TLC and Column Chromatography Workshop

TLC and Column Chromatography Workshop
Air Sensitive Techniques 1: Using Schlenk Lines Advanced Research Techniques Workshop April 20th 2011 James Watson

Outline Why use air-sensitive techniques? Common air-sensitive compounds Equipment Specialist glassware The Schlenk line Purge and refill Cycling tubing Handling Reagents Dry solvents and distillation Measuring and weighing Spills/Fires Clean-up Reagents Glassware Syringe/cannula Workshop 20/04/11

Why Use Air Sensitive Techniques? Many commonly used chemical reagents are extremely reactive towards water and/or oxygen and will oxidise, decompose, ignite or even explode Air sensitive techniques have been developed to avoid contact with air and moisture from the atmosphere at every step !! these techniques do NOT protect from explosive or shock sensitive materials, and provide limited protection from toxic compounds !!

Common Air Sensitive Compounds Examples of pyrophoric compounds: - Metal alkyls and aryls e.g. RMgX, RLi, RNa, R3Al, R2Zn - Metal hydrides e.g.. NaH, KH, LiAlH4 - Hydrides e.g.. B2H6, PH3, AsH3 - Boranes, phosphines, arsines Examples of chemicals which react violently with water - Metal hydrides - Metal amides - Alkali metals e.g.. Na, K, Cs - Many main group halides e.g.. BCl3, BF3, AlCl3, PCl3, SiCl4

Specialist Equipment Glove Box – for solid reagents that must be handled under completely dry conditions Glove Bags are also available cheaply for less sensitive compounds (not suitable for pyrophoric materials)

Air Sensitive Techniques Apparatus Cap N2 inlets Tipper funnel Schlenk flask Dropping funnel Ampoule/graduated ampoule

Air Sensitive Techniques Apparatus Glass syringe and plunger Condenser Frit Cannula Filter stick

The Schlenk Line (or Double Manifold)
G H F

The Schlenk Line (or Double Manifold) A. Metre-long nitrogen bubbler E. Two-way double oblique manifold taps B. Nitrogen line F. Vacuum tubing C. Tubing to nitrogen supply G. Trap surrounded by liquid N2 dewar D. Vacuum line H. Tubing to vacuum pump After assembly, the Schlenk line must be purged with nitrogen as follows (only suitable for Schlenk lines with 1m long bubbler and sufficient Hg in the reservoir): Connect 2 taps with one piece of tubing, ensuring the nitrogen supply is off Turn one of the taps to vacuum, then slowly open the other tap to N2 to pull the mercury up the exhaust line, whilst the air is sucked from the manifold Close the tap turned to the N2 manifold, and turn on the nitrogen supply to purge Repeat steps 2 – 4, twice

The Schlenk Line (or Double Manifold) Before switching on the vacuum pump, do a visual check of the line: check all taps on the manifold are closed check all joints are streak-free ensure the trap is empty and connected to the line with no streaks at the joint check the nitrogen supply by turning on the tap until there is a good flow through the mercury bubbler

Liquid Nitrogen Liquid nitrogen is a cryogenic fluid which is extremely cold and boils at -196 oC Small volumes of liquid vaporise into large volumes of gas, ~700 times expansion !! wear safety goggles and protective clothing at all times !! !! never draw air through traps containing liquid N2, as explosive liquid O2 can condense !! Hazards Cold burns can occur very rapidly and over a wide area – always wear thermoprotective gloves (available from Gas Safety UK, product code , £16) !! do not wear cloth gloves of any kind !! Asphyxiation can occur as liquid nitrogen rapidly expands to large volumes of nitrogen gas. !! never accompany a dewar containing liquid N2 in a lift !! Over pressure will occur if the rapidly boiling liquid N2 is used in a closed system. Beware of an “ice-dam” forming in apparatus. !! never screw a lid on a thermos flask containing liquid N2 !!

