Analytica Chimica Acta 488 (2003) 243–254

Analyser of chromium and/or cobalt

L.A. Tortajada-Genaro, P. Campı́ns-Falcó∗ , F. Bosch-Reig
Departament de Quı́mica Analı́tica, Facultad de Quı́mica, Universitat de València, C/Dr. Moliner 50, E-46100 Burjassot, Valencia, Spain
Received 9 January 2003; received in revised form 14 April 2003; accepted 27 May 2003

Abstract
Two stopped-flow manifolds have been proposed for individual or simultaneous determination of chromium and cobalt in
water samples. Automated procedures based on multicommutation systems have emphasised the differences of their catalytic
effect in luminol–hydrogen peroxide chemiluminescence reaction. A more rapid decay of signal was observed for Co for
both configurations (flow injection or continuous injection). The influence of chemical and hydrodynamic variables has been
studied in order to establish the robustness of method. The analysis rate was lower 1.5 min per replicate.
Chemometric tools have been employed for the resolution of their contributions. Partial least squares (PLS) and H-point
standard additions method (HPSAM) were used as multivariate calibration models. The percentages of explained variance were
97–99% (two factors). PLS and HPSAM obtained similar results. HPSAM provided a simple calibration model contributing
to develop an analyser for chromium and/or cobalt.
Standard mixtures, spiked samples and a certified reference material validated the proposed strategy. The applicability has
been demonstrated by the determination of Cr and Co concentration in different water samples. The best results have been
obtained for continuous injection providing more robust predictions. The achieved detection limit was 0.2 ␮g/l for both metals.
© 2003 Elsevier B.V. All rights reserved.
Keywords: Multicommutation; Chromium and cobalt; Simultaneous determination; Chemiluminescence; Water matrix; HPSAM; PLS

1. Introduction solenoid valve. The approach has been used to per-

Computer-controlled switching systems are widely
used in some analytical areas. It supposes a flow switc-
hing technique in order to achieve automation of steps
of an analytical process. On-line sample washing,
preconcentration, extraction, dilution or hyphenated
techniques have been proposed. Many of the strategies
described come from the field of chromatography [1].
Nevertheless, multicommutation is a novel ap-
proach in flow analysis [2]. It can be implemented by
using discrete commutation devices, such as three-way
∗ Corresponding author. Tel.: +34-96-35-43-002;
fax: +34-96-35-44-322.
E-mail address: pilar.campins@uv.es (P. Campı́ns-Falcó).
form binary sampling, masking feasible the reduction
of reagent consumption, sequential determinations
and sequential management of incompatible reagents
[3–7].
Some applications have been proposed for water
analysis: majority metals [8], phenols [9,10] and an-
ions [11,12]. However, the solenoid-activated rotatory
valves have only been employed as sample introduc-
tion system based on time. So, simple flow registers
are obtained by using these manifolds.
Stopped-flow techniques have also been employed
in order to emphasise kinetic differences between
some analytes in different detection systems. Some
compound mixtures have been resolved obtaining
time-intensity curves [13–19]. Kinetic methods based

