior to roasting. Activated carbon is used to remove the caffeine from the recovered solvents (35pp.92–135). Pharmaceuticals Pharmaceuticals account for 6% of the liquid-phase acti- vated carbon consumption (36pp.731.2000V–731.2000L). Many antibiotics, vitamins, and steroids are isolated from fermentation broths by adsorption onto carbon followed by solvent extraction and distillation (58pp.87–125,274–292). Other uses in pharmaceutical production include removal of chromogenic impurities, process water purification, and removal of impurities from intravenous solutions prior to packaging (35pp.92–135). Mining The mining industry accounts for only 4% of liquid-phase activated carbon use, but this figure may grow as low- grade ores become more common (36pp.731.2000V– 731.2000L). Gold, for example, is recovered on activated carbon as a cyanide complex in the carbon-in-pulp extrac- tion process (58pp.87–125, 274–292). Activated carbon serves as a catalyst in the detoxification of cyanides con- tained in wastewater from cyanide stripping operations (46pp.8–19). Problems caused by excess flotation agent concentrations in flotation baths are commonly cured by adding powdered activated carbon (58pp.87–125,274– 292). Miscellaneous Uses Several relatively low-volume activated carbon uses com- prise the remaining 6% of liquid-phase carbon consump- tion (36pp.731.2000V–731.2000L). Small carbon filters are used in households for purification of tap water. Oils, dyes, and other organics are adsorbed on activated carbon in dry cleaning recovery and recycling systems. Electroplatingso- lutions are treated with carbon to remove organics that can produce imperfections when the thin metal layer is depos- ited on the substrate (58pp.87–127,274–292). Medical ap- ACTIVATED CARBON, DECOLORATION OF PHARMACEUTICAL PRODUCTS 9 Table 6. Gas-Phase Activated Carbon Uses in the United States in 1987 Application Consumption (103 t) Solvent recovery 4.5 Automotive/gasoline recovery 4.1 Industrial off-gas control 3.2 Catalysis 2.7 Pressure swing separation 1.1 Air conditioning 0.5 Gas mask 0.5 Cigarette filters 0.5 Nuclear 0.3 Total 17.4 plications include removal of toxins from the blood of pa- tients with artificial kidneys (35pp.92–135), and oral ingestion into the stomach to recover poisons or toxic ma- terials (58pp.87–125,274–292;72). Activated carbon also is used as a support for metal catalysts in low-volume pro- duction of high-value specialty products such as pharma- ceuticals, fragrance chemicals, and pesticides (73). GAS-PHASE APPLICATIONS Gas-phase applications of activated carbon include sepa- ration, gas storage, and catalysis. Although only 20% of activated carbon production is used for gas-phase appli- cations, these products are generally more expensive than liquid-phase carbons and account for about 40% of the total dollar value of shipments. Most of the activated carbon used in gas-phase applications is granular or shaped. Ac- tivated carbon use by application is shown in Table 6 (74). Separation processes comprise the main gas-phase ap- plications of activated carbon. These usually exploit the differences in the adsorptive behavior of gases and vapors on activated carbon on the basis of molecular weight and size. For example, organic molecules with a molecular weight greater than about 40 are readily removed from air by activated carbon. Solvent Recovery Most of the activated carbon used in gas-phase applica- tions is employed to prevent the release of volatile organic compounds into the atmosphere. Much of this use has been in response to environmental regulations, but recovery and recycling of solvents from a range of industrial processes such as printing, coating, and extrusion of fibers also pro- vides substantial economic benefits. The structure of activated carbons used for solvent re- covery has been predominantly microporous. Micropores provide the strong adsorption forces needed to capture small vapor molecules such as acetone at low concentra- tions in process air (75). In recent years, however, more mesoporous carbons, specifically made for solvent recovery, have become available and are giving good service, espe- cially for the adsorption of heavier vapors such as cumene and cyclohexanone that are difficult to remove from micro- pores during regeneration (75). Regeneration of the carbon is performed on a cyclic basis by purging it with steam or heated nitrogen. Gasoline Emission Control A principal application of activated carbon is in the capture of gasoline vapors that escape from vents in automotive fuel systems (76). Under EPA regulations, all U.S. motor vehicles produced since the early 1970s have been equipped with evaporative emission control systems. Most other auto-producing countries now have similar controls. Fuel vapors vented when the fuel tank or carburetor are heated are captured in a canister containing 0.5–2 L of activated carbon. Regeneration of the carbon is then ac- complished by using intake manifold vacuum to draw air through the canister. The air carries desorbed vapor into the engine where it is burned during normal operation. Activated carbon systems have also been proposed for cap- turing vapors emitted during vehicle refueling, and acti- vated carbon is used at many gasoline terminals to capture vapor displaced when tank trucks are filled (77). Typically, the adsorption vessels contain around 15 m3 of activated carbon and are regenerated by application of a vacuum. The vapor that is pumped off is recovered in an absorber by contact with liquid gasoline. Similar equipment is used in the transfer of fuel from barges (78). The type of carbon pore structure required for these applications is substan- tially different from that used in solvent recovery. Because the regeneration conditions are very mild, only the weaker adsorption forces can be overcome, and therefore the most effective pores are in the mesopore size range (79). A large adsorption capacity in these pores is possible because va- por concentrations are high, typically 10–60%. Adsorption of Radionuclides Other applications that depend on physical adsorption in- clude the control of krypton and xenon radionuclides from nuclear power plants (80). The gases are not captured en- tirely, but their passage is delayed long enough to allow radioactive decay of the short-lived species. Highly micro- porous coconut-based activated carbon is used for this ser- vice. Control by Chemical Reaction Pick-up of gases to prevent emissions can also depend on the chemical properties of activated carbon or of impreg- nants. Emergency protection against radioiodine emis- sions from nuclear power reactors is provided by isotope exchange over activated carbon impregnated with potas- sium iodide (81). Oxidation reactions catalyzed by the car- bon surface are the basis for several emission control strat- egies. Sulfur dioxide can be removed from industrial off-gases and power plant flue gas because it is oxidized to sulfur trioxide, which reacts with water to form nonvolatile sulfuric acid (82,83). Hydrogen sulfide can be removed from such sources as Claus plant tail gas because it is con- verted to sulfur in the presence of oxygen (84). Nitric oxide can be removed from flue gas because it is oxidized to ni- trogen dioxide. Ammonia is added and reacts catalytically on the carbon surface with the nitrogen dioxide to form nitrogen (85). 