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An observation chamber for a high-pressure stopped-flow apparatus
Journal of Biochemical and Biophysical Methods, 6 (1982) 173-177 Elsevier Biomedical Press
173
Short note
An observation chamber for a high-pressure stopped-flow apparatus * Paul D. Smith ~, Ernest E. Beile ~ and Robert L. Berger 2 I Biomedical Engineering and Instrumentation Branch, Division of Research Services, and e Laboratory of Technical Development, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20205, U.S.A.
(Received 2 December 1981) (Accepted 2 February 1982)
A new design of the obseravtion window to observation chamber seal is described for the Berger rapid stopped-flow apparatus. This design permits reliable operation of the apparatus and retains the features of a square non-deviating observation path with flush windows. Incorporation of fiber optic light guides for the observation path has made the apparatus insensitive to vibration artifacts. A demonstration reaction of calcium binding to EGTA is presented; the reaction, which is 90% complete within 2 ms, illustrates the capability of this apparatus for examining fast reactions. Key words: stopped flow; fiber optics; calcium chelation.
In the study of the rates of reaction, stopped-flow is a widely employed technique. One of the more important criteria by which stopped-flow apparatuses are judged is the dead-time of the instrument. This parameter determines the rate of reaction which may be studied; the smaller the dead-time, the faster the reaction rate that may be investigated. The design described by Berger et al. [1] has a dead-time of 200 /~s; this apparatus maintains the solutions under a drive pressure of approximately 400 lb/inch 2, and mixing of the reactants is achieved in the turbulent wake of a ball mixer. This apparatus has the shortest dead-time of those instruments where direct observation of the reaction is made after transportion of the reactants a finite distance from the point of mixing to the point of observation. Smaller dead-times can be achieved, for example, by observing through the mixer itself and integrating along the flow path [2]; these techniques are limited, however, owing to the fact that the reaction mechanism must be well understood to allow analysis of the resulting integrated observation. The content of this report describes modifications made to the Berger apparatus to overcome a major operating drawback and illustrates the utility of the apparatus for the measurement of a fast reaction.
* The naming of specific products or companies in this paper does not imply an endorsement by either the authors or the Federal Government. 0165-022X/82/0000-0000/$02.75 ~ 1982 Elsevier Biomedical Press
174
The stopped-flow apparatus used in this study was constructed by Science Products Corp., Dover, N.J. after an original design built in collaboration with Berger [1]. The observation chamber described in this work is used in a smaller version of the apparatus which is also built by Science Products Corp. The observation windows, which are core-cut from 4.5 mm thick polished plate glass or quartz, were obtained from Vitro Dynamics, Rockaway, N.J. The casting material, Skycast resin 2651 MM, was obtained from Emmerson and Cummings, Canton, Mass. Fiber optic bundles from Edmund Scientific, Barrington, N.J., were index matched using index matching oil, n --- 1.466, obtained from Cargille, Cedar Grove, N.J. The ethylene glycol bis(aminoethyl ether) tetraacetic acid (EGTA) used in the demonstration reaction was supplied by Geigy; the indicator bromthymol blue was supplied by BDH (British Drug House); the remaining chemicals were analytical grade and were purchased from normal supply houses. The ball mixer and observation chamber, as originally designed, are shown in Fig. 1. Solution A passes between the surfaces S l and S2 and enters the base of the ball mixer through four radial ports oriented at 90 ° to each other. Solution B, on the other hand, flows through the center of the surface S~ and exits through four jets also oriented at 90 ° to each other. The two solutions come into contact with each other at the base of the ball mixer and are thoroughly mixed in the turbulent wake downstream from the ball as they flow into the observation chamber. After mixing, the reactants are rapidly brought to a stop by a valve which closes in 25 #s; a short duration pressure pulse (ca. 1000 lb/inch2) is produced on closing. The distance from the ball mixer to the observation chamber is 4.5 mm which, at flow velocities of 20 m/s, corresponds to a dead-time of 225 /~s. To achieve the high-flow velocities and the rapid wash out of previous reactants, the apparatus is maintained at a pressure of 350-400 lb/inch2. The apparatus thus remains in a 'ready state' at all times, and the solution flow is totally controlled by the valve. The observation chamber has a square cross-section at the windows; this provides a known optical pathlength for monitoring purposes, and a non-deviating path for the monitor light. These are significant advantages over the more common circular cross-section for the monitor port. To accommodate this square cross-section, the observation chamber must provide a smooth transition between the various surfaces involved. These are, the outer surface of the ball mixer, the transition from this surface to the square cross-section, and the transition to a circular geometry for connection to the control valve. The only practical means for providing these varied topologies is to mold the piece. In the molding process, the observation windows are held in place against the mold stem, which is fashioned to the varied shapes discussed above. The windows are then cast into place using the Stycast resin; this resin is dyed black and is optically dense thus avoiding stray light problems associated with light not passing through the observation region. The resin is degassed during casting, and the assembled mold cured for several hours before the cell is released from the mold. The observation windows are flush with the flow path which avoids cavitation problems during flow. A major problem of the observation chamber was leakage of solution at the
175 TO CONTROLVALVE
]
!
