QUALITATIVE AND QUANTITATIVE ANALYSIS BY INFRARED SPECTROSCOPY AND MOLECULAR FLUORESCENCE (VERSION 1.0)

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1 REVISED Infrared Spectroscopy/Molecular Fluorescence, Page 1 QUALITATIVE AND QUANTITATIVE ANALYSIS BY INFRARED SPECTROSCOPY AND MOLECULAR FLