LABORATORY EXPERIMENT pubs.acs.org/jchemeduc
A Portable, Low-Cost, LED Fluorimeter for Middle School, High School, and Undergraduate Chemistry Labs Benjamin T. Wigton,† Balwant S. Chohan,† Cole McDonald,‡ Matt Johnson,‡ Doug Schunk,‡ Rod Kreuter,‡ and Dan Sykes*,‡ † ‡
School of Science, Engineering, Technology, The Pennsylvania State University, Harrisburg, Pennsylvania 17057, United States Department of Chemistry and Forensic Science Program, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
bS Supporting Information ABSTRACT: A low-cost portable fluorimeter was constructed using a 360 nm LED for excitation and a silicon photodiode for detection. The instrument is simple to operate and has been used to investigate fluorescent whitening agents extracted from various brands of paper, to determine the linear range and limit of detection of quinine in various commercial tonic water samples, and to quantify the amount of vitamin B1 in commercial multivitamin supplements. The instrument is straightforward to construct and to use and facilitates the introduction and hands-on application of spectroscopy, fluorescence, and related topics in middle school, high school, and undergraduate laboratory courses. KEYWORDS: First-Year Undergraduate/General, High School/Introductory Chemistry, Upper-Division Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Applications of Chemistry, Fluorescence Spectroscopy, Laboratory Equipment/Apparatus, Quantitative Analysis
I
n 1981, the U.S. Secretary of Education created a commission whose seminal report, A Nation at Risk1 called for widespread, systemic reform, including stronger graduation requirements, more rigorous and measurable standards, more time in school, and significantly improved teaching. Since the 1980s, there have been numerous federal- and state-sponsored programs designed to strengthen secondary and postsecondary science education and encourage more students to enter science, technology, engineering, and mathematics (STEM) related fields. Among the most notable federal legislation is the America Competes Act of 20072 that promotes investment in innovation through research and development to improve the competitiveness of the United States. Of the many provisions, this particular act refocuses on the need to strengthen the teaching and learning of STEM at the secondary-school levels. The more recent Race to the Top 2009 program3 is designed to spur reforms in state and local district K12 education, with the quality of teacher training being the main criterion for funding, followed by student achievement, and implementing high-quality assessments to gauge success and failures. The Higher Education Opportunity Act (HEOA) of 19984 consisted of many important updates to the Higher Education Act of 1965, including sections that dealt with improvements and expansion of the scientific and technological capacity of the United States, and better preparation of scientists, engineers, and technical experts, by enhancing professional development activities, with a particular focus on underrepresented minorities. Such acts and educational initiatives call for Copyright r 2011 American Chemical Society and Division of Chemical Education, Inc.
providing school and college undergraduate students with the opportunity to engage in challenging, thought-provoking STEMrelated projects. It is our firm belief that a healthy introduction to advanced projects, research, and hands-on use of the scientific method is the best way to integrate the book-knowledge acquired in courses. It is with meaningful lab experiments and research that students can develop, explore, and fail-rebound with their own projects, just as it happens in the world outside of school or college. Challenging projects and research integrate the many components of the general curriculum into a unified picture and help students acquire a spirit of inquiry, independence, confidence, sound judgment, and dedicated persistence.5 These experiences will help students develop a deeper understanding of the subject, and also critical-thinking and problem-solving skills that they can then use in the profession they pursue and in every facet of their lives.68 The analytical sciences have changed significantly in the last 25 years from the classical test tube spot test analysis to modern instrumental analysis. With a plethora of sensitive and selective techniques, modern analytical chemistry has significantly contributed to the public awareness of chemical problems and issues relating to quality-control systems in industrial production, in the pharmaceutical area, in the food industry, and in the environment.
Published: May 31, 2011 1182
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Figure 1. The circuit diagram for the 360 nm fluorimeter.
