1. Take the cell assembly and the loose parts referred to in step 14 from the oven and allow them to cool down (this should take about 30-60 min). Close all the valves. Cover the mouth of the needle of syringe S1 and the orifice on valve V7 with parafilm. (The tubing soldered to the needle for syringe S2 will be connected later.)
2. Place the Buna M O-rings on the piston and screw the metal rod onto it. Mark the rod with a fine pen at points corresponding to the desired initial and final volumes of the cell, Vi and Vf (see also steps 3,5, and 9). Push the piston into the cell, from the back, so that the rear end of the piston stays about 5 mm below the bed of the Teflon O-ring. Put the Teflon O-ring in place. Allow the rod to go through the small nut with Teflon O-rings and screw on the rear end of the cell, with the cell held tightly on a bench vice (do not bend the tubing for connection to the backpressure fluid too close to the soldered joint). Adjust the torque on the small nut to avoid leaks of the back-pressure fluid, while allowing the rod to move with the piston.
3. Connect the back of the cell to the back-pressure fluid line via valve V9. Open V9. Pressurize the back-pressure fluid with a manual pump to push the piston forward to a position corresponding to mark on the rod. Keep valve V9 open.
4. Hold the cell in the vice so that its front end is slightly tilted upward. Introduce the enzyme, all substrates but one, any additional reaction mixture components (e.g., salt hydrate pairs), and a stirring bar. Put the Teflon O-ring in place. Put the polyacetal washer in position on the front end screw of the cell where it should have a tight fit and let the sapphire window rest on it. Screw on the front end screw. Do not connect syringe S2 to valve V7. Do not connect the KF line of valve V8 to the Karl Fischer apparatus. Do not connect syringe S1 to valve ADM. Place the cell assembly in a thermostatted bath. Connect the cell to the solvent admission line via valve V5.
5. Open valves V1, V2, and V4 and pressurize the solvent gas to a pressure about 50 bar higher than the required reaction pressure. Make sure valve ADM is in position 1 in Fig. 3. Open valve V5 gently, and admit the liquefied solvent up to valve V6. Close valve V5. Open valve V6 gently to admit a small amount of solvent into the cell. Keep valve V6 open. Open valve V5 and admit the solvent until the reading in the pressure transducer PT1 drops to the vapor pressure of the gas solvent. (The piston tends to go slightly backward.) Close valve V5. Immediately raise the pressure of the back-pressure fluid to keep the piston in its original position (see Note 1). Pressurize the solvent gas circuit, to a pressure of about 50 bar higher than the required reaction pressure. Open valve V5 to admit more solvent into the cell until PT1 gives a reading of about 20 bar higher than that of the back-pressure fluid on PT2. Once again, check the mark on the rod. Repeat the process until the cell is filled with the solvent at the desired pressure and volume, as indicated by the pressure transducer PT2 and the mark V, on the metal rod. Close valve V6. Start stirring.
6. Check for leaks, in particular for gas bubbles from parts immersed in the thermostated bath and for a pressure drop on PT2 (care should be taken to avoid overtightening the nut through which the rod is allowed to move). Final adjustments should be made once the temperature of the cell has stabilized, which takes place about 1 h after starting the admission of the solvent. Valve V9 is kept open to monitor the pressure via PT2. The reaction mixture should be allowed to equilibrate with water before taking samples; in the case of aw control via salt hydrate pairs, the rate of water transfer depends on the salts chosen.
7. To take a sample for Karl Fischer titration, connect valve V7 to the nitrogen line. Adjust the pressure regulator of the nitrogen bottle to a relative pressure of 0.5 bar. Open valves V7 and V8-to-KF. Allow the nitrogen to flow through the lines and into the atmosphere for about 10 min to eliminate any humidity in the system. With nitrogen still flowing, immerse the KF line in the solution of the Karl Fischer apparatus and allow the nitrogen to bubble through the solution until a constant drift is reached (the drift is the amount of water (in ^g) that enters the titration chamber per minute). Register the drift (the average drift if the values fluctuate slightly) — drift 1. Stop stirring the reaction mixture to allow solids to settle down. While this takes place, close valves V7, SAMP-to-V7, and V8-to-KF and wait until the drift reaches a constant value — drift 2. Also see Notes 2-4.
