TITLE: Distillation of total reduced inorganic sulfur from marine sediments
AUTHORS: Patrick D Larkin, Sebastian  Rubiano-Rincon

[DESCRIPTION]
This procedure can be used to precipitate H2S as Ag2S from the Total Reduced Inorganic Sulfur (TRIS) fraction of marine sediments. 

[GUIDELINES]
This procedure takes 6 - 8 hours, from distillation setup to post-takedown clean-up. It takes an additional 1-2 hours on the following day to clean and dry glassware that has soaked overnight.

[STEPS]
SECTION: Distillation Apparatus Set-Up
4. Connect a 250 ml Buffer (Gas washing) Bottle (D) to the Connection Adapter for Condenser (C). Attach the glassware to the framework with clamps (not shown):
  

SECTION: Distillation Apparatus Set-Up
5. Insert a Trap Orifice Tube (F) into one of a Smog Bubbler Trap Bottle (E).Insert a Trap Fritted-Tip Tube (G) into the second Smog Bubbler Trap Bottle (E’). Connect Trap 1 (F+E) to the Buffer Bottle (D) and Trap 2 (G+E’) to Trap 1. Attach the glassware to the framework with clamps (not shown):
  

SECTION: Distillation Apparatus Set-Up
7. 
  

SECTION: Distillation Procedure
8. Carefully remove from the distillation apparatus the Four-Necks Round Bottom Flask (A), the Buffer Bottle (D), and Traps 1 (F+E) and 2 (G+E’).
SECTION: Distillation Procedure
9. Fill Buffer Bottle (D) with 200 mL of 0.05 M KHP Buffer. Secure the gas dispersion tube to the wash bottle using metal springs (S). Re-attach to the distillation apparatus. 
SECTION: Distillation Procedure
10. Fill traps 1 (E + F) and 2 (E' + G) with 15 ml of 0.1 M AgNO3. Secure the orifice (F) and fritted (G) tubes to the traps with metal clips (R). Re-attach to the the distillation apparatus.

SECTION: Distillation Procedure
11. Secure the connections between the Connection adapter (C), Gas Washing Bottle (D), Orifice Tube (F), Fritted Tube (G) and Hose Connection Adapter (L) with stainless steel Pinch-Type Clamps (Q) (see Figure 8).
SECTION: Distillation Procedure
12. Thaw the sediment sample without opening its container (oxygen contamination). Shake to mix.
SECTION: Distillation Procedure
13. Transfer the sediment sample to a glove box, if available. If not, place a glove bag (T) on the benchtop and connect it to a source of Nitrogen gas using the plastic coupler provided and a length of plastic tubing. 

SECTION: Distillation Procedure
14. Purge the glove bag for 3-5 minutes with N2 gas while inserting the following equipment: the four-neck round bottom flask from the distillation apparatus (A), a beaker for supporting the thawed sediment sample tube, a balance, a spatula, four rubber septa (P), and a stir bar.
SECTION: Distillation Procedure
15. Close the sleeve of the glove bag and inflate it with nitrogen gas. Once inflated. stop the N2 flow. Make sure the bag is not fully inflated as it can rupture. 
SECTION: Distillation Procedure
16. Using the gloves on the bag, place the Four-Necks Round Bottom Flask (A) on the balance and zero it. 
SECTION: Distillation Procedure
17. Carefully transfer ~10 g of thawed sediment sample to the zeroed Four-Neck Round Bottom Flask (A) through one of the middle stems. Record weight to the nearest 0.1 mg. 
SECTION: Distillation Procedure
18. Add the stir bar to the Four-Necks Round Bottom Flask (A) and close all four openings with rubber septa (P).
SECTION: Distillation Procedure
19. Open the nitrogen-filled glove bag and take the closed Four-Necks Round Bottom Flask (A) back to the fume hood. 
SECTION: Distillation Procedure
20. Insert the Temperature Sensor (N) all the way into the bushing associated with the mini-adaper (J). Add a #008 FETFE O-ring to the tip of the sensor and push up until it makes contact with the bushing. Screw the probe/bushing/O-ring combo into the #7 (24/40) glass mini-adapter (J). Connect the plug of the sensor into the temperature controller. 
SECTION: Distillation Procedure
21. Take the N2 Gas Dispersion Tube (H) and insert its fritted tip into the bushing associated with the midi-adapter (I). Add a #012 FETFE O-ring to the fritted end and push it up until it makes contact with the bushing. Screw the tube/bushing/O-ring combo into the #11 glass Midi-Adapter (I). Attach the other end of the tube (H) to the hose connected to a N2 source. Start a very light gas flow. (Our standard for very light is one where we can barely feel the gas pressure from the dispersion tube on our cheek).
SECTION: Distillation Procedure
22. To minimize the exposure of the sediment to oxygen, insert the Midi-Adapter (I) with the N2 Gas Dispersion Tube into one of the openings of the Four-Necks Round Bottom Flask (A). At the same time, remove the septa (P) from the opening where the Coiled Condenser (B) is inserted.
  

SECTION: Distillation Procedure
23. Quickly connect the Four-Necked Round Bottom Flask (A) to the Coiled Condenser (B). Next, insert the Mini-Adapter (J) with the Temperature Sensor into one of the two remaining openings of the Four-Necks Round Bottom Flask (A). Leave the remaining opening closed with a rubber septa (P). Secure the Mini-adapter (J), Midi-adapter (H) and Coiled Condenser (B) to the necks of the flask with metal clips (R).
SECTION: Distillation Procedure
24. Connect water in and out hoses to the corresponding inlets from the Coiled Condenser (B) (see Figure 8). Start the water flow.
SECTION: Distillation Procedure
25. Flush the distillation apparatus for 10 min with N2 gas with a continuous flow rate of approximately 5 to 10 bubbles per second observed in traps 1 and 2. 

If necessary, connections may be wrapped with PTFE (plumber’s) tape to minimize loss of gas during the distillation. 

At this point, the distillation apparatus is ready for use 
SECTION: Distillation Apparatus Set-Up
2. On the left side of the framework, place on top of a hot/stir plate a hemispherical heating mantle (O) and and a 250 ml Four-Necked Round Bottom Flask (A):
  

SECTION: Distillation Apparatus Set-Up
1. Set up the framework for the distillation using two support stands and three metal bars under a fume hood:  

SECTION: Distillation Apparatus Set-Up
3. Insert a Coiled Condenser (B) into the top-middle opening of the Four-Necked Round Bottom Flask (A). Insert the Connection Adapter (C) in the top-opening of the Condenser. Secure the Adapter (C) to the Condenser (B) with a metal clip (R). Attach the glassware to the framework with clamps (not shown):
  

SECTION: Distillation Apparatus Set-Up
6. Assemble a NaOH trap as follows
  

SECTION: Distillation Apparatus Set-Up
6.1. Obtain a large rubber stopper and make two holes for fitting a disposable glass pipette and a polypropylene hose.
SECTION: Distillation Apparatus Set-Up
6.2. Obtain a polypropylene hose and attach to one of its ends the Gas Dispersion Tip (M). 
SECTION: Distillation Apparatus Set-Up
6.3. Obtain a disposable glass pipette and cut a 5 cm section off of its stem.
SECTION: Distillation Apparatus Set-Up
6.4. Insert the 5cm glass section into one of the holes of the rubber stopper.
SECTION: Distillation Apparatus Set-Up
6.5. Insert the free end of the polypropylene hose into the other hole of the rubber stopper.
SECTION: Distillation Apparatus Set-Up
6.6. Add 250 mL of 1M NaOH solution to the 500 mL Erlenmeyer flask (K). 
SECTION: Distillation Apparatus Set-Up
6.7. Insert the Gas Dispersion Tip (M) into the Erlenmeyer flask (K) with the NaOH solution. Close the trap with the stopper.
SECTION: Distillation Apparatus Set-Up
6.8. Insert the Hose Connection Adapter (L) into the other end of the polypropylene hose. Connect to trap 2 with a stainless steel pinch-type clamp (Q). 
SECTION: Distillation Procedure
26. Collect 10 mL of 50% EtOH solution with a plastic syringe/stainless steel needle (U) and inject into the Four-Necked Round Bottom Flask (A)  through the opening covered by the septum (P). Turn the stir plate on to mix with the sediment. This shall facilitate reflux condensation during the distillation. 
SECTION: Distillation Procedure
27. Collect 50 mL of 6M HCl degassed solution with a plastic syringe/stainless steel needle (U).
SECTION: Distillation Procedure
28. Inject into the Four-Necked Round Bottom Flask: (i) 50 mL of 6M HCl, followed by (ii) 50 mL of 1M reduced Cr2+ solution (dx.doi.org/10.17504/protocols.io.b2b9qar6). Be careful with the positive pressure induced by the nitrogen flow into the system when injecting the reagents. 
SECTION: Distillation Procedure
29. Set the temperature of the Controller (N) to 100-105°C and gently boil the sediment solution for 2 hours, while continuing to stir and maintaining a constant N2 flow.
SECTION: Distillation Procedure
30. After 5 to 15 min of having injected the reagents, a black precipitate (Ag2S) should start forming in the AgNO3 trap 1 (F + E). As the precipitate begins to form, the solution will darken. Distillation progress can be monitored by a clearing of such darkened solution. When most of the H2S has been liberated and precipitated as Ag2S, the solution will become clear. 
SECTION: Distillation Procedure
31. Once the distillation is complete, stop the N2 flow and carefully dismount Traps 1 and 2 containing the solid Ag2S.
SECTION: Distillation Procedure
32. Collect the precipitated Ag2S by vacuum filtration, using a Büchner funnel, a (pre-weighed) disk of WhatmanTM 42filter paper, a rubber bung, and a vacuum flask. Use a DI water squirt bottle to aid transfer of the precipitate from the trap bottle, and to wash the precipitate.
  

SECTION: Distillation Procedure
33. Transfer the Ag2S-containing filter paper to a small ceramic crucible. Cover with aluminum foil and punch 8-12 holes using a pen knife or 16-gauge syringe.
SECTION: Distillation Procedure
34. Lyophilize for 2 hours to dry.
SECTION: Distillation Procedure
35. Weigh the precipitate-containing dried filter paper. Subtract the weight of the filter paper to estimate the amount of Ag2S recovered. Carefully transfer the Ag2S from the filter paper into a small (3 ml) glass vial. 
SECTION: Distillation Procedure
36. Store Ag2S sample in a dessicator cabinet until isotopic analysis
SECTION: Cleaning Instructions
37. Rinse each piece of glassware with DI water to dispose of any solid or liquid into a waste bottle. Make sure every chemical is listed on the waste bottle.
SECTION: Cleaning Instructions
38. Non-glassware items may be washed in 1% solutions of Alconox or Citranox. Rinse with Distilled H2O (3x) followed by DI H2O (1x). Air dry overnight.
SECTION: Cleaning Instructions
39. Submerge glassware overnight in warm, 1% Citranox solution. In the morning wash and rinse glassware with Distilled H2O (3x), followed by a DI H2O rinse (1x). Dry in oven for 2 hours at 100oC or air dry.