Standard Operating Procedures for Laboratory Operations in the Bell Research Group

University of Nevada, Reno

Revised 9/09

Bell Research Group

Standard Operating Procedures

TABLE OF CONTENTS Page Parr Hydrogenator ............................................................................. 1 Atmospheric Hydrogenator ............................................................... 5 Rotary Evaporator .............................................................................. 8 Tetrahydrofuran (THF) Still ............................................................ 10 Ozonizer .................................................................................... 12

Vacuum Manifolds and Traps ......................................................... 14 High Vacuum Pumps ....................................................................... 16 Gas Cylinders and Regulators ......................................................... 18 Fluorescence Spectrometer .............................................................. 19 High-Pressure Liquid Chromatography (HPLC) Instrument .......... 23 UV-Visible Spectrophotomer .......................................................... 26 FT-IR Spectrophotomer ................................................................... 27 Solvent systems ............................................................................... 29

ii

Bell Research Group

Standard Operating Procedures, page 1

PARR HYDROGENATOR

Procedure A. Initial reaction bottle set up: 1. Put all reaction materials in the glass reaction bottle (do not fill the bottle more than one third of its capacity). 2. Place the glass reaction bottle in the center of the cage and screw down the green stopper assembly as tight as possible. 3. Thread the 4 (four) thumbscrews to reassemble the cage and tighten them. B. Reservoir tank H2 fill procedure: 1. Make sure all valves (A, B, C, D, E, F, G, H, I and J in the figure) are closed and the Parr rocking motor is off. 2. Open valve A a few turns (the main valve on the H2 tank) and adjust the regulator pressure to the appropriate value (40-50 psi) with valve B. 3. Open valve C a few turns and slowly open valve H. Make sure that the H2 gas pressure on the right hand gauge increases. Allow the system to equilibrate briefly (ca. 30 sec.) and close valve H, then valves C, B and A. C. Evacuation and filling of the reaction bottle: 1. Make sure all valves (A, B, C, D, E, F, G, H, I and J in the figure) are closed and the Parr rocking motor is off. 2. Turn on the water faucet aspirator and evacuate the line and water trap by closing the air valve on top of the Erlenmeyer flask. 3. Open valve I a few turns and allow the reaction bottle to be evacuated (about 3-5 minutes required). 4. With the aspirator still running, close valve I. 5. Very slowly open valve J by about 1/4 turn to reach a pressure of no more than 5 psi on the left-hand gauge, then close valve J. 6. Repeat steps 3, 4 and 5 at least two more times. Caution: Incomplete replacement of air by hydrogen could cause the reaction bottle to explode during operation. 7. Open valve J slowly a few turns. The left-hand pressure gauge should increase while the right hand gauge should decrease. Wait until they come to the same value and then close valve J. 8. Shut off the aspirator by opening the air valve on top of the Erlenmeyer flask first and then turning off the water faucet. 9. Now you are ready to turn on the rocking motor to begin reaction. Record the initial hydrogen pressure in your notebook.

Bell Research Group

Standard Operating Procedures, page 2

D. Refilling procedures: 1. For reservoir tank refilling proceed with all steps in section B (Reservoir tank H2 fill procedure). 2. For reaction bottle refilling proceed with step 7 only in section C (Evacuation and filling of the reaction bottle). E. Reaction bottle removal after completion of the reaction: 1. Make sure all valves (A, B, C, D, E, F, G, H, I and J in the figure) are closed and the Parr rocking motor is off. 2. Turn on the water faucet aspirator and evacuate the line and water trap by closing the air valve on top of the Erlenmeyer flask. 3. Open valve I very slowly (1/8 of a turn at the beginning) to allow the reaction bottle to be evacuated at a speed of about 2 psi per 10 seconds on the left hand pressure gauge. (Note: The right hand pressure gauge shouldnt change. If it does, you havent closed valve J, so please close it now.) 4. After the pressure on the left-hand gauge drops to zero, open valve I a few turns and wait for the system to be fully evacuated (about 4 minutes required). 5. Shut off the aspirator by opening the air valve on top of the Erlenmeyer flask first and then turning off the water faucet. 6. Carefully remove the 4 thumbscrews from the cage, remove the screen, unscrew the green stopper assembly, and remove the reaction bottle. 7. Make sure all valves (A, B, C, D, E, F, G, H, I and J in the figure) are closed, the green stopper is clean, and the cage is reassembled. Safety precautions 1. Wear safety goggles! 2. Hydrogen tank must be secured by means of a tank strap or cradle. 3. Vacuum tubing for water aspirator should be secured with hose clamps; make sure there are no leaks and the aspirator is working. 4. Water aspirator exit hose should be safely located in a drain keep debris away from drain to avoid obstruction! 5. Use at least 3 evacuation/filling cycles to insure complete removal of air from the glass reaction bottle. 6. Avoid hydrogen leaks by keeping all connections tight; report immediately any loose connections, loose valves or breaks in the hydrogen line. 7. Make sure reaction cage is securely fastened and close hood door during Parr operation; inspect reaction bottle carefully for defects (e.g., cracks) before using it. 8. Keep all debris, clothing, hair and fingers away from moving parts. 9. Do not allow air to enter the H2 reservoir tank (e.g., via valve J during step 5 of section H at end of reaction). If this happens, immediately report the problem to the

Bell Research Group

Standard Operating Procedures, page 3

person responsible for maintaining the Parr apparatus. Hazards 1. Explosion of glass reaction due to overpressure (>50 psi) or defects (e.g., star cracks); do not use a defective reaction bottle. 2. Hydrogen explosion caused by residual oxygen in the reaction bottle or reservoir. 3. Damage to hand or head caused by catching fingers or hair in moving parts. 4. Violent rocketing of hydrogen tank if it falls over and top is severed. 5. Flooding caused by aspirator exit hose leaving drain or by clogging of drain by debris.