OPTICAL PATH.
I,'F t
A
~iS j~
t
A
Fig. I. Observation chamber and ball-mixer of Berger stopped-flow apparatus. The stainless-steel body $2 incorporates the observation chamber (shaded), the optical path, and provides the outer surface of the flow path for reactant A. The stainless-steel element St provides the inner surface for the reactant A flow and also the path for reactant B. Mixing occurs at the base of the ball-mixer shown schematically as the diamond shaped piece,
windows. The pressures involved in the use of this apparatus exploit any weakness in the window to resin seal. Leakage would occur at random times from the manufacture of the observation chamber; a range of lifetimes of a single shot to a month was observed. The leakage, when it occurred, was minor, only affecting the optical path; the solutions were still retained in the apparatus except for the small drop size leak. A redesign of the observation chamber was undertaken retaining the features of the molding technique and providing a secondary seal to prevent solution leakage past the windows. The design shown in Fig. 2 consists of a shallow 'O'-ring groove, at the
SOLUTION FLOW
Fig. 2. Construction detail of the observation chamber/window seal. The window, W, is cast into the resin, P.; the sealing O-ring is compressed by the insert I, which also holds the fiber light guide.
176 outer end of the window, large enough to accommodate a Parker No. 2 - 6 O-ring with 125 ~m of compression. The window itself is retained in place by the Stycast resin. To verify that the 'O'-ring would prevent leakage of solution, a small (No. 80 drill) hole was drilled alongside the window; no solution leakage occurred from this hole which, in comparison to the usual leakage, represented a massive leak. The cell has remained viable for many months of experimental work with this modified arrangement. The new 'O'-ring retainers were designed to accommodate 3 mm diameter fiber optic cable. The polished ends of the fiber are held flush against the window and are in optical contact with it by using an index matching oil. This has the advantage of making the apparatus immune to vibration artifacts associated with the start and the stop of flow, which were observed with the previous design.
Fig. 3. Reaction profile of I mM calcium chloride and 1 mM EGTA at pH 7.0. 20--C. The trace represents the change in transmission of the pH indicator dye bromthymol blue in response to the hydrogen ion released in the action. Increasing transmission is downward. Vertical sensitivity is 0.005 A units/division.
