How to elute nitrate from 5 mL anion exchange columns and prepare eluant for d¹⁵N and d¹⁸O analysis. (For 2 mL columns, changes in the procedure are denoted with an asterisk *)

Note: Anion resin has the capacity of 1.2 meq/L so 5 mL can hold 6 meq or 6,000 M of NO₃. The cation resin, 50WX has the capacity of 1.7 meq/mL, so 5 mL will retain 8.5 meq. Remember that the anion column will fill up with other anions besides NO₃. The relative selectivity for SO₄ and Cl and NO₃ is 15, 2.3, and 8 (higher number means greater selectivity). But when trying to determine which anions will fill the column first, one must also take into account the concentration. This relationship is complicated, and we do not have a formula that will allow us to predict the exact behavior of the column. 
 

Reagents and Equipment: For faster, easier ordering, please see the Resource Guide.

1. Clean silver oxide until there is < 0.5 ppm of nitrate in rinse water. 

Silver nitrate needs to be rinsed with DI water to remove nitrate. Take about 500 grams of silver oxide, and put this in a large container, such as a 4 L flask or a large plastic bottle. Stir or shake the silver in DI water, and after about 2 hours, the water will be ready to change. Before pouring the rinse water down the drain, the residual silver must be removed. To do this, pour the rinse water through large paper filters and capture the water in a flask. We use Whatman filter circles. Make a cone out of it and pour the water through the cone. Large coffee filters also work. Add NaCl to the rinse water to precipitate the silver as silver chloride. Filter  this water through 0.45 micron filters, then discard  the water. Save the filters and dispose of as waste. It will take about 30 rinses for the silver to be "nitrate free". Initially, the nitrate concentration of the rinse water will be high.Gradually, after many rinses, the nitrate concentration will equilibrate around 2 ppm. Frequent rinsing is the key. Longer rinsing with the same water does not pull off more nitrate.

1. 3N HCL
2. Racks for the columns.
3. 1 M BaCl₂ solution.
4. Sturdy glass stirring rods about 8 inches long,  with the ends smashed down.
5. Isopropanol/ dry ice slushes. 
6. Vacuum pressure bulb with a rubber stopper fitted on the end.  The stopper should fit over the top of the column because it will be used to push the residual acid out from the column.
7. Small stopcocks that fit the bottom of the anion column.
8. No. 1 and no. 4 Whatman filter circles. Fold and make into cones.  Funnels for the circles.
9. Tri cornered plastic beakers.  Glass beakers. 
10. Liquid nitrogen.
11. pH paper, parafilm, rubber bands.
12. Quartz combustion tubes for the d¹⁸O portion of the sample, silver sample boats for the d¹⁵N portion.
13. Metal block with holes drilled in it for the sample boats.  A metal tray that the block will fit in.  Used for freeze drying samples.
14. A 1.5 or 2 quart metal sauce pan used for freeze drying centrifuge tubes.
15. Plastic 50 mL centrifuge tubes with a conical bottom.  Rack for tubes. 
16. Styrofoam sheet or cutting board that is used to insulate the counter top from the container holding the liquid nitrogen. Prevents the counter from cracking due to thermal stress. 

Stripping the 5 mL column:

1. If column is dry, add enough Distilled Deionized water to moisten the resin.  Do not shake because shaking will disturb the layering of isotopic species.
2. Place sample column on rack above a waste beaker and allow excess water to drip out.
3. Place a labeled 100 mL beaker below the column and a stopcock on the bottom of the column.
4. Blow column dry with vacuum pressure bulb Fisher cat# 14-085 . 
5. Close stopcock of column and add 3 mL of 3 M HCl.
6. Wait 30 seconds, allow and column to gravity drip into a beaker.  When dripping stops, blow out remaining eluant trapped in pore space.

