The Characterization of an Easy-to-Operate Inexpensive Student-Built

LABORATORY EXPERIMENT pubs.acs.org/jchemeduc

The Characterization of an Easy-to-Operate Inexpensive Student-Built Fluorimeter Benjamin T. Wigton,† Balwant S. Chohan,*,† Rod Kreuter,‡ and Dan Sykes*,‡ † ‡

School of Science, Engineering, and 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 compact, rugged, and portable fluorimeter was designed and constructed using a 460 nm light-emitting diode for the excitation and a silicon photodiode for detection. The total cost of such an instrument is less than $110, thus allowing for deployment of multiple instruments at low cost. The fluorimeter is easy to operate and has been successfully used to quantify fluorescein in commercial-brand antifreeze and chlorophyll a pigment in spinach leaves. The mini instrument and the associated experiments have provided for a novel approach to teaching the principles and applications of spectroscopy and fluorescence to a wide academic variety of students. KEYWORDS: First-Year Undergraduate/General, High School/Introductory Chemistry, Second-Year Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Consumer Chemistry, Fluorescence Spectroscopy, Food Science, Quantitative Analysis

I

n the past 25 years, technological gadgets have transformed the chemistry lab enormously. The traditional scientific paraphernalia such as test tubes, watch glasses, spectroscopes, and microscopes now sit alongside high-resolution coupled mass spectrometers, Fourier-transform IR spectrometers, superconducting electromagnetic NMR spectrometers, and fiber-optic UV
vis-NIR spectrometers. At the college level, the percentage of instructional time spent on electronic instruments has increased substantially. Students are required to gain experience on computer-interfaced instrumentation and computer-assisted data acquisition and manipulation. However, the strong emphasis on computer-controlled instrumentation has been at the expense of knowing exactly how the instrument works, the basic principles of the measurement, and the limitations of the method. The analytical chemistry program at Penn State University requires students to build a functional instrument as part of a semester-long class research project. The students build, from scratch, calibrated and quantitative instruments, using basic electronic and soldering skills. The basic design of the analytical instruments are comparable to commercial instruments, the biggest difference is the lack of software and expensive circuitry that is designed to give superior sensitivity. Some of our current projects include a colorimeter, a dissolved-oxygen probe, a 2H NMR probe, a cyclic voltammetry instrument, and several labon-a-chip instruments. The semester-long projects provide an intensive guided-inquiry learning experience. Students must submit a literature-based research proposal, work as a team to complete the project, present their research at a chemistry-wide poster symposium, and write a final report. The success or failure Copyright r 2011 American Chemical Society and Division of Chemical Education, Inc.

of any project is based on the students’ initiative, motivation, organization, and work ethic. Introducing the projects into the curriculum was a substantial risk because 75% of the course grade would be attributed to electronics-based topics. To our surprise and glee, these projects have become a positive life-defining experience for our students, and over the past three years, 80% of the students consistently indicated that the electronic projects are by far their favorite activities in the course. Although most analytical chemistry curricula at the undergraduate level teach students the core principles of luminescence, few students have the opportunity to appreciate the power of this phenomena and simplicity of the instrumentation involved. Commercial hand-held type fluorimeters with limited excitation wavelengths enable highly accurate analyses, but are often costly for multiple users in an undergraduate lab setting. The fluorimeter instruments that we describe are built by upper-level students, are low-maintenance, lightweight, reliable, and rugged, and could be made widely available at a cost of no more than $110 per instrument. Sophisticated commercial fluorimeters employ monochromators, optical filters, and lasers to select excitation and emission wavelengths. The instrument that has been designed and tested in this article contains the minimum electrical circuitry to generate and detect fluorescence with relatively good accuracy. To illustrate the fundamental concepts and applications of this fluorimeter within high school and undergraduate chemistry lab courses, upper-level students were Published: May 20, 2011 1188