177
To illustrate the performance of the apparatus, the reaction at pH 7.0, 20°C, of 1 mM calcium chloride with 1 mM ethylene glycol bis(aminoethyl ether) tetraacetic acid (EGTA) is shown in Fig. 3. When calcium binds to EGTA, hydrogen ion is released; a 10 mM cacodylate buffer limits the pH change of the solution to 0.1 pH units. The reaction is observed optically using the absorbance change at 615 nm of the indicator dye bromthymoi blue as it responds to the release of the hydrogen ion as the reaction proceeds. Fig. 3 represents the change in transmission for this reaction and it can be seen the reaction is 90% complete within 2 ms (the total absorbance change is 0.024 A units). A clean break, indicated by the arrow, is seen at the point of the stop of flow and the start of the reaction. The use of stopped-flow techniques in the study of chemical and biochemical reactions has been well documented in the literature. In conventional approaches to this technique, (i.e. those in which direct transport of the mixed reagents occurs to the point of observation downstream), the design first reported by Berger in 1968 achieves this transport in the shortest time and remains the fastest of the stopped-flow apparatus. However, wide acceptance of the apparatus has been thwarted, primarily by operating difficulties. This present study demonstrates that a reliable apparatus has been developed and allows reactions to be observed which would fall within the dead-time of other apparatus. The demonstration reaction shown here is part of a larger study of calcium binding to EGTA [3] performed using this apparatus and the Aminco-Morrow. There are several points arising from this work which are worth mentioning in the context of this report. Firstly, not a single failure of the observation chamber occurred. Secondly, it was observed that for reactions having half lives of greater than 7 ms. (i.e., well above the Aminco-Morrow dead-time), good agreement was obtained between the Aminco-Morrow and the Berger stopped-flow apparatus. Thirdly, as can be seen from Fig. 3, a clean break between the flow and stopped-flow condition is observed with little rounding observable. This is in contrast to the observations made by Berger et al. [4] on hemoglobin, where a rounding of this transition was seen. The authors concluded this was due to the oxygen-binding process, and considerable controversy arose about this point. The observation reported here shows that a sharp transition on this timescale is possible. Finally, the reaction is complete within 2 ms, which is shorter than the dead-time of other stopped-flow devices of which the Aminco-Morrow is an example. The rate of reaction for the uptake and calcium by EGTA was found to be 2 . 1 0 6 M - I . s-1 at 25°C; the details of this reaction are complex and are beyond the scope of this paper.
References 1 Berger, R.L., Balko, B., Borcherdt, W. and Friauf, W. (1968) Rev. Sci. Inst. 39, 486-492 2 Owens, G.D., Taylor, R.W., Ridley, T.Y. and Margerum, D.W. (1980) Anal Chem. 52, 130-138 3 Smith, P.D., Liesegang, G.W., Berger, R.L., Czerlinski, G. and Podolsky, R.J., Submitted for publication 4 Berger, R.L., Antonini, E., Brunori, M., Wyman, J. and Rossi-Fanelli, A. (1967) J. Biol. Chem. 242, 4841-4843
173
Short note
An observation chamber for a high-pressure stopped-flow apparatus * Paul D. Smith ~, Ernest E. Beile ~ and Robert L. Berger 2 I Biomedical Engineering and Instrumentation Branch, Division of Research Services, and e Laboratory of Technical Development, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20205, U.S.A.
(Received 2 December 1981) (Accepted 2 February 1982)
A new design of the obseravtion window to observation chamber seal is described for the Berger rapid stopped-flow apparatus. This design permits reliable operation of the apparatus and retains the features of a square non-deviating observation path with flush windows. Incorporation of fiber optic light guides for the observation path has made the apparatus insensitive to vibration artifacts. A demonstration reaction of calcium binding to EGTA is presented; the reaction, which is 90% complete within 2 ms, illustrates the capability of this apparatus for examining fast reactions. Key words: stopped flow; fiber optics; calcium chelation.
In the study of the rates of reaction, stopped-flow is a widely employed technique. One of the more important criteria by which stopped-flow apparatuses are judged is the dead-time of the instrument. This parameter determines the rate of reaction which may be studied; the smaller the dead-time, the faster the reaction rate that may be investigated. The design described by Berger et al. [1] has a dead-time of 200 /~s; this apparatus maintains the solutions under a drive pressure of approximately 400 lb/inch 2, and mixing of the reactants is achieved in the turbulent wake of a ball mixer. This apparatus has the shortest dead-time of those instruments where direct observation of the reaction is made after transportion of the reactants a finite distance from the point of mixing to the point of observation. Smaller dead-times can be achieved, for example, by observing through the mixer itself and integrating along the flow path [2]; these techniques are limited, however, owing to the fact that the reaction mechanism must be well understood to allow analysis of the resulting integrated observation. The content of this report describes modifications made to the Berger apparatus to overcome a major operating drawback and illustrates the utility of the apparatus for the measurement of a fast reaction.