Bell Research Group

Standard Operating Procedures, page 4

Figure 1: Hydrogenator and its schematic diagram.

Bell Research Group

Standard Operating Procedures, page 5

ATMOSPHERIC HYDROGENATOR Procedure 1. Put all reaction materials and a magnetic stirring bar into the reaction flask. 2. Attach the reaction flask to the hydrogenator. 3. Make sure all valves (A, B, C, D, E, F, G, H, I and J) are closed and turn on the water aspirator faucet and evacuate the line and water trap by closing the air valve on top of the Erlenmeyer flask. 4. Start stirring the contents of the reaction flask and open the 3-way valve connecting the hydrogenator to the aspirator. 5. Make sure that the H2 gas-measuring cylinder is completely filled with water. If air is present in the cylinder, then substitute the air with water by slowly opening the valve connecting the cylinder to the hydrogenator under vacuum. Close this valve quickly as soon as the cylinder is filled with water; avoid sucking water into the hydrogenator manifold. 6. Continue evacuation of the hydrogenator and reaction flask for about 3 minutes. 7. Open valve A a few turns (the main valve on the H2 tank) and adjust the regulator pressure to the appropriate value (about 10 psi) with valve B (see figure [p 3a] in Parr Standard Operating Procedure [SOP]). 8. Open valve C a few turns, as well as valves F and G, connecting the bubbler to the hydrogen tank (see figure [p 3a] in Parr SOP). Make sure that H2 gas is bubbling vigorously through the bubbler. 9. Stop the stirring in the reaction flask and rotate the 3-way valve, which connects the hydrogenator to the aspirator, by 180 to connect the hydrogenator with the H2 gas line. 10. Carefully open the valve connecting the bubbler with the hydrogenator (between the bubbler and the 3-way valve). (Caution: If this valve is opened too fast, the oil from the bubbler can be sucked into the system, and air can get into the hydrogenator.) 11. After the system is filled with H2 gas, apply vacuum to the hydrogenator by rotating the 3-way valve by 180. 12. Resume stirring in the reaction flask and close the valve connecting the bubbler with the hydrogenator. 13. Repeat steps 6, 9, 10, 11 and 12 at least two more times. 14. Fill the hydrogenator with H2 gas (follow steps 6, 9 and 10). 15. Fill the measuring cylinder with H2 gas by opening the valve connecting the cylinder to the hydrogenator. Close this valve when the cylinder is filled with required amount of H2 gas. 16. Isolate the whole system by rotating the 3-way valve 90.

Bell Research Group

Standard Operating Procedures, page 6

17. Close valves A, B, C, F and G and the valve connecting the bubbler to the hydrogenator (see figure [p 3a] in Parr SOP). 18. Shut off the aspirator by opening the air valve on top of the Erlenmeyer flask first and then turning off the water faucet. 19. Open the valve connecting the measuring cylinder with the hydrogenator and resume stirring. 20. After completion of the reaction, turn on the aspirator (step 3), and then evacuate the whole system for about 5 minutes (step 4). 21. With the aspirator still running, fill the system with air by opening the air valve on top of the Erlenmeyer flask. 22. Turn off the water aspirator faucet and detach the reaction flask. 23. Make sure all valves are closed and the flask adapter and connecting tubing are clean. Safety precautions 1. Wear safety goggles! 2. Hydrogen tank must be secured by means of a tank strap or cradle. 3. Vacuum tubing for aspirator should be secured with hose clamps. 4. Water aspirator exit hose should be safely located in a drain keep debris away from drain to avoid obstruction. 5. Use at least 3 evacuation/filling cycles to insure complete removal of air from the apparatus and reaction flask. 6. Avoid hydrogen leaks by keeping all connections tight. 7. Close hood door during operation of hydrogenator. 8. Avoid spark sources in hood in case of H2 buildup. Hazards 1. Hydrogen explosion caused by residual oxygen in the reaction flask or hydrogenation apparatus. 2. Violent rocketing of hydrogen tank if it falls over and top is severed. 3. Flooding caused by aspirator exit hose leaving drain or by clogging of drain by debris.

Bell Research Group

Standard Operating Procedures, page 7

Figure 2: Schematic diagram showing atmospheric hydrogenation setup.

