Hydralazine Hydrochloride: An Alternative Complexometric Reagent for Total Iron Spotometric Determination

doi:10.4236/ajac.2011.27089

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American Journal of Anal yt ical Chemistry, 2011, 2, 776-782

doi:10.4236/ajac.2011.27089 Published Online November 2011 (http://www.SciRP.org/journal/ajac)

Hydralazine Hydrochloride: An Alternative

Complexometric Reagent for Total Iron

Spectrophotometric Determination

Arlan de Assis Gonsalves1, Cleônia Roberta Melo Araújo1, Cristiane Xavier Galhardo2,

Marília Oliveira Fonseca Goulart3, Fabiane Caxico de Abreu3

1Colegiado de Ciências Farmacêuticas, Universidade Federal do Vale do São Francisco, Petrolina, Brazil

2Colegiado de Engenharia Agronômica, Universidade Federal do Vale do São Francisco, Petrolina, Brazil

3Instituto de Química e Biotecnologia, Universidade Federal de Alagoas, Maceió, Brazil

E-mail: [email protected]

Received July 14, 2011; revised August 22, 2011; accepted September 3, 2011

Abstract

An alternative spectrophotometric method has been developed for total iron determination using flow injec-

tion analysis (FIA). The procedure is based on the coordination reaction between hydralazine and Fe2+ ions,

which results in the formation of a purple complex monitored at 538 nm. For determination of total iron, Fe3+

ions were reduced using ascorbic acid. Under optimized conditions, a linear calibration graph (0.1 - 6.0

g·ml–1; n = 6) was obtained. The method allows LOD (3



of blank/slope = 0.06 g·ml–1) and LOQ (10



blank/slope = 0.22 g·ml–1). The RSD ((s/x) × 100) for a mixed standard containing 0.60 g·ml–1 Fe2+ and

Fe3+ was 0.10% (n = 10). Recoveries of spiked samples were 94.3% - 106.0%. The analytical frequency was

60 h–1. The effect of possible interferences has been studied. The procedure was successfully applied for

analysis of environmental samples. The real samples results were comparable with those obtained by the of-

ficial method considering a paired t-test and 95% of confidence level.

Keywords: Hydralazine, Total Iron Determination, Spectrophotometry, Flow Injection Analysis

1. Introduction

The iron element (Fe) is the fourth most abundant

chemical specie of the planet and is present in nature in

the following oxidation states: Fe2+ and Fe3+ [1].

Nowadays the determination of iron in some real sam-

ples cannot be considered an analytical challenge but the

introduction of alternative complexing agents for this

purpose it is very attractive. Some analytical procedures

already employed commercial drugs to determine iron,

for example: the antibiotics chlortetracycline and nor-

floxacin were used by Ruengsitagoon and Pojanagaroon

respectively to quantify Fe3+ in real samples [1,2]. Using

the same strategy, this work comes demonstrate the use

of hydralazine hydrochloride for total iron determination

in real samples. A list of some complexometric organic

reagents not usual used for iron determination employing

spectrophotometry is shown in Table 1.

Hydralazine hydrochloride (Figure 1) is an antihyper-

tensive drug that acts as a potent arteriolar dilator and

Table 1. Some complexometric organic reagents not usual

used for iron determination employing spectrophotometry.

Organic Reagent Type of Iron λMAX (nm) Ref.

DPQHa Fe2+ 504 [3]

1,10-Phenantroline Totalb 510 [4]

Thioglycolic acid Fe3+ 535 [5]

DPPHa Fe2+ and Fe3+c 535 [6]

5-Br-PSAAa Totalb 558 [7]

Nitro-PAPSa Totalb 582 [8]

Tirona Totald and Fe3+ 635 [9]

DPKBHa Totalb 686 [10]

DPFTHa Totalb 738 [11]

TLCRa Fe2+ 741 [12]

aSee list of acronyms in section 10 of this paper; bTotal iron expressed as

Fe2+ after a reduction of Fe3+; cConversion of Fe3+ to Fe2+ with a reduction

agent; dTotal iron expressed as Fe3+ after an oxidation of Fe2+.

