Universidade de So Paulo

2011

Direct Solid-Phase Optical Measurements in
Flow Systems: A Review


ANALYTICAL LETTERS, v.44, n.1/Mar, p.528-559, 2011
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Direct Solid-Phase Optical Measurements
in Flow Systems: A Review
Fbio R. P. Rocha
a
, Ivo M. Raimundo Jr.
b
& Leonardo S. G. Teixeira
c
a
Centro de Energia Nuclear na Agricultura, Universidade de So
Paulo, So Paulo, Brazil
b
Instituto de Qumica, Universidade Estadual de Campinas, So
Paulo, Brazil
c
Instituto de Qumica, Universidade Federal da Bahia, Campus
Universitrio de Ondina, Salvador, Brazil
Version of record first published: 18 Feb 2011.
To cite this article: Fbio R. P. Rocha, Ivo M. Raimundo Jr. & Leonardo S. G. Teixeira (2011): Direct
Solid-Phase Optical Measurements in Flow Systems: A Review, Analytical Letters, 44:1-3, 528-559
To link to this article: http://dx.doi.org/10.1080/00032719.2010.500790
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Flow and Sequential InjectionGeneral Approaches
DIRECT SOLID-PHASE OPTICAL MEASUREMENTS
IN FLOW SYSTEMS: A REVIEW
Fabio R. P. Rocha,
1
Ivo M. Raimundo, Jr.,
2
and
Leonardo S. G. Teixeira
3
1
Centro de Energia Nuclear na Agricultura, Universidade de Sa o Paulo,
Sa o Paulo, Brazil
2
Instituto de Qumica, Universidade Estadual de Campinas, Sa o Paulo, Brazil
3
Instituto de Qumica, Universidade Federal da Bahia, Campus Universita rio
de Ondina, Salvador, Brazil
Measurements based on absorption, reectance, or luminescence of molecular species or
complex ions can be carried out directly on a solid support simultaneously to the retention
of the analyte. The use of this strategy in ow-based systems is advantageous in view of the
reproducible handling of solutions in retention and elution steps of the analyte. This
approach can be exploited to increase sensitivity, minimize reagent consumption as well
as waste generation, improve selectivity or for simultaneous determination based on selec-
tive retention or differences in sorption rates of the analytes. This review focuses on the
main characteristics of direct solid-phase measurements in ow systems, including the dis-
cussion of advantages and limitations and practical guidelines to the successful implemen-
tation of this approach. Selected applications in diverse elds, such as pharmaceutical,
food, and environmental analysis are discussed.
Keywords: Flow analysis; Flow-through optosensors; Infrared spectroscopy; Luminescence; Reectance;
Review; Solid-phase spectrophotometry
INTRODUCTION
Flow analysis in its different modalities (e.g., FIA, SIA, and MCFA) is gener-
ally used for processing samples in solution, aiming mechanization, or automation of
analytical procedures. In this sense, the main aim is to replace operations usually
carried out by the analyst, improving precision and reducing the analysis time.
Sensitivity in ow-based methodologies is usually lower than those achieved in batch
procedures, due to the dispersion process and the incomplete conversion of the
analyte to the species to be measured, due to the reduced sample residence time.
Received 13 March 2010; accepted 5 May 2010.
This paper was submitted as part of a Special Issue on Flow Injection Analysis.
Address correspondence to Leonardo S. G. Teixeira, Instituto de Qu mica, Universidade Federal
da Bahia, Campus Universitario de Ondina, Salvador, Bahia, 40170-290, Brazil. E-mail: lsgt@ufba.br
Analytical Letters, 44: 528559, 2011
Copyright # Taylor & Francis Group, LLC
ISSN: 0003-2719 print=1532-236X online
DOI: 10.1080/00032719.2010.500790
528
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However, heterogeneous systems can be also adapted to ow analysis, sometimes
contributing to the improvement of the analytical performance. A typical example
is the use of solid-phase reactors, which are exploited for analyte concentration, deri-
vatization (e.g., using of immobilized reagents or enzymes), as well as on-line gener-
ation of reagents (Luque de Castro 1992; Calatayud 1996).
Direct measurements on solid-phase are another interesting alternative involv-
ing application of heterogeneous systems, in which the analytical signal is recorded
simultaneously to the retention of the analyte on a suitable solid-support (Teixeira
et al. 2001; Rocha and Teixeira 2004). Measurements can be carried out by electro-
chemical or, more typically, by spectrometric techniques. The approach called solid
phase spectrophotometry (SPS) or optosensing, which exploits measurements by
UV-vis spectrophotometry (transmission or reectance modes), is clearly the most
frequently used detection technique for direct measurements on solid surfaces.
However, luminescence and infrared spectroscopy have also been exploited (Rocha,
Teixeira, and Nobrega 2009). The main advantages of this approach are: (1) increase
in sensitivity and achievement of lower detection limits due to the possibility of
accumulation of a large amount of the analyte in a reduced amount of the solid-
support; (2) improvement of selectivity by separation of the analyte from the sample
matrix or by exploiting differences in the rates of reaction or retention of the analyte
and concomitant species on the solid-support; (3) simultaneous determinations by
kinetic discrimination; and (4) minimization of reagent consumption and waste gener-
ation by the reversible retention of the analyte and reuse of the solid-phase reagent.
In spite of several applications of direct measurements on solid-phase carried
out in batch, the use of ow systems present some advantages. The differences in
packing of the solid-phase in the cell between the measurements, commonly observed
in batch procedures, are avoided. In addition, sample processing in ow systems are
less time-consuming and precision is improved. On the other hand, enrichment
factors are usually lower than in batch mode due to the lower sample volumes.
A schematic representation of the process is presented in Figure 1.
Some requirements need be attained for direct solid-phase measurements in
ow systems:
1. Thermodynamic of the retention process: The coefcient of distribution of the
retained species needs to be considered, i.e., analyte retention on the solid support
needs to be favorable. With this aim, non-polar materials are used for the reten-
tion of hydrophobic species, and so on. When reaction occurs at the solid surface
(e.g., complex formation with an immobilized ligand), the equilibrium constant
and the reaction conditions (e.g., pH and presence of masking agents) should also
be taken into account. However, in ow based systems the residence time is
usually lower than those required to achieve the steady state, and the thermodyn-
amic values are only indicative;
2. Selectivity: the solid-support and reactional conditions should allow separation of
the analyte from interfering species. This is often achieved by chemical derivatiza-
tion with a selective reagent immobilized at the solid support or previously to the
retention of the species at the solid surface. Control of the composition of the
medium (e.g., pH) and use of selective eluents are other commonly employed
approaches;
DIRECT SOLID-PHASE MEASUREMENTS IN FLOW SYSTEMS 529
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3. Kinetic aspects: analyte retention needs to be compatible with the short residence
time characteristic of the ow methodologies. Fast retention of the analyte
in comparison to that of interfering species can also be exploited to improve
selectivity.
4. Reversibility: the solid-phase needs to be efciently regenerated by the eluent sol-
ution after sample measurement by the eluent solution, making feasible the use of
the same solid support for several measurements. One alternative is replacement
of the whole solid support in each measurement cycle, which can be also
efciently carried out in ow systems;
