In the Laboratory edited by
Topics in Chemical Instrumentation
David Treichel Nebraska Wesleyan University Lincoln, NE 68504
Automated Flow-Injection Instrument for Chemiluminescence Detection Using a Low-Cost Photodiode Detector An Interdisciplinary Project in Chemical Instrumentation, Graphical Programming, Computer Interfacing, and Analytical Chemistry
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A. Economou,* D. Papargyris, and J. Stratis Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki 541 24, Greece; *
[email protected] Chemiluminescence (CL) involves the production of light as the result of a chemical reaction. The best-known CL reactions are the oxidation of luminol (1) and the peroxalate reaction (2). A useful Internet resource, including theory, demonstrations, and bibliography on chemiluminescence can be found in ref 3. The main attractions of CL for chemical analysis are its high sensitivity and the simple and inexpensive instrumentation required (4, 5). Photomultiplier tubes (PMTs) are commonly used as light detectors in CL, although advances in solid-state devices have enabled photodiodes to compete with PMTs (6). Flow injection analysis (FIA) is ideally suited to applications involving CL (7): in this case, a sample solution is injected into a flowing carrier stream, mixed with the appropriate reagents, and the resulting CL solution flows into a cell positioned in front of a photodetector where the intensity of light is measured. With the recent advances in the field of graphical instrumentation software, exemplified by the LabVIEW software package (8–11), automatic instrument control and data acquisition in FIA can be greatly simplified. The development of an FI analyzer for CL detection using a low-cost photodiode is presented. This work was the subject of an undergraduate final-year project in our laboratory. The project was composed of three independent phases and now forms part of the laboratory courses in chemical instrumentation and instrumental chemical analysis in our department: i. Construction and optimization of an inexpensive photodetector based on a photodiode. The goal is for students to become familiar with some aspects of optical instrumentation (photodetectors, photodiodes, and selection of their performance characteristics), operational amplifiers, analog filtering, and frequency domain representation of signals, and to demonstrate the utility of this knowledge in a real-world chemical experiment. ii. Design, construction, and automation of a simple FIA apparatus using graphical programming. The goal is to introduce students to the implementation of various modes of computer interfacing (DAC, ADC, digital I兾O operations) by providing a typical example of control and data acquisition in chemistry. We also intend
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to provide hands-on experience in graphical programming using LabVIEW. iii. Optimization of the instrumental and chemical parameters for the detection of Co(II) by chemiluminescence. Students make use of experimental optimization schemes of the instrumental and chemical parameters and are faced with a typical case of analytical calibration for quantitative analysis.
This experiment clearly demonstrates in a single project how different aspects in chemical instrumentation fit together to produce a working analytical system. This work, due to its multidisciplinary nature, requires familiarity with, and theoretical knowledge in, several areas of chemical instrumentation. It is suitable as teamwork exercise at the end of the second term of the academic year when students have been exposed to the topics involved in this work. In our laboratory this experiment is scheduled in five to six three-hour lab sessions, but, as the experiment is modular, work can be easily tailored to the structure of each course and the time availability. Reagents and Equipment Luminol, hydrogen peroxide, sodium hydroxide, and cobalt(II) sulfate heptahydrate are analytical grade. Deionized water is used to prepare all solutions. Stock solutions of 0.01 mol L᎑1 luminol, 1000 mg L᎑1 Co(II), 1 mol L᎑1 sodium hydroxide, and 0.1 mol L᎑1 hydrogen peroxide are prepared. The items of equipment used are: a peristaltic pump (Gilson Minipuls 3, France), a 10-way multiposition valve (Valvo-Vici, Switzerland), two passive T mixers (Jour Research, Sweden), and a Pentium 133 MHz computer equipped with a multifunction interface card (6025E from National Instruments, Austin, TX). The interface card features 16 ADCs, 2 DACs and 32 I兾O lines. PTFE tubing of 0.75 mm i.d. was used for all the flow lines. The flow cell is made of perspex in a spiral configuration. The photodetector is based on a PIN photodiode (OPT 301, Burr-Brown) and is soldered on a printed circuit board. The software is LabVIEW 5.1.1 (National Instruments) running under Windows 98 (LabVIEW programs are platform-independent and may run under other popular operating systems such as the MacOS).
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Construction of the Detector
Rf = 10 MΩ
According to eq 1, the larger the resistor the more sensitive the detector will be. The OPT 301 has an internal 1-MΩ feedback resistor that can be externally overridden. Initially, the unmodified detector is tested using the chemical system described later in this work and it is found that measurements suffer from a noisy background. Photodiodes are particularly susceptible to thermal noise and to a lesser degree to shot noise, flicker noise, and environmental noise (more details on each type of noise can be found in ref 15), which leads to a low signal-to-noise, S兾N, ratio. The simplest approach to improve the S兾N ratio is analog low-pass filtering (16). To filter high-frequency noise, a capacitor, Cf, serving as an analog low-pass filter is inserted across the output of the detector and ground, as illustrated in Figure 1. The cutoff frequency, fc, of such a low-pass filter is given by: fc =
1
2 π Rf C f
(2)
Using this analog filter, frequencies higher than fc are attenuated while frequencies lower than fc are not affected. Obviously, the choice of the cutoff frequency, fc, is critical. Too high a value of fc will allow high frequency noise components to contaminate the signal and the S兾N ratio will deteriorate. Too low a value of fc will narrow the frequency response of the detector (i.e., the detector will be slow to respond to transient light pulses) and the CL signal could be distorted. Therefore, a compromise must be reached between sensitivity, noise rejection, and speed of response. A program is developed in LabVIEW that produces the power spectrum of the CL signal in the frequency domain. The power spectrum is a graph of the signal power versus frequency and illustrates graphically how the total power contained in the signal is quantitatively distributed among the individual constituent frequency components. During the development of this program, students have the opportunity to learn LabVIEW programming basics (17) and some notions of frequency domain analysis using the LabVIEW Analysis libraries (18). The power spectrum suggests that most of the signal power resides at low frequencies while higher frequency components are mainly due to noise. Using the information in the power spectrum students can design an
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1 MΩ 4
40 pF
i PL
75 Ω 5
+
(1)
Vout = iRf
2
–
A useful starting point on photodiode technology, optical characteristics, electrical properties, and modes of operation can be found in ref 12. The purpose of any light detector is the proportional conversion of the light intensity into a measurable electrical quantity (usually voltage). The photodetector currently supplied and recommended for use is the hybrid OPT 301 from Burr-Brown (13), although other similar devices are commercially available. The photodiode first converts the photoluminescence intensity, PL, produced in the course of the CL reaction into a current, i. The current is converted into a voltage, Vout, using an operational amplifier connected as a current-to-voltage (i兾V ) converter (14). The value of the feedback resistor, Rf, controls the gain of the i兾V converter and the sensitivity of the photodetector since:
8
1 +9V
OPT 301 3 −9V
Vout = iRf (to DAC) Cf = 10 nF
9-V batteries
Figure 1. Schematic diagram of the photodetector used for CL measurements (numbers indicate the pins in the detector chip).
appropriate filter (by calculating the product RfCf) to achieve the desired cutoff frequency, fc. They can experiment with different combinations of values of the resistors Rf (ranging from 1 MΩ to 100 MΩ) and capacitors Cf (ranging from 5 nF to 400 nF) and observe the effect that these changes have on the CL signal (magnitude of the signal, S兾N ratio, preservation of the peak shape). In our case the best combination was achieved for Cf = 10 nF and Rf = 10 MΩ (as shown in Figure 1) but the values may vary considerably since they depend on temperature, physical environment, quality of grounding, type of detector, et cetera. Two 9-V batteries connected in series are used to power the operational amplifier with a supply voltage of ± 9 V. The use of batteries, instead of a plug-in power supply, provide some degree of immunity from interference and also make the detector portable. Construction and Control of the Flow System In this part of the project, students have a hands-on experience and familiarize themselves with different aspects of control, data acquisition, and analysis (DAC and ADC operations, digital lines, sampling frequencies, the Nyquist criterion, and digital filtering; further reading on these topics can be found in refs 18, 19). The mechanism of the CL reaction between luminol and hydrogen peroxide is illustrated in Scheme I: oxidation of luminol produces the 3aminophthalate anion in its excited state, which returns to its ground state by producing light at 425 nm. The reaction is catalyzed by Co(II). The experimental setup of the FIA–CL apparatus is shown in Figure 2. A continuous flow of reactants is maintained in the system. The luminol and hydrogen peroxide (oxidant) merge at the first mixer (M1). Injection of the sample (a solution of cobalt) is made into the carrier by switching the valve to the sample line for a predetermined period of time. Then, the valve is switched back to the car-
