the reduction of nitrate to nitrite using the natural electron donor NADH.

The nitrite reacts with color reagents (dyes) under acidic conditions to produce

a visible color. All reaction steps occur in the microplate. The concentration of

nitrate in the unknown sample is determined by measuring absorbance with a

microplate reader using a 540 nm filter (or a filter as close to 540 nm as possible),

and then comparing the absorbance to a standard curve generated from

nitrate standards.

Nitrate can be determined in water samples and extracts of plant tissues, soils

and foods. The test is designed to measure nitrate in the range of 0.5 to 10 ppm

nitrate-N using a 96-well microplate. The nitrate concentration can also be expressed

page 4).

Prepare on day of use. Discard any unused NADH and NaR solutions at end of day.

desired, the Assay Buffer may be more quickly warmed in a 30oC water bath.

with a vortex-type mixer. Allow to stand at room temperature for 20 minutes, with 3

sec of vortex-mixing at 10 and 20 minutes. Keep on ice until use.

and replace cap. Mix for 3 sec with a vortex-type mixer. Keep on ice until use.

6 microtubes (provided in kit) to prepare nitrate standards as shown below.

to the other basin.

each well. Shake on a plate mixer for ~10 minutes @ 800 rpm.

filter within 20 nm of 540 nm).

analysis and printing.

1 To correct for any background absorbance due to the reagents, subtract the

mean absorbance of the reagent blanks from the mean absorbance of each nitrate

standard and unknown sample:

Corrected mean sample A-540 nm = (mean A-540 nm for samples) - (mean A-540 nm for reagent blanks)

2 Generate a standard curve for the nitrate standards (see example at left on

page 2). Using linear graph paper or computer plotting program such as Sigma

Plot® or spreadsheet such as Excel®, plot the ppm nitrate-N on the x-axis, and

the A-540 nm for each nitrate standard on the y-axis. If plotting by hand, draw

a straight line through the points for the nitrate standards. If plotting by computer,

the slope of the line can be calculated for determining nitrate-N ppm in the

unknown sample.

3 Using the standard curve, determine the ppm nitrate-N for the sample: (a)

Find the corrected A-540 nm for the sample on the y-axis of the standard curve.

(b) Follow over along a horizontal line to where the line intersects the standard

curve. Trace down to the x-axis and read the ppm of nitrate-N on the x-axis.

4 The Nitrate Standard Curve shown at the left (on page 2) was prepared using

a plate reader with a 550 nm filter. The slope is 0.085 Abs/ppm Nitrate-N, or

0.019 Abs/ppm Nitrate, or 0.0012 Abs/μM Nitrate.

The following procedure uses 2 replicates of each standard, unknown sample

wells.

rpm.

This NECi Nitrate Test Kit is capable of determining nitrate levels of up to 10

ppm Nitrate-N (44 ppm Nitrate or 710 μM Nitrate). If an unknown sample is

found to have more than 10 ppm Nitrate-N, the sample may be diluted with diwater1:

10 to allow an exact determination. For example, take 100 μl of sample

and add 900 μl of deionized water to make a 1:10 dilution and then assay 10 μl

of the diluted sample. After finding the amount of nitrate in the diluted sample,

multiply the nitrate concentration by 10 to find the nitrate concentration in the

original sample. NOTE: Keep the sample volume constant by diluting the sample

rather than using a smaller volume of sample in the assay.

with 90 μl of di-water and eliminating Step 4.

• Assay Buffer - 25 mM K-PO4, 0.025 mM EDTA; pH 7.5

• Color Reagent No. 1 - 1% Sulfanilamide in 3 N HCl

• Color Reagent No. 2 - 0.02% N-Naphthylethylenediamine in di-water

• NADH - ~1.1 mM NADH

• Nitrate Standard - 1 vial of 100 ppm Nitrate-N

• Quench Agent - mild, non-toxic oxidizing chemical to remove excess NADH.

Follow all local guidelines and regulations. If no local guidelines apply to your