This section deals with some of the nitty gritty details of performing reactions in the lab.

Table of Contents

1. General Degassing Techniques
   • Flushing Reaction Vessel with Nitrogen
   • Degassing empty tube/tube with solids (no solvent)
   • Degassing a solvent via sparging with nitrogen
   • Freeze Pump Thaw
2. Weighing
   • Weighing solids onto weighing paper
   • Weighing solids into flask
   • Weighing liquids into flask
   • Weighing product samples
3. Starting the Reaction
   • Adding reagents under air
   • Adding reactants/solvent under strict conditions (via syringe)
   • Stirbars and stirring
   • Setting up condenser, balloon and water flow for reflux
   • Heating
4. Stopping Reactions
5. Glovebox Usage

General Degassing Techniques

Many organic reactions tend to be sensitive to air or water, so it is good practice to degas the flask and reaction mixture before running the reaction. These techniques are more or less in order from least to most strict.

Flushing reaction vessel with nitrogen

This is pretty simple. Seal the reaction vessel with a rubber stopper, and insert the nitrogen flow needle as well as an exit needle. Allow the system to purge for 5 minutes, and most of the gas in the reaction vessel would be nitrogen.

Degassing empty tube/tube with solids (no solvent)

1. Connect Schlenk line to Schlenk tube/reaction tube via stiff rubber tubing
2. Turn on the vacuum, and turn the Schlenk line knob to connect reaction vessel to vacuum line
   • If Schlenk tube: Slowly open the cap of the Schlenk tube
     • When turning the cap, there will be two ‘stops’:
     • First stop: To switch between the actual ‘turning mode’ of the Schlenk tube cap and the ‘non turning mode’
     • Second stop: When reached, will actually open/close the Schlenk tube cap
   • If reaction tube: Open the knob
   • If strict reaction conditions/no moisture: Use heat gun to heat the tube to ensure that almost all of the air is removed by vacuum pump
   • NOTE: If knob/cap is opened too fast, and there is solid inside the tube, the solid will fly up the tube and into the rubber tubing, making you very sad (and you will need to clean it). Open the knob/cap SLOWLY.
3. Slowly turn the Schlenk line knob to refill the reaction vessel with nitrogen/argon (depending on what your lab uses)
4. Check the bubbling of the oil tube at the end of the Schlenk line – the oil should NOT burst through the trap (the tube thingy that is connected at the end of the Schlenk line) or not air will go inside the reaction vessel
5. Repeat the degassing process at least 3 times, leaving the system under vacuum for a longer period during the third repetition

Degassing a solvent via sparging with nitrogen

1. Should be done after stirbar and almost all reagents are added in (with exception of any reagents that have to be added last, depending on the reaction)
2. Make sure that most solids are dissolved by the solvent
3. Use a rubber stopper (Suba-Seal) to close the mouth of the flask and invert the stopper to ensure a tight seal
4. Insert an exit needle into the rubber stopper to act as an air outlet – I usually like to use a larger one
5. Connect a syringe adapter and long bendy needle to the Schlenk line, and turn the knob on the Schlenk line so that nitrogen is coming through
   • NOTE: Make sure that the gas pressure is sufficient, but not too high. If pressure is too high, the exit needle can fly out, which would be very dangerous.
6. Switch on the stirring and put the needle into the solvent. Make sure that bubbles can be seen and that the stirring is vigorous enough to stir the bubbles inside. Leave it to sparge for about 15 minutes.
7. Remove needles and you’re done!

Freeze Pump Thaw

This is only used when the reaction requires extremely strict exclusion of air from the reaction mixture; usually this would be overkill.

1. Fill a shallow dewar with some liquid nitrogen.
2. Connect Schlenk tube to Schlenk line. Make sure that at this stage, the tube is not connected to nitrogen and tha the knobs are all closed. Make sure that solvent/stirbar/reagents have already been added into the Schlenk tube.
3. Ensure that Schlenk tube cap is closed. Put the end of the tube into the dewar to freeze the solvent and reagents using the liquid nitrogen until the entire thing is fully solid.
4. With the tube still inside the dewar of liquid nitrogen, VERY SLOWLY open the Schlenk tube cap to allow the vacuum to remove the air. Let it go for about a few minutes.
5. With the tube still inside the dewar of liquid nitrogen, slowly turn the knob on the Schlenk line to recharge the tube with nitrogen.
6. Remove the tube from the dewar after recharging is complete and allow the solid solvent and regents to thaw. You may see bubbling in the solvent when the tube is thawing – this is normal.
7. Repeat the entire process for 3 times. Remember to close the cap of the schlenk tube before freezing and only open the cap when the solvent is completely solid.

