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Catalog No. M-NTK-301

Microplate Nitrate Test Kit - Standard Range

This nitrate test kit is based on the enzyme

Nitrate Reductase (NaR), catalyzing

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

as μM nitrate, where the range is 36 to 710 μM nitrate.

Nitrite can also

be determined by omitting NaR and NADH from the test (see

Notes on the Tests,

page 4).

IMPORTANT

- Keep contents at 4°C until day of use. See expiration date on box.

Supplied in Nitrate Test Kit

Assay Buffer in liquid form - (1) 15 ml amber bottle.

Color Reagent No. 1 in solid form - (1) 15 ml amber bottle.

Color Reagent No. 2 in solid form - (1) 15 ml amber bottle.

NADH - 1 tube in amber bag.

Nitrate Reductase (NaR) - 1 tube in amber bag.

Nitrate Standard (100 ppm Nitrate-N) in liquid form - 1 tube.

Quench Agent - 1 tube in amber bag.

Microplate - (1) 96-well microplate with flat bottom wells.

Pipetter Reservoir Basins (2) clear plastic containers for reagents.

Microtubes - 6 tubes for preparing working nitrate standards.

Supplied by User

25 ml graduated cylinder.

Pipetters - variable pipetters (10/100 μl and 1 ml) or multi-well pipetter.

Several test tubes.

Vortex-type mixer.

Microplate mixer.

Microplate reader capable of reading at 540 nm +/- 20 nm.

Timer (0 to 20 minutes) - a clock or stop watch is adequate.

Deionized or distilled water (di-water) - Nitrate and Nitrite free!

2.5 ml of concentrated hydrochloric acid (HCl).

Ice and Ice Bucket.

A. Reagent Preparation

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

1

Assay Buffer - ready to use from kit. Warm to room temperature for nitrate tests. If

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

2 Remove tube of

NaR from amber bag and add 1.0 ml Assay Buffer. Mix for 3 seconds

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.

3 Prepare

3 N HCl by adding 2.5 ml concentrated HCl to 7.5 ml di-water. Mix.

4 Add

10 ml 3 N HCl to Color Reagent No. 1 bottle. Mix by inverting several times.

5 Add

10 ml di-water to Color Reagent No. 2 bottle. Mix by inverting several times.

6 Remove tube of

NADH from amber bag and tap tube to settle contents. Add 0.6 ml di-water

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

7 Add

di-water to the 5 ml line of the clear tube labeled Quench Agent. Mix thoroughly.

8 In pipetter reservoir basin (included in kit), add

9 ml Assay Buffer, 1 ml NaR solution

(prepared in Step 2), and

0.5 ml NADH solution (prepared in Step 6). Mix thoroughly.

B. Preparation of Working Nitrate Standards

Transfer

1 ml of 100 ppm Nitrate-N standard (provided in kit) to a test tube. Dilute

with

9 ml di-water to make a 10 ppm Nitrate-N standard. Use Nitrate standard and the

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

 

Typical Nitrate Std Curve with Slope = 0.085 A-550 nm/ppm Nitrate-N

2

Microplate Nitrate Test Kit (Catalog No. M-NTK-301)

Microplate Nitrate Test Kit (Catalog No. M-NTK-301)

3

5 If using a multi-pipetter, rinse the 2 pipetter reservoir basins with

di-water.

Transfer

Color Reagent No. 1 to one basin and Color Reagent No. 2 solution

to the other basin.

6 Add

50 μl Color Reagent No. 1 and 50 μl Color Reagent No. 2 solution to

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

7

Zero the plate reader with a Reagent Blank well using a 540 nm filter (or a

filter within 20 nm of 540 nm).

8

Read absorbance of all wells. Transfer results to your computer system for

analysis and printing.

D. Calculations

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.

C. Nitrate Test Procedure

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

and reagent blank. Using the

Microplate Sample Template, assign and record a

set of 2 wells for

each Nitrate Standard, Sample and Reagent Blanks.

1 Pipet

10 μl di-water into 2 wells for Reagent Blanks.

2 Pipet

10 μl of Working Nitrate Standards and Samples into designated

wells.

3 Pipet

90 μl NaR-Assay Buffer-NADH solution (prepared in Step 8 of Reagent

Preparation

) to each well. Shake on a plate mixer for ~20 minutes @ 800

rpm.

4 Pipet

30 μl Quench Agent to each well. Shake plate for ~10 min.

4

Microplate Nitrate Test Kit (Catalog No. M-NTK-301)

NOTES ON THE TEST

Unknown Samples with High Nitrate

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.

Testing-for Nitrite?

Nitrite can be determined by replacing the solution in Step 3 of the

Procedure

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

Notes on the Reagents

• 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 Reductase (

NaR) - approx. 0.5 unit of NaR per tube

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

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

Caution

Use care when handling

hydrochloric acid (HCl). Gloves are recommended.

Waste Disposal

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

situation, wash waste down a sink using large amounts of running water.

 
 

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