Close stopcock and add the second 3 mL addition of 3 M HCl. Wait 30 seconds and give a light push with the pressure bulb. Allow to gravity drip and then blow remaining acid out of the column.
7. Repeat step 7 until 30 mL of 3 M HCl has been run through the column for a total of 10 additions of 3 mL 3 M HCL. *( use 15 mL of acid for 2 mL resin column)

Neutralizing sample:

1. Prepare Dewar flask with a slush made from isopropanol and dry ice.
2. Weigh about 16 g pure silver oxide (which has been rinsed and tested for background nitrate).  * (use ~6 g of silver oxide for a 2-mL column).
3. Add the silver oxide in 4 portions to the sample while holding the sample beaker immersed in the slush to the level of the liquid.  Mix the sample between each addition with a flat bottomed stirring rod.  Be sure to allow the heat of reaction to dissipate between additions of Ag₂O.
4. Mix the sample until reaction with the Ag₂O is no longer apparent and un-reacted (black colored) Ag₂O remains in the beaker. Continue crushing and stirring for another minute.  In most cases the milky consistency will diminish leaving the solution clear.
5. Check pH by paper. It should be approximately pH 5 or 6.  If solution is neutral and clear, filter sample through a rinsed no.1 or 4 Whatman filter paper into a labeled 100 mL beaker.  If the solution is neutral but remains cloudy, vacuum filter through 0.45 micron filter nylon in labeled 100 mL beaker.

1. Add 4.5 mL of 1 M BaCl₂ for every 5 mL of resin in the original column.  4.5 mL of 1M BaCl₂ is more than enough to precipitate 3000 µMoles of Sulfate or as much sulfate as 5 mL of resin can hold. *(for 2-mL columns, use 2 mL of BaCl2)
2. Recap containers, shake, and place in refrigerator over night.
3. Pink or white precipitate should form.
4. If solution is clear filter sample through a rinsed no.4 or 1 Whatman filter paper into a labeled 100 mL beaker.  If the sample solution remains cloudy, vacuum filter through 0.45 micron filter in labeled 100 mL beaker.
5. Gravity drain and blow dry a cation column with 9 mL of resin (2 mL resin for every 1 mL of 1M BaCl₂).  *(for 2-mL columns, use 4 mL of cation resin)
6. Pour the sample through a cation column collecting eluant in a labeled 100 mL beaker. Rinse with 5 mL DI water.
7. Add 1 g pure silver oxide to sample to remove the Cl which has been added to the sample. Stir with flat bottomed glass rod at least 1 minute. In most cases the milky consistency will diminish leaving the solution clear.
8. Check pH using pH paper. The pH should be 5 or 6. 
9. Filter sample through a rinsed no. 1 or 4 Whatman filter paper into a labeled and pre- weighed plastic bottle for storage in freezer.

Note: Procedure for stripping 2-mL anion columns is the same except the column is  stripped with 5 additions of 3 mL each of 3M HCL, 6 g of silver oxide is used, 2 mL of 1M BaCl₂ and 4 mL cation resin columns are used.
 

Removing the reserve and splitting d¹⁵N and d¹⁸O samples:

1. Weigh sample bottle and sample. Calculate total volume of sample. 
2. Multiply requested ¹⁵N split percentage by the total volume of sample to get the volume of  the ¹⁵N split.
3. Place a 50 mL plastic centrifuge tube in a beaker on the balance, label and tare.  Pipet the ¹⁵N volume calculated in step 2 from the sample bottle to the plastic beaker.
4. Multiply requested ¹⁸O split percentage by the total volume of sample to get the volume of  the ¹⁸O split
5. Place 100 mL Pyrex beaker on the balance, label and tare.  Pipet the ¹⁸O volume calculated in step 4 from the sample bottle to the shaker flask.
6. The volume of sample in the sealable plastic container is your reserve. Tightly cap and label with ID number, volume and concentration. Place in labeled Ziplock bag with any similar sample reserves. Place bag in freezer.

d¹⁵N sample processing:

1. Place 50 mL centrifuge tube in a rack and freeze slowly from the bottom with several additions of liquid nitrogen to the tray. Place a sheet of Styrofoam beneath the container holding the liquid nitrogen to avoid cracking the counter top. After freezing, cover beakers with Parafilm, secure with rubber band and poke three small holes in the Parafilm near the rim of the beaker. This is necessary because some of the solid will spatter on the middle of the parafilm, and it is less likely to exit the container from the side.
2. Place in freeze drier and freeze.
3. Redissolve sample in 2 mL DI water, washing sides and scraping bottom. 
4. Freeze tube slowly from the bottom with several additions of liquid nitrogen. Be sure the tube does not tip over as they are very light. Cover tube with Parafilm, secure with a rubber band, and poke three small holes into the Parafilm.
5. Freeze dry.
6. Redissolve in 800 µL DI water, washing sides and scraping bottom.
7. Pipet 200 µL sample into each of four 11 x 7 mm silver boats held in a metal block. Note:  Aluminum boats cause the glass in the Optima to melt and tin boats are corroded by silver nitrate).
8. Lightly pinch the tops of the boats, place metal block containing silver boats into a metal tray and slowly freeze samples from bottom up with several additions of liquid nitrogen to the  tray. Be sure not to get liquid nitrogen in the boats or above the top of  the metal block.
9. Freeze dry.
10. Un-pinch boats and add approximately 2 to 5 mg of commercially produced dry granulated sugar.
11. Fold tops of boats closed and fold boats into a small cube making sure to remove all the air.  If the boat breaks place the broken boat into another clean boat and crimp. Place sample cubes into labeled plastic sample box.
12. Submit for mass spec analysis. 

d¹⁸O sample processing

1. Place beaker into tray and freeze slowly from bottom with several additions of liquid nitrogen into the tray. Cover beaker with Parafilm with three holes at the edges.
2. Freeze dry.
3. Redissolve sample with 2 mL DI washing sides and scraping bottom of beaker.
4. Transfer into top labeled quartz tube, freeze sample in tube slowly from the bottom by lowering it into a Dewar containing a slush of dry ice and isopropanol (tubes must be labeled near the top to avoid removal by alcohol), cover with Kimwipe secure with a rubber band and freeze dry.
5. Add 4-5 mg powder graphite to tube through long tube funnel. Label the bottoms of tubes so the will not be lost when torched
6. Place on vacuum line and pull down to 20 millitorr for 30 minutes. Cover bottom of tubes to block light.
7. Torch tubes.
8. Fill out muffle furnace tracking sheet so samples will not be confused when the label is combusted off.
9. Place samples in furnace, program furnace for ¹⁸O combustion (heating to 875^oC for 4 minutes and then cooling slowly). Run furnace.
10. Extract CO₂ on line and submit for Mass Spec analysis.

1. Redissolve the sample in 2 mL and out into combustion tubes.
2. Cut a small square of Kimwipe and use it to cap the top of the tube so that material doesn't blow out of the tube when freeze drying.  Use a rubber band to fasten the Kimwipe to the tube. 
3. Freeze dry.
4. Put in the following combustion ingredients into the tubes in the order listed:  1.5 g CuO, 350g of  CaO, 2 g CuO, 2.5 g Cu.   Use long stem funnels.  Preparation of these ingredients is described by Kendall and Grim, 1990. 
5. The lower halves of the tubes are covered with paper or foil jackets to protect them from light (silver oxide is photo reactive).

Appendix A

Preparation of Cation columns:

1. Prepare large plastic columns with frit disk and end cap. Mark a 9 mL line on the side of the column. 
2. Rinse AG 50 W-X 8 100-200 mesh hydrogen form resin (cat.#. 142-1441) in a 500-1000 mL beaker with DI water until the rinse water no longer turns a red color or at least 5 times.
3. Add sufficient DI water so that the resin can be pipetted. 
4. Pipet two 4.5 mL portions of resin into a large column.
5. Allow resin to settle, and then pipet a little more resin into the column until the 9 mL is reached.
6. Tightly cap the top of the column. 

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The URL of this page is: http://wwwrcamnl.wr.usgs.gov/isoig/guidelines/nitrate/Recovery.htm/
Page created by Carla Chamberlain, Daniel Steinitz and Cecily Chang.
Please contact Carol Kendall (ckendall@usgs.gov) for questions and comments regarding this page.
This page was last changed on July 19, 2001.
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