Weighing

• Make sure that the scale is clean before weighing anything out
• Do not weigh with the stirbar in the flask – this will mess with the reading on the scale
• Make sure that you clean up after using the scale – not only does a dirty scale make everyone sad, some chemicals can corrode and destroy the scale if not promptly removed.

Weighing solids onto weighing paper

1. Fold the weighing paper into half diagonally, then into half again (this is to make sure that the powder stays on the “line” on the weighing paper and doesn’t go everywhere). This also makes it easier to tip the solid into the reaction vessel later on.
2. Place weighing paper onto scale. Make sure that no part of the weighing paper is outside of the weighing plate or touching other parts of the scale.
3. Tare the weighing scale.
4. Wash spatula with solvent and wipe it down to make sure it is clean.
5. Open both side doors of the weighing scale.
   • One hand: Holding opened bottle of solid powder
   • The other hand: Holding spatula
6. Transfer the solid from the bottle onto the weighing paper using the spatula
7. If the weight on the scale is close enough but not exactly the weight that you want, that is okay. Just remember to write down the actual weight used in your lab journal.

Weighing solids into flask

1. Place flask onto a cork ring or plastic container (preferred, mass of cork ring may change if there is solvent/whatever on it), and place them onto the weighing plate. Make sure that none of it is touching other parts of the scale.
2. Tare the weighing scale.
3. Transfer the solid in the same way in which you would transfer solids onto weighing paper.

Weighing liquids into flask

Note: It is preferable to just add the liquid to the reaction mixture by volume (use the density of the liquid to calculate). However, sometimes you would have no other choice but to weigh a liquid (e.g. crude liquid reaction mixture).

1. Place flask onto a cork ring or plastic container (preferred, mass of cork ring may change if there is solvent/whatever on it), and place them onto the weighing plate. Make sure that none of it is touching other parts of the scale.
   • Cork ring: Tilt flask onto one side so that opening is facing one of the side doors
   • Plastic container: You will add liquid from the top door of the scale.
2. Tare the weighing scale.
3. Take a clean glass pipette, suck up some of the liquid, then expel it. Then suck up the liquid again. The liquid level on the pipette should be kept low (because some of the liquid will be stuck onto the walls of the pipette and would lead to a loss of material if there is a lot of it there).
   • If the liquid is viscous, suck it up slowly, or not it may splat everywhere in the glass pipette, making you very sad.
4. Add the liquid into the flask through the side/top door.
5. After weighing, hold the pipette down for a short while to allow some of the liquid stuck onto the glass walls to flow down. Expel that liquid back into the storage container for that liquid. Fill an Eppendoff tube with some solvent and wash the last bit of the liquid on the glass pipette down into it (and label and keep this sample for TLC).

Weighing product samples

1. Ensure sample is dry. Ideally:
   • Rotavap it once to remove most of the solvent
   • Empty the rotavap solvent trap, and rotavap it for another 5 minutes at low pressureto get rid of most of the residual solvent
   • Leave the sample on high vacuum overnight
2. Tare the weighing balance to make sure that it is zeroed.
3. Weigh the sample two times
   • If sample has gas replaced with nitrogen: Directly place sample vial/flask onto weighing plate and wait for mass to stabilize
   • If sample has gas replaced with argon: Gently wave sample vial/flask around a bit for the argon to be displaced with air or just wait for a short while for diffusion to occur. Then place sample vial/flask onto weighing plate and wait for mass to stabilize. This is however not ideal if the sample is air/moisture sensitive.
4. Take the average of the two values

NOTE: If the mass of the product keeps decreasing, the sample is not dry. Dry it on the vacuum pump for a little longer.

