A Spectrophotometric Flow InjectionMethod for Determination of Ultratrace Amounts of Phenylhydrazine by Its Inhibition Effect on the Reaction of Thionin and Nitrite

A simple �ow injection colorimetric procedure for determining phenylhydrazine was established. It is based on the reaction of phenylhydrazine in sulfuric acid with thionin and sodium nitrite. Reaction was monitored spectrophotometrically by measuring thionin absorbance at λλmax = 602 nm. A standard or sample solution was injected into the sulfuric acid stream, which was then merged with sodium nitrite stream and thionin stream. Optimum conditions for determining phenylhydrazine were investigated by univariate method. Under the optimum conditions, a linear calibration graph was obtained over the range 0.05–0.60 μμmol L, and the detection limit was 0.027 μμmol L(sssss = ss. e proposed method has been satisfactorily applied to the determination of phenylhydrazine in human serum and water samples.


Introduction
Phenylhydrazine has many side effects.Its absorption through skin causes erosion, burns, and contact dermatitis and if exposures be in toxic dosage, skin contact can produce symptoms in other organ systems.Its inhalation in the form of combustible solid, liquid, and vapor produces cough and dyspnea.Its swallowing causes vomiting, faint, jaundice, and dizziness.Phenylhydrazine too can breakdown red blood cells (RBCs) and produce hemolytic anemia and consequential involvement of other tissues, such as spleen, liver, and kidney injury.Chronic exposure causes adverse effects in Bone Marrow (BM), liver, kidney, and it also increases the cancer risk [1].
is paper studies a rapid, sensitive, and cost-effective �ow injection method for the determination of phenylhydrazine based on the spectrophotometric detection by the reaction of phenylhydrazine standard in sulfuric acid, thionin, and sodium nitrite as a sensitizer.ionin and phenylhydrazine have the following structure (Figure 1).Measurements were made at 602 nm.

Experimental
2.1.Apparatus.e 8-channel peristaltic pump (ismatec, MCP process, IP 65,) was �tted for pumping solutions.Silicon rubber tube with 0.8 mm i.d. was used for delivery of the solutions.A mixed solution of thionin, nitrite, and sulfuric acid as a carrier stream was delivered through silicon rubber tubing (at 35 ∘ C). e thermostatic water bath (Gallen Kamp Griffin, BGL 240 V) was used at a given temperature of 35 ± 0.1 ∘ C. e standard solution of phenylhydrazine was injected into a carrier stream with a sample injector (Rhedyne, model 9125).An UV-Visible spectrophotometer (2501 C�CIL) equipped with a �ow-through cell with 10 mm path length connected a recorder was used for monitoring the variation in the absorbance spectrum.

Reagents and Solutions.
All chemicals were of analytical reagent grade and were used without further puri�cations.A 1.0 × 10 −3 mol L −1 stock standard solution of phenylhydrazine was prepared by dissolving 0.0147 g of phenylhydrazinine chloride (Merck, M = 144.6 g/mol) in distilled water and diluting it to 100 mL.Solutions of the desired concentrations were obtained by diluting the stock solution to volume with distilled deionize water.A 100 mL of 2.0 × 10 −3 mol L −1 sodium nitrite solution was prepared by dissolving 0.0138 g of NaNO 2 (Merck, M = 69.0g/mol) in distilled water and diluting it to mark in a 100 mL volumetric �ask.A 4.0 × 10 −4 mol L −1 thionin solution was directly prepared by dissolving 0.0115 g of thionin (Merck, M = 287.34g/mol) in distilled water and diluting it to mark in a 100 mL volumetric �ask.Sulfuric acid solution (2.0 mol L −1 ) was prepared by diluting a known volume of its concentrated solution (Merck).All laboratory glasswares were cleaned by soaking in a detergent solution and acidi�ed solution, followed by washing with concentrated nitric acid and rinsing several times with distilled deionize water.

