1. Analytica Chimica Acta 409 (2000) 275–282
Kinetic spectrophotometric determination of V(IV) in the presence of
V(V) by the H-point standard addition method
A. Safavi ∗
, H. Abdollahi, F. Sedaghatpour, S. Zeinali
Department of Chemistry, College of Science, Shiraz University, Shiraz, 71454, Iran
Received 4 June 1999; received in revised form 21 October 1999; accepted 7 November 1999
Abstract
The H-point standard addition method was applied to kinetic data for simultaneous determination of V(IV) and V(V)
or selective determination of V(IV) in the presence of V(V). The method is based on the difference in the rate of complex
formation between vanadium in two different oxidation states and xylenol orange at pH = 2. Vanadium(IV) can be determined
in the range 0.2–5 ␮g ml−1 with satisfactory accuracy and precision in the presence of excess vanadium(V) and other metal
ions that rapidly form complexes with xylenol orange under working conditions. The proposed method was successfully
applied to the simultaneous determination of V(IV) and V(V) and also the selective determination of V(IV) in the presence
of V(V) in several synthetic mixtures with different concentration ratios of V(IV) to V(V). ©2000 Elsevier Science B.V. All
rights reserved.
Keywords: Vanadium; H-point standard addition method; Kinetic method
1. Introduction
Vanadium is an important element in environmental
and biological studies. Vanadium’s role in physiolog-
ical systems includes normalization of sugar levels,
participation in various enzyme systems as an inhibitor
and a co-factor [1] and catalysis of the oxidation of
various amines [2]. Although vanadium can exist in
oxidation states from II to V in aqueous solution, most
methods have concentrated on its determination in the
states IV and V, as these are the most common forms
encountered in inorganic and biological systems. Also
in vitro studies showed that the vanadium (IV) and
(V) are essential for cell growth at ␮g dm−3 levels,
but can be toxic at higher concentrations. The toxi-
∗ Corresponding author: Fax: +98-71-20027.
E-mail address: safavi@chem.susc.ac.ir (A. Safavi).
city of vanadium is dependent on its oxidation state
[3], with vanadium(V) being more toxic than vana-
dium(IV). Recently, there has been renewed interest
in the determination of V(IV) in the presence of V(V),
since V(IV) is produced by various industrial redox
processes (e.g. the Linde Sulpholin process [4]), and
is less toxic than V(V) in environmental systems [3].
UV-visible spectrophotometry is the most common
technique used for vanadium determination, owing to
the sensitivity and selectivity achieved in these reac-
tions [5]. With the current choice of reagents, tech-
niques and conditions, it is also possible to distinguish
between the various oxidation states of vanadium. The
analytical applications of vanadium (IV) have been
reviewed; previously; it was concluded that V(IV) in
aqueous solutions forms complexes most easily with
reagents containing oxygen or sulfur donor ligands
[6–10]. It is thus clear that the presentation of simple,
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2. 276 A. Safavi et al. / Analytica Chimica Acta 409 (2000) 275–282
selective, precise and inexpensive methods for the de-
termination of V(IV) in the presence of V(V) is very
important.
The H-point standard addition method (HPSAM)
is a modification of the standard addition method
that transforms the incorrigible error resulting from
the presence of a direct interference in the determi-
nation of an analyte into a constant systematic error.
This error can then be evaluated and eliminated. This
method also permits both proportional and constant
errors produced by the matrix of the sample to be
corrected directly. The basis of the method was es-
tablished previously [11,12]. Absorbance increments
as analytical signals were used when only the ana-
lyte concentration was required [13]. The method has
been applied to eliminate the blank bias error due
to the use of the absorbant blank [14,15], to liquid
chromatography [16] and to the analysis of kinetic
data [17], with time as an additional variable.
Two variants of HPSAM can be used for the treat-
ment of kinetic data [17]. One is applied when the
reaction of one component is faster than that of the
other or the latter does not take place at all. This vari-
ant of the method is based on the assumption that only
analyte X evolves with time and the other species Y
or interferents do not affect the analytical signal with
time. In this case the variables to be fixed are two
times t1 and t2 at which the species Y, which does
not evolve with time or over the range between these
times, should have the same absorbance. The other
variant of the method is used when the rate constants of
the two components are time-dependent. In this case,
the two species in a mixture, X and Y, evolve with time,
Cx (concentration of analyte) and Ay (the absorbance
of interference) can be calculated by plotting the ana-
lytical signal At1−t2 against the added concentration
of X at two wavelengths λ1 and λ2, provided that the
absorbances of the Y component at these two wave-
lengths are the same (AY ) and so are thus the At1−t2
values.
