The ROXI Colorimeter & Fluorimeter. Laboratory Application I. Colorimetric measurements via Beer’s Law. Required Supplies & Costs: RGB LED; $1.95 Light Sensors; $3.95 ea 3-way switch; $6.54 300 ohm resistor; $0.10 Arduino Uno; $24.95 1. Sensor Description. The sensor consists of a common cathode RGB light emitting diode (Sparkfun Electronics, https://www.sparkfun.com/products/105 ), and two logarithmic scale analog light sensors (Adafruit Industries, https://www.adafruit.com/products/1384 ). Peak wavelengths for emission by the LED are specified as 467.5, 520, 625 nm by the manufacturer. The light sensors are powered by 2.3-6 VDC, and produce an output voltage (Vout) that is linear with the logarithm of the light intensity incident upon the sensor. The light sensors are glued onto the side of a cuvette holder that was 3D printed. Two light sensors (shown in blue in figure below) are present. The first at 180 deg. to the incident beam is for transmittance / absorbance measurements. The second detector is placed at 90 deg. angle for fluorescence or light scattering measurements. A circuit diagram for the apparatus is shown below. A 3-way switch (NKK Switch, HS13Y) distributes power to the RGB LED with current controlled via a 300-ohm resistor at the common cathode. Each light detector is powered via 5VDC, and ground is common. Two analog input channels of the Arduino are devoted to voltage measurements from the sensors. light detectors cuvette holder Vcc GND Ω Ω Ω Ω Signals Vcc GND V GND S = 5V R= 300 R= 3-way switch to distribute 5V to anode of desired color Figure 1. Schematic of the colorimeter / fluorimeter setup. D) A) B) C) Figure 2. Photograph of (A) the Adafruit light sensor chip; (B) view from above of the 3D printed cuvette holder, (C) the cuvette holder with LED and light detectors affixed with JB Weld, (D) the colorimeter installed within the ROXI case. The label was printed and laminated prior to gluing to the plexiglass. Three thru holes are designed into the cuvette holder to allow for placement of the LED and two light detectors. Peak wavelengths for emission by the LED are specified as 467.5, 520, 625 nm by the manufacturer. 2. Calibration of the Colorimeter. After assembly, we desired to devise a method to calibrate, or check the accuracy of the light sensor by using samples of known transmittance. To accomplish this, various plastic filters manufactured by Lee Filters were used. The manufacturer of these filters kindly distributes transmittance spectra for their products (shown below), allowing a simple means to achieve a calibration standard. Several optical filters could be placed into the optical path sequentially and the change in detector signal observed. The accepted transmittance values and measured voltages were then plotted and an exponential fit applied to the data. The resulting best-fit function was subsequently used to determine % transmittance for samples. This allows a transmittance standard to establish the relationship between voltage and transmittance. Data collected for an example trial experiment is shown on the next two pages. Information Regarding the Optical Filters LED color Measured Known (courtesy of Lee Filters) Voltage (V) Transmission Green 1.6023 1.5 % Red 2.2295 83% Blue 0.98 0% Green 0.5488 0% Red 1.5092 8% Blue 0.4949 0% Green 1.9845 12 % Red 0.3577 0% Blue 1.744 5% Green 2.5431 84 % Red 2.1021 47% Blue 2.2687 n.a. Green 2.4794 60 % Red 2.1021 12% Blue 2.6019 79% Green 1.9649 8% Red 0 0% Blue 2.4206 42% Green 2.3373 50% Red 0 0% Blue 2.5774 72% Green 1.9943 8% Red 2.0139 35% Blue 2.4059 38% Green 2.2589 30% Red 1.911 22% Blue 2.5235 59% Blank – 100% T Green -530 nm 2.5921 100% Blank – 100% T Blue -467 nm 2.6558 100% Blank – 100% T Red -630 nm 2.2785 100% Blue Channel 120 100 y = 0.0172e 3.2352x 80 R² = 0.9975 60 40 % Transmission % 20 0 0 1 2 3 sensor voltage (V) Green Channel 120 100 y = 0.0022e 4.1755x 80 R² = 0.9832 60 40 % Transmission % 20 0 0 1 2 3 sensor voltage (V) Red Channel 120 100 y = 0.0339e 3.4571x 80 R² = 0.9683 60 40 Transmission 20 0 0 1 2 3 sensor voltage (V) 3. Experiment: Demonstration of Beer’s Law. Introduction. The purpose of this experiment is to demonstrate the Beer-Lambert law relationship using the colored dye bromthymol blue and the ROXI colorimeter. Transmittance is defined as the fraction of incident electromagnetic power that is transmitted through a sample: ͽ Transmittance: T = ͽt where I, I 0 are the transmitted and incident radiant powers, respectively. All spectrometers measure transmittance, but chemists frequently convert to absorbance since this variable is linearly related to the concentration of the absorbing substance: Absorbance: A= -log (T) A= 2 - log (%T) The Beer-Lambert law of optical absorption describes the linear relationship between absorbance (A) and concentration (c). Beer’s law: A = ϵ × b × c In this equation, ε is a wavelength dependent number that describes the ability of the substance to absorb light, and b is the pathlength (cm) light travels through the sample. There is flexibility with what units of concentration can be used, however, if molarity is chosen ε is known as the molar absorptivity (L / mol cm). Procedure. 