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Journal of Chemical Education also charged with the task of developing a series of laboratory experiments that provide for an investigation of real-life problems. Our belief is that these types of laboratory experiments encourage students to take part in the activity with more enthusiasm, commitment, and thoughtfulness. The developed fluorescein and chlorophyll a labs also expose students to common extraction and purification techniques and provide for a thorough exercise in spectrometry and the Beer
Lambert law. In addition, these experiments are used to test, evaluate, and compare the detection limits afforded by the custom student built 460 nm fluorimeters. Fluorescence is a spectrochemical method of analysis where the molecules of the analyte are excited by irradiation at a certain wavelength and emit radiation at a different longer wavelength, Fluorescence spectroscopy is an important investigational tool in many areas of analytical science owing to its extraordinary sensitivity, selectivity, and specificity. Fluorimetry is ordinarily 1000-fold more sensitive than absorbance measurements. Fluorescent substances have characteristic excitation and emission spectra that make up the material’s fluorescence signature. In a sample which contains a fluorescent compound, excitation at any wavelength within the excitation spectrum range will cause emission radiation to be detected across the entire emission spectrum.1,2 No two compounds have the same excitation and emission spectra and so, by careful selection of the excitation wavelength and the emission wavelength measured, excellent detection of the target compound may be achieved. Such characteristics follow the Beer
Lambert law, and the intensity of emitted light is proportional to the concentration of the analyte being measured. The 460 nm fluorimeters built by upper-level students were incorporated into two introductory undergraduate labs. The fluorescein and chlorophyll a experiments can be carried out in a 3 h lab session. Laboratory time constraints were carefully taken into account while developing the protocols at each stage. The first experiment involves the preparation of a calibration curve and subsequent analysis of fluorescein in a commercial brand of antifreeze. The second experiment is more involved and requires students to generate a calibration curve and then isolate and measure chlorophyll a from spinach. A short thin-layer chromatography (TLC) procedure provides an opportunity for students to distinguish and discuss the many components in their extract and appreciate the methodologies involved in purifying natural products.

’ EXPERIMENTAL MATERIALS Instrument

The circuitry of the 460 nm fluorimeter is similar in design to a 360 nm instrument originally built by the students (detailed information about the 360 nm instrument will be published in a separate article in this Journal). There are two major changes from the 360 nm design: the source and the presence of a second optical filter. The source light-emitting diode (LED) was purchased from Avago Technologies (part number ASMT-MB00). The LED is forward biased at 3.6 V and 48 mA. The spectral emission curve has a peak wavelength at 460 nm and an asymmetric spectral bandwidth of
30 nm/þ60 nm (95% of total emission intensity). A minor change to the original circuit diagram was required to accommodate the 460 nm LED; this included replacement of the 220 Ω load resistor with a 75 Ω resistor. Two optical filters are also required for the 460 nm

LABORATORY EXPERIMENT

fluorimeter. The first, a bandpass filter, is placed between the source LED and the cuvette holder to minimize scattering of the source at wavelengths between 470 and 530 nm. Scattering of the source emission at these wavelengths significantly overlaps and masks fluorescence emission from many samples. The bandpass filter was purchased from Edmund Optics, (part number NT62-084). The center of the bandpass and full-width half-maximum are 458 ( 10 nm. The second optical filter is placed between the cuvette holder and the detector. For this cutoff filter (part number NT54-653), the stop-band limit is 440 nm, the cutoff position is 515 nm, and the pass-band limit is at 580 nm. The addition of the bandpass filter increases the cost of the instrument by approximately $30 over the cost of the 360 nm fluorimeter. Chemicals

Hexane, distilled water, acetone, anhydrous MgSO4, 95% ethanol, fresh spinach leaves (purchased from local grocery stores, no stems), 30 ppm chlorophyll a (from spinach, purchased in solid form from Sigma-Alrich), Prestone Antifreeze/ Coolant, and fluorescein (Standard Fluka from Sigma-Aldrich).