10 ACTIVATED CARBON, DECOLORATION OF PHARMACEUTICAL PRODUCTS Protection against Atmospheric Contaminants Activated carbon is widely used to filter breathing air to protect against a variety of toxic or noxious vapors, includ- ing war gases, industrial chemicals, solvents, and odorous compounds. Activated carbons for this purpose are highly microporous and thus maximize the adsorption forces that hold adsorbate molecules on the surface. Although acti- vated carbon can give protection against most organic gases, it is especially effective against high molecular weight vapors, including chemical warfare agents such as mustard gas or the nerve agents that are toxic at parts per million concentrations. The activated carbon is employed in individual canisters or pads, as in gas masks, or in large filters in forced air ventilation systems. In air-conditioning systems, adsorption on activated carbon can be used to control the buildup of odors or toxic gases such as radon in recirculated air (86). Inorganic vapors are usually not strongly adsorbed on activated carbon by physical forces, but protection against many toxic agents is achieved by using activated carbon impregnated with specific reactants or decomposition cat- alysts. For example, a combination of chromium and cop- per impregnants is used against hydrogen cyanide, cyan- ogen, and cyanogen chloride, whereas silver assists in the removal of arsine. All of these are potential chemical war- fare agents; the Whetlerite carbon, which was developed in the early 1940s and is still used in military protective filters, contains these impregnants (87). Recent work has shown that chromium, which loses effectiveness with age and is itself toxic, can be replaced with a combination of molybdenum and triethylenediamine (88). Oxides of iron and zinc on activated carbon have been used in cigarette filters to absorb hydrogen cyanide and hydrogen sulfide (89). Mercury vapor in air can be removed by activated carbon impregnated with sulfur (90). Activated carbon im- pregnated with sodium or potassium hydroxide has long been used to control odors of hydrogen sulfide and organic mercaptans in sewage treatment plants (91). Alkali- impregnated carbon is also effective against sulfur dioxide, hydrogen sulfide, and chlorine at low concentrations. Such impregnated carbon is used extensively to protect sensitive electronic equipment against corrosion by these gases in industrial environments (92). Process Stream Separations Differences in adsorptivity between gases provides a means for separating components in industrial process gas streams. Activated carbon in fixed beds has been used to separate aromatic compounds from lighter vapors in pe- troleum refining process streams (93) and to recover gas- oline components from natural and manufactured gas (94,95). Molecular sieve activated carbons are specially made with restricted openings leading to micropores. These ad- sorbents are finding increasing use in separations utilizing pressure swing adsorption, in which adsorption is en- hanced by operation at high pressure and desorption oc- curs upon depressurization (96). Larger molecules are re- stricted from entrance into the pores of these carbons and, therefore, are not retained as strongly as smaller mole- cules. The target product can be either the adsorbed or unadsorbed gases. Examples include separation of oxygen from air and recovery of methane from inorganic gases in biogas production. Hydrogen can be removed from gases produced in the catalytic cracking of gasoline, and carbon monoxide can be separated from fuel gases. Use of pres- sure swing techniques for gas separation is an area of growing interest in engineering research. The hypersorption process developed in the late 1940s used a bed of activated carbon moving countercurrent to gas flow to separate light hydrocarbons from each other and from hydrogen in refinery operations. The application is of interest because of its scale, treating up to 20,000 m3/h of gas, but the plants were shut down within a few years, probably because of problems related to attrition of the rapidly circulating activated carbon (97). It should be noted, however, that in recent years moving-bed and fluid- bed adsorption equipment using activated carbon has been successfully employed for solvent recovery (98). Gas Storage Adsorption forces acting on gas molecules held in micro- pores significantly densify the adsorbed material. As a re- sult, activated carbon has long been considered a medium for lowering the pressure required to store weakly ad- sorbed compressed gases (99). Recent work with modern high-capacity carbons has been directed toward fueling passenger cars with natural gas, but storage volume tar- gets have not yet been attained (100). Natural gas storage on activated carbon is now used commercially in portable welding cylinders (101). These can be refilled easily at about 2,000 kPa and hold as much gas as a conventional cylinder pressurized to 6,000 kPa (59 atm). Catalysis Catalytic properties of the activated carbon surface are useful in both inorganic and organic synthesis. For exam- ple, the fumigant sulfuryl fluoride is made by reaction of sulfur dioxide with hydrogen fluoride and fluorine over ac- tivated carbon (102). Activated carbon also catalyzes the addition of halogens across a carbon-carbon double bond in the production of a variety of organic halides (73) and is used in the production of phosgene from carbon monoxide and chlorine (103,104). BIBLIOGRAPHY 1. H. Jüntgen, Carbon, 15, 273–283 (1977). 2. Brit. Pat. 14,224 (1900), R. von Ostrejko; Fr. Pat. 304,867 (1900); Ger. Pat. 136,792 (1901); U.S. Pat. 739,104 (1903). 3. J.W. Hassler, Forest Products J. 8, 25A–27A (1958). 4. J.W. Hassler, Activated Carbon, Chemical Publishing Co., Inc., New York, 1963, pp. 1–14. A comprehensive account of the development and use of activated carbon products to about 1960. 5. R.V. Carrubba, J.E. Urbanic, N.J. Wagner, and R.H. Zan- itsch, AIChE Symp. Ser. 80, 76–83 (1984). 6. B. McEnaney and T.J. Mays, in H. Marsh ed., Introduction to Carbon Science, Butterworths, London, 1989, pp. 153–196. A good introduction to carbon science in general. ACTIVATED CARBON, DECOLORATION OF PHARMACEUTICAL PRODUCTS 11 7. H. Marsh and J. Butler, in K.K. Unger, J. Rouquerol, K.S.W. Sing, and H. Kral eds., Characterization of Porous Solids, Proceedings of the IUPAC Symposium (COPS I), Bad Soden a.Ts., FRG, Apr. 26–29, 1987, Elsevier, Amsterdam, The Netherlands, 1988, pp. 139–149. 8. K.S.W. Sing et al., Pure Appl. Chem. 57, 603–619 (1985). 9. S. Brunauer, P.H. Emmett, and E. Teller, J. Am. Chem. Soc. 60, 309–319 (1938). 10. S.J. Gregg and K.S.W. Sing, Adsorption, Surface Area, and Porosity, 2nd ed., Academic Press, London, 1982. An indis- pensable text on the interpretation and significance of ad- sorption data. 11. H. Marsh, D. Crawford, T.M. O'Grady, and A. Wennerberg, Carbon 20, 419–426 (1982). 12. Jpn. Chem. Week 30, 5 (Mar. 16, 1989). 13. M.M. Dubinin, J. Colloid Interface Sci. 46, 351–356 (1974). 14. K.S.W. Sing, Carbon 27, 5–11 (1989). 15. J. Zawadzki, in P.A. Thrower ed., Chemistry and Physics of Carbon, vol. 21, Marcel Dekker, New York, 1989, pp. 147– 380. Chemistry and Physics of Carbon, published in 23 vol- umes through 1991, is a primary source of excellent review articles on carbon, many relevant to activated carbon. 16. D. Atkinson, A.I. McLeod, K.S.W. Sing, and A. Capon, Car- bon 20, 339–343 (1982). 17. J.M. Solar, C.A. Leon y Leon, K. Osseo-Asare, and L.R. Ra- dovic, Carbon 28, 369–375 (1990). 18. K.D. Henning, W. Bongartz, and J. Degel, 19th Biennial Con- ference on Carbon, Penn State University, Pa., June 25–30, 1990, extended abstracts, pp. 94–95. 19. Product data bulletins from activated carbon manufacturers, Calgon Carbon Corp., 1990, American Norit Co., 1990, and Westvaco Corp., 1988. 20. T. Wigmans, Carbon 27, 13–22 (1989). 21. F. Derbyshire and M. Thwaites, Proc. 4th Australian Coal Science Conf., Brisbane, Australia, Dec. 3–5, 1990, pp. 372– 379. 22. U.S. Pat. 3,976,597 (Aug. 24, 1976), A.J. Repik, C.E. Miller, and H.R. Johnson (to Westvaco Corp.). 23. W. Gerhartz, Y.S. Yamamoto, and F. Thomas Campbell eds., Ullmann's Encyclopedia of Industrial Chemistry, 5th ed., vol. A5, VCH, New York, 1986, pp. 124–140. Good descriptions of activation processes. 24. Product literature on Pittsburgh activated carbon, Pitts- burgh Coke & Chemical Co. (now Calgon Carbon Corp.), Pittsburgh, Pa., ca 1960. 