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In the Laboratory
rier that transports the sample plug to the second mixer (M2) where it mixes with the combined luminol–hydrogen peroxide solution and flows through the cell while the light produced is recorded by the photodetector. As the sample plug travels through the system, the sample is diluted near the edges of the plug as a result of dispersion. The shape of the FIA peaks reflects the Co(II) distribution in the sample plug (i.e., high concentration at the center of the plug and lower concentration at the edges). The peak height is proportional to the Co(II) concentration in the sample. The detector and the cell are placed in a lighttight plastic box in which holes are drilled for solution inlet or outlet and for electrical connections. Connections to the inlet and outlet of the cell are made with black tubing to prevent light from entering the box. As illustrated in Figure 2, the pump is controlled by a TTL line (1 = start, 0 = stop) and a DAC (0 V = minimum, 5 V = maximum flow rate). Automation of the injection valve requires binary-coded-decimal (BCD) code in 14 TTL lines. An ADC channel acquires the analog output voltage produced by the detector. From the instructor’s point of view it is important to point out that the selected application makes use of all the interface operations (DAC, ADC, TTL digital lines) available on the 6025E card. The front panel of the control and acquisition program of the FIA apparatus is illustrated in Figure 3 in the process of acquiring typical FIA peaks after injection of a 10 mg L᎑1 Co(II) solution. The program consists of 4 subprograms or sub-VIs: the PUMP_CONTROL program controls the pump (on versus off, flow rate), the CHEMILUMINESCENCE program performs the data acquisition from the detector, the
NH2
O
Figure 2. Schematic diagram of the FIA–CL instrument.
INJECTION_PROGRAMME controls the injection valve (injection time, delay time between injections, number of injections), and the STATUS program indicates the current status of the valve (carrier or sample). To reduce noise, signal averaging is employed in the CHEMILUMINESCENCE program (20): this operation involves acquiring 100 samples (samples to average) at a fast scan rate of 1000 samples per s (this operation required 0.1 s), computing their average value, and plotting the average value at a rate of 5 samples per s (sample interval). Another subprogram was developed in LabVIEW to perform digital filtering on the signal acquired.
NH2
C NH NH
NH2
O C
+ 2OH
C
N N
O N
C
C
C
O
O
O
luminol
+ 2 H 2O
N
dianion of luminol
catalyst 2H2O2
NH2
*
O C
O O
NH2 release of photon
O C
O O
C
C
O
O
excited state of 3-aminophthalate ion
+ h ν + 2 H 2O + N2
ground state of 3-aminophthalate ion
Scheme I. Pathway of the CL oxidation of luminol.
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CL signal / mV
30
20
10
0 0
50
100
150
200
[Co(II)] / (mg/L) Figure 3. The front panel of the LabVIEW control and acquisition program of the FIA instrument in the course of acquiring the CL response of a 10 mg L᎑1 Co(II) solution.
Figure 4. Typical calibration curve for Co(II) in the range 0.5–200 mg L᎑1 using the FIA apparatus with CL detection.
Chemical Measurements
Acknowledgment
This part of the project involves work on a Simplex experimental optimization procedure (21) and calibration (22). Students can optimize the instrumental parameters (typical starting values—total flow rate: 1–8 ml min᎑1; injection time: 1–10 s) and the chemical parameters (typical starting concentrations—luminol: 1–10 mmol L᎑1; hydrogen peroxide: 2–20 mmol L᎑1; NaOH: 0.01–0.1 mol L᎑1) so that the CL signal is maximized. The students are asked to construct a calibration graph using Co(II) solutions in the concentration range 0.5–200 mg L᎑1 and analyze a liquid or solid sample containing cobalt to determine the Co(II) concentration or percent cobalt content. A typical calibration curve for Co(II) is illustrated in Figure 4.
The financial assistance of the Royal Society of Chemistry, through the provision of a research fund grant to A.E., is gratefully acknowledged.
Hazards Luminol and hydrogen peroxide are irritating to eyes, respiratory system, and skin. Sodium hydroxide is caustic and causes burns. Cobalt(II) sulfate heptahydrate is toxic to organisms, harmful if swallowed, a probable carcinogen, and may cause sensitization by skin contact and inhalation. All the electrical equipment involves high voltages and is potentially dangerous. Conclusion The response of the students to this project is considered positive as it allows them to “build their own instrument”, provides them with the opportunity to translate the theoretical knowledge they gain into a practical application, and improves their skills. This work is also useful to instructors by providing information and feedback on the degree of assimilation of the taught material by the students and can, therefore, assist in enhancing the relevant courses in instrumental analysis and chemical instrumentation.
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Supplemental Material
Instructions for preparing the solutions, notes for the instructor, approximate cost of the expendables, and a table of Simplex optimization procedure are available in this issue of JCE Online. Literature Cited 1. Chalmers, J. H., Jr.; Bradbury, M. W.; Fabricant, J. D. J. Chem. Educ. 1987, 64, 969. 2. Hadd, A. G.; Lehmpuhl, D. W.; Kuck, L. R.; Birks, J. W. J. Chem. Educ. 1999, 76, 1237–1239. 3. The Chemiluminescence Homepage of the Sam Houston State University. http://www.shsu.edu/~chm_tgc/chemilumdir/ chemiluminescence2.html (accessed Nov 2003). 4. Chemiluminescence in Analytical Chemistry; Garcia-Campana, A. M., Baeyens W. R. G., Eds.; Marcel Dekker: New York, 2001. 5. Garcia-Campana, A. M.; Baeyens, W. R. G.; Zhang, X. In Chemiluminescence in Analytical Chemistry; Garcia-Campana A. M., Baeyens, W. R. G., Eds.; Marcel Dekker: New York, 2001; pp 42–61. 6. Advanced Photonics Homepage. http://www.advancedphotonix. com/papers_frameset.htm (accessed Nov 2003). 7. Hansen, E. H.; Ruzicka, J. J. Chem. Educ. 1979, 56, 677. 8. Allerhand, A.; Dobie-Galuska, A. Chem. Educator 2000, 5, 71–76. 9. Economou, A. S.; Volikakis, G. J.; Efstathiou, C. E. J. Autom. Methods Manage. Chem. 1999, 21, 33–38. 10. Ogren, P. J.; Jones, T. P. J. Chem. Educ. 1996, 73, 1115.
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In the Laboratory 11. Drew, S. M. J. Chem. Educ. 1996, 73, 1107–1111. 12. Centrovision Homepage. http://www.centrovision.com/ tech2.htm (accessed Nov 2003). 13. Texas Instruments Homepage. http://focus.ti.com/docs/prod/ productfolder.jhtml?genericPartNumber=OPT301 (accessed Nov 2003). 14. Smith, R. J. Electronics: Circuits and Devices; Wiley: New York, 1993; pp 357–445. 15. Horowitz, P.; Hill, W. The Art of Electronics; Cambridge University Press: Cambridge, 1980; pp 286–313. 16. Horowitz, P.; Hill, W. The Art of Electronics; Cambridge University Press: Cambridge, United Kingdom, 1980; pp 29–34. 17. Wells, L. LabVIEW for Everyone: Graphical Programming Made
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Even Easier; Prentice Hall: Upper Saddle River, NJ, 1996. 18. Chugani, M. L. LabVIEW Signal Processing; Prentice Hall: Upper Saddle River, NJ, 1998. 19. Beyon, J. Y. LabVIEW Programming, Data Acquisition and Analysis; Prentice Hall: Upper Saddle River, NJ, 2001. 20. Horowitz, P.; Hill, W. The Art of Electronics; Cambridge University Press: Cambridge, United Kingdom, 1980; pp 624– 626. 21. Shavers, C. L.; Parsons, M. L.; Deming, S. N. J. Chem. Educ. 1979, 56, 307. 22. Miller, J. C.; Miller, J. N. Statistics for Analytical Chemistry, 2nd ed.; Ellis Horwood: Chichester, United Kingdom, 1988; pp 101–134.
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