777

A. de A. GONSALVES ET AL.

Figure 1. Chemical structure of hydralazine hydrochloride

(1-hydrazinophthalazine monohy dr oc hloride ) .

also is used in the treatment of congestive heart failure

[13]. It is a white powder, soluble in water with a pKa

value of 7.3 [14]. This drug demonstrates redox proper-

ties (acting as a reducing agent) [14], antioxidant activity

[15] and coordination capacity toward some metal

cations [16]. Up to now and at the best of our knowledge,

no report of spectrophotometric procedure using hydra-

lazine as the chromogenic reagent for determination of

metal ions has been available in literature.

Before the facts stated, this paper describes the devel-

opment of a simple, rapid and sensitive flow injection

method for total iron determination using hydralazine as

an alternative chromogenic reagent. The proposed pro-

cedure is based on the spectrophotometric detection of

the purple complex formed by the coordination reaction

between hydralazine and Fe2+ ions in neutral media. The

resulting complex is monitored at 538 nm. The devel-

oped method was successfully applied for determination

of total iron, after the reduction of Fe3+ into Fe2+ ions

using ascorbic acid, in samples of drinking, tap and la-

goon waters, besides lagoon sediments.

2. Equipments

An UV/Vis spectrophotometer (Femto®, model 700 Plus,

Brazil) equipped with a 20 mm “U” glass flow cell was

used as a detector in all FIA experiments, and the ab-

sorbance signal was obtained directly from the instru-

ment. A multichannel peristaltic pump (Watson Mar-

low®, model 400 Sci-Q, United States), a manual injec-

tor (made of acrylic, with two fixed sidebars and a slid-

ing central bar), pump tubes (Tygon®, model R-3603,

1.02 mm i.d.) and polyethylene tubes (1.0 mm i.d.) were

also used in the proposed flow injection system.

3. Reagents and Solutions

All chemicals were of analytical reagent grade and were

used without further purifications. Hydralazine hydro-

chloride was acquired from Sigma (St. Louis, USA).

Deionized water from a Milli-Q system (resistivity 18

Mcm) was used for preparation of reagents and buffers.

A stock solution containing 5.0 mmol·l–1 HCl, prepared

from concentrate hydrochloric acid (Vetec, Rio de Ja-

neiro) was used for preparation of all standard solutions,

and for dissolution and dilution of all real samples.

A stock solution containing 0.01 mol·l–1 hydralazine

hydrochloride was prepared by dissolving 0.1000 g of

C8H8N4.HCl in 50 ml of deionized water.

A stock solution containing 0.15 mol·l–1 buffer

NaH2PO4/Na2HPO4 (pH 7.0) was prepared by dissolving

5.2000 g of NaH2PO4.H2O (Vetec, Rio de Janeiro) and

6.7000 g of Na2HPO4.2H2O (Vetec, Rio de Janeiro) in

250 ml of deionized water. After that, the buffer working

solution was prepared by dilution of 50 ml of the stock

solution in 100 ml of deionized water with pH previously

adjusted to 7.0 with 1.0 mol·l–1 HCl solution using a

pHmeter.

Finally, the reagent solution (chromogenic reagent)

was prepared by mixing 10 ml of the hydralazine stock

solution and 20 ml of the buffer working solution into

100 ml of deionized water. So, the final concentration of

the hydralazine at this solution was 1.0 mmol·l–1.

A stock solution containing ascorbic acid 1.0% w·v–1

was prepared by dissolving 0.5000 g of this reagent

(Sigma, St Louis) in 50 ml of deionized water. So, the

ascorbic acid 0.1% w·v–1 working solution was obtained

by appropriate dilution of the stock solution in deionized

water. The ascorbic acid stock solution was prepared

every week and was kept in dark bottles and under re-

frigeration.