25. U.S. Pat. 4,014,817 (Mar. 29, 1977), B.C. Johnson, R.K. Sinha, and J.E. Urbanic (to Calgon Corp.). 26. A. Cameron and J.D. MacDowall, in J.M. Haynes and P. Rossi-Doria eds., Principles and Applications of Pore Struc- tural Characterization, Proceedings of the RILEM/CNR In- ternational Symposium, Milan, Italy, Apr. 26–29, 1983, J.W. Arrowsmith, Ltd., Bristol, U.K., 1985, pp. 251–275. 27. R.C. Bansal, J.-B. Donnet, and F. Stoeckli, Active Carbon, Marcel Dekker, New York, 1988, p. 8. A modern treatise on activated carbon based on a comprehensive review of the lit- erature. 28. U.S. Pat. 4,082,694 (Apr. 4, 1978), A.N. Wennerberg and T.M. O'Grady (to Standard Oil Co.). 29. T. Kasuh, D.A. Scott, and M. Mori, Proceedings of an Inter- national Conference on Carbon, The University of Newcastle upon Tyne, U.K., Sept. 18–23, 1988, pp. 146–148. 30. Product literature on activated carbon cloth, Charcoal Cloth Ltd., U.K., 1985, and on C-tex products, Siebe Gorman & Co., Ltd., U.K., 1985. 31. Product literature on KYNOL activated carbon fibers and cloths, GUN EI Chemical Industry Co., Ltd., Japan, 1987; product literature on AD'ALL activated carbon fibers, Uni- tika, Ltd., Japan, 1989. 32. Product literature on KURASHEET activated carbon foam sheets, Kuraray Chemical Co., Ltd., Japan, 1987. 33. AWWA Standard for Granular Activated Carbon, ANSI/ AWWA B604, American Water Works Association, Denver, Colo., 1991. 34. AWWA Standard for Powdered Activated Carbon, ANSI/ AWWA B600, American Water Works Association, Denver, Colo., 1990. 35. The Economics of Activated Carbon, 3rd ed., Roskill Infor- mation Services, London, 1990. 36. J. Goin, V. von Schuller-Goetzburg, and Y. Sakuma, Chemi- cal Economics Handbook—SRI International, Menlo Park, Calif., 1989. 37. Wertheim Schroder, Calgon Carbon Corp., Company Report No. 955346, Mar. 19, 1990. 38. S. Irving-Monshaw, Chem. Eng. 97(2), 43–46 (1990). 39. W.G.P. Schuliger, Waterworld News 4(1), 15–17 (1988). 40. R.M. Clark and B.W. Lykins, Jr., Granular Activated Carbon—Design, Operation, and Cost, Lewis Publishers, Chelsea, Mich., 1989, pp. 295–338. 41. P.N. Cheremisinoff and F. Ellerbusch, Carbon Adsorption Handbook, Ann Arbor Science Publishers, Ann Arbor, Mich., 1978, pp. 539–626. An excellent reference book on activated carbon, ranging from theoretical to applied aspects. 42. Product literature on PACT systems, Zimpro/Passavant, Inc., Rothschild, Wis., 1990. 43. W.E. Koffskey and B.W. Lykins, Jr., J. Am. Water Works As- soc. 82(1), 48–56 (1990). 44. A. Yehaskel, Activated Carbon—Manufacture and Regener- ation, Noyes Data Corporation, Park Ridge, N.J., 1978, pp. 202–217. A dated, but still useful summary of key patent literature. 45. P.N. Cheremisinoff, Pollut. Eng. 17(3), 29–38 (1985). 46. R.G. Rice and C.M. Robson, Biological Activated Carbon— Enhanced Aerobic Biological Activity in GAC Systems, Ann Arbor Science Publishers, Ann Arbor, Mich., 1982, 611 pp. 47. Department of Defense Military Specifications, MIL-C- 0013724D(EA), Sept. 22, 1983; MIL-C-0013724D(EA) Amendment 1, Mar. 5, 1986; and MIL-C-17605C(SH), Mar. 22, 1989. 48. S.J. Gregg and K.S.W. Sing, Adsorption, Surface Area, and Porosity, 1st ed., Academic Press (London) London, 1967, pp. 308–355. 49. H.M. Rootare, Advanced Experimental Techniques in Powder Metallurgy, Plenum, New York, 1970, pp. 225–252. A com- prehensive review of the use of mercury penetration to mea- sure porosity. 50. G. Horvath and K. Kawazoe, J. Chem. Eng. Jpn. 16, 470– 475 (1983). 51. D. Dollimore and G.R. Heal, J. Appl. Chem. 14, 109–114 (1964). 52. M.M. Dubinin and H.F. Stoeckli, J. Colloid Interface Sci. 56, 34–42 (1980). 53. C. Orr, Jr., Powder Technol. 3, 117–123 (1970). 12 ACTIVATED CARBON, DECOLORATION OF PHARMACEUTICAL PRODUCTS 54. Annual Book of ASTM Standards, 15.01, Section 15, Amer- ican Society for Testing and Materials, Philadelphia, Pa., 1989. 55. 1985–1986 Registry of Toxic Effects of Chemical Substances, vol. 2, National Institute for Occupational Safety and Health, U.S. Department of Health and Human Services, Washington, D.C., 1987, p. 1475. 56. National Research Council, Assembly of Life Sciences, Di- vision of Biological Sciences, Food and Nutrition Board, and Committee on Codex Specifications, Food Chemicals Codex, 3rd ed., National Academy Press, Washington, D.C., 1981, pp. 70, 71. 57. American Society of Civil Engineers, American Water Works Association and Conference of State Sanitary Engineers, Water Treatment Plant Design, American Water Works As- sociation, New York, 1969, p. 297. 58. J.W. Hassler, Purification with Activated Carbon, 3rd ed., Chemical Publishing, New York, 1974. Contains much of the information given in Ref. 4 but with more emphasis on the commercial uses of activated carbon. 59. U.S. Environmental Protection Agency, Process Design Man- ual for Carbon Adsorption, Swindell-Dressler, Pittsburgh, Pa., 1971, pp. 3–68. 60. Material safety data sheets on activated carbon products, available from the manufacturers, 1991. 61. R.A. Hutchins, Chem. Eng. 87(2), 101–110 (1980). A particu- larly useful paper on liquid-phase adsorption. 62. M. Suzuki, Adsorption Engineering, Kodansha Ltd., Tokyo and Elsevier Science Publishers B.V., Amsterdam, The Neth- erlands, 1990, pp. 11, 35–62. 63. T.F. Speth and R.J. Miltner, J. Am. Water Works Assoc. 82(2), 72–75 (1990). 64. F.L. Slejko ed., Adsorption Technology, Marcel Dekker, New York, 1985, pp. 23–32. A good account of the theory, design, and application of adsorption systems. 65. American Water Works Association, Water Quality and Treatment, 3rd ed., McGraw-Hill, New York, 1971, pp. 1–216. 66. W.J. Weber, Jr. and B.M. Van Vliet, in I.H. Suffet and M.J. McGuire eds., Activated Carbon Adsorption of Organics from the Aqueous Phase, vol. 1, Ann Arbor Science Publishers, Ann Arbor, Mich., 1980, pp. 15–41. A comprehensive, two- volume treatise with many key references. 67. J.L. Oxenford and B.W. Lykins, Jr., J. Am. Water Works As- soc. 83(1), 58–64 (1991). 68. I.N. Najm and co-workers, J. Am. Water Works Assoc. 83(1), 65–76 (1991). 69. L.W. Canter and R.C. Knox, Ground Water Pollution Control, Lewis Publishers, Chelsea, Mich., 1985, pp. 89–125. 70. Environmental Science and Engineering, Inc., Removal of Volatile Organic Chemicals from Potable Water—Technolo- gies and Costs, Noyes Data Corp., Park Ridge, N.J., 1986, pp. 23–40. 71. G. Culp, G. Wesner, R. Williams, and M.V. Hughes, Waste- water Reuse and Recycling Technology, Noyes Data Corp., Park Ridge, N.J., 1980, pp. 343–432. A useful review of wastewater treatment with activated carbon. 72. M. Smisek and S. Cerny, Active Carbon—Manufacture,Prop- erties, and Applications, Elsevier Publishing, New York, 1970, pp. 290–294. 73. A.J. Bird, in A.B. Stiles ed., Catalyst Supports and Sup- ported Catalysts, Butterworths, Stoneham, Mass., 1987, pp. 107–137. 74. pp. 731.2000W, 731.2001M-731.2001P. 75. P.J. Luft and P.C. Speers, Paper 52c, AIChE Summer Na- tional Mtg., Aug. 19–22, 1990. 76. P.J. Clarke and co-workers, SAE Trans. 76, 824–837 (1967). 77. Product literature on hydrocarbon vapor recovery systems, John Zink Co., Tulsa, Okla., 1990. 78. J. Hill, Chem. Eng. 97, 133–143 (1990). 79. H.R. Johnson and R.S. Williams, S.A.E. Technical Paper No. 902119, International Fuels and Lubricants Exposition, Tulsa, Okla., Oct. 23, 1990. 80. D.W. Moeller and D.W. Underhill, Nucl. Saf. 22, 599–611 (1981). 81. M.L. Hyder, Comm. Eur. Communities [Rep.] EUR 1986, EUR 10580, Gaseous Effluent Treat. Nucl. Install., pp. 451– 462 (1986). 82. F.J. Ball, S.L. Torrence, and A.J. Repik, APCA J. 22, 20–26 (1972). 83. P. Ellwood, Chem. Eng. 76, 62–64 (1969). 84. J. Klein and K.-D. Henning, Fuel 63, 1064–1067 (1984). 85. E. Richter, Catal. Today 7, 93–112 (1990). 86. M.A. Brisk and A. Turk, Proc. APCA Ann. Meetg., 77th 2, 84– 93 (1984). 87. U.S. Pat. 2,920,050 (Jan. 5, 1960), R.J. Grabenstetter and F.E. Blacet (to U.S. Dept. of Army). 