* The naming of specific products or companies in this paper does not imply an endorsement by either the authors or the Federal Government. 0165-022X/82/0000-0000/$02.75 ~ 1982 Elsevier Biomedical Press
174
The stopped-flow apparatus used in this study was constructed by Science Products Corp., Dover, N.J. after an original design built in collaboration with Berger [1]. The observation chamber described in this work is used in a smaller version of the apparatus which is also built by Science Products Corp. The observation windows, which are core-cut from 4.5 mm thick polished plate glass or quartz, were obtained from Vitro Dynamics, Rockaway, N.J. The casting material, Skycast resin 2651 MM, was obtained from Emmerson and Cummings, Canton, Mass. Fiber optic bundles from Edmund Scientific, Barrington, N.J., were index matched using index matching oil, n --- 1.466, obtained from Cargille, Cedar Grove, N.J. The ethylene glycol bis(aminoethyl ether) tetraacetic acid (EGTA) used in the demonstration reaction was supplied by Geigy; the indicator bromthymol blue was supplied by BDH (British Drug House); the remaining chemicals were analytical grade and were purchased from normal supply houses. The ball mixer and observation chamber, as originally designed, are shown in Fig. 1. Solution A passes between the surfaces S l and S2 and enters the base of the ball mixer through four radial ports oriented at 90 ° to each other. Solution B, on the other hand, flows through the center of the surface S~ and exits through four jets also oriented at 90 ° to each other. The two solutions come into contact with each other at the base of the ball mixer and are thoroughly mixed in the turbulent wake downstream from the ball as they flow into the observation chamber. After mixing, the reactants are rapidly brought to a stop by a valve which closes in 25 #s; a short duration pressure pulse (ca. 1000 lb/inch2) is produced on closing. The distance from the ball mixer to the observation chamber is 4.5 mm which, at flow velocities of 20 m/s, corresponds to a dead-time of 225 /~s. To achieve the high-flow velocities and the rapid wash out of previous reactants, the apparatus is maintained at a pressure of 350-400 lb/inch2. The apparatus thus remains in a 'ready state' at all times, and the solution flow is totally controlled by the valve. The observation chamber has a square cross-section at the windows; this provides a known optical pathlength for monitoring purposes, and a non-deviating path for the monitor light. These are significant advantages over the more common circular cross-section for the monitor port. To accommodate this square cross-section, the observation chamber must provide a smooth transition between the various surfaces involved. These are, the outer surface of the ball mixer, the transition from this surface to the square cross-section, and the transition to a circular geometry for connection to the control valve. The only practical means for providing these varied topologies is to mold the piece. In the molding process, the observation windows are held in place against the mold stem, which is fashioned to the varied shapes discussed above. The windows are then cast into place using the Stycast resin; this resin is dyed black and is optically dense thus avoiding stray light problems associated with light not passing through the observation region. The resin is degassed during casting, and the assembled mold cured for several hours before the cell is released from the mold. The observation windows are flush with the flow path which avoids cavitation problems during flow. A major problem of the observation chamber was leakage of solution at the
175 TO CONTROLVALVE
]
!
OPTICAL PATH.
I,'F t
A
~iS j~
t
A
Fig. I. Observation chamber and ball-mixer of Berger stopped-flow apparatus. The stainless-steel body $2 incorporates the observation chamber (shaded), the optical path, and provides the outer surface of the flow path for reactant A. The stainless-steel element St provides the inner surface for the reactant A flow and also the path for reactant B. Mixing occurs at the base of the ball-mixer shown schematically as the diamond shaped piece,
windows. The pressures involved in the use of this apparatus exploit any weakness in the window to resin seal. Leakage would occur at random times from the manufacture of the observation chamber; a range of lifetimes of a single shot to a month was observed. The leakage, when it occurred, was minor, only affecting the optical path; the solutions were still retained in the apparatus except for the small drop size leak. A redesign of the observation chamber was undertaken retaining the features of the molding technique and providing a secondary seal to prevent solution leakage past the windows. The design shown in Fig. 2 consists of a shallow 'O'-ring groove, at the
SOLUTION FLOW
Fig. 2. Construction detail of the observation chamber/window seal. The window, W, is cast into the resin, P.; the sealing O-ring is compressed by the insert I, which also holds the fiber light guide.
