Application: For waters with nitrate concentrations >20 micromoles and when total DOC load is  <30 mg. 

Small columns:  This method is suited for waters where nitrate 100 mmoles of nitrate can be collected with <3L of water, or where DOC concentrations are such that the DOC load of the sample does not exceed 30 mg of C.  This method has been successfully used for groundwater samples. Samples are collected on anion exchange columns with 2 mL of resin. 

Equipment:  For faster, easier ordering, please see the Resource Guide that follows this instructional paper. The first three items are available from BIO-RAD Laboratories.
 

1. Anion exchange resin AG1-X8 200-400 mesh cat.# 140-1451
2. Anion exchange columns (2 mL), cat.# 731-1550 $52/ pkg . of 50
3. Pre-filled columns are also available. cat.# 731-621 $130/pkg. of 50
4. If the sample volume is <2 liters, use Econo-Column Funnel 250 mL, cat.#731-003 $52/pkg.of 5.
5. If the sample volume is >2 liters, you will want to use a collapsable 'cube container to hold your water sample. You will also need rubber stoppers to fit on the top of the column and flexible tubing to connect the 'cube container to the anion column. Drill a hole in the stopper, insert a short piece of inflexible tubing through the stopper and use the flexible tubing to connect the sample to the anion column. A disposable 1 mL pipet tip with the end snipped off works nicely.  Use a clamp to secure the stopper to the column. 
6. Small stopcocks that fit on the bottom of the column. Cole Parmer, cat.# E-30600-01. 

1. Rinse the resin. Put some resin in a beaker and add ~10 excess volume of  DI water. Let the resin sink to the bottom of the beaker and discard the water. Repeat 3 times to rinse the resin. Example for 50 mL of resin, rinse with 500 mL of water. 
2. Put the columns in a rack and pack columns with 2 mL of resin.We find it convenient to fill a lot of columns at once and then store them in the refrigerator until we need to use them. Mark off the 2 mL level with a pen.  Pour the resin slurry into the column or use a 5 mL pipet.The column is ready to use when the resin fills up to the 2 mL mark, after the water has drained out of the resin.
3. If you are going to store the columns, cap the bottom of the column and add DI water to the 6 mL mark.This keeps the resin from drying out. Cap the top and store columns upright.

The sample should flow through the anion column no faster than 2 L per hour. Use the small stopcocks to adjust the flow rate.  With time it, may be necessary to open the stopcock in order to keep the flow rate up. 

If your sample volume is <2 L use the column funnels.  Attach the funnels to the column and slowly pour the sample through.  The funnels hold about 250 mL, so you will have to pour more sample in as the water drips through the column.To verify that nitrate did not bleed through the column, collect he eluant and measure for nitrate. 
 
 

Large columns: This method was designed for waters where nitrate concentrations range from 1-10 mM, necessitating the collection of large volumes of water to collect 100 mM of nitrate.  It was originally designed for alpine waters (snow, snowmelt).  Even though the DOC concentration of these waters is low, the load of DOC can be appreciable if 20 or more liters are needed.  To accommodate the larger volume of water and the DOC load, we use larger columns with more resin (5 mL), a courser mesh size of resin so that the sample will flow through faster, and we put a 5 mL cation column in front of the anion column to minimize DOC accumulation on the anion column.  To verify that nitrate did not bleed through the column, collect the eluant and measure for nitrate. See Nitrate Collection Poster for a graphic example of Method II. 
 

Equipment - Column equipment: For faster, easier ordering, please see the Resource Guide that follows this instructional paper. Items 1-4 available from BIO-RAD Laboratories.

1. 20 mL columns - Econo-Pac Column  cat.# 732-1010, $85/pkg. of 50.
2. Fill with 5 mL of AG2X-8 anion resin (100-200 mesh), cat.# 140-2441, $210/500g per bottle. 
3. Econo-Column Funnel, 250 mL, cat.# 731-003,  $52/pkg. of 5. May be useful for loading 1 or 2  liter samples.
4. Biorad cation exchange resin- AG 50W-X8, (100-200 mesh), cat.# 142-1441, $210/500g per bottle. 
5. Stopcocks to regulate the flow coming out of the cation column. Nalgene stopcocks, cat.# 6460-002, $111/pkg. of 6.
6. Small stopcocks for the end of the anion column. Source: Cole Parmer, cat.# E-30600-01, $16.25/pkg. of 10. Cole Parmer, ph.1-800 323 4340.
7.  Number 6 rubber stoppers with a hole.These fit on top of the columns. Kjheldahl stoppers, Fisher cat.# 14-14913.
8. Joint clamps, stainless steel, size 35/20. Used to clamp stopper to column. Makes a tight seal.Crimp in one side of the clamp so that it grips the stopper. Fisher cat.# 05-8856, $151/pkg of 12.
9. Straight connectors, used to attach the columns. Daigger cat.# LX28000B, $2.95/pkg of 10. Diager ph.1-800-621-7193.  (Note: 1 mL pipet tips with the end snipped off are also useful)
10. Nalgene tubing, 3/8 I.D., to fit onto the valve of the Cube Container. Neoprene tubing, with 1/4 ID, used to connect the cation column to the anion column, using a straight connector.
11. Collapsible Cube containers to put the water in when loading the columns. 
12. HCL 1.2 N (10% when taking straight from a 12 N bottle) for rinsing the plasticware used to collect and store your water samples. 
13. If you have the good fortune to do this in the lab: Plastic garbage cans (20 gal) with lids to melt water in, for processing samples. 

DOC equipment

1. Silver and GF/F filters for DOC filtration. Filters must be combusted prior to filtration.(see step 2, in loading the columns). Glass fiber filters serve as a prefilter.Order from PORETICS,111 A Lindbergh Ave.  Livermore, CA, 94550.  Ph: 1-800-922-6090. Silver Filters, cat.# 53014(0.2 micron,47mm diameter),$244.80/pkg. of 100 (government pricing). Also available in pkgs.of 25.GF/F type filters, glass fiber, cat.# 88231, $21./pkg.of 100.Combust silver filters at 200 C for half an hour. Do not over bake. Note that there is a wrong and right side, dull side up! Combust glass filters 2 hours at 400 C.Combust filters in a glass container, a beaker covered with tin foil or a civered glass petri dish . Store in said containers. 
2. Filtration unit that will accept 47 mm diameter filters. Unit should be stainless steel or Teflon, to avoid contamination that can be introduced with a glass unit. 
3. Glass bottles for the DOC sample. Boston round bottles that hold 100 mL are good.Obtain bottles with Teflon cap liners. Remove the cap and place a piece of tin foil on the top of the bottle.Combust the bottles with the foil tops in a furnace, 400 C for 2 hours.  Do not combust the caps as they will melt.
4. Combust about twenty, one and a half inch squares, of aluminum foil in a glass container as in step 1.Combust with bottles. 
5. Filter forceps.

Use a Hach Kit to make field measurements or have your samples analyzed by a lab. If you decide to have your samples analyzed by a lab, you must take a small sample. 

1. HDLPE bottles, 100 mL.One bottle for each sample. 

If  your nitrate concentrations are high enough so that you need only a few liters of water, then you can filter your sample with a disk filter. Some people like to use the large,147 mm filters (0.45 mm). Use a nylon filter or a polycarbonate filter. Do not use a filter made of cellulose nitrate. If the filter clogs, you can use a GF/F type glass fiber filter (0.7mm) as a prefilter. Put the glass filter on top of the 0.45mm filter. The filter must not have a binder in it. Poretics makes a GF/F type filter for a more affordable price than Whatman.  If you have a lot of material in your sample, then use a glass fiber filter with a larger pore size, (1 or 2 mm) but be sure it does not contain a binder. If you must pass large volumes of water (>20 L) through a column, because your NO₃ concentration is low, use the Gelman groundwater capsule. Filtration is rapid, thereby saving you a lot of time. 

1. Gelman "groundwater sampling capsules"  (0.45m m filter) polystyrene, cat.# 12176,  $150/for a box of 10. Order from Fisher Scientific.
2. Peristaltic pump, used with the capsules. 
3. Silicone or neoprene tubing, 3/8" ID, with walls that are 1/16 or 1/32 for the peristaltic pump or some type of tubing that won' t adsorb or disturb nitrate. Necessary only if you are using the Gelman capsules. 
4. Ring stands and clamps. Support rack for columns.
5. Vacuum Pressure bulb, Fisher cat.#14-080.

Determine your nitrate concentrations!

1. Rinse each plastic bottle, the collapsible containers, and garbage cans, in the HCL solution.Rinse three times with DI water.If you are collecting a small sample for nitrate determinations, then the small sample bottles must be washed and rinsed too.With the smaller bottles, we advise that you fill them with DI and let them soak overnight. 
2. Currently, we are requesting a minimum of a 100 mmoles of nitrate on each column.To determine the volume of sample required to flow through the column, you must know the nitrate concentration in each of you samples.To do this, filter the sample through a 0.45 micron nylon or polycarbonate filter to remove particulates. 

Rinse your sample bottle with a few mL of your sample first, to condition the surface of the bottle, discard, and then fill with your sample. Fill you small sample bottle (100 mL) with your sample.Use this sample to determine your nitrate concentration and calculate the volume of water you will need to load 100 mmoles of nitrate. If nitrate concentrations are low, 2 mM or less, it may be too difficult to obtain 100 mmoles nitrate. In this case, collect as much as you can.The sample will probably be analyzed for N-15 only. Record the nitrate concentration, liters of sample passed through the column, and mass of nitrate loaded onto each column in the spreadsheet. Do this for each sample.

1. Determine your Cl, SO₄ and PO₄ concentrations and calculate the load in micromoles (concentration times vol) that will be loaded onto the anion column.These can interfere with our analysis. We have to remove them, before the sample can be analyzed.  Estimate these concentrations, if you don't know them. Enter this data in the appropriate place on the spreadsheet. Note, that the masses of these constituents, will be calculated on the spreadsheet once you enter the appropriate information. One mL of resin will absorb 1.2 meq of anions, so 5 mL is 6 meq or 6000 mmoles. If only nitrate were absorbed, the column could hold 6000 mmoles of nitrate. Remember, the column will adsorb all anions. If your samples contains large quantities of other anions, Cl in particular and DOC, you will have to use more than one column to obtain the required 100 mmoles of nitrate. 
2. Determine DOC concentrations. The DOC concentrations of the sample, along with the mass in mg loaded onto the column, should be known.This is because dissolved organic carbon (DOC) can adsorbing onto the sites that the nitrate would adsorb to, potentially clogging the anion column.  This would lower the ability of the column to retain nitrate, and possibly interfere with the d¹⁸O. 

Filter about 100 mL of your sample through the silver filter, directly into the combusted glass bottle. (Rinse the bottle out with a few mL of your sample, to condition the bottle. Discard the sample and fill). If your sample seems to clog the silver filter, put a glass fiber filter on top, as a prefilter.  Remember, the silver filter goes dull side up. Save the silver filters, and combust again for reuse. Replace the foil and screw the lid on. Refrigerate and send to a lab for DOC analysis. Do not freeze.  The silver acts as a bactericide. If your samples can be analyzed within 24 hours, they can be filtered just with the glass filter. Enter the information about DOC on the spreadsheet. 

