Larry D. Bowers Department of Laboratory Medicine and Pahlogy University of Minnesota Minneapolis. Minn. 55455
Immobilized Biocatalvsts in Chemical Analysis
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In 1974, Weetall published a REPORT in ANA. LYTICAL CHEMISTRYdo umenting the increasing interest in a relatively new concept in catalysis involving enzymes physically or covalently hound to a solid support ( I ) . This article and a subsequent at ticle (2) discussed the ap plications of immobilized enzymes over the 60 years since it was first observed that enzymes could be adsorbed onto a surface and retain their activity. The decade of the seventies produced an enormous proliferation of literature on the technology and analytical applications of these materials, including more than 20 monographs and more than 1000 research publications. Immobilized enzymes have now moved beyond the novelty stage and have become a mature area of research. Very few reports of a new immobilization chemistry can stir enthusiasm, and the descriptions of new analysis systems have become less and less frequent. With these advances in mind, it seems appropriate to evaluate the use of immobilized enzymes as routine laboratory tools, a prediction made in 1976 (2),and the reasons for the success or failure of the technique. The advantaces of immobilized enzymes are numerous. The moat obvious is that the enzyme can he readily 0003-2700186/0358-513A$01.50/0 0 1986 American Chemical Societv
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separated from the reaction mixture and reused. Thus the effort and expense of preparing immobilized enzymes are offset hy their extended use and greater economy. In some cases, there have been assertions that immobilized enzymes are more stable than their soluble analogues. It would be particularly useful if immobilization permitted the use of enzymes in organic solvents or a t temperatures above those that would ordinarily cause a loss of activity. This wiU be discussed in greater detail below. The cost of these advantages is an increase in complexity of the system. When the enzyme is physically localized on a matrix, the substrate for the reaction must diffuse to the enzyme for reaction to occur. This convolution of diffusion and reaction rate makes quanti-
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fication of catalytic activity a function of a number of nonenzymic factors. As a result, two types of activity can he measured. Operational or apparent activity is the measured catalytic rate (pmollmin) under conditions similar to those used in the analytical system. This type of activity can be used to optimize the system for an analysis. Inherent or intrinsic actiuity is the catalytic rate of the enzyme when the physical limitations of mass transport are removed. In order to characterize the properties of the enzyme, compare recovery of enzyme activity from the immobilization mixture, investigate the effects of immobilization on enzyme behavior, or assess the economic and catalytic efficiency of the enzyme, a measure of the inherent activity is required. Clearly, a complete description of the enzyme system requires measurement, and understanding, of both parameters. Elfects ol irnmobllization on enzyme
P-b Physically confining an enzyme to a specific region of a carrier material has a number of effects on the characterktics of the enzymic catalysis. These effects may arise as a result of a specific interaction between the support and the enzyme such that the mechanism
ANALYTICAL CHEMISTRY, VOL. 58. NO. 4, APRIL 1986
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Flgun 1. External (a) and internal (b) mass transfer processes for an lmmoblllred enzyme contained in a spherical support
partlcle of catalysis is affected. One example of this type of behavior would be an interaction of the support with the active site of the enzyme. A second potential cause of a change in enzyme characteristics might be the result of a difference between the bulk properties of solution and the microenvironment near the enzyme. The introduction of a negatively or positively charged group onto the support material shifts the optimum pH for enzyme catalysis due to partitioning of protons either toward or away from the surface. Thus the pH of the bulk solution is either greater than or less than the pH experienced by the enzyme. A similar partitioning of substrate toward or away from the surface can also be induced by introduction of a charge or a hydrophobic group onto the support. The possibility of tailoring the enzyme mi-
croenvironment to allow the use of enzymes in an otherwise hostile environment has not received much attention in the development of analytical systems. Effect of diffusion on kinetics. As mentioned above, the use of heterogeneous catalysts introduces the complication of diffusion into any consideration of catalytic rates. The rate of conversion of substrate to product is influenced by both external and internal resistance to mass transfer. External mass transfer is the process of movement of the substrate from the bulk of solution to the surface of the support matrix. Internal mass transfer resistance is the result of diffusion of the substrate within the support matrix (Figure 1). These phenomena exist for all forms of immobilized enzymes commonly used in analytical
applications. The form of the equations describing the mass transfer differs depending on whether the immobilized enzyme matrix is in the shape of a spherical particle, an open tube, or a membrane. The mathematical relationships relating the enzyme reaction rate to the parameters that affect it are summarized in Table I. External mass transfer is partly attributable to diffusion. If the fluid to be analyzed is moving near the surface of the support because of bulk flow or stirring, convective transport will also contribute to the delivery of substrate to the enzyme. As illustrated in Figure la, in the case of external mass transfer resistance control, the rate of reaction is a function of the particle diameter ( d p ) ,the diffusion coefficient of the substrate (DAand the superficial flow velocity ( p ) . Clearly this presents
Membraneb
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[PI. Is poduct concentration at me sensor surface; a is he enzymeloadingfactw (ks[EllD,Km);# is me pwmeabiiity ot the membrane tw substrate: b" is the volume of me membrane: Lis the membrane thickness. Relamattedfrom RelmllCo 3 l a an ampemmebic electrode; n is the number of electrons and F is Faraday's constant. [PI. is the $wduct COncBmration of the exit Of the reactor; V, is the reactor volume; 0 is me volumetric now rata: dp Is the 6uppm diameter.