176 outer end of the window, large enough to accommodate a Parker No. 2 - 6 O-ring with 125 ~m of compression. The window itself is retained in place by the Stycast resin. To verify that the 'O'-ring would prevent leakage of solution, a small (No. 80 drill) hole was drilled alongside the window; no solution leakage occurred from this hole which, in comparison to the usual leakage, represented a massive leak. The cell has remained viable for many months of experimental work with this modified arrangement. The new 'O'-ring retainers were designed to accommodate 3 mm diameter fiber optic cable. The polished ends of the fiber are held flush against the window and are in optical contact with it by using an index matching oil. This has the advantage of making the apparatus immune to vibration artifacts associated with the start and the stop of flow, which were observed with the previous design.
Fig. 3. Reaction profile of I mM calcium chloride and 1 mM EGTA at pH 7.0. 20--C. The trace represents the change in transmission of the pH indicator dye bromthymol blue in response to the hydrogen ion released in the action. Increasing transmission is downward. Vertical sensitivity is 0.005 A units/division.
177
To illustrate the performance of the apparatus, the reaction at pH 7.0, 20°C, of 1 mM calcium chloride with 1 mM ethylene glycol bis(aminoethyl ether) tetraacetic acid (EGTA) is shown in Fig. 3. When calcium binds to EGTA, hydrogen ion is released; a 10 mM cacodylate buffer limits the pH change of the solution to 0.1 pH units. The reaction is observed optically using the absorbance change at 615 nm of the indicator dye bromthymoi blue as it responds to the release of the hydrogen ion as the reaction proceeds. Fig. 3 represents the change in transmission for this reaction and it can be seen the reaction is 90% complete within 2 ms (the total absorbance change is 0.024 A units). A clean break, indicated by the arrow, is seen at the point of the stop of flow and the start of the reaction. The use of stopped-flow techniques in the study of chemical and biochemical reactions has been well documented in the literature. In conventional approaches to this technique, (i.e. those in which direct transport of the mixed reagents occurs to the point of observation downstream), the design first reported by Berger in 1968 achieves this transport in the shortest time and remains the fastest of the stopped-flow apparatus. However, wide acceptance of the apparatus has been thwarted, primarily by operating difficulties. This present study demonstrates that a reliable apparatus has been developed and allows reactions to be observed which would fall within the dead-time of other apparatus. The demonstration reaction shown here is part of a larger study of calcium binding to EGTA [3] performed using this apparatus and the Aminco-Morrow. There are several points arising from this work which are worth mentioning in the context of this report. Firstly, not a single failure of the observation chamber occurred. Secondly, it was observed that for reactions having half lives of greater than 7 ms. (i.e., well above the Aminco-Morrow dead-time), good agreement was obtained between the Aminco-Morrow and the Berger stopped-flow apparatus. Thirdly, as can be seen from Fig. 3, a clean break between the flow and stopped-flow condition is observed with little rounding observable. This is in contrast to the observations made by Berger et al. [4] on hemoglobin, where a rounding of this transition was seen. The authors concluded this was due to the oxygen-binding process, and considerable controversy arose about this point. The observation reported here shows that a sharp transition on this timescale is possible. Finally, the reaction is complete within 2 ms, which is shorter than the dead-time of other stopped-flow devices of which the Aminco-Morrow is an example. The rate of reaction for the uptake and calcium by EGTA was found to be 2 . 1 0 6 M - I . s-1 at 25°C; the details of this reaction are complex and are beyond the scope of this paper.
References 1 Berger, R.L., Balko, B., Borcherdt, W. and Friauf, W. (1968) Rev. Sci. Inst. 39, 486-492 2 Owens, G.D., Taylor, R.W., Ridley, T.Y. and Margerum, D.W. (1980) Anal Chem. 52, 130-138 3 Smith, P.D., Liesegang, G.W., Berger, R.L., Czerlinski, G. and Podolsky, R.J., Submitted for publication 4 Berger, R.L., Antonini, E., Brunori, M., Wyman, J. and Rossi-Fanelli, A. (1967) J. Biol. Chem. 242, 4841-4843