sop lab operations

8/3/2019 SOP Lab Operations

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Standard Operating Procedures

for

Laboratory Operations

in the

Bell Research Group

University of Nevada, Reno

Revised 9/09


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ii

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


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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 theParr 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 closingthe air valve on top of the Erlenmeyer flask.

3. Open valve I a few turns and allow the reaction bottle to be evacuated (about 3-5minutes 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 ofno 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 valueand then close valve J.

8. Shut off the aspirator by opening the air valve on top of the Erlenmeyer flask firstand then turning off the water faucet.

9. Now you are ready to turn on the rocking motor to begin reaction. Record theinitial hydrogen pressure in your notebook.


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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 ofabout 2 psi per 10 seconds on the left hand pressuregauge. (Note: The right hand pressure gauge shouldnt change. If it does, youhavent 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 andwait for the system to be fully evacuated (about 4 minutes required).


6. Carefully remove the 4 thumbscrews from the cage, remove the screen, unscrew thegreen 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, thegreen stopper is clean, and the cage is reassembled.

Safety precautions1. 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


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Figure1:Hydroge

natoranditsschematicdiagram

.


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ATMOSPHERIC HYDROGENATOR

Procedure1. 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 valveon top of the Erlenmeyer flask.

4. Start stirring the contents of the reaction flask and open the 3-way valve connectingthe 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 thevalve connecting the cylinder to the hydrogenator under vacuum. Close this valvequickly as soon as the cylinder is filled with water; avoid sucking water into thehydrogenator 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] inParr 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 gasline.

10. Carefully open the valve connecting the bubbler with the hydrogenator (between thebubbler 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.


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17. Close valves A, B, C, F and G and the valve connecting the bubbler to thehydrogenator (see figure [p 3a] in Parr SOP).


19. Open the valve connecting the measuring cylinder with the hydrogenator andresume stirring.

20. After completion of the reaction, turn on the aspirator (step 3), and then evacuate thewhole system for about 5 minutes (step 4).

21. With the aspirator still running, fill the system with air by opening the air valve ontop 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 precautions1. 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.

Hazards1. 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.


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Figure 2: Schematic diagram showing atmospheric hydrogenation setup.


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ROTARY EVAPORATOR

Procedure1. 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


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Hazards1. 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.


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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 round-

bottomed flask:

Purple > Blue > Green > Yellow(DRY) (WET)

The solution should turn blue/purple after 1-12 hours of reflux; if not, add another25-30 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 inthe condenser head.

2. When enough THF collects in the condenser head, insert the needle of an oven-dryhypodermic syringe through the rubber septum and the stopcock.

3. Flush the syringe with nitrogen from the condenser head 3-4 times beforewithdrawing the dry THF.

4. Release the excess dry THF back to the round-bottomed flask SLOWLY byreopening 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 partialvacuum 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.


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Safety precautions1. 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 thestills are in frequent use.

3. At no time should the volume of the THF in the still be allowed to fall low enoughto 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 beforeusing 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.

Hazards1. 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.


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OZONIZER

Procedure1. 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 highlytoxic.

6. When the reaction is complete, usually indicated by the appearance of a bluish colorin 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 precautions1. 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.


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Figure 4: Ozonizer.

Magnetic stirrer

Dewar

From ozonizer

Pasteur pipetThermometer

Dry ice/ acetonebath


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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 endswith hose clamps.

2. Use vacuum grease (Apiezon) on glass joints and stopcocks; use metal clamps tohold ball-and-socket joints together.

3. Test the vacuum manifold, traps and tubing connections for leaks by measuring thepressure at the end of the system (most distant from the vacuum pump) by means ofa mercury-filled gauge (McCleod gauge); if the measured pressure is much higherthan 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 efficientlycondenses vapors. Use liquid nitrogen if there is any possibility of pumpcontamination by reactive gases, such as ammonia, HCl, etc. If dry ice is used,donot 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 dryice that is just covered by acetone.

3. Cover traps with cloth or glass wool to reduce loss of coolant. Do not operatepump 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


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and water, if necessary.8. Dry the traps at room temperature overnight or in an oven.9. Regrease clean traps before re-use.

Safety precautions1. 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, causingdanger of explosion. If this bluish liquid is seen, turn off the vacuum pump, ventthe vacuum system, evacuate the laboratory and notify Prof. Bell and/or safetypersonnel.

Hazards1. 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.


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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 thepump.

5. Open stopcock beneath fill window to drain used oil into waste container; the pumpshould 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


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procedure! If solvents remain in the pump during operation, it could be severelydamaged. This procedure should be followed only under unusual circumstances.

7. Refill pump chamber to proper level, as observed in the glass window; do notoverfill.

8. If previous contamination is suspected, run pump with new oil briefly (10-30minutes), 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 otherchemicals) and contact EH&S for pick-up.

Safety precautions1. 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 becomesunusually noisy, or if a leak develops in the system it is attached to.

Hazards1. 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.


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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 ofthe tank.

2. Secure the cylinder to a tank cart with the chain and move it carefully to the newlocation.

3. Secure the cylinder at the new site against a wall or bench by means of a chain, tankstrap 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, holdingthe gauges on the regulator upright.

4. Close the small regulator exit valve and turn the regulator off by rotating the mainhandle 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 precautions1. 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.

Hazards1. Violent rocketing of gas cylinder if main valve is severed from top.2. Injury to arm or leg caused by falling cylinder.


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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 downthe computer.

2. On the lower box (LPS-220) to the right of the computer, press the POWER buttonso it lights up; the LED should give a reading.

