In the Laboratory

edited by
Topics in Chemical Instrumentation David Treichel
Nebraska Wesleyan University
Lincoln, NE 68504

Sequential-Injection Analysis: Principles, Instrument

Construction, and Demonstration by a Simple Experiment W
A. Economou,* P. D. Tzanavaras, and D. G. Themelis
Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki 541 24, Greece; *aeconomo@chem.auth.gr

Sequential-injection analysis (SIA) is an approach to detectable species. The detectable species gives rise to a tran-
sample handling that enables the automation of manual wet- sient peak when it passes through the flow cell of the detec-
chemistry procedures in a rapid, precise, and efficient man- tor.
ner. SIA was developed by Ruzicka and Marshall at the SIA, on the other hand, (Figure 1, bottom) makes use
University of Washington (1) in response to an industry-ini- of a multi-position selection valve rather than an injection
tiated requirement for a more robust automated wet-chem- valve. Also, an additional coil (the holding coil in SIA termi-
istry technique than traditional flow-injection analysis (FIA) nology) is added between the pump and the selection valve.
(2). Today, SIA is widely applied for the determination of Initially, the holding coil is filled with the carrier solution.
several species in a variety of matrices (3). Then, the selection valve is advanced to a port that is con-
Consider a FIA experiment as depicted in Figure 1 (top). nected to the sample line. A small volume of sample is drawn
A volume of sample is inserted into the sample loop of a six- up as a zone into the holding coil by the pump. The volume
way, two-position injection valve while a stream of carrier and of sample that is aspirated into the holding coil is determined
a stream of reagent are mixed in a mixer and are flowing con- by the product of the flow rate of the pump and the time
stantly through the detector. The length of the sample loop that sample is drawn. The selection valve is then advanced
determines the volume of sample injected. After the sample to a port that is connected to the reagent line. A small vol-
loop is filled with the sample (loaded in FIA terminology) ume of reagent is drawn up into the holding coil directly ad-
the valve is switched, the sample is introduced into a flow- jacent to the sample (this procedure is called stacking in SIA
ing carrier stream (injected in FIA terminology) and physi- terminology). Then, the selection valve is advanced to a po-
cally transported by the carrier to the mixer where it mixes sition that is connected to the detector line. The pump de-
with the reagent. In the course of its travel through the reac- livers the stacked sample and reagent zones to the detector.
tion coil, the sample zone is spread and diluted (is dispersed As the adjacent sample and reagent zones move through the
in FIA terminology) and reacts with the reagent to form a reaction coil, the zone are mixed together. Again, a detect-
able species is formed and is registered as a peak by the de-
tector. In SIA, the larger the extent of zone overlap, the more
FIA sensitive the measurement will be.
sample Comparing SIA and FIA, the following points can be
carrier injection
valve and
made:
pump
sample loop • SIA makes use of a single flow-channel even with mul-
detector ticomponent chemical systems. In FIA, additional
flow-channels are required if more than one reagent is
reaction to be used.
mixer coil
• With SIA the sample and reagent consumptions are
drastically reduced.
waste
reagent
• The single-channel operation of SIA enables the use
of the same manifold for the implementation of wide
range of determinations.
SIA • In SIA, the selection valve provides a means for per-
multi-position forming convenient automated calibration.
pump detector
selection
valve • In SIA, accurate measurements of sample and reagent
reaction zones necessitate computer control and, therefore, au-
coil
tomation becomes essential.
holding
coil waste The first goal of this work was the construction and au-
tomation of a SIA apparatus using the graphical program-
carrier sample reagent
ming environment of LabVIEW. It has been shown that using
this programming tool results in extremely flexible and user-
Figure 1. Comparison of typical FIA and SIA manifolds. friendly applications (4–6). Recently, we reported on an au-

1820 Journal of Chemical Education • Vol. 82 No. 12 December 2005 • www.JCE.DivCHED.org

 In the Laboratory

tomated FIA apparatus controlled by a program developed been adopted as an official method of analysis (10). Further
in the graphical programming environment of LabVIEW (7). information on automation of this analytical method can be
We decided to use the same hardware as the basis for our found in (11, 12).
SIA system but using a different control program. This ap- The analysis sequence consists of the following steps:
proach of using the same basic hardware for both FIA and 1. Aspirate the Sn(II)–hydrazine solution (solution T)
SIA (with different software and experimental configuration) (step 1)
is advantageous in terms of pedagogical value, cost-effective-
2. Aspirate the sample solution (solution S) (step 2)
ness and ease of operation and, also, gives the opportunity
to compare the two techniques. The second goal was to make 3. Aspirate the molybdenum solution (solution M) (step 3)
use of this system to introduce students to SIA using a simple 4. Pump the stacked zones to the detector while record-
and well-known chemical system, namely the photometric ing the absorbance (step 4)
determination of phosphate using the “molybdenum blue” In a typical SIA system, the figures of merit that must
method. Using the system developed, students could famil- be considered are the sensitivity, the reagent consumption,
iarize themselves with the operational principles of the tech- the sample throughput, the linear range, and the precision.
nique and learn how to develop an analytical method for
“real-world” measurements.
This experiment fits well in the course of Instrumental
Chemical Analysis and especially in the section of Automatic
Methods of Analysis provided by chemistry departments in
most universities. The sections on instrument interfacing and
software development can also be used in courses of chemi-
cal instrumentation. The work is expected to occupy a mini-
mum of two four-hour laboratory sessions. However, the
structure of this experiment is flexible, allowing modifica-
tions and extensions by the instructor.