’ EXPERIMENTAL PROCEDURES A detailed set of procedures for each of the experiments is provided in the Supporting Information. All solutions should be kept in the dark because they are photosensitive (wrap aluminum foil around the containers). Concentrated solutions are stable in the refrigerator for up to two days. A glass 1.0 cm cuvette was used for each measurement, and each measurement was repeated three times so as to obtain a mean average and standard deviation. Lab 1: Fluorescein Experiment

Sodium fluorescein, also known as uranine, color additive D&C Yellow #8, or acid yellow #73, has a high quantum yield (Φ = 0.85 in 0.1 M NaOH) such that low concentrations yield intense fluorescence in alkaline solutions.3 Sodium fluorescein is commonly added to commercial antifreeze ethylene glycol solutions as a colorant to aid in the detection of leaks in automobile cooling systems. A set of five fluorescein standard solutions, varying in concentration from 2.50 to 0.156 ppm (2.50, 1.25, 0.625, 0.313, and 0.156 recommended) in 95% ethanol, were carefully prepared from a 5.00 ppm fluorescein stock solution. The upper concentration limit is determined by the limitation (2.000 mV) of the fluorimeter. A cuvette containing the ethanol blank was first inserted into the cell holder, and the fluorimeter was zeroed. Intensity measurements on each fluorescein solution, given as millivolts (mV)readings, were then obtained and used to generate a standard calibration curve. An initial intensity measurement was obtained on a sample of commercial antifreeze. Appropriate dilutions were then made to ensure that the readings were within the working curve. Fluorescence readings were taken for nine different concentrations prepared by successive dilution of the antifreeze sample. This data set was then pooled to determine the concentration and perform error analysis, including confidence limits, of fluorescein in the original commercial antifreeze. Students were required to tabulate their data, convert parts per million (ppm) to molarity, generate plots, and to calculate the fluorescein concentration in the original antifreeze sample. In addition, students were asked to comment on the instrument 1189

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Journal of Chemical Education with suggestions on how to improve its design and functionality and to address their understanding of fluorescence by answering a number of postlab questions. Lab 2: Chlorophyll a Experiment

Chlorophyll is the most important pigment involved in photosynthesis, the process by which light energy is converted to chemical energy by green plants and algae. Chlorophyll occurs in several distinct forms, and chlorophyll a and b are the major types found in the chloroplasts of green plants. A molecule of chlorophyll contains one Mg2þ ion coordinated to a nitrogencontaining porphyrin ring. Chlorophyll a acts as a reaction center, trapping light energy and converting it to electrical and chemical energy. Chlorophyll b constitutes 25% of the total chlorophyll content,4,5 and similar to carotenoid and xanthophyll pigments, it acts as an accessory pigment that broadens the spectrum of light absorbed during photosynthesis, transferring its energy to the chlorophyll a center. During senescence, the leaves of a green plant begin to decompose, the chlorophylls are converted to pheophytin, which changes the color of the green leaves to an olive
gray. In this experiment, the concentration of chlorophyll a was determined in an extract of fresh spinach (Spinacia oleracea). In addition, students developed a thin-layer chromatogram (TLC) of the spinach extract and identified the various components present: Carotenes, pheophytin a and b, chlorophyll a and b, and xanthophylls. A number of reviews and procedures have appeared on laboratory methods for preparation of chlorophyll from plant materials.6
13 The method that we have chosen to adapt involves the least number of steps and can be conveniently and cost-effectively done within a period of 40 min in an introductory chemistry lab setting. A calibration curve was constructed from five separate chlorophyll a concentrations made in hexane solvent. For economical reasons, each separate standard concentration came from serial dilution of a 30.0 ppm stock solution (15.0, 7.50, 3.75, and 1.88 recommended). A cuvette containing the hexane blank was first inserted into the cell holder, and the fluorimeter was zeroed. Intensity measurements on each chlorophyll solution were then obtained to generate a calibration curve. Fresh spinach leaves (3.00 g) were ground in 1 mL of acetone with a ceramic pestle and mortar. The green paste was diluted with 10 mL of water and 10 mL of hexane, and gravity-filtered. The hexane layer was separated from the green-colored filtrate, dried with anhydrous MgSO4, filtered, and then analyzed with the 460 nm fluorimeter. Successive dilutions of this extract allowed for fluorescence readings at four different concentrations to be taken. As a short exercise in chromatography, students spotted the spinach extract onto a silica TLC plate (Sigma-Aldrich, silica gel 60), which was then placed in a beaker containing a 7:3 mix of hexane/acetone developer (mobile phase). The students worked with the instructor to identify the pigment components using reference colors and Rf values from the literature.6 Students were required to tabulate their data, generate Excel plots, and convert the parts per million (ppm) readings to percent (%), weight per weight (w/w), and micrograms per gram (μg/g) concentration for the chlorophyll a in spinach. Upon completing the lab exercise, students were asked to comment on the instrument with suggestions on how to improve it and to address their understanding of fluorescence and chromatography separations by answering a number of postlab questions.