5. Stability of the solid support: the solid-phase should not change signicantly when
submitted to the different media required in the retention and elution steps. This is
more critical when a reagent is immobilized on the solid support and leaching can
reduce the lifetime of the solid-phase sensor. This is often minimized by proper
immobilization of the reagent at the solid support and selection of suitable carrier
and eluent solutions. The stability of the solid-phase under irradiation should also
be evaluated, especially for measurements based on uorescence, when the solid
surface is submitted to high power radiation favoring photo-degradation.
6. Compatibility between the solid support and the measurement system: materials
which cause excessive attenuation of the radiation beam (by absorption or
scattering) are not suitable for spectrophotometric measurements based on trans-
mission, but can be used for measurements by reectance or uorescence;
7. Backpressure: the measurement cell, particle size, and packing need to be care-
fully selected to avoid uid leakage. This aspect generally limits the total ow-rate
that can be employed, also affecting system design. As an alternative, reagents
can be immobilized in porous membranes, minimizing the drawbacks related to
the increase of the hydrodynamic impedance.
Figure 1. Schematic representation of a typical FI-SPS procedure and the corresponding signal: a)
establishment of baseline and insertion of the sample aliquot; b) retention of the analyte at the solid
support yielding the analytical signal; and c) elution process and regeneration of the solid support. In
the scheme, a transient signal is obtained because the eluent reaches the solid support immediately after
the whole sample zone cross the ow cell.
530 F. R. P. ROCHA ET AL.
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The ow manifold is designed to implement distinct conditions for analyte
retention and elution. A common strategy is preparation of the sample in a medium
suitable for analyte retention and injection on an eluent stream (Lazaro, Luque de
Castro, and Valcarcel 1989). With this approach, the sample volume should be large
enough to avoid mixing with the eluent in the central part of the sample zone. More
ingenious alternatives are the sequential injection of sample and eluent (Teixeira et al.
1999) or the intermittent addition of the eluent solution (Molina-D az et al. 2002).
Both strategies are represented in the scheme in Fig. 1, in which a transient signal
is obtained because the eluent reaches the solid support immediately after the whole
sample zone cross the ow cell.
Applications have exploited the direct retention of the analyte (Ruiz-Medina,
Fernandez de Cordova, and Molina-D az 1999; Ortega-Barrales et al. 2002), reten-
tion of a reaction product formed in solution (Frenzel and Krekler 1995; Cassella
et al. 2000), or direct formation of the product on solid-phase (Ayora-Canada,
Pascual-Reguera, and Molina-D az 1998; Teixeira et al. 1999).
This review focuses on the main characteristics of direct solid-phase measure-
ments in ow systems, including the discussion of advantages and limitations and
practical guidelines to the successful implementation of this approach. Selected
applications in diverse elds, such as pharmaceutical, food, and environmental
analysis are discussed.
APPLICATIONS OF DIRECT SPECTROMETIC MEASUREMENTS ON SOLID
PHASE IN FLOW SYSTEMS
UV-Vis Spectrophotometry
Most of the applications of direct optical measurements on solid-phase are based
on transmission spectrophotometry. In solid-phase spectrophotometry (SPS), the absor-
bance due to the retention of the analyte is measured directly on the solid surface, which
can be modied by immobilization of a suitable chromogenic reagent. In the pioneer
work, presented by Yoshimura et al. in 1976, an ion-exchange resin modied with
1,5-diphenylcarbazide, 1,10-phenantroline, ammonium thiocyanate or Zincon was
employed for the determination of chromium, iron, cobalt, or copper, respectively.
After analyte retention, the solid support was transferred to a cuvette, resulting in a
10-fold increase in sensitivity in comparison to the analogous procedures in solution.
Batch procedures are usually based on dual-wavelength spectrophotometry, being mea-
surements simultaneously carried out at the absorption maximum and at a wavelength
taken as reference, in order to compensate differences in packing of the solid-phase
between measurements. On the other hand, coupling of solid phase spectrophotometry
to ow systems (FI-SPS) allows the use of simple mono-channel spectrophotometer.
This coupling was rst evaluated for retention of copper in a cation-exchange resin
placed at a ow cell designed for measurements in solution (Yoshimura 1987). Most
of the applications of FI-SPS are aimed at determination of metal ions (Table 1) or spe-
cies of pharmaceutical interest (Table 2). Applications on this subject were previously
revised (Molina-D az et al. 2002; Matsuoka and Yoshimura 2010). The potential of ow
systems coupled to solid-phase spectroscopy to the development of Greener Analytical
procedures was also emphasized (Garc a-Reyes, Gilbert-Lopez, and Molina-D az 2009).
DIRECT SOLID-PHASE MEASUREMENTS IN FLOW SYSTEMS 531
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The solid support and the design of the measurement cell are critical aspects for
SPS based on transmission. The solid material should ideally be transparent at the
measurement wavelength, and the thickness of the solid phase should be carefully
adjusted in order to minimize the attenuation of the radiation beam, which can occur
by absorption, scattering, and reection. On the other hand, limited amounts of the
solid support can hinder the response range, by limiting the number of sites for ana-
lyte retention. Measurements by FI-SPS have been carried out with conventional
ow-cells partially lled with the solid support (Yoshimura 1987; Yoshimura,
Matsuoka, and Waki 1989) or by specially designed cells. These are projected to
increase the amount of solid at the observation area and to reduce the backpressure
(Reis et al. 2000).
The increase in sensitivity by SPS depends mainly on the optical path and the
sample volume. Measurements in ow systems usually are carried out with sample
volumes lower than 1 mL and optical paths of ca. 1 mm. As a consequence, sensi-
tivity is usually lower than that achieved in batch, in which sample volumes, some-
times as high as 1000 mL, are employed. However, mechanization of the process,
improvement of precision, and an increase of the sample throughput make ow
based measurements attractive.
Direct measurements on solid phase are an effective way to increase sensitivity,
as demonstrated by comparing three procedures for preconcentration in determi-
nation of total phenols by the reaction with 4-aminoantipyrine (Frenzel and Krekler
1995). The direct measurement of the product by FI-SPS yielded better sensitivity,
with a detection limit of 0.4 mg L
1
, against 8 mg L
1
for liquidliquid extraction of
the reaction product in chloroform and 11 mg L
1
for preconcentration of the pro-
duct on a C
18
-bonded silica mini-column followed by elution with methanol. The low
detection limit by SPS in comparison to the last procedure was achieved because
sample dilution at the elution step is avoided, illustrating one of the advantages of
FI-SPS.
Several procedures have been proposed for determination of metal ions in low
concentrations (Table 1). This can be carried out after complex formation in solution
or directly on the solid-phase. The former approach can be exemplied by the deter-
mination of Sn(IV) in fruit juices after complex formation with Pyrocatechol Violet
(Capitan-Vallvey, Valencia, and Miron 1994). The reaction product was retained on
Sephadex QAE A-25 gel and reagent concentration was selected in order to minimize
the blank values.
The complex formation with an immobilized reagent is more interesting in view
of the reuse of the ligand for sequential determinations. In this sense, a solid-phase
formed by 1-(2-pyridylazo)-2-naphthol (PAN) immobilized on a cation-exchange
resin was employed for zinc determination in water, human hair, pharmaceutical,
and cosmetic preparations (Ayora-Canada et al. 1998). Variation of the sample vol-
ume (100 or 2000 mL) was exploited to extend the working ranges to 20500 mg L
1
or 0.24.0 mg L
1
, with sampling rates of 38 or 15 determinations per hour, respect-
ively. An EDTA solution prepared in buffered medium at pH 4.0 was used to elute
the metal ions without removing PAN from the solid support. A similar approach
was adopted for determination of nickel in alloys, petroleum crude, mineral oil,
and industrial wastewater, using H
2
SO
4
as eluent (Ayora-Can ada, Pascual Reguera,
and Molina-D az 1999). Other examples of applications of FI-SPS for metal ions are
536 F. R. P. ROCHA ET AL.
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the determinations of iron in natural waters and wines by formation of complex with
thiocyanate (Lazaro et al. 1989); molybdenum in natural waters or rocks through
reaction with Tiron (Yoshimura et al. 1989); V(V) in mussel, oyster and toadstool
tissues, petroleum crudes and water samples (Ayora-Can ada, Pascual-Reguera,
and Molina-D az 2000); and chromium(VI) after reaction with 1,5-diphenylcarba-
zide (Yoshimura 1988). In the last application, sensitivity increased 160-fold in com-
parison to the measurements in solution.
Reversible analyte retention and reuse of the immobilized reagent were
achieved in applications exploiting 1-(2-tiazolylazo)-2-naphthol (TAN) adsorbed
on C18 bonded silica (Baliza, Ferreira, and Teixeira 2009). A specially designed
ow-cell containing the solid-phase was coupled to ow injection systems for deter-
mination of zinc in pharmaceutical preparations (Teixeira et al. 1999) and iron in
natural waters (Teixeira and Rocha 2007). Elution of the metal ions was implemen-
ted with a low volume of a diluted acid solution without removing the TAN reagent,
which was used for at least 100 determinations. This corresponds to reagent con-
sumption lower than 1 mg per determination, which is 1000-fold lower than those
required in batch determination of zinc by conventional spectrophotometry (Ferreira
et al. 1995). The procedures are, thus, inherently more environmentally friendly, in
addition to the increase in sensitivity (e.g., 10-fold higher sensitivity for iron deter-
mination in comparison to measurements in solution).
The C
18
-TAN system was also employed for the sequential determination of
nickel and zinc in copper-based alloys, exploiting differences in the sorption rates
of the metal ions (Teixeira et al. 2000). Zinc retention is relatively fast and inde-
pendent of the ow rate in the range 0.70 to 2.2 mL min
1
, whereas nickel complexa-
tion at the solid-phase is hindered at higher ow rates. A ow injection system with
intermittent ows was employed to carry out measurements at different ow rates
(0.85 and 1.9 mL min
1
) for sequential sample injections.
Chemometric data treatment by Partial Least-Squares (PLS-2) and the differ-
ences in the retention rates at the solid support were explored for the simultaneous
determination of iron, nickel, and zinc, whose TAN complexes yield strongly over-
lapped spectra (Teixeira et al. 2002). The PLS was also applied for data treatment
aimed at the simultaneous determination of caffeine, dimenhydrinate, and acetami-
nophen (Ayora-Canada, Pascual Reguera, Molina-D az, and Capitan-Vallvey 1999)
or caffeine, acetaminophen, and acetylsalicylic acid (Ruiz-Medina et al. 1999) by UV
measurements directly at the solid phase.
Sulde was determined by FI-SPS by formation of Methylene Blue in reaction
with N,N-dimethyl-p-phenylenediamine and iron(III) (Cassella et al. 2000). The reac-
tion product was adsorbed on C
18
-bonded silica, and the elution was carried out with
a mixture of methanol and diluted hydrochloric acid. A linear response was achieved
from 5 to 50 mg L
1
, with a sampling rate of 12 determinations per hour. The
same solid support was employed for determination of pyridoxine (vitamin B
6
) after
derivatization with N,N-diethyl-p-phenylenediamine and potassium hexacyanoferra-
te(III) (Portela et al. 2004). The reaction product showed maximum absorption at
633 nm and the procedure presented linear response up to 4.0 or 10.0 mg L
1
for
860 or 235 mL sample volumes, respectively.
An alternative to the solid materials used in FI-SPS is reagent immobilization
in porous membranes. This strategy often simplies the coupling to the ow system
DIRECT SOLID-PHASE MEASUREMENTS IN FLOW SYSTEMS 537
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and overcomes the drawbacks caused by backpressure. Reagent immobilization on
Naon
1
, for example, has found several applications, such as determination of
nickel based on immobilization of 2-(5-bromo-2-pyridylazo)-5-(diethylamino)phenol
(Amini et al. 2004) or of selenium with p-amino-p
0
-methoxydiphenylamine or varia-
mine blue (Coo and Martinez 2004). In the former, the chromogenic reagent was
directly dissolved in a 5% Naon
1
solution which originated a transparent and
mechanically stable sensing membrane. The immobilization process minimized
reagent leaching and increased the sensor lifetime, which was applied to nickel deter-
mination in samples of vegetable oils and chocolate. For selenium determination, the
sensing phase was prepared by immersing the membrane in a concentrated reagent
solution. The optode was coupled to a ow injection system for selenium determi-
nation in natural waters. Another approach was the development of an optical sen-
sor based on the physical entrapment of 4-(2-pyridylazo)resorcinol (PAR) in solgel
thin lms (Jero nimo et al. 2004). The sensor was applied for copper determination in
urine samples in a multicommuted ow system, yielding results in agreement with
ICP-MS.
Spectrophotometric determination without chemical derivatization (measure-
ment of the intrinsic absorption by the analytes) can be implemented in FI-SPS
due to the capacity to separate the analyte from the sample matrix. This also contri-
butes to the development of greener procedures by avoiding the use of toxic reagents.
As an example, ascorbic acid and paracetamol were both determined in pharmaceu-
tical formulations by absorption at 264 nm (Ruiz-Medina et al. 2000). An anion
exchanger gel (Sephadex
1
QAE A-25) placed in an 1-mm optical-path quartz ow
cell was used as solid support for at least 200 determinations. The strategy for deter-
mination of both species was the selection of suitable solutions employed as carrier=
elution streams in a single line manifold. When acetate buffer at pH 5.6 was used as a
carrier, only ascorbic acid was temporally retained on the solid support, which is
quantied without interference from paracetamol or other components of the sample
matrix. The carrier was then replaced to NaCl at pH 12.5 for the selective retention
of paracetamol in its ionic form (phenolic group ionized). Linear responses were
observed from 0.3 to 20 mg L
1
ascorbic acid and from 0.4 to 25 mg L
1
paraceta-
mol, with a mean sampling rate of 20 determinations per hour.
Intrinsic absorption of radiation was also exploited for the determination of
salicylamide and caffeine in pharmaceutical preparations (Llorent-Mart nez et al.
2005). A multicommuted ow system was employed to reduce up to 85% the con-
sumption the carrier and eluent solutions. Other applications of ow-through opto-
sensors coupled to multicommutation for environmental analysis were previously