88. U.S. Pat. 4,801,311 (Jan. 31, 1989), E.-D. Tolles (to Westvaco Corp.). 89. U.S. Pat. 3,460,543 (Aug. 12, 1969), C.H. Kieth, V. Norman, and W.W. Bates, Jr. (to Ligget & Meyers Corp.). 90. R.K. Sinah and P.L. Walker, Carbon 10, 754–756 (1972). 91. W.D. Lovett and R.L. Poltorak, Water and Sewage Works 121, 74–75 (1974). 92. G.N. Brown, M.A. Lunn, C.E. Miller, and C.D. Shelor, Tappi J. 66, 33–36 (1983). 93. S. Dunlop and R. Banks, Hydrocarbon Process. 56, 147–152 (1977). 94. G.F. Russell, Petrol. Refiner. 40, 103–106 (1961). 95. T. Scott, Gas. J. 303, 300–307 (1960). 96. E. Richter, Erdol Kohle, Erdgas, Petrochem. 40, 432–438 (1987). 97. C. Berg, Chem. Eng. Prog. 47, 585–590 (1951). 98. Gastak Solvent Recovery System, product literature, Kureha Chemical Industry Co., Ltd., New York, 1990. 99. H. Briggs and W. Cooper, Proc. R. Soc. Edinburgh 41, 119– 127 (1920–1921). 100. J. Braslaw, J. Nasea, and A. Golovoy, Alternative Energy Sources: Proceedings of the Miami Int. Conf. on Alternative Energy Sources, 4th ed., Ann Arbor Science Publishers, Ann Arbor, Mich., pp. 261–270, 1980. 101. U.S. Pat. 4,817,684 (Apr. 4, 1989), J.W. Turko and K.S. Czer- winski (to Michigan Consolidated Gas Co.). 102. U.S. Pat. 4,102,987 (July 25, 1978), D.M. Cook and D.C. Gus- tafson (to The Dow Chemical Company). 103. H. Jüntgen, Fuel 65, 1436–1446 (1986). 104. H. Jüntgen, Erdol Kohle, Erdgas, Petrochem. 39(12), 546– 551 (1986). ADDITIONAL READING "Active Carbon" under "Carbon" in Encyclopedia of Chemical Technology (ECT) 1st ed., Vol. 2, pp. 881–899, by J.W. Hassler, Nuchar Active Carbon Division, West Virginia Pulp and Paper Co., and J.W. Goetz, Carbide and Carbon Chemicals Corp. ADSORBENTS, INORGANIC 13 "Activated Carbon" under "Carbon" in ECT 2nd ed., Vol. 4, pp. 149–158, by E.G. Doying, Union Carbide Corp., Carbon Prod- ucts Division. "Activated Carbon" under "Carbon (Carbon and Artificial Graph- ite)" in ECT 3rd ed., Vol. 4, pp. 561–570, by R.W. Soffel, Union Carbide Corp. ADSORBENTS, INORGANIC KAMI BEYZAVI Bioprocessing Limited Consett, County Durham, United Kingdom KEY WORDS Alumina Chromatography CPG (controlled pore glass) Hydroxyapatite Rigid matrices Separation Silica Titania Zirconia OUTLINE Introduction Choice of Matrices for Bioprocess Separations Inorganic Matrices Controlled Pore Glass Silica Alumina Zirconia and Titania Hydroxyapatite Controlled Pore Glass Manufacturing Process Physical Characteristics Surface Modification Applications Future Trends List of Suppliers Bibliography INTRODUCTION The design of a process for the large-scale purification of biological molecules is one of the several complex tasks that must be successfully completed to bring a product to market. Most downstream processes include some chro- matography steps as well as other conventional separation methods. Chromatography is generally recognized as the technique that enables the highest degree of purification of biomolecules. For example, the U.S. Food and Drug Administration (FDA) recommends that at least two chromatography steps be used in the purification of therapeutic-grade monoclonal antibodies. Chromatography separations are sophisticated and can be expensive processes, which explains the continuous development of effective approaches by scientists and en- gineers to improve the productivity of chromatography technologies. Fluidized bed adsorption, various column technologies, computerized optimization strategies, and continuous and semicontinuous processes are some of the recent approaches that have been proposed. However, one of the most important aspects of chromatography technol- ogy is the choice of matrix. In this article, we examine the use of inorganic rigid matrices in chromatography separation processes of bio- logical molecules, with emphasis on the preparation, prop- erties, and applications of controlled pore glass (CPG). CHOICE OF MATRICES FOR BIOPROCESS SEPARATIONS The subject of matrices for process chromatography and the requirements of an ideal matrix have been reviewed by many authors (1–3). In process-scale purification, the important characteristics of a matrix are chemical and physical stability, rigidity of the matrix to withstand high flow rates and differing hydrodynamic pressure, uniform porosity, and high surface area. In addition, for most chro- matography applications, surface modification of the ma- trix will be necessary to facilitate ligand immobilization, which requires the presence of suitable functional groups on the matrix. Finally, the cost and commercial availability of the matrix are also important considerations in selecting a matrix. The majority of matrices used in chromatography pro- cesses can be divided into two categories: natural and syn- thetic. Natural matrices include glass, silica, ceramic, alu- mina, agarose, chitosan, and cellulose. Synthetic matrices are prepared by polymerization of functional monomers. The incorporation of monomers with suitable functional groups can provide activation sites on these supports for ligand attachments. There are also the composite matri- ces, which are mixed networks constituted of at least two components. Generally, one component acts as a rigid skeleton, and the other component has active groups that interact with the biological molecule to be separated. Dextran-coated silica and polyacrylamide-agarose are ex- amples of composite matrices that have been applied to the separation of biological molecules. INORGANIC MATRICES A large selection of inorganic matrices are commercially available for incorporating homogenous catalysts, and some have been applied to chromatography separations. These matrices include silica, alumina, glass, zeolite, clay, celite, zerconia, titania, magnesium oxide, and carbon. Only a limited number of these matrices with suitable pore 14 ADSORBENTS, INORGANIC Table 2. Properties of Alumina Matrix (Selecto Scientific) Nominal pore diameter (nm) Surface area (m2/g) Particle size range (lm) 6 120–150 60–120 10 100 60–120 15 75 60–120 30 80 40 50 50 40 100 8 40 180 6–8 20 Table 1. Properties of Matrex� Silica Nominal pore diameter (nm) Surface area (m2/g) Pore volume (ml/g) Particle size range (lm) 6 540 0.93 20–500 10 320 1.20 45–300 25 160 1.10 45–70 50 90 1.10 50 100 45 1.10 50–130 150 25 1.10 50 structure, particle size, and mechanical stability can be used for chromatography separation of biomolecules. Sil- ica, alumina, and glass are available with a large variety of surface areas, pore structure, and particle sizes, and some have been used extensively in both analytical and preparative chromatography of biological molecules. Controlled Pore Glass Controlled pore glass (CPG) has excellent mechanical sta- bility and can be manufactured with a wide range of pore diameters, a high surface area, and a range of particle sizes. The material is almost pure silica and has a very narrow pore size distribution. Surface-modified CPG with the trade name PROSEP manufactured by Bioprocessing Limited has been extensively used in large-scale purifica- tion of biomolecules. (See later sections on CPG for a more detailed discussion of this material.) Silica Silica is extensively used in the analytical field (HPLC) and to a much lesser extent for preparative chromatogra- phy of biomolecules. Silica matrices for chromatography applications are generally produced by a wet process, that is, by acidification of sodium silicate in water (4,5), and are available in particle sizes of up to 40 lm and pore sizes of up to 30 nm. In most cases, these materials are unsuitable for large-scale purification processes. The small particle size causes excessive backpressure in preparative chro- matography columns, and the pore size of 30 nm is not large enough for the separation of most proteins, especially in the field of affinity chromatography, where both the li- gand and the ligate could be large proteins. In our laboratories, we