A stock solution containing 100 g·ml–1 Fe2+ was pre-

pared by dissolving 0.4980 g of FeSO4.7H2O (Vetec, Rio

de Janeiro) in 1000 ml of HCl 5.0 mmol·l–1. In the same

way, a stock solution containing 100 g·ml–1 Fe3+ was

also prepared by dissolving 0.2904 g of FeCl3 (Vetec,

Rio de Janeiro) in 1000 ml of HCl 5.0 mmol·l–1. The

standard working solutions were prepared by appropriate

dilution of these stock solutions in HCl 5.0 mmol·l–1. All

the stock solutions were prepared every week and the

working solutions every day.

4. Flow Injection Manifold and Procedure

The flow injection manifold used for total iron determi-

nation can be seen in Figure 2. At this flow system, the

Figure 2. Flow injection manifold for total iron determina-

tion. Experimental conditions: reagent 1 (hydralazine 1.0

mmol·l–1 in buffer NaH2PO4/Na2HPO4 pH 7.0), reagent 2

(ascorbic acid 0.1% w·v–1), peristaltic pump (0.9 ml·min–1),

sample volume (200 l) and mixing coil (30 cm).

A. de A. GONSALVES ET AL.

778

chromogenic reagent acts as the proper carrier solution.

The peristaltic pump drives the solutions forward, at the

same time, the reagent 1 (hydralazine 1.0 mmol·l–1 in

buffer NaH2PO4/Na2HPO4 pH 7.0) into the injection

valve and the reagent 2 (ascorbic acid 0.1% w·v–1) into

the confluence. After this, an aliquot of the standard or

the real sample is injected into the carrier stream and

meets the reagent 2 in the confluence before the mixing

coil. From this point, the resulting stream follows to de-

tector and the analytical signal generated by the

Fe2+-hydralazine complex is finally observed. After at-

taining the signal maximum, the central bar of the injec-

tion valve is moved back to the sampling position to start

another measurement cycle.

5. Samples Preparation

Drinking water was acquired in local markets. Tap water

was collected in some points of the Federal University of

Alagoas (Maceió, Alagoas, Brazil). Samples of water

and sediments were from Mundaú Lagoon (Maceió,

Alagoas, Brazil) and were collected in different points of

this estuary. Before the analysis of the water samples, all

of them were previously acidified and preserved during

24 h with HCl 1.0 mol·l–1. In this procedure, the water

sample was initially filtered through a 45 m glass fiber

membrane filter. In a 100 ml volumetric flask the water

sample was acidified with 500 l of HCl 1.0 mol·l–1, and

the final volume was completed with the real sample.

Some of these samples were adequately diluted before

the determination of total iron.

Approximately 1.20 g of dry sediment of the Mundaú

lagoon was treated with HCl 0.1 mol·l–1. The resulting

acid solution was shaken during 2 hours at 200 rpm, and

filtered through a 45 m glass fiber membrane filter into

a 50 ml volumetric flask, which final volume was com-

pleted with deionized water.

6. Results and Discussion

6.1. Absorption Spectra and Metal:Ligand Ratio

The absorption spectrum of the purple complex obtained

by coordination reaction between Fe2+ ions and hydra-

lazine was measured over the range of 400 - 600 nm us-

ing a spectrophotometer. The absorption maximum of

the complex was 538 nm. In order to achieve the greatest

sensitivity, measurements were made at this wavelength.

The metal-to-ligand ratio (Fe2+:hydralazine) was de-

termined by the Job’s method (continuous variation

method) and was found to be 1:2 at pH 7.0, as shown in

Figure 3. The stoichiometry was verify remained the

plus of concentrations of the metal and ligand constant,

however each plus differ one each other (CFe + Chid = 0.4

mmol·l–1; CFe + Chid = 0.3 mmol·l–1; CFe + Chid = 0.2

mmol·l–1; CFe + Chid = 0.1 mmol·l–1).

6.2. Optimization of the Flow Injection System

The physical parameters optimization of the flow injec-

tion system was conducted employing standard solutions

of Fe2+ (1.0 g·ml–1), since the proposed method deter-

mines total iron by reduction of Fe3+ to Fe2+. In these

studies, the effect of sample volume and mixing coil

length were evaluated to reach the best conditions of

analysis. Different sample loop lengths were tested: 10,

15, 20, 25, 30 and 40 cm with volumes of 100, 150, 200,

250, 300 and 400 µL, respectively. Various mixing coil

tubing lengths: 20, 30, 40, 50 and 70 cm was also studied.

