From Janet Hurley - last modified 090610

Preparation procedures for 14C analysis of Cocaine

NOTE - recent changes to procedure

  • Janet B. has slightly changed placement of the Zn/Ti tube and the small Fe tube that fit within the reaction tube.
  • Also, there is a three strikes target pounding procedure.

Precombustion procedures

  • Cut 6 mm quartz tubes at 8 inches long (200 mm). To cut, score tube at 8 inches and snap along score.
  • Turn on the torch by turning on gas supply at wall valve and oxygen supply at tank regulator. Be sure the torch is pointed away from you and any combustible equipment or materials. Turn the red knob on the torch for the gas feed and ignite with spark. Once the gas flame is steady and about 5 ½ inches in length, delicately turn the green knob to introduce oxygen. Adjust until there is a bright blue triangle at the base of the flame. The more oxygen, the smaller and hotter the triangle. Adjust to get a 7 inch flame with at least ¼ inch bright blue.
  • Holding a quartz tube at an angle and wearing #6 protective eyeshades (to prevent retinal burning) and leather gloves (to prevent finger burns), seal one end of each quartz tube while continually rotating the tube in the flame of the torch. Rotate slowly and hold just above the small blue triangle in the flame. Be sure that the hole is sealed and there is a solid glass bottom to the tube.
  • Place hot sealed tubes upside down in a beaker to cool.
  • Once the tubes are cooled, fire polish the open end of each in the flame.
  • Place hot sealed tubes in a beaker to cool.
  • Turn off the torch by turning the green knob to stop oxygen flow, then the red knob to stop gas flow. Turn of the gas supply at the wall valve and the oxygen supply at the tank regulator.
  • Dry the tubes by placing in a muffle furnace at 500ºC for about a half hour. Allow to cool slowly and completely (a few hours or overnight). Cover with aluminum foil until ready to preload.
  • Preload 200 mg CuO (about 2 spatulas full) into tubes using precut plastic funnel.
  • Precombust quartz tubes loaded with CuO at 900ºC for 2 hours. Place tubes in a steel basket or other high-temperature container for precombustion. Do not place them in Pyrex beakers, which will begin to melt at about 800ºC, or aluminum baskets or racks, which will melt at around 650ºC.
  • Wear gloves to prevent contaminating precombusted tubes.
  • To minimize static problems, store precombusted quartz tubes in batches wrapped in aluminum foil in bubble desiccator with water in bottom.

Loading combustion tubes

  • Wear gloves for loading combustion tubes to prevent contamination.
  • Clean all surfaces and tools with a methanol wipe. Clear tools of any dust particles after methanol wiping with an aerosol duster.
  • Standards are loaded for combustion at 0.8 to 1.2 mg carbon. Standards should be kept in individual zip lock bags with designated spatulas or curettes. Spatulas and curettes should not be exchanged between standards and unknowns and should be cleaned with methanol and cleared with aerosol duster prior to each use. Samples are also loaded for combustion at 0.8 to 1.2 mg carbon – for cocaine HCl this is about 1.7 mg. Load into 3 x 5 mm silver capsules (precombusted at 900ºC for 1 hour).
  • Record weight and crimp loaded silver capsule slightly so that it will drop into the precombusted 6 mm quartz tube.
  • Label precombusted quartz tubes with high temperature marker.
  • Once tubes are loaded, fill the large dewar flask with liquid nitrogen and freeze the trap between the pump and vacuum line.
  • Secure tubes to vacuum line and open valves on each tube. Once vacuum gauge indicates all tubes are at baseline pressure, they can be sealed.
  • Wear #6 protective eyeshades and seal each tube in turn with a torch. Light the torch as described above. At about 1 to 1 ½ inch below the Ultra Torr fitting, hold the flame so that the blue triangle almost touches the quartz. Rotate the flame around the tube until the quartz becomes soft enough to turn. Twist the tube and pull down until the two pieces separate. Melt the tips of the tube and the vacuum stub into smooth blobs to seal each, preventing tip leaks and breakages. Set sealed tubes on the Hardibacker board to cool.
  • Turn off the torch as described above.
  • Place tubes into protective steel sleeves and into the muffle furnace. Place tubes in systematic order and record sample names on ordered worksheet in case tube labeling is baked off.
  • Combust at 900ºC for 2 hours and allow to cool overnight.
  • Once combustion is complete and the tubes have cooled, remove from steel sleeves and retrace labels with a Sharpie marker. Wear gloves to handle combusted sample tubes, and wipe steel dust off of tubes with Kimwipes.
  • If not performing gas transfer right away, store combusted samples in a beaker in a clean cabinet for up to one week.