0003-2670/03/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0003-2670(03)00677-9
244 L.A. Tortajada-Genaro et al. / Analytica Chimica Acta 488 (2003) 243–254
on flow assemblies and chemiluminescence detection
have been described using temporal difference in
activator effect [20,21] and temporal injection [22].
In this paper, two automated assemblies are pro-
posed for simultaneous determination of chromium
and cobalt. Computer controls a six-way valve and
a rotatory valve in order to register time-intensity
curves. The chemiluminescence (CL) registers from
luminol–hydrogen peroxide reaction are obtained
according to stopped-flow theoretical background.
In the first manifold, sample introduction is discon-
tinuous. The time-intensity register is composed from
two regions divided where the flow is stopped. Previ-
ous to maximum intensity, the register is obtained from
a flow injection system. After maximum intensity, the
register is obtained from a stopped-flow system. In this
region, the response is result of decomposition of CL
species. In the second manifold, sample introduction
is continuous. Previous to maximum intensity, the reg-
ister is obtained from a system of continuous sample
injection and after a decay of CL intensity is observed.
For the resolution of binary mixtures, partial least
squares regression (PLS) and H-point standard addi-
tion method (HPSAM) have used. HPSAM [23] is
other useful multivariate calibration method in equi-
librium and in kinetic studies. The method has been
developed for the determination of unbiased analyte
concentrations in the event that the presence of a direct
interferent and/or the total Youden blank is known.
HPSAM has been also applied for the correction of
measured signal in FIA systems [24].
The usefulness of the new proposed method is
demonstrated by applying it to simultaneous determi-
nation of cobalt and chromium in some water samples.
These metals are widespread in water, existing in vari-
ous chemical, physical and morphological forms. Very
low levels are presented in underground and surface
waters but industrial wastes often introduce them into
hydric cycle. Their release must be carefully moni-
tored because many of their forms are toxic. Then,
laws in the most of the countries have fixed the max-
imum concentration for Co and Cr in drinking water.
2. Theorical background
Let us consider a sample S which is a binary mix-
ture of species X and species Y. If the responses are
additives, the signal of sample at time ti will be a con-
tribution of both species (SS,i = SX,i + SY,i ), where
SS ,i , SX,i and SY ,i are the sample signal, signal of
species X and signal of species Y at ti , respectively.
2.1. HPSAM for flow injection
In this paper, HPSAM [24] has been modified for
stopped-flow data. The time-intensity register is com-
posed from two regions, according to point where the
flow is stopped. Previous to maximum intensity, the
register is obtained from a flow injection system. The
response is result of dispersion of sample zone within
the carrier and the chemical process of formation
of a chemical species. After maximum intensity, the
register is obtained from a stopped-flow system. In
this region, the response is result of decomposition of
chemiluminescence species. In Fig. 1a, an example
of injected stopped-flow registers is shown.
For the determination of species X, HPSAM is based
on selection of two times ti and tj in such a way that
the interferent signals of species Y are equal to SY,i =
SY,j ( SY,i,j = 0). ti corresponds to FI region and tj
corresponds to stopped-flow region.
The difference of signals at two times ti and tj will
be for the sample:
SS,i,j = SS,j − SS,i = SX,j + SY,j − SX,i − SY,j
= SX,i,j + SY,i,j = SX,i,j (1)
Therefore, standard addition calibrations of species
X provides values of slopes Mi and Mj at two previ-
ously selected times, ti and tj , In absence of matrix
effect, the Mi and Mj values can be calculated from
standard calibration curves, directly. These calibration
lines intersect at H-point, with (−cH , SH ) coordinates.
The H-point is a function of the analyte concentration
(cX ) expressed in the following equation:
SS,i,j SX,i,j
−cH = = = cX (2)
Mj − M i Mj − M i
The species Y concentration is calculated by inter-
polation of the SH value in a conventional calibration
graph. Other possibility is to develop another equation
similar to Eq. (1) for species Y where SX,i,j = 0.
The method can generally be performed with more
than one pair of variables (ti , tj ), in which case it can
be considered a multivariate method. The selection of
 L.A. Tortajada-Genaro et al. / Analytica Chimica Acta 488 (2003) 243–254
Fig. 1. Hypothetical analytical signal for stopped-flow manifold:
(a) flow injection and (b) continuous injection.
variables is based on the best ratio between noise and
analyte increments.
2.2. HPSAM for continuous injection flow
In this paper, K-ratio [25,26], a variant of HPSAM,
has been modified for injected stopped-flow data.
The time-intensity register is composed from two re-
gions, according to point where the continuous flow
is stopped. Previous to maximum intensity, the regis-
ter is obtained from a system of continuous sample
injection. The response is only the chemical process
of formation of a chemical species. After maximum
intensity, the register is obtained from a stopped-flow
system. In this region, the response is result of decom-
245
position of chemiluminescence species. In Fig. 1b,
an example of stopped-flow registers for continuous
injection is shown.
The method is based on the well-known property
for most of analytical registers. For instance, in spec-
trophotometry, the ratio between absorbance values at
two wavelengths is a constant for a pure compound.
This fact is used for the detection of overlapped peaks
[27,28]. The study of chemiluminescence registers has
demonstrated that the ratio of signals at two times is
also a constant. For times values ti and tj , KX,i,j ratio
is defined for the X species as:
SX,i
KX,i,j = (3)
SX,j
It means the ratio between the signal at time ti (SX,i )
and the signal at time tj (SX,j ) for X species. So, the
difference SX,i − KX,i,j SX,j will be equal to zero.
Considering this weighted difference of signals at
both time values ti and tj for the sample, the following
equation is obtained:
SS,i − KX,i,j × SS,j
= SX,i + SY,i − KX,i,j × SX,j − KX,i,j × SY,j
= SY,i − KX,i,j × SY,j (4)
Therefore, the weighted increment of signals only
depends on Y species. A calibration procedure based
on standard addition of Y species will be provided
the calibration slope at the selected time values ti
and tj . In the absence of matrix effect, such values
can be calculated from standard solution of Y species
directly.
The following step is the estimation of Y species
concentration in samples. The concentration can
be calculated by interpolation of the corresponding
SS,i − KX,i,j SS,j values in the calibration curves
SY,i − KX,i,j SY,j versus cY .
3. Experimental section
3.1. Apparatus and reagents
A Jasco CL-1525 (Tokyo, Japan) chemilumines-
cence detector, a LabPro Rheodyne six-way injection
valve and a LEA six-position valve were used for the
246 L.A. Tortajada-Genaro et al. / Analytica Chimica Acta 488 (2003) 243–254