Bell Research Group

Standard Operating Procedures, page 8

ROTARY EVAPORATOR Procedure 1. Attach a suitable splash-head between combi clip, A, and the flask. Use a Keck clip to fasten the flask to the splash-head. 2. Bath immersion angle can be adjusted with the help of screw B. 3. Place an ice bath under the receiving flask (optional, but preferred if solvent bp <80C at 760 mm). 4. Turn on the Teflon diaphragm vacuum pump or water aspirator (maximum water flow). 5. Circulate water through the condenser. 6. Close stopcock C. 7. Turn on the power switch and set the speed of rotation to approx. 240 rpm. 8. Allow the flask to first spin in air (not immersed in heating bath); support it with your hand if it is very heavy. 9. If bumping of the liquid in the evaporation flask is observed, increase the pressure (reduce the vacuum) by briefly admitting some air through stopcock C. 10. Repeat step 9 till liquid stops bumping. 11. Immerse the flask in the water bath with the help of the quick action jack D. Turn on the heating switch and set the knob to the desired temperature. Repeat step 9 if necessary. 12. After evaporation of solvent, raise the flask from the water bath using D. Turn off the heating switch. 13. Stop rotation, turn off the power switch, open stopcock C to air, turn off the water aspirator and cooling water. Then disconnect the splash head and the flask. 14. Discard the solvent collected in the receiving flask into the correct container (halogenated or nonhalogenated solvent). Safety precautions 1. Wear goggles or safety glasses! 2. Water hoses should be secured with hope clamps or wire. 3. Cracked or old hoses should be replaced. 4. Vacuum hoses should be kept as short as possible and should have an internal diameter of at least 6 mm. 5. Insure that cooling water and aspirator water exit hoses are safely located in sinks and that drains are not obstructed by debris, such as Kimwipes, pipette bulbs, stoppers, glassware, etc. 6. Inspect evaporating flask carefully, especially for star cracks; do not use flasks with any defects. It is preferable to use a pear-shaped heavy-walled, plastic-coated flask. Do not use flat-bottomed (Erlenmeyer) or thin-walled flasks!

Bell Research Group

Standard Operating Procedures, page 9

Hazards 1. Flooding caused by leaky or loose water hoses or hoses not properly located in sinks; caution, the force of water flow can cause a hose to eject from the sink! 2. Implosion of glass parts (condenser, receiving flask or evaporation flask), causing lacerations and eye injury.

Figure 3: Rotary evaporator.

Bell Research Group

Standard Operating Procedures, page 10

TETRAHYDROFURAN (THF) STILL Procedures A. Charging the still: 1. The 3 L round-bottomed flask should be clean and dried in an oven. 2. Pour 2.5 L of anhydrous THF (hplc grade) into the 3 L round-bottomed flask. 3. Cut 50 g of sodium metal (immersed in hexane) into peanut-size pieces, exposing as much new metal surface as possible. 4. Add the cut sodium metal pieces to the THF in the flask, connect the condenser head, and heat the contents under reflux for 15 minutes. 5. Add 10 g of benzophenone to the warm solvent-sodium metal mixture and replace the condenser head. 6. Turn on the nitrogen valve and adjust the flow rate to about 10 bubbles per minute through the vent bubbler. 7. Resume heating under reflux. 8. The following colors indicate the degree of dryness of the solvent in the roundbottomed flask: Purple (DRY) > Blue > Green > Yellow (WET)

The solution should turn blue/purple after 1-12 hours of reflux; if not, add another 25-30 g of cut sodium metal and resume heating under reflux. B. Withdrawing anhydrous THF: 1. Turn up the Variac dial to 70 V and turn the stopcock to trap the refluxing solvent in the condenser head. 2. When enough THF collects in the condenser head, insert the needle of an oven-dry hypodermic syringe through the rubber septum and the stopcock. 3. Flush the syringe with nitrogen from the condenser head 3-4 times before withdrawing the dry THF. 4. Release the excess dry THF back to the round-bottomed flask SLOWLY by reopening the stopcock closed in step 1 above. 5. Increase the nitrogen pressure to about 20 bubbles per minute as the excess dry solvent drains from the condenser head into the flask to avoid generating a partial vacuum in the still. 6. Turn down the voltage control dial back to about 30 V or to ZERO when there is no immediate need for dry solvents. 7. REMEMBER to turn down the nitrogen pressure to about 5 bubbles per minute before you leave the still.

Bell Research Group

Standard Operating Procedures, page 11

Safety precautions 1. Wear goggles or safety glasses. 2. When THF is not needed, the voltage control dial on the Variac should be set to about 30 V to keep the THF warm. DO NOT SET THE DIAL TO ZERO if the stills are in frequent use. 3. At no time should the volume of the THF in the still be allowed to fall low enough to expose the sodium metal pieces. 4. Avoid the use of water during the cleaning of the stills and disposal of still residues. 5. Do not allow THF (or other flammable solvent) to spill into the heating mantle. If this happens, turn off the Variac immediately to allow the heating mantle to cool. Remove heating mantle from apparatus, allow to dry thoroughly and test before using again. 6. Dispose of excess sodium, and any paper towels or Kimwipes used to cut sodium, by adding to a beaker of isopropanol in a fume hood (Caution: bubbling of H2 gas). After the reaction subsides, add a few drops of water to complete the reaction. 7. Water hoses should be attached to the faucet, condenser and water flow monitor with hose clamps. Hazards 1. Fire and/or explosion caused by burning of THF or hydrogen produced by reaction of sodium with water. 2. Flooding caused by leaky or loose water hoses or by exit hose not properly located in a sink. 3. Burns caused by contact of sodium metal (or sodium hydroxide on surface) with the skin. 4. Poisoning caused by inhalation of THF or hexane.

Bell Research Group

Standard Operating Procedures, page 12

OZONIZER Procedure 1. Set the oxygen tank regulator to approximately 15 psi and open the small valve on the regulator to begin oxygen flow through the ozonizer. 2. Turn the switch to STANDBY, making sure O2 is flowing through the instrument (reading in flow meter is > 0 SCFH). 3. Attach the exit tube to the Pasteur pipet and begin bubbling O2 into the cooled solution. 4. Turn the voltage dial to 85 volts. 5. Close the fume hood doors as much as possible and turn the switch to RUN. Adjust the flow meter to 8 SCFH. CLOSE FUME HOOD DOORS!!! Ozone is highly toxic. 6. When the reaction is complete, usually indicated by the appearance of a bluish color in the reaction mixture, turn the switch to STANDBY and the voltage dial back to 0 volts. Allow O2 to flow for 5 minutes to cool the instrument. 7. Turn off O2 at the main tank valve and detach ozone exit tube from Pasteur pipet. 8. Purge the reaction flask by bubbling nitrogen gas through the reaction mixture. 9. Turn ozonizer switch to OFF. 10. Turn off N2. Safety precautions 1. Wear goggles and gloves. 2. Oxygen and nitrogen tanks must be secured by means of tank straps or cradles. 3. Keep hood doors closed during ozonization reaction. 4. Do not exceed 85 volts on the voltage dial. 5. Do not switch ozonizer to Standby or Run unless O2 is flowing through the instrument. 6. Do not allow dry ice or dry ice/acetone mixture to contact skin. 7. Purge ozone with N2 flow at end of reaction and insure ozonide is destroyed before removing solvents from the reaction mixture. Hazards 1. Fire or explosion caused by ignition of oxygen mixture with flammable gases (e.g., H2 or solvent vapors). 2. Violent rocketing of oxygen or nitrogen tank if it falls over and top is severed. 3. Inhalation of ozone (toxic!). 4. Skin freezing and tearing upon contact with dry ice or very cold surfaces. 5. Explosion of solid ozonide after solvent evaporation. 6. Overheating of ozonizer if operated without cooling action provided by O2 flow.

Bell Research Group

Standard Operating Procedures, page 13

From ozonizer Thermometer Pasteur pipet

Dewar

Dry ice/ acetone bath

Magnetic stirrer

Figure 4: Ozonizer.

Bell Research Group

Standard Operating Procedures, page 14

VACUUM MANIFOLDS AND TRAPS Procedures A. Installation: 1. The vacuum pump is connected to the traps leading to the vacuum manifold by means of heavy-walled rubber or Tygon vacuum tubing; secure tubing at both ends with hose clamps. 2. Use vacuum grease (Apiezon) on glass joints and stopcocks; use metal clamps to hold ball-and-socket joints together. 3. Test the vacuum manifold, traps and tubing connections for leaks by measuring the pressure at the end of the system (most distant from the vacuum pump) by means of a mercury-filled gauge (McCleod gauge); if the measured pressure is much higher than that measured directly at the pump (not connected to traps and manifold) then there is a leak. 4. If there is a leak, try regreasing joints and stopcocks and tightening hose clamps. 5. If leak persists, check individual components by isolating others from the system and remeasuring pressure at various points. 6. Support Dewar containers around the traps by means of clamps, rings or platforms underneath them. B. Operation: 1. To start up the system, close all stopcocks on the manifold and turn on the vacuum pump. 2. Cool the traps by means of dry ice/acetone (-78C) or liquid nitrogen (-196C). Liquid nitrogen is more hazardous (see below) but dry ice/acetone less efficiently condenses vapors. Use liquid nitrogen if there is any possibility of pump contamination by reactive gases, such as ammonia, HCl, etc. If dry ice is used, do not fill trap with acetone first (it will boil over when dry ice is added). Instead, alternately add small amounts of dry ice and acetone until the trap is fitted with dry ice that is just covered by acetone. 3. Cover traps with cloth or glass wool to reduce loss of coolant. Do not operate pump without coolant in traps. 4. Turn stopcocks slowly to avoid sudden pressure changes and to avoid breaking them off by applying too much force. 5. To shut down the system, turn off the vacuum pump, then open a stopcock to release the vacuum and vent the system to the air. 6. Withdraw the Dewars from the traps and allow them to warm to room temperature (liquid oxygen hazard, see below). 7. Clean the traps by washing them with acetone or other solvents, followed by soap

Bell Research Group

Standard Operating Procedures, page 15

and water, if necessary. 8. Dry the traps at room temperature overnight or in an oven. 9. Regrease clean traps before re-use. Safety precautions 1. Wear goggles or safety glasses. 2. Keep hood doors closed while vacuum operations are conducted. 3. Avoid contact of skin with dry ice/acetone, liquid nitrogen or cold surfaces. 4. Do not operate vacuum system if there is a leak, especially if liquid nitrogen is used as the coolant; this could cause liquid oxygen to condense in the traps, causing danger of explosion. If this bluish liquid is seen, turn off the vacuum pump, vent the vacuum system, evacuate the laboratory and notify Prof. Bell and/or safety personnel. Hazards 1. Implosion of evacuated glassware causing injury, including lacerations and blindness. 2. Glass cuts on hands and fingers caused by breakage of stopcocks. 3. Damage to skin caused by contact with coolants or cold surfaces. 4. Explosive reaction of liquid oxygen with combustible materials in traps cooled by liquid nitrogen.

