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Nitrite Assay (Griess)


Technical Overview

(from Wikipedia): "When sulphanilic acid is added (in the picture its sulphonamide is shown instead), the nitrites [present in solution] form a diazonium salt. When the azo dye agent (N-alpha-naphthyl-ethylenediamine) is added, a pink color develops. This diamine is generally used in place of the simpler and cheaper alpha-naphthylamine because this latter is a potent carcinogen and moreover the diamine forms a more polar and hence a much more soluble dye in acidic aqueous medium." 
Griess Test Reaction.svg

A typical Griess reagent may contain 0.2% naphthylethylenediamine dihydrochloride (NEDD), and 2% sulphanilamide in 5% phosphoric acid. The Griess reagent can be prepared in-house by purchasing and combining these chemicals individually. 

Commercial Griess reagent currently used (containing 2-Naphthylamine) is classified as Acutely Toxic (GHS07). Care should be taken when working with this reagent.

Primary References

General Review of nitrate and nitrite measurement techniques for biological systems, Tsikas 2005:
http://dx.doi.org/10.1080/10715760500053651
Overview of Griess reagent chemistry, as well as other methods for measuring nitrite and nitrate.

Original publication reporting use of Griess reaction, Griess 1879:
http://dx.doi.org/10.1002/cber.187901201117
(This paper is in German)

Ref3

Ref4

Materials

Griess' Reagent for nitrite
Specification; MSDS (Acutely toxic)
Sigma-Aldrich (Fluka Analytical): link to product
Product number: 03553-100ML ($30-40 per 100mL)
Formula:                Contains sulfanilic acid (C6H7NO3S), 2-Naphthylamine (C10H9N), Acetic acid (30-50%)

Sulfanilic acid:                

2-Naphthylamine (carcinogen):
         

Griess reagent components (NEDD and sulfanilic acid) are kept upstairs in Clark Lab (Sharmin's bench).

Protocol

Safety:
- Griess reagent is acutely toxic. (GHS Classification: Acute toxicity)
        Oral (Category 5)
        Skin irritation (Category 2)
        Eye irritation (Category 2A)
        Specific target organ toxicity - single exposure (Category 3) 
***USE ONLY IN FUME HOOD, WITH PPE*** 


Materials:

Griess reagent: commercial solution; lives in the Graves Lab refrigerator. It is light sensitive to return to the refrigerator immediately after use. 


Procedure:

1. After treatment, immediately add 100ul Griess reagent per 100ul sample volume. The sample+reagent may be mixed directly in the wells of a 96-well plate.* For samples containing >200uM NO2, dilute your sample such that the nitrite concentration is less than 200uM (otherwise the color will saturate). 

***If unsure of concentration, I recommend making several dilutions and developing them simultaneously to save time. 

*Because the Griess reagent is rough on cuvettes, using the plate reader for this assay is recommended. A calibration curve for 200ul (1:1 sample to Griess reagent ratio) added to the wells in a 96-well plate is attached to this page.
 
2. Add 100ul deionized water + 100ul Griess reagent to another well to use as a blank. 
 
3. Allow the sample/reagent mixture and blank to develop in the dark for 30 min. (The mixture will continue to develop past 30 minutes although at a relatively slow rate. Absorbance-concentration curves for longer periods of time are available; ask Carly if needed.)

3. Measure the absorbance of sample mixture and blank solutions at 548nm using the spectrophotometer. The absorbance of the blank and sample can be measured simultaneously if using the plate reader. Make sure the window heading in the program says “plate”. Then click “Read”. *Note: you can select which wells you want read under the setup menu. 

4. Subtract the blank absorbance from the absorbance of the sample(s). For 30 minutes developing time, multiply the blank-subtracted absorbance by 0.1305 (revised 14-02-2014 for new batch of Griess reagent) to determine the nitrite concentration in mM. 

5. Dispose of waste containing Griess reagent in the chemical waste containers in the Clark or Graves lab fume hoods. Record what you added and the approximate volume on the label of the waste bottle. 

Notes and Calculations





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