Pump Maintenance Oil Change: every 6 months or when necessary 1. open outlet valve and decant oil into waste bottle/drum via funnel 2. pour fresh oil into the inlet valve using a clean funnel to flush out old oil 3. close outlet valve and fill pump Measuring Pressure using a McLeod gauge 1. attach to the Schlenk line using rubber tubing 2. turn the two-way double oblique manifold tap to vacuum slowly 3. carefully rotate the gauge to the vertical position to read the vacuum pressure 4. turn back to horizontal and repeat step 3 until the reading is constant ideal pressure should be 10-2 torr or lower

Pump Maintenance Poor Vacuum: cannot achieve a good vacuum 1. check vacuum tubing for degradation and replace if necessary 2. check oil level and for discolouration – change if necessary 3. check joints and taps in line and ensure there are no streaks in grease 4. if adequate vacuum cannot be achieved, take the pump to the workshop to be tested and cleaned !! ensure liquid N2 is always topped up around traps to prevent volatiles contaminating pump oil !!

Purge-and-Refill: How to Cycle Tubing Cycling tubing and glassware using the purge-and-refill method ensures all air is removed and replaced with inert gas 1. turn on the vacuum pump and have liquid N2 around the solvent traps 2. turn on the nitrogen supply and ensure the bubbler is at a gentle rate 3. connect glassware to a tap on the line using thick-walled rubber tubing 4. cycle the tubing only (the tap on the glassware remains closed): a. turn the two-way double oblique manifold taps to vacuum to purge b. after a few seconds, rotate the tap to refill with nitrogen (the mercury/oil in the bubbler will be sucked up). If there are already other taps in use under nitrogen, close these before turning new taps to nitrogen, and only re-open when the pressure is equalised c. wait for the pressure to equalise and the bubbler to begin bubbling d. repeat steps a – c twice more to purge-and-refill

Purge-and-Refill: How to Cycle Tubing 5. with tubing under nitrogen, the stopcock on the glassware can now be opened 6. when the bubbler begins to bubble again, the pressure is equalised and the flask and tubing are ready for further manipulation n.b. if connecting more than one piece of glassware and tubing to the line, the above steps must be carried out simultaneously for each piece two-way double oblique manifold taps stopcock Schlenk line tap

Positive Nitrogen Flow at all times during a reaction, the system should be under a slight positive pressure of nitrogen (positive flow) which can be visually indicated by the mercury or oil bubbler when removing stoppers or caps from apparatus, there must be a positive flow of N2. This provides a protective atmosphere “blanketing” the glassware from entry of air and moisture

Glass-Aware Plan ahead! 1. Plan the whole reaction ahead of time (a diagram for each step can be helpful at first!) how many Schlenk flasks will I need? will I need a dropping funnel/condenser/frit? Laboratory glassware contains a thin film of adsorbed moisture which must be removed Pre-heat all glassware in an oven (>100 oC, > 30mins), then assemble whilst hot using silicone grease at the joints (see next slide), and allow to cool under vacuum. Open joints must be closed with stoppers or glass caps. Schlenks may also be flamed under vacuum with great care using a Bunsen burner or heat gun !! never heat a sealed system !! When cool, purge the glassware with nitrogen and cycle. Schlenk flasks can then be weighed with their stoppers and stirrer bars. Mark Schlenks and stoppers with corresponding symbols to avoid mix-up

Greasing Joints when assembling glassware using grease there should be no streaks in the grease at the joints. This ensures that the joints will not seize and that no air can enter. 1. apply a small amount of grease to the male joint and spread along the length 2. assemble the joint and rotate carefully to distribute the grease. If streaking occurs, add an additional small amount of grease to cover this area 3. the joint is correctly greased if no streaks appear streaked joint streak-free joint

Dry Solvents Dry solvents must always be used, as standard bench top solvents can contain up to 30% water. These must be dried and handled carefully. Distillation stills apparatus and drying agents Storage molecular sieves Transferring to reaction vessel cannulae / syringes