The parameters were compared in terms of peak height

and precision. Among the evaluated parameters the most

suitable injection loop volume was 200 l, and because

of the higher signal the mixing coil of 30 cm was also

chosen for next studies.

The following chemical parameters of the flow injec-

tion system were also studied: hydrochloric acid concen-

tration, hydralazine concentration, ascorbic acid concen-

tration, effect of the Fe3+ concentration in the reduction

step and pH of the reaction media. In all of these cases,

the chosen of the concentration and the pH was made

considering the studied value that produced the maxi-

mum absorbance signal (in order to obtain greatest sensi-

tivity) and the lowest Schlieren effect. The Table 2

summarizes the optimum conditions of all studied pa-

rameters in the proposed flow injection method for total

iron determination.

-0,10,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 1,1

-0,1

0,0

0,1

0,2

0,3

0,4

0,5

0,6

Absorbance

CFe / (CFe + CHid)

Figure 3. Job’s method for the study of complexation of

hydralazine and Fe2+ ions at pH 7.0. (CFe + Chid = k) wi t h k =

0.4 mmol·l–1 (); k = 0.3 mmol·l–1 (); k = 0.2 mmol·l–1 ();

k = 0.1 mmol·l–1 ().

779

A. de A. GONSALVES ET AL.

Table 2. Physical and chemical parameters for flow injec-

tion determination of total iron.

Parametersa Studied

Range

Optimum

Value

Physical:

Wavelength (nm) 400 - 600 538

Mixing coil length (cm) 20 - 70 30

Sample injection volume (l) 100 - 400 200

Chemical:

Hydralazine concentration (mmol·l–1) 0.5 - 8.0 1.0

Ascorbic acid concentration (% w·v–1) 0.01 - 0.2 0.1

HCl concentration (mmol·l–1) 1.0 - 100 5.0

pH (NaH2PO4/Na2HPO4 buffer) 6.2 - 8.2 7.0

aStudied only at room temperature (25˚C).

7. Method Validation

The calibration curve was obtained employing mixed

standards containing the same amounts of Fe2+ and Fe3+.

Under the optimum conditions, the graph was found to

be linear over the range of 0.1 - 6.0 g·ml–1. This short

range is due to the previously discussed reduction step.

To determine total iron in real samples, six mixed stan-

dards were employed, containing 0.05, 0.1, 0.2, 0.4, 0.7

and 1.0 g·ml–1 of Fe2+ and Fe3+, so leading to concen-

trations of 0.1, 0.2, 0.4, 0.8, 1.4 and 2.0 g·ml–1 of total

iron. The regression plot obtained for total iron determi-

nation fitted the equation: A = 0.0325 C – 0.0013 (r =

0.9990, n = 6), where A is the signal (in absorbance) and

C the concentration of total iron (g·ml–1). The limit of

detection (LOD) obtained by the proposed method de-

fined as 3



/0.0325 and the limit of quantification (LOQ)

defined as 10



/0.0325 was 0.06 and 0.22 g·ml–1 respec-

tively, where



is the standard deviation of the blank

signal (n = 10) and 0.0325 is the slope of the calibration

curve [17].

The relative standard deviation (peak height in ab-

sorbance; (s/x) × 100) calculated from 10 replicate in-

jections of a mixed standard containing 0.60 g·ml−1 Fe2+

and Fe3+ was 0.10%. Considering the optimum condi-

tions of the proposed flow injection system, a maximum

sample throughput of 60 h–1 was obtained without any

carryover effect.

7.1. Interference Studies

The effects of potential interfering ions were examined

by using solutions containing 1.0 µg·ml–1 Fe2+ and the

ionic species evaluated at different concentrations. The

ions selected for this study were those usually found in

water samples besides some metal cations. The tolerable

concentration of each different ion was taken as a highest

concentration causing a relative error of ±5.0%. The re-

sults were summarized in Table 3. Most of the ions ex-

amined did not interfere with the determination of iron.