measuring. It was controlled by Borwin chromatog-
raphy software and the interface Hercule lite (JMBS,
France). A Gilson Miniplus peristaltic pump was used
to drive the reagents to the detection cell. Tygon tubing
(i.d. = 0.8, 2.0, 2.5 mm) was used for the peristaltic
pump. Other tubing was made of PTFE with i.d. =
0.5 mm. The light emission intensity was recorded as
function of time.
The following reagents were used: chromium (III)
nitrate (Panreac, Spain), cobalt nitrate (Panreac), hy-
drogen peroxide (Merck, Germany), luminol (Fluka,
Switzerland), sodium carbonate (Merck), chlorhydric
acid (trace pur, Merck) and nitric acid (trace pur). The
solutions were prepared in water (nanopure, Sybron,
Barnstead, Spain). The concentrations of stock stan-
dard solutions were 100 mg/l of chromium (III) or
cobalt (II).
3.2. Procedures
Working standard solutions were prepared in HCl,
2 × 10−3 mol/l, daily. Luminol (1.2 × 10−4 mol/l)
was prepared in carbonate solution (0.01 mol/l, pH
10.8). The concentration of hydrogen peroxide was
0.01 mol/l.
3.2.1. Assembly I. FI procedure
The carrier was composed of carbonate (0.05 mol/l,
pH 10.8) and EDTA (2 × 10−3 mol/l). The scheme of
FI manifold is shown in Fig. 2a. The luminol reagent
(L) was mixed with peroxide reagent (P). The stan-
dard solutions or samples (S) were injected in carrier
(C) by action of valve 1. The reagent flow met with
carrier flow in T-piece. Chemiluminescence emission
was registered in the detection system. Valve 2 was
always operating in position V2A, then the solution
was driven to detector position.
3.2.2. Assembly II. Stop-flow procedure by flow
injection
The manifold was the same than described above
and shown in Fig. 2a. The mixing of reagents and
injection of sample were the same than FI procedure.
However, switching of valve 2 stopped the flow in the
detection cell because solution was driven to waste
outlet.

Fig. 2. (a) Scheme of FI system and flow injected stopped-flow system and (b) Scheme of continuous stopped-flow system. V1: valve 6-ways,
V2: valve 2-ways, PP: peristaltic pump, D: detector, W: waste, C: carrier stream, S: sample stream, P: peroxide stream, L: luminol stream.
 L.A. Tortajada-Genaro et al. / Analytica Chimica Acta 488 (2003) 243–254 247

