Standard Operating Procedure for the
Analysis of Nitrate/Nitrite and Nitrite
in Fresh Waters
CCAL 31C.1
Cooperative Chemical Analytical Laboratory
College of Forestry
Oregon State University
3015 Western Blvd
Corvallis, Oregon
Prepared by Kathryn Motter
And Laura Hartley
Revised June 2019
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Standard Operating Procedure for the
Determination of Nitrate/Nitrite and Nitrite in Fresh Waters
CCAL 31C.1
Table of Contents
1.0 Scope and Application .......................................................................................... 3
2.0 Summary of Method ............................................................................................. 3
3.0 Definitions .............................................................................................................. 3
4.0 Interferences .......................................................................................................... 4
5.0 Safety ...................................................................................................................... 4
6.0 Equipment and Supplies ...................................................................................... 5
7.0 Reagents and Standards ....................................................................................... 5
7.1 Preparation of Reagents ...................................................................................... 5
7.2 Preparation of Cadmium Column ....................................................................... 6
7.3 Preparation of Standards ..................................................................................... 6
8.0 Sample Handling and Storage ............................................................................. 7
9.0 Quality Control ..................................................................................................... 7
10.0 Calibration and Standardization ......................................................................... 8
11.0 Procedures ............................................................................................................. 8
11.1 Calibration and Analysis Procedure ................................................................... 8
11.2 System Notes ...................................................................................................... 8
12.0 Data Analysis and Calculations ........................................................................... 9
13.0 Method Performance ............................................................................................ 9
14.0 Pollution Prevention ............................................................................................. 9
15.0 Waste Management............................................................................................. 10
16.0 References ............................................................................................................ 10
17.0 Tables, Diagrams, Flowcharts, and Validation Data ....................................... 11
17.1 Nitrate/Nitrite Nitrogen Reaction Manifold ..................................................... 11
17.2 Nitrate-Nitrite Manifold Specifications ............................................................ 11
17.3 Data System Parameters ................................................................................... 12
18.0 Document Revision History ............................................................................... 12
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Standard Operating Procedure for the
Determination of Nitrate/Nitrite and
Nitrite in Fresh Waters
CCAL 31C.1
1.0 Scope and Application
1.1 This method details the determination of nitrate/nitrite and nitrite in fresh
waters by automated colorimetric analysis. The practical range of
determination for this method is 0.001 to 0.100 mg/L as N. Method
detection limit for this analysis is 0.001 mg/L N.
2.0 Summary of Method
2.1 An automated analysis method is used for the colorimetric determination
of nitrate/nitrite and nitrite in fresh waters. Nitrate is reduced to nitrite
when passed through a copperized cadmium reduction column. For the
analysis of nitrite exclusively, the column is taken out of line. The nitrite
is diazotized with sulfanilamide and coupled with N-(1-napthyl)-
ethylenediamine dihydrochloride to form a highly magenta colored azo
dye, the absorbance of which is measured colorimetrically at 520 nm.
Concentration of nitrate/nitrite or nitrite is determined by comparison of
absorbance signal with calibration results obtained from prepared
standards of varying concentrations.
3.0 Definitions
3.1 DI water: Water that has been through a deionization system to produce
water similar to ASTM Type I reagent with 16.7 Mohms resistivity
(ASTM) (Reference 16.3).
3.2 Method Detection Limit (MDL): The minimum concentration of an
analyte that can be measured and reported with 99% confidence, based on
a one-sided 99% confidence interval (t-value at a significance level of 0.01
and n-1 degrees of freedom) from at least seven repeated measurements of
a low concentration standard measured within an analysis run.
MDL = ts
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Where,
t = Student’s t value at a significance level of 0.01 and n-1 degrees of
freedom
s = standard deviation of at least seven repeated measurements of a low
level standard
4.0 Interferences
4.1 Oxidizing and reducing agents and metal ions that could potentially cause
interference are generally found at negligible concentrations in unpolluted
surface and groundwaters.
4.2 High concentrations of iron, copper or other metals result in negative
interference. EDTA is added to the buffer to minimize this effect.
4.3 Turbidity and color that persists in the sample after filtration may absorb at
the 520 nm. Reanalyze samples without the color reagent as
turbidity/color blanks. Any response from the turbidity/color blanks must
then be subtracted from the initial analysis response.