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ANALYTICAL CHEMISTRY. VOL. 58, NO. 4, APRIL 1986
Flgure 2. Rate of generation of product as a function of flow rate for Immobilized &giucuonidase using a gradient-
less recirculating reactor Repinted wlth prmissimfrm Rehwenoe 4
several problems. If the amount of active enzyme that has been immobilized is to be measured accurately, the effects of external diffusion must he eliminated, as the activity measurement is based on the rate of reaction. This has been accomplished with microreactor systems in which the rate of conversion of substrate can be measured as a substrate solution is rapidly flowing through a thin bed of catalyst. The reaction rate as a function of flow
rate for immobilized 8-glucuronidase is shown in Figure 2. Note that the "activity" is threefold greater in the absence of external mass transfer limitations (e.g., at high flow rates). The reaction rates observed even at the lowest flow rates used in this study are sixfold greater than those observed for particles suspended in bulk stirred solution. Substantial differences in the recovery of enzyme activity would be calculated based on these various experimental conditions. Even greater difficulty would be encountered if the recovery of different-sized particles were compared under diffusion-controlled conditions. A second problem is chat if diffusion limits the rate of reaction. the enzyme is not being used efficiently. Again, this has economic rather than scientific implications. In addition to the effect of external mass transfer limitations on the rate of reaction, the other enzyme characteristics are affected as well. Slow mass transfer invariably increases the apparent Michaelis constant, K f f P P . (The Michaelis constant is the concentration of substrate at which half of the maximal reaction rate is observed.) Thus, mass transfer limitations have a significant impact on the evaluation of the characteristics of immobilized enzymes. However, this increase in K I p P P has the analytical ben-
efit of increasing the linear dynamic range of the immobilized enzyme system relative to its soluble counterpart. I t should be pointed out that the apparent activity and K n p p p are the operational parameters that should be optimized in an analysis system. Unfortunately, much of the work reported in the analytical literature fails to take mass transfer limitations into account. This makes valid comparison of various immobilized enzyme systems extremely difficult, if not impossible. Internal or intraparticle mass transfer occurs within the interior of the support matrix, a region not accessible to convection from the hulk solution. As shown in Figure lb, the reaction rate will be a function of the radius of the particle, the substrate concentration, the diffusion coefficient of the substrate, and the amount of enzyme present. For the analytically useful case in which the reaction rate is first order with respect to substrate, the reaction rate is given by
R, = o.[Sla/dp X [Rcoth((@)/Z)
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where R, is the reaction rate, [SI0 is the concentration of substrate in the bulk of solution, and m is the enzymeloading factor (k#3]/D&p'). The loading factor is a function of the in-
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herent dissociation constant of the product from the enzyme, the concentration of enzyme in the matrix, the diffusion coefficient of the substrate, and the apparent Michaelis constant. The derivation of the equation for internal mass transfer limitations in spherical particles assumes that the enzyme is uniformly distributed throughout the particle. Recent evidence indicates that this may not always be the case. It should be noted that the internal mass transfer limitations on the reaction rate are most deleterious a t low substrate concentrations. The above equation would seem to indicate that with increasing internal mass transfer limitations the enzyme that has been immobilized is used less and less effectively; that is, to achieve a twofold increase in reaction rate one must increase the enzyme concentration at least fourfold. An effectiveness factor can be derived for the various circumstances limiting enzyme catalysis in each of the forms in which enzymes are used analytically. This is basically the rate observed in the presence of the ratelimiting factor relative to the rate of reaction in the absence of the limitation. T h e enzyme-loading factor, a , is of fundamental importance in both t h e response of the analytical system to substrate and the effectiue use of
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the enzyme. An increase in the loading factor results in an increase in the analytical response but may result in a decrease in the efficiency of enzyme use. An increase in the loading factor might also result in a desirable increase in the amount of time the enzyme preparation can be used. It is not uncommon to set the experimental conditions to maximize the loading factor and accept any consequences. Clearly there are commercial implications of this trade-off that will have a significant impact on the design of immobilized-enzyme instrumentation. Effect on stability. One of the advantages cited when immobilized enzymes are discussed is an increase in stability. However, we have observed, as have others, that this so-called advantage is ambiguous. There are a number of reasons for these equivocal findings. First, there are several types of stability that can be measured. Storage stability is simply the ability of the enzyme to retain its activity under a specified set of storage conditions. Thermal stability is a measure of the ability of the enzyme-matrix system to withstand elevations in temperature, frequently in excess of those that would denature the native protein. In principle, an enzyme system capable of withstanding autoclaving and other types of sterilization could
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be developed. Finally, operational stability reflects the ability of the enzyme system to function in the analysis system. It is a function not only of the enzyme, but also the carrier durability, the inhibitor concentrations in the analysis stream, the pH, and other physical characteristics of the analyte solution. Unfortunately, the storage stability is most frequently reported, and comparison of the various types of stability is difficult. A second consideration is the manner in which the stability of the enzyme is measured. As noted above, diffusion can have a significant effect on the observed enzyme kinetics. It can also have a substantial influence on the stability measurements. When the enzyme loading is high and the system is diffusion controlled, the rate of reaction is independent of the enzyme concentration. Thus, a significant amount of enzyme could be inactivated before the diffusional limitations were removed and a decrease in “activity” was measured. Although this is an operational increase in stability, no inherent stabilization of the enzyme has occurred. In economic terms, there may have been no cost savings. Inexplicably, systematic study of the experimental factors leading to increased, controlled stabilization has
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been lacking-possibly because enzyme inactivation itself is a complex and poorly understood phenomenon. Althoueh the inactivation nrocess mav be different for each individual enzyme, the generally accepted mecbanism of loss of activity involves the unfolding of the three-dimensional structure of the protein, which disassembles the active site of the enzyme. Processes that would prevent this unfolding would be expected to stabilize the enzyme activity, and in fact the results support this hypothesis. Much work remains to be done in this area. Investigations into the possibility of stabilizing enzymes by immobilization for use in organic solvents or in organic-aqueoua solvent mixtures have, in general, met with limited success. Our own studies have indicated that under conditions in which mass transfer limitations are removed, the immobilized enzyme is not significantly more stable than the soluble enzyme (4).The inactivation problem is exacerbated by certain solvents such as acetonitrile and is less troublesome in bydrogenbonding solvents such as methanol. Martinek and co-workers have taken a different approach, using reversed micelles of enzyme solution to perform reactions in water-immiscible solvents (5). Interestingly, the use of either miscible (4) or immiscible (5) solvents can increase the observed rate of reaction because of improved solubilization of either substrate or product. Another interesting point is that the enzyme inactivation process seems to affect only some of the enzyme molecules because once the initial inactivation is observed, the enzyme seems to function in the mixed-solvent environment without further loss of activity. Clark and Bailey recently reported on the detection by electron paramagnetic resonance of two subpopulations of a-chymotrypsin that became inactive at different rates (6).This may explain our observations of a differential 520).
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siderations in developing an immobilized enzyme analysis system. It must be emphasized that there is no best technique for immobilization.