have used Matrex� silica (Ami- con) for the large-scale purification of biomolecules. This material is available in a range of pore diameters and par- ticle sizes (Table 1) and is manufactured by first forming a hydrosol from organosilicic acid. Hydrosol is then allowed to set to a jelly-like mass called hydrogel. After setting, the hydrogel is thoroughly washed to remove salts resulting from the reaction. Temperature and pH of the washing me- dium and the washing time influence the specific surface area, pore volume, and purity of the hydrogel. The hydro- gel is subsequently dried to give a structurally stable xero- gel. This material is 99.5% silica, with impurities including Na�, Ca2�, Fe3�, , and Cl� ranging from 600 to 602�SO4 ppm. Alumina Alumina-based materials have a much wider pH stability than silica matrices and therefore are of great interest in chemical separations and industrial-scale chromatogra- phy. Commercially available alumina matrices are all al- uminium oxides prepared by thermal dehydration of alu- minum hydroxide (6,7). The origin of the starting material together with other factors, such as the heating rate, dry- ing time, and temperature, influence the properties of the final product. Alumina matrices can be obtained in a wide range of particles and pore sizes (Table 2). The physical stability of alumina particles in chromatography columns, even under moderate flow rates and pressure, has been a major prob- lem in our laboratories. Generally, the particles break up and cause a substantial increase in the backpressure gen- erated by the column. Furthermore, any risk of contami- nation of the purified product with aluminum may not be acceptable to the pharmaceutical and biotechnology indus- tries, particularly when the reports relating to Alzheimer's disease to aluminum are considered (8). Zirconia and Titania Recently, there has been an increasing interest in zirconia- and titania-based matrices in chromatography applica- tions, mainly because of their chemical stability over a relatively wide pH range and their physical characteris- tics. Zirconia or titania supports with properties suitable for preparative chromatography or HPLC are not commer- cially available, although zirconia and titania powders can be obtained commercially from a number of sources and have been used by researchers to prepare matrices with a small particle size (1–10 lm) and pore sizes of up to 100 nm, suitable for HPLC applications (9). More recently, larger porous zirconia particles (30–150 lm) have been prepared and used in packed and fluidized beds for protein purification (10–13). Zirconia and titania as base stable matrices not only enable separation of biomolecules at high pH values, but they can also be sanitized with alkaline media, which is the standard sanitization procedure in downstream pro- cessing of biological molecules. Various adsorbents such as affinity, ion-exchange, and reversed phase can be prepared from these matrices for protein purification by either poly- mer coating or reaction with organosilanes (14–17). ADSORBENTS, INORGANIC 15 Hydroxyapatite Hydroxyapatite is a special crystalline form of calcium phosphate that has been used in the separation of biologi- cal molecules. The adsorbent consists of a mosaic of crys- tals that result from a combination of phosphate and cal- cium salts after a number of steps of wet alkaline treatment and heat aging. The application of hydroxyapa- tite to the field of protein separation was first introduced by Tiselius et al. (18). The standard procedures for the operation of hydroxyapatite columns were developed by Bernardi and Gorbunoff (19,20) using a number of differ- ent proteins in several solvent systems, Bernardi also pro- posed a mechanism for protein adsorption to, and desorp- tion from, hydroxyapatite. Proteins bind reversibly to hydroxyapatite by a complex ionic mechanism different from the principle of ion- exchange chromatography (19,20). Both the amino and carboxyl groups on the protein molecules act in the ad- sorption mechanism. The adsorption of proteins to hy- droxyapatite via the amino groups is the result of their positive charges and the negative charge on the adsorbent when the column is equilibrated in phosphate buffer. The surface of hydroxyapatite crystals consists of sites with both positive (calcium) and negative (phosphate) charges. When the hydroxyapatite columns are equilibrated in phosphate buffers, normally at pH 6.8, the surface of the adsorbent can be regarded as negative because of partial neutralization of the positive calcium loci by phosphate ions. The carboxyl groups act in two ways. First, they are repelled electrostatically from the negative charge of the adsorbent; second, they bind specifically by complex for- mation with the calcium sites. The elution of basic proteins from hydroxyapatite is generally carried out by specific displacement with Ca2� and Mg2� ions that neutralize the negative charges of the hydroxyapatite phosphate sites by complex formation. Acidic proteins are eluted by displacement of their car- boxyl groups from hydroxyapatite calcium sites by ions, such as fluoride or phosphate, which form stronger com- plexes. The difficulty with using hydroxyapatite in medium- and large-scale purification of biological molecules is the physical nature of the material. The crystals are highly fragile, generating small particles resulting in column clogging and precluding any long-term usage of the col- umn. An improved form of hydroxyapatite crystals has re- cently been prepared and is commercially available from a number of sources, including Bio-Rad. The new ceramic material from Bio-Rad is spherical and has been reported to be stable both chemically and physically. CONTROLLED PORE GLASS The method used for the preparation of CPG was originally invented by Hood and Nordberg (21). They discovered that by heat treating a certain sodium borosilicate glass com- position, the glass separated into two intermingled and continuous glassy phases. One phase, rich in boric oxide, was soluble in acids; the other phase was high in silica and stable toward acid solutions. This process was used by the inventors to prepare nonporous silica particles by first re- moving the boron phase with an acid solution to obtain porous particles and then revitrifying this material by heating to 900 �C or above to yield transparent homogen- ous nonporous particles having a composition of greater than 95% silica. The porous glass material obtained by this method typ- ically had an average pore diameter of about 4 to 5 nm. A procedure for enlarging the pore size to approximately 20 nm was invented by Chapman and Elmer (22). This was carried out by impregnating a porous glass body with an aqueous solution of a weakly reactive fluorine-containing compound, reacting the compound in situ with a mineral acid to release hydrofluoric acid at a temperature sufficient to dissolve a portion of the glass body and washing the body to remove the soluble constituents. Ammonium bifluoride and nitric acid at 95 �C were applied to a porous glass disc to enlarge the pore size from 4 to 24 nm. The process for making porous glass was modified by Haller (23) to produce the material with a range of con- trollable pore sizes. This was achieved by varying the tem- perature and duration of the heat treatment to obtain po- rous glass with pore sizes from 12 to 250 nm. Larger pore sizes were also obtained by the addition of a sodium hy- droxide leaching process. Manufacturing Process CPG can be commercially obtained from a number of sources, and the manufacturing processes are all based on the process invented by Haller (23). The composition of the starting metal oxides mixture and the exact conditions used for the heat treatment and leaching of the glass, which influence the quality and physical properties of the porous glass, are the propriety technology