4.4 Residual chlorine may interfere by oxidation of the cadmium column.
5.0 Safety
5.1 The toxicity or carcinogenicity of each reagent has not been precisely
determined; however, each chemical should be regarded as a potential
health hazard. Exposure to these chemicals should be reduced to the
lowest possible level. Cautions are included for known extremely
hazardous materials.
5.2 The following chemicals have the potential to be highly toxic or
hazardous. For detailed explanations, consult the MSDS.
5.2.1 Hydrochloric acid
5.2.2 Phosphoric acid
5.2.3 Sodium hydroxide
5.2.4 Ammonium hydroxide
5.2.5 N-(1-naphthyl)-ethylenediamine dihydrochloride
5.2.6 Cadmium
5.2.7 Sufanilamide
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6.0 Equipment and Supplies
Note: Brand names, suppliers and part numbers are for illustrative purposes only. No
endorsement is implied. Equivalent performance may be achieved using apparatus and
materials other than those specified here, but demonstration of equivalent performance
that meets the requirements of this method is the responsibility of the laboratory.
6.1 Analytical balance with resolution to 0.1 mg
6.2 Glassware: including volumetric flasks and pipettes as required
6.3 Lachat QuikChem 8500 Flow Injection Analyzer
6.3.1 Multichannel proportioning pump (peristaltic)
6.3.2 XYZ Autosampler
6.3.3 Reaction Manifold
6.3.4 Colorimetric Detector
6.3.5 Data System
6.4 Safety glasses
6.5 Nitrile gloves
6.6 Lab coat or apron
6.7 Laboratory exhaust fume hood
6.8 High density polyethylene (HDPE) bottles
7.0 Reagents and Standards
7.1 Preparation of Reagents
All reagents need to be filtered and degassed with He
7.1.1 15 N Sodium Hydroxide:
Slowly add 150 g sodium hydroxide to 250 mL DI water. Swirl
until dissolved. Cool and store in plastic bottle.
7.1.2 Sulfanilamide Color Reagent:
Slowly add 100 mL conc phosphoric acid (H3PO4) to
approximately 800 mL DI water in a 1 L volumetric flask. Add
40.0 g sulfanilamide, and 1.0 g N-(1-naphthyl)-ethylenediamine
dihydrochloride. Stir for 30 minutes or until complete dissolution.
Dilute to the mark and invert to mix. Store in a brown bottle and
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keep in the dark when not in use. This solution is stable for one
month.
7.1.3 Ammonium Chloride Buffer:
To 500 mL of DI water in a 1 L volumetric flask, add 105 mL
concentrated hydrochloric acid. Swirl to mix. Add 95 mL
ammonium hydroxide and 1.0 g disodium EDTA. Stir until
dissolution complete. Dilute to the mark. Adjust pH to 8.5 with 15
N NaOH.
7.2 Preparation of Cadmium Column
7.2.1 Check the size of the coarse cadmium granules before washing and
discard exceptionally large granules.
7.2.2 Wash granules first with acetone; then with 1N HCl (color should
be silvery). At this point the cadmium may be dried and stored in
an air-tight container.
7.2.3 Wash the clean cadmium with 50 to 100 mL of 2% CuSO4 •
5H2O, until no blue color remains in the solution and the semi-
colloidal copper particles begin to enter the supernatant liquid.
Rinse with DI water.
7.2.4 Continue to wash with water until cadmium appears black; the
number of water rinses is not critical.
7.2.5 Fill the column with ammonium chloride buffer (7.1.3) and
transfer the granules to the column.
7.2.6 Condition the column in-line by running Ammonium Chloride
Buffer through for 1 hour. Check the column conditioning by
analyzing a minimum of six 0.100 mg N/L standards. Results
should agree to within 1 %.
7.3 Preparation of Standards
7.3.1 Calibration Standard:
Standards are prepared by dilution of a standard purchased from a
vendor that provides traceability to NIST standards. A stock
standard is prepared by dilution of the purchased reagent to an
intermediate concentration. The stock standard is used to prepare
working standards in the table below.
mg NO3-N/L
1 0.005
2 0.025
3 0.050
4 0.100
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For preparation of nitrite standards use the concentrations in the
table below.
mg NO2-N/L
1 0.003
2 0.015
3 0.030
4 0.061
7.3.2 Second Source Check Standard:
Standards are prepared by dilution of a standard purchased from a
vendor that provides traceability to NIST standards; this standard is
from a source other than that of the calibration standards. A stock
standard is prepared by dilution of the purchased standard to an
intermediate concentration. Check standard concentration is the
same as calibration standard 3.
