colorimetric cyanide.jpg

Application Benefits

  • Robustness from gas diffusion principle
  • Operator workload reduced due to the use of automated in-line digestion 
  • Increased operator safety and environmental awareness the use of isonicotinic acid instead of pyridine


  • Cyanide, isonicotinic acid, pyridine, flow injection analysis, FIA, gas diffusion, ISO 14403, flow analysis, colorimetric, UV digestion, digester, digestor


The traditional colorimetric cyanide assay is known for the use of pyridine as a key ingredient. Handling such a toxic, volatile and pungent reagent creates many safety issues for reagent preparation, as well as for day-to-day use of the method. An alternative method would be desired to avoid analyst exposure to pyridine and thus create operation conditions compliant with modern-day health and safety regulations. With such a strong motivation, it is not surprising that an alternative color reagent – isonicotinic acid - has indeed been identified and incorporated into a robust, optimized method. More than that, an official method (ISO 14403) has been compiled for measurement of free and total cyanide, based on the principles of in-line digestion, gas diffusion and colorimetric detection by a color reagent-containing isonicotinic acid.


Cyanide in a sample solution is made volatile by in-line mixing with citric acid buffer. The resulting solution is then directed to a gas diffusion cell where it comes in contact with a gas-permeable membrane. Volatile cyanide migrates through the membrane into an acceptor solution of sodium hydroxide. The cyanide-containing acceptor solution is directed into a manifold where cyanide is combined with the various color-producing ingredients (Chloramine-T, isonicotinic acid and 1,3-diethyl barbituric acid).

The resulting reaction product is a blue dye that can be detected at 590-610 nm. The use of gas diffusion greatly improves method robustness by minimizing detrimental effects from the sample matrix. As only gaseous components can pass through the membrane, many potential interfering agents in the sample matrix are completely excluded from the detector. For total cyanide measurements, the UV source on the in-line digestion module is turned on. When the sample passes through the digestion module, it is subjected to UV radiation that breaks metal-cyanide complexes, resulting in liberation of bound cyanide. For free cyanide, the UV source is turned off. The sample still passes through the digestion module, but in the absence of UV radiation only free cyanide is converted into volatile form.


Experiments were carried out using the FIAlyzer-FLEX, equipped with a spectrometric detector.

Sampling TImes

  • 15 sec (sample) + 65 sec (wash)

Detector Settings

  • Primary Wavelength: 605 nm
  • Secondary Wavelength: 670 nm

Reagent Composition

  • Reagent 1 (R1): Citric acid buffer, pH 3.8
  • Reagent 2 (R2): Sodium hydroxide
  • Reagent 3 (R3): Phthalate buffer, pH 5.2
  • Reagent 4 (R4): Chloramine T solution
  • Reagent 5 (R5): Color reagent (isonicotinic acid + 1,3-dimethyl barbituric acid) 

The instrument and its accompanying methods are applicable to:

  • Free/available cyanide determination by ISO14403
  • Total cyanide determination by ISO 14403



Figure 2: Instrument setup and fluidic schematic

Colorimetric cyanide schematic-01.png


Example calibration run. The samples containing free cyanide result in a signal both with the digester on and off. The samples containing bound cyanide (in form of ferricyanide) only result in a signal when the digester is on. 


Figure 3: Digestor on

Calibration_digester on.PNG


Figure 4: Digestor off

Calibration_digester off.PNG


Table 1: Method performance parameters


* If desired, the range can be extended to higher concentrations by decreasing the sampling volume.


The colorimetric cyanide assay is a sensitive, selective and robust method for measuring cyanide in water samples. Robustness is optimized by incorporating a gas diffusion unit to isolate the detector from potentially interfering components in the sample solution.


[1] Nagashima et al. “Spectrophotometric determination of cyanide with isonicotinic acid and barbituric acid”, International Journal of Environmental Analytical Chemistry 10, 1981, p. 99-106.