Department of Soil Science Standard Operating Procedure
NITRATE / NITRITE – NITROGEN ANALYSIS FOR TECHNICON AUTOANALYZER
(Automated Cadmium Reduction Method)
INTRODUCTION
In water and wastewater, the forms of nitrogen of greatest concern are (in order of
decreasing oxidation state) nitrate, nitrite, ammonia and organic nitrogen.
In soil, available Nitrogen is derived from various sources. In production agriculture
it is dominantly from fertilizer, biological N2 fixation and mineralization of organic N
wastes, crop residues and soil organic matter.
In most soils and climates, a significant amount of soil organic N is mineralized
during the growing season. Surface soils commonly contain between 0.08 and 0.4 %
total N (almost entirely in the form of Organic N. If 1 to 3 % of this N is mineralized in a
growing season from 8 to 120 Kg of N/ha may be available for crop utilization. This is
insufficient for most agricultural needs and it is slowly released. Most crops require the N
early in the growing season.
Organic nitrogen and ammonia can be determined together, and are referred to as
"Kjeldahl nitrogen".
Nitrite is an intermediate oxidation state of nitrogen, both in the oxidation of
ammonia to nitrate and in the reduction of nitrate to ammonia.
Ammonia is present naturally in surface and ground water. It is generally present in
low concentration because it is readily absorbed to soil particles and is not easily
leached. It is produced largely by deamination of organic nitrogen containing compounds
and by hydrolysis of urea.
Organic nitrogen includes such natural materials as proteins and peptides, nucleic
acids and urea and numerous synthetic organic materials.
Total oxidized nitrogen is the sum of nitrate and nitrite nitrogen.
Nitrate generally occurs in trace quantities in surface waters, but may attain high
levels in ground water. An upper limit of 10 mg/L of nitrate is imposed on drinking water
to prevent the illness known as methemoglobinemia in infants.
PRINCIPLE
In this method, nitrate (NO3-) is reduced to nitrite (NO2-) and is measured
combined with any nitrite already present in the sample. A correction can be made for
the nitrite originally present in the sample by analyzing the sample a second time without
this reducing step.
Nitrite ions form a reddish-purple azo dye at pH 2.0-2.5 by coupling diazotized
sulfanilamide with N-(1-naphthyl)-ethylenediamine dihydrochloride (NED). The colour is
measured at 520 nm.
The nitrate is reduced to nitrite by passing the sample through a column of granular
cadmium coated with copper.
Original Author: Rob Ellis, May 16, 2001
Last Revised By: Rob Ellis, July 2010
NITRATE / NITRITE – NITROGEN ANALYSIS
(Automated Cadmium Reduction Method)
Page 2 of 9
SCOPE
This method details two operating ranges: the low calibration range is 2 -20 mg/L
NO3/NO2-N with a detection limit of 0.2 mg/L N and the high calibration range is 5 - 40
mg/L NO3/NO2-N with a detection limit of 0.5 mg/L N. The above ranges are applicable
to soil, saturated paste, manure, feed and plant tissue extracts.
The operating range can be prepared at lower ranges if low end peak resolution is
required. Greater than a 40 mg/L upper end can only be set up if a dilution coil is added
to the manifold, otherwise the calibration curve enters a non linear section.
A trace range would be necessary to analyze surface, ground and wastewaters.
All customized ranges should be confirmed as being linear prior to beginning regular
analysis.
INTERFERENCES
Nitrogen trichloride gives a false red colour, but chemical incompatibility makes it
unlikely that nitrite, free chlorine and nitrogen chloride will coexist.
The following ions interfere by causing precipitation under test conditions, and
should be absent: Sb3+, Au3+, Bi3+, Fe3+, Pb2+, Hg2+, Ag+, PtCl62- and VO32-.
Cupric ions may cause low results by catalyzing decomposition of the diazonium salt.
Coloured ions and particulate matter which may alter the colour should be removed.
Oil and grease can affect the analysis by coating the cadmium column surface.
Acidic samples may cause accelerated deterioration of the reduction column but the
analysis has been shown to operate satisfactorily at the acidic level of the preserved
nutrient water samples. The nutrient bottle is preserved with 1 ml of 3% H2SO4 per 100
ml of sample.
EQUIPMENT
a. Autosampler II
b. Proportioning pump III (with nitrate lines)
c. Nitrate manifold
d. Colorimeter with 520 nm interference filters (flow cell 15 mm x 1.5 mm)
e. PC with Labtronics software and programming
REAGENTS
Preparation Information
-fill in the Reagent Preparation log with: date, reagent name, ingredient name,
manufacturer, product #, lot#, amount, final volume and Technician’s initials.