revised (Llorent-Mart nez, Ortega-Barrales, and Molina-Diaz 2006).
An ingenious alternative was proposed for the simultaneous determination of
thiamine and ascorbic or acetylsalicylic acids in pharmaceutical preparations based
on direct measurements at UV (Ortega-Barrales et al. 2002). A mini-column and a
ow-through cell, both lled with a cation-exchange gel (Sephadex
1
SP-C25), were
placed in sequence in the ow system. Vitamin B
1
was retained at the mini-column,
while the other analyte was carried out to the ow cell being measured in the inter-
stitial solution. Thiamine was then eluted by acetate buffer yielding a transient signal
for analyte quantication. Increase of sensitivity by direct measurements on
solid-phase and reduction of the apparent absorptivity by measurements in the
538 F. R. P. ROCHA ET AL.
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interstitial solution enabled the determination of the analytes in different concen-
tration ranges. Linear responses were then observed in the ranges 350, 25400,
and 3003000 mg L
1
for thiamine, ascorbic acid, and acetylsalicylic acid, respect-
ively. In another work, a mini-column and a ow-through cell both lled with
C
18
-bonded silica were placed in series in a ow system for the determination of
paracetamol, acetylsalicylic acid, and caffeine. The analytes were determined in a sin-
gle sample injection and discrimination was due to the different interactions with the
solid upport (Dom nguez-Vidal et al. 2002a). A similar strategy was later used for
determination of sulfamethoxazole and trimethoprim by using a mini-column and
a ow-through cell lled with Sephadex SP C-25 ion-exchanger gel and different elu-
ent solutions (Fernandez de Co rdova et al. 2003).
In another approach for biparametric determination, ow cells packed with
cationic (Sephadex SP C-25) and anionic (Sephadex QAE A-25) exchange gels were
used in a ow system for conjunct determination of ascorbic acid and thiamine
(Ruiz-Medina 2002). The cells were placed in a double beam spectrophotometer
and suitable solutions were used as carrier and eluent for sequential determination
of both analytes. For a sample volume of 1000 mL, linear responses were obtained
in the ranges 0.515 and 350 mg L
1
for thiamine and ascorbic acid, respectively.
In FI-SPS, the eluent should efciently regenerate the solid-phase in order to
assure the same experimental conditions for analyte retention in subsequent samples.
However, this is not ease to achieve for species strongly retained on the solid-phase,
requiring the use of a solid support with high retention capability or renovation of
the solid-phase in each measurement cycle. An ingenious alternative is the bead injec-
tion approach, which explores the reproducible handling of suspensions in ow sys-
tems for the regeneration of the solid support. The bead suspension can interact with
the sample during the transport to the ow-through cell or after retention of the solid
(Ruzicka and Scampavia 1999). After measurement, the whole solid-phase is dis-
carded and the process is repeated for a new sample aliquot. Bead injection was suc-
cessfully implemented with a commercially available ow cell by using ferrozine
immobilized in an anion exchanger (Ruedas-Rama, Ruiz-Medina, and Molina-D az
2003). The procedure was applied to the determination of iron in wine, natural
waters and pharmaceutical preparations as well as of ascorbic acid in fruit juices,
pharmaceuticals and conservatives.
Luminescence
Analytical methods based on luminescence are less widely applicable than
absorption methods because of the relatively limited number of species which pre-
sents radiative relaxation. However, these techniques show attractive features such
as better sensitivity and selectivity and wide linear range when compared with
absorption spectrometry (Skoog, Holler, and West 1994). This also holds for direct
measurements in solid-phase, as demonstrated by recent applications.
As absorbance measurements directly on solid supports, the solid phase uori-
metry (SPF) have the merit of combining separation and preconcentration steps with
in situ surface uorescence detection (Zhu, Chen, and Zhou 2003). The association of
ow analysis with SPF combines the advantages of both approaches: the selectivity
and sensitivity inherent to solid phase spectrouorimetry and the capability for
DIRECT SOLID-PHASE MEASUREMENTS IN FLOW SYSTEMS 539
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mechanized sample processing with high sampling rate usually achieved in ow-
based methodologies. In addition, retention on a solid surface can increase the quan-
tum yield of the luminescence processes by minimizing the losses of energy by
vibrational relaxation. This is especially relevant for measurements of phosphor-
escence at room temperatures. Table 3 summarizes the characteristics of some appli-
cations of this combined approach.
A procedure based on the combination of multi-commutation and SPF has
been developed for the determination of thiabendazole in citrus fruits (Garcia-Reyes
et al. 2006). The native uorescence of the target pesticide, retained on-line on C
18
-
bonded silica packed in the detection ow cell, was exploited for analytical purposes.
Under the optimized conditions, the analytical response was linear in the range
0.310 mg kg
1
with a detection limit of 0.09 mg kg
1
and coefcient of variation
better than 2%.
An analytical procedure for determination of dipyridamole consisted of a sin-
gle channel ow-injection system using Sephadex
1
QAE A-25 resin as a sorbing sub-
strate, placed into a uorimetric ow-through cell. The uorescence of the analyte
retained on the solid phase was continuously monitored followed by elution with
a phosphate buffer solution at pH 6.0 allowing resin regeneration. The system
responds linearly in the range 10500 mg L
1
with a detection limit of 0.94 mg L
1
and coefcient of variation of 0.82%. The method was applied to the determination
of dipyridamole in pharmaceutical preparations and human plasma (Ruiz-Medina,
Fernandez de Co rdova, and Molina-D az 2001).
Sometimes, it is necessary the derivatization of the analyte to a uorescent species
before sorption at the solid-support. An example of this strategy is the uorimetric
determination of thiamine after its conversion to the uorescent thiochrome by oxi-
dation with hexacyanoferrate(III) in alkaline medium (Zhu et al. 2003). The thio-
chrome formed was concentrated and separated from the sample matrix by sorption
on octadecyl-alklylated poly-[styrene=divinylbenzene] (C
18
-PS=DP) emitting strong
uorescence on the solid surface. Based on this observation, a sequential injection sys-
tem coupled to a ow-through cell was employed for sample processing, including
chemical derivatization, sorption of the reaction product and removal of the
solid-support after measurement. Detection was realized by coupling the ow-through
cell to a spectrouorimeter by means of optical bers. Under the optimized conditions,
a detection limit of 0.03 mg mL
1
was achieved with a sample throughput of 30 h
1
and
consumption of 1 mg C18-PS=DP microbeads per run. The developed approach was
applied for the determination of thiamine contents in pharmaceutical preparations.
Another useful way to convert a non-uorescent analyte to a uorescent com-
pound is through photochemical derivatization. The photochemically induced uor-
escence (PIF) detection was also adopted for determination of thiamine. In the
procedure, the uorescent photoproduct generated on-line was retained on C
18
-
bonded silica and the emission was continuously monitored on the solid surface.
The elution of the analyte from the solid support was carried out with a 60% (v=v)
acetonitrile solution. Concentrations of the vitamin in the range 0.0950 ng mL
1
were determined with a detection limit of 28 ng L
1
for a 600 mL sample. The method