Such changes in the real world demand that schools and colleges appropriately train students in the analytical skills and knowledge required to cope, understand, and develop these techniques. However, in many school districts and small colleges across the United States, there is little or limited provision for such experiences given the high cost of instrumentation and lab supplies. For large enrollment institutions, providing each individual student with hands-on access to a commercial instrument requires either a multi-week laboratory rotation through a set of experiments or the deployment of multiple instruments and has therefore been, either time- or cost-prohibitive. To address these problems, we have been developing low-cost, low-maintenance, rugged analytical instruments in our undergraduate courses for the past four years. Students in the analytical chemistry courses design and build the instruments in kit form, which are then donated to other courses or local schools for use in their science labs. Our efforts to develop an $80 fluorimeter instrument are described. To encourage greater student participation, a series of tested labs are included that can be conducted independently and address real-world problems to help students realize the usefulness and the limits of their fluorescence measuring instrument.
’ EXPERIMENTAL SECTION Instrumental Design
Several versions of low-cost fluorimeters and portable fluorimeters have been described in the literature.916 Some of these employ elaborate design features that include diode- and nitrogen-based lasers as the excitation source and that are intended to optimize measurements for specific chemical systems. Others lack durability, or require the ability to program and control every feature of the instrument through LabView, or require computers and associated software, which prohibits their broad distribution and implementation in a variety of chemistry curricula (middle school through upper-level undergraduate). The custom-built fluorimeter described here is less expensive than those described
in literature, simple in design, quick to construct, rugged, and capable of performing accurate measurements. The circuit diagram for the fluorimeter is given in Figure 1. The power supply is a 110:12 VAC converter connected to an acto-dc converter (not shown). The output voltage is regulated using two 9.1 V Zener diodes. The source LED was purchased from FOX Group optoelectronics (part number FG360-TO18BL010). The LED is forward-biased at 3.6 V and 23 mA. The spectral emission curve is approximately Gaussian with a peak wavelength at 361 nm and a spectral bandwidth of (10 nm (95% of total emission intensity). The ultraviolet cutoff filter was purchased from Edmund Optics, (part number NT54-049). For this filter, the stop-band limit is 360 nm, the cutoff position is 400 nm, and the pass-band limit is at 450 nm. A 90° configuration between source and detector is used to minimize the amount of scattered excitation radiation from hitting the detector. The silicon photodiode detector was purchased from Edmund Optics (part number NT53-372). Under 9 V reversed-bias conditions, the radiant sensitivity of the photodiode is approximately 100 mA/W at 450 nm. Similar to most silicon photodiodes, the peak sensitivity is ∼960 nm; however, the detector used here is manufactured to have enhanced sensitivity in the blue region of the spectrum. The output of the photodiode is converted to a voltage (02 V) using a transimpedance amplifier. An offset null adjustment is placed between the current to voltage converter and the first amplifier stage. The analog signal voltage is converted to digital format by the digital meter and displayed using a liquid crystal display (not shown). A complete schematic is provided in the Supporting Information. The external and internal configurations of the fluorimeter are shown in Figure 2. Some machine work is required to drill holes in the printed circuit board and cuvette holder, and to mount the liquid crystal display onto the housing. The cuvette assembly is shown in Figure 3. The cuvette holder is assembled from three pieces of machineable plastic. Two of these pieces are glued (or, alternatively, screwed) together to form the cuvette holder. The LED fits into a drill hole on one side of the cuvette assemble. The third piece, the detector 1183
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Table 1. Cost To Construct the Fluorimeter Part
Vendor
Pricea/U.S. $
12 V ac Adaptor
Power Stream
4.50
Capacitors
MCM Electronics
4.00
Circuit Board
JameCO Electronics
1.00
Cuvette Holder
Quadrant Engineering
2.50
Detector, Silicon
Edmund Optics
Digital Panel Meter
MPJA
Diodes Filter, UV-Longpass
Radio Shack Edmund Optics
Plastic Products 22.00 8.95 2.00 16.90
34 mm Diameter
Figure 2. The external (A) and internal (B) configurations of the custom-built fluorimeter.
Housing
Hammond Manufacturing
LED, 360 nm
Fox Group Incorporation
8.00 4.50
Operational
JameCO Electronics
0.29
Regulator, 5 V
JameCO Electronics
0.25
78L05 Resistors
MOJO
5.00
Amplifier TL084
Total
79.89
a
The prices given are for bulk purchase. Retail price for individual components is 25% greater. Part numbers are provided in the Supporting Information.
