Trends in Analytical Chemistry, Vol. 22, No.

4, 2003

Trends

Sequential injection lab-on-valve:

the third generation of ow
injection analysis
Jianhua Wang, Elo Harald Hansen
Termed the third generation of ow-injection analysis, sequential injection (SI)-lab-on-valve (LOV) has specic advantages and allows novel,
unique applications not least as a versatile front end to a variety of
detection techniques. This review presents and discusses progress to date
of the SI-LOV approach as well as its applications in the automation and
the micro-miniaturization of on-line sample pre-treatment. Special
emphasis is placed on using SI-LOV in conjunction with bead injection
(BI) for on-line separation and pre-concentration of ultra-trace levels of
metals by exploiting the renewable micro-column approach. With detection by ETAAS and ICP-MS, it is shown, as illustrated by recent results in
the authors laboratory, that this methodology eliminates the problems
encountered in conventional on-line column pre-concentration systems,
improves the overall operational eciency and yields the robustness
necessary for routine assays. Also discussed is the future potential of the
SI-LOV approach as a front end to various analytical protocols.
# 2003 Published by Elsevier Science B.V.
Keywords: Bead injection (BI); Electrothermal atomic absorption spectrometry
(ETAAS); Inductively coupled plasma mass spectrometry (ICP-MS); Lab-on-valve
(LOV); Renewable micro-column; Separation and pre-concentration; Sequential
injection (SI)

Jianhua Wang,
Elo Harald Hansen*
Department of Chemistry,
Technical University of
Denmark, Building 207,
Kemitorvet, DK-2800
Kgs. Lyngby, Denmark

*Corresponding author.
Tel.:+45-4525-2346;
Fax:+45-4588-3136;
E-mail: ehh@kemi.dtu.dk

1. Introduction
Appropriate sample pre-treatment prior
to analysis remains inevitably the bottleneck of the entire analytical procedure
with modern instrumentation [1,2].
Flow-injection analysis (FIA) has led to
signicant progress in this eld, not only
in terms of eciency, but also in reliability, rapidity and robustness. Since its
introduction in 1975 [3], this technique,
or rather analytical concept, has grown
into a discipline covered by a series of
monographs and more than 12,500 scientic publications [4]. All this clearly
shows that in the past 27 years FIA has
grown almost exponentially. In fact, it
has been characterized as having gone

through three generations [5,6], that is:

(1) ow injection (FI); (2) sequential
injection (SI) [7]; and, (3) laboratory-onvalve (lab-on-valve, LOV) [8].
The rst and the second generations of
ow injection and their hyphenations
have played, and indeed continue to play,
important roles in the automation and
the miniaturization of on-line sample pretreatment, resulting in the acceleration of
analysis with considerable reductions in
sample/reagent consumption and waste
production [1,2,6,7]. However, even
more importantly, they have allowed
implementation of procedures that used
to be dicult or even not feasible by conventional approaches [9].
It is especially noteworthy that these
rst two generations have been the main
tools for replacing labor-intensive manual operations in various separation/preconcentration techniques through clever
on-line manipulations. However, in
reagent-limited assays, in handling
highly hazardous chemicals, or where
waste production is a critical parameter,
further downscaling of solution handling
is warranted [8]. In addition, the most
widely employed on-line, sample-processing approach (i.e., solid-phase extraction (SPE) based on liquid^solid
interactions) is often impaired by the irreversible changes of the reactive surface of
the solid phase and/or the creation of ow
resistance in the packed reactor. In order
to overcome these drawbacks, the third
generation of ow injection techniques,
SI-LOV, was introduced [8]. The miniaturized LOV system potentially oers
facilities to allow any kind of chemical
and physical processes, including uidic

0165-9936/03/$ - see front matter # 2003 Published by Elsevier Science B.V. doi:10.1016/S0165-9936(03)00401-1

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Trends in Analytical Chemistry, Vol. 22, No. 4, 2003

and microcarrier bead control, homogenous reaction,

liquid-solid interaction and in-valve, real-time optical
detection of various reaction processes with optical bers.
Although still in its infancy, SI-LOV has already
proved to be an attractive, eective front end to various
detection devices with micro-miniaturized sample processing along with improved eciency and ruggedness.
The present review presents a brief outline of progress
in development of SI-LOV, with special emphasis on the
application of SI-LOV-bead injection (BI) to on-line
separation/pre-concentration of ultra-trace levels of
metals by means of the renewable micro-column concept, as used in conjunction with detection by electrothermal atomic absorption spectrometry (ETAAS) and
inductively coupled plasma mass spectrometry
(ICP-MS).

