NECi Nitrate Reductase Nitrate Analysis Method 

Revised and Updated: 03/30/2011                                                               1.888.NITRATE  www.nitrate.com


Introduction:
   The NECi Nitrate Reductase Nitrate Analysis Method has been perfected based on more than 30 years of academic and commercial experience and in collaboration with colleagues at the US Geological Survey (USGS), US Dept of Agriculture (USDA) and other laboratories.  The Method is based on NECi Superior Stock Nitrate Reductase which are of two types YNaR1 & AtNaR2 (Campbell et al., 2004; Campbell et al, 2006a).  Using Superior Stock NaR, the NECi Nitrate Analysis Method has been shown to match cadmium reduction in precision and accuracy with equal sensitivity, and the results published (Patton et al., 2002; Campbell et al., 2004; Patton et al., 2006; Campbell et al., 2006a; Campbell et al., 2006b).  The certification of the method is currently in process at the USGS and other certifying agencies.  A key step toward certification of the method has now been made by "qualifying" NaR Nitrate Analysis on two discrete analyzers - the Kone Lab 60 and related instruments (Patton et al., 2006) and Lachat AP300 (sold by Hach Co. - see Campbell et al., 2006b). [Kone Lab is now called Konelab AquaKem and Konelab Arena.]  Cadmium reduction method for nitrate is difficult to implement in discrete analyzers, which are the wave of the future in environmental chemistry. 

The NECi NaR Nitrate Analysis Method,
which is well suited to discrete analyzer technology,
is the answer!

The Method:
   Nitrate analysis is accomplished by reduction of nitrate to nitrite, and reaction of the resulting nitrite with the Griess reagents to yield a highly colored compound which is quantified using a colorimeter or spectrophotometer. In the Method the enzyme Nitrate Reductase (Superior Stock NaR) catalyzes the reduction of nitrate to nitrite with the natural reducing agent of this enzyme, NADH (reduced nicotinamide dinucleotide), to drive the conversion (Campbell et al., 2006a). Since all the reagents for the NECi NaR Nitrate Analysis Method are water soluble and stable in solution, the method is ideally suited for discrete analyzers, which are pipet driven devices and the traditional continuous flow and flow injection analyzers (Patton et al., 2002; Campbell et al., 2006b; Patton et al., 2006). However, the Method is also easily used in manual nitrate analysis which has been commercialized by NECi in a series of Nitrate Test Kits (NTK). NaR NTK produced highly accurate and reproducible results like those shown below.

Detailed Protocol for Manual Nitrate Analysis

Nitrate Analysis Results

The NECi NaR Nitrate Analysis Method was recently carried out Superior Stock AtNaR2, at 37°C in the manual protocol recently published (Campbell et al., 2006a)
and the following results were obtained :

The NaR Nitrate Standard Curve:


Each data point is mean of 5 determinations with a standard error of less than 5%.

Comparison of cadmium reduction and NaR Nitrate Analysis:


Water samples were analyzed using the NECi Manual Nitrate Reductase Nitrate Analysis Method to compare nitrate content to that found by the cadmium reduction method nitrate content determined by USGS - samples provided by C. J. Patton. Each data point is mean of 10 determinations with a standard error of less than 5% for the NaR Nitrate Analysis Method, while no error range was reported for the USGS cadmium reduction determination of nitrate content.

Clearly, these results show that the NECi NaR Nitrate Analysis Method matches the cadmium reduction method for nitrate content in real world water samples.

References:

Campbell, WH, T Kinnunen-Skidmore, MJ Brodeur-Campbell & ER Campbell (2004) New and improved nitrate reductase for enzymatic nitrate analysis.  American Laboratory 22(10): 12. http://new.americanlaboratory.com/982-Archives/  Read it

Campbell, Wilbur H, P Song., GG Barbier (2006a) Nitrate Reductase for Nitrate Analysis in Water. Environmental Chemistry Letters, 4: 69-73. Abstract.

Campbell, Wilbur H., Ellen R. Campbell, Lynn Egan (2006b) Green Chemistry Nitrate Determination: An Alternative Nitrate Analysis Method. American Laboratory, February, 2006.  http://new.americanlaboratory.com/982-Archives/

Patton CJ, AE Fischer, WH Campbell & ER Campbell (2002) Corn leaf nitrate reductase: A nontoxic alternative to cadmium for photometric nitrate determinations in water samples by air-segmented continuous-flow analysis. Environmental Science and Technology, 36: 729-35.  Abstract.

Patton, Charles J., et al. (2006) Discrete Analyzer Nitrate Analysis with Nitrate Reductase. For more information.