Spring 2008
Determination of Nitrite
Purpose:
To determine the concentration of nitrite (NO2-) in various water samples.
Materials:
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polyethylene bottles
pipettes (various sizes)
graduated cylinders (various sizes)
volumetric flasks
9 cuvettes
Spectrophotometer
Access to the following reagents:
Reagents: These reagents will be mixed for you. The recipes are included for your
benefit.
1. Sulfanilamide solution
Dissolve 5 grams of sulfanilamide in a mixture of 50mL concentrated
hydrochloric acid (HCl) and about 300mL of deionized distilled water (DIW).
Dilute to 500mL with DIW. The solution is stable for many months.
2. N-(1-naphthyl)ethylenediamine dihydrochloride
Dissolve 0.5 grams of the dihydrochloride in 500mL of DIW. Store the solution in
a dark bottle. The solution should be remade monthly or sooner if a strong brown
coloration develops.
3. Primary Standard
Dissolve in 1000mL DIW 0.345 grams of Sodium Nitrite (NaNO2) that has been
previously dried at 110ºC for 1 hour.
Methods:
1. Make Secondary Standard:
a. Volumetrically dilute 5 mL of the Primary Standard to 500 mL using DIW. For
use the same day only, do not store. Make less as needed.
This is a dilution of the Primary Standard, and will be used to make your
Run Standards.
2. Make 3 Run Standards:
a. Pipette 2.0mL of the Secondary Standard into three 50mL plastic graduated
cylinders. Fill each to 50mL with DIW. LABEL them.
These will be used to determine the Calibration (F) factor. Essentially,
we know the concentration of nitrite in these solutions; therefore we can
calibrate our unknown samples (Greenfield Lake, Cape Fear River, etc.)
with this known amount.
Spring 2008
3. Make 2 Reagent Blanks:
a. Fill two plastic graduated cylinders with 50 mL of DIW. LABEL them.
These will also be used to determine the Calibration (F) factor.
4. Water Samples:
a. Fill 4 plastic graduated cylinders with 50 mL of EACH of your water samples.
LABEL them.
5. Sample Analysis:
a. Add 1.0 mL sulfanilamide to each 50 mL plastic graduated cylinder.
Stopper & mix (invert 5-10 times). Allow to react for more than 2
minutes but less than 10 minutes.
Sulfanilamide (in an acid solution) reacts with nitrite to produce a
diazo compound (a light sensitive salt).
b. Add 1.0 mL of N-(1-naphthyl) ethylenediamine dihydrochloride solution
to each 50 mL plastic graduated cylinder. Re-stopper & mix (invert 5-10
times).
N-(1-naphthyl) ethylenediamine dihydrochloride reacts with the
diazo compound to create a pink azo dye. This dye has an
absorbance of 543 nm.
6. Spectrophotometer Measurements:
a. Measure the absorbance of each of your 9 plastic graduated cylinders: 3
Run Standards, 2 Reagent Blanks, and 4 water samples. Record these on
your data sheet.
b. Find the Average Absorbance of your 3 Run Standards (ABSstnd) and
record on your data sheet.
c. Find the Average Absorbance of your 2 Reagent Blanks (ABSrb) and
record on your data sheet.
d. Determine your Calibration (F) factor using the formula below. Record
this value on your data sheet (it should be around 2.00).
F1 cm = 2.00 / [(ABSstnd - ABS rb) x 10]
e. Measure the absorbance of the Turbidity Blanks for EACH of your water
samples. These cuvettes will be next to the spectrophotometer. Record
these values on your data sheet.
7. Data Sheet:
a. Subtract the Average Reagent Blank ABS and the Turbidity Blank ABS
from the water sample absorbance to obtain the Corrected Sample ABS.
Corrected Absorbance = ABSsample - ABSrb - ABSturbidity
b. Use the following formula to obtain the amount of microgram atoms of
nitrogen per liter of sample (g-at N/L):
g-at N/L = Corrected Sample ABS x F