A Fluorometric Analysis of Quinine in Tonic Water CHEM 329 Professor Vogt TA: Sam Rosolina Allison Poget Date Performed: March 29, 2016 Date Submitted: April 5, 2016 ABSTRACT In this experimental, various solutions containing a stock quinine solution will be prepared and examined using fluorescence spectroscopy. The resulting fluorescence intensities will be plotted against their respective concentrations to develop a calibration curve. The resulting line equation from this calibration curve will be used to analyze the fluorescence intensity of an unknown concentration of commercial quinine. This type of instrumentation has many applications ranging from organic chemical analysis, to forensics, to medical uses. Additionally, quinine is a common and useful chemical encountered. Understanding more about both quinine and fluorescence spectroscopy is vital to daily life and the future of technology.
Introduction: Fluorescence spectroscopy is a very sensitive instrumentation used to analyze samples that fluoresce in Analytical Chemistry. This instrumentation requires that the analytical sample is able to fluoresce. The process of fluorescence is necessary for this type of spectroscopy to exist. When a molecule within a sample is excited by some type of radiation, it will move from an unexcited ground state to a higher excited energy state. Within the realm of the excited energy state, there are many different vibrational levels. When the initial ground state molecule is excited by absorbing a photon of energy from the applied radiation, it will excite to the excited energy state. From there, the excited molecules will collide with one another and lose vibrational energy until it reaches the lowest vibrational energy within the excited energy state. When the molecule drops from the lowest vibrational state within the excited energy to one of the numerous vibrational states within the ground energy state, the molecule will emit a photon of energy, and this process is called fluorescence. In this experimental, quinine will be examined with fluorescence spectroscopy because quinine is a good fluorescer and possesses excitation wavelengths at 250 and 350 nm [1]. Though the concentrations are kept within a legal limit by the Food and Drug Administration (FDA), quinine has many used ranging from malaria cure agent, to the popular gin and tonic drink. In this particular instrumentation, a beam of light will pass through a filter or a monochromator and come into contact with the analytical sample. The beam of light will then excite the sample molecules from a ground state to an excited state, and the sample will fluoresce. Since the fluorescence will occur in all directions, there is another monochromator, which will angle the fluoresced particles to reach the detector and generate a signal to be analyzed. A diagram of instrumentation is seen below. [4] Because some of the molecular energy is lost within the excited state from vibrational collisions with other molecules, the energy of excitation will always exceed that of the relaxation. An additional consideration of a competing luminescent process of phosphorescence must also be recognized. Phosphorescence occurs when a molecule is excited to an excited energy state from a ground state and then experiences intersystem crossing into a triplet state before relaxing back down to the ground state and emitting the photon of energy. All of the aforementioned topics can are visually displayed in the Jablonski diagram below.
[1] Figure 2: Jablonski diagram Within the instrument, many different light or radiation sources may be used including lasers and LEDs [2]. The radiation source needs to be selected with consideration to how it will interact with the sample present and one must be selected that will generate the most useful spectra for analysis [2]. The monochromator used is most commonly a grating which will select for the wavelength of light desired for instrumentation [3]. An additional consideration is that of polarizers. Polarizer filters are necessary within the instrument following the wavelength selection monochromator and prior to the emission monochromator [3]. Oftentimes in chemical analysis, the results from fluorescence spectrometry can be used to determine concentrations of analyte solutions by mathematically comparing the major spectra peak value to the known concentrations. The known concentrations and their respective peak values are plotted against one another and used to develop a calibration curve. The linear trendline of this value can be used to analyze peak values of analytes of unknown concentration. This is the method that will be used in this experimental. If the concentrations get to be too high, the some of the fluoroesecnce will self- absorb and the relationship will no longer be linear, so this needs to be considered in analyte preparation and avoided by proper dilution [1]. Experimental error could occur if the concentration of Cl-, a quenching agent for quinine is not kept low enough. A quenching agents is one that can absorb radiation during this type of spectroscopy experimental and decrease the resulting fluorescence intensity. [4]
The applications of this technology and instrumentation include analysis of organic compound in research sciences, biomedical labs, and other medical applications. One common medical application is analysis of cancer biopsies for confirmation of benign or diagnosis of malignant tumors [4]. This type of spectroscopy can additionally be used in forensic sciences to confirm DNA analysis matching in crime scenes or in identifying the DNA of a Jane or John Doe [4]. Additionally, the success of organic synthesis experimentals can be inspected using fluorescence spectroscopy [4]. Experimental: Using a 0.10 ppm stock solution previously prepared by the TA, various dilutions were made. The TA prepared the quinine stock solution by weighing 120.7 mg of quinine sulfate dehydrate and 50 ml of 1M H2SO4 to a 1L volumetric flask and diluted to the line. The TA prepared this solution just prior to experimentation in order to protect the solution from exposure to light. Five dilutions were prepared in 25 ml volumetric flasks at varying concentrations of 0.010 ppm, 0.015 ppm, 0.020 ppm, 0.025 ppm, and 0.030 ppm. The fluorescence intensity of each of the respective concentrations of stock solution prepared was recorded. A graph of the log of the relative fluorescence intensity versus the log of the concentration in ppm was generated from the aforementioned data collected. A linear trendline was fit to the plot of the data and the line and R 2 value were reported. Additionally, a linear least squares analysis was performed on the plot of data and the resulting line equation. Next, 5 ml of tonic water was added to a 250 ml volumetric flask and diluted to the line with 0.05 M H2SO4 and mixed. Then 1 ml of this solution was added to a 100 ml volumetric flask and once again diluted to the line with 0.05 M H2SO4. The fluorescence intensity of this solution was measured and plugged into the line of best fit equation. The TA instructed on the performance of the instrument along with mentioning that either of the two excitation wavelengths of 250 or 350 nm could be used in this experimental. One factor that must be kept in mind is that the maximum fluorescence for quinine is 450 nm, as seen in the resulting spectra from fluorescence spectroscopy.
Data: Figure 4: This is the resulting spectrum from the analysis of the prepared quinine stock solutions and the unknown quinine solution. The maximum wavelength of quinine used in this experimental was ~450 nm. Conc (ppm) F. Intensity log(conc) log(rel. Fluoro. Intensity) 0.010 252.40-2 2.402089351 0.015 382.89-1.823908741 2.583074024 0.020 513.01-1.698970004 2.710125831 0.025 618.88-1.602059991 2.791606448 0.030 730.87-1.522878745 2.863840136 unknown 85.50 1.931966115 Table 1: Data values of respective solution concentrations and resulting fluorescent intensity log( Rel. Fluoro. Intensity) 2.9 2.8 2.7 2.6 2.5 2.4 log(rel. Fluoro. Intensity) vs. log(concentration) y = 0.9683x + 4.3449 R² = 0.99843 2.3-2.5-2 - 1.5-1 - 0.5 0 log(concentration)
Figure 5: Resulting calibration curve from the above data including a line of best fit with equation and R 2 value included for further analysis slope 0.968311704 std dev of slope 0.022175171 R^2 0.998429123 Fisher F Statistic 1906.761691 Regression ss 0.133094937 intercept, b 4.344903734 std dev of intercept 0.038534932 std dev of y 0.008354733 degrees of freedom, n- 2 3 error ss; unexplained variation 0.000209405 Table 2: Linear Least Squares Analysis of the data in Figure 5 Calculations: Calibration preparation calculations: Stock quinine solution concentration: 0.100 ppm Volume of dilution chamber: 25 ml Desired concentration: 0.010 ppm C1V1 = C2V2 1. (0.010 ppm)*(25 ml) = (0.100 ppm)*(x ml of original solution x = 2.5 ml of 0.100 ppm quinine solution to prepare 0.010 ppm quinine calibration 2. (0.015 ppm)*(25 ml) = (0.100 ppm)*(x ml of original solution x = 3.75 ml of 0.100 ppm quinine solution to prepare 0.015 ppm quinine calibration 3. (0.020 ppm)*(25 ml) = (0.100 ppm)*(x ml of original solution x = 5.0 ml of 0.100 ppm quinine solution to prepare 0.020 ppm quinine calibration 4. (0.025 ppm)*(25 ml) = (0.100 ppm)*(x ml of original solution x = 6.25 ml of 0.100 ppm quinine solution to prepare 0.025 ppm quinine calibration 5. (0.030 ppm)*(25 ml) = (0.100 ppm)*(x ml of original solution x = 7.5 ml of 0.100 ppm quinine solution to prepare 0.030 ppm quinine calibration Determination of the concentration of the unknown commercial quinine solution: y = 0.9683x + 4.3449 log of unknown fluorescent intensity: 1.932 85.50 = 0.9683x + 4.3449 log of unknown concentration (x) = - 2.492