Recommended
Procedure.Using of this the three channels manifold as shown in Figure 2, a 200 L sample or standard solution containing phenylhydrazine was injected into the reagent stream consisting of 2.0 mol L −1 of sulfuric acid and 0.9 × 10 −4 mol L −1 of sodium nitrite, which were then merged with 2.5 × 10 −4 mol L −1 thionin at the same optimum �ow rate of 0.3 mL min −1 .Subsequently, the sample zone �owed through the mixing coils no. 1 and 2 with 75 and 125 cm in reaction coil length, respectively, where the reagents to be mixed and �owed through the detection unit.e signal was monitored by the spectrophotometric detector at 602 nm, and the FI signal was recorded on a chart recorder.
samples were analyzed directly aer neutralization with sodium hydroxide solution and dilution with doubly distilled water to a suitable volume.
e water samples were collected in 1.0 L polyethylene bottles from Zayandeh Rood River, Isfahan province and analyzed immediately aer sample digestion.e water samples were �ltered through Whatman no.41 �lter paper, then 5 mL of each �ltered water sample was accurately transferred into a 25 mL round bottom �ask and made up to the mark with distilled deionize water, mixed, subsequently analyzed by the proposed FI method.

Results and Discussion
e proposed �ow system was undertaken for the development of FI procedure for analysis of phenylhydrazine based on the phenylhydrazine with thionin in sulfuric acid and sodium nitrite resulting in having an absorption maximum at 602 nm.e present work was developed and optimized by an univariate method.e variable by variable methods was applied to select the appropriate conditions for the �ow injection spectrophotometric determination of phenylhydrazine.To have more signals, the effect of reagent concentrations and manifold variables on the analytical signal was studied.

Choice of FI Manifold. Preliminary investigation has
been carried out to obtain the most suitable FI manifold for phenylhydrazine thionin in a sodium nitrite/sulfuric acid.ree FI manifold have been designed namely a threechannel FI manifold two channels designed FI various and a single-channel FI manifold.
ey were tested for determining phenylhydrazine in standard solutions.It was preferable to use the three-channel FI manifold for phenylhydrazine determination since it provided a greatest sensitivity, reproducibility, and relative high sample throughput.

Effect of Sulfuric
Acid, ionin, and Sodium Nitrite Concentration.e effect of varying concentration of H 2 SO 4 solutions between 0.2-5.0mol L −1 was examined.e highest peak height was recorded when the concentration H 2 SO 4 solution was 2.0 mol L −1 and was therefore chosen as optimum concentration.Further increasing of the sulfuric acid concentration makes the peak height decreased gradually up to 5.0 mol L −1 (Figure 3).e concentration of thionin solution was optimized.Various concentrations over the range 0.5 × 10 −4 -4.0 × 10 −4 mol L −1 were investigated.It was found that the peak height increased with increasing thionin concentration and reached a maximum peak height at 2.5 × 10 −4 mol L −1 , above which the peak height decreased.us, 2.5 × 10 −4 mol L −1 of thionin was used subsequently (Figure 3).e effect of various concentrations of sodium nitrite solution (0.1 × 10 −4 -1.5 × 10 −4 mol L −1 ), on the absorption of the sodium nitrite (as peak height) was examined.e sodium nitrite concentration which exhibited the greatest peak height was found to be 0.9 × 10 −4 mol L −1 and was therefore chosen as optimum concentration.Further increasing of the sodium nitrite concentration makes the peak height decreased gradually up to 1.5 × 10 −4 mol L −1 (Figure 3).