In this work the first variant of the H-point standard
addition method is suggested as a simple and selective
method for the determination of V(IV) in the presence
of V(V). This method is based on the difference in the
rate of complex formation of V(IV) and V(V) with
xylenol orange under acidic conditions.
The well-known metallochromic indicator, xylenol
orange (XO) [18,19] has been proposed for the colori-
metric determination of a number of metals [20–23].
It is evident that XO shows only slight selectivity,
so that direct determination of metal ions with XO
usually suffers from serious interferences. Although
XO has been used for the determination of vanadium
previously [23], to the best of our knowledge, the
complexation kinetics of V(IV) and XO were not
considered previously for selective determination of
V(IV) in the presence of V(V).
2. Experimental
2.1. Reagents
All chemicals used were of analytical reagent grade.
Triply distilled water was used throughout. Stock
vanadium(V) and (IV) solutions (1000 ␮g ml−1), were
prepared from ammonium metavanadate and vanadyl
sulfate monohydrate (Fluka), respectively, and stan-
dardized [24,25]. XO stock solution (0.01 M) was
prepared from its disodium salt (Fluka). The working
solutions were diluted with water to the appropriate
concentrations. 1000 ␮g ml−1 solutions of the studied
interfering ions were prepared from appropriate salts.
A buffer of pH 2 was prepared by using sodium citrate
and hydrochloric acid at appropriate concentrations
[26].
2.2. Apparatus
UV-visible absorbance spectra were recorded on a
Philips 8750 spectrophotometer. Measurements of pH
were made with Metrohm 691 pH meter using a com-
bined glass electrode. The absorbance measurements
as a function of time, at fixed wavelength, were made
with a Philips PU875 spectrophotometer attached to a
Pentium 200 MHz computer.
2.3. Procedure
2 ml of buffer solution, 0.2 ml of stock XO solu-
tion and appropriate volumes of V(IV) and V(V) were
added to a 5 ml volumetric flask and made up to the
mark with water. For each measurement, ca. 2 ml of
the above solution was transferred to a spectropho-
tometer cell and the variation of absorbance vs. time

3. A. Safavi et al. / Analytica Chimica Acta 409 (2000) 275–282 277
was recorded immediately. The absorbance was mea-
sured at 562 nm with 5 s time intervals for each sam-
ple. Simultaneous determination of V(IV) and V(V)
with HPSAM was performed by measuring the ab-
sorbances at just 60 and 240 s for each sample solution.
The concentration ranges of V(IV) and V(V) for the
construction of a HPSA calibration graph were 0.2–5
and 0.1–3.0 ␮g ml−1, respectively. The same proce-
dure was repeated with 2 ml of samples such as blood
serum, river water, tap water or synthetic samples.
3. Results and discussion
HPSAM as a modified standard addition method
was proposed to obtain an unbiased analyte concentra-
tion when both analyte and interference are present in a
sample. This method can be applied to kinetic data for
the simultaneous determination of binary mixtures or
calculation of analyte concentrations completely free
from bias.
The rate of complex formation between XO and
V(IV) is slower than V(V) complex formation under
acidic conditions. Fig. 1 shows the change in visi-
ble absorption spectra of the XO–V(IV) system as a
function of time. The study on the influence of pH on
the rate of V(IV)–XO complex formation showed that
Fig. 1. Variation of visible absorption spectra of XO (3.0 × 10−4 M) solution in the presence of 2.0 ␮g ml−1 V(IV), as a function of time.
Time interval 1 min.
the maximum change in absorbance in the fixed time
period (30–60 s) occurred at ca. pH = 2. The rate of
complex formation above pH 3 was so fast that the
complex formation reaction was almost complete be-
fore 30 s and thus the change in absorbance between
30 and 60 s was very small (Fig. 2). So, 562 nm and
pH 2 were selected for monitoring the kinetics of the
system. However, under the same condition, the com-
plexation of V(V) was very fast.