allowed the determination of the vitamin in pharmaceutical preparations and biologi-
cal samples without chemical derivatization (Lopez-Flores, Fernandez de Cordova,
and Molina-Diaz 2005).
540 F. R. P. ROCHA ET AL.
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A ow-through optosensor combined with PIF was also employed for
simultaneous determination of binary mixtures of sulfamethoxazole=sulfanilamide
and sulfathiazole=sulfanilamide. The procedure exploited the native uorescence
of sulfanilamide and that of the photoproduct generated from sulfamethoxazole
(or sulfathiazole). The strategy used for simultaneous determination was the on-line
separation of both sulfonamides in a mini-column lled with Sephadex
1
QAE A-25,
placed before the ow cell containing the same resin. While one of the sulfonamides
passed through the mini-column and was determined by its native uorescence, the
other was retained and photochemically converted into a uorescent photoproduct
after elution, yielding a transient signal on the sensing support. The selected resin
was able to retain sulfamethoxazole and sulfathiazole in pH above 4.0, whereas
sulfanilamide only was retained in pH values above 10.0, making possible their
simultaneous determination. The detection limits for the determination of sulfa-
methoxazole, sulfanilamide, and sulfathiazole were 8.1, 2.9, and 5.7 ng mL
1
,
respectively, and the method was applied to pharmaceuticals, milk, and human urine
(Flores, Fernandez de Co rdova, and Molina-D az 2007).
Simultaneous determination of thiabendazole and benomyl was possible with a
solid phase spectrouorimetric ow injection method. In the procedure, C
18
silica gel
beads were placed in the ow cell and the pesticides were separated using different
retention-desorption kinetics in their interaction with the solid support. After thiaben-
dazole was totally eluted from the solid phase by a 20% (v=v) CH
3
OH solution and its
transient uorescence signal was obtained, a 65% (v=v) CH
3
OH solution was used for
eluting benomyl. For a sample volume of 3200 mL, the detection limits were 0.06 and
3.6 ng mL
1
for thiabendazole and benomyl, respectively, and coefcient of variation
were lower than 5% for both analytes. The proposed method was applied to pesticide
formulations and water samples (Garcia-Reyes, Llorent-Mart nez et al. 2004).
Other approach for simultaneous determination is the combination of a ow-
through optosensor spectrouorimetric system with partial least-squares (PLS)
calibration. In this way, a procedure was developed for the resolution of mixtures
of three pesticides: a-naphthol, o-phenylphenol, and thiabendazole. The sensor
was developed in conjunction with a single channel ow-injection manifold with
detection using C
18
silica gel as solid support. The different retention rates of the
analytes on the sensing zone allowed the selection of a time matrix for each analyte
providing better results in the PLS calibration. The method was successfully applied
to natural water samples (Dom nguez-Vidal et al. 2007; Garcia-Reyes et al. 2006).
Others strategies employing uorescence based optosensors were used for simul-
taneous determination of fuberidazole, carbaryl, and benomyl (Garcia-Reyes,
Llorent-Mart nez et al. 2004); and benomyl and carbendazim (Garcia-Reyes,
Ortega-Barrales, and Molina-D az et al. 2003).
The use of molecularly imprinted polymers (MIPs) is an interesting alternative
to obtain new materials that can be used in the development of optical chemical sen-
sors, considering the highly selective molecular recognition properties, physical
robustness, and good thermal, chemical, and mechanical stability. These facts render
them particularly suitable for use as recognition elements in sensor technology. In
this way, advantages of uorescent ow-through sensors were improved employing
a solid matrix based on MIPs (Valero-Navarro, Damiani, et al. 2009; Valero-
Navarro, Salinas-Castillo, et al. 2009).
DIRECT SOLID-PHASE MEASUREMENTS IN FLOW SYSTEMS 543
D
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A MIP solid-phase extraction ow-injection uorescence sensor was developed
for determination of orfenicol in animal tissues (Gea et al. 2010). In the procedure,
after adsorption of the analyte and uorescence response, the clean step was performed
with a methanol:acetic acid:SDS solution. Under optimum conditions, the intensity of
uorescence was directly proportional to the concentration of the antibacterial agent
over the range from 1.2 10
6
to 2.6 10
5
g mL
1
. The proposed sensor was satis-
factorily applied to the determination of the analyte in liver and meat samples.
As reported previously, ow systems required for developing analytical meth-
ods based on the measurement of native uorescence on a solid phase are very sim-
ple, normally employing a single channel conguration (Ruiz-Medina et al. 2001).
On the other hand, when non-uorescent species can be transformed into a uor-
escent compound by means of derivatizing reactions or photochemically (Zhu et al.
2003; Lopez-Flores et al. 2005), more complex manifolds (exploiting FIA, SIA, or
multicommutation) are combined with the uorescence detection on the solid phase.
Spectrouorimetry seems to be one of the most suitable analytical techniques
for pharmaceuticals, due to its simplicity and sensitivity. This methodology (batch
mode) has been successfully applied to the sensitive uorimetric determination of
various analytes. However, a low sample throughput is obtained, making this tech-
nique unsuitable for routine analysis. The combination with ow system has been
employed to determine low concentrations of various chemical species improving
sampling throughput and providing low consumption of reagents and samples
(Molina-D az et al. 2002).
Others luminescence processes, as phosphorescence and chemiluminescence,
can be used analytically for direct detection on a solid support. However, applica-
tions are scarce in comparison to the use of uorimetry, because phosphorescence
and chemiluminescence are less common phenomena. For example, a ow-through
optosensor for determination of tetracyclines, based on formation of a chelate with
europium and room temperature phosphorescence, was developed (Alava-Moreno,
Diaz-Garcia, and Sanz-Medel 1993). The method was based on the transient reten-
tion of the chelate on a non-ionic resin (Chelex
1
l00) packed in a ow-through cell.
The detection limits for tetracycline, oxytetracycline, and chlortetracycline were 0.25,
0.30, and 0.40 ng mL
1
, respectively, with a coefcient of variation of 1% for a 0.24
p g mL
1
concentration. The procedure was applied to urine samples and pharma-
ceutical preparations.
A method for determination of thiabendazole in water samples was developed
based on phosphorescence obtained when the analyte was retained in a solid support
(Piccirilli and Escandar 2009). While thiabendazole does not phosphoresce in pack-
ing materials commonly used in ow-through optosensors, emission signals were
observed after retention on nylon powder in the presence of iodide and sulte. A
ow-injection manifold coupled to a phosphorescence detector containing the solid
phase packed into a conventional ow-cell was employed. After the phosphorescence
emission was registered, the analyte was eluted with a 65% (v=v) methanolwater
mixture. Using a sample volume of 2000 mL, the analytical signal showed a linear
response in the range 12.9110 ng mL
1
, with a detection limit of 4.5 ng ml
1
and
a sample throughput of 14 samples per hour.
A single ow-through phosphorescence optosensor for simultaneous determi-
nation of the pesticide N-1-naphthylphthlamic acid and its metabolite 1-naphthylamine
544 F. R. P. ROCHA ET AL.
D
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o
a
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e
d