8.0 Sample Handling and Storage
8.1 Unfiltered samples are filtered upon receipt through glass fiber filters into
clean HDPE bottles and stored at 4°C in the dark. Samples are analyzed
within 48 hours to ensure sample integrity. If samples must be held prior
to analysis, they are stored frozen at -15ºC.
9.0 Quality Control
9.1 Preparation of stock standards is recorded on worksheets and documented
by weight of standard added to a given flask before dilution to volume
with DI water. All records of certification are kept on file at CCAL
Laboratory.
9.2 Blank: DI water run after the calibration and before and after each check
standard.
9.3 Quality Control Check Standard: Calibration standards run in rotation
every ten samples to monitor stability and validate the calibration.
9.4 Method Detection Limit (MDL): Established for each analyte. Based on a
one-sided 99% confidence interval (t-value) from at least seven repeated
measurements of a low concentration standard. The t-distribution value is
multiplied by the standard deviation of the population (n-1) to obtain the
MDL.
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9.5 Analytical Duplicate: Separate analysis from the same sample aliquot.
Run a minimum of once every ten samples.
10.0 Calibration and Standardization
10.1 Balances: calibrated yearly by external vendor.
10.2 Pipette delivery checked by weight to within 2% of theoretical weight of
aliquot volume.
10.3 Calibration curve with r2 ≥ 0.995. (See 17.3 for calibration data set-up.)
10.4 Calibration verification with check standards, monitored throughout the
run. If measurement exceeds +/- 10% of the theoretical value, the analysis
should be terminated and the instrument recalibrated. The calibration
must be verified before continuing analysis.
11.0 Procedures
11.1 Calibration and Analysis Procedure
11.1.1 Prepare reagents and standards as outlined in Section 7.
11.1.2 Set up manifolds as shown in Section 17.1.
11.1.3 Samples are injected into the reaction path at a fixed time interval.
Setup or confirm data system parameters as detailed in Section
17.2.
11.1.4 Pump DI water through all reagent until stable flow. Check for
leaks, even flow, and monitor baseline. Pump reagents through all
lines until the system equilibrates. Monitor baseline until stable.
11.1.5 Record sample ids in the data template.
11.1.6 Calibrate the instrument with standards. Calibration regression
equations must have r2 ≥ 0.995, or calibration is discarded and
repeated.
11.2 System Notes
11.2.1 If the baseline is excessively noisy, clean the manifold using the
following procedure:
• Place all reagent and carrier lines in rinse water and pump
to clear reagents.
• Place all lines in 1.2N HCl solution for several minutes.
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• Place lines in DI water and pump until thoroughly rinsed.
• Be certain that no acid passes through the cadmium
column.
11.2.2 If the baseline has a large number of air spikes, check pump tubes
for excessive wear and replace as necessary.
11.2.3 To minimize the potential for microbial growth in the ammonium
chloride reagent, rinse the reagent bottle thoroughly with DI water
between mixes.
11.2.4 Turbidity and color that persists in the sample after filtration may
absorb at the 520 nm. Reanalyze samples without the color reagent
as turbidity/color blanks. Any response from the turbidity/color
blanks must then be subtracted from the initial analysis response.
11.2.5 Nitrite standards are unstable at low concentrations. Standards
older than seven days should be prepared fresh prior to analysis.
12.0 Data Analysis and Calculations
12.1 The data system prepares a calibration curve by plotting response of
injected standards versus known standard concentration. The resulting
regression equation is used to calculate the sample concentration.
12.2 All results and sample information are filed in the analysis data system by
analysis run. Details specific to the instrumental analysis are noted in the
Instrument Run Log created and maintained for the AAII. Analytical
results are entered into electronic format and entries are verified by a
second person.
13.0 Method Performance
13.1 This method was validated through inter-laboratory studies. The CCAL
Water Analysis Laboratory participates in the USGS Standard Reference
Water QA program and the National Water Research Institute’s (NWRI)
Environment Canada Proficiency Testing (PT) Program.