Experimental
The equipment consists of a peristaltic pump (Gilson
Minipuls 3, France), a 10-way multi-position selector valve
(Valvo-Vici, Switzerland), a 5023 FI star double-beam spec-
trophotometer (consisting of a 5032 detector controller and
a 5023-011 spectrophotometer optical unit from Tecator,
Hoganas, Sweden). Note that any other UV–vis spectropho-
tometer equipped with a flow-through cell and analog out-
put can be used. The hardware is controlled by a Pentium
133 MHz computer equipped with a multifunction inter-
face card (6025E from National Instruments, Austin, TX).
The software is LabVIEW 5.1.1 (National Instruments) run-
ning under Windows 98. PTFE tubing of 0.75-mm i.d. is
Figure 2. Schematic diagram of the SIA instrument and order of
used for all the flow lines. The experimental configuration is
zones aspiration: T is the Sn(II)–hydrazine solution, S is the sample
illustrated in Figure 2. solution, and M is the molybdenum solution.

Chemical Measurements
The determination of free phosphate in human urine 2.0
has important diagnostic value in some clinical cases (8). The
standard chemical system selected for this work is based on
1.6
the “molybdenum blue” reaction (i.e., the reaction of phos-
phate and molybdate ions to form molybdophosphoric acid
Absorbance

that is then reduced to a blue product—“molybdenum 1.2

blue”—by a mixture of Sn(II) and hydrazine) with photo-

metrical detection at 690 nm according to the reaction shown 0.8
in Scheme I (9). Owing to its robustness, this reaction has
0.4

+
PO43− + 12MoO42− + 24H
0.0
2Sn(II) 0 1 2 3 4
[PMo12O40]3− + 12H2O [PSn2Mo12O40]3− c (PO4 3ⴚ
) / (10 ⴚ4
mol L ⴚ1
)
hydrazine
Figure 3. Calibration curve for the determination of phosphate by
Scheme I. Reaction scheme of the formation of “molybdenum blue”. SIA and photometric detection.

www.JCE.DivCHED.org • Vol. 82 No. 12 December 2005 • Journal of Chemical Education 1821

 In the Laboratory
These figures are affected by both geometrical and chemical
parameters. In our case, these variables include: the mixing
order of sample and reagents; the mass concentration (mass
per unit volume) of the molybdate solution,
γ[(NH4)6Mo7O24⭈4H2O]; the mass concentration of the
Sn(II) solution, γ(SnCl2⭈2H2O); the amount concentration
(molarity) of the H 2 SO 4 in the molybdate solution,
c(H 2SO 4)兾Mo 7O 246−; the amount concentration of the
H2SO4 in the Sn(II) solution, c(H2SO4)兾Sn(II); the volume
of the molybdate solution, V(Mo7O246−); the volume of the
Sn(II) solution, V(Sn(II)); the volume of the sample solution,
V(S); the length of the reaction coil, l(RC); the delivery flow
rate, QV.
The selection of these parameters is made using the
univariate approach in the order mentioned in the previous
paragraph (13). After selection of the most appropriate con-
ditions, the effect of common interfering species is investi-
gated. A calibration curve for phosphate is constructed in the
range 1 × 10᎑5 mol L᎑1 to 3.5 × 10᎑4 mol L᎑1; a typical cali-
bration curve is shown in Figure 3. Finally, the absorbance
in urine samples is measured, the concentration of phosphate
is calculated by means of the calibration curve, and the
method is validated by recovery tests (typical recovery values
are in the range 100 ± 5 %).
Hazards
Skin and eye contact with concentrated sulfuric acid
causes burns. SnCl2 is harmful if swallowed or absorbed
through the skin and is irritating to respiratory system. It
causes burns. Hydrazine is flammable, toxic by inhalation,
in contact with skin, and if swallowed. It causes burns and
may cause sensitization by skin contact and cancer.
(NH4)6Mo7O24⭈4H20 is irritating to the eyes, respiratory sys-
1822 Journal of Chemical Education • Vol. 82 No. 12 December 2005
tem, and skin. All electrical equipment involves high volt-
ages and is potentially dangerous.
W
Supplemental Material
Instructions for the students and notes for the instruc-
tor are available in this issue of JCE Online.
Literature Cited
1. Ruzicka, J.; Marshall, G. D. Anal. Chim. Acta 1990, 237, 329–
343.
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81, 406.
8. Phosphorus, Urine. http://www.labcorp.com/datasets/labcorp/
html/chapter/mono/pr007100.htm (accessed Sep 2005).
9. Karlberg, B.; Pacey, G. E. Flow Injection Analysis, A Practical
Guide; Elsevier: New York,1989; p 156–162.
10. Standard Methods for Examination of Water and Wastewater,
20th ed.; American Public Health Association: Washington,
DC, 1998.
11. Mas Torres, F.; Estela, J. M.; Miró, M.; Cladera, A.; Cerdà, V.
Anal. Chim. Acta 2004, 510, 61.
12. Munoz, A.; Mas Torres, F.; Estela, J. M.; Cerdà, V. Anal. Chim.
Acta 1997, 350, 21.
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