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was developed (Salinas-Castillo et al. 2004). The system was based on the on-line
immobilization of the analytes onto a non-ionic resin solid support (Amberlite
TM
XAD 7) in a continuous ow system, followed by the measurement of their native
phosphorescence. Nitric acid was used to strip the retained analytes from the solid
phase before proceeding with the next injection. The developed sensor was applied
to the determination of the pesticides in drinking and mineral waters. Table 4 sum-
marizes the characteristics of some applications of sensors based on phosphorescence
measurements.
The use of ow-injection system associated with chemiluminescence (CL)
detection on a solid support is another way to congure a ow-through optical
sensor (Llorent-Mart nez et al. 2009). There has been a deal of interests in the
development of CL-based sensors due to their high sensitivity compared with
photoluminescence-based sensors. This promoted sensitivity of CL-based sensing
system is ascribed to the avoidance of noise caused by light scattering, and features
simpler set-up with lower background emissions in comparison with photolumines-
cence detection. However, one of the major deciencies in applying CL sensors to
routine analysis was the short lifetime and signal drift due to the irreversible con-
sumption of CL reagents that have limited the application of CL sensors in practice
(Zhang, Zhang, and Zhang 2005).
Ion-exchange resin has been widely used to immobilize CL reagents to develop
a series of CL sensors by analyte reaction with the immobilized reagents. Generally,
the solid phase with immobilized reagents is packed into a ow cell and positioned in
front of the detection window of a photomultiplier (Zhang et al. 2005). With this
purpose, a chemiluminescence ow-through sensor for the determination of pyrogal-
lol based on the reaction between the analyte and potassium hexacyanoferrate(III) in
sodium hydroxide solution was developed (Chen and Bai 2008). In the procedure,
potassium hexacyanoferrate(III) was immobilized on anion-exchange resin packed
in a column. Pyrogallol was sensed by the CL reaction with the potassium hexacya-
noferrate(III) and sodium phosphate was employed as eluent. The CL emission
allowed the determination of pyrogallol in the range 0.013.8 mg mL
1
, with a detec-
tion limit of 0.003 mg mL
1
, being applied to water analysis.
As well as ow-injection uorescence sensor applications, the use of MIPs as
recognition element in ow injection CL sensors has been also developed in recent
years, improving the selectivity of the analysis. The MIP-CL sensors have been
designed for determination of hydralazine (Xiong et al. 2006), maleic hydrazide
(Fang et al. 2009), salbutamol (Zhou et al. 2005) and isoniazid (Xiong et al.
2007), for example. Applications on the developments and applications of chemi-
luminescence (CL) sensors dated from 1999 to 2005 were previously revised by
Zhang et al. (2005).
Reectance
Reectance measurements are a convenient alternative to transmittance mea-
surements in SPS as they circumvent the problems regarding the transparency of
the solid support as well as the length of the optical path. This approach usually
employs a bifurcated optical ber bundle (one arm to conduct the radiation from
the light source to the sample and other to conduct the radiation diffusively reected
DIRECT SOLID-PHASE MEASUREMENTS IN FLOW SYSTEMS 545
D
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546
D
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towards the detector). This provides higher exibility as the optical ber permits to
place the ow cell outside of the spectrophotometer. In addition, multiparametric
measurements can be straightforwardly implemented, as several ow cells with solid
phases selective to different analytes can be incorporated to the ow manifold.
Diffuse reectance has been widely explored in detection systems based on
optodes (or optrodes), which employ a (selective) reagent immobilized in an appro-
priate support coupled to a bifurcated optical ber. In this area, measurements are
usually performed in continuous ow systems, that is, sample is continuously
pumped through the sensor until the steady state is reached. As a consequence, a
short response time is not an essential gure of merit and values up to 15 minutes
can be found in the literature. Of course, response times as high as 15 min are
impracticable in ow injection analysis, thus restricting this review to optosensors
that are appropriate as detectors in ow systems. Table 5 summarizes the applica-
tions addressed in the present work.
As aforementioned, the signal intensity depends on the injected sample volume,
the ow rate, as well as the mass transfer rate of the analyte from the solution to the
solid phase. As the mass transfer rate also depends on the concentration gradient,
decreasing the sample dispersion can enhance the signal intensity. In this aspect,
Sotomayor et al. (1998) demonstrated that a monosegmented ow system (MSFA)
provides higher signals than an unsegmented ow system, reaching up to 90% of
the steady state signal for a pH optosensor based on bromocresol purple immobi-
lized on the distal end of a bifurcated optical ber. Unfortunately, the MSFA
approach is difcult to use in SPS based on a packed reactors, as the air bubbles
can perturb the arrangement of the solid particles in the measuring cell, decreasing
the repeatability of the measurements.
Plasticized PVC membranes have been used in SPS for immobilizing different
reagents. The membrane is cast (by manual deposition, spin coating or dip coating)
on an appropriate support (e.g., polymeric lm or glass slide) from a THF solution
containing PVC (usually ca. 33% m=m), a plasticizer (ca. 66% m=m) and a selective
reagent (ca. 1% m=m). Sometimes an ion-exchanger is incorporated to the mem-
brane, in order to keep the charge balance as well as to decrease the response time.
A PVC membrane plasticized with 2-nitrophenyl-octyl-ether (NPOE) was used to
immobilize PAN (1-(2-pyridylazo)-2-naphthol) for detection of Cu(II) in water
and ore samples (Sanches-Pedren o et al. 2000). A sample volume of 1.0 mL in
ammonia buffer at pH 9.0 was injected in a water carrier stream at a ow
rate of 0.44 mLmin
1
, providing a linear response from 5.0 10
5
to 1.0
10
3
mol L
1
and a detection limit of 5.0 10
6
mol L
1
. A 1.0 10
2
mol L
1
EDTA solution was employed to regenerate the solid phase for the next measure-
ment. A similar PVC membrane with PAN immobilized was employed for deter-
mination of Zn(II) in pharmaceuticals (Albero et al. 2002). A sample solution
prepared in ammonia buffer pH 10.2 was injected (90 mL) into an acetate buffer
pH 3.9 carrier stream, which also acted as eluent. A detection limit of
0.10 mg L
1
of Zn(II) was obtained for a linear response range from 0.16 to
3.27 mg L
1
. The main disadvantage of optosensors based on PAN is the poor sel-
ectivity of the reagent, which reacts with several metal ions. However, for specic
applications and controlled conditions, this drawback can be circumvented, as
demonstrated in the aforementioned papers.
DIRECT SOLID-PHASE MEASUREMENTS IN FLOW SYSTEMS 547
D
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m
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(
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)
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(
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C
1
8
:
C
1
8
-
b
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d
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d
s
i
l
i
c
a
.
548
D
o
w
n
l
o
a
d
e
d