Figure 3. The cuvette assembly of the custom-built fluorimeter.
mounting plate, has a drill hole into which the detector is snugly fit. The optical filter is placed into a 2.54 cm diameter by 0.1 cm deep circular cut-out that is end-milled into the plate on the side facing the cuvette holder. Once the filter is placed in position, the third piece is glued (or, alternatively, screwed) to the other two. Once assembled, the filter sits between the cuvette and the detector. Material costs to construct the entire device are less than $80. A list of components and their costs are provided in Table 1. The prices reflect discounts obtained for bulk purchases. Our analytical chemistry course enrollment exceeds 50 students per semester. Together with various outreach programs that we are involved with, we have been making at least 60 of these instruments per year for 3 years. On average, the retail price for individual components is 25% greater, which would place these fluorimeters at around $110 each. A detailed list of the components and a close-up of the circuit board layout are provided as Supporting Information. Chemicals
For the three experiments described, the following chemicals are needed: ethanol (g95%), quinine sulfate dihydrate (J.T. Baker), commercial tonic water, sulfuric acid (0.05 M), hydrochloric acid (0.001 M), thiamine hydrochloride (Alfa Aesar), mercuric chloride, trisodium phosphate, and sodium phosphate dibasic (all from Sigma-Aldrich), commercial multivitamin. Laboratories
The fluorimeter has been used to investigate three real-world problems: (i) to compare the relative fluorescence of whitening
agents extracted from various brands of paper, (ii) to determine the linear range and limit of detection of quinine in various commercial tonic water samples, and (iii) to quantify the amount of vitamin B1 in commercial multivitamin supplements. A complete and detailed set of procedures for each of the experiments below is provided as Supporting Information.
’ HAZARDS Dilute sulfuric and hydrochloric acids are corrosive and may cause damage to skin and eyes. Dispose any of the working solutions containing these acids down the drain with plenty of water. Mercuric chloride, HgCl2, is corrosive and highly toxic by ingestion and skin absorption. Use caution and handle the mercuric chloride solution with appropriate skin and eye protection. All mercurycontaining solutions should be disposed of in a metals-only waste container. Ethanol is flammable and harmful if inhaled or absorbed through skin. Quinine sulfate dihydrate may cause irritation to skin, eyes, and respiratory tract and may be harmful if swallowed or inhaled. Appropriate precautions need to be taken when constructing the apparatus, especially with hot soldering irons and live circuits. Students should be advised to not stare directly at the 360 nm LED. When soldering and assembling the device, the power supply should be left unplugged. Prior to final assembly, power should only be supplied during brief intervals for circuit testing and only under instructor supervision. ’ RESULTS AND DISCUSSION Lab 1: Fluorescent Whitening Agents
This semiquantitative experiment was developed for eighthgrade students at a local middle school. Fluorescent whitening agents (FWAs) are large, conjugated, anionic fluorescent compounds that are added to products, such as paper and clothing, to make them appear brighter and whiter.17 Natural fibers absorb 1184
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Figure 4. The relative fluorescence intensities of fluorescent whitening agents extracted from commercial paper: paper A = No Name brand, paper B = Boise Aspen 30, paper C = OfficeMax Copy Paper No. 06012030, and paper D = Georgia Pacific Everyday Copy & Print Paper.
