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; *
[email protected] Sequential-injection analysis (SIA) is an approach to sample handling that enables the automation of manual wetchemistry procedures in a rapid, precise, and efficient manner. SIA was developed by Ruzicka and Marshall at the University of Washington (1) in response to an industry-initiated requirement for a more robust automated wet-chemistry technique than traditional flow-injection analysis (FIA) (2). Today, SIA is widely applied for the determination of several species in a variety of matrices (3). Consider a FIA experiment as depicted in Figure 1 (top). A volume of sample is inserted into the sample loop of a sixway, two-position injection valve while a stream of carrier and a stream of reagent are mixed in a mixer and are flowing constantly through the detector. The length of the sample loop determines the volume of sample injected. After the sample loop is filled with the sample (loaded in FIA terminology) the valve is switched, the sample is introduced into a flowing carrier stream (injected in FIA terminology) and physically transported by the carrier to the mixer where it mixes with the reagent. In the course of its travel through the reaction coil, the sample zone is spread and diluted (is dispersed in FIA terminology) and reacts with the reagent to form a FIA carrier
sample pump
injection valve and sample loop
• SIA makes use of a single flow-channel even with multicomponent chemical systems. In FIA, additional flow-channels are required if more than one reagent is to be used.
detector
mixer
reaction coil waste
reagent
multi-position selection valve
pump
sample
• In SIA, the selection valve provides a means for performing convenient automated calibration.
detector
• In SIA, accurate measurements of sample and reagent zones necessitate computer control and, therefore, automation becomes essential.
reaction coil
holding coil
waste reagent
Figure 1. Comparison of typical FIA and SIA manifolds.
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• With SIA the sample and reagent consumptions are drastically reduced. • The single-channel operation of SIA enables the use of the same manifold for the implementation of wide range of determinations.
SIA
carrier
detectable species. The detectable species gives rise to a transient peak when it passes through the flow cell of the detector. SIA, on the other hand, (Figure 1, bottom) makes use of a multi-position selection valve rather than an injection valve. Also, an additional coil (the holding coil in SIA terminology) is added between the pump and the selection valve. Initially, the holding coil is filled with the carrier solution. Then, the selection valve is advanced to a port that is connected to the sample line. A small volume of sample is drawn up as a zone into the holding coil by the pump. The volume of sample that is aspirated into the holding coil is determined by the product of the flow rate of the pump and the time that sample is drawn. The selection valve is then advanced to a port that is connected to the reagent line. A small volume of reagent is drawn up into the holding coil directly adjacent to the sample (this procedure is called stacking in SIA terminology). Then, the selection valve is advanced to a position that is connected to the detector line. The pump delivers the stacked sample and reagent zones to the detector. As the adjacent sample and reagent zones move through the reaction coil, the zone are mixed together. Again, a detectable species is formed and is registered as a peak by the detector. In SIA, the larger the extent of zone overlap, the more sensitive the measurement will be. Comparing SIA and FIA, the following points can be made:
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The first goal of this work was the construction and automation of a SIA apparatus using the graphical programming environment of LabVIEW. It has been shown that using this programming tool results in extremely flexible and userfriendly applications (4–6). Recently, we reported on an au-
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tomated FIA apparatus controlled by a program developed in the graphical programming environment of LabVIEW (7). We decided to use the same hardware as the basis for our SIA system but using a different control program. This approach of using the same basic hardware for both FIA and SIA (with different software and experimental configuration) is advantageous in terms of pedagogical value, cost-effectiveness and ease of operation and, also, gives the opportunity to compare the two techniques. The second goal was to make use of this system to introduce students to SIA using a simple and well-known chemical system, namely the photometric determination of phosphate using the “molybdenum blue” method. Using the system developed, students could familiarize themselves with the operational principles of the technique 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 chemical instrumentation. The work is expected to occupy a minimum of two four-hour laboratory sessions. However, the structure of this experiment is flexible, allowing modifications and extensions by the instructor.
been adopted as an official method of analysis (10). Further information on automation of this analytical method can be found in (11, 12). The analysis sequence consists of the following steps: 1. Aspirate the Sn(II)–hydrazine solution (solution T) (step 1) 2. Aspirate the sample solution (solution S) (step 2) 3. Aspirate the molybdenum solution (solution M) (step 3) 4. Pump the stacked zones to the detector while recording the absorbance (step 4)
In a typical SIA system, the figures of merit that must be considered are the sensitivity, the reagent consumption, the sample throughput, the linear range, and the precision.
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 spectrophotometer (consisting of a 5032 detector controller and a 5023-011 spectrophotometer optical unit from Tecator, Hoganas, Sweden). Note that any other UV–vis spectrophotometer equipped with a flow-through cell and analog output can be used. The hardware is controlled by a Pentium 133 MHz computer equipped with a multifunction interface card (6025E from National Instruments, Austin, TX). The software is LabVIEW 5.1.1 (National Instruments) running under Windows 98. PTFE tubing of 0.75-mm i.d. is used for all the flow lines. The experimental configuration is illustrated in Figure 2.
Figure 2. Schematic diagram of the SIA instrument and order of zones aspiration: T is the Sn(II)–hydrazine solution, S is the sample solution, and M is the molybdenum solution.
Chemical Measurements 2.0
1.6
Absorbance
The determination of free phosphate in human urine has important diagnostic value in some clinical cases (8). The standard chemical system selected for this work is based on the “molybdenum blue” reaction (i.e., the reaction of phosphate and molybdate ions to form molybdophosphoric acid that is then reduced to a blue product—“molybdenum blue”—by a mixture of Sn(II) and hydrazine) with photometrical detection at 690 nm according to the reaction shown in Scheme I (9). Owing to its robustness, this reaction has
1.2
0.8
0.4
PO43− + 12MoO42− + 24H
+
[PMo12O40]3− + 12H2O
0.0
2Sn(II)
[PSn2Mo12O40]3−
hydrazine
Scheme I. Reaction scheme of the formation of “molybdenum blue”.
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0
1
c (PO4
2
3ⴚ
) / (10
3
ⴚ4
mol L
4
ⴚ1
)
Figure 3. Calibration curve for the determination of phosphate by SIA and photometric detection.
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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 conditions, the effect of common interfering species is investigated. 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 calibration 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-
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tem, and skin. All electrical equipment involves high voltages and is potentially dangerous. W
Supplemental Material
Instructions for the students and notes for the instructor are available in this issue of JCE Online. Literature Cited 1. Ruzicka, J.; Marshall, G. D. Anal. Chim. Acta 1990, 237, 329– 343. 2. Hansen, E. H.; Ruzicka, J. J. Chem. Educ. 1979, 56, 677. 3. Lenehan, C. E.; Barnett, N. W.; Lewis, S. W. Analyst. 2002, 127, 997. 4. Gostowski, R. J. Chem. Educ. 1996, 73, 1103. 5. Drew, S. M. J. Chem. Educ. 1996, 73, 1107. 6. Ogren, P. J.; Jones, T. P. J. Chem. Educ. 1996, 73, 1115. 7. Economou, A.; Papargyris, D.; Stratis, J. J. Chem. Educ. 2004, 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. 13. Miller, J. C.; Miller, J. N. Statistics for Analytical Chemistry, 2nd ed.; Ellis Horwood: Chichester, United Kingdom, 1988; pp 28–29, 175–187.
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