Bell Research Group

Standard Operating Procedures, page 16

HIGH VACUUM PUMPS Procedures A. Installation: 1. Insure the following before pump installation. a) Pump is clean and not leaking oil. b) Belt between motor and pump chamber is tight and in good condition. c) Belt cover (metal or plastic) is installed securely over belt. d) Oil is clean (yellow or light brown) and chamber is filled to proper level (above line in glass window); do not overfill! e) Power cord (and switch) are in good working condition; replace cord if insulation or plug is damaged. 2. Position pump where needed, preferably in a metal oil pan and where it will not be exposed to water. On/off switch should be operable without reaching over pump. 3. Fasten heavy-walled rubber or Tygon tubing to the pump inlet tube by means of a hose clamp. B. Operation: 1. Close all stopcocks on the manifold attached to the pump. 2. Turn on vacuum pump. 3. See SOP on gas/vacuum manifold for other procedures. 4. Before turning pump off, vent system (release vacuum) to avoid pump oil being sucked into manifold or other apparatus attached to pump. 5. Periodically (at least monthly), check items a)-d) listed under A. Installation. Carefully monitor condition of pump oil and change it if you believe that it has been contaminated by organic solvents or reactive vapors, such as CH2Cl2, ether, HCl, amines, etc. C. Changing pump oil: 1. Change pump oil in case of contamination or after daily use of pump for 2 months or more. 2. Disconnect pump from vacuum system. 3. Elevate pump and position it so the used oil will drain into a waste container. 4. Remove fill-cap, usually a large round disk screwed into a hole on the top of the pump. 5. Open stopcock beneath fill window to drain used oil into waste container; the pump should be tilted to drain the oil as completely as possible. 6. In case of chemical contamination only, the pump can be rinsed with CH2Cl2, followed by hexane and new pump oil. Consult Prof. Bell before using this

Bell Research Group

Standard Operating Procedures, page 17

procedure! If solvents remain in the pump during operation, it could be severely damaged. This procedure should be followed only under unusual circumstances. 7. Refill pump chamber to proper level, as observed in the glass window; do not overfill. 8. If previous contamination is suspected, run pump with new oil briefly (10-30 minutes), then drain and replace with new oil, repeating steps 3-7. 9. Collect all waste oil in a single container (do not mix with solvents or other chemicals) and contact EH&S for pick-up. Safety precautions 1. Wear goggles when working with pumps and vacuums. 2. Insure that belt cover remains fastened securely in place; keep loose clothing, long hair and fingers away from belt, wheels and other moving parts. 3. Turn off pump and check problem if it fails to start when power is turned on, if sparking occurs, if the oil becomes viscous, black or heterogeneous, if it becomes unusually noisy, or if a leak develops in the system it is attached to. Hazards 1. Implosion of evacuated glassware can cause serious injury, especially lacerations and blindness. 2. Loose clothing, long hair or fingers can get caught in vacuum pump, causing serious injury. 3. Faulty power cords and switches cause danger of electrical shock.

Bell Research Group

Standard Operating Procedures, page 18

GAS CYLINDERS AND REGULATORS Procedures A. Transport and installation: 1. If a regulator is attached to the top of the cylinder, turn off the main tank valve, remove the regulator assembly with a wrench, and screw a metal cap on the top of the tank. 2. Secure the cylinder to a tank cart with the chain and move it carefully to the new location. 3. Secure the cylinder at the new site against a wall or bench by means of a chain, tank strap or cradle. B. Regulator: 1. Use the correct regulator for the type of gas cylinder; thread size and type (right or left) is standardized for each type of gas to avoid mixing of reactive gases (e.g., H2 and O2). An exit valve should be attached to the regulator before installation on the gas cylinder. 2. Unscrew the metal cap on the gas cylinder. 3. Apply a piece of Teflon tape to the threads of the regulator and use an open-end or crescent wrench (not a pipe wrench!) to secure it to the main cylinder valve, holding the gauges on the regulator upright. 4. Close the small regulator exit valve and turn the regulator off by rotating the main handle fully counterclockwise (until no more resistance to turning is felt). 5. Open the main cylinder valve and check for leaks by means of a soap solution; tighten further if a leak is detected. Safety precautions 1. Wear safety glasses or goggles. 2. Do not move a cylinder with a regulator attached or without a metal cap installed over the main valve. 3. Never open the main valve unless a regulator is attached. 4. Always secure a gas cylinder (even if empty!) to a wall, bench or tank cart. Never lay it on its side or allow it to fall to the floor. Hazards 1. Violent rocketing of gas cylinder if main valve is severed from top. 2. Injury to arm or leg caused by falling cylinder.

Bell Research Group

Standard Operating Procedures, page 19

FLUORESCENCE SPECTROMETER Procedure A. Turning on the spectrometer: 1. Make sure the computer has been turned off. If it is on, turn it off with the START button in the lower left-hand corner of screen, and reply yes to shut down the computer. 2. On the lower box (LPS-220) to the right of the computer, press the POWER button so it lights up; the LED should give a reading. 3. Press the IGNITE button, holding it down until you see the LED value jump; this is usually accompanied by a small sound (like an electrical discharge) from the photomultiplier tube. 4. Note in the logbook your name, date and time the lamp is turned on. 5. If not on already, turn on the switch on the power strip to the left of the computer. 6. Turn the computer on (password: receptor). 7. Press the POWER button on the middle box (MD-5020) to the right of the computer, so that the button lights up. Check the reading on the photomultiplier detector to the far right of the spectrometer (810). It should read 1050. If there is no reading, turn it on by flicking the switch on the bottom. If the reading is not 1050 after 15 seconds, carefully adjust the grey knob to set it to 1050. 8. Wait five minutes for lamp power supply to stabilize. If not already set, on the LPS220 unit set the DISPLAY to WATTS. It should read 75. If not, carefully adjust the CURRENT knob to set it to 75. 9. The instrument is now ready to use. B. Sample preparation: 1. Prepare a solution of the sample in a suitable solvent at a concentration of 10-9 to 107 M. 2. Add the solution to the cuvette, which should be filled to a height of about 2.5 cm (ca. 2/3 of the cuvette height). C. General operation: 1. For test reproducibility of results, check the slit widths. Thus, close them (there are three) by turning each one clockwise until you meet some resistance. TO NOT FORCE THEM you will break them. Then open the desired amount anticlockwise. Usually each one is opened one full rotation, which is 2 nm (2-4 nm is recommended). 2. When you open the FELIX software and tell it to ACQUIRE an emission scan, it will calibrate the spectrometer. Note the dials rotating at this time. You will see

Bell Research Group

Standard Operating Procedures, page 20

3.