Effect of Mixing Coil Length, Injection Loop Volume, and
Flow Rate.ese studies were carried out at various mixing coil lengths between 50 and 275 cm for mixing coil 1 (H 2 SO 4 , sodium nitrite) and mixing coil 2 (H 2 SO 4 , sodium nitrite, thionin), injection loop volumes between 50 and 400 L were investigated.It was found that the peak height increased with the mixing coil 1 and mixing coil 2 lengths up to 75 and 125 cm, respectively.Both mixing coil lengths of 50, 75, 125, 175, 225, and 275 cm provided the peak height (Δ) of 1.35, 1.90, 1.81, 1.76, 1.74, 1.70 and 1.42, 1.69, 2.09, 1.88, 1.86, 1.81, respectively.e optimum mixing coil lengths of no. 1 and no. 2 for subsequence studied were 75 and 125 cm, respectively.
e in�uence of the sample/standard volume on the absorbance was recorded by injecting volumes in the range 50 and 400 L of phenylhydrazine standard solution (0.1 mol L −1 ).It was shown that the peak height (Δ) increased from 1.51 to 2.33 on increasing the injection volume from 50 to 400 L.It was found that the peak height increased with the injection volumes up to 200 L, and the injection volume of 50, 100, 200, 250,300, and 400 L produced the peak height (Δ) of 1.51, 1.92, 2.87, 2.32, 2.29, and 2.25, respectively.e appropriate peak height was reached at 200 L.e most suitable injection loop volume value for further use was 200 L.
e effects of sulfuric acid, thionin, and sodium nitrite solutions �ow rates were investigated on the determination of phenylhydrazine standard solution (0.1 mol L −1 ).e peak heights were monitored from the �ow rate of 0.2-0.6 mL min −1 for all solution streams.e peak height increased with increasing �ow rate of each stream up to 0.3 mL min −1 for sulfuric acid and sodium nitrite solutions, which were then merged with the �owing stream of thionin solution with the �ow rate of 0.3 mL min −1 above which the peak height slightly decreased.us, 0.3 mL min −1 of sulfuric acid, thionin, and sodium nitrite solutions was regarded as the optimum �ow rates.

Analytical Characteristics.
Analytical characteristics for the determination of phenylhydrazine were studied under the optimum conditions (Table 1).

Calibration Curve.
Using this the proposed FI manifold for the determination of phenylhydrazine under the optimum conditions, the linear calibration graph over the range of 0.05-0.6mol L −1 of phenylhydrazine standard solution was established, which could be expressed by the regression equation   12  2 ( 2  ) where  represents the peak height (Δ) and  was phenylhydrazine concentration in mol L −1 aer subtraction of blank.us, the amounts of phenylhydrazine in samples can be quanti�ed according to the above regression lines of equation.e detection limit was de�ned as the concentration of analyte that gives the signal that is different from the blank by an amount equal to three times the standard deviation of the blank signal (s/  ).It was found to be 0.027 mol L −1 .e quantitation limit (de�ned as ten time standard deviation) was studied and found to be 0.091 mol L −1 .It is shown that the present method was very suitable for determining relatively large amounts of phenylhydrazine in human serum and Zayandeh Rood water.
3.6.Reproducibility and Accuracy.e relative standard deviation of the proposed method (peak height (Δ)) calculated from 5 replicate injections of 0.2 and 0.4 mol L −1 of phenylhydrazine was 0.62% and 0.56%, respectively.e recoveries were determined with the standard addition method, in which phenylhydrazine (0, 0.1, 0.2, 0.4, and 0.5 mol L −1 ) was added and mixed with human serum and Zayandeh Rood water samples the human serum and water samples were analyzed using the proposed method.e percentage recoveries of 0.1, 0.2, 0.4, and 0.5 mol L −1 (  ) of phenylhydrazine were found to be between 92.50%-108.00%,showing that the proposed method could provide acceptable T 1: Variable ranges study and optimum conditions for the determination of phenylhydrazine.

Interferences.
In this stage, the in�uence of contaminant species presented in various samples for the determination of phenylhydrazine, 0.1 mol L −1 , was investigated.e tolerance limit was de�ned as the concentration of added ions causing a relative error less than 3% (Table 3).e results show that the developed method is very selective.

Conclusions
e proposed FI spectrophotometric method has proven to be simple and sensitive for phenylhydrazine determination.e linearity of the calibration graph is in the useful concentration range for quantitation of phenylhydrazine in human serum and water samples.e method developed simple, economic, rapid, providing a good sample frequency of 40 h −1 and is especially suitable for routine analysis.