Distillation Apparatus 4 Round bottom flasks (still pot) Heating mantle Condenser Inert gas via bubbler Solvent collector Solvent outlet Syringe port Overflow 3 5 7 6 8 1 2

Drying Agents Solvent Drying agent Diethyl ether* / petroleum ether* Distil over sodium pieces (0.5% w/v) and benzophenone (1 spatula per litre) Dichloromethane Calcium hydride 5% w/v Toluene* Distil over sodium pieces (1% w/v) DMF* Distil over calcium hydride (1% w/v) THF* Distil over potassium pieces (1% w/v) Acetonitrile* * these solvents can be pre-dried over sodium. !! never add sodium to chlorinated solvents – an explosion may occur !!

Collecting and Storing Dry Solvent Once collected, dry solvent must be stored in a suitably dry manner. This can be achieved by using 4Å molecular sieves which have a cage-like crystalline structure which captures water. To activate molecular sieves: Pre-heat molecular sieves in hot oven overnight. Leave ampoule in oven for at least 1h !! Young's taps will be damaged if placed in the oven !! Pour sieves into ampoule and insert Young’s tap (but do not close)and connect to Schlenk line. Turn the Schlenk line tap to vacuum. Place ampoule in a heating mantle, using a clamp stand. Turn heat dial to 7. Heat for at least two hours. Turn off heat, and allow ampoule to cool under vacuum When cool, cycle ampoule, and leave under nitrogen until ready to use

Collecting and Storing Dry Solvent Collecting solvent from distillation apparatus turn on the heating mantle, water supply and check N2 flow. If the collector has a tap close it. 2. when collector is full, turn off heating mantle and allow solvent to cool connect ampoule with sieves to an inert gas line using PVC tubing; turn the gas on first insert a needle through the tubing to purge air from the Young's joint 5. remove Young's tap and place ampoule under solvent outlet. Turn off N2 supply to prevent blow-back. collect solvent into the ampoule !! distillation apparatus can be dangerous, do not use without proper training !!

Transferring Dry Solvent from Ampoule to Schlenk connect your solvent ampoule and dried Schlenk flask (see Glass-Aware) to the Schlenk line and cycle tubing unscrew (anticlockwise) Young's tap and remove. Maintain a positive pressure of N2, creating an inert blanket over the solvent. quickly and carefully insert the correct sized subaseal (usually no. 21) containing a bleed needle (to purge the cavity with N2) into the neck of the ampoule 4. open the stopcock of the Schlenk flask. Maintain a positive pressure of N2 and remove the stopper. Insert a subaseal containing a bleed needle. Be sure to rotate this bleed needle to purge the large cavity, then remove the needle.

Transferring Dry Solvent
5. Insert one end of a cannula/double-tipped needle into the ampoule and check the nitrogen is flowing through against your hand. Insert the other end into the Schlenk flask. Put a bleed needle through the subaseal of the Schlenk flask, and check nitrogen flow against your hand. Close the Schlenk flask tap and lower the cannula/double-tipped needle into the solvent to transfer. The pressure of nitrogen will now force the solvent into the empty Schlenk. After transferring the desired amount, lift the cannula out of the solvent in the ampoule. Now open the tap to restore N2 flow, remove the bleed needle and cannula. Clean cannula as described later (Syringe/ Cannula/ Needle Clean Up slide) Cannula

Measuring and Weighing Dry Reagents Dry reagents must be carefully measured or weighed to ensure that their potential contact with air and moisture is minimised. Dry Solid Reagents: from the bottle powder funnels Dry Solid Reagents: from a Schlenk flask transfer of solids from Schlenk flask to Schlenk flask Dry Liquid Reagents: from the bottle cannulae/syringes

Weighing Out: Dry Solid from a Bottle prepare a dry, pre-weighed Schlenk flask (see Glass-Aware) and a dried powder funnel (long-necked funnels are better to avoid compound sticking to greasy joint!) clamp the Schlenk flask and connect to the Schlenk line. Cycle the tubing. while under a gentle flow of nitrogen, remove the stopper. Test the flow against your hand (too strong a flow will blow your solid back out of the flask) do not block the joint completely with funnel (solid will blow out), hold it just above or use a ring clamp add solid to flask. Remove any solid sticking to the grease with a tissue and re-grease stopper. Ensure all solid is removed, it can cause the joint to crack. replace the stopper and weigh the flask.

