680 Fermentation and biochemical engineering Handbook 5.0 CHEMICAL COMPOSITION The most widely used method for determining chemical composition is chromatography. Several categories have been developed depending upon the species being separated. These include gas chromatography and several varieties of liquid chromatography including low pressure(gel permeation) and high pressure liquid chromatography and thin layer chromatography The basic principle behind these is the separation of the constituents traveling through a porous, sorptive material such The degree of retardation of each molecular species is based on its particular affinity for the sorbent. Proper selection of the sorbent is the most critical factor in determining separation. Other environmental factors such as temperature and pressure also play a key role The chemical basis for separation may include adsorption, covalent bonding or pore size of the material Gas chromatography is used for gases and for liquids with relatively low boiling points. Since many of the constituents in a biochemical reaction are of considerable molecular weight, high pressure liquid chromatography is the most commonly used. Specialized apparatus is needed for performing his analysis since chromatograph pressures can range as high as 10, 000 psi Thin layer chromatography requires no pressure but instead relies on the capillary action of a solvent through a paper-like sheet of sorbent. Each constituent travels a different distance and the constituents are thus separated Analysis is done manually, typically using various coloring or fluorescing Gel permeation chromatography utilizes a sorbent bed and depends on gravity to provide the driving force but usually requires a considerable time to effect a separation All of these analyses are typically performed in a laboratory; therefore hey require the removal of samples. As the reaction is conducted in a sterile environment, special precautions and sample removal procedures must be utilized to prevent contaminating the contents of the reactor 6.0 DISSOLVED OXYGEN Dissolved oxygen is one of the most important indicators in a fermen ss. It determines the potential for growth. The measurement of dissolved oxygen is made by a sterilizable probe inserted directly into the aqueous solution of the reactor. Two principles of operatic
680 Fermentation and Biochemical Engineering Handbook 5.0 CHEMICAL COMPOSITION The most widely used method for determining chemical composition is chromatography. Several categories have been developed depending upon the species being separated. These include gas chromatography and several varieties of liquid chromatography including low pressure (gel permeation) and high pressure liquid chromatography and thin layer chromatography. The basic principle behind these is the separation ofthe constituents traveling through a porous, sorptive material such as a silica gel. The degree of retardation of each molecular species is based on its particular affinity for the sorbent. Proper selection of the sorbent is the most critical factor in determining separation. Other environmental factors such as temperature and pressure also play a key role. The chemical basis for separation may include adsorption, covalent bonding or pore size of the material. Gas chromatography is used for gases and for liquids with relatively low boiling points. Since many of the constituents in a biochemical reaction are of considerable molecular weight, high pressure liquid chromatography is the most commonly used. Specialized apparatus is needed for performing this analysis since chromatograph pressures can range as high as 10,000 psi. Thin layer chromatography requires no pressure but instead relies on the capillary action of a solvent through a paper-like sheet of sorbent. Each constituent travels a different distance and the constituents are thus separated. Analysis is done manually, typically using various coloring or fluorescing reagents. Gel permeation chromatography utilizes a sorbent bed and depends on gravity to provide the driving force but usually requires a considerable time to effect a separation. All of these analyses are typically performed in a laboratory; therefore they require the removal of samples. As the reaction is conducted in a sterile environment, special precautions and sample removal procedures must be utilized to prevent contaminating the contents of the reactor. 6.0 DISSOLVED OXYGEN Dissolved oxygen is one of the most important indicators in a fermentation or bioreactor process. It determines the potential for growth. The measurement of dissolved oxygen is made by a sterilizable probe inserted directly into the aqueous solution ofthe reactor. Two principles of operation
Instrumentation and Control Systems 681 re used for this measurement the first is an electrochemical reaction while the second employs an amperometric(polarographic) principle The electrochemical approach uses a sterilizable stainless steelprobe with a cell face constructed of a material which will enable oxygen to permeate across it and enter the electrochemical chamber which contains two elec- trodes of dissimilar reactants(forming the anode and cathode)immersed in a basic aqueous solution(Fig. 2). The entering oxygen initiates an oxidation reduction reaction which in turn produces an EMF which is amplified into a signal representing the concentration of oxygen in the solution Figure 2. Sterilizable polarimetric dissolved oxygen probe. (Courtesy of ingold Elec- trodes, Inc, wilmington, Mass)
Instrumentation and Control Systems 681 are used for this measurement: the first is an electrochemical reaction while the second employs an amperometric (polarographic) principle. The electrochemical approach uses a sterilizable stainless steel probe with a cell face constructed of a material which will enable oxygen to permeate across it and enter the electrochemical chamber which contains two electrodes of dissimilar reactants (forming the anode and cathode) immersed in a basic aqueous solution (Fig. 2). The entering oxygen initiates an oxidation reduction reaction which in turn produces an EMF which is amplified into a signal representing the concentration of oxygen in the solution. Figure 2. Sterilizable polarimetric dissolved oxygen probe. (Courtesy of Ingold Electrades, Inc., Wilmington,Mass.)