2. Micro SI-LOV (SI-LOV) system

The rst LOV microsystem was a single, monolithic
fabricated Perspex component mounted atop a six-port
selection valve (hence the name lab-on-valve).
Designed to integrate all necessary laboratory facilities
for a variety of analytical schemes, it is made to include
connecting ports, working channels and a owthrough cell. The conduits are situated in three layers
in order to facilitate as many uidic operations as
possible [8,10]. The central ow-through channel, via
which the other individual ports are connected, is used
to communicate with a syringe pump.
A multi-purpose ow cell, which is permanently
incorporated into port 2 (Fig. 1, close up), allows a series
of miniaturized uidic operations to be performed,
including in-valve sample dilution and reagent addition/
mixing, as well as incubation. The ow cell is furnished
with optical bers, communicating with an external
light source and a detection device to facilitate real-time

Figure 1. Schematic diagram of a SI-LOV microsystem incorporating a

multi-purpose ow cell congured for real-time measurement of
absorbance (adapted from [11], with permission of The Royal Society of
Chemistry).

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monitoring of the reactions taking place inside the cell.

The ow cell can also be congured as a jet-ring-cell,
to execute BI and real-time monitoring of the changes
in the optical properties of the beads after liquid-solid
interaction by means of absorbance, uorescence and/
or reectance spectroscopy.
This version of the LOV system is operated with
microliter and sub-microliter levels of sample and
reagents. Therefore, this approach is also known as the
micro sequential injection (mSI) protocol [8,11,12].
Fig. 1 shows a typical mSI-LOV system with a close-up
of the multi-purpose ow cell congured for real-time
measurement of absorbance.

3. Applications of SI-LOV system

The mSI-LOV system has been employed in a variety of
applications of micro-miniaturized in-valve uids and
micro-carrier beads, as well as appropriate in-valve
interactions followed by real-time detection. It has also
been used as a front end to capillary electrophoresis
(CE). Table 1 summarizes various applications of the
mSI-LOV to date.
In the mSI-LOV system, the multi-purpose ow cell
depicted in Fig. 1 is employed to accommodate the sample/
reagent zone and to facilitate real-time detection by
recording the absorbance (facing bers, as shown in
Fig. 1) or uorescence (bers at 90 , not shown in Fig. 1).
To increase the sensitivities for kinetically slow reactions, selected segments of the sample zone can be monitored by adopting the stopped ow technique. The
eectiveness of the stopped ow technique in mSI-LOV
systems has been illustrated in the determination of
phosphate [8,12], enzymatic assays [8], and monitoring of ammonia, glycerol, glucose and free iron in fermentation broths [11].
To carry out on-line measurement of nitrate and
nitrite, a cadmium reduction column can be incorporated into the LOV system to reduce nitrate to nitrite
before merging the stream of nitrite with chromogenic
reagents (sulfanilamide and N-(1-naphthyl)ethylenediamine) [12]. With a miniaturized column, an appropriate period of stopped ow of the sample zone in the
cadmium column is employed to increase the contact
time and thus improve reduction eciency.
In the mSI-BI-LOV mode, the ow cell is congured
into the jet-ring-cell approach, or more precisely an
adaptation of the renewable column concept, in order
to perform bioligand-interaction assays. In this case, an
appropriate suspension of beads is injected into the ow
cell, whereupon the beads are trapped and then perfused with analyte solution. Afterwards, the loaded
beads are exposed to various stimuli, and the (bio)chemical reactions taking place on the bead surface are
recorded in real time. This has been demonstrated by

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Trends

Table 1. The applications of mSI-LOV system for micro-miniaturized in-valve uid and microcarrier bead handling as well as appropriate in-valve
interactions followed by real-time monitoring and/or as a front end to CE
LOV system

Applications

Detection

Ref.

mSI-LOV

Phosphate assay
Enzymatic activity assay of protein
Savinase
Fermentation monitoring of ammonia,
glycerol, glucose and free iron

Absorbance

[8,12]

Fluorescence

[8]

Absorbance

[11]

mSI-LOV with
reduction column

Environmental monitoring of NO3 and

NO2 (NO3 on-line reduced to NO2 )

Absorbance

[12]

mSI-BI-LOV
(jet-ring-cell)

Bioligand interaction assay of

immunoglobulin (IgG) on protein G
immobilized Sepharose beads

Fluorescence
Absorbance

[8]
[8]

mSI-LOV-CE

Separation of anions. The mSI-LOV is used

as a sample pre-treatment unit and a sample
introduction system to CE

Absorbance

[13]

the bioligand-interaction assay of immunoglobulin

(IgG) on protein G immobilized Sepharose beads [8].
Very recently, the mSI-LOV system was interfaced to
CE for anion separation. In this case, the multi-purpose
ow cell was recongured as a front end between the
mSI-LOV system and the CE system [13]. The microuidic property of the mSI-LOV system not only provided
an ecient means of delivering sample to the CE system
with various sample-injection modes, including
electrokinetic, hydrodynamic and head column eld
amplication (HCFA) sample stacking, but also served
as a versatile means of sample pre-treatment to facilitate the ensuing CE separation.