Weighing Out: Dry Solid from Schlenk flask to Schlenk flask Solids likely to decompose over time may be better stored in a dried Schlenk under N2 Prepare a dry pre-weighed Schlenk flask (see Glass-Aware), and a tipper funnel capped on one end, with an N2 inlet tap at the other end. Clamp all glassware and connect your pre-weighed Schlenk, your reagent Schlenk, and the tipper funnel to the Schlenk line. Cycle tubing simultaneously. 3. Under a positive flow of nitrogen, remove the stopper from your pre-weighed flask and the cap from the tipper funnel, and connect the two. Rotate the flask to ensure a streak-free joint. Close the N2 inlet tap on the tipper funnel and remove this, along with the stopper from the reagent Schlenk, and connect the funnel to the Schlenk flask. Rotate joint to remove streaks.

Weighing Out: Dry Solid from Schlenk flask to Schlenk flask 5. Secure the joints in place with elastic banding so they stay together while tipping. Close the tap of your reagent Schlenk flask, and carefully turn your pre-weighed flask to vacuum (if there is a stirrer bar in your pre-weighed flask, use a magnet on the outside of the flask to secure it) Carefully tip an estimated amount of solid into the pre-weighed Schlenk. Clamp the reagent Schlenk flask. Carefully turn the tap of the pre-weighed Schlenk back to N2. Open the tap of the reagent Schlenk flask to restore the flow of N2.

Weighing Out: Dry Solid from Schlenk flask to Schlenk flask
9. Under positive nitrogen pressure, remove the pre-weighed flask and replace the stopper. Also replace the cap on the tipper funnel and turn this flask back to vacuum. 10. Now re-weigh your prepared Schlenk and calculate how much solid was transferred. 11. If there was too much or too little, connect to the Schlenk line again and cycle the tubing. Turn the reagent flask and tipper back to nitrogen, and repeat from step 4. 12. When you are happy with the mass of solid transferred, remove the tipper under positive nitrogen pressure and place stoppers in both flasks.

Measuring Dry Liquid Reagents: Syringes Syringe transfer tips: Small quantities (up to 20 mL) of air-sensitive reagents and dry solvents may be transferred with a syringe equipped with a thin, 12-18inch long needle. This avoids having to tip reagent bottles and storage flasks. Tipping often causes the liquid to come in contact with the septum/subaseal causing swelling, deterioration and spills Using syringes: Pressurise the vessel with N2 (for bottles see next slide). Subaseals can be inserted into Schlenks/ampoules (as described in transferring solvents). Purge the syringe with N2 by inserting into the septum/subaseal and filling with N2 only; the N2 pressure should push the plunger back, so that the syringe fills, and then remove from the bottle and push N2 out into fume hood. Repeat step 2 twice more

Measuring Dry Liquid Reagents Sure/Seal bottles Sure/Seal bottles have a crown cap and liner crimped in place which gives some protection against air and moisture from the atmosphere. Needles and cannulae can be inserted through this seal. Pressurise the bottle with N2 by inserting a needle connected to a Schlenk line via a Luer adapter (check the N2 flow first) The reagent can then be dispensed using a syringe or double- tipped needle inserted through the hole in the metal cap. Liquids likely to decompose over time may be better transferred to a dried ampoule under nitrogen for storage. Crown cap with hole Luer adapter

Measuring Large Volumes of Dry Liquid Reagents When measuring large volumes of liquid, it is not advisable to use a syringe. Instead use a graduated ampoule and a cannula. Connect a dry graduated ampoule and your reagent flask or bottle to the Schlenk line. Cycle tubing simultaneously. Have ampoules and Schlenks under N2, and replace stoppers/ Young's taps with subaseals. Reagent bottles can be pressurised with N2 as described on the previous slide. Transfer liquid from a reagent bottle/ampoule to the graduate ampoule using an oven dried cannula/double-tipped needle, as described in Transferring Dry Solvents. When you have the desired amount, return nitrogen flow and remove the bleed needle Then transfer the liquid reagent from the graduated ampoule to reaction vessel, using the cannula/double-tipped needle, and opening/closing the stopcock to alter the nitrogen flow and adjust rate of addition.