b
y

[
I
n
s
t
i
t
u
t
o

d
e

P
e
s
q
u
i
s
a
s

e

E
s
t
u
d
o
s

F
l
o
r
e
s
t
]

a
t

0
9
:
0
9

2
5

S
e
p
t
e
m
b
e
r

2
0
1
2

The PVC membranes plasticized with NPOE have also been employed for
determination of perchlorate ions in water (Garc a et al. 2003). The membrane
was used to immobilize the lipophilic pH indicator 5-octadecanoyloxy-2-(4-
nitrophenylazo)phenol (ETH 2412) and the ionic additive methyltridodecylammo-
nium chloride, being the response mechanism based on the co-extraction of per-
chlorate and proton from a buffered solution of the anion. The extracted
hydrogen ion then reacts with the pH indicator, generating a signal proportional
to the perchlorate concentration. A 1.0 10
2
mol L
1
NaOH solution was
employed as carrier in a single line manifold, in which 200 mL of sample solution
buffered with 5.0 10
2
mol L
1
TRIS pH 8.2 was injected. A linear response was
obtained from 7.0 10
5
to 2.0 10
2
mol L
1
, with a detection limit of 6.2 10
5
mol L
1
ClO

4
. The optosensor presented a severe interference of bicarbonate ion,
which would be eliminated by acidifying the sample previously the addition of the
buffer solution. Recovering tests performed in tap and spring waters demonstrated
the adequate performance of the optosensor for determination of perchlorate in
this kind of matrix.
Solid phases have been used to retain a product formed in solution in order to
improve sensitivity. In this aspect, nitrite was determined in waters by injecting
2500 mL of the sample solution into a Shinn reagent carrier stream (Miro et al.
2001). The product formed was retained by an octadecyl covalently bonded silica
gel (C
18
disk) coupled to an open sandwich-shape ow cell furnished with a bifur-
cated optical ber for reectance measurements. The product adsorbed onto the
C
18
disk was removed by injecting 150 mL of a 80% (v=v) methanol aqueous solution.
The optimized conditions provided an enrichment factor of 140 and a detection limit
of 0.1 mg L
1
, with reproducibility and repeatability better than 3.2%. The sample
throughput was 11 h
1
and the membrane can be used for up to 40 runs. A C
18
disk
has also been used in the determination of 1-naphthylamine in water samples by the
Griess reaction performed in a multisyringe ow injection system (Mar et al. 2006).
The diazonium salt formed in the reaction was retained by the disk, which was
cleaned up with an 80% (v=v) methanol aqueous solution. By using a sample
volume of 2000 mL, a working linear range from 10 to 160 mg L
1
was obtained, with
a detection limit of 1.1 mg L
1
and repeatability better than 4.7%. A sampling rate of
14 injections per hour was also attained.
Three different strategies based on multisyringe ow injection analysis have
been proposed for the determination of sulde by solid phase spectrophotometry
(Ferrer et al. 2005a; Ferrer et al. 2005b; Ferrer, Estela, and Cerda` 2006). All of them
employed the Fischer reaction, in which sulde reacts with N,N-dimethyl-p-pheny-
lenediamine in the presence of Fe(III) as oxidizing agent. The methylene blue
formed in the reaction was then adsorbed onto an octadecyl-chemically modied
silica (C
18
disk), which was cleaned up and regenerated for a next measurement
with a 80% (v=v) methanol solution containing 0.01 mol L
1
HCl, followed by
an 80% (v=v) methanol aqueous solution. The rst strategy was applied to the deter-
mination of low concentrations of sulde in water samples and processed waste-
waters (Ferrer et al. 2005a). With the injection of 2900 mL of sample into a
0.14 mol L
1
HCl carrier stream at 8 samples per hour, a linear dynamic range from
20 to 200 mg L
1
was achieved, providing a detection limit of 2.9 mg L
1
. A repeat-
ability of 0.7% and a reproducibility of 4.5% were also obtained and the addition
DIRECT SOLID-PHASE MEASUREMENTS IN FLOW SYSTEMS 549
D
o
w
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l
o
a
d
e
d

b
y

[
I
n
s
t
i
t
u
t
o

d
e

P
e
s
q
u
i
s
a
s

e

E
s
t
u
d
o
s

F
l
o
r
e
s
t
]

a
t

0
9
:
0
9

2
5

S
e
p
t
e
m
b
e
r

2
0
1
2

of known amount of sulde in different water samples permitted recoveries from
95% to 112%, demonstrating the feasibility of the method. The same authors (Ferrer
et al. 2005b) expanded the use of the MSFIA system by coupling a gas diffusion
unity, which permitted the determination of sulde in more complex matrices, con-
taining suspended material, without the necessity of preliminary treatment. The gas
diffusion step was performed by injecting a sample volume of 5000 mL into a
0.14 mol L
1
HCl carrier stream, which merged with a 1.5 mol L
1
HCl solution.
The H
2
S diffused through the membrane towards a 0.001 mol L
1
NaOH acceptor
solution, which was then conducted to the reaction coil for production of methylene
blue, as described previously. The ow system allowed a linear response range from
20 to 500 mg L
1
, with a detection limit of 1.3 mg L
1
. Finally, a fully automated
smart MSFIA system was proposed for the determination of sulde in a wide con-
centration range (Ferrer et al. 2006). In this system, three different procedures based
on the gas-diffusion step can be automatically elected to perform the determination:
(a) a direct procedure, in which absorbance measurements were carried out for
the determination; (b) a procedure involving a dilution step followed by the direct
measurements; and (c) a preconcentration procedure, which employed a C-
18
disk.
The optosensing procedure provided a detection limit of 4.6 mg L
1
and a linear
response from 50 to 1000 mg L
1
, values that do not differ considerably from the
previous work.
The MSFIA systems have been also employed to the determination and
speciation of iron in water samples by employing extraction disks (Pons, Forteza,
and Cerda` 2005a, 2005b). The use of a chelating disk modied with iminodiacetic
groups (Pons et al. 2005a) permitted to preconcentrate Fe(III), which subsequently
reacted with ammonium thiocyanate for reectometric determination of the com-
plex in the solid phase. Total iron was determined by on-line oxidation of Fe(II)
with hydrogen peroxide prior to the preconcentration step. A detection limit of
0.0012 mg was achieved for Fe(III) ions, which is 10-fold better in comparison to
a similar MSFIA system that employed the same chelating disk, performing, how-
ever, the determining reaction in solution (Pons, Forteza, and Cerda` 2004). With
the use of an anion-exchanger disk, which retains the FeSCN
3
6
previously
formed in the MSFIA system, a detection limit of 0.0004 mg was reached (Pons
et al. 2005b).
A biosensor employing a pH optical transduction was described for the deter-
mination of the pesticides carbaryl and propoxur in vegetables (Xavier et al. 2000). A
controlled pore glass was used to covalently immobilize the enzyme acetylcholines-
terase, whose activity in the hydrolysis of the acetylcholine to produce choline and
acetic acid is inhibited by the pesticides. The pH variation due to the formation of
acetic acid was measured by a reectometric optosensor based on the acid-base indi-
cator chlorophenol red also immobilized on CPG. A substrate solution was initially
injected into a 5.0 mmol L
1
phosphate buffer pH 8.5 carrier solution, providing an
analytical signal proportional to the enzyme activity. Subsequently, 50 mL of sample
was injected and the ow was stopped for 6 minutes after the sample zone reached
the enzymatic column. Afterward, another injection of the substrate was carried
out, in order to determine the percentage of inhibition of the enzyme, which was
proportional to the pesticide concentration. Detection limits of 0.008 mg L
1
and
0.5 mg L
1
were achieved for propoxur and carbaryl, respectively.
550 F. R. P. ROCHA ET AL.
D
o
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o
a
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e
d

b
y

[
I
n
s
t
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t
u
t
o

d
e

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e
s
q
u
i
s
a
s

e

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s
t
u
d
o
s

F
l
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r
e
s
t
]

a
t

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9
:
0
9

2
5

S
e
p
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m
b
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r