4. Solving a dilemma
Column-based SPE is among the most ecient and
widely employed on-line separation/pre-concentration
techniques [6,14,15], and the interfacing of SPE with
detection by ETAAS and ICP-MS has received extensive
attention.
In conventional FI, on-line column, pre-concentration systems, the column is used as a permanent
component. For reliable application, the break-through
capacity of the column, the extent of interfering eects
and the particle size of the sorbent material must be
carefully balanced. Although smaller particle sizes
result in higher break-through capacities, ner particles tend to cause progressively tighter packing and
hence create ow resistance in the column. Moreover,
some sorbent materials undergo volume changes, i.e.,
swelling or shrinking, at dierent experimental conditions, which make the situation even worse, not to
mention complicated [16].
Although some approaches to alleviate the ow resistance have been quite successful, such as use of bidirectional ows during loading and elution sequences

[16^18], it is very dicult to eliminate its adverse

eects completely. Besides, the deterioration of the analytical results of an on-line column, pre-concentration
procedure may also be associated with the malfunction
of the reactive surface of the sorbent itself. This can be
attributed to contamination or deactivation of the surface after processing a large number of samples, and/or
even the loss of some of the functional groups or active
sites present.
In this respect, a superb alternative for eliminating
any problems associated with the changes of the surface
properties of the sorbent materials and/or the creation
of ow resistance in a column reactor is a surfacerenewal scheme, i.e., the column is simply renewed or
replaced for each analytical run. The renewable-surface concept, proposed by Ruzicka, has been well established by using the jet-ring-cell in a variety of
(bio)chemical studies [19^21]. This concept is well
suited to on-line SPE separation/pre-concentration in a
LOV incorporating a miniaturized renewable column,
i.e., SI-BI-LOV [22].

5. The SI-BI-LOV system

This version of the LOV is fabricated using hard PVC
containing (1 + 6) channels and is mounted atop a
6-port selection valve, as shown in Fig. 2. Two of the
channels, including the central one, are used as microcolumn positions (C1 and C2). To trap the beads within
the channel cavities serving as micro-columns and to
prevent the beads from escaping during the operations,
the outlets of these two channels are furnished with
small pieces of PEEK tubing, which t into, and are
axed by the screws in, the outlets. The diameter of this
tubing is slightly smaller than the diameters of the
columns; it provides an internal channel which thus
allows liquid to ow freely through the channel and
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Figure 2. Diagram of a LOV system for bead injection (BI) incorporating

two micro-column positions (C1 and C2), along with a close-up of a
packed renewable micro-column.

along the walls, but entrap the beads. A close-up of the

packed renewable micro-column is shown in Fig. 2.
The sorbent beads are suspended in an appropriate
amount of water or buer solution, usually within the
range of 1:10^1:20 (m/v). In operation, the suspension is
rst aspirated into a 1.0 ml plastic syringe that is afterwards mounted on port 6 of the LOV. To pack the column
precisely and reproducibly for each analytical run, the
aspiration rate of the suspension of beads from the plastic
syringe into micro-column position C1 should be carefully
controlled (2^5 ml/s is appropriate). After aspiration into
the LOV system, the beads can then be readily transferred
back and forth between the two micro-column positions
(C1 and C2), as the protocol requires. These manipulations facilitate both sample loading and the treatment of
the analyte-loaded beads according to the scheme that is
to be followed for the post-analysis, as illustrated in the
following examples taken from the authors own work.

6. Trace-metal examples
In the experiments described below, an aqueous suspension of SP Sephadex C-25 cation-exchange resin (drybead size 40^125 mm; H+- or K+-form) was employed [22].
When using ETAAS as the detection device, two different schemes for dealing with the analyte loaded
beads can be exploited (Fig. 3) [22^24]:
1. after the resin beads have been exposed to a certain amount of sample solution, the loaded beads
are eluted with a small, well-dened volume of
an appropriate eluent, which is ultimately
transported into the graphite tube for quantication, whereupon the used beads are discarded.
New beads are aspirated and fresh columns are
generated for the ensuing sample cycles.

