Demonstrating Basic Properties of Spectroscopy Using a Self

Article pubs.acs.org/jchemeduc

Demonstrating Basic Properties of Spectroscopy Using a SelfConstructed Combined Fluorimeter and UV-Photometer Eivind V. Kvittingen,† Lise Kvittingen,*,‡ Thor Bernt Melø,§ Birte Johanne Sjursnes,∥ and Richard Verley† †

Overlege Kindts gate 16a, 7052 Trondheim, Norway Department of Chemistry, NTNU, Norwegian University of Science and Technology, 7491 Trondheim, Norway § Department of Physics, NTNU, Norwegian University of Science and Technology, 7491 Trondheim, Norway ∥ Faculty of Engineering, Østfold University College, 1757 Halden, Norway ‡

S Supporting Information *

ABSTRACT: This article describes a combined UV-photometer and fluorimeter constructed from 3 LEDs and a few wires, all held in place with Lego bricks. The instrument has a flexible design. In its simplest version, two UV-LEDs (355 nm) are used as light source and to detect absorption, and a third LED, in the visible spectrum (e.g., 525 nm), is used to detect fluorescence. Various experiments are described: fluorescence as a function of concentration and light source intensity; quantitative measurements of quinine in tonic water, using absorption and fluorescence measurements, with results in line with those obtained on scientific instruments; a demonstration of the inner filter effect reducing fluorescence at higher concentrations; investigation of the effect of drinking tonic water on the fluorescence of quinine in urine; temperature effects on fluorescence; chloride-ion quenching of fluorescence with result comparable to those previously reported; and, finally, a demonstration of phosphorescence. KEYWORDS: High School/Introductory Chemistry, First-Year Undergraduate/General, Second-Year Undergraduate, Upper-Division Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Fluorescence Spectroscopy, Spectroscopy

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INTRODUCTION Absorption spectroscopy is usually students’ first encounter with spectroscopy. Simple instruments that can display the basic principles are desired, and some suitable colorimeters1−11 and a UV-photometer12 using LED technology have been reported. Today, fluorescence spectroscopy is used in many areas. In Suhling’s words, fluorescence is13 “[U]sed from physical chemistry to clinical and pharmaceutical applications, drug discovery, forensic science, fluorescence microscopy in cell biology and life sciences in general, but also in environmental monitoring and art conservation. There are no signs that its role is going to be diminished in the future.” Students would therefore benefit from encountering fluorescence spectroscopy early in their studies. Previously, we reported the design of a colorimeter11 and a UV-photometer,12 both of which were used for absorption measurements. Here a flexible instrument that can be used for both absorption and fluorescence measurements is presented. The simplest version is shown in Figure 1. This instrument has a design similar to that of the instruments reported previously, but the emphasis here is not on the design, but is rather on application of the device for exploring fluorescence. We © XXXX American Chemical Society and Division of Chemical Education, Inc.

Figure 1. Simplest version of the instrument.

Received: February 13, 2017 Revised: July 30, 2017

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DOI: 10.1021/acs.jchemed.7b00121 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

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evaluate various LEDs for measuring fluorescence (1); investigate sensitivity of the instrument and fluorescence as a function of intensity (2); perform quantitative measurements of fluorescence in tonic water (3); visualize the inner filter effect, i.e., the effect of concentration on fluorescence (4); demonstrate a biological application, fluorescence of urine (5); measure fluorescence at different temperatures (6); investigate quenching (7); and demonstrate phosphorescence (8). A well-known fluorescent compound, quinine, in the form of quinine sulfate dissolved in 0.5% H2SO4, has been used as a model compound. Quinine is found in tonic water. Other possible compounds are fluorescein or chlorophyll; however, care must be taken considering the different solvents required. The instrument, quinine sulfate, and 0.5% H2SO4 comply with HSE requirements for use in nonlaboratory settings. Small-scale photometers using an LED as light source have been made for pedagogical1−11 and purely analytical14−18 purposes, but they operate in the visible light spectrum. One reason is the cost. Another is the relatively low quantum efficiency of ultraviolet (UV) LEDs compared to visible (VIS) LEDs. Self-constructed and relatively simple fluorescence instruments have been reported for more than 50 years.19−25 However, it is only since about the year 2000 that LEDs have been used (as light sources), making the instruments both cheaper and simpler.26−33 Photometers using an LED as a light detector generally use the LED in reverse biased mode, requiring an external voltage and some additional circuitry.3,4,17,18 The use of the LED in photovoltaic mode has also been reported.14,34,35 An LED is a current source; the current generated is in proportion to the incident radiation. When the current (I) flows through a resistor (R), the developed voltage (V) across the resistor is in proportion to the current, through Ohm’s law: V = RI. The input resistance of the voltmeter can be used for this purpose. The measured voltage is then proportional to the light intensity. The size of the resistance scales the signal up to a convenient level in the millivolt range. The current through a diode increases exponentially with voltage, so to operate in the (near) linear range, the voltage should be in the millivolt range. In our previously reported colorimeter11 and UV-photometer,12 the LED used as detector functions in photovoltaic mode with no circuitry. This reduces the number of components and simplifies understanding. In the present instrument, the photometer and fluorimeter detectors (a UV and a VIS LED, respectively), are each connected directly to a millivoltmeter which has an internal resistance of 10 MΩ.

