NECi Standard Operating Procedure
AMD
Revision #
Original
2
4
11-13-13
Total Nitrogen Analysis by Alkaline Persulfate
Digestion Using Nitrate Reductase for Nitrate
Determination
Date
11-13-13
Initials
AMD
Revision #
1
3
5
Date
Initials
Chemicals
Item
Sodium Hydroxide
Potassium Persulfate
100ppm Nitrate Standard
Manufacturer
Sigma Aldrich
Sigma Aldrich
NECi
Catalog #
S8045-1KG
37,982-4
Labware
Item
15mL Centrifuge Tubes
with Flat Top Cap
Ultrasonic Water Bath
Autoclave
NECi Standard Range
Nitrate Test Kit
I.
Manufacturer
Biologix Research
Company
Fisher Scientific
NECi
Catalog #
10-9151
FS15
L-NTK-201
Purpose
This method is intended to be used in conjunction with NECi Standard Range
Nitrate Test Kit to provide a means of assessing the total amount of nitrogen in
a water sample. Total nitrogen=organic nitrogen + ammonium + nitrate + nitrite.
Alternatively, Nitrate + Nitrite can be determined by the NECi Discrete Analyzer
Nitrate Analysis Method.
II.
Stock Solutions
1. 500 mL 1.5M Sodium Hydroxide
30 g NaOH
500mL DI H2O
2. 50mL Alkaline Persulfate Solution
2 g K2S2O8
50mL DI H2O
III.
IV.
Sample composition – 1.5mL total volume
1 mL Standard or sample
500 µL Persulfate solution
Reagent Preparation
1. Sodium Hydroxide Solution (1.5M):
a. Dissolve 30 g of sodium hydroxide (NaOH, MW: 40.0) in approximately
350mL of DI H2O. Be cautious as NaOH releases heat when dissolved in
water.
b. After complete dissolution, bring the volume to 500 mL with additional
DI H2O. The solution has a shelf life of 6 months at room temperature.
2. Alkaline Persulfate Solution:
a. Add 2 g of potassium persulfate (K2S2O8, MW: 270.33) and 5 mL 1.5M
NaOH in approximately 35mL of DI H2O.
b. Place into ultrasonic bath to dissolve the K2S2O8 (~30 minutes)
c. Bring volume to 50mL with additional DI H2O. Prepare fresh solution
before every set of assays. This is sufficient reagent for ~70 Total
Nitrogen Assays.
3. Potassium Nitrate Standard:
a. Prepare stock solution (100mg N/L) by dissolving 16.31 mg of
potassium nitrate (KNO3, MW: 101.1) in 100 mL of HPLC water. The
stock solution is stable for 6 months when stored at 4°C. To create a
working solution (10 mg N/L), dilute stock solution 10x with HPLC water.
Further dilute the working solution to create standard solutions, using the
volumes listed in the chart below:
NO3—N mg N/L
0
2.5
5
7.5
10
V.
10 ppm Nitrate
HPLC water
Standard
0
5mL
1.25mL
3.75mL
2.5mL
2.5mL
3.75mL
1.25mL
5mL
0mL
Table 1. Preparation of Nitrate Calibrants
Procedure
1. Mix the samples and persulfate in a 2:1 ratio (1mL Standard or sample:
500µL persulfate solution) in autoclavable culture tubes with caps.
2. Autoclave the mixture at 250°F (121°C) and 17lb/in2 (117.2 kPa) for one hour.
Set the autoclave for slow release of pressure, to ensure that no samples rupture
due to a drastic change in pressure.
3. Remove samples from the autoclave and allow samples to cool to room
temperature. These samples can be stored at room temperature for several
days.
4. Analyze samples for total nitrate concentration using one of these methods:
a. NECi Standard Range Nitrate Test Kit: follow the test kit instructions
using the Calibrants shown in Table 1.
VI.
VII.
b. Discrete Analyzer (DA) Nitrate Analysis Method: Use the NECi DA
Reagent Pack for your instrument and apply standard QC/QA practices
for Nitrate analysis.
Calculations
1. Calculate the Total Nitrogen in the sample using a Standard Curve and the
linear regression fit to the line. The values for the Total Nitrogen in the samples
will be in units of mg N/L.
2. The Kjeldahl Method reports Total Nitrogen as mg N/L. Since the NECi
Nitrate Test Kit is calibrated as mg N/L, the results of the analysis done here will
be in the same units as the traditional Kjeldahl Method. Thus, there is no need
to convert the results to any other form.
Alternate Procedure to Correct for Nitrate and Nitrate in the Samples:
If the initial Nitrate + Nitrite, as mg H/L, is determined before the sample is
digested, then the Total Nitrogen in the form of Organic Nitrogen and Ammonium
can be calculated.
1. In a portion of the sample, analyze for Nitrate + Nitrite using either the NECi
Nitrate Test Kit or the NECi Discrete Analyzer Analysis Method. This will yield
the Total Nitrate+ Nitrite in mg N/L.
2. In the other portion of the sample, carry out the persulfate digestion as
described above. Determine the Nitrate in the digested sample by either the
NECi Nitrate Test Kit or the NECi Discrete Analyzer Nitrate Analysis Method.
This will yield the Total Nitrogen (Organic Nitrogen + Ammonium + Nitrate +
Nitrite) in mg N/L.
3. To calculate the NET Total Nitrogen as Organic Nitrogen + Ammonium in mg
N/L, subtract the Total Nitrate + Nitrite in mg N/L in the sample from the Total
Nitrogen + Ammonium + Nitrate + Nitrite) in mg N/L in the sample.
VIII.
Waste Handling:
Follow all local regulations with regard to disposal of all toxic waste generated in
using this method.