-there is a Standards Preparation Log (White Binder). All details concerning
calibration or quality control standards should be documented in this binder.
-ensure a WHMIS workplace label is in place on solution bottles.
NITRATE / NITRITE – NITROGEN ANALYSIS
(Automated Cadmium Reduction Method)
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List of Chemicals for MSDS reading
Ammonium chloride, D2
Ammonium Hydroxide, D1, E
Brij-35, D2
Cadmium, D1, D2
Cupric Sulphate Pentahydrate D1
Ethylenediaminetetraacetic acid, tetra-sodium salt, D2
Hydrochloric acid, E
N-(1-naphthyl)-ethylenediamine dihydrochloride (NED), D1
Phosphoric acid, E
Potassium Nitrate, C, D2
Sodium Nitrite, C D2
Sodium Nitrate, C D2
a.
Nitrate Calibration Stock Solution (1000 mg/L KNO3-N, 250 mL)
- dissolve 1.8045 g potassium nitrate (KNO3) which has been dried at 105C and
desiccated, in deionized water and bring volume to 250 mL.
b.
Nitrate Calibration Standard Solutions
i.
Low range (2 - 20 mg/L KNO3-N)
Standard number
1
2
3
4
5
MSVS = M1V1
1000 VS = M1100
VS = 0.1M1
Standard concentration
(mg/L)
2.0
5.0
10.0
15.0
20.0
Aliquot volume of
1000 mg/L stock sol'n
0.2 mL in 100 mL
0.5 mL in 100 mL
2.0 mL in 200 mL
1.5 mL in 100 mL
2.0 mL in 100 mL
- make standards up to assorted volumes with
2 N KCl or extraction solution
ii. High range (5 - 40 mg/L KNO3-N)
Standard number
1
2
3
4
5
MSVS = M1V1
1000 VS = M1500
VS = 0.5M1
Standard concentration
(mg/L)
5
10
20
30
40
Aliquot volume of
1000 mg/L stock sol'n
5 mL/L
5 mL/500 mL
20 mL/L
15 mL/500 mL
20 mL/500 mL
- make standards up to assorted volumes with 2N KCl
NITRATE / NITRITE – NITROGEN ANALYSIS
(Automated Cadmium Reduction Method)
c.
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Nitrate QC Stock Solution (1000 mg/L NaNO3-N, 250 mL)
- dissolve 1.5170 g sodium nitrate (NaNO3) which has been dried at 105C and
desiccated, in deionized water and bring volume to 250 mL.
d.
Nitrate QC LO (4 mg/L NaNO3-N, 100 mL)
- add 0.4 mL of 1000 mg/L NaNO3-N Stock to the same solution that the Calibration
standards, bring final volume to 100 mL.
e.
Nitrate QC HI (18 mg/L NaNO3-N, 100 mL)
- add 1.8 mL of 1000 mg/L NaNO3-N Stock to the same solution that the Calibration
standards, bring final volume to 100 mL.
Nitrite Stock Solution (100 mg/L NaNO2-N, 100 mL)
- dissolve 0.0493 g sodium nitrite (NaNO2,) which has been dried at 105C and
desiccated, in deionized water and bring to a volume of 100 mL.
f.
10 mg/L NaNO2-N Cadmium Column Check Sol’n, (200 mL)
- add 20 mL of 100 mg/L nitrite stock solution to deionized water and bring to a
volume of 200 mL
g.
Colour Reagent (2L)
- add 200 mL of 85% phosphoric acid (H3PO4) and 20 g sulfanilamide to 800 mL of
deionized water and stir to dissolve (this solution is light sensitive, do not stir longer
than necessary)
- add 1 g N-(1-naphthyl)-ethylenediamine dihydrochloride, stir to dissolve
- bring volume to 1L with deionized water
- transfer to a dark glass bottle and add 2 mL of Brij 35 (surfactant)
- the solution is stable until it starts to develop a red colour (approximately one
month)
- at busy times make a 4 L batch
h.
Ammonium Chloride Stock Solution (1%, 2L)
- dissolve 20 g ammonium chloride (NH4Cl) in deionized water and bring volume to
the 2L mark on the solution bottle, with deionized water.
i.
Ammonium Chloride Working Solution
- add 0.1 g imidazole per 100 mL ammonium chloride solution
- 250 mL of solution will run for approximately 2.5 hours
NITRATE / NITRITE – NITROGEN ANALYSIS
(Automated Cadmium Reduction Method)
j.