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EXPERIMENTAL SECTION Standard solutions of quinine sulfate [CAS 207671-44-1] (structure and absorption and fluorescence spectra are given in Supporting Information) were made in the range 0.5−100 mg/ L in 0.5% H2SO4 (see Supporting Information). Tonic water samples (Schweppes) were diluted 10, 20, 30, 50, and 100 times with 0.5% H2SO4. For the quenching experiments, aliquots of NaCl solution (600 mM) were added directly to the cuvettes which already held 2.5 mL of quinine sulfate solution. This resulted in NaCl solutions in the range 2−25 mM (see Supporting Information). Urine samples were collected over 200 h after ingesting a 500 mL bottle of tonic water and stored in a freezer. Before analysis the urine samples were equilibrated at room temperature and diluted 7.5 times with 0.5% H2SO4. For demonstrating phosphorescence, the cuvette with content was held with wooden pincers in liquid nitrogen for ∼5 min, before being placed in the instrument and radiated with 355 nm from two sides. The cuvettes must not absorb light at 355 nm and must withstand 0.5% H2SO4; poly(methyl methacrylate) (PMMA) cuvettes fulfill these requirements. For fluorescence measurements, the cuvettes must have four clear sides. Fluorescence is measured directly (in mV) and absorption as transmittance (see Supporting Information and refs 11 and 12). Control measurements were made with a Hitachi 2000 spectrophotometer and a Fluorolog 3 from Horiba Jobin Yvon.

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HAZARDS The hazards are mainly the same as those for refs 11 and 12 but are included here for completeness. To cut the Lego brick (2 × 2) a hacksaw can be used. The Lego brick should be held in place in a vice and safety glasses worn to reduce hazards. UV light should not be looked at directly. However, the LED used here emits at 355 nm, a range close to VIS and far from shortwave UV. The design of the instrument makes it also nearly impossible to look directly at the LED, reducing the hazard even further. Sulfuric acid with a concentration of 0.5% is not classified as dangerous according to European Union Legislation and needs no hazard labeling according to Regulation (EC) No. 1272/2008. The experimenter should, however, consult local HSE regulations. Preparing the solution must be undertaken with care and in accordance with HSE regulations, as higher concentrations of H2SO4 are highly corrosive. For the experiment involving urine, HSE regulations applied in the particular institution should be followed. Some institutions may also require approval from a review committee as urine samples are collected from human subjects. Details are found in Supporting Information. If phosphorescence is to be demonstrated, liquid nitrogen must be handled with care according to HSE regulations.

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EQUIPMENT SETUP Figure 1 illustrates the simplest arrangement of the instrument. One UV LED (355 nm) is used as the light source and a second, aligned with the incident light, as the detector for absorption measurements. Perpendicular to the incident light is a VIS LED (of any wavelength >450 nm; in our case 525 nm) used to measure fluorescence. The use of Lego bricks to support the LEDs, the battery to power the light source, the multimeters to make readings, and the considerations to take for measurements are the same as those described in refs 11 and 12. Abbreviated versions are described in Supporting Information.