NMR tube holder Some reactions can be monitored by 31P and 11B NMR spectroscopy, by preparing an NMR sample in an NMR tube holder, as shown. The NMR tube is placed in the holder and dried in the same fashion as a Schlenk flask. Transfer a sample of the reaction solution in the same fashion as transferring solvent.

Attaching Glass Apparatus Connecting condensers and dropping funnels Dry glassware and assemble as described in Glass-Aware. Cap the bottom joint of the glassware with a greased cap and insert a greased N2 inlet into the top joint, ensuring there are no streaks. Connect the N2 inlet to the Schlenk line using PVC tubing, and turn the two-way manifold tap vacuum. Allow to cool under vacuum. Elastic band the cap and inlet, and cycle the glassware. Attach reaction vessel to Schlenk line and cycle tubing. Under positive N2 flow, remove stopper from flask and the cap from the apparatus, and connect the two

Attaching Glass Apparatus Connecting a frit and filtering solutions Dry glassware and assemble as described in Glass-Aware. Cap both joints of the glassware with greased caps and insert a greased tap into the stopcock, ensuring there are no streaks. Connect the N2 inlet to the Schlenk line using PVC tubing, and turn the two-way manifold tap vacuum. Allow to cool under vacuum. Elastic band the caps and cycle the glassware. Attach reaction vessel to Schlenk line and cycle tubing. Under positive N2 flow, remove stopper from flask and bottom cap from the frit, and connect the two. Transfer reaction solution by removing the top cap and inserting a subaseal containing a bleed needle. Transfer using cannula techniques as described in Transferring Dry Solvents

Removing Solvent Solvent must be removed on the high-vac line using an external trap Connect external trap to the Schlenk line by the top outlet. Connect the external trap to the Schlenk flask with a piece of tubing from the side-arm. The Schlenk flask must be closed. Place a Dewar flask around trap and fill with liquid N2. Turn the Schlenk line to vacuum, and agitate the solution with a stirrer bar or swirling. Slowly open the Schlenk stopcock. Solvent will begin to bubble/bump When the bumping subsides the tap can be fully opened. The flask will become cold and solvent may freeze, warm very gently heating with a heat gun. 7. When all solvent is removed close the Schlenk stopcock, and close the Schlenk manifold tap. Remove the tubing and take the trap out of the Dewar. Connect the flask to the line, and cycle the tubing. Open the flask to N2.

Elastic Banding Elastic bands are required to secure joints during flushing, as the nitrogen pressure may open the seals of unsecured joints. Rubber bands are more secure than clips which are quite flexible. Stoppers could easily come out of their joints if there is a build up of vapour pressure inside the Schlenk, exposing your material to the atmosphere. Schenk flask taps can also be secured with an elastic band. Open: during a reaction to prevent accidental removal Closed: Schlenk can be left in a basket with tap secured and cannot be knocked out of position

Spills / Fires Spills Phosphines/ mercaptans must be cleaned using bleach Lithium aluminium hydride can be wiped up using barrel roll, and can be quenched by immediately and quickly submerging tissue in a sink full of water Sodium hydride can be destroyed with alcohol Fires Always alert someone else first! Small fires can often be allowed to burn out In case of larger fires, use a dry powder extinguisher or fire blanket

Reagent Clean Up Empty Sure/Seal bottles – remove the crown cap and leave in the fume hood to allow the last traces of reagent to hydrolyse or oxidise Destroy phosphines/mercaptans with bromine solution (IMS:bromine 100:1 approx.)/ bleach, followed with IMS, water and acetone (in that order). Destroy organometallic compounds with IPA/IMS followed by water, then acetone Alkali metals must be placed in a beaker filled with toluene, and destroyed using a isopropanol:toluene solution (1:4). When doing this on a large scale (i.e. Still pot) do this under an inert atmosphere.