3.2.3. Assembly III. Stop-flow procedure by 3.2.5. Sampling

continuous injection
The carrier was composed of carbonate (0.125 mol/l,
pH 10.8) and EDTA (5 × 10−3 mol/l). The scheme of
FI manifold is shown in Fig. 2b. The luminol reagent
(L) was mixed with peroxide reagent (P). The stan-
dard solutions or samples (S) were mixed with carrier
(C) in T-piece when the valve 2 was in position V2A.
The reagent flow met to carrier flow in other T-piece.
Chemiluminescence emission was registered in the
detection system. The flow stopped in the detection
cell by switching of valve 1, because solution was
driven to waste outlet.
In order to obtain the registers, the following con-
trol sequences were performed. The sequences permit-
ted to control the sample injection and/or stop-flow.
Switching of valves was realised by methodology se-
quences in Borwin software. In Table 1, the general
step in accordance with the number of replicates (r) is
shown, being r = 1, 2, 3, . . . , n. In all studies, three
replicates were performed at least, r ≥ 3.
3.2.4. Calibration set
Different calibration sets were assayed. For uni-
variate calibration, standard solutions of chromium or
cobalt were measured 0–4 ␮g/l for instrumental gain
of 10 and 0–20 ␮g/l for instrumental gain of 1. Stan-
dard bicomponent mixtures were also prepared. The
composition of solutions followed a partial design
52 .
Samples were collected in different points of Va-
lencia area (Spain) and were filtered using a cellu-
lose acetate filter (0.45 ␮m). Natural water samples
were acidified with HCl, 5 × 10−3 mol/l, but the acid-
ification of metallurgic industrial wastewater was not
performed (presence of S2− ). Sampling and storage
containers and other glassware or plastic material was
acid-washed (nanopure water washing, bath HCl 10%
for 12 h and nanopure water washing).
Recovery assays were performed in a river water
sample. Spiked samples were prepared by addition of
1 + 0, 0 + 1 and 1 + 1 ␮g/l of Cr and Co, respectively.
The certificate reference material SRM 1640 (NIST,
USA), composed of natural fresh water, was also anal-
ysed.
In order to calculate the total concentration of
chromium, a reduction treatment procedure was per-
formed. To the samples 0.08% H2 O2 was added and
heated (80 ◦ C, 30 min).
For all calculations, Unscrambler (CAMO, Norway)
and Visual Basic routines for Excel were used.
4. Results and discussion
4.1. Optimisation of chemiluminescence signal
Different chemical and physical parameters were
optimised for the three configurations. According to

Table 1
Protocol sequences for controlling of valves for different procedures
Step Time (min) Valve Action Description

Flow injection
2(r − 1) + 1 1.1r + 0.1 V1 A→B Sample insertion (replicate r)
2r + 2 1.1r + 0.4 V1 B→A Filling of sample coil
Stopped-flow and flow injection
2r + 1 1.4(r − 1) + 0.1 V2 A → B Sample insertion (replicate r)
2r + 2 1.4(r − 1) + 0.3 V1 A → B Stop-flow
2r + 3 1.4(r − 1) + 0.45 V1 B → A Filling of sample coil
2r + 4 1.4(r − 1) + 1.0 V2 B → A Activation of flow
Stopped-flow and continuous injection
1 0.1 V2 A → B Sample stream insertion
2r + 2 1.1(r − 1) + 0.5 V1 B → A Stop-flow (replicate r)
2r + 3 1.1(r − 1) + 0.5 V1 A → B Activation of flow
n – V2 B → A Reinitiating
V2 in position for the entire sequence.
248 L.A. Tortajada-Genaro et al. / Analytica Chimica Acta 488 (2003) 243–254

initial experiences, most critical variables were cho-
sen. The studied range was selected based on the in-
fluence of variable in chemiluminescence signal o its
evolution in time. The optimisation criterion was the
maximum signal and maximum repeatability. So, CL
signal of standard solutions (0.5–2 ␮g/l of Cr or Co)
was registered as function of time.
The chemical variables of studied reaction were:
luminol concentration, carbonate concentration, pH
(stream L and stream C), H2 O2 concentration and
EDTA concentration. Table 2 shows the optimal val-
ues. EDTA concentration was chosen using selectivity
criteria, it means, a selective signal of Cr or Co against
other metals and similar intensity of signal for both
metals. It is important to use high quality reagents, be-
cause the presence of impurities involves bad results
(interferences or high base-line).
The hydrodynamic variables were optimised for
three configurations. Flow rate of sample stream, car-
rier stream and reagent streams were studied. The
length of reaction coil and volume of sample coil
were also optimised, considering the distance between
consecutive peaks. The selected values are shown in
Table 2.
The automation of procedure involved the optimi-
sation of control sequence. Then, the time values for
switching of valves were studied, the objective was to
stop the flow in the point maximal response. It made
possible to register some replicates avoiding over-
lapped peaks and guaranteeing to fill the sample coil
between replicates. Stopped-flow times were 0.15 min
for flow injection manifold and 0.4 min for continuous
injection manifold. The injection frequency expressed
in function of time for each replicate were 1.1 min
for flow manifold, 1.4 min for stopped-flow and flow
injection manifold and 1.1 min for stopped-flow and
continuous injection manifold. Therefore, the opti-
mised sequences is shown in Table 1.
In order to assess the robustness of method, some
experiments were performed. The optimal conditions
were varied in a factor of 10–20%. The objective was
to study the influence of the effect of experimental
variations or error sources in the response. The critical
variables were pH of carbonate buffer and EDTA con-
centrations. Specially, variations of cobalt signal were
observed. For the rest of variables the signal changes
were acceptable according to precision and accuracy
criteria.
4.2. Unicomponent and univariate determination
Analytical parameters for the determination of
chromium and cobalt were calculated in conditions
where only one analyte was measured. However,
the determination in water sample with both metals
in measurable forms was not possible because the
interference effect is mutual.
Height peak was chosen as analytical signal for
flow manifold and stopped-flow manifold using flow
injection (Fig. 3). The parameters of univariate lin-
ear calibration are summarised in Table 3 (interval
0–4 ␮g/l).