LABORATORY EXPERIMENT

’ HAZARDS Hexane, acetone, and ethanol may cause irritation to skin, eyes, and respiratory tract and may be harmful if swallowed or inhaled. The organic solvents are also flammable. If students come in contact with these solutions, it is important to rinse the affected area thoroughly with soap and water. The active ingredient in antifreeze is ethylene glycol, a fatal toxin if swallowed. Avoid spilling antifreeze on the ground. Use sand, vermiculite or other inert materials to absorb and collect into a waste container. Appropriate precautions need to be taken when constructing the apparatus, especially with hot soldering irons. Instructors and students alike need to take appropriate precautions when dealing with open live electrical circuits. ’ EXPERIMENTAL RESULTS The results for the fluorescein calibration curve are provided in Table 1. A plot of the relative fluorescence intensity versus concentration is shown in Figure 1. The linear range of response for the student-built fluorimeter, based on the data for fluorescein, appears to fall between 0.30 and 2.50 ppm; values greater than 3.80 ppm were off-scale. The instrument is designed to give readings down to the microvolt (μV) level. Additional experiments are necessary to ascertain how far the linear range extends at the lower limits, but initial results suggest that reasonable and reliable data is obtainable down to a signal of 10 μV, equivalent to a fluorescein concentration of 0.06 ppm (about 0.13 μM). Limits of detection down to the picomolar range are possible for sophisticated commercial instruments. The results for the antifreeze sample are given in Table 2. Using the calibration curve in Figure 1, the fluorescein contents in Prestone Antifreeze/ Coolant were calculated to be 5.2 ppm with a standard deviation of 0.4. For example, for the 25% diluted sample from Table 2 (dilution factor = 4), using the equation of best-fit line (Figure 1): mV = (0.796  ppm)
0.034, the value of 1.3 ppm Table 1. Experimental Data for the Fluorescein Calibration Standards Concentration (ppm)

Fluorescence/mV

5.00 2.50

not on scale 1.929

1.25

1.003

0.625

0.509

0.313

0.194

0.156

0.052

Figure 1. Calibration curve for fluorescein using the 460 nm fluorimeter. The equation of best-fit line is y = 0.7960x
0.0336, R2 = 0.999. 1190

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Table 2. Experimental Data for the Commercial Antifreeze Antifreeze Concentration (% original)

Fluorescence/mV

100.0

not on scale

50.0

1.843

25.0

1.009

12.5

0.519

6.25

0.275

3.13

0.140

1.56 0.781

0.070 0.037

0.391

0.021

0.195

0.012 Fluorescein Concentration

Antifreeze Dilution

(ppm)

1/2

4.7

1/4 1/8

Figure 2. Calibration curve for chlorophyll a using the 460 nm fluorimeter. The equation of best-fit line is y = 0.00330x þ 0.00462, R2 = 0.999.