1. Dissolve approx. 150 mg of bromthymol blue (M.W. = 624.38 g/mol) in a 250.00 mL volumetric flask (water is solvent). Compute the exact molarity of the solution. 2. Obtain five 100 mL volumetric flasks. Transfer 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of the stock solution to volumetric flasks 1-5, respectively. 3. Fill the volumetric flasks to the mark using pH=10 ammonium chloride / ammonia buffer and mix well. Compute the exact concentrations of these solutions by using the dilution factors. 4. Turn on the ROXI colorimeter and set the color dial to red. Confirm the 100% T has been set. Recalibrate using transmittance standards if necessary. Zero the blank (buffer solution). 5. Use the ROXI colorimeter to measure the absorbance of the solutions using the red channel. 6. Use the data and concentrations to prepare a Beer’s law plot of the data. 7. Obtain a solution of unknown concentration and test the unknown solution and measure the absorbance using the colorimeter. 8. If desired, the absorbance values measured with the ROXI colorimeter can be compared to measurements made with a laboratory spectrophotometer for quality control. Sample Data. Mass of Bromthymol Blue used (g) 0.161 Molar mass of Bromthymol blue (g/mol) 624.38 Moles of Bromthymol blue (mol) 0.0002578 Molarity of solution (mol/L) 0.001031 standard concentration Absorbance Absorbance measured via laboratory solutions (mol/L) measured via ROXI spectrophotometer (reference method) A 0.00001031 0.48 0.48 B 0.00002062 0.83 0.67 C 0.00003093 1.41 0.96 D 0.00004124 2 1.28 E 0.00005155 2.47 1.60 Roxi Colorimeter Spectrophotometer @ 630 nm 3 y = 49989x - 0.11 2 y = 27430x + 0.148 2.5 R² = 0.9934 R² = 0.9902 2 1.5 1.5 1 Abs./ A Abs./ 1 Abs./ A Abs./ 0.5 0.5 0 0 0 0.00002 0.00004 0.00006 0 0.00002 0.00004 0.00006 Conc. / M Conc. / M laboratory Unknown analysis ROXI spectrophotometer (reference method) Absorbance for unknown solution 1.1141 0.8110 Unknown concentration from 0.00002445 0.00002417 calibration curve (mol/L) 4. Conclusion & Discussion. From the results of the analysis and the graph, it can be seen that the concentration was linearly proportional to absorbance as expected. The unknown solution tested had a true concentration of 2.47 x 10 -5 M, very close to the measured values of 2.445 x 10-5 M and 2.417 x 10 -5 M obtained by the ROXI colorimeter and the laboratory spectrometer. The concentration of an unknown sample could be determined with good accuracy (here 1%) using the ROXI colorimeter. However, the ROXI colorimeter data yielded a slope of the Beer’s law plot considerably different from the laboratory spectrophotometer. The cause of the discrepancy is likely that the laboratory spectrophotometer was operated at 630 nm, a slightly larger wavelength than the LED. The slightly larger wavelength is on the rapidly falling edge of the absorption spectrum for the dye used. This will lead to a smaller slope of the Beer’s law plot. Laboratory Application II. Fluorescence measurements. Introduction. For a demonstration of the ROXI fluorescence measurement we have prepared several different standard solutions of sodium fluorescein dye, placed them within a cuvette and illuminated the samples using the blue LED channel at λ=467 nm. The #735 Velvet Green (Lee Filters) plastic filter matches the emission spectrum of fluorescein well, and this filter was placed in front of the fluorescence detector to reduce excitation light. It was found that the observed signal voltage plotted vs. the logarithm of the concentration of fluorescein produced a linear response that could be used for simple analysis. Emission Filter Spectrum Sodium Fluorescein excitation and emission spectra Wavelength (nm) Procedure. 1. Preparation of standard solution. Place approx. 0.025g sodium fluorescein in a 0.25L volumetric flask. Dilute with D.I. water (approx. conc.= 100 µg / ml). 2. Preparation of standards for analysis. Transfer 0.01mL, 0.02 mL, 0.1 mL, 0.2 mL, 0.35 mL, 0.5 mL, 1mL from the stock solution to a 10 mL volumetric flask labeled A, B, C, D, E, F, G, respectively.