Preparation of reaction tubes

  • Cut 9 mm Pyrex tubing at 6-inch lengths. To cut, score tube at 6 inches and snap along score.
  • Seal the end of each tube with the torch. Note that the torch cannot be as hot as with quartz tubing. Use the glass pulling technique to form an evenly sealed bottom.
  • Fire polish the open end of each tube with the torch.
  • Burn a small dimple into the side of each tube at ¾ inch (2 cm) from the base of the tube.
  • Precombust the 9 mm Pyrex tubes and the 6 mm Kimble culture tubes at 500ºC for 3 hours and 550ºC for 4 hours.
  • Store precombusted Pyrex tubing wrapped in aluminum foil in a bubble desiccator with dry desiccant. Always handle precombusted reaction tubes with gloves to prevent contaminating them.

Loading reaction tubes

  • Wear gloves while handling reaction tubes and reagents to prevent contamination.
  • Place a white sheet of paper under the glass work surface. Clean all surfaces and tools with methanol wipe. Clear tools after methanol wipe with aerosol duster.
  • With the designated curette, load 30 mg of zinc powder into the 9 mm Pyrex tube (1 rounded scoop). Try not to get Zn on the sides of the tube. Knock any powder to the bottom of the tube by tapping the tube on the work surface. Do this for as many tubes as the number of samples to be prepared, plus a few extra.
  • With the curette designated for titanium hydride, add 10 to 15 mg of TiH2 to each of the 9 mm tubes (2 packed scoops). Avoid getting powder on the sides of the tube by tapping it on the work surface to knock any TiH2 to the bottom.
  • Now, using the small curette designated for iron, load 5 mg (exactly) of Fe powder into the 6 mm Kimax tubes (1 rounded scoop).
  • Place an iron-loaded 6 mm tube into each preloaded 9 mm tube, by tilting the 9 mm tube and allowing the 6 mm tube to slide gently to rest on the dimple.
  • Bake the tubes and loaded reagents at 300ºC for 1 hour.
  • Cover the preloaded reaction tubes with foil to prevent contamination.
  • Store preloaded tubes under vacuum in bubble dessicator for up to 2 weeks before use. If not used before two weeks, tubes may be prebaked again at 300ºC for 1 hour and used.