RESULTS AND DISCUSSION

1. Evaluation of Detector LEDs for Fluorescence

An LED can only detect radiation of a wavelength equal or shorter than it emits itself. The 355 nm LED will therefore only detect the 355 nm radiation that is transmitted through the solution and not the fluorescence which is at ∼450 nm. In our previous reports,11,12 we have shown that absorbance is proportional to concentration of a sample (in the mV range used here) for both the UV and the green LED. The LEDs’ photovoltaic responses are therefore linear to the light intensity striking them. B



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Figure 2 shows standard curves for fluorescence measurements using several different detector LEDs, as well as results

Figure 5 shows that using either two light sources and one detector (Figure 4b) or one light source and two detectors (Figure 4c) approximately doubles the sensitivity. Furthermore, using two light sources and two detectors (Figure 4d) increases the sensitivity to nearly 4 times that of using one light source and one detector (Figure 4a). This demonstrates the approximate proportionality between irradiation intensity and fluorescence, as well as the increase in sensitivity by using two detector LEDs in series. 3. Quantitative Measurements Using Fluorescence and Absorbance

Standard curves for absorption and fluorescence of quinine in the range 0−100 mg/L were made using the configuration in Figure 4c, i.e., two fluorescence detectors and one absorption detector (see Supporting Information). These curves were used to find the concentration of 5 samples of quinine in commercial tonic water diluted from 10 to 100 times. From the absorption measurements, the concentration of quinine was calculated to be 82, 82, 73, and 80 mg/L from the samples diluted 10, 20, 30, and 50 times, respectively. Absorption was too low to give meaningful results when diluted 100 times. Fluorescence measurements gave similar results for all dilutions (10−100 times) with values of 76, 81, 84, 87, and 81 mg/L. The results demonstrate that fluorescence is an important method for measuring low concentrations. Student results were in line with ours, and this is discussed in Supporting Information.

Figure 2. Standard curves from fluorescence measurements of quinine with various LEDs and using a Fluorolog 3 from Horiba Jobin Yvon.

obtained with the Fluorolog 3 laboratory instrument. As can be seen the blue LED (428 nm) and the UV-LED (355 nm), both emitting at less than 450 nm, do not detect any fluorescence. The remaining LEDs and the Fluorolog 3 instrument exhibit qualitatively similar curves, at least for lower concentrations, as is evident in Figure 3 where data have been normalized by values at 25 mg/L quinine solution and all the curves are very similar.

4. Fluorescence and Concentration

Fluorescence measurements are linear at low concentrations, but not at high concentrations, as can be seen from Figure 2. The reason is, among others, the inner filter effect whereby light is absorbed at higher sample concentrations, so that little penetrates further into the cuvette where fluorescence is detected. This characteristic of fluorescence and absorption interaction can be observed directly when looking at the fluorescence in the solution in the cuvette (see Figure 6). Simply disconnect the multimeter from the 355 nm detector LED and instead apply a voltage as shown in the diagram in Figure 4b (without the detector LED), so that two light sources irradiate the solution. Figure 6b shows the most concentrated solution (1000 mg/L). Fluorescence is visible only at the edges of the cuvette. With less concentrated solution (Figure 6a,c, 750 mg/L), fluorescence can be observed penetrating further into the cuvette, but still not through to the center as in the very diluted solutions (Figure 6d, 12.5 mg/L). Having observed this directly, students understand better that fluorescence measurements should be made at low concentration, and that the dimensions of the cuvettes are important. With our instrument, we found a linear relationship for fluorescence in the concentration range from 0 to 10 mg/L.

Figure 3. Curves from Figure 2, for concentrations of quinine up to 25 mg/L, normalized by values at 25 ppm.

Fluorescence is linear to about 10 mg/L quinine concentration, increases up to around 100 mg/L, and thereafter decreases (not shown). The falloff is mainly due to the inner filter effect (described in more detail in section 4 below), which is dependent on the dimensions and other characteristics of the instrument used. Some LEDS are more sensitive than others. The most sensitive investigated here (red 826-442 and green L5-G81NGUV) both yield about 15 mV for a quinine concentration of 50 mg/L in the design shown in Figure 1. In the remaining experiments (including those with our students) we have used a green LED (L5-G81N-GUV) as a detector for fluorescence measurements.

5. Biological Application of Fluorescence

Figure 7 shows fluorescence measurements of the diluted urine samples after a bottle of tonic water was consumed. Urine production and constituent components depend upon a range of variables. Several of its naturally occurring chemicals fluoresce around 450 nm.36 The metabolism of quinine is complicated, but approximately 20% is excreted unchanged in the urine.37 Figure 7 illustrates an expected peak in fluorescence (∼6 mV), in this case some 12 h after the intake of tonic water, followed by a slow decline to the basal fluorescence of urine (2−3 mV).