82 Fermentation and Biochemical Engineering Handbook In the amperometric(polarographic)approach, oxygen again perme ates a diffusion barrier and encounters an electrochemical cell immersed in basic aqueous solution. a potential difference of approximately 1.3 V is maintained between the anode and cathode as the oxygen encounters the cathode. an electrochemical reaction occurs 02+ 2H20+ 4e+40H(at cathode) The hydroxyl ion then travels to the anode where it completes the electro- chemical reaction process 40H+>O2+ 2H20+ 4e(at anode) The concentration of oxygen is directly proportional to the amount of current passed through the cell 7.0 EXHAUST GAS ANALYSIS Much can be learned from the exchange of gases in the metabolic process such o2, CO2, N2, NH3, andethanol. In fact, most ofthe predictive analysis is based upon such calculations as oxygen uptake rate, carbon dioxide exchange rate or respiratory quotient. This information is best obtained by a component material balance across the reactor. a key factor in determining this is the analysis of the bioreactor offgas and the best method for measuring this is with a mass spectrometer because of its high resolution Two methods of operation are utilized. These are magnetic deflection and quadrapole. The quadrapole has become the primary commercial system because of its enhanced sensitivity and its ability to filter out all gases but the one being analyzed Magnetic deflection mass spectrometers inject a gaseous sample into an inlet port, bombard the sample with an electron beam to ionize the particle and pass the sample through a magnetic separator. The charged particles are deflected by the magnet in accordance with its mass-to-energy(or charge) ratio-the greater this ratio, the less the deflection. Detectors are located on the opposing wall of the chamber and are located to correspond to the trajectory of specific components as shown in Fig 3. As the ionized particles strike the detectors, they generate a voltage proportional to their charge. Thi information is used to determine the percent concentration of each of the gass
682 Fermentation and Biochemical Engineering Handbook In the amperometric (polarographic) approach, oxygen again permeates a diffusion barrier and encounters an electrochemical cell immersed in basic aqueous solution. A potential difference of approximately 1.3 V is maintained between the anode and cathode. As the oxygen encounters the cathode, an electrochemical reaction occurs: 0, + 2H,O + 4e- + 40H- (at cathode) The hydroxyl ion then travels to the anode where it completes the electrochemical reaction process: 40H- + 0, + 2H20 + 4e- (at anode) The concentration of oxygen is directly proportional to the amount of current passed through the cell. 7.0 EXHAUST GAS ANALYSIS Much can be learned from the exchange of gases in the metabolic processsuchas O,, CO,,N,,NH,,andethanol. Infact, mostofthepredictive analysis is based upon such calculations as oxygen uptake rate, carbon dioxide exchange rate or respiratory quotient. This information is best obtained by a component material balance across the reactor. A key factor in determining this is the analysis ofthe bioreactor offgas and the best method for measuring this is with a mass spectrometer because of its high resolution. Two methods of operation are utilized. These are magnetic deflection and quadrapole. The quadrapole has become the primary commercial system because of its enhanced sensitivity and its ability to filter out all gases but the one being analyzed. Magnetic deflection mass spectrometers inject a gaseous sample into an inlet port, bombard the sample with an electron beam to ionize the particles and pass the sample through a magnetic separator. The charged particles are deflected by the magnet in accordance with its mass-to-energy (or charge) ratio-the greater this ratio, the less the deflection. Detectors are located on the opposing wall of the chamber and are located to correspond to the trajectory of specific components as shown in Fig. 3. As the ionized particles strike the detectors, they generate avoltage proportional to their charge. This information is used to determine the percent concentration of each of the gasses
