This note describes the collection and preparation of nitrate samples for isotopic analysis using both the original Silva et al. (2000) method and the modified Chang et al. (1999) method.  Information about interpretation of nitrate isotopes can be found in Kendall (1998) and Kendall and Aravena (2000). The references are given below.

Using  d¹⁵N and  d¹⁸O of nitrate to identify nitrate sources and to determine whether certain biological transformation have occurred can be more effective than using d¹⁵N alone.  This is because of the overlap between nitrate sources when relying on  d¹⁵N alone (Table 1). 

For instance,  it is easier to determine if denitrification has occurred using both isotopes.  This is because as denitrification occurs, the d¹⁵N and  d¹⁸O of the remaining nitrate becomes heavier, in a ratio of about 2:1 for d¹⁵N/d¹⁸O.  This technique is also useful when nitrate has been produced by nitrification, as two of the oxygens are derived from H₂O and one from dissolved O₂.
 
 

In this paper we describe two methods for the collection, concentration and procesessing of nitrate for d¹⁵N and  d¹⁸O analysis. In the first section, we describe how to collect the sample. In the second section, we describe how to process the sample in the laboratory.  Instructions are also provided for researchers who are sending us samples for laboratory analysis. 

Sampledata.htm   See example of sample analysis sheet and link for a blank sample analysis sheet.

One method, hereto referred to as the CAT AG2X, (Method I Collection) is appropriate for water samples with either low nitrate concentrations (~1-20 mM NO₃), or with dissolved organic carbon (DOC) concentrations (>15 ppm). This method uses 5 mL anion and cation exchange columns. The cation column offsets DOC accumulation on the anion column (see below). 

Typically, stream, rain, and snow melt waters have low nitrate and low DOC (1-8 ppm) concentrations.  If your samples have nitrate concentrations >20 µM, or if the total load of  DOC  (the cumulative mass of DOC) in your sample is >30 mg then you should follow the instructions for high nitrate concentration samples, which uses 2 mL exchange columns (AG1X method or Method I Collection).  The idea is straight forward: filter the water through a filter (pore size 0.45 micron) to remove most particulates, determine the NO₃ concentration of your water sample, and  pass enough water through the anion column to collect a minimum of 100 mmoles NO₃ on the column while preventing DOC accumulation on the anion column. (Note: 1 mg of  N-NO₃ micromoles of N). 

When nitrate concentrations are low, large volumes of water are often required (20-80 L) to obtain 
100 micromoles NO₃. With large sample volumes, the cumulative DOC load can be appreciable. The worst case is for summer soil waters, where NO₃ concentration may be only 1 µM and the DOC load is high (DOC 45 ppm).  Such a sample would require 50 L of sample, resulting in 2250 mg C!   Dissolved organic carbon gums up the columns and reduces the ability of the resin to retain NO₃, because DOC competes for exchange sites that would otherwise be available for NO₃.  For this reason, if the DOC load is >30 mg, we recommend the CAT AG-2X method. 

Both the CAT AG-2X (5 mL column) and AG1X (2 mL column) methods use anion resin with an anion retention capacity of 1.2 meq/mL.  In order to decide which method is suitable, you must calculate the total anion load in your sample and compare the load against the capacity of the anion column.  It is very important not to overload the anion exchange column since the resin retains anions in addition to NO₃ anions.  Therefore, if you need to use 4 liters of water to collect 100 micromoles of NO₃, but the concentration of other anions is high enough so that the collective load of total anions exceeds the capacity of a 2 mL resin column (2.4 meq), then you must use the CAT AG-2X method, which uses 5 mL of resin. 

To determine the appropriate method to be used for your study, you must know the concentrations of nitrate, DOC and other major competing anions (e.g., Cl and SO₄ ) a priori.  Use existing historical data if recent chemical information is not available.

For instructions on how to collect a sample for DOC analysis, please refer to the DOC section.
 
 

1. To remove particulates that will clog the exchange column, use a filter that has a pore size of  0.45 microns.  If your sample has a high concentration of particulates, use a courser grade filter first, then allow the sample to flow through the 0.45 micron filter.  This can be done most conveniently in line, using Gelman water capsules (see equipment list below).  Failure to use the proper pore size filters will result in extended sample collection times and frustration.
2. If the sample flow-through rate slows to less than 1 liter an hour, use another anion column.  It's perfectly ok to use more than one column for a given sample.  You will simply combine the eluted nitrate from your multiple columns. 
3. Even if DOC concentration is low, a large sample volume means that you can potentially accumulate a large mass of DOC.  Never underestimate the potential mischief that DOC accumulation inflicts on your sample.
4. Problems occur when the column is overloaded.  Precautions must be taken not to exceed the exchange capacity of the column.  This can lead to isotope fractionation. Overloading the column will cause the heavier nitrate to exit, resulting in an isotopically "light" sample.
5. Incomplete elution of nitrate from the column usually results in an isotopically heavy sample, because the heavy nitrate exits first. 

 If you are sending us samples to be processed and run in our lab, we will need to following information: 

1. A brief description of  the question or processes you are trying to understand.
2. Scope of the study.  How many samples, how long the study will be done, and whether you will be collecting samples over a period of time.
3. The best estimates you have for the chemistry of your samples.  Send us this information in micromole units, except for DOC which can be in ppm.  Include the volume of sample needed to obtain the necessary 100 micromoles of NO₃.  You can collect more, as long as the total loading capacity of the anion column is need exceeded (6 meq). In fact, we recommend that you collect ~ 200 mmoles of nitrate so that you can run duplicates. Calculate the load of all anions that will be put through the column and stay within the loading capacity of the column  Please follow the format shown in the data table at the end of these instructions.  Concentrations of all dissolved solutes should be in micromoles except for DOC which is in ppm.  Do not send us data in units of ppm or meq.

For more methods details please refer to: 

Chang, C.C.Y., Langston, J., Riggs, M., Campbell, D.H., Silva, S.R., and Kendall, C., 1999. A method of  nitrate collection for  d15N and d18O analysis from waters with low nitrate concentrations.  Can. J. Fish. Aquat. Sci. v. 56, p. 1856-1864.

Silva, S.R., Kendall, C., Wilkison, D.H., Ziegler, A.C., Chang, C.C.Y., and Avanzino, R.J., 2000. A new method for collection of nitrate from fresh water and analysis of the nitrogen and oxygen isotope ratios. J. Hydrology. v. 228, p. 22-36.
 

For more information about nitrate isotopes see:

Kendall, C., 1998. Tracing nitrogen sources and cycling in catchments, Chapter 16, In: C. Kendall and J.J. McDonnell (Eds.), Isotope Tracers in Catchment Hydrology, Elsevier, Amsterdam, p. 519-576. 

Kendall, C., and Aravena, R., 2000. Nitrate isotopes in groundwater systems, Chapter 9, In: P. Cook and Herczeg, A.L. (Eds.), Environmental Tracers in Subsurface Hydrology, Kluwer Academic Publishers, p. 261-297.
 

On-line resources:

Excerpts from Kendall (1998)

Excerpts from Clark and Fritz (1997)
 

Return to Index.

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