2. Sensitivity of the Instrument and Fluorescence Dependence on Irradiation Intensity

The use of Lego bricks makes the design flexible. In Figure 4 several configurations for fluorescence measurement are presented. In Figure 4a,c, optional detector LEDs (355 nm) for absorption measurements are indicated with dashed lines. C



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Figure 4. Various configurations for fluorescence measurement. (a) One light source (UV-LED, 355 nm) and one detector (green LED, 525 nm, for fluorescence measurement). (b) Two light sources and one detector. (c) One light source and two detectors. (d) Two light sources and two detectors. Parts a and c also show optional detector LEDs (355 nm) for absorption measurements.

Figure 5. Sensitivity as a function of number of sources and/or detectors used.

Figure 6. Photos of fluorescence. (a) Cuvette with 750 mg/L quinine sulfate. (b−d) 1000, 750, and 12.5 mg/L quinine sulfate, respectively.

7. Fluorescence and Quenching

Figure 9 shows results from a classic quenching experiment of chloride (from NaCl) on quinine sulfate (9.9 mg/L). Fo and Fl are measures of fluorescence in absence and presence of quencher. This is used to portray a Stern−Volmer plot, where the quencher constant is here measured to 152 M−1 at 25 °C. This result may be compared with 155 M−1 from ref 39 (obtained by fitting to data given in the table on p 611 in the reference) and 72.7 M−1 from ref 23. However, neither of these references

6. Fluorescence and Temperature

Figure 8 illustrates fluorescence of quinine sulfate at 23 °C and ∼6 °C and shows that throughout the quinine concentration range fluorescence is 6−8% higher in the colder condition; i.e., the effect of temperature on fluorescence is ∼0.4%/°C. This temperature quenching of fluorescence is due to increased molecular motions at elevated temperatures, resulting in more molecular collisions with subsequent loss of energy.38 D



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Figure 7. Fluorescence after drinking tonic water.

Figure 10. Quenching of fluorescence by NaCl. Still from a video clip (see Supporting Information) showing the effect on fluorescence of adding solid NaCl (cuvette on rhs).

Figure 8. Fluorescence and temperature.

Figure 11. Fluorescence (a) and phosphorescence (b) of sample precooled in liquid nitrogen. Stills from video clip.

transition from a singlet excited state (fluorescence) is greater than that from a triplet excited state (phosphorescence). This is illustrated with the blue (shorter wavelength) fluorescence and green (longer wavelength) phosphorescence. Again, a theoretical concept becomes a physical experience, which hopefully eases understanding and learning.

Figure 9. Fluorescence and quenching at 25 °C.

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states the temperature at which the quencher constant was obtained. Figure 10 is a photo (a film clip is shown in Supporting Information) of a solution of quinine sulfate in 2 cuvettes, radiated by LEDs (355 nm) from two sides, as solid NaCl is added to the cuvette on the right-hand side (rhs). It is a simple and fascinating experiment; a theoretical concept becomes clearly visible and not simply presented graphically.

SUMMARY A simple, flexible fluorimeter and UV-photometer is made from two UV LEDs (355 nm), one (or two) VIS LED(s), a few wires, a single resistor, a battery, a few Lego bricks, and one or two digital millivoltmeters. This instrument is used to find concentrations of quinine sulfate in tonic water by measuring fluorescence and absorption; to visualize fluorescence as a function of concentration, light intensity, and temperature; and to demonstrate the quenching effect of NaCl, as well as to give a glimpse of phosphorescence.

8. Phosphorescence

Figure 11 shows fluorescence and phosphorescence of quinine solution after cooling a cuvette (which did not crack) in liquid nitrogen. Figure 11a shows fluorescence of the cooled sample when subjected to incident UV light. When the incident UV light is switched off (Figure 11b), the sample continues to phosphoresce. The phosphorescence lasts only a few seconds; nevertheless, it is clearly visible in a dark room. As can be seen, the phosphorescence is green (Figure 11b) while the fluorescence is blue (Figure 11a). A Jablonski diagram illustrates that the energy release accompanying the electron

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.7b00121. Instructor/students notes with supplementary experimental details (PDF, DOCX) Video of fluorescence quenching (AVI) E



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Article

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Lise Kvittingen: 0000-0002-0151-1852 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Mickaël Fontaine for technical help and to an unknown reviewer for useful comments. REFERENCES

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Demonstrating Basic Properties of Spectroscopy Using a Self

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