of the manufac- turing companies. An outline of the general procedure used for preparing CPG is shown in Figure 1. CPG is manufactured in a range of particle sizes, 40 to 200 lm, and different pore sizes, 7 to 300 nm (Table 3). The manufacturing batch sizes are generally much smaller than silica particles, possibly because of the heat treat- ment process. The largest batch size commercially avail- able is in the region of 50 L (20 kg). Physical Characteristics Most commercially available CPG granules are irregularly shaped and highly porous (Figs. 2 and 3). CPG has a nar- row pore size distribution, with 80 to 90% of the pores showing a deviation less than �10% from the nominal pore diameter (Fig. 4). CPG has a very high surface area that varies according to the pore diameter and pore vol- ume. The surface area increases with increasing pore vol- ume and decreases with increasing pore diameter, as is shown by the data in Table 3. CPG is a rigid insoluble ma- trix, compatible with most reagents used in the purifica- tion of biological molecules. Because CPG is essentially pure silica, it generally exhibits a high degree of solubility in alkali solutions. The solubility of CPG is a function of temperature, time, and pH of the solution. The surface of native CPG is slightly hydrophobic and contains acidic hydroxyl groups that could denature bio- 16 ADSORBENTS, INORGANIC Glass mix; Na2O, B2O3 and SiO2 Homogenous glass melt furnace at 1,200–1,500 °C, continuous stirring Chill the melt to form glass discs Heat treat in furnace 500–700 °C, 4–16 h, two-phase separation Crush the glass discs and fractionate the particles Dry in oven at > 100 °C Acid wash; 3 M HCl Water wash Alkali leach to remove colloidal silica 0.5 M NaOH, 25–30 °C Water wash Acid leach to remove silica poor phase 1–3 M HCl, 50–90 °C Second alkali leach to enlarge pores 0.5 M NaOH, 25–30 °C Figure 1. Process for the preparation of porous glass. Table 3. Properties of Controlled Pore Glass (CPG, Inc.) Nominal pore diameter (nm) Surface area (m2/g); pore volume � 1.0 cm3/g Surface area (m2/g); pore volume � 0.9 cm3/g 7.5 340 240 12 210 150 17 150 100 24 110 75 35 75 50 50 50 35 70 36 25 100 25 18 140 18 13 200 13 9 300 9 7 logical molecules and cause strong, often irreversible bind- ing of proteins. For most separation applications, surface modification of CPG with organosilanes is necessary, al- though native CPG has been used for the purification of a number of biological molecules, such as factor VIII (24). Surface modification also enhances the stability and du- rability of CPG. Surface Modification Siliceous materials such as CPG can be reacted with the organosilane compounds in a number of different ways to obtain the desired bonding through hydrolyzable groups of the organosilane. Depending on the type of organosilane, the reaction can be performed in organic, aqueous, or va- pour phase, resulting in matrices suitable for different ap- plications. The majority of the work cited on this subject relates to the surface modification of silica material (25), which is not always applicable to CPG because of the dif- ference in the surface characteristics of the two matrices. Surface modification of CPG is carried out by treating the material with an organosilane containing an organic functional group at one end and a silylalkoxy group at the other. Attachment of the silane compound to the matrix is through the surface silanol or oxide groups to the silyl- akoxy groups with polymerization between the adjacent silanes. The result is an inorganic matrix with available organic functional groups that can be used in the prepa- ration of chromatography adsorbents. A large number of different organosilanes are commercially available with functional groups such as epoxy, amine, and sulfhydryl (Table 4). The first step in the surface modification of porous glass is to clean the surface by washing the matrix in nitric acid at elevated temperature. The matrix is then washed with water and dried in the oven at temperatures not exceeding 180 �C. The dry matrix is reacted with the silane compound dissolved in water or an organic solvent at temperatures ranging from 40 �C to 120 �C, depending on the solvent. After removing the excess silane by washing the matrix with suitable solvents or water, the functional groups on the matrix can be further modified to obtain various ad- sorbents. The exact conditions used in the surface modifi- cation of CPG will depend on the nature of the silane com- pound and the application of the resulting adsorbent. Extensive optimization of the surface modification pro- cedure is usually required to obtain a suitable adsorbent for a specific application. At Bioprocessing, special tech- ADSORBENTS, INORGANIC 17 Figure 2. The irregularly shaped granules of CPG matrix. Figure 3. Scanning electron micrograph of CPG matrix. P en et ra ti on ( cm 3 /g ) Pore diameter (µm) 0.001 0.01 0.1 1 10 100 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Figure 4. Pore size distribution of CPG matrix as determined by mercury porosimetery method. Average pore diameter was deter- mined to be 0.1125 l. Table 4. Surface Modification of CPG Organosilane Reactant Activated CPG 3-Aminopropyltriethoxysilane — CPG-amine 3-Aminopropyltriethoxysilane Glutaraldehyde CPG-aldehyde 3-Aminopropyltriethoxysilane Succinic anhydride CPG-carboxyl 3-Glycidoxypropyltrimethoxysilane — CPG-epoxy 3-Glycidoxypropyltrimethoxysilane Acid solution CPG-diol 3-Glycidoxypropyltrimethoxysilane Sodium periodate CPG-aldehyde Mercaptopropyltrimethoxysilane — CPG-sulfyhyryl niques for preparing surface modified CPG (PROSEP�) have been developed for applications in affinity and other types of separation processes. PROSEP� has been de- signed for effective immobilization of both large and small ligands, resulting in affinity adsorbents with a high capac- ity for the target molecules, minimum nonspecific binding of contaminating proteins, and low ligand leakage (26). Applications CPG has been effective in the separation of a range of bio- logical molecules, using a variety of chromatography tech- niques that include adsorption, size exclusion, affinity, and ion exchange. It has also been used extensively in the field of enzyme immobilization for the modification of biological molecules. The rigidity and incompressibility of the matrix allows it to be used in large columns at very high flow rates generating low backpressure (Fig. 5), which makes this matrix especially useful for processing large volumes of feedstock in short cycle times. Application of native CPG in adsorption chromatogra- phy has been generally limited because of its denaturing surface characteristics and irreversible binding of pro- teins. A number of proteins, such as factor VIII (24), have been purified from plasma by first blocking the surface of CPG with albumin. The use of CPG in ion exchange chro- matography applications has also been very limited be- cause of the instability of the matrix in sodium hydroxide solution, which is the standard cleaning and sanitization solution for ion exchange adsorbents. Furthermore, ion ex- change adsorbents prepared with CPG as the matrix have generally shown low functional capacity when compared to the polymetric matrices. Nevertheless, some applications of surface-modified CPG in ion exchange chromatography have been reported (27–29). The use of CPG in size-exclusion chromatography has generally been for the separation of macromolecules such 18 ADSORBENTS, INORGANIC P re ss ur e (p si ) Linear velocity (cm/h) 0 1000 2000 3000 4000 0 2 4 6 8 10 12 14 16 18 20 Figure 5. Backpressure generated from a column (11.3 � 10.0 cm) packed with PROSEP�. as viruses, cell components, and polymers (30,31). Sepa- ration of these species from low molecular weight contam- inants has been achieved by taking advantage of the very sharp exclusion limits and narrow pore size distribution of