Copper was found to seriously interfere in the determina-

tion of iron when its concentration reaches 0.5 µg ml–1.

The positive interference observed when Cu2+ is present

in samples is probably due to the formation of a complex

between this metal cation and hydralazine that also ab-

sorbs electromagnetic radiation next to 538 nm.

To eliminate the Cu2+ interference in analyses was

used a liquid-liquid extraction method developed by

Faquim and Munita [18]. Adapting this procedure for the

purposes of this work, an organic solution of dithizone

(diphenylthiocarbazone) was used to extract selectively

Cu2+ ions from aqueous standard solutions containing

Fe2+, Fe3+ and Cu2+ ions. The procedure consisted in treat,

previously of analyses, 50.0 ml of the aqueous standard

solutions with 5.0 ml of an extraction solution containing

1.0 mmol–1 of dithizone in chloroform. The standard

solutions were prepared always containing 1.0 µg·ml–1

Fe2+ e Fe3+ and different concentrations of Cu2+ (1.0, 2.0,

3.0 e 4.0 µg·ml–1). In treatment, the aliquots of the ex-

traction solution were added to the standards and then,

Table 3. Effect of chosen ions on the peak height of 1.0 µg

ml–1 Fe2+ standard solution (n = 3).

Ionic Species

(concentration in µg·ml–1)

Relative Percentage of

Peak Height (%)

None 100.0

Cations:

Na+ (150) 94.2

K+ (150) 102.8

Mg2+ (300) 98.5

Ca2+ (25) 102.0

Cu2+ (0.5) 105.4

Cd2+ (5) 103.5

Pb2+ (10) 102.7

Anions:

Cl– (50) 98.9



(10) 102.8



(50) 101.5



(10) 101.0



(5) 102.4



(25) 98.3

A. de A. GONSALVES ET AL.

780

the resulting mixture remained under vigorous agitation

during 5 minutes. Finalized the time, waited the organic

phase decant and then, the aqueous phase was collected

with a syringe to be analyzed in the proposed flow sys-

tem. Comparing the spectrophotometric signals in

analyses of the standards before and after the treatment

with dithizone was verified that the procedure was effi-

cient to eliminate or reduce the interference of Cu2+ ions

present until the concentration of 3.0 µg·ml–1, a fact veri-

fied by checking the relative percentage of peak height of

the standards containing 1.0 (100%), 2.0 (101.2%), 3.0

(104.8%) and 4.0 (108.6%) µg·ml–1 of Cu2+ ions.

7.2. Analysis of Total Iron in Real Samples

The proposed flow injection method was applied to tap

and drinking water and also natural water besides sedi-

ments from Mundaú lagoon. When the obtained results,

showed in Table 4, were compared with those obtained

by using 1,10-phenanthroline spectrophotometric method

[19], it was seen that the proposed procedure provide

good results, and no statistical difference was found con-

sidering the paired t-test at the 95% confidence level [17].

The good agreement between the results of the con-

centration of total iron measured as Fe2+ using the stan-

dard and proposed methods is showed in Figure 4. The

value obtained from linear regression showed that the

intercept including 0 (0.002 ± 0.02) and the slope in-

cluding 1 (0.98 ± 0.02) [17]. The proposed method using

hydralazine as chromogenic reagent was efficient for the

determination of tap and lagoon water.

Table 4. Determination of total iron by the proposed flow

injection method and standard me thod.

Total Iron Found (µg·ml–1 or g·kg–1)

