TKN & Ammonia by OPA Method
FIAlab Instruments, Inc. Method 100, ‘‘Determination of Inorganic Ammonia by Continuous Flow Gas Diffusion and Fluorescence Detector Analysis,’’ dated April 4, 2018 (FIAlab Instruments, Inc. 2018a). FIAlab Method 100 uses automated flow injection analysis with gas diffusion and fluorescence detector analysis to determine concentrations of ammonia in wastewater, ambient water, and Kjeldahl digestates. FIAlab Method 100 can be obtained from FIAlab Instruments, Inc., 4259 23rd Ave W, Seattle, WA 98199. See the full validated method here.
Introduction & Principle
The o-phthalaldehyde (OPA) indicator, in connection with a sulfurous reducing agent, has been used extensively for the derivatization and detection of various analytes containing an amine group. The choice of sulfite as a 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.
Figure 1: instrument setup with FIAlyzer-1000, heater, and PMT fluorescence detector (L to R)
Application Benefits:
High sensitivity due to fluorescence detection
High selectivity from OPA/sulfite reagent chemistry
Gas diffusion cell and chelating agent assist in robustness
Excellent reagent stability
EPA-approved July 19th, 2021
Figure 2: Method Schematic for OPA Ammonia/TKN method
Experimental
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.
Instrument Parameters
Reagent Composition
Carrier (C): Water
Reagent 1 (R1): 0.3M NaOH with 50mM DTPA
Reagent 2 (R2): 6mM OPA in 0.05M sodium tetraborate
Sample Volume
35 µL
Heater Temperature
65°
Detector Settings
Excitation: 365 nm
Emission: 430
Results
Figure 3: Example data of Ammonia/TKN analysis via OPA method
Figure 4: Example calibration of Ammonia/TKN analysis via OPA method
| Method Performance Parameters | Value | Sample Matrices |
|---|---|---|
| Detection Limit | 5 µg N/L* | |
| USEPA Reporting Limit | 50 µg 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 effluent, pre-UV disinfection) | |
| 105% | POTW**(Primary clarifier effluent) | |
| Sample Throughput | 35 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
Conclusions
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 potential 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 injected sample solution.
References
[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. CRC Press, Boca Raton, 2009.