Table 2
Experimental conditions for chemical and hydrodynamic variables
Variables Unity Stream Assembly I Assembly II Assembly III

Luminol concentration mol/l L 1.2 × 10−4 1.2 × 10−4 1.2 × 10−4

Carbonate concentration mol/l L 0.01 0.01 0.01
C 0.05 0.05 0.125
Buffer pH – L 10.8 10.8 10.8
– C 10.8 10.8 10.8
H2 O2 concentration mol/l P 0.01 0.01 0.01
EDTA concentration mol/l C 2 × 10−3 2 × 10−3 5 × 10−3
Flow-rate ml/min L 1.2 1.2 1.2
P 1.2 1.2 1.2
C 3.5 3.5 1.2
S 1.2 1.2 2.8
Reactor length cm – 10 10 10
Sample coil volume ␮l – 200 200 –
 L.A. Tortajada-Genaro et al. / Analytica Chimica Acta 488 (2003) 243–254 249

Fig. 3. Normalised analytical signal for standard solutions of chromium (0–2 ␮g/l) and cobalt (0–2 ␮g/l) and for certificate reference
material (1:25) obtained stopped-flow configurations by flow injection (a) and continuous injection (b).

Detection limits were calculated from three-time
derivation of blank solution (LDN/S) and from cal-
ibration curve (LD 3syx /b). The repeatability (RSD)
was calculated from the measurement of standard so-
lutions (1 ␮g/l, replicates = 5).
It can be seen, the sensibility values, detection lim-
its and repeatability values were similar for three man-
ifolds. Additionally, the standard solutions for linear
response interval were measured using a lower in-
strumental gain (factor 10). The regression parameters
were calculated. Detection limits and sensibility val-
ues were 10 times higher than values show in Table 3.
4.3. Multivariate calibration models: PLS and
HPSAM
Fig. 3 shows the normalised registers obtained for
standard solutions of chromium and cobalt and for a
sample. It can be seen the decay of chemilumines-
cence signal is most significant for Co standards than
Cr standards, the sample has an intermediate decay.
This behaviour was observed for both configurations
of stopped-flow. The signal curve is the same for dif-
ferent concentrations of each metal ion as it can be
seen in this figure.
The application of HPSAM needed the selection
of pairs of variables according to some requirements
depending on the option studied.
For the configuration of flow injection, the oper-
ative criteria was based on a distance between vari-
ables higher than 4, a difference of interferent signals
lower than 5% and a determination coefficient (r2 ) for
(Si − Sj versus c) curve higher than 0.99.
For the configuration of continuous injection, the
variables were chosen according to a suitable K-ratio
for the corresponding interferent and analyte. It sup-
posed a distance between variables higher than 4, a
weighted increment higher than 10% for the analyte

Table 3
Analytical parameters for unicomponent and univariate determination
Parameter Assembly I Assembly II Assembly III

Cr Co Cr Co Cr Co
a 32 ± 8 14 ± 14 8±9 8±4 19 ± 3 26 ± 8
b 149 ± 3 188 ± 6 132 ± 4 144 ± 4 187 ± 2 174 ± 4
r2 0.992 0.99 0.991 0.994 0.999 0.993
syx 20 29 19 9 9 22
LD N/S 0.2 0.2 0.1 0.1 0.1 0.1
LD 3syx /b 0.4 0.5 0.4 0.2 0.1 0.4
RSD 5.5 5.3 5.2 4.2 2.0 3.3
250 L.A. Tortajada-Genaro et al. / Analytica Chimica Acta 488 (2003) 243–254