10 log(concentration) = 10 (x) = 10-2.492 = 0.00322 ppm of diluted commercial quinine solution (tonic water) Reverse Dilution #2: (0.00322 ppm)*(100 ml) = (1 ml)*(c2) C2 = 0.322 ppm Reverse Dilution #1: (0.322 ppm)*(250 ml) = (5 ml)*(c1) C1 = 16.11 ppm original concentration of quinine in tonic water solution Results and Discussion: When examining the resulting fluorescence intensity from the respective known concentrations from the prepared solutions, a trend was observed that as the concentration of the solution increases, so does the intensity of the fluorescence peak. In Figure 4, this trend is once again observed. The only line that does not have a peak at ~450 nm is that of the blank because the blank will not have a peak at this wavelength but will still exhibit some important elastic scattering that must be considered as well. When the log of the relative fluorescence intensity is plotted against the log of the concentration of the known calibration quinine solutions, a linear trend is observed. When fit for a line of best fit, a resulting line equation of y = 0.9683x + 4.3449 was determined. Using this line, the known concentration of quinine in the tonic water solution was determined by plugging the value obtained for the log of the unknown solution s resulting intensity from fluorescence spectroscopy into the line of best fit equation and solving for the log of the unknown solution s concentration. The accuracy and precision of measurements can be seen by analyzing the R 2 value and the resulting values from the linear least squares analysis performed on the data group. The closer the R 2 value is to unity, the better representative the resulting line equation of the data which it aims to represent. In this case the R 2 value is 0.998 which is extremely close to unity indicating that the line of best fit is very linear and representative of the trend which it summarizes. This aids in adding confidence and quality to future calculations performed using this equation. Additionally the errors in the slope and the intercepts for the data are extremely small, 0.02 and 0.03 respectively. This, once again, indicates that there is much accuracy and precision in this experimental. After this, the resulting value could be used as the exponent in the equation 10 x in order to remove the log function. This will generate the concentration of the doubly diluted tonic water solution in ppm. From there, the two reverse dilutions must be calculated using C1V1 = C2V2 in order to find the concentration in ppm of the quinine in the tonic solution used in experimentation. The value calculated from the other data and values in this lab was determined to be 16.11 ppm. Some error in this experimental could arise from many places. If the preparation of the quinine stock solution by the TA is incorrect, than every resulting solution prepared would contain increasing amounts of error. If any calculations for the preparation of the s of quinine for the calibration curve development are incorrect, than an incorrect will be prepared and will negatively impact the validity of the calibration curve. Errors of this type could result from improper usage of pipets, improper dilutions, and improper mixing of solutions before and after dilution. These errors could mean that the solution prepared is not at the concentration that it is believed to be.
Conclusion: In this experimental fluorescence spectroscopy techniques and instrumentation were introduced as the concentration of quinine in tonic water was determined to be 16.11 ppm. Using a pre- prepared stock solution of 0.100 ppm quinine, 5 s of 0.010, 0.015, 0.020, 0.025, and 0.030 ppm were prepare in 25 ml volumetric flasks. These s were examined under fluorescence spectroscopy and the respective intensities were recorded. The log of both the intensities and the concentrations in ppm were plotted against each other to determine a calibration curve line equation that could be used to determine the concentration of quinine in the tonic water solution used in the experimental. The amount of error in this experimental is low as seen in near unity of the R 2 value and the low errors in the slope and the intercept values of the resulting calibration curve. This type of instrumentation is highly useful because of its ease in sample preparation, ease in analysis performance, and ease in calibration curve development. Since it is relatively easy to generate a calibration curve, the line of this resulting curve will be used to determine the unknown concentration in analysis. This same technique could be used in biomedical research, scientific experimentation, analysis of concentrations of drugs in pharmaceuticals, forensic sciences, and even in development of any commercial product containing chemicals. References: [1] Rosolina, Sam. A Fluorometric Analysis of Quinine in Tonic Water. Knoxville, TN, 2016. Print. [2] Sharma, Ashutosh, and Stephen G. Schulman. Introduction to Fluorescence Spectroscopy. New York: Wiley, 1999. Print. [3] An Introduction to Fluorescence Spectroscopy. Buckinghamshire, UK: PerkinElmer, 2000. Print. [4] Bright, Frank V. Bioanalytical Applications of Fluorescence Spectroscopy. Buffalo, NT: American Chemical Society, 1988. Print.