The chemical and physical methods of immobilization have not changed much over the years. There are basically four methods used in immobilization: chemical cross-linking of the enzyme to itself and to other protein molecules, entrapment of the enzyme within the pores of a support, adsorption onto a rigid matrix, and covalent coupling to a matrix. An additional technique, called encapsulation, has not been used in analytical applications because of the diffusional limitations of substrate entering a “globule” of enzyme. No universally useful immobilization technique bas emerged; hence the approach taken to immohilization is frequently bit or miss. The chemistry of immobilization has been well reviewed in the monographs listed in the suggested reading list at the end of this REPORT. The decision about which immobilization technique to use depends on the expected use of the preparation. Covalent attachment of the enzyme to the matrix is generally the most irreversible of the immobilization techniques. A few of the most commonly used reactions are summarized in Figure 3. I t has been our experience that for some enzymes the binding chemistry may have a substantial effect on the recovery and stability of activity, so a single immobilization scheme may not always yield optimal results. The choice of the immobilization method depends larzelv on the final application intendea for the enzyme. The rapacity of the matrix or technique to bind enzyme, the mechanical and chemical stability of the matrix, the expense and the difficulty in artivation of the support, the availability of the support material, and the flow characteristics are all important con-
Analytical applicatiaar Although hundreds of analytical systems using immobilized enzymes have been described, basically they can be divided into two types. In one approach, the immobilized enzyme is in direct contact with the sensor or transducer. In the second approach, the enzyme resides in a flowing stream into which the analyte is inserted, and detection occurs in the stream itself. The differences in these two approaches arise from the limitations imposed by the controlling pbenomena in the analytical system. Thus, the two approaches can be viewed as extremes of a continuum of system design rather than as totally independent approaches. Figure 4 illustrates the physical layout of the various approaches. Transducer-bound systems. The transducer-bound system is primarily limited hy diffusion of substrate to the immobilized-enzyme membrane and by the loading of enzyme in the membrane. It can be viewed as a reactor attached to the sensor in which diffusion is the primary form of mass transfer. This approach has been the most popular as reflected by the number of papers in the literature. The most prominent approach in this category has been the enzyme electrode, in which an amperometric or potentiometric electrode is used as the sensor. The transducer-bound approach accentuates the importance of certain enzyme characteristics while minimizing others. For example, recovery of activity is relatively unimportant because in a typical membrane less than one unit of enzyme activity will be used. This assumes a membrane thickness of 100-300 p m and a concentration of enzyme sufficient to deliver
loss of activity. As was mentioned earher, this area is relatively unexplored.
ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL IS86
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99%of the substrate to the sensor surface as product. Hence, it should be possible to use even an expensive enzyme in this type of system. The response time of the device is also a strong function of the thickness of the membrane. If one considers a substrate with a diffusion coefficient of 10-5 em%, the steady-state response time of the electrode will range from 14 to 216 s for the aforementioned membrane thicknesses. The limit of detection of the probe will be a function of the limit of detection of the sensor and the relative rates of diffusion and enzyme reaction. Thus, there are a variety of trade-offs that must be explored. For example, the enzyme rate can be increased relative to the rate of diffusion by increasing the enzyme concentration in the membrane, by increasing the membrane thickness, and by decreasing the permeability of the membrane to substrate. The second approach bas an effect on the response time as well as on the magnitude of the response. The last approach will affect the limit of detection as well as the magnitude of the response. The behavior of singlesubstrate and pseudo-single-substrate immobilized-enzymemembranes in contact with sensors has been described and should assist in the design of transducer-bound immobilized-enzyme systems. Recent research in the area of immobilized-enzymetransducers has involved approaches directed a t specific problems in the analysis system. For example, a functional implantable infusion pump has been developed that could be used to release drugs or hormones such as insulin on physiological demand if a suitable sensor could be developed. Clearly, the constraints on development of an enzyme electrode 522A