Instrumentation and Control Systems 683 MAGNETIC SEPARATOR FOCUSING DETECTOR SOURCE Figure 3. Magnetic deflection principle. The quadrapole mass spectrometer also employs an electron beam to ionize the particles using the quadrapole instead of a magnet to deflect the path of the particles and filter out all but the specific component to be analyzed. The quadrapole is a set of four similar and parallel rods(see Fig 4)with opposite rods electrically connected. A radio frequency and dc charge of equal potential, but opposite charge, is applied to each set of the rods. By varying the absolute potential applied to the rods, it is possible to eliminate all ions except those of a specific mass-to-energy ratio. Those ions which successfully travel the length of the rods strike a Faraday plate which releases electrons to the ions thereby generating a measurable change in EMF. For a given component the strength of the signal can be compared to references to determine the concentration The quadrapole, when used in conjunction with a gas chromatograph to separate the components, can measure a wide range of gases, typically fror 50 to 1000 atomic mass units(amu) mass spectrometers are relatively expensive, the exhaust gas of three or more bioreactors is typically directed to a single analyzer. This is possible because the offgas analysis is done outside the bioreactors them- selves. However, the multiplexing of the streams results in added complexity with regard to sample handling and routing, particularly if concerns of cross contaminationneed be addressed The contamination issue is usually handled by placing ultrafilters in the exhaust lines. Care, however, must be taken to
Instrumentation and Control Systems 683 Figure 3. Magnetic deflection principle. The quadrapole mass spectrometer also employs an electron beam to ionize the particles using the quadrapole instead of a magnet to deflect the path of the particles and filter out all but the specific component to be analyzed. The quadrapole is a set of four similar and parallel rods (see Fig. 4) with opposite rods electrically connected. A radio frequency and dc charge of equal potential, but opposite charge, is applied to each set of the rods. By varying the absolute potential applied to the rods, it is possible to eliminate all ions except those of a specific mass-to-energy ratio. Those ions which successfidly travel the length ofthe rods strike a Faraday plate which releases electrons to the ions thereby generating a measurable change in EMF. For a given component the strength of the signal can be compared to references to determine the concentration. The quadrapole, when used in conjunction with a gas chromatograph to separate the components, can measure a wide range ofgases, typically from 50 to 1000 atomic mass units (amu). As mass spectrometers are relatively expensive, the exhaust gas of three or more bioreactors is typically directed to a single analyzer. This is possible because the offgas analysis is done outside the bioreactors themselves. However, the multiplexing ofthe streams results in added complexity with regard to sample handling and routing, particularly if concerns of cross contamination need be addressed. The contamination issue is usually handled by placing ultrafilters in the exhaust lines. Care, however, must be taken to
68 Fermentation and Biochemical Engineering handbook ensure that these filters don' t plug resulting in excessive backpressure Periodic measurement calibration utilizing reference standards must be sent to the spectrometer to check its calibration NONRESONANT ION ELECTI ION COLLECTOR RESONANT ION dc AND r VOLTAGES ELECTRON BEAM 8.0 MEASUREMENT OF pH Metabolic processes are typically highly susceptible to even slight changes in pH, and therefore, proper control of this parameter is critical Precise manipulation of pH can determine the relative yield of the desired species over competing by-products. Deviations of as little as 0. 2 to 0.3 may adversely affect a batch in some cases. Like the cell mass probe and dissolved oxygen probes described earlier, the pH probe(see Fig. 5)is packaged in a sterilizible inert casing with permeable electrode facings for direct insertion into the bioreactor. The measurement principle is the oxidation reduction potential of the hydrogen ion and the electrode materials are selected for that purpose
684 Fermentation and Biochemical Engineering Handbook ensure that these filters don’t plug resulting in excessive backpressure. Periodic measurement calibration utilizing reference standards must be sent to the spectrometer to check its calibration. NONRESONANT ION m W IONIZING ELECTRON BUM dc AND rl VOLTAGES Figure 4. Quadrapole principle 8.0 MEASUREMENT OF pH Metabolic processes are typically highly susceptible to even slight changes in pH, and therefore, proper control of this parameter is critical. Precise manipulation of pH can determine the relative yield of the desired species over competing by-products. Deviations ofas little as 0.2 to 0.3 may adversely affect a batch in some cases. Like the cell mass probe and dissolved oxygen probes described earlier, the pH probe (see Fig. 5) is packaged in a sterilizible inert casing with permeable electrode facings for direct insertion into the bioreactor. The measurement principle is the oxidation reduction potential of the hydrogen ion and the electrode materials are selected for that purpose