CPG. Generally, surface-modified CPG has been used for size-exclusion chromatography to eliminate the problem of nonspecific and irreversible binding of biological molecules to native CPG. The surface of the matrix is modified with glycidoxypropyltrimethoxysilane, converting the solid phase to diol-CPG (Table 4). This is a nonionic, hydrophilic coating compatible with aqueous and most solvent systems (32). Surface-modified CPG has been widely applied in the area of affinity chromatography for the purification of bio- logical molecules. Biologically active molecules will often bind to other molecules in a reversible and highly specific manner. The formation of these stable, specific, and dis- sociable complexes forms the basis of this powerful sepa- ration technique. Affinity adsorbents prepared using PROSEP� and other surface-modified CPG have been used in many applica- tions for the purification of biological molecules, such as antibodies (33,34) and enzymes (35). PROSEP� adsorbents have been used in packed-bed chromatography columns and in fluidized or expanded bed mode (Fig. 6) for the direct capture of biological molecules from feedstocks containing particulate or colloidal solids (36,37). Table 5 shows the application of PROSEP� adsorbents in the field of affinity purification. FUTURE TRENDS The progress of inorganic matrices such as silica and CPG as adsorbents in the downstream processing of biological molecules is hampered by their instability toward alkaline solutions. Sodium hydroxide solution up to 0.5 M concen- tration is the standard cleaning and sanitizing reagent for chromatography adsorbents used in the separation pro- cesses of biomolecules. Furthermore, certain chemistries used in the surface modification of matrices and some pu- rification protocols require alkaline conditions. Alkaline-resistant inorganic matrices are under devel- opment using different approaches. Development of zirco- nia and titania matrices by sol-gel techniques have already been mentioned. Surface coating of silica and CPG with an inert metal oxide such as zirconium oxide and preparation of glass–ceramic matrices may result in suitable products that satisfy all the requirements of an ideal matrix for the purification of biomolecules. Methods for coating silica matrices with zirconium ox- ide were studied by Meijers et al. (38) and applied to CPG at Bioprocessing. Great improvement in the stability of the Figure 6. Principle of fluidized-bed adsorption. From left to right: PROSEP� adsorbent is packed into a column specially adopted for fluidized-bed operation. The bed is gradually expanded by an up- ward flow of solution through the column until full expansion is reached. Table 5. Application of Surface-Modified CPG (PROSEP�) in Separation Processes Product Trade name Application Protein A adsorbent PROSEP�-A High Capacity Purification of IgG from all species and subclasses except rat IgG Protein G adsorbent PROSEP�-G Purification of IgG from all species and subclasses Heparin adsorbent PROSEP�-Heparin Purification of antithrombin III and blood clothing factors Lysine adsorbent PROSEP�-Lysine Purification of plasminogen and tPA Metal-chelating adsorbent PROSEP�-Chelating A metal-binding adsorbent used in purification of metal-binding proteins and metal removals from dilute solutions Thiophilic adsorbent PROSEP�-Thiosorb Purification of antibodies Immobilized antibodies — Purification of specific antigens and second antibodies Note: IgG, immunoglobulin G; tPA, tissue-type plasminogen activator. ADSORBENTS, INORGANIC 19 coated CPG to alkaline solutions were observed; however, total stability has not yet been achieved. A preferred approach for making alkaline-stable inor- ganic matrices is the glass–ceramic route. Porous glass– ceramic with a skeleton of CaTi4(PO4)6 crystals has been prepared by controlled crystallization of glasses in the CaO-TiO2-P2O5-Na2O system and subsequent acid leach- ing of the resulting dense glass–ceramic (39). The medium pore diameter has been reported to be controllable in the range of 40 to 150 nm by changing the temperature of the heat treatment. Such an approach is being examined at Bioprocessing in collaboration with a glass–ceramic re- search center for developing alkaline stable inorganic ma- trices suitable for downstream processing of biological mol- ecules. LIST OF SUPPLIERS Bioprocessing Limited, Medomsley Road, Consett, Co Durham, DH8 6SZ, UK Amicon Products–Millipore Corporation, 80 Ashby Road, Bedford, MA 01730-2271, USA Selecto Scientific, 5933 Peachtree Industrial Blvd., Bldg. B, Norcross, GA 30092, USA Bio-Rad Laboratories, 2200 Right Avenue, Richmond, CA 94804, USA CPG Inc., 3 Borinski Road, Lincoln Park, NJ 07035, USA BIBLIOGRAPHY 1. E. Boschetti, J. Chromatogr. A 658, 207–236 (1994). 2. S.R. Narayanan, J. Chromatogr. A 658, 237–258 (1994). 3. G.B. Wisdom, Peptide Antigens: A Practical Approach, IRL Press at Oxford Univ. Press, London, 1993, pp. 117–136. 4. R.K. Iler, The Chemistry of Silica, John Wiley, New York, 1979. 5. K.K. Unger, Porous Silica, Elsevier, Amsterdam, 1979. 6. M.A. Coletti-Previero and A. Previero, Anal. Biochem. 180, 1– 10 (1989). 7. L.K. Huston, C. Misra, and K. Weters, Ulmann Encyclopedia of Industrial Chemistry, Volume A1, VCH, Weinheim, 1985, p. 557. 8. Lancet 343, 993 (1994). 9. J. Nawrocki, M.P. 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See also ADSORPTION, EXPANDED BED; ADSORPTION, PROTEIN, BATCH; ADSORPTION, PROTEINS WITH SYNTHETIC MATERIALS. 20 ADSORPTION, EXPANDED BED ADSORPTION, EXPANDED BED ANN-KRISTIN BARNFIELD FREJ Amersham Pharmacia Biotech Uppsala, Sweden KEY WORDS Capture Expanded bed Chromatography Downstream process Fluidized bed Protein adsorption Protein purification Recovery OUTLINE Introduction Background Theory Column Adsorbent Equipment and Methodology Column Adsorbent Method Development Operation of an Expanded Bed Feedstocks Industrial-Scale Applications Purification of Recombinant Human Serum Albumin from Whole Yeast Culture Purification of Monoclonal Antibodies from Whole Mammalian Cell Culture Purification of Bovine Serum Albumin from Whole Yeast Culture Potential Industrial-Scale Applications Bibliography Additional Reading INTRODUCTION Expanded-bed adsorption is a technique based on fluidiza- tion for recovery of proteins directly from unclarified feed- stocks such as fermentation broths, cell homogenates, and other solutions containing suspended particulates. An ex- panded bed is a chromatography bed with an increased void volume between the adsorbent particles, as compared to a packed chromatography bed. An expanded bed is formed when high-density adsorbent particles are lifted by an upward liquid flow through a column specially designed for expanded bed adsorption. When the bed has expanded and is stable, the particulate-containing feedstock is ap- plied, and the protein of interest can bind to the adsorbent particles while the particulates in the feedstock can pass unhindered through the void of the bed. Expanded-bed ad- sorption is a one-passage technique designed for large- scale recovery processes. Traditionally, when conventional packed-bed chromatography is used to recover proteins, the feedstock has to be clarified by centrifugation or filtra- tion prior to application or the particulates can block the bed. Expanded-bed adsorption can handle particulate- containing feedstocks; as a result, clarification, concentra- tion, and initial purification are accomplished in one unit operation. Reducing the number of unit operations in a downstream processing scheme decreases the risk of prod- uct loss, decreases the processing time, and lowers the overall production costs. The basic principles of expanded- bed adsorption and some applications are described in this article. BACKGROUND The biotechnology industry produces proteins for thera- peutic and diagnostic use by using biological systems such as bacteria, yeast, and