CO2 gas transfer

  • Fill two 4 liter dewars with liquid nitrogen.
  • Wear leather work gloves to protect hands and fingers from freezes and burns throughout the process. Wear safety glasses as well.
  • Freeze the trap between the pump and vacuum line by filling the large dewar flask with liquid nitrogen. This trap prevents oil from entering the vacuum line and water from entering the pump. The dewar may require topping-off with liquid nitrogen throughout the gas transfer procedure.
  • Once the trap is frozen, open the entire vacuum line to the pump. Check the line for any leaks by monitoring the gauge, which should show a baseline reading after a short time.
  • Prepare an ethanol and dry ice slush and freeze the water trap. The water trap will remain frozen throughout the extraction procedure and will require refreshening with dry ice every few hours.
  • Isolate the tube cracker and clean the tube cracker interface. Add grease if necessary.
  • Score a quartz sample tube at the appropriate height and place it in the tube cracker apparatus.
  • Open the tube cracker to the vacuum.
  • Isolate the reaction tube section of the line, remove Pyrex stub, and replace with preloaded and labeled reaction tube.
  • Open the newly placed reaction tube to the vacuum slowly so that reagents are not mobilized and mixed.
  • Open all valves to the pump and wait for gauge to return to baseline.
  • Close the valve from the tube cracker to the pump. Once vacuum gauge reads baseline, close valves to isolate the water trap and CO2 trap.
  • Freeze the bottom 3 inches of the CO2 trap with a dewar flask full of liquid nitrogen.
  • Once the bottom of the CO2 trap is frozen, crack open the sample tube and open the valve to the water trap.
  • Close the valve between the CO2 trap and the reaction tube.
  • Open the valves from the water trap to the CO2 trap and allow the CO2 to freeze in the CO2 trap.
  • Monitor N2 or other combusted sample gases on the analog pressure gauge. Open the valve from the CO2 trap to the vacuum pump to draw off these gases. Once the gauge has returned to baseline, isolate the CO2 trap by closing the valves on either side.
  • Isolate the tube cracker and water trap by closing the valves on either side of the water trap.
  • Open the valve between the reaction tube and the vacuum pump, and the cold finger and the reaction tube.
  • Once the gauge shows a baseline reading, close the valve leading to the vacuum pump.
  • Fill a mini-dewar with liquid nitrogen and place on the cold finger.
  • Remove the liquid nitrogen from the CO2 trap and allow to warm. Use a water bath to speed the warming process.
  • Open the valve between the CO2 trap and the reaction tube, allowing the CO2 sample to collect in the cold finger. Wait for the gauge to reach baseline. If baseline is not achieved, open valve to vacuum pump to draw off excess gasses.
  • Isolate the cold finger by closing the valve between the cold finger and reaction tube. Also isolate the reaction tube at this time by closing the valve between the reaction tube and CO2 trap.
  • Remove the liquid nitrogen from the cold finger and use a water bath to warm the finger.
  • Reaction tubes are prepared to accept 1 milligram of carbon for graphitization. The cold finger gauge will have been calibrated (and labeled) to indicate a pressure reading that is equivalent to 1 mg C. If indicating about 1 mg C, record the pressure reading. If there is excess CO2 gas, some may need to be partitioned so that the reaction tube accepts only 0.8 to 1.2 mg C. Use Boyle’s law, p1V1 = p2V2.
  • Freeze the reaction tube with liquid nitrogen, then open the valve between the cold finger and reaction tube.
  • Wearing safety glasses to protect from sodium flare and once the CO2 has frozen in the reaction tube, seal-off the reaction tube at about 11 cm with the torch. The torch should not be too hot. Prepare a 3 ½-inch gas flame and then add oxygen to get a steady triangle. Do not subject the Pyrex to the hottest part of the flame. As one point on the tube softens, move the flame to another point until the tube is soft enough to turn. Twist the tube and pull to seal.
  • Anneal the tube by turning off the oxygen and allowing the tube to cool slowly at the top of the gas flame until the top of the tube is black with carbon residue.
  • Once cooled, be sure each sealed reaction tube is clearly labeled and place into a 20-place aluminum block.
  • Repeat steps 6 through 30 until finished collecting CO2 samples for the set.
  • Open all valves to the vacuum and allow gauges to return to baseline.
  • Remove the ethanol slurry from the water trap and secure the dewar flask cover over the slurry mixture. The mixture may remain cold enough for use the next day.
  • Remove dewar flasks containing liquid nitrogen and replace any remaining into one of the 4-liter storage dewars.
  • Close the main valve (large red valve) between the pump and the vacuum line.

Graphitization

  • Once 2 aluminum blocks are stocked with 40 sealed reaction tubes, complete a graphitization worksheet listing sample names for each position in each block. Each set of 40 samples must contain at least one 14C-dead blank such as acetanilide, six primary standards (OX2), and three secondary standards (2 ANU-sucrose, 1 IAEA C3).
  • Place loaded aluminum blocks into the muffle furnace and bake at 500ºC for 3 hours, then 550ºC for 4 hours.
  • Allow reaction tubes to cool overnight.