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EDTA Buffer Solution (1L, for cleaning purposes)
- dissolve 20 g tetrasodium ethylenediaminetetraacetate and 3.38 g
ammonium chloride in 400 mL of deionized water
- add 28.5 mL of concentrated ammonium hydroxide (NH4OH)
- adjust pH to approximately 10.5 using NH4OH or HCl
- bring volume to 500L with deionized water
k. Copper Sulphate Solution 2%
- dissolve 20 g CuSO4∙5H2O in 600 ml deionized water and bring final volume to
1L with deionized water.
SAMPLE HISTORY REQUIREMENTS
PROCEDURE
All extracts and samples must be free of turbidity. Centrifuge and/or filter as necessary.
For equipment maintenance and instrument optimization, refer to the instrument manual.
Autoanalyzer Startup
1. Turn on the colorimeter bulb, then the computer and all the auxiliaries.
2. Inspect the condition of the pump tubes. Look at the wear condition of the tubes as
well as for any disconnected fittings.
3. Change the deionized water in the Wash/Dilution container.
4. Check waste containers (lines correctly placed and not too full)
5. Close the platen on the proportioning pump and turn on the switch.
6. Check levels of reagents in containers, refill as required. Prepare fresh Ammonium
Chloride Working Solution.
7. Remove reagent lines from the rinse containers and place in the reagent bottles,
ensure the line is sitting on the bottom of the container. Cover the bottle openings for
the colour reagent and the Ammonium Chloride with parafilm. All other containers
are covered with an inverted plastic cup.
8. Wait for any extra air bubbles to pass through the system, install the Cd reducing
column. The taped end of the column is attached to the input injection fitting first.
This will allow carrier solution to enter the column. Then attach the exit end of the
column.
9. When the solutions have worked through the system, remove the line from the “pull”
connection on the colorimeter and use the dedicated squirt bottle to back flush the
flow cell with water..
10. Reconnect the line and pinch a full bubble through the flow cell.
11. With Labtronics loaded into the computer, turn the appropriate channel on standby
and monitor the baseline.
12. When the baseline has stabilized, set it between 2 - 4 % fullscale.
13. Standard calibration dial settings are posted on the colorimeter. These values may
change slightly as pump tubes and the bulb age. Verify the correct setting by running
a high standard. The high std should be between 80 and 95 % fullscale.
NITRATE / NITRITE – NITROGEN ANALYSIS
(Automated Cadmium Reduction Method)
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14. Set up the standards solutions on the autpsampler carousel using the 4 mL plastic
sample cups. Mark the cup ID’s with a felt pen. Then fill the cup 3/4 full with the
appropriate solution.
Cup
Marking
Contents
1
H
std
2
3
CO
B
dH2O dH2O
4
0
dH2O
5
1
std
6
2
std
7
3
std
8
4
std
9
5
std
10
3A
NO2
15. Before starting the analysis, inspect the bubble pattern through the entire system.
Look for air bubbles to be uniform in shape and that they are moving at a smooth
constant speed.
16. Load the carousel using the tray protocol for this particular set of samples. The tray
protocol establishes the frequency of samples, duplicates, gains and rinse cups. It
should be confirmed for each set of project samples, prior to the first analysis being
performed.
17. Switch the Labtronic’s channel from “standby” to “on”. Place the carousel so that cup
#1 is aligned with the sampling probe and then turn on the power to the autosampler.
AUTOSAMPLER PROCEDURE
Control the sample rate and wash time with the Labtronics or with the instrument cam.
The fastest the system should be operated at is:
Sample time = 35 seconds, Total time = 75 seconds
The system can be run slower if it is being run combined with the Ammonia system. The
combined rate is no faster than Sample time 45 seconds, Total time = 90 seconds
1. Run the calibration standards first, followed by the blanks, samples and duplicates.
The quality control standards are run in accordance with the quality assurance
section.
2. Make any adjustment to probe height with the first sample.
3. Place the Autosampler stopping pin with the last cup.
SHUTDOWN
1. Once peaks are through, a final baseline is established and QC is verified, shut off
the Labtronics channel.
2. Disconnect the exit line from the cadmium reduction column exit and reattach the c
0.110 storage tubing. Allow solution to fill the line then remove the entire column
from the system. Attach the storage tubing to the other end of the column, making a
closed loop. Store in an upright position.
3. Remove reagent lines from reagent bottles. Wash each line in to the waste
container and then place them in the rinse container.
4. Run deionized water through the system for 3 minutes.
5. Place the NH4Cl and the NO3 lines in the EDTA cleaning solution bottle for 5
minutes. (NOTE: steps 4 and 5 are performed only when the glass tubing becomes
discoloured)
6. Rinse the lines and return to the deionized water rinse container. Run the system for
an additional 15 minutes.