Fig. 4. Increment of signal for standard solutions of chromium and cobalt obtained stopped-flow configurations: (a) flow injection Cr,
47–115 increment, (b) flow injection Co, 51–80 increment, (c) continuous injection Cr, 27–80 increment and (d) continuous injection Co,
27–120 increment.

and a determination coefficient (r2 ) for (Si − K Sj ver-

sus c) curve higher than 0.99.
An example of increment selected for each config- Table 4
uration is shown in Fig. 4. Using a selected time pair PLS and HPSAM models for calibration set
with the same signal of Y species (Co or Cr), it is pos- Model Parameter Assembly Assembly
sible to verify than the increment is independent to II III
concentration of this species. Five concentrations of HPSAM Increments Assayed 12246 12246
Co or Cr between 0 and 2 ␮g/l were included in the Cr selection 25 32
Co selection 20 12
plots of Fig. 4. A tendency of the points in function SEP Cr 0.27 0.18
of the Y concentration was not observed. Then, it is calibration Co 0.69 0.18
demonstrated that the cancellation of the Y species is
PLS Factors Assayed 5 5
satisfactory. Selected 2 2
The predictions were calculated as an average of % Explained X-block 99.7 99.7
results provided for the pairs of variables with the Variance Y-block cal 97.9 99
highest slope of calibration curves. Table 4 shows the Y-block val 97.3 98.8
SEP Cr 0.16 0.1
assayed increments, selected increments and calcu-
calibration Co 0.14 0.16
lated errors of prediction for calibration set.
 L.A. Tortajada-Genaro et al. / Analytica Chimica Acta 488 (2003) 243–254 251

Fig. 5. Predictions of binary mixtures measured in stopped-flow assemblies with different injection configurations: (a) Cr assembly II, (b)
Co assembly II, (c) Cr assembly III and (d) Co assembly III.

In order to evaluate the prediction of previous mod-
els, binary standard solutions were determined. The
composition of these binary solutions described a par-
tial 52 design for both metals. The prediction results
are plotted in Fig. 5. It can be observed that the best re-
sults were obtained by using the assembly II. Compar-
ing both metals, the prediction of chromium is more
accurate and precise. Any difference was detected be-
tween both calibration models.
PLS-2 models were obtained for both configura-
tions from unicomponent standard solutions of Cr or
Co. X-block was composed of CL registers versus
time (column centred) for 160 variables (0–0.08 min
interval). Percentages of explained variance, number
of factors and root means standard errors of prediction
(SEP) are summarised in Table 4. SEP values were
calculated for calibration set.
The results were similar to provided for both meth-
ods, then HPSAM can be considered as an alternative
to PLS calibration. Although, the advantage of HP-
SAM lies in the simplicity and the correction of matrix
effect when it is present.
4.4. Determination in water samples
The proposed configurations were applied for the
simultaneous determination of chromium and cobalt
in different water samples. Previous to measure-
ment step, reduction treatment (Cr(VI) to Cr(III))
was performed in order to determine total chromium
252 L.A. Tortajada-Genaro et al. / Analytica Chimica Acta 488 (2003) 243–254

Fig. 6. Recovery percentages for spiked water sample measured in stopped-flow assemblies by flow injection (a) and continuous injection
(b) for both calibration models.

concentration. This procedure was based in the reac-
tion with H2 O2 in acidic conditions and T = 80 ◦ C.
Fig. 6 shows the recovery values obtained for a wa-
ter sample spiked with 1 ␮g/l of Cr, 1 ␮g/l of Co or
1 ␮g/l of Cr and 1 ␮g/l of Co. Both calibration mod-
els (PLS and HPSAM) and assemblies II and III were
also compared.
The percentages of recovery were better for assem-
bly III. Similar prediction values were observed for
PLS and HPSAM models.
A certificate reference material (SMR 1640) was
analysed. It is a fresh water sample in which chromium
and cobalt contents are 38.6 ± 1.6 ␮g/l and 20.28 ±
0.31 ␮g/l, respectively. In Fig. 3 are represented the
CL registers obtained for both configurations. The pre-
dicted concentrations are included in Table 5.
Samples collected in different locations in Valen-
cia area (Spain) were analysed. They are river water,
coastal water, harbour water and wastewater of met-
allurgic industries. The water parameters and the con-
centrations of Cr and Co for both configurations and
both calibration models are summarised in Table 5.
In order to compare both stopped-flow configura-
tions (assemblies II and III), a t-test for paired samples
was performed. The following t values were obtained:
0.57 (Cr-PLS), −1.29 (Cr-HPSAM), −3.75 (Co-PLS)
and −0.07 (Co-HPSAM). Therefore, the predictions
were similar except for Co-PLS (ttab = 2.57, 95%
and d.f. = 5). However, the predictions were more
robust and precise for continuous injection. The con-
centrations obtained for certificate reference material
indicated that the prediction errors for continuous