Preparation of aluminum cathodes

  • Clean undrilled aluminum cathodes with acetone in a beaker by sonication for 30 min. Decant the acetone and repeat the process until the decanted acetone is clear.
  • Spread the cathodes in a tray lined with a Durx cleanroom wiper towel, and let them dry in the laminar flow hood.
  • Wear safety glasses while drilling cathodes.
  • Check that the drill press is set to the correct depth (0.160”). The drill press has an electronic depth reading mechanism that is very sensitive to o-ring compression on the face cover. Tightening or loosening the screws may be necessary if the depth reader is not reading 0.160” at fully pulled handle position. If the reading is unstable and adjusting the face cover does not resolve it, the battery made need to be replaced.
  • Place a clean cathode funnel-side up under the drill bit, and secure the cathode with spring arm.
  • Test positioning of the stage by pulling down on the arm. The drill bit should come to the center of the cathode funnel. If not properly centered, manipulate the stage to center the drill bit on the funnel.
  • Once properly centered, turn on the drill press and slowly pull the arm down until the depth reading is 0.156 to 0.158” (we have found that this gives us a depth of 0.160”), release the arm, and turn off the drill press.
  • Remove the cathode from the drill press and tap it upside-down on the clean work surface to expel aluminum ribbons, then use aerosol duster to remove any residual aluminum from the hole.
  • Remove aluminum ribbons from the drill bit and stage before proceeding to drill additional cathodes.
  • Inspect drilled cathodes under a dissection microscope to be sure that the holes are centered in the funnel.
  • Clean the work area after desired amount of cathodes are drilled.
  • Store clean cathodes in a clean plastic container with tight fitting lid.

Pressing graphite into cathodes

  • We submit pressed graphite targets in sets of 40 to the Keck AMS lab at UC-Irvine. Pressed targets will be placed into 96-well plates and can therefore contain up to 2 sets of 40 samples per plate. As noted above, each set must contain 1 blank, 6 primary standards, and 3 secondary standards. Before pressing, prepare sample worksheets showing placement, cathode number, target number, sample ID, estimated mg carbon, d13C values and any relevant comments for each target.
  • The order of pressing for each sample set is as follows: Blanks, OX2, ANU-Sucrose, C3, and unknowns. Clean in between each sample type, and change gloves as they become contaminated with graphite material. NOTE: while pressing multiple targets of the same material, it is only necessary to dust the pin head with the aerosol duster in between targets.
  • Turn on the laminar flow hood and clean all surfaces with methanol and Durx cleanroom wipes.
  • To clean the portable press pin, sand the pin head in a circular motion on 600 grit silicon carbide sandpaper, then sand the sides of the pin by folding a small piece of the sandpaper and rotating the pin against the paper. Finally, wipe the pin and pin holder with methanol and dust with aerosol duster. Notice that the pin will need replacing after every few sets of 40 samples are hammered.
  • Clean the cathode holder, sleeve, and magnetic forceps with methanol and the aerosol duster.
  • Check the target number and write it on a drilled cathode using an ultra fine Sharpie marker.
  • Place the labeled cathode into the cathode holder.
  • Score the 9 mm reaction tube about 2.5 cm above the dimple. Hold the tube horizontally, gently scoot the Kimax tube above your score line and break the 9 mm tube to access the 6 mm tube containing the graphite-Fe powder. Discard the bottom of the tube containing the zinc and TiH2 into the plastic waste container under the flow hood. Discard the empty top of the tube in a large beaker containing glass waste.
  • Carefully wipe the outsides of the Kimax tube with a clean Kimwipe.
  • Knock the graphite-Fe powder from the 6 mm tube into the funneled cathode.
  • Using the portable pin, gently crush the graphite into a powder (if necessary), and work it into the hole.
  • Once the graphite powder has been worked into the hole, slide the pin sleeve over the cathode and insert the pin through the top to fit into the drilled cathode hole.
  • Place the assembly on the lab bench and give it 3 firm strikes with the hammer.
  • Disassemble the cathode holder, remove the cathode and tap it upside down firmly against the top surface of the cathode holder to eliminate any residual graphite from the cathode funnel.
  • If a flat shiny surface is not apparent, inspect the graphite target under a dissection microscope. Each graphite target should be flat and shiny, and contain no loose powder. Repeat pressing if necessary. Excessive loose powder can be picked up with clean magnetic forceps and returned to the cathode hole.
  • Once a flat and shiny surface is achieved, place the cathode into the appropriate position in the 96-well plate.
  • Before moving on to the next sample, clean the cathode holder, the sleeve, forceps, and the press pin as above.
  • Clean and put away all equipment when finished pressing and turn off the laminar flow hood.