8. Taking a sample causes a pressure drop in the cell. The magnitude of the drop depends on the volume of the sampling loop and the compressibility of the reaction mixture. When operating sufficiently close to the critical point of the latter and for loops with a volume of 1-2% of the volume of the cell, the pressure drops should not exceed 20 bar. Thus, increase the pressure of the back-pressure fluid, P1, by about 20 bar (AP) and open valve SAMP-to-V8. If AP was chosen correctly, the pressure should recover to the value of P1; if not, do the necessary readjustment. Close valve SAMP-to-V8. Press the start button on the Karl Fischer titrator and slowly open valve V8-to-KF so that the content of the sampling loop is discharged directly into the titration chamber. Once the gaseous solvent stops bubbling through the solution, open valves SAMP-to-V7 and V7 to rinse the expansion zone with nitrogen. Register the time interval between the opening of valve V7 and the end point of the titration — At. The drift will eventually stabilize (drift 1). In the second titration, drift 1 is usually, but not necessarily, similar to that determined as described in step 7. Further water titrations are done in the same way. Use the adequate AP and keep stirring off.
9. To initiate the reaction, close valves V7 and V8 and resume stirring. Prepare an adequate mixture of substrates — the missing substrate plus appropriate amounts of other substrate(s) that have already been added to the solvent (see Note 5). About a fivefold excess of this mixture over the volume of the loop of valve ADM should be prepared. Make sure valves V6 and SAMP are closed and that the pressure reading in PT1 exceeds that in PT2 by at least 50 bar. Open and immediately close valve V5; the latter condition should remain. Connect syringe S1 to valve ADM. Introduce the mixture in syringe S1, wash, and then fill up the loop with it. Turn valve ADM to position 2. Open valve V6 slowly, thereby releasing the contents of the loop into the cell with solvent. This marks the start of reaction. As before, alternately admit solvent and back-pressure fluid so as to reach the desired pressure and volume — final volume of the cell, mark Vf on the rod — at the selected temperature (see Note 1). Keep valve ADM in position 2 (the solvent in the loop is released at the end of the experiment). Wash the substrate admission lines (this is particularly important in the case of solid substrates that may plug the lines once the liquid in which they were dissolved evaporates).
10. Close valves V6, V5, V4, V2, and V1 while keeping valve V9 open. Periodically, stop stirring, wait until all solids settle down, and take a sample to follow the reaction. To do this, connect syringe S2 to valve V7. Increase the back-pressure fluid pressure by AP as described in step 8, open valve SAMP-to-V8, immediately make any necessary pressure adjustments, and close valve SAMP-to-V8. Take a volumetric flask with some of the collecting solvent and immerse the
GC/HPLC line in it. By slowly opening valve V8-to-analysis, allow the gaseous solvent to bubble through the collecting solvent (it is important that the gas be released very gently). Once the gas stops bubbling, introduce an appropriate amount of the collecting solvent into the syringe S2, open valve V7, and wash the contents of the sampling loop into the volumetric flask. Disconnect syringe S2, fill it up with air, reconnect it, and push the collecting solvent that remained in the lines into the flask. Add the solvent directly to the flask to reach the marked volume. Close valves V7 and V8. Typically, six samples are taken for analysis. The volume of collecting solvent chosen must be sufficient for quantitative recovery of the content of the loop.
11. At the end of experiment (see Note 6), with valve V9 open, depressurize the back-pressure fluid down to atmospheric pressure. The piston will move backward and a meniscus that indicates the presence of vapor and liquid phases should form. Close valve V9. Disconnect the cell assembly from valves V5 and V9 and take it out of the thermostated bath.
12. To avoid frequent disassembling and reassembling of connections to the cell, the latter is emptied through valve V7. In a hood, disconnect syringe S2, open valve V7, and then slowly open valve SAMP-to-V7 to release the solvent. After that, unscrew the nut with Teflon ferrules that holds the metal rod so that it will be possible to unscrew the rear end screw of the cell. Remove this screw and the Teflon O-ring, which may or may not be reutilized, depending on the pressure at which the experiment was performed. (O-rings are often too deformed after being extruded at 300 bar and hence discarded.) Unscrew the front end-screw of the cell, remove the Teflon O-ring (the same consideration applies), the polyacetal washer, and the sapphire window. Use the Teflon bar referred to in Subheading 2.4., item 4 to push the piston backward, out of the cell. Remove the Buna M O-rings carefully with a stylus and discard them.
13. Wash the interior of the cell, all the lines of the cell assembly, loops, piston, O-rings, washer, and sapphire window, first with water and then with acetone, using a syringe. Make sure no solids remain in the tubing. Blow out the remaining acetone, first with a syringe, then with nitrogen (compressed air usually has a higher level of humidity).
14. Place the cell assembly with all valves open and the loose parts — sapphire window, polyacetal washer, piston, Teflon O-rings, Buna M O-rings, stir bar, tubing, and needle for connecting syringe S2, syringe S1 — in an oven at 60°C and leave them there to dry overnight. Make sure the liquid substrates are stored over molecular sieves.
15. It is important that there are no leaks; hence, the apparatus should be pressure tested with nitrogen after replacing parts.
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