Samplesa Proposed FI

Method

Standard

Methodb

Tap water 1(w·v–1) 1.09  0 1.07  0.02

Tap water 2 (w·v–1) 0.75  0 0.73  0

Tap water 3 (w·v–1) 1.27  0 1.28  0

Tap water 4 (w·v–1) 1.67  0 1.62  0

Lagoon water 1 (w·v–1) 1.26  0.02 1.22  0.02

Lagoon water 2 (w·v–1) 0.83  0 0.82  0.01

Lagoon water 3 (w·v–1) 0.70  0.02 0.68  0.01

Lagoon water 4 (w·v–1) 0.98  0.04 0.95  0

Lagoon sediment 1 (w·w–1) 42.5  0.83 44.0  0.52

Lagoon sediment 2 (w·w–1) 70.8  1.02 68.5  0.95

Lagoon sediment 3 (w·w–1) 65.8  0.67 66.0  0.80

Lagoon sediment 4 (w·w-1) 48.9  1.61 47.0  0.72

7.3. Recovery Studies

In order to estimate the accuracy of the procedure, dif-

ferent amounts of Fe2+ and Fe3+ were spiked in samples

of drinking and tap water. Samples of water and sedi-

ments from Mundaú Lagoon (Maceió, Alagoas, Brazil)

were also investigated. The results are given in Table 5.

A good agreement was obtained between the added and

measured analyte amounts. The average recoveries of

total iron for added standards were superior to 99%, thus

confirming the accuracy of the proposed procedure. Re-

coveries above and below 105% and 95%, respectively,

may be due to interference from other elements in the

samples, since complex matrices were analyzed.

8. Conclusions

A spectrophotometric method using FIA for total iron

determination employing hydralazine was developed.

0,6 0,8 1,01,2 1,4 1,6 1,8

0,6

0,8

1,0

1,2

1,4

1,6

1,8

Total Iron (Fe2+, g ml-1) - Standard Method

Total Iron (Fe2+, g ml-1 ) - Proposed Method

Figure 4. Correlation between the proposed and standard

methods for total iron determination in water samples.

Table 5. Recoveries of total iron from drinking, tap and

lagoon water and extracts of lagoon sediments.

Samples Added (µg·ml–1)

Fe2+ Fe

Founda

(µg·ml–1)

Recovery

(%)

Drinking water 1 1.00 0.80 1.88 104.4

Drinking water 2 0.80 1.00 1.83 101.7

Drinking water 3 0.80 0.80 1.65 103.1

Tap water 1 0.60 0.80 1.32 94.3

Tap water 2 0.50 0.50 1.06 106.0

Lagoon water 1 0.60 0.40 1.05 105.0

Lagoon water 2 0.20 0.40 0.58 96.7

Extract of lagoon

sediment 1 0.50 0.50 1.06 106.0

781

A. de A. GONSALVES ET AL.

The procedure showed to be accurate, precise and sensi-

tive for the analyte determination in some environmental

samples. Disadvantages of the proposed method were the

difficult to make the speciation between Fe2+ and Fe3+

and the significant interference of Cu2+ ions when it is

present up to 0.5 g·ml–1 in samples. Despite these dis-

advantages, the use of hydralazine as an alternative

chromogenic reagent, a commercial drug of low cost and

easily acquisition, still ensures the applicability of the

procedure in chemical analysis of total iron.

9. Acknowledgements

Financial support from CNPq, CNPq/CTHIDRO, CAPES

and FAPEAL (Brazil) is gratefully acknowledged. A

DTI/CNPq fellowship for C. X. Galhardo is acknowl-

edged.

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A. de A. GONSALVES ET AL.

782

Acronyms

The Table 6 shows a list of acronyms and its respective

meanings used in this paper.

Table 6. List of acronyms used in this paper.

Acronyms Meanings

DPQH 2,2’-Dipyridyl-2-quinolylhydrazone

DPPH 2,2’-Dipyridyl-2-pyridylhydrazone

5-Br-PSA

2-(5-Bromo-2-p yri d ylazo)-5 -[N-n-propyl-N-(3

-sul-fopropyl)-amino]aniline

Nitro-PAP

2-(5-Nitro-2-pyridylazo)-5-[N-n-propyl-N-(3-

sul-fopropyl)-amino]-phenol disodium salt

dihydrate

Tiron 4,5-Dihydroxy-1,3-benzenedisulfonic acid

disodium salt

DPKBH Di-2-pyridyl ketone benzoylhydrazone

DPFTH 2,2’-Dipyridyl-2-furancarbothiohydrazone

TLCR 4-(2-Thiazolylazo)-6-chlororesorcinol