blue light and appear yellow in color, so FWAs are often embedded in the fibers during processing. FWAs are dyes that absorb light in the UV region (340 370 nm) and re-emit light in the 420470 nm region of the electromagnetic spectrum. This fluorescence enhances the emission in the violet and blue region of the spectrum causing the product to appear “whiter-than-white” in color. Students in the lab extract the fluorescent whitening agents from various brands of copy paper and compare the relative fluorescence of each (Figure 4). There are about 400 fluorescent whitening agents but fewer than 90 are produced commercially on any significant scale.18 For students to appreciate the variety of chemicals that are available as FWAs, they perform thin-layer chromatography (TLC) to determine if different brands of paper contain different agents. Students conduct a controlled experiment following the scientific method, use proper laboratory techniques, analyze the data, and present their data in graphical form. The FWA experiment and the subsequent experiment (quantitative determination of quinine) are used as a foundation to discuss the major differences between qualitative and quantitative investigations in analytical chemistry. The FWA lab is a qualitative investigation for several reasons. First, neither the fluorimeter nor TLC precisely identifies the FWAs. The TLC results merely suggest two different whitening agents are used as does the fluorescent data; that is, the same or similar FWA for paper samples A and C and a different FWA for paper samples B and D (but the same or similar with respect to each other). Second, extraction efficiencies may differ between samples, an issue that is related to the inherent chemistry of the paper. Third, the distribution of FWAs in each sheet of paper (and between papers of the same brand) may not be homogeneous. There are numerous other lines of inquiry to pursue with the students, and the lab can be readily expanded to include these. Lab 2: Quinine in Tonic Water
This particular lab is a standard analytical chemistry1921 lab, which has been adapted for use with our customized fluorimeter. The primary objective of this experiment was to determine the concentration of quinine in one or more brands of tonic water. Quinine is a natural product that was originally extracted from the bark of the cinchona tree (Cinchonae cortex) and used as a remedy for malaria. Quinine is not a cure for the disease, but it does suppress parasitic activity. Quinine is a crystalline white powder, and because of its characteristic bitter flavor, it is used in foods, particularly in soft drinks (tonics, bitter lemon drinks). The United States Food and Drug Administration regulates the
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Figure 5. A calibration curve expressing the relationship between concentration of quinine and signal intensity in a set of standard solutions. Line equation, y = (0.073 ( 0.003)x þ (0.07 ( 0.04), coefficient of correlation, r2 = 0.994.
quantity of quinine in carbonated beverages to no more than 83 ppm. For the middle school students, the quinine experiment was employed as one tool in a set of co-curricular exercises between the science and mathematics teachers to inform students of the practical applications of algebra in science. Students in the college quantitative analysis course perform a more advanced version of this lab, calculating linear least-squares fits to the data with appropriate standard errors, comparing calibration curves to the method of standard addition, calculating limits of detection and quantitation, performing t tests, learning about propagation of uncertainties, and the general precautions one must take when statistically treating data sets. Typical results for the calibration curves are shown in Figure 5. From the linear least-squares fit to the data, a minimum detectable concentration of 0.5 ppm and a lower limit of quantitation (LOQ) of 1.5 ppm were calculated. The linear range of response for the fluorimeter falls between 2 and 20 ppm. It should be noted that the 1 ppm standard used in the experiment falls below the LOQ. Lab 3: Vitamin B1 in Commercial Vitamins
This particular lab is a standard analytical chemistry22,23 lab, which has also been carefully adapted for use with our customized fluorimeter. The primary objective of this experiment is to determine the concentration of Vitamin B1 (thiamine) in one or more brands of multivitamin supplements. Vitamins are necessary in small quantities for normal health and metabolism. Unlike other biologically important compounds such as proteins and carbohydrates, vitamins cannot be synthesized by the body in sufficient quantities and therefore must be obtained through a regular diet or from dietary supplements. Thiamine is a watersoluble vitamin involved in the metabolism of carbohydrates and fats and is found abundantly in cereal grains, peanuts, meat, potatoes, egg-yolk, bananas, lentils, and tuna fish. Thiamine deficiency can lead to edema, muscular atrophy, and beriberi, a disease that impairs the nervous and cardiovascular system. Thiamine does not fluoresce, but can be detected indirectly by oxidizing thiamine to thiochrome, as shown in Scheme 1. To date, three oxidizing agents have been used: potassium ferricyanide,24,25 cyanogen bromide,26 and mercuric chloride.22,23,27 There is considerable fluorescence quenching and spectral interference by the ions in the ferricyanide method.2831 Cyanogen bromide is toxic and has a relatively short (3 h) serviceable life after preparation. 1185
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lab have been enthusiastic, excited, and hungry for more fun labs partly due to the instrument’s ease of use, the fact that it was built by student peers, and the relevance of the labs to the real world.
’ ASSOCIATED CONTENT
bS
Supporting Information Complete circuit diagram for the fluorimeter; a detailed list of the components and a close-up of the circuit board layout; a complete and detailed set of procedures for each of the experiments. This material is available via the Internet at http://pubs. acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
Figure 6. Fluorescence versus time plots for the various thiamine to thiochrome oxidations (300, 200, 150, 100, 50 ppm initial concentrations). Coefficient of correlation, r2 = 0.996, 0.997, 0.997, 0.999, 0.999, respectively.