4.

5.

6.

7. 8. 9. 10. 11.

that the one at the back says 273. Change the value on the computer screen from 274 to 273. DO NOT ROTATE THE DIAL FROM 273 to 274. Note the step size, integration time and averages. Changing them will change the quality of your spectrum obtained. For consistency you should use the same values every time. YOU can decide your own values (recommendation: step size = 0.5; integration = 0.1; average = 1). Before acquiring the emission scan, it is suggested that you obtain a UV-spectrum to find the maxima where you would like to excite the sample in order to obtain the emission spectrum. Set the excitation wavelength, as obtained from step 4, and scan the emission wavelength detection from low to high, starting at 10 nm greater than the excitation wavelength. If the emission spectrum flattens out at the top of a peak, i.e., if the number of 6 counts exceed ca. 3 10 , the sample is too concentrated. Stop scanning and use a more dilute sample (preferable) or use a smaller slit width. After obtaining the emission spectrum, find the maxima using the data pointer (you will see a gun target, or toggle it on with DISPLAY/DATA POINTER). Then run the excitation scan, setting the emission wavelength, as from step 7, and scan up to a wavelength that is 10 nm lower in value. The maxima in the excitation spectrum should be similar to that from step 4. If not, you should reacquire the emission spectrum using this new value. IF YOU SPILL ANYTHING IN THE SAMPLE CHAMBER CLEAN IT UP! Clean them thoroughly with the same solvent used for the sample solution. Recommendation: wash 10-20 times, rinse with reagent grade acetone, then dry in air. BE CAREFUL WITH THE FLUORESCENCE CELLS, THEY COST MORE THAN $100 PER CUVETTE!

D. Turning off the spectrometer: 1. First enter the time in the logbook, and write in how much time the machine was on. Then on the lower unit (LPS-220) turn the DISPLAY knob to VOLTS, note the reading in the lab book, and then do the same for AMPS. Reset to WATTS when finished. 2. Press the POWER button on both units, in any order, such that both units are turned off not lighted up. E. Processing data: 1. To change the color of a curve, click on it in the legend on the left. If not present, use DISPLAY/LEGEND, the DISPLAY/COLOR and make your selection. 2. To put text on your printout, use DISPLAY/ANNOTATION. 3. To print, make sure data transfer switch (box between IR and Fluorimeter) is set to

Bell Research Group

Standard Operating Procedures, page 21

Fluorimeter. 4. Turn computer off as in step 1 of section A (Turning on the spectrometer).

Figure 5: Flurescence Spectrometer.

Bell Research Group

Standard Operating Procedures, page 22

Figure 6: Flurescence Spectrometer.

Bell Research Group

Standard Operating Procedures, page 23

HIGH-PRESSURE LIQUID CHROMATOGRAPHY (HPLC) INSTRUMENT Components 1. Two (2) Perkin-Elmer Series 10 pumps for separate aqueous and organic solvents. Both pumps are appropriately labeled. The plastic tubing connecting the pumps to the solvent reservoirs have distinguishing markers: a solid black-colored tape to indicate aqueous and another black tape with a white band to indicate organic. 2. Cole-Parmer plotter. 3. UV detector, Perkin-Elmer model LC-15B. CAUTION! UV light source. It is recommended that the detector be turned on and warmed-up for about 2 hours before use. Record the time in the logbook. 4. Chromatography column. These are normally not connected to the HPLC pumps when the HPLC is not in use.

Mobile Phase Preparation 1. Mobile phase solvents need to be pre-filtered through 0.45 m nylon or cellulose filters before use. 2. Degassing of solvents is also required prior to use. Apply a water-aspirator vacuum to the solvent reservoir while it is immersed in an active sonicating water bath for 4 minutes or more, depending on the nature of solvent/solvent mixture. Aqueous buffer solvents with volatile components may be degassed in less time to prevent loss of components. Sample Preparation The sample is dissolved in an appropriate solvent, usually the mobile phase, at a concentration of 1-10 mg in 0.5 to 10 mL solvent. It is recommended that the sample solution be filtered and degassed to prevent clogging of the system and gas bubble formation. General Operation 1. Turning on the instrument: Turn on the power switches on both pumps. Connect the pump solvent tubing to the appropriate solvent reservoirs. Make sure that the metal pre-filters are completely immersed in the solvent. It is recommended that they be washed with the appropriate solvent before immersion to avoid solvent cross-contamination in the case of change to a different solvent. 2. Priming and purging the pumps: After connection to solvent reservoir, the pump should be purged to remove air trapped in the tubing during changing of solvents. Use a plastic Luer-type syringe to draw solvent. Attach the syringe with the

Bell Research Group

Standard Operating Procedures, page 24

3.

4.

5. 6.

7.

8.