Glassware Clean-Up Disassemble glassware Destroy phosphines, mercaptans and organolithiums as described on the previous slide. Dispose of reagents as appropriate and rinse the glassware with water Wipe all grease from joints, stoppers, taps, caps etc. Place glassware in a base bath (KOH in IMS) ensuring there are no air bubbles and the glassware is completely submerged, for a minimum of 4 hrs; 16hrs max Rinse glassware with water and place in an acid bath (HCl in water) for 1 hr min. Rinse glassware with water then acetone and put back in oven !! always use thick nitrile gloves or marigolds !! !! never put greasy glassware in the oven !!

Syringe/ Cannula/ Needle Clean Up Clean all syringes and needles immediately after use (hydrolysis and oxidation of reagents can cause needles to plug) Syringes rinsed at the sink , using the plunger to clean the needle. To rinse a cannula/double tipped needle, use a Buchner funnel as shown with the Subaseal clamped into place. Apply vacuum to pull solvent through cannula. Order of rinse: Bromine solution (phosphines); IMS; Water; Acetone Dilute acid can be used to unblock a needle Dry plunger and syringe separately in the oven Organolithiums must be destroyed using IMS Phosphines/mercaptans must be rinsed with IMS into bromine solution (IMS:bromine roughly 100:1)

Summary Using air- and moisture- sensitive techniques is a necessary requirement for handling many types of reagents Dry glassware is essential When removing stoppers/caps from Schlenk flasks and other apparatus ensure that the inlet taps are open to nitrogen/inert gas flow Sure/seals found on reagent bottle gradually degrade. Store liquid reagents in ampoules for prolonged use Solid reagents can be stored in Schlenk flasks for prolonged use Remember to label all flasks and co-ordinating stoppers

Workshop 20th April 2011 Weigh zirconocene dichloride (0.5g) into a sample vial on the balance and transfer to a separate Schlenk flask using a powder funnel. Add dry THF (15mL) to the zirconocene using a syringe, then cool to -78oC. Weigh out dry potassium tbutoxide from a Schlenk flask via tipper funnel into a pre-dried, pre-weighed Schlenk flask. Weigh out approx. 0.5g Add dry toluene from an ampoule using a syringe. Use 15mL. Add dry nbutyl lithium from an ampoule using a syringe. Use 2.8mL of 1.6M solution. Now transfer the benzyl potassium into the zirconcene solution using a cannula. Concentrate the solution to around 1/3 of the volume and filter through a frit.

References Sigma-Aldrich Technical Bulletin AL-134 Advanced Practical Organic Techniques. J. Leonard, B. Lygo, G. Procter Advanced Practical Inorganic and Metalorganic Chemistry. R.J. Errington

Acknowledgements Dr K. Izod Mr J. Dyson Miss L. Cotterill The Postgraduate Committee

Other Workshops in the Series Workshop Date Using Mestre-Nova and Delta to analyse NMR data 23rd March 2011 Air Sensitive Techniques 1: Using Schlenk Lines 20th April 2011 Effective Quenching and Working-up of Reactions 18th May 2011 Recrystallisation and Growing Samples for X-Ray 15th June 2011 Industry Techniques 1: Flash Chromatography 13th July 2011 Air Sensitive Techniques 2: Using a Glove box 10th August 2011 Industry Techniques 2: Microwave Reactions 7th September 2011 Dates, locations and titles to be confirmed Sign-up sheet for each workshop will be available on the Postgraduate Notice board two weeks in advance PhD students: record attendance at each workshop in your e-portfolio 1 school PGRDP credit is available per workshop

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