Fig. 3 shows the possible applicability of HPSAM
for the simultaneous determination of V(IV) and V(V)
by the proposed system. The absorbances correspond-
ing to V(IV) at 562 nm and at two selected times 60
and 240 s are bi and Ai, respectively, while those cor-
responding to V(V) under the same condition are b
and A . A and b are equal because of the fast kinetics
of complex formation between XO and V(V). They
are related, in the simplest assumption, through the
following equations [17]:
for V(IV) Ai = bi + miti 60 ≤ tj ≤ 240
i = 0, 1, ..., n (1)
for V(V) or interferent A = b + m tj (m = 0) (2)
where the subscripts i and j denote the different solu-
tions for n additions of V(IV) concentration prepared
in order to apply the HPSAM and the time compris-

4. 278 A. Safavi et al. / Analytica Chimica Acta 409 (2000) 275–282
Fig. 2. Effect of pH on the change in absorbance of the XO–V(IV) system in a fixed time (between 30–60 s).
ing the 60–240 s range, respectively. These two times
were selected because the change in absorbance re-
lated to V(V) concentration was negligible in this time
interval and also there was an appropriate difference
between the slopes of the calibration lines for V(IV)
at these two times. Thus, the overall absorbances of
the V(IV)–V(V) mixture at 60 and 240 s are:
at 60 s A60 = bi + b (3)
at 240 s A240 = Ai + A (4)
However, applications of HPSAM at the two
aforesaid times are:
Fig. 3. Kinetic curves for (a) 1.0 ␮g ml−1 V(IV) (b) 1.0 ␮g ml−1 V(V) (c) mixture of 1.0 ␮g ml−1 V(V) and 1.0 ␮g ml−1 V(IV).
A60 = b0 + b + M60Ci (5)
A240 = A0 + A + M240Ci (6)
which intersect at point H(−CH, AH) ≡ (−CV(IV),
AV(V)) (Fig. 4). M60 and M240 are the slopes of the
calibration lines at 60 and 240 s, respectively.
At the intersection point:
b0 + b + M60CH = A0 + A + M240CH (7)
hence
CH = (A − b) +
(A0 − b0)
(M60 − M240)
(8)

5. A. Safavi et al. / Analytica Chimica Acta 409 (2000) 275–282 279
Fig. 4. Plot of H-point standard addition method for simultaneous determination of V(IV) (1.05 ␮g ml−1) and V(V) (1.85 ␮g ml−1).
Table 1
Results of four replicate experiments for the analysis of V(IV)–V(V) mixtures
A–C equation r Present in the sample (␮g ml−1) Found (␮g ml−1)
V(IV) V(V) V(IV) V(V)
A240 = 0.304 Ci + 0.520 0.9998 0.62 1.85 0.63 1.82
A60 = 0.192 Ci + 0.451 0.9988
A240 = 0.300 Ci + 0.401 0.9997 0.21 1.85 0.23 1.83
A60 = 0.190 Ci + 0.576 0.9989
A240 = 0.304 Ci + 0.520 0.9998 1.86 0.92 1.76 0.90
A60 = 0.188 Ci + 0.464 0.9999
A240 = 0.300 Ci + 0.317 0.9997 0.74 0.78 0.74 0.73
A60 = 0.188 Ci + 0.234 0.9998
As the V(V) complex is assumed not to evolve over
the considered range of time, then A = b and
CH =
(A0 − b0)
(M60 − M240)
(9)
which is equivalent to the existing CV(IV)(= b0/M60
= A0/M240). So, as shown in Fig. 5(a), the value of
CH is independent of the concentration of V(V) in the
sample.
Substitution of CV(IV) into Eq. (5), yields AH = b.
The overall equation for the absorbance at the H-point
is simply:
A = b = AH = AV(V) (10)
So, again as shown in Fig. 5(b) the value of AH is
independent of the amount of V(IV) in the sample.
Therefore, the intersection of the straight lines by
Eqs. (5) and (6) directly yields the unknown V(IV)
concentration (CV(IV)) and the analytical signal of the
Table 2
Determination of V(IV) in different samples
Sample Concentration of V(IV) (␮g ml−1)
Spiked Founda
Blood serum 0.50 0.45 ± 0.08
Blood serum 0.75 0.78 ± 0.07
River water 0.50 0.52 ± 0.05
Tap water 0.25 0.27 ± 0.04
Syntheticb 0.25 0.22 ± 0.04
a Mean ± s.d. (n = 5).
b The composition of the synthetic sample was, V(V) 2.5 ␮g
ml−1, Cu(II) 5.1 ␮g ml−1, Cd(II) 40 ␮g ml−1, Pb(II) 12 ␮g ml−1,
Ca(II) 6 ␮g ml−1, Zn(II) 2.0 ␮g ml−1, Na(I) 276 ␮g ml−1.