that can be implanted in people are unique. The sensor must be extraordinarily reliable, must be totally dependent on the physiological milieu for supply of all of the substrates required for analysis, and must be biocompatible. Leypoldt and Gough (7,B) have been extending the theory of enzyme electrodes to the two-substrate case as would be encountered for a glucose oxidase electrode (glucose, 02)By . evaluating the model with regard to the various trade-offs, they concluded that limiting the permeability of the membrane for glucose would yield the best results for an electrode that is limited by physiological amounts of oxygen. More recently, Gougb and coworkers (9)have described an electrode for glucose in which oxygen can diffuse into the electrode either axially or radially, whereas the glucose can enter the electrode axially only. This may prevent the oxygen depletion observed in previous electrode designs and has important implications for an implantable electrode. Castner and Windgard (10) have been evaluating treatment of the platinum electrode itself to standardize the response to glucose relative to the product of the glucose oxidase reaction. These important advances should lead to the development of an effective treatment for diabetes mellitus if tight control of blood glucose is found to ameliorate the secondary pathology of this disease. The recent use of microorganisms, both native and genetically engineered, for the commercial production of a variety of substances from ethanol to drugs requires frequent analysis of nutrients in the bioreactor containing the microorganisms. This application, of course, has different constraints than those described. above. If the sen-
ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986
sor is to be placed inside the bioreactor, there is a need to sterilize it so that the contents of the reactor are not contaminated by another organism. If the microbe used is anaerobic, a source of oxygen would be required if the enzyme used were an oxidase. Cleland and Enfors (11)recently described an externally buffered electrode that uses a continuously flowing stream of fluid within the electrode body. This approach allows 02,H+, or a buffer solution to be added on the inside of the enzyme membrane, thus allowing its use under a variety of conditions. The authors report that the electrode can be sterilized and used in anaerobic sites. Another application of immobilizedenzyme membranes has been the development of a sensor for monitoring air for organophosphates and carbamates. Invented by workers a t the Midwest Research Institute ( E ) the , sensor is a pad containing immobilized acetylcholinesterase. When pesticides are present, the enzyme, which is similar to the enzyme causing toxic symptoms in humans, is irreversibly inhibited. The activity of the enzyme on the pad can be monitored with a small instrument and the degree of exposure quantified. Similar approaches are being applied to the detection and detoxification of neurotoxins. In addition to the investigation of specific conditions in particular applications, a unique approach has been taken in the development of microorganism and tissue electrodes. Rechnitz and his eo-workers (23.14) have entrapped various bacteria and plant and animal tissue a t the surface of an electrode. These electrodes work amazingly well and have unique specificity. Perhaps most interesting is the lack of effort required to prepare the
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electrode. Schubert et al. reported on an electrode system in which a 0.1-mm slice of potato was fixed on the surface of an immobilized glucose oxidase electrode to measure phosphate and fluoride by inhibition of the alkaline phosphatase in the potato slice. The response time of 25 s was amazing in light of the discussion above (15).The ability to measure various chemicals that affect the rate of the enzyme reaction, such as inhibitors and activators, presents a wide variety of opportunities for extending the role of sensor analyses. Siegopaul and Rechnitz have reported on an electrode for measurement of thiamine phosphate (16). The analyte activates apo-pyruvate decarboxylase, which in the absence of this cofactor is inactive. This activation results in production of a potentiometrically active product. Because one molecule of the cofactor results in the production of many molecules of the detected species, a significant amplification can be obtained. Electrodes are not the only transducers that have been used in conjunction with enzyme membranes. Fiber-optic probes have been used to conduct the light generated in a chemiluminescent enzyme reaction to a photomultiplier tube. Enzyme membranes have also been affixed to thermistors to monitor the heat gener-
ated by the enzyme reaction, albeit with mixed success. Perhaps one of the most novel systems reported involves the use of the change in enzyme conformation upon binding of the substrate to cause a chemomechanical change sensed by a force transducer! These specific examples are only a few of the numerous inventive analyses reported. Immobilized-enzyme reactors. In contrast to transducer-bound enzyme systems, immobilized-enzyme reactors (IMERs) generally consist of a relatively large amount of enzyme immobilized on a support in the form of a bed or annulus. The advantage of using a large amount of enzyme is that all of the substrate contained in a sample can be converted to product in the relatively short time that the sample is in the reactor. Under first-order conditions, the fractional conversion, X, is given by the relation
X = 1 - exp - (aD,V,./Q) where a is the enzyme-loading factor, V, is the volume of the reactor, and Q is the volumetric flow rate. The enzyme-loading factor is very important because increasing it will allow an increase in flow rate or a decrease in the reactor volume. The disadvantage of requiring a large amount of enzyme is
