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Instrument Testing : Colorimetric Detection of a Blue Dye


Colorimetric detection is an important detection method in flow injection analysis (FIA). Here a simple FIA manifold has been used to introduce a 20 ppm bromothymol blue dye solution (made up in 0.01M borax) into a borax carrier stream (also 0.01M). A VODS unit fitted with a high brightness red LED and a TSL235 light-to-frequency converter is used for the measurements.

Dye solution was first loaded into a 50 μl injection loop. On injection the sample and carrier streams merge and pass through a mixing coil (300 mm x 0.5 mm i.d.) before entering a 31 μl internal volume flow cell (Starna).

The data opposite shows the absorbance readings computed from the measured intensities as a result of four dye injections. The readings for each injection are extremely consistent and have a peak absorbance value of slightly less than 0.08. The standard deviation of the noise in the baseline in this trace (measured between channels 500 and 600) is only 50 microAbs. Drift in the baseline is also minimal.

Colorimetric Determination of Ammonia using a Gas-Diffusion Flow Cell

The ammonium concentration in an unknown sample can be determined using a gas-diffusion flow cell with colorimetric detection. By injecting a sample containing ammonium ions (NH4+) into a carrier stream of sodium hydroxide (NaOH) at pH 12, NH3 gas is produced.

The NH3 is detected by allowing it to pass by diffusion through a gas-permeable membrane (which can be a piece of Teflon tape) into an acceptor stream containing (pH sensitive) bromothymol blue dye, whose pH has been previously adjusted to 6.3.

The presence of ammonia gas in the acceptor stream will cause the dye to change colour from green to an intense blue, which is monitored using a red LED light source, emitting at 620nm.

Results at left show FIA data for 4 replicate sample injections of solutions containing 10 ppm (NH4+).

Ammonia Calibration Curve


At left is the calibration curve obtained by repeating the experiment above to a cover an ammonium ion concentration spanning the range 0.02 – 10 mg/L.

The detection limit for ammonium ions using the method described here is 1 µg/L, or 1 ppb.

Colorimetric Determination of Nitrite via FIA


In the data shown at left, successive injections of a 10 ppb nitrite (NO2-) solution are made into a carrier stream of ultra-pure water.

The carrier then mixes with a colour reagent, containing 1% sulfanilamide and 0.1% N-(1-Naphthyl)ethylenediamine (NED) in 0.1M hydrochloric acid.

Reaction of nitrite with the colour reagent produces an intense pink colour, detected by a green LED emitting at 525 nm (here, a Chameleon C-525-C). The peak absorbance seen in each of the traces is only ~0.001.

Based on this data the nitrite detection limit (the concentration at a which the peak height is at least three times the standard deviation of the baseline signal) is less than 2 ppb.

Colorimetric Determination of Chloride via FIA


Using the following simple chemistry, the concentration of chloride ions in an unknown sample can be assessed colorimetrically :

(a) Hg(SCN)2 + 2Cl- HgCl2 + 2SCN-

(b) 2SCN- + Fe3+ Fe(SCN)2+

In reaction (a), chloride ions displace SCN- ions from Hg(SCN)2. These go on to react with Fe(III) ions in (b), to produce the bright red Fe(SCN)2+ complex, which strongly absorbs blue-green light at 480nm.

The measured absorbance resulting from the presence of this complex is proportional to the concentration of chloride ions present in the original sample. In the four replicate injections shown here the chloride concentration is 1 ppm.

The “glitches” seen in the purple trace in-between the peaks are due to manual switching of the injection valve.