9.

plunger pushed all the way. While holding the syringe in place, carefully open the PRIME PURGE valve. Making sure the syringe does not detach, pull the syringe plunger to draw solvent. A non-frothy solvent flow indicates purge is complete. With the syringe still attached, turn the PRIME PURGE clockwise to close it. Then release the syringe and dispose of the solvent. Repeat if necessary. Changing solvent: Prior to attaching a column, the pumps should be run with the solvents to be used, changing over from methanol used for storage of pumps. For the aqueous solvent, use filtered, degassed double-distilled water initially. Recommendation: ~15 minutes of ~1-2 mL/min flow rate (combined pumps). Make sure that the solvent outlet is connected to a waste receiver. Attaching the column: Locate and disassemble the joint to which the column will be connected. Make sure that the column is consistent with the direction of solvent and sample flow. Use the appropriate-sized Allen wrench to loosen and tighten the connections. Some parts of the connection may only need to be finger-tight. Caution: Do not over-tighten connections to avoid damaging them. If in doubt, finish tightening after turning pump on, using only enough torque to stop leakage. A column pre-filter should be attached to the column between the sample injection port and the column. Setting flow rate: Set the appropriate flow rate on the flow rate switches. Operation: Press the RUN switch on both pumps, a green light will be seen on the switch. Check for leaks in the connections. Make sure that the pressure gauge shows that the internal pressure is within the pressure specifications of the column (under 5000 PSI) and that it stabilizes to constant value. If not, the column may need to be regenerated or the pre-filter replaced. Using the plotter: a) Turn the plotter on. Set to 20 mV input and 30 cm/hr chart speed. Turn the REC switch to start recording, or to STBY to pause. b) Adjust the Zero knobs on both UV detector (to read 0.000 absorbance) and plotter, with REC switch selected, to set the location of the recording pen on the paper. c) Set the ABSORBANCE RANGE on the detector to an arbitrary initial value, e.g., 0.256, which later can be changed depending on sample concentration to adjust the plotting scale. Set TIME CONSTANT to 0.5 sec (typical value). Solvent change: Change the aqueous solvent from water to appropriate aqueous buffer solution. Run this new solvent mixture for at least 15 minutes at 1.2 mL/min flow rate. Injecting the sample: Turn the sample injector port lever to the left (marked LOAD), without pushing the plunger insert syringe (20 L sample volume) all the way, push the plunger to introduce the sample, turn the lever to the right (marked

Bell Research Group

Standard Operating Procedures, page 25

10.

11. 12.

13.

INJECT), push EVENT MARK and then remove syringe. Monitoring the chromatogram: Monitor the progress by checking the absorbance reading on the UV detector in conjunction with the chromatogram being drawn by the plotter. A stopwatch may be used to check the chart speed. At 30 cm/hr chart speed, each centimeter of plotter paper corresponds to 2 minutes. Cleaning the injection port: Wash the sample port with fresh blank mobile phase solvent after every sample. Turning off the HPLC: After the last sample has been analyzed, prepare the HPLC for cleaning and storage. Turn off the plotter and then the UV detector. Run blank mobile phase for at least 15 minutes or double the retention time of the last peak and then switch aqueous solvent to double-distilled water and run for another 15 minutes. Press the STOP switch and allow the internal pressure to decrease to normal pressure. Detach the column and reconnect the tubing. The column should be capped with the correct screws and stored in its case in a cool, dry drawer. Press RUN and allow pre-filtered methanol to flow throughout the system for at least 10 minutes. Press STOP and then turn the POWER SWITCH off on both pumps. Logbook: Do not forget to complete the information in the HPLC logbook. Record name, date and time, solvents used, column used, problems and comments.

Bell Research Group

Standard Operating Procedures, page 26

UV-VISIBLE SPECTROPHOTOMETER Procedure 1. Find the UV software icon on the desktop of the computer, Emerson, attached to the instrument. 2. With the mouse, press lamp on (F7) and confirm the lamp is operating by observing light at the back of the instrument. Around one hour should be allowed for the lamp to warm up to avoid fluctuations in the readings. 3. Press F1 for general scanning of samples. 4. The UV cuvettes having two clear sides are stored near the instrument (do not use fluorescence cells, having 4 clear sides). Wash the UV cell thoroughly and rinse 3 with the solvent to be used. 5. Put the solvent-filled cuvette in the instrument and run a blank spectrum by pressing F8. 6. Place the sample solution in the cuvette and press F7 to run the spectrum. 7. To analyze the spectrum, use F2 to mark peaks, F3 to alter the scale, F6 to save and F10 to leave. 8. When saving, use F5 to change the directory and the type in C:\UV\Data\your name. You should have already made a folder. You can also save the file on a diskette (drive B). 9. Type in a name and press F6 to save. To print, see below. 10. Press F10 to leave and then T to get to the top level. 11. Turn off the lamp and close the program 12. All files are saved as wavefiles and require the use of a UV converter program to convert to MS Excel for printing. 13. The UV computer cannot perform this action. Use either the fluorometer computer (Maccabee) or the FT-IR computer (Cyclops). 14. The files can be transferred through the network connection or by diskette. 15. Open the Grams UV converter program (desktop icon). 16. Go to HP 8542 and click. 17. Import data file. 18. Select file or all files and click OK. 19. Select automatic and click OK. 20. The file should now be a .UV file. 21. Go to ASCIIXYP and click export. 22. Select file or files and click OK. 23. The files should now be .prn files. 24. Open MS Excel. 25. Open file(s) and click next 2X. 26. Use chart wizard to graph.