Starting the Reaction

Adding reagents under air

1. Turn on stirring so that solvent/reactants are stirred.
2. If reaction is sensitive to moisture but not oxygen: Reaction should be done in 2-necked flask. Turn on the nitrogen/argon gas (but not too much to prevent solid from flying out) to allow gas to flow into the flask to push out the air and prevent moisture from going in while reagents are being added.
3. Take another piece of weighing paper and fold into a cone. Place the cone into the mouth of the flask.
4. Tip previously weighed out solid reagent on folded weighing paper into the cone weighing paper so that it is transferred into the flask.
5. Use your finger to flick the back of the folded weighing paper/give the folded weighing paper a little shake to make sure that almost all the powder has been transferred
6. Tip the folded weighing paper which previously held the solid reagent up, then carefully open it to check if there is any more unadded reagent
7. If there is more unadded reagent, add it
8. Slowly shake the cone weighing paper while opening it, then flick it with your finger to make sure that almost all the solid reagent stuck onto it is transferred into the flask.
9. Slowly remove the cone weighing paper to check if there is any more unadded reagent
10. If there is more unadded reagent, add it
11. Stopper the flask.

NOTE: Solid and liquid reagents should never be directly added to each other – they are both in high concentration, we don’t know what reactions can and will occur

Adding reactants/solvent under strict conditions (via syringe)

1. Strict degas procedures should already be performed by this point, and the flask should be stoppered with an overturned rubber stopper
2. Degas the syringe
   • Insert syringe into flask, and draw out some nitrogen/argon gas (the flask should be under positive inert gas pressure)
   • Pull syringe out of flask, and eject gas
   • Repeat 3 times
3. Add the reagent/solvent
   • The reagent/solvent bottle should be under positive inert gas pressure (nitrogen line in, exit needle out)
   • Reactant/solvent can then be drawn directly from the bottle
4. Insert needle of syringe into rubber stopper of reaction flask, and inject solvent/reactant into flask

Stirbars and stirring

• Round-bottomed flask: Almond stirbar
• Schlenk tube/reaction tube: Rod stirbar or small almond stirbar
• Stirring: Should always be at the highest possible intensity that allows for consistent stirring.
• Schlenk tube/reaction tube: Don’t use a very high rpm or not the rod stirbar will not be able to stir properly
• Dual phase solvent systems: Stirring should be high enough so that the separation between phases cannot be seen

Aside: Should stirbars be clean? Yes, you should always, always, ALWAYS use a clean stirbar. You don’t know whether the crap from a dirty stirbar would affect your reaction. Ideally, once the reaction is complete and it is time for clean up, you should dunk your stirbar in aqua regia to get rid of random metal crap in addition to the usual washing.

BUT… It has been found that bits of leftover metal catalyst on improperly cleaned stirbars could potentially catalyze reactions. The Ananikov group managed to show that Palladium-catalyzed Suzuki coupling reaction could be performed by simply chucking in a dirty stir bar – in one case, the yield even matched that of a reaction run with the proper catalyst! You can read more about this case here.

Another interesting story involving Suzuki couplings (no stirbars this time) was the alleged “metal-free Suzuki coupling” published (now retracted) by the Xu group. They claimed have performed the reaction without the catalyst, which instantly made many people in the synthetic chemistry community highly suspicious as this was something that many people have tried and failed. It was later found that palladium was used to make a starting material for their coupling reaction, and that the palladium compound somehow hung around and catalyzed the Suzuki when it was run. Another “metal-free Suzuki coupling” by the Leadbeater group was debunked when they realized that the sodium carbonate they used as the base was contaminated by trace amounts of palladium which happened to be just enough to catalyze their reaction. You can read about the metal-free Suzuki coupling saga here.

Anyways, back to experimental synthetic chemistry!

Setting up condenser, balloon and water flow for reflux

When should I heat things under reflux? In general, heating under reflux is used if the temperature required for the reaction is near or at the boiling point of the solvent used. Heating under reflux would prevent the loss of solvent via evaporation as any solvent in gaseous form would condense back to liquid on the surface of the condenser and drip back into the reaction mixture.

For detailed descriptions and pictures of how to do a reflux, refer to this article on Chemistry Libretexts.