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the premium it places on the development of a stable preparation of enzyme. This is because a significant amount of time and money is invested in the reactor. A second disadvantage of the IMER is that the capability of complete conversion is offset by the dispersion of the product band, which occurs in the reactor and connecting tubing before the detector. Thus, evaluation of an IMER will depend on both the enzyme activity and the dispersion inherent in the reactor system. In general, three types of reactors have been used-open-tubular gassegmented, packed-bed, and STRING reactors. The dispersion in open-tubular flow systems has been discussed in detail by Snyder and co-workers (17). Basically, the axial dispersion is limited by the gas segmentation, and what dispersion does occur is the result of a thin film of liquid adhering to the wall. It should be pointed out that extrareactor broadening during the removal of the gas segments from the stream was a major contribution to the relatively poor characteristics of the first generation of autoanalyzers. Incorporation of an electronic debubbler has resulted in significant improvement in the dispersion characteristics. The major limitation of gassegmented reactors is the fact that for
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a 1-mm4.d. tube, the surface area available for immobilization is on the order of 30 cm2per meter of length. This compares with hundreds of square meters per gram of inorganic support material and, as mentioned above, the amount of enzyme immobilized is related to the available surface area. To circumvent this problem, a number of methods to produce organic or inorganic annuli have been reported. Unfortunately, the presence of the annulus thickens the stagnant layer at the wall of the tube, requiring greater diffusion of the product to re-enter the fluid stream and resulting in greater dispersion of the product. Nevertheless, a large number of successful demonstrations of open-tubular reactors have been reported. Dispersion in packed-bed IMERs is analogous to the dispersion seen in chromatographic columns. Approaches to minimizing dispersion include improved packing structure, decreased support particle diameter, and optimization of the linear fluid velocity. At present, typical particle diameters in IMERs are 37-74 pm, and packing techniques are archaic. In our experience, there are other factors that limit the utility of decreasing the particle size and achieving lower dispersion, although the theoretical basis and techniques are well established by
modern high-performance liquid chromatography (HPLC). Most of the work done on the enzyme kinetics in IMERs has assumed a continuous introduction of substrate, when in actuality a sample for analysis is introduced as a pulse. Adachi and his coworkers (18) have studied the elution profile for an impulse injection for both a single enzyme and consecutive first-order reactions. The most important characteristic of the reactor in minimizing dispersion was the ratio of the time required to diffuse into the support to the time required to be eluted from the column. When this parameter approaches one, very broad and asymmetric peaks were observed. Fortunately, for the conditions normally used in analytical reactors (100-pm particles, 0.5 X 5-cm reactor), the peaks should be symmetrical and relatively narrow. In essence, we have decoupled the dispersion and reaction rate processes. We have also shown that if the reaction is not totally first order, “kinetic” broadening can occur. Recently a third type of reactor has been described in which solid beads are packed single file in a tube whose inside diameter is one to five times the diameter of the beads. Reijn, Poppe, and van der Linden have studied dispersion in the single-bead STRING reactor (SBSR) (19). The authors con-
clude that there is a residence timereaction rate component and a flow rate-reactor length-bead diameter component to the dispersion. The reactor can be optimized using either one of the two components, and the authors conclude that they are independent of each other. Using nonreactive compounds, they conclude that the SBSR has similar dispersion, reagent consumption, and residence time as a more conventional packedbed reactor. Gnanasekaran and Mottola have reported on an immobilized penicillinase SBSR, but no comparison to a packed-bed system was included (20). Because the issue of comparable enzyme loading cannot be easily addressed, it is unclear what the role of the SBSR in immobilized-enzyme technology will be. The possibility that the enzyme itself can cause peak broadening has recently been described. Because the substrate must bind to a specific site on the enzyme to facilitate the reaction and the product(s) must then be released, it is not inconceivable that the enzyme could influence the peak shape. Slow release of product, for example, would be analogous to a kinetically slow dissociation from a binding site in adsorption chromatography and would result in asymmetric, broad peaks. This phenomenon has been ob-
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526A
ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986
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Do All Anall, Need To Be Watched?
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So there is no longer any need to push buttons or monitor every chromatogram when your valuable time could be spent doing all your other important functions. For additional information on the D-2000,655A-40 and L-5000 or to arrange for a demonstration. call or write: 111 Woodcrest Rd. Circle 52 lor literature. Cherry Hill, NJ 08034-0395 Circle 53 for a dernmslration (609)354-9200 (800) 222-0342 EM SCIENCE A Division oiEM Indurrner, In
