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TKN & Ammonia by OPA Method

Application Benefits

  • Included in 40 CFR Pt 136 with the 2021 CWA Methods Update Rule.
  • High sensitivity due to fluorescence detection
  • High selectivity due to OPA/sulfite reagent chemistry
  • Robustness due to use of gas diffusion cell and chelating agent
  • Excellent reagent stability



OPA, ammonia, TKN, flow injection analysis, FIA, EPA, gas diffusion, matrix interference, fluorescence

Introduction and Principle

The o-phthalaldehyde (OPA) indicator, in connection with a sulfurous reducing agent, has been used extensively for derivatization and detection of various analytes containing an amine group. The choice of sulfite as reducing agent makes the reaction highly specific to ammonia, forming the basis of a sensitive and selective fluorimetric ammonia assay. [1] The OPA method has substantial advantages over other ammonia assays in terms of simplicity, high sensitivity, reagent stability, low toxicity, minimal refractive index bias, low salt effects and immunity towards turbidity and the presence of colored natural substances. [2]

Ammonia in the sample solution is made volatile by in-line mixing with a solution of sodium hydroxide base. The solution also contains diethylenetriaminepentaacetic (DTPA) chelating agent to sequester metals that could otherwise interfere with the assay and result in poor spike recoveries. The alkalized solution is directed to a gas diffusion cell where it comes in contact with a gas-permeable membrane. Volatile ammonia migrates through the membrane into an acceptor solution of o-phthalaldehyde (OPA). The acceptor solution flows from the gas diffusion unit to an in-line heater to facilitate the reaction between OPA and ammonia, forming a fluorescent product. The resulting fluorescence intensity is measured in a photomultiplier detector using 365 nm excitation and 430 nm emission.


Experiments were carried out using the FIAlyzer-1000 Flow Injection Analyzer, equipped with the PMT-FL Fluorescence Detector. The FIAlyzer-1000 is a single-channel flow injection analyzer capable of running this assay and a host of others.

Reagent Composition

  • Carrier (C): Water
  • Reagent 1 (R1): NaOH with DTPA
  • Reagent 2 (R2): OPA in sodium tetraborate 

 FIAlyzer 1000 equipped with photomultiplier for TKN & Ammonia OPA method

Figure 1: the instrument setup with the FIAlyzer-1000, heater, and PMT fluorescence detector (left to right).

Instrument Schematics

TKN ammonia OPA method schematics.jpg

Figure 2: A schematic view of the instrument setup.


OPA calibration data

Figure 3: Example calibration run. 

Method Performance Parameters 

    Sample Matrices
Detection Limit 12μg N/L*  
USEPA Reporting Limit 50μg N/L*  
Range Upper Limit 10,000 μg N/L  
Spike Recovery 99.3% POTW** (Anaerobic digester sludge***)
  97.3% Industrial discharge (food processor) 
  109% Industrial discharge (Metal finisher) 
  96.9% River water
  102% POTW* (Final affluent, pre-UV disinfection)
  105% POTW* (Primary clarifier effluent)
Sample Throughput 50 samples / h  
  • * If desired, limits can be lowered by increasing the injected sample volume. 
  • ** POTW = Publicly Owned Treatment Works (U.S. Sewage Treatment Plant)
  • ***TKN digestate


The OPA assay is a sensitive, selective and robust method for measuring ammonia in wastewater and TKN samples. Robustness is optimized by incorporating a gas diffusion unit to isolate the OPA reagent from potentially interfering components in the sample solution. Most importantly, the use of gas diffusion allows easy and reliable determination of ammonia in highly acidic TKN digestate solutions. The performance of the method was validated by determining ammonia in several real-life sample matrices, showing good recoveries. It should be noted that the method performs well even on wastewater samples originating from metal finishers, where most traditional ammonia methods can exhibit quite poor spike recoveries.

The presented configuration addresses the concentration range desired for most environmental analysis needs. For ultra-low level requirements (e.g. oceanography applications), it is possible to enhance sensitivity and attain lower detection/quantization limits by increasing the volume of the injected sample solution.


[1] A. Aminot et al. “A flow injection-fluorometric method for the determination of ammonium in fresh and saline waters with a view to in situ analyses”, Water Research, 35 (7), 2001, p. 1777-1785.

[2] A. Aminot et al. in O. Wurl (ed.) “Practical Guidelines for the Analysis of Seawater”, p. 166-169. CRCPress, Boca Raton, 2009.