1. Balloon set-up:
   • Preparation of balloon attachment:
     • Attach balloon to a short segment of hard tube and secure with a rubberband
     • Degassing and filling of balloon:
       • Attach the tube with the balloon to an adapter, and connect that to the tube of the Schlenk line.
       • Switch on the vacuum to remove all the air, then recharge with nitrogen/argon.
       • Repeat the process 3 times, allowing the vacuum to run for about 5 minutes on the 3rd time.
       • To fill the balloon, turn the Schlenk line so that nitrogen/argon flows into the balloon, then slightly close the tap connecting the Schlenk line to the oil tube. The balloon should fill very fast. Remember to open the tap connecting the Schlenk line to the oil tube once done.
     • Transfer of balloon to attachment:
       • Twist the bottom part of the balloon, then remove it from the tube adapter and connect it to an adapter for the condenser. Close the tap of the adapter, then release the balloon.
2. Condenser set-up (assuming you are using a 2 necked flask):
   • Remove the stopper on the main opening of the flask while switching on the nitrogen/argon flow to the flask via the Schlenk line and adapter (connected to the second neck), then add on the condenser.
   • Allow the nitrogen/argon gas to flush through the condenser for about 15 seconds
   • Add the balloon and balloon adapter to the top of the condenser, then turn the tap on that adapter to connect the balloon to the system
   • Close the tap on the adapter connected to the second neck of the flask, and switch off the nitrogen/argon gas flow from the Schlenk line.
3. Water to condenser set-up:
   • Connect the water pipes to the condenser pipes, with the input going from the bottom of the condenser, and the output coming out from the top of the condenser
   • Make sure that all attachments are secure – this is very important to prevent flooded fumehoods!
   • Turn the water so that the flow is very very small (because water pressure increases at night when no one uses it, and if pressure is too high you will get a flood!)
4. Clamping:
   • Adapter used to connect to Schlenk line should be clamped to smaller neck of 2-necked flask with metal C-clip
   • Condenser does not need to be clamped to big neck of flask, it should sit on the flask nicely
   • Balloon adapter needs to be clamped to condenser using metal C-clip

Heating

1. Make sure that thermometer is submerged in the oil bath, but not touching the bottom or side to ensure accurate temperature readings
2. Oil bath should be preheated prior to starting reaction
3. Set the oil bath to the desired temperature, and turn it to PRECISE (instead of FAST, for Heidolph instruments) heating. Turn the stirring to about 200-400 rpm.
4. When reaction system is fully set up (with condenser and water and all), carefully transfer the reaction system to the oil bath. HOLD THE MAIN FLASK WITH THE REAGENTS WHILE TRANSFERRING THE SETUP, NOT THE CONDENSER. If you grab the system by the condenser while transferring, the main flask may drop off, making you lose all your hard work and you will be very sad. :<
5. Clamp the system above the oil bath tightly, before lowering the system into the oil bath. The level of the oil should be the same as the solvent level in the flask.
6. Make sure all clamps are tight before you leave the lab – the heat may cause the rubber on the clamps to expand/become loose and your flask may drop into the oil bath, which will also make you very sad. :(

Stopping Reactions

1. Switch off the heating and stirring.
2. Lift the flask/tube out of the oil bath by lifting up the clamp attached to the stirplate. Allow the silica oil to drip back into the oil bath for a while and for the reaction mixture to cool back to room temperature.
3. After the flask/tube has cooled, unclamp it from the stirplate stand. Rinse the part of the flask/tube that has been inside of the oil bath with hexane until all the silica oil has been removed. Wipe the tube down with a tissue to clean it.
4. It is good practise to take a crude TLC, NMR, and LCMS/MALDI as necessary, as well as to save a tiny bit of the crude sample just in case it is required for further analyses in the future.

Glovebox Usage

In some cases, a glovebox may be required to deal with particularly air or moisture sensitive reagents.

1. Check that the pressure, O2 and H2O levels are normal before use.
2. Open the transfer chamber hatch and put all the things you need inside. Push all the things deep into the chamber so that they are easier to remove later.
3. Degas the chamber and the things inside
   • Schlenk tube: Should be slightly opened when placed into glovebox chamber
   • Turn the gas knob to switch to vacuum, then to refill with nitrogen gas. Use the pressure gauge to judge if the chamber has been properly degassed.
   • Repeat degassing step 3 times, then finally refill the chamber with nitrogen gas.
4. (Depending on your lab’s practice) Wear the first glove layer, before putting on the glovebox gloves. The pressure from the glovebox gloves may be high enough to push you away and make using the glovebox difficult. Use one hand to help the other hand put on the gloves.
5. Open the hatch to the transfer chamber from the inside the glovebox and remove the required equipment.
6. Perform required operations inside glovebox (e.g. weighing moisture sensitive solids). Be careful not to pierce a hole in the glovebox gloves.
7. After finishing operations, place everything back into the transfer chamber and close the hatch from the inside of the glovebox tightly.
8. Remove the glovebox gloves, then remove items from the transfer chamber.
9. Degas the transfer chamber and refill with nitrogen gas.
10. Check the glovebox pressure, O2 and H2O levels to make sure that all is nominal before leaving.

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