11
W
dH2O
NITRATE / NITRITE – NITROGEN ANALYSIS
(Automated Cadmium Reduction Method)
Page 7 of 9
7. Shut off the proportioning pump and disengage the platen.
8. Release the plastic tubing holder from the pin at the corner diagonally opposite from
the air bar.
CADMIUM REDUCTION COLUMN PREPARATION
1. Cadmium granules are cleaned by swirling in 1N hydrochloric acid (HCl). When
clean, the cadmium should have a shiny appearance (no blackness).
2. After pouring off the acid, rinse and swirl vigorously with deionized water to remove
all traces of the acid.
3. Coat the cadmium with copper by soaking it in 2% copper sulfate (CuSO4) at a ratio
of 50 mL CuSO4 per 10 g cadmium.
4. The blue colour will disappear and brown flakes of free copper will appear when the
coating process is complete.
5. Rinse the coated cadmium with deionized water, swirling gently so as not to remove
the copper coating.
6. Keep the coated cadmium under water until the columns are packed.
All cadmium rinse water is stored in one container, and the water is removed by
evaporation. Used cadmium is saved and regenerated for further use. Do all cadmium
work with gloves on and over top of the plastic bucket.
CADMIUM COLUMN PACKING
The columns are made from purple/purple pump tubes, cut to be approximately 30
cm in length.
Wear gloves when handling cadmium and during the packing process. Only hold one
side of the column at all times.
1. Fill the clean, empty column with deionized water. Keep one end of the column
sealed with a fingertip, and keep a water head on the other end.
2. Using a spatula, add cadmium in small amounts. As long as a water head is
maintained, the cadmium granules will tumble into the column. Tap the column to
pack the granules. Alternate into which end of the column the cadmium is added.
3. When the column is filled to within 1/8" of each end of the column, maintain water
heads at both ends and insert wet, rolled plugs of glass wool at each end.
4. On the entrance end of the column, install a 3/8" collar made from a 0.110 Technicon
line. On the exit end, install a 2" piece of 0.110 line with a large plastic nib in the
end.
5. For storage of the column, the line is filled with deionized water and the nib is
inserted into the collar to create a closed system. The closed columns are then
stored under deionized water as there is a tendency for solution to leak out of the
column creating air bubbles in the column.
6. Before storing the columns, condition them by installing them on the N manifold and
running NH4Cl through them.
7. Before a column is put into service run a number of high calibration standards. This
is done to confirm the standard calibration setting as well as to see if the column is
NITRATE / NITRITE – NITROGEN ANALYSIS
(Automated Cadmium Reduction Method)
Page 8 of 9
producing stable peak heights. The peaks should be at the same height with good
inter peak resolution. If a column does not meet the above criteria then re-prepare
the cadmium and re-pack the column.
QUALITY ASSURANCE
The tray protocol set up will determine the amount of QC that a particular project will
have in place.
Duplicate samples of the same sample vial will monitor the reproducibility of the
analytical system.
Blank samples, reference soils and replicates monitor the quality control of the
procedure prior to the analysis step.
Blank samples can be used to monitor background levels and if necessary be subtracted
from sample values.
Duplicates values should be examined before the daily run is completed and should be
charted. The charting should contain two ranges, :% difference and absolute difference.
The acceptable ranges and required action steps should be established for each project.
CALCULATIONS
Technicon value (mg/L) x dilution ratio = concentration of Nitrate/Nitrite-N.
SAFETY
Operators should have read and be aware of the information contained in the Material
Safety Data Sheets pertaining to all chemicals used in this analysis.
The cover sheet for each applicable MSDS needs to be signed, before work begins with
that chemical.
Safety Glasses are to be worn at all times when working in the Autoanalyzer Lab, Rm
300.
First Aid Kits are located in Rm 304 and 357.
An initial chemical spill kit is located at the doorway of Rm 300. A full spill kit is located at
the Link stairwell.
A Fire Blanket is located in the hallway by Rm 302.
The Eyewash and Safety Shower are located in the hallway outside of Rm 359.
The nearest (BC) CO2 Fire Extinguisher is located in the hallway by Rm 300. The
nearest (ABC) Fire Extinguisher is located in the hallway by Rm 359.
NITRATE / NITRITE – NITROGEN ANALYSIS
(Automated Cadmium Reduction Method)
REFERENCES
Standard Methods for Examination of Water and Wastewater
A.E. Greenberg et al. 18th edition, 1992.
4500 - NO3- F (Automated Cadmium Reduction Method)
Technicon Autoanalyzer II Continuous Flow Instrument Manual
Technicon Instrument Corporation, 1975.
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