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Figure 3. Illustration of the two possible schemes for dealing with analyte-loaded beads in the renewable micro-column approach. (a) The
analyte-loaded beads are eluted and the eluate is exploited for quantication by ETAAS and/or ICP-MS. (b) The analyte-loaded beads are
transported directly into the graphite tube of the ETAAS, where, following pyrolysis of the beads, the analyte is quantied.

2. alternatively, as a novel, unique approach, the

loaded beads can be transported directly into the
graphite tube, where advantage can be taken of
the fact that the beads primarily comprise
organic materials, that is, they can be pyrolyzed,
thereby allowing ETAAS quantication of the
analytes. While the former approach can be used
with ICP as well, the latter is obviously prohibitive for this detection device. Outlines of operational sequences for these approaches are given
below.
To execute on-line separation/pre-concentration in
the renewable column, a set volume of sample solution
is rst aspirated and stored within the holding coil (HC),
followed by aspiration of a small amount of the suspension of beads, usually 15^20 ml, which is initially captured in column position C1. Thereafter, the central
channel is directed to communicate with column position C2, and, while the syringe pump moves slowly forward, the beads are thereby transferred to C2 followed
by sample solution, i.e., separation/pre-concentration
takes place in this column position (Fig. 4, top). Afterwards, the analyte retained within the micro-column
can be dealt with according to the two schemes mentioned above for post-analysis.
In the rst scheme (Fig. 4, top), both sample-loading
and analyte-elution processes take place in the same
column position C2, the eluent having been aspirated from
port 2 in an intermediate step and stored in HC, before
being pumped forward and through C2. Eventually, via

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Figure 4. The SI-BI-LOV on-line renewable column pre-concentration

system. The beads are eluted and the eluate is introduced into the
graphite tube for quantication. SP: syringe pump; HC: holding coil;
IV: two-position injection valve.

air segmentation, the eluate is transported into the graphite tube [23].
In the second approach (Fig. 5), the separation/preconcentration is carried out in column position C2, and,
on completion, the analyte-retaining beads are, along
with a certain amount of carrier solution, usually
30^40 ml, transferred back to column position C1. (This
is necessary because all external communication is
eected via syringe pump SP). Afterwards, via air segmentation and through port 4, the beads and the carrier zone are transported to the graphite tube of the
ETAAS instrument, where the beads are pyrolyzed and
the analytes quantied [22]. The validity of both
schemes has been demonstrated by the determination
of Ni in biological and environmental samples.
It should be emphasized that, in the bead-pyrolysis
scheme, a temperature of more than 1000 C and a
holding time of ca. 40 s is required to pyrolyze the
beads. These conditions are obviously not applicable for
the determination of metals with a low atomization
temperature, particularly Cd, Bi and Pb. In such cases,
it is necessary to make use of the rst approach comprising an elution step, since the eluate can be pyrolyzed at
substantially lower temperatures as compared to the
beads themselves, which helps to avoid analyte loss
before atomization. This was demonstrated by the
determination of trace levels of Bi in biological and
environmental samples [24].
Table 2 clearly shows that the procedures based on
the employment of renewable columns give much

Trends

Figure 5. The process of the SI-BI-LOV on-line renewable column

pre-concentration system: Transportation of the analyte-loaded beads
into the graphite tube followed by pyrolysis and quantication.
IV: two-position injection valve.

better precisions (relative standard deviations, RSDs)

and lower limits of detection (LODs) than those of the
conventional processes, i.e., by using a permanent
column either with repetitive uni-directional or bidirectional operations. While the two schemes for
employing renewable columns exhibit comparable performance, the precision of the elution-based procedure
is much better than that of pyrolyzing the beads
directly, because it is easier to handle and transport
homogeneous solutions than heterogeneous ones.
The interface of the SI-BI-LOV on-line, renewable
column, pre-concentration system to ICP-MS is, in
principle, quite straightforward as compared to that to
ETAAS. In addition, considering that nitric acid can
be used as an eluent for the retained analytes, the protocol used for ETAAS is, after appropriate modication, readily transferable for interfacing with ICPMS, with due consideration to the continuous feature
of the ICP-MS system and the characteristics of the
renewable micro-column and the LOV system. The
manifold is illustrated in Fig. 6. The interface of the
renewable column-based pre-concentration protocol
to ICP-MS opens a route to multi-element screening of
ultratrace metals in complex matrices, such as saline
samples, as demonstrated in the simultaneous determination of Ni and Bi in biological and environmental
samples (Table 2).
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Table 2. Comparisons of the performance data for the pre-concentration of Ni and Bi in the SI-BI-LOV system using a renewable column or a permanent
column with uni-directional and bi-directional repetitive operation
With detection by ETAAS
Conventional

RSDHa (%, 0.3 mg/l)

RSDK (%, 0.3 mg/l)
LODH (ng/l)
Enrichment factor

Bead pyrolysis

Elution

Ni

Ni
1.5
1.7
10.2, 11.4c
71.1

3.4
9c
72.1

uni-direct.

bi-direct.