6. 280 A. Safavi et al. / Analytica Chimica Acta 409 (2000) 275–282
Fig. 5. Plots of H-point standard addition method for (a) fixed V(IV) concentration (1.24 ␮g ml−1) and different concentration of V(V);
(b) fixed V(V) concentration (1.85 ␮g ml−1) and different concentration of V(IV).
V(V) species (AV(V)) corresponding to t60 and t240 in
the original samples, as the two times were chosen in
such a way that the latter species had the same ab-
sorbance at both times. This analytical signal enables
the calculation of the concentration of V(V) from a
calibration graph. Several synthetic samples with a
different concentration ratio of V(IV) and V(V) were
analyzed by HPSAM . As can be seen from Table 1,
the accuracy of the results is satisfactory in all cases.
In addition, for the validation of the method, environ-
mental and biological complex matrix samples were
spiked with V(IV) and the proposed method was ap-
plied for determination of the analyte. The results are
shown in Table 2; it can be seen that the results are
satisfactory.
An absorbance increment as an analytical signal can
be employed in another version of HPSAM to allow
the analyte concentration, CV(IV), to be calculated with
no systematic, constant or proportional error thanks to
the intrinsic feature of the HPSAM and the nature of

7. A. Safavi et al. / Analytica Chimica Acta 409 (2000) 275–282 281
Fig. 6. A vs. added V(IV) concentration at different time intervals and 562 nm for synthetic mixtures with (a) 0.77 ␮g ml−1 V(IV) and
0.74 ␮g ml−1 V(V) (b) 1.53 ␮g ml−1 V(IV) and 2.50 ␮g ml−1 V(V).
Table 3
Application of signal increment version of HPSAM to two synthetic mixtures
Time interval (s)
100–240 150–240 100–150 100–200 80–200 60–240 60–200 90–150 110–170 80–200
V(IV) Found (␮g ml−1) 0.73 0.71 0.71 0.76 0.70 1.57 1.57 1.54 1.57 1.56
Actual V(IV) (␮g ml−1) 0.77 1.53
Relative standard deviation (%) 2.6 (n = 5) 0.6 (n = 5)

8. 282 A. Safavi et al. / Analytica Chimica Acta 409 (2000) 275–282
the method of standard addition. This variant should
be of use whenever only the analyte is to be deter-
mined or when the A–t curve rather than the compo-
sition of the matrix is known. It should also be useful
to apply the single-standard calibration method, which
features greater simplicity and rapidity. This method
can also be used to diagnose the occurrence of inter-
ferences with a given analytical procedure as, in the
absence of errors, the plot of any At1−t2 against the
added analyte concentration will have a constant point
(−CH, 0) [17]. Thus, for the determination of V(IV)
in the presence of V(V), the absorbance increment
as an analytical signal was used. The application of
the HPSAM in the At1−t2 –Cadded variant yields the
concentration of V(IV) directly from the intercept on
the y-axis. However, in order to ensure the absence
of constant and proportional errors from the calcu-
lated concentration, all the possible At1−t2 –Cadded
lines for V(IV) should intersect at the same point,
namely, that corresponding to the unknown concen-
tration, CH, as this would indicate that the time eval-
uation of the matrix would be a horizontal line. Fig.
6 and Table 3 show the results obtained from em-
ploying this version of HPSAM on the synthetic mix-
tures of V(IV) and V(V). The results obtained by this
procedure were in good agreement with those given
above.
The behavior of the system in the presence of some
ions was also studied. As (III), Cd2+, Sn (II), Pb2+,
Ca2+, Na+ and K+ did not complex with XO un-
der working conditions and thus up to 100 ␮g ml−1
of these ions did not interfere with the determination
of 0.50 ␮g ml−1 V(IV). Cu2+, Hg2+, Zn2+, Mg2+,
Co2+, Se(IV), Te(IV) and Fe3+ instantly form com-
plexes with concentrations up to ␮g ml−1 level, and
because complex formation is fast, no interference was
observed from their presence in the determination of
V(IV). However, only Al3+, Ni2+ and Fe2+ showed
an interference, due to slow complex formation at the
␮g ml−1 level.
Acknowledgements
The authors gratefully acknowledge the support of
this work by the National Research Council of I.R.
Iran (NRCI) as a National Research Project under the
grant number 1081.
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