Mercury salts are poisonous, but a 1% salt solution is much easier to handle than cyanogen bromide. The protocol described in the Supporting Information is simple to use, reasonably accurate, and significantly reduces the time it would take using conventional ferricyanide methods, and the rate for the reaction and production of the fluorescent signal is proportional to the thiamine concentration (Figure 6). Thiamine hydrochloride salt solution was used as a calibrant in an experiment that quantified thiamine in a variety of commercially available vitamin B1 supplement tablets. The oxidation of thiamine to thiochrome was carefully monitored. Fluorescence increased as thiochrome was produced (Figure 6), and the rate of increase in fluorescence is based on the amount of thiamine that was originally present and corresponds to first-order kinetics. The experiment strongly emphasizes techniques in quantitative dilution, collection and graphical treatment of kinetic data, and the importance of rate equations and rate constants. In addition it stresses the importance of good organization and patience. All of our students were able to complete the lab within the 3 h allotted time.
’ CONCLUSIONS A low-cost miniaturized fluorimeter was successfully created and constructed. The instrument circuitry is straightforward and easy to construct and use. The instrument has been used to facilitate the understanding and application of spectroscopy to a wide variety of groups from middle school science students to undergraduate chemistry majors specializing in analytical chemistry and instrumental analysis at college. The fluorescent whitening agent, quinine, and vitamin B1 labs are low cost and highly rewarding lab experiments for students. Each of these lab experiments can be customized for the level of student and can easily be expanded and elaborated. Students in
’ ACKNOWLEDGMENT We would like to thank the Penn State Schreyer Institute for Teaching Excellence and the Summer Experience in the Eberly College of Science program (SSECoS) for financial support. The SEECoS program is supported by the Upward Bound Math and Science Center (UBMS) at Penn State and a USDoE TRIO grant. The authors are thankful to the entire staff of the Electronics Research Instrumentation Facility at Penn State. The project is very much based on student effort and feedback, and we are very grateful for the time and patience that each of our undergraduate, high school, and middle school student participants has shown as we developed, tested, retested, and piloted the instruments. The authors also thank the editors and reviewers for their generous assistance. ’ REFERENCES (1) The National Commission on Excellence in Education, D. P. Gardner (Chair), “A Nation at Risk: The Imperative for Educational Reform” 1983, Report to the Secretary of Education, US Department of Education. http://www2.ed.gov/pubs/NatAtRisk/index.html (accessed May 2011). (2) America Competes Act of 2007. http://www.nist.gov/mep/ upload/PL110-69_8907.pdf (accessed May 2011). (3) Race to the Top Executive Summary. http://www2.ed.gov/ programs/racetothetop/executive-summary.pdf (accessed May 2011). (4) 1998 Amendments to the Higher Education Act of 1965. http:// www2.ed.gov/policy/highered/leg/hea98/index.html (accessed May 2011). (5) The Committee on Education of the American Physical Society, Peter J. Collings (Chair), “Statement On Undergraduate Research” April 4, 2009. http://www.aps.org/programs/education/upload/undergrad_ research_statement.pdf (accessed May 2011). (6) Hutchinson, A. R.; Atwood, D. A. J. Chem. Educ. 2002, 79, 125–12. (7) Seymour, E.; Hunter, A. B.; Laursen, S. L.; Deantoni, T. Sci. Educ. 2004, 88, 493–534. (8) Hunter, A. B.; Laursen, S. L.; Seymour, E. Sci. Educ. 2007, 91, 36–74. (9) Hadley, F. J.; Mahloudji, A. J. Chem. Educ. 1990, 67 (9), 806–807. (10) Jones, B. T.; Smith, B. W.; Leong, M. B.; Mignardi, M. A.; Winefordner, J. D. J. Chem. Educ. 1989, 66, 357–358. (11) Algar, W. R.; Massey, M.; Krull, U. J. J. Chem. Educ. 2009, 86, 68–71. (12) Chen, G. Rev. Sci. Instrum. 2005, 76, 0631071–0631077. 1186
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