Bell Research Group

Standard Operating Procedures, page 27

FT-IR SPECTROPHOTOMETER Procedure A. KBr sample preparation using the KBr press: 1. All materials needed should be on top of the acid cabinet and/or next to the instrument. The KBr, shiny metal plates and agate mortar and pestle should be in the dessicator. 2. Grind the sample with KBr in the mortar and pestle. 3. Take the top of the KBr die (silver color) with the metal plunger and o-ring and invert it in your hand. The surface of the metal rod (plunger) should be flat. If it is tapered, invert the plunger. 4. Place one of the polished metal plates (shiny side up) on top of the plunger. 5. Take one of the paper ring sample holders and place it on top of the plate. 6. Lower the plunger so the plate and sample holder are inside the die. 7. Place enough of the sample on the sample holder to cover the hole in the paper ring. 8. Place the other metal plate (shiny side down) on top of the sample. 9. Take the black colored base of the die and gently reassemble the two parts. 10. Remove the protective shield from the KBr press, normally next to the instrument, and place the die inside with the extended metal plunger on top. 11. Replace the protective shield and lock the sample in place with the screw wheel. 12. Turn the silver knob on the bottom right side of the press (pressure relief valve) all the way to the right. 13. Place the bar into the socket on the lower right side and apply pressure to the die by pumping the lever. Do not exceed ten tons. Try to obtain 8-9 tons for a few minutes and then release the pressure by turning the silver knob to the left. 14. Return the bar to the holder and release the screw wheel from the die. 15. Remove the sample from the press. 16. Invert the die and remove the base. Push the plunger up gently and obtain the sample from between the shiny metal plates. The sample should appear as a glassy solid and not a powder. If it is a powder, try again. 17. Place the sample on the appropriate sample holder and use. 18. Clean all materials used, polish the silvery die surface, and return them to their original places. B. Use of salt plates: 1. The salt plates are usually stored in the desiccator. 2. Clean with acetone or hexane. NEVER USE WATER!

Bell Research Group

Standard Operating Procedures, page 28

3. If the sample is to be run neat, place a drop in the center of one plate and use the other plate to spread the sample around between the two plates. When placed in the appropriate holder, the sample should be spread wide enough to cover the beam area. 4. If using mineral oil, place a little bit of sample in the middle of one plate and then add a drop of mineral oil. Mix thoroughly and then use the other plate to spread the sample around as above. 5. When done, clean plates as described above and return them to the desiccator. C. Spectrometer operation: 1. Before using, sign the FT-IR logbook. 2. The FT-IR software program, Omnic ESP, is on the computer named Cyclops next to the instrument. An icon should be on the desktop. 3. Open the program and click on collect on the menu bar, followed by experiment set up. 4. Click on the bench tab and record the maximum value in the logbook. 5. If you want to alter the number of scans, resolution or format, click on the collect tab. 6. Close the setup screen and click on collect from the menu bar and choose collect background. 7. When done, place the sample in the instrument and choose collect sample from the collect menu on the menu bar. 8. To use peak pick, click on the analyze menu and find peaks. To correct the baseline and to smooth out noise, use the process menu. 9. To print or save the file, use the icons or the file menu. 10. When done, remove the sample, close the software and record the time used in the logbook.

Bell Research Group

Standard Operating Procedures, page 29

SOLVENT SYSTEMS (THF, N,N-dimethylformamide, Toluene)

Procedure (Instruction has been written for Toluene. The procedure is same for other two also). Start the nitrogen flow before 5-10 min. 1. Make A on (on means parallel to outlet). 2. Begin the nitrogen flow (1-2 psi; recommended <2 psi) and control its rate with the help of B regulator (notice the C bubbler for the rate and D arrow should be in the direction of E hose). 3. Wait for 5-10 min. 4. To collect the solvent make F arrow down. (for less than 75 mL of solvent, its ok to withdraw from G outlet with the help of syringe or else withdraw from H outlet using 24 joint round bottom flask.) 5. Once you get desired amount of solvent in the I receiver make F up again. 6. After withdrawal of solvent, drain the excess solvent in the J collector and close the nitrogen flow with the help of B again. 7. Close A (perpendicular to outlet). Maintenance of 5 psi pressure in the solvent tank (Recommended pressure in the solvent tank is 5 psi, if it is <5 please, make it 5.) 1. Turn D all the way 180 0 (arrow should be in the direction of K hose). 2. Open the L knob all the way. 3. Set the pressure to 5 psi with the help of B (Notice M pressure gauge). 4. Close L. 5. Turn D arrow again all the way to 180 0 (arrow should be in the direction of E). 6. Release the excess pressure through C N2 bubbler. Safety Precautions 1. Wear safety goggles and gloves. 2. Nitrogen tank must be secured by means of tank straps or cradles. 3. Nitrogen flow should be between 1-2 psi (recommended <2 psi; notice the nitrogen bubbler). 4. Avoid contact of skin with any solvent (THF, N,N- dimethylformamide or toluene). 5. Handle every parts of the solvent system very gently. Hazards 1. Implosion of evacuated glassware causes injury, including laceration and sometimes blindness. 2. Damage to skin caused by contact with solvent. 3. Explosion of receiver may be caused due to high pressure (above 10 psi). 4. Poisoning caused by inhalation of any solvent. 5. Violent rocketing of gas cylinder if main valve is severed from top. 6. Injury to arm or leg caused by falling cylinder.

Bell Research Group

Standard Operating Procedures, page 30

Figure 7: Solvent withdrawal system.

Bell Research Group

Standard Operating Procedures, page 31

Figure 8: Schematic diagram of solvent withdrawal system.