Table 5
Prediction of samples
Sample pH Sma Model Assembly II Assembly III

Cr Co Cr Co

CRM (1.25)b – – PLS-2 1.87 ± 0.10 0.59 ± 0.09 1.6 ± 0.3 0.69 ± 0.17
HPSAM 1.3 ± 0.2 0.9 ± 0.3 1.6 ± 0.4 0.67 ± 0.16
Harbour 8.52 2.8 PLS-2 0.61 ± 0.16 1.7 ± 0.6 0.75 ± 0.05 1.79 ± 0.10
HPSAM 0.6 ± 0.3 1.46 ± 0.11 0.52 ± 0.11 1.70 ± 0.09
Industrial (1:10) 8.22 17 PLS-2 1.1 ± 0.3 1.5 ± 0.3 1.46 ± 0.09 1.8 ± 0.4
HPSAM 1.51 ± 0.15 1.6 ± 0.3 1.43 ± 0.14 1.6 ± 0.2
Industrial (1:10) 7.80 10 PLS-2 1.86 ± 0.04 0.43 ± 0.12 1.6 ± 0.3 0.58 ± 0.05
HPSAM 1.10 ± 0.05 0.35 ± 0.06 1.4 ± 0.2 0.66 ± 0.07
Replicates n = 3.
a SM: suspended material (mg/l).
b Reference values: Cr, 1.54; Co, 0.81.
 L.A. Tortajada-Genaro et al. / Analytica Chimica Acta 488 (2003) 243–254
assembly were lower than 10% for Cr and Co, but
higher for flow assembly.
The comparison of the calibration models (PLS and
HPSAM) involved a t-test for paired samples. The fol-
lowing t values were obtained: 1.47 (Cr, discontinu-
ous), 1.85 (Cr, continuous), −0.40 (Co, discontinuous)
and 1.76 (Co, continuous). Therefore, any difference
of prediction was found between both methods (ttab =
2.57, 95% and d.f. = 5). The standard deviation for
replicates of samples were also similar for PLS and
HPSAM.
The programming of HPSAM calibration for the
procedure described is easy. Then, it is possible to
propose an analyser of chromium and/or cobalt. A
sequence of calibration standards and samples is
programmed. The concentrations of Cr and Co are
provided direct and automatically.
5. Conclusions
In this paper, the combination of two multicommu-
tated assemblies, the stopped-flow technique and two
multivariate calibration models has been proposed.
The proposed methodology has achieved the simulta-
neous determination of Cr and Co in water samples
by a chemiluminescence detection system.
The proposed assemblies were based on differ-
ent injection configurations. The critical variables
(chemical or hydrodynamic) of the systems have been
optimised. The robustness of method has been also
studied. Although both configurations have presented
similar analytical parameters for unicomponent de-
termination, some differences have been observed for
simultaneous determination. The best results have
been obtained for continuous injection providing
more robust predictions for standard solutions and
samples.
Respect to the comparison of calibration models,
PLS and HPSAM have provided similar results. Both
models can be used without distinction between cal-
culation times, predictive error of calibration or val-
idation, recovery percentages or prediction in water
samples. Therefore HPSAM can be an alternative to
PLS in the chemiluminescence analysis.
The proposed methods have been validated by us-
ing standard mixtures, spiked samples and a certified
reference material. The applicability has been demon-
253
strated by the determination of Cr and Co concentra-
tion in different water samples.
Comparing to other described methods, these strate-
gies have some advantages like low cost, rapid re-
sponse and no limitations of specialised personnel.
These characteristics are very important for periodical
monitoring of water quality.
Acknowledgements
The authors are grateful to the DGICYT (Project no
PB 97-1387) for financial support. L.A.T.G. expresses
their gratitude to Ministerio de Eduación y Cultura
(Spain) for the predoctoral grant.
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