Table 4. Experimental Data for Chlorophyll a in Spinach Concentration (% original extract)

Fluorescence/mV

5.2

100.0

0.088

5.6

50.0

0.050

25.0

0.027

12.5

0.014

Table 3. Experimental Data for the Chlorophyll a Calibration Standards Concentration (ppm)

Fluorescence/mV

30.500

0.104

15.250 7.625

0.057 0.031

3.813

0.017

1.906

0.009

is calculated, when the voltage is set at 1.009 mV. This value multiplied by the dilution factor gives 5.2 ppm. Fluorescein is a minor component of antifreeze, and therefore is not required to be listed on the contents or on the material safety data sheets. The manufacturers are unwilling to share this propriety information, so it has not been possible to evaluate the accuracy of the results. Our data has relatively good precision. However, our calculated fluorescein content appears to be a little lower than typical values that are reported (15
20 ppm) in the scientific literature for a Prestone antifreeze product from 20 years ago.14 The results for the chlorophyll a calibration curve are provided in Table 3. A plot of the relative fluorescence intensity versus concentration is shown in Figure 2. The linear range of response for the student-built fluorimeter, based on the data for chlorophyll a, appears to fall between 2 and 30 ppm. Additional experiments are necessary to ascertain how far the linear range extends at the upper end. Our results indicate that reasonable and reliable data can be obtained down to a lower limit signal of 10 μV with this instrument; this lower voltage limit would be equivalent to 1.7 ppm (about 1.2 μM) of chlorophyll a. Fresh spinach leaves were the source of the spinach extract. The spinach (3.00 g) was crushed and pulverized using a mortar and pestle, with a little acetone. Hexane was then used to separate the cholorophyll a from the mixture. The results of the hexane (10.0 mL) extract containing the chlorophyll a are given in Table 4. Using the calibration curve in Figure 2, the chlorophyll a concentration was calculated to be 24.0 ppm (about 17.3 μM)

Chlorophyll a Concentration Chlorophyll a Extract Dilution

(ppm)

undiluted 1/2

27.7 30.0

1/4

29.2

1/8

24.0

with a standard deviation of 2.2. For example, for the 12.5% diluted sample from Table 4 (dilution factor = 8), using the equation of best-fit line (Figure 2): mV = (0.003  ppm) þ 0.005, the value of 3.0 ppm is calculated, when the voltage is set at 0.014 mV. This value multiplied by the dilution factor gives 24.0 ppm. The mean average value obtained in this lab (27.7 ppm, 20.3 μM) corresponds to a chlorophyll a concentration of 60.5 μg/g fresh weight. This value is on the lower end of the various values reported for fresh spinach leaves in the literature: 25,15 691,16 440,17 614,18 and 380
610 μg/g.19 The variations in literature are due to the solvent extraction and purification techniques employed. The low value obtained in this lab is possibly due to the short period of time available for the extraction process. The chlorophyll a content for spinach is highly dependent on factors such as freshness, brand, whether the leaves or stems were used in the extract, temperature, solvent, degree of luminescence, exposure to oxygen, and time required for extraction.20
22 Considerable analyte losses can occur during sample preparation, and it is important to maintain low temperature and light conditions. However, this is challenging in an introductory lab setting with limited time. Literature data are scarce, and are occasionally given as a percentage content in isolated chloroplasts, or protein content, or as dry weight.23
25 It is apparent that literature procedures dealing with chlorophyll extraction from spinach take considerably longer than what was employed in this lab and involve multiple solvent extractions, column chromatography, and strict physical conditions. It is therefore not surprising that the value we obtained is lower than many of the values reported for fresh spinach leaves.15
19 1191

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Journal of Chemical Education The pigments extracted from a plant leaf are a mixture of chlorophylls and carotenoids. As an additional exercise in chromatography, students performed TLC to separate the various pigments. It is possible to use basic paper chromatography but to separate plant pigments a more efficient method such as TLC, column chromatography, or HPLC is required. The chlorophyll content is dependent on many factors, with temperature and lighting playing a big factor in its stability and degradation. Chlorophyll degradation has a number of pathways, leading to many products, not all of which are detectable by TLC analysis. Fresh spinach tends to have the most intact chlorophyll a and the least pheophytin a. Student calculated Rf values for chlorophyll a varied from 0.41 to 0.50 and for pheophytin a from 0.63 to 0.69. These TLC results were comparable to literature data.6,13 The results of TLC analysis allowed students to qualitatively assess the freshness of the samples and the various factors one needs to consider during the extraction and preparation process.

’ DISCUSSION Commercial laboratory instruments are becoming increasingly more sophisticated and more streamlined. These advances for the most part have closely followed the extraordinary leaps taken by the semiconductor and computational industry. The computerized instruments have become very convenient for routine use in industrial and scientific laboratories but less suitable for teaching purposes. Though the technology is fascinating and the instruments much simpler, many of us in the educational profession feel that the objective has shifted to knowing how to insert a sample and quickly learning the instrument software. As chemistry changes from traditional fields to multidisciplinary, it is imperative we prepare our students accordingly for successful careers by updating the curricula. There is an alarming trend that chemists have become more reliant on automation and lack the knowledge about both the instruments and the science behind the measurements they depend upon. The tendency is to view instruments as black boxes and data as statistically meaningful regardless of the many limitations of the analytical method. Students are unable to appreciate or consider the practical limitations that influence instrument design and data acquisition and, therefore, never have any reason to question the quality or significance of the data. This trend is further propagated in use of outdated analytical methods and simply teaching techniques, rather than the science behind the methodology. The goal of this project has been to “strip-away” some of the veils that get in the way of understanding the science behind the measurements. The construction of fluorimeters have been described with varying complexity in this Journal;26
28 however, the student-built fluorimeter that was constructed came about from an initiative within the instrumental analysis courses to require students to build a functional instrument as part of a semester-long class research project. The students build the instruments using their rudimentary electronic skills. They are then required to obtain data from the instrument; failure to get good data requires them to go back and adjust or repair the instruments. The basic design and methods for probing chemical systems between commercial instrument and the student-built instrument are identical. The most substantial difference is the lack of computer control, software, and a variety of the high-tech devices that give superior sensitivity. The use of these studentbuilt instruments promotes student competency in the sciences and engineering. We have found that it is a natural conduit for

LABORATORY EXPERIMENT

co-curricular experiences and strongly fosters student ownership of their program’s curriculum.

’ CONCLUSION We have shown that it is possible to teach upper-level undergraduate students the science behind the measurements they use in the chemistry laboratory by building their own instrument. The 460 nm fluorimeter described in this article has been used in a college general chemistry laboratory, a college quantitative analysis course, and an eighth-grade science class at a local high school to introduce the subject of luminescence in a fun and effective manner at a fraction of the cost of purchasing commercial instruments. The instrument is easy to use and each of the experiments reported here can be performed in a one 3-h laboratory session. The most significant challenge is focused on the instructor and his or her ability to cover the various concepts in an effective manner during the lab session. It is important to coordinate the lab with the classroom course, thereby saving prelab time. Student evaluations and feedback have highlighted the ease of use of the instrument, the advantage of not having to line up to use the instrument, and the highly relevant and real-life lab experiments. The students were amused to learn that the instrument costs so little, less than 1% the price of commercial fluorimeters. Currently, we use 12 instruments (a combination of the 360 and 460 nm instruments) in our quantitative analysis and general chemistry labs; all built for a total cost of less than $1000. ’ ASSOCIATED CONTENT

bS

Supporting Information Student handouts for both laboratories. This material is available via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: (B.S.C.) [email protected]; (D.S.) [email protected].

’ ACKNOWLEDGMENT We are most grateful to Joe Keiser at Penn State University Park, to John Bylander and Richard Papez at Penn State Harrisburg, and to Matthew Johnson at West Branch Area School District, each of whom were kind enough to allow their classrooms to be used as test sites for the fluorimeter instruments. The Schreyer Institute for Teaching Excellence provided initial funding for the project. We appreciate the time, energy, effort and comments of the reviewers. ’ REFERENCES (1) Skoog, D. A.; Holler, F. J.; Nieman, T. A. Principles of Instrumental Analysis, 5th ed.; Brooks/Cole Thomson Learning: Belmont, CA, 1997. (2) O’Hara, P. B.; Engelson, C.; St. Peter, W. Turning on the Light: Lessons from Luminescence. J. Chem. Educ. 2005, 82, 49–52. (3) Umberger, J. Q.; La Mer, V. K. The Kinetics of Diffusion Controlled Molecular and Ionic Reactions in Solution As Determined by Measurements of the Quenching of Fluorescence. J. Am. Chem. Soc. 1945, 67, 1099. (4) Leong, T.-Y.; Anderson, J. M. Adaptation of the Thylakoid Membranes of Pea Chloroplasts to Light Intensities. I. Study on the Distribution of Chlorophyll Protein Complexes. Photosynth. Res. 1984, 5, 105–115. 1192

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66. (11) Iriyama, K.; Ogura, N.; Takamiya, A. A Simple Method for Extraction and Partial Purification of Chlorophyll from Plant Material Using Dioxane. J. Biochem. 1974, 76, 901–904. (12) Silveira, A.; Koehler, J. A.; Beadel, E.; Monroe, P. A. HPLC Analysis of Chlorophyll a, Chlorophyll b, and β-Carotene in Collard Greens. J. Chem. Educ. 1984, 61, 264. (13) Reiss, C. Experiments in Plant Physiology; Prentice Hall: Englewood Cliffs, NJ, 1994. (14) Winter, M. L.; Ellis, M. D.; Snodgrass, W. R. Urine Fluorescence Using a Wood’S Lamp to Detect the Antifreeze Additive Sodium Fluorescein: A Qualitative Adjunctive Test in Suspected Ethylene Glycol Ingestions. Ann. Emerg. Med. 1990, 19, 663–667. (15) Walker, S. Investigations Get Real. Educ. Chem. 2009, 46, 1. (16) Bohn, T.; Walczyk, T. Determination of Chlorophyll in Plant Samples by Liquid Chromatography Using Zinc
Phthalocyanine As an Internal Standard. J. Chromatogr., A 2004, 1024, 123–128. (17) Omata, T.; Murata, N. Preparation of Chlorophyll a, Chlorophyll b and Bacteriochlorophyll a by Column Chromatography with DEAE-Sepharose CL-6B and Sepharose CL-6B. Plant Cell Physiol. 1983, 24, 1093–1100. (18) Rapsch, S.; Ascaso, C. Effect of Evernic Acid on Structure of Spinach Chloroplasts. Ann. Bot. 1985, 56, 467–473. (19) Khalyfa, A.; Kermasha, S.; Alli, I. Extraction, purification, and characterization of chlorophylls from spinach leaves. J. Agric. Food Chem. 1992, 40, 215–220. (20) Schwartz, S. J.; Vonelbe, J. H. Kinetics of Chlorophyll Degradation to Pyropheophytin in Vegetables. J. Food Sci. 1983, 48, 1303. (21) Mangos, T. J.; Berger, R. G. Determination of Major Chlorophyll Degradation Products. Z. Lebensm.Unters. Forsch. A 1997, 204, 345. (22) Lopez-Ayerra, B.; Murcia, M. A.; Garcia-Carmona, F. Lipid Peroxidation and Chlorophyll Levels in Spinach during Refrigerated Storage and after Industrial Processing. Food Chem. 1998, 61, 113–118. (23) Smith, E. L. The Chlorophyll Protein Compound of the Green Leaf. J. Gen. Physiol. 1941, 24, 565–582. (24) Teng, S. S.; Chen, B. H. Formation of Pyrochlorophylls and Their Derivatives in Spinach Leaves during Heating. Food Chem. 1999, 65, 367–373. (25) Turkmen, N.; Poyrazoglu, E. S.; Sari, F.; Velioglu, Y. S. Effects of Cooking Methods on Chlorophylls, Pheophytins and Colour of Selected Green Vegetables. Int. J. Food Sci. Technol. 2006, 41, 281–288. (26) Delorenzi, N. J.; Araujo, C.; Palazzolo, G.; Gatti, C. A Simple Device to Demonstrate the Principles of Fluorometry. J. Chem. Educ. 1999, 76, 1265–1266. (27) Wickliff, J. L.; Wickliff, D. E. Instrumentation for Measuring in Vivo Chlorophyll Fluorescence Induction. J. Chem. Educ. 1991, 68, 963–965. (28) White, C. E. The Use of Fluorescence in Qualitative Analysis. J. Chem. Educ. 1951, 28, 369–372.

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