3. Press the IGNITE button, holding it down until you see the LED value jump; thisis usually accompanied by a small sound (like an electrical discharge) from thephotomultiplier 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 photomultiplierdetector to the far right of the spectrometer (810). It should read 1050. If there isno reading, turn it on by flicking the switch on the bottom. If the reading is not1050 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 LPS-220 unit set the DISPLAY to WATTS. It should read 75. If not, carefully adjustthe 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 10-

7 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 NOTFORCE THEM you will break them. Then open the desired amount anti-clockwise. Usually each one is opened one full rotation, which is 2 nm (2-4 nm isrecommended).

2. When you open the FELIX software and tell it to ACQUIRE an emission scan, itwill calibrate the spectrometer. Note the dials rotating at this time. You will see


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that the one at the back says 273. Change the value on the computer screen from274 to 273. DO NOT ROTATE THE DIAL FROM 273 to 274.

3. Note the step size, integration time and averages. Changing them will change thequality of your spectrum obtained. For consistency you should use the same valuesevery time. YOU can decide your own values (recommendation: step size = 0.5;integration = 0.1; average = 1).

4. Before acquiring the emission scan, it is suggested that you obtain a UV-spectrum tofind the maxima where you would like to excite the sample in order to obtain theemission spectrum.

5. Set the excitation wavelength, as obtained from step 4, and scan the emissionwavelength detection from low to high, starting at 10 nm greater than the excitationwavelength.

6. If the emission spectrum flattens out at the top of a peak, i.e., if the number of

counts exceed ca. 3 106, the sample is too concentrated. Stop scanning and use a

more dilute sample (preferable) or use a smaller slit width.7. 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).8. 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.9. 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.10. IF YOU SPILL ANYTHING IN THE SAMPLE CHAMBER CLEAN IT UP!11. 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 MORETHAN $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 thereading in the lab book, and then do the same for AMPS. Reset to WATTS whenfinished.

2. Press the POWER button on both units, in any order, such that both units areturned 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


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

Figure5:FlurescenceSpectrometer.


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Figure6:F

lurescenceSpectrometer.


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HIGH-PRESSURE LIQUID CHROMATOGRAPHY

(HPLC) INSTRUMENT

Components1. Two (2) Perkin-Elmer Series 10 pumps for separate aqueous and organic solvents.

Both pumps are appropriately labeled. The plastic tubing connecting the pumps tothe solvent reservoirs have distinguishing markers: a solid black-colored tape toindicate 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 hoursbefore use. Record the time in the logbook.

4. Chromatography column. These are normally not connected to the HPLC pumpswhen the HPLC is not in use.

Mobile Phase Preparation1. 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 4minutes or more, depending on the nature of solvent/solvent mixture. Aqueousbuffer solvents with volatile components may be degassed in less time to preventloss of components.

Sample PreparationThe sample is dissolved in an appropriate solvent, usually the mobile phase, at aconcentration of 1-10 mg in 0.5 to 10 mL solvent. It is recommended that the samplesolution be filtered and degassed to prevent clogging of the system and gas bubbleformation.

General Operation1. 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 thatthey be washed with the appropriate solvent before immersion to avoid solventcross-contamination in the case of change to a different solvent.

2. Priming and purging the pumps: After connection to solvent reservoir, the pumpshould 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


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INJECT), push EVENT MARK and then remove syringe.10. Monitoring the chromatogram: Monitor the progress by checking the absorbance

reading on the UV detector in conjunction with the chromatogram being drawn bythe plotter. A stopwatch may be used to check the chart speed. At 30 cm/hr chartspeed, each centimeter of plotter paper corresponds to 2 minutes.

11. Cleaning the injection port: Wash the sample port with fresh blank mobile phasesolvent after every sample.

12. Turning off the HPLC: After the last sample has been analyzed, prepare the HPLCfor cleaning and storage. Turn off the plotter and then the UV detector. Run blankmobile phase for at least 15 minutes or double the retention time of the last peak andthen switch aqueous solvent to double-distilled water and run for another 15minutes. Press the STOP switch and allow the internal pressure to decrease tonormal pressure. Detach the column and reconnect the tubing. The column shouldbe 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 10minutes. Press STOP and then turn the POWER SWITCH off on both pumps.

13. Logbook: Do not forget to complete the information in the HPLC logbook. Recordname, date and time, solvents used, column used, problems and comments.


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UV-VISIBLE SPECTROPHOTOMETERProcedure

1. Find the UV software icon on the desktop of the computer, Emerson, attached to theinstrument.

2. With the mouse, press lamp on (F7) and confirm the lamp is operating by observinglight at the back of the instrument. Around one hour should be allowed for the lampto 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 3with the solvent to be used.

5. Put the solvent-filled cuvette in the instrument and run a blank spectrum bypressing 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 program12. 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.


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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 inthe 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 istapered, 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 fewminutes 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 glassysolid 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!


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3. If the sample is to be run neat, place a drop in the center of one plate and use theother plate to spread the sample around between the two plates. When placed in theappropriate holder, the sample should be spread wide enough to cover the beamarea.

4. If using mineral oil, place a little bit of sample in the middle of one plate and thenadd a drop of mineral oil. Mix thoroughly and then use the other plate to spread thesample 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.


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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 (onmeans parallel to outlet).2. Begin the nitrogen flow (1-2 psi; recommended


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Figure7:Solventwit

hdrawalsystem.


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Figure8:Schematicdiagramo

fsolventwithdrawa

lsystem.

sop lab operations

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