2
0
1
2

Vibrational Spectroscopy
The use of infrared spectrometry (MID or NIR) for direct determination of
organic compounds in aqueous samples is limited due to the low sensitivity of
the technique as well as the intense absorption bands of water in this region of the
spectrum. Thus, the extraction of the analyte from the aqueous solution seems to
be a valuable alternative, as this procedure avoids the interference of water, besides
pre-concentrating the analyte in the solid phase. In addition, once the extraction is
performed, the interfering species can be separated from the analyte, improving
the selectivity. Early procedures employed a solid phase extraction cartridge to
pre-concentrate the analyte, followed by its elution with an appropriate organic
solvent towards the detection cell (Garrigues 1994). The rst ow-through sensor
was constructed by using a conventional MID-IR transmission cell furnished with
a 55 mm polymeric spacer modied in order to hold DEAE Sephadex
1
A-25
anion exchange resin beads, which swelled in contact with water, forming a gel-like
disc between the two CaF
2
windows (Lendl and Schindler 1999). The optosensor
was applied to the determination of acetic and malic acids in aqueous solutions
by sequential injection analysis. The addition of NaOH to the sample solutions
produced the corresponding anions, allowing their retention by the anion exchange
resin. Thus, by injecting 500 mL of sample a linear response up to 1 mmol L
1
of
acids was obtained, with standard deviations of 0.032 and 0.031 mmol L
1
for acetic
and malic acids, respectively.
A mid-IR ow-through sensor for determination of carbohydrates in beer
was constructed by immobilizing amyloglucosidase on agarose beads (Haberkorn,
Hinsmann, and Lendl 2002). Maltose standard solutions were automatically
prepared in the SIA system and injected towards the detector. Once the solution
reached the optosensor, the ow was stopped and spectra were run for 10 min in
order to monitor the formation of the product. A water carrier stream was employed
to ush the system and the content of carbohydrates in beer samples, expressed as
maltose, was successfully determined by standard addition method.
The C
18
modied silica particles were employed to develop a FTIR optosensor
for determination of caffeine in soft drinks (Armenta and Lendl 2009). The C
18
particles were initially activated by pumping 0.8 mL of methanol, followed by
3 mL of water. Afterward, a reference spectrum was recorded and 1.5 mL of sample
solution was impelled through the sensor. The beads were subsequently rinsed with
2 mL of water for elimination of all adsorbed compounds except caffeine, whose
spectrum was then acquired. Finally, the optosensor was regenerated by injecting
0.8 mL of methanol to completely remove the adsorbed analyte, followed by
3.0 mL of water to prepare the system for the next determination. A linear response
up to 115 mg L
1
of caffeine was obtained, with a detection limit of 1.8 mg L
1
and a
relative standard deviation of 4.1%. The results obtained in the determination of
caffeine in soft drinks showed good agreement with those obtained by the HPLC
reference method.
Raman spectroscopy has been also applied in SPS, presenting the advantage
that water weakly scatters Raman radiation. In this aspect, the determination of
caffeine in energy drinks by a FT-Raman ow-through sensor based on a C18 solid
phase was also described in the literature (Armenta et al. 2005). An anionic
DIRECT SOLID-PHASE MEASUREMENTS IN FLOW SYSTEMS 551
D
o
w
n
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o
a
d
e
d

b
y

[
I
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i
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o

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]

a
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0
9
:
0
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2
5

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T
a
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a
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(
s
)
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(
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b
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552
D
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o
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d

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y

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I
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o

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e

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0
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:
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solid-phase reactor located before the C
18
column was used to avoid matrix inter-
ference, improving the selectivity of Raman measurements. The activation of the
solid support was accomplished by pumping 2 mL of methanol, followed by 2 mL
of water. Afterward, 10 mL of sample was passed through the sensor at a ow rate
of 7.5 mL min
1
, followed by 20 mL of distilled water. The solid phase was pre-
viously dried with a nitrogen stream before acquiring the spectrum. The calibration
graph was linear from 100 to 600 mg L
1
, providing a detection limit of 18 mg L
1
,
with a relative standard deviation of 3%. An analytical throughput of 13 samples per
hour was obtained, higher than those provided by HPLC procedure (7.0 h
1
). Sepha-
dex
1
QAE A-25 anion exchanger gel has also been used to determination of sulfo-
namides in pharmaceutical preparations (Ruedas-Rama et al. 2005) by FT-Raman.
In this approach, a ow cell was packed with the gel and baseline signal was obtained
by pumping 0.010 mol L
1
NaOH solution at a 1.0 mL min
1
. A volume of 1000 mL
of sample solution (sulfathiazole or sulfamethoxazole) was injected into the system
and the ow was stopped at the maximum intensity signal for acquiring spectral data
(as an average of 20 scans). Afterward, 2000 mL of a 0.10 mol L
1
NaCl solution con-
taining 0.010 mol L
1
NaOH was injected for the elution of the analyte retained by
the solid phase, thus recovering the baseline signal. A detection limit of 0.1 g L
1
was
obtained for both sulfonamides, with a sampling rate of 10 samples per hour and
relative standard deviations lower than 4.1%.
An attenuated total reection-based optosensor was also employed for the
determination of caffeine in soft drinks (Alcudia-Leo n et al. 2008). A ow cell was
specially designed to accommodate a small amount of a polystyrene-divinylbenzene
sorbent material at the sensitive zone of the ATR crystal, with the aid of a cotton frit.
A SIA system was employed to manage the solutions. Initially, 2 mL of water was
pumped through the system for acquiring a reference spectrum. Three milliliters of
sample was then passed through the optosensor, followed by 2 mL of water, for
retention of caffeine and elution of the interfering species. Once a spectrum was
acquired, the sorbent material was regenerated with 0.25 mL of acetone. The use
of a ow rate of 0.5 mL min
1
allowed a whole measuring cycle in 14 min. A relative
standard deviation of 4% and a detection limit of 7 mg L
1
were estimated.
Near infrared (NIR) was also employed as detection technique in solid phase
spectroscopy for the determination of a-naphthylamine in water samples (Ortega-
Barrales et al. 1999). A commercial cell of 1 mm pathlength was lled with a C
18
-
bonded silica gel solid phase, which was conditioned=regenerated with 1 mL of a
20% (v=v) methanol aqueous solution. For measurements, 17 mL of naphthlamine
sample solution was pumped through the optosensor at a ow rate of 2.1 mL
min
1
, providing a dynamic range from 4 to 30 mg L
1
, with a detection limit of
0.6 mg L
1
and a relative standard deviation of 5.2%. Table 6 lists the applications
of MID and NIR ow-through optosensors.
CONCLUSIONS AND TRENDS
The combination of ow analysis with detection on optically active solid phases
packed in a ow-through cell (optosensor) has demonstrated to offer important
advantages due to its precision, simplicity, selectivity, and high sensitivity, provided
by the in situ preconcentration of analyte(s) on the solid sensing support. In addition,
DIRECT SOLID-PHASE MEASUREMENTS IN FLOW SYSTEMS 553
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they exhibit notable analytical features that contribute to a greener analytical
chemistry by saving reagents and sample, and reducing waste generation. Further
developments of these optosensing techniques have shortened analysis time consider-
ably and reduced costs, being adequate for routine control, since automated systems
accelerated the operational sequence and can reduce manipulation by the analyst.
Despite measurements by UV-vis spectrophotometry (transmission or reec-
tance modes) is the most frequently used detection technique in SPS for inorganic
or organic analytes, luminescence, infrared and Raman spectroscopies can also be
the basis of measurements on solid phase. The high sensitivity and selectivity pro-
vided by luminescence-based techniques are favorable features, while vibrational
spectroscopy can be an alternative for organic analytes determination. Additionally,
considering that some application of spectrometric methods is often limited by
selectivity, development of new sorbents, such as MIP, with high afnity, specic
recognition, and high stability is of great signicance for use in direct measurements
on solid surface.
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