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
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  of
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
2
NO

(10) 102.8
3
NO

(50) 101.5
2
3
CO

(10) 101.0
2
4
SO

(5) 102.4
3
4
PO

(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
3+
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
A
2-(5-Bromo-2-p yri d ylazo)-5 -[N-n-propyl-N-(3
-sul-fopropyl)-amino]aniline
Nitro-PAP
S
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