1. Filter the sample. If you have a lot of samples and/or large volumes, store your water in a cool place to discourage bacterial growth. 
2. Put the sample in a cube container. 
3. Note, the sample contains gases that can cause bubbles to form in the resin. Bubbles slow the flow through the resin and reduce your efficiency. Gases can be removed by allowing the sample to come to room temperature. We have found that stirring the sample for half an hour, under a vacuum, helps to remove gas. Insert a piece of tubing through the stopper hole. Put a piece of saran wrap or parafilm over the stopper so that the sample will not contact the rubber directly. Attach tubing from the container spigot, to the tubing, in the stopper. Push the stopper down firmly so that it makes a tight seal, clamp down. 
4. Insert a stopper with a hole, into the cation and anion columns.To connect the cation column to the anion column, use a straight connector and a piece of 1/4 ID tubing. Connect a short piece (7 cm) of tubing to the bottom of the cation column and insert a straight connector to the other side of the tubing. Insert the straight connecter into the stopper hole of the anion column. 
5. Attach a piece of 3/8 inch, ID tubing, to the spigot of the cube container. 
6. Connect the cation column to the tubing using your connectors or Nalgene stopcocks. Remember the longer the head distance, the more pressure there will be on the column, and the water will flow through more quickly.
7. Attach the small stopcock on the bottom of the anion column to regulate the outflow.
8. Stabilize the column (use a rack or ring stand with clamps) to prevent it from swaying. If you are using large volumes of water, you may want to put a large stopcock between the columns so that you can stop the flow from the cation column, when you need to put on a fresh anion column. 
9. Collect the eluent and measure the nitrate concentration. You want to verify that there was no bleed-out of nitrate. 
10. Fill each column with about 5 mL of DI water or with the sample, to prime the flow. Otherwise, when you open the stopcock of the cube container, it will be difficult for the sample to flow through (back pressure). 
11. Allow the sample to flow through the cation and anion columns. As the sample flows through the columns, bubbles may develop in the resin. Insert a small glass rod or a straightened paper clip and tease out the bubbles. Bubbles cause the water to move around the them, causing sample flow to slow down, thus decreasing the efficiency of the resins ability to absorb nitrate. Do not stir the resin or shake the column! 
12. Gravity drip the sample through. We think a rate of 2 L per hour is good. Make a note of how fast your samples drip.Whether the sample can drip faster, has yet to be determined. If the flow rate is too fast, use the stopcock to slow the flow. As the sites fill, the flow rate will slow. Adjust as needed.
13. As the sample is loaded on the column, the resin will begin to discolor.When all the resin is discolored or when the column ceases to drip, change to a new anion column. Discoloration usually indicates a DOC problem. Remember, you may need to use more than one column per sample to obtain your 100 µmoles of nitrate. 
14. After samples have passed through the column(s), blow or vacuum excess water from column. Fill the column to the top with DI water, cap top and bottom and refrigerate. Seal the top and bottom with parafilm, so they don't open upon shipping. A one-way bulb attached to an appropriately sized stopper works well to blow the columns dry.

1. Store columns refrigerated. 
2. When shipping, place columns in double "Zip-Lock," plastic bags and ship in coolers.
3. That's it! You are finished.  Send the columns and your spreadsheet (Quattro Pro or Excel)  to us (see instructions for spreadsheet Sampledata.htm). Include a hard copy of the file, as well as a disk, when you send us the information.

1. BIO-RAD Laboratories, 200 Alfred Nobel Drive, Hercules, CA 94547, PH:1-(800)424-6723. BIO-RAD
2. Cole-Parmer Instrument Co., 625 East Bunker Court, Vernon Hills, IL 60061-1844, PH: 1-800-323-4340. COLE-PARMER
3. Daigger, 675 Heathrow Drive, Lincolnshire, IL 60069-4206,PH:1-800-621-7193. DAIGGER
4. Fisher Scientific, U.S. Headquarters, 585 Alpha Drive, Pittsburg, PA 15238, PH: 1-800-766-7000. FISHER
5. Gelman Sciences, 600 Wagner Road, An Arbor, MI 48103-9019,PH: 1-800-521-1520. GELMAN SCIENCES
6. HACH Company, World Headquarters, P.O. Box 389, Loveland, CO 80539-0389, PH: 1-800-227-4224. HACH
7. Poretics Corporation, Order Service, 111 Lindbergh Avenue, Livermore, CA 94550-9261, PH: 1-800-922-6090. PORETICS
8. Whatman LabSales, P.O. Box 1359, Hillsboro, OR 97123-9881, PH: 1-800-942-8626. WHATMAN

 

 
 
 
 
 
 
 

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The URL of this page is: http://wwwrcamnl.wr.usgs.gov/isoig/guidelines/nitrate/collection.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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