mammalian cells. A common bot- tleneck in the production of these proteins is the initial purification, during which the proteins are recovered from large volumes of cell- and debris-containing feedstocks, such as fermentation broths and homogenates. The puri- fication processes are usually based on packed-bed chro- matography techniques in which the protein is bound to an adsorbent packed in a column. A packed bed (fixed bed) cannot handle particulates very well and would eventually become totally blocked if an unclarified feedstock was passed through it. Another concern with these types of feedstocks is that because of the sometimes low production level of the protein of interest, the feedstock volume is large. A large volume takes a long time to pass through a chromatography column, and a long processing time in- creases the risk of proteolytic breakdown of the protein. Unit operations to clarify and reduce the volume of these feedstocks are therefore vital for a successful purification process. It is not uncommon for a purification process to consist of a sequence of unit operations just to achieve ini- tial recovery. The techniques traditionally used for feed- stock clarification are centrifugation and filtration (1). Centrifugation is well suited to industrial processes be- cause the throughput is high and it can be performed in a contained environment (a requirement when handling re- combinant host organisms). The efficiency of the centrifu- gation process will depend on the properties of the feed- stock, but it is usually difficult to remove all particulates by centrifugation, and the resulting fluid may therefore not be sufficiently free of particulates for direct application to a packed chromatography bed (2). Whole cells are rela- tively easy to remove by centrifugation but disrupted cells are not, and cells (especially mammalian) may even be damaged by the centrifugation process (3) because of shear stress and centrifugal forces. New and improved centri- fuges that are more gentle can be used for mammalian cells (4), but often the clarification problem remains be- cause at harvest, culture fluids usually contain relatively large amounts of subcellular particulates, such as debris. When the feedstock is secreting bacteria or a bacterial ho- ADSORPTION, EXPANDED BED 21 mogenate, for example, clarification is even more difficult and centrifugation is not very efficient. When filtration is used for clarification of feedstocks, it is usually performed in cross-flow mode (5,6). The fluxes that can be used will depend on the properties of the feed- stock, but it is often difficult to reach an acceptable eco- nomic limit (100 L/m2/h) (7) for a cross-flow filtration pro- cess because fluxes are typically less than 50 L/m2/h. Cross-flow filtration can easily be operated as a completely contained system, and it yields a particulate-free solution. The fouling of the filtration membranes is a major concern, and the resulting decrease in flux per filter area often leads to long processing times. In addition, the cost of mem- branes must be taken into account in a process where membrane fouling is severe because the membranes have to be replaced frequently. Because of the drawbacks asso- ciated with both the centrifugation and the filtration tech- niques, they are often used in combination in a purification process to achieve the best clarification of the feedstock. The volume of the feedstock is not significantly reduced by either centrifugation or filtration, which is why product concentration does not usually increase until the protein has been adsorbed to a chromatography adsorbent in a packed-bed column. Because the feedstock volume is not reduced before application to the packed bed, processing time will be long. Thus, for a number of reasons, the best option for successful initial recovery of a protein from a large-volume cell- or debris-containing feedstock is direct adsorption of the protein. A variety of methods for direct recovery have been tried. One such method is to use a batch procedure in which an adsorbent is added directly to the unclarified feedstock in a stirred tank (8,9). The advantage of using a batch pro- cedure is that the feedstock does not have to be clarified. The disadvantage is that adsorption of protein to the chro- matography adsorbent is inefficient, because the stirred tank acts as one single theoretical plate in a separation process (10). Several attempts, however, have been made to improve batch adsorption, for example, continuous- batch adsorption processes (11). Another alternative for direct recovery is fluidized bed adsorption. This technique uses high-density adsorbent particles in an upflow fluidized bed column (12,13). As with a batch procedure, a fluidized bed behaves like a column with a low number of theoretical plates. The adsorbent particles and the feedstock are constantly "back-mixed," which usually results in poor adsorption of the protein by the adsorbent particles, making it necessary to recirculate the feedstock through the column, leading to long process- ing times. A number of solutions to improve the efficiency of fluidized beds have been suggested, such as columns fit- ted with perforated horizontal plates (14). This creates a column with a number of smaller, each completely back- mixed, fluidized beds. The number of fluidized beds de- pends on the number of plates inserted. The end result is a column with more than one theoretical plate and conse- quently a more efficient process. Another way to improve the efficiency of a fluidized bed is to use magnetically sta- bilized fluidized beds (15). The adsorbent particles are magnetically susceptible and are fluidized while being sta- bilized by a magnetic field. Stabilizing the particles in this way prevents back-mixing and gives the column several theoretical plates. There are, however, drawbacks that have prevented this technique from being used at indus- trial scale, such as the heat generated by the magnetic field. The concept of expanded-bed adsorption (or expanded- bed chromatography) was introduced after another at- tempt to stabilize and prevent back-mixing in a fluidized bed. An expanded bed is a chromatography bed in which the bed voidage is larger than in a settled or packed bed. Furthermore, it displays no or very low back-mixing of ad- sorbent and liquid. An expanded bed can be also be de- scribed as a stable fluidized bed because the liquid flow shows a constant velocity profile over the whole cross- section of the bed (i.e., plug flow) (Worth mentioning is that the terms fluidized bed and expanded bed are used differ- ently in the literature, and sometimes authors do not differ between the expressions.) The early expanded bed exper- iments were performed using Sepharose� Fast Flow (Amersham Pharmacia Biotech, Uppsala, Sweden) as the adsorbent and a sintered glass filter as the liquid flow dis- tributor at the bottom of the column (16). The results showed that if the bed expansion was limited to approxi- mately twice the settled bed height, the liquid flow through the column was close to plug flow and the protein adsorp- tion efficiency was similar to that of a packed bed. These promising results showed that in an expanded bed it is possible to combine the fluidized bed's ability to handle unclarified feedstocks with a packed bed's characteristic of efficient protein adsorption. The adsorbent used in these early experiments is intended for use in packed beds. The density of the particles is relatively low, which meant that the flow through the expanded bed column also had to be low, otherwise the bed would expand to an extent that ad- sorbent would start to pack at the top of the column. For this technique to be attractive at industrial scale, the flow velocities must be significantly increased. The sintered glass filter that was used as a flow distributor is not ap- propriate f