14.0 Pollution Prevention
14.1 The chemicals used in this method pose little threat to the environment
when properly managed.
14.2 All standards and reagents should be prepared in volumes consistent with
laboratory use to minimize the volume of disposable waste.
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14.3 For further information on pollution prevention, consult Less is Better:
Guide to Minimizing Waste in Laboratories, available from the American
Chemical Society at www.acs.org.
15.0 Waste Management
15.1 It is the laboratory’s responsibility to comply with all federal, state and
local regulations governing waste management, and to protect the
environment by minimizing and controlling all releases from fume hoods
and bench operations. Compliance with all sewage discharge permits and
regulations is required.
15.2 For further information on waste management, consult Less is Better:
Guide to Minimizing Waste in Laboratories, available from the American
Chemical Society at www.acs.org, and Environmental Management Guide
For Small Laboratories (233B00001) from the US Environmental
Protection Agency at https://nepis.epa.gov.
16.0 References
16.1 Standard Methods For The Examination of Water and Wastewater,
Method 4500-NO3, Nitrate Nitrogen Automated Cadmium Reduction
Method. American Public Health Association. 21st Edition, 2005.
16.2 Code of Federal Regulations. Protection of Environment. Section 40,
Appendix B to Part 136. Definition and procedure for the determination
of the method detection limit. Revision 1.11. Revised July 1, 1990.
Office of the Federal Register, National Archives and Records.
16.3 ASTM. American Society for Testing and Materials. Standard
Specifications for Reagent Water. D1193-77 (Reapproved 1983). Annual
Book of ASTM Standards, Vol. 11.01. ASTM: Philadelphia, PA, 1991.
16.4 Lachat Instruments Methods Manual. Determination of Nitrate/Nitrite in
Surface and Wastewaters by Flow Injection Analysis 10-107-04-1-B.
Lachat Instruments, 5600 Lindburgh Drive, Loveland CO 80539.
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17.0 Tables, Diagrams, Flowcharts, and Validation Data
17.1 Nitrate/Nitrite Nitrogen Reaction Manifold
17.2 Nitrate-Nitrite Manifold Specifications
Carrier: DI water
Interference Filter: 520 nm
Pump tubing: PVC
Manifold tubing: 0.8 mm i.d.
Mixing coils: 135 cm tubing on a 7 cm coil support
10 mm flow cell
Backpressure coil, 200 cm x 0.5 mm id tubing
Note: Cadmium Column Switching Valve used to place the column in-
line.
nitrite only nitrate + nitrite
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17.3 Data System Parameters
Cycle throughput: 80 samples/hr
Cycle Period: 45 s
Analyte Data:
Concentration Units mg N/L
Calibration Data:
Level Nitrate Nitrogen 1 2 3 4
Concentration mg/L 0.005 0.025 0.050 0.100
Level Nitrite Nitrogen 1 2 3 4
Concentration mg/L 0.003 0.015 0.030 0.061
Calibration Fit Type: 1st Order polynomial
Sampler Timing:
Minimum Probe in Wash Period: 9s
Probe in Sample Period: 20 s
18.0 Document Revision History
Original Document: March 2006
Version: 31A.0
Title: Standard Operating Procedure for the Analysis of Nitrate/Nitrite in Fresh Waters
Edit Date: February 2010
New Version: 31B.0
Address update
Add Nitrite procedure and change document title to reflect the addition
Section 7.3: change preparation of standards from raw materials, to serial dilution of
purchased standards. Change standard concentrations. Add Second Source
Check Standard.
Section 13.1: add Environment Canada Proficiency Testing Program participation.
Section 17.2: change standard concentrations and order of standards.
Edit Date: April 2014
New Version: 31B.1
Section 1: change working range of method with appropriate significant figures.
Section 7.1: update reagent preparation storage for alkaline water.
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Section 7.3: add mixed intermediary standard solution and provide a table of final
standard concentrations. Add table for nitrite standards.
Section 11.2: add system notes for nitrite standards.
General editing.
Edit Date: June 2019
New Version: 31C.1
Change in instrumentation from Technicon AutoAnalyzer II to Lachat Quikchem 8500
Flow Injection Analyzer, effective 5/1/2019.
Updates throughout to reflect changes in chemistry and instrumentation.
Title Page: new author, address change
General editing throughout