Bi
2.3b

Ni
5.8

Ni
4.9

27.0
33.4

42.0

24.0

With detection by ICP-MS (Elution)

RSDH (%, 0.8 mg/l)
LODH (ng/l)
Enrichment factor

Ni
2.9
15.0
35.2

Bi
1.7
4.0
28.4 (1.63.2 mg/l)
9.1 (0.041.6 mg/l)

The subscript stands for the form of ion exchanger;

RSD value obtained at the 2.0 mg/l level;
c
LOD value obtained with the K+ form of ion exchanger.
b

The micro-miniaturized renewable column volume in

the LOV results in a correspondingly smaller breakthrough capacity for the analytes, and consequently a
very limited amount of eluent suces for the complete
elution of the retained analytes. It is, therefore, critical
to minimize the dispersion of the eluate zone during its
transport to the ICP, as this might cause severe loss of
sensitivity. The introduction of small air segments at
both ends of the eluate zone, as used successfully with

detection by ETAAS, might help to minimize the dispersion [25^27]. Yet, in the experience of the present
authors, with interfacing FI/SI on-line sample pretreatment systems with an ELAN 5000 ICP-MS
[28,29], the use of air segments tend to extinguish the
plasma.
However, dispersion is particularly critical when
using a conventional nebulization system. The low
sample transport eciency of a cross ow nebulizer,
typically 1^2%, might cause an unduly large dispersion of the eluate zone. Therefore, it is clearly not
appropriate for delivering such a small volume of
eluate.
To overcome these drawbacks, a direct-injection,
high-eciency nebulizer (DIHEN) was found to be eective [30]. The eluate zone was sandwiched between
carrier segments, and transport was eected by a continuous infusion pump (IP). Under such conditions, no
signicant dispersion was encountered.

7. Conclusions and future outlook

Figure 6. The SI-BI-LOV on-line renewable column pre-concentration

system with detection by ICP-MS: The beads are eluted and the eluate
is introduced into the ICP via a DIHEN. SP: syringe pump; HC: holding
coil; IV: two-position injection valve; SL: sample loop; IP: infusion pump;
DIHEN: direct injection high eciency nebulizer.

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The SI-BI renewable micro-column, pre-concentration

scheme using a miniaturized LOV system is proposed as
preferable to the conventional procedure employing a
permanent column. Interfacing this approach to both
ETAAS and ICP-MS has been shown to have a number
of merits in comparison with its traditional counterpart, including the complete elimination of ow resistance, high precision and a low LOD. In addition, the
expense of column renewal for each analytical cycle
is very limited, i.e., the bead suspension obtained from

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1 gram of dry bead of SP Sephadex C-25 can be used for
ca. 500 operations.
The studies based on the miniaturized SI-BI-LOV
renewable column approach have focused on pre-concentration of trace metals and its interfacing with
ETAAS and ICP-MS. However, this scheme is equally
applicable to virtually any kind of liquid-solid interactions, including SPE as well as precipitation/coprecipitation separation/pre-concentration protocols with
detection by various techniques. Further investigations
are required in this eld. In particular, the LOV-based
SI-BI surface-renewal approach has opened a promising avenue for immunoassay and/or bio-chemical
assays employing immobilized enzymes, although
rather few studies hitherto have been directed to this
subject by employing the BI scheme [19^21,31^33].
However, considering its vast potential, it is expected
that the SI-BI-LOV protocol will gain considerable
momentum in the very near future.

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Elo Harald Hansen, who is one of the two inventors of FI, is Professor of Analytical Chemistry at the Department of Chemistry, Technical University of Denmark. His research interests are instrumental
and automated analysis with special emphasis on the development
and application of procedures based on FI/SI. In recent years, he has
focused particularly on developing FI/SI schemes for the determination of trace levels of metals in complex matrices by hyphenation with
atomic spectrometric techniques and ICP-MS.
Jianhua Wang obtained his MSc from Nankai University, China.
After employment at Yantai Normal University, he spent one year at
the University of Delaware, USA, before commencing his PhD studies
at the Technical University of Denmark in 1999. He was awarded the
PhD degree in 2002. He is currently pursuing his interests in the
development and use of FI/SI procedures at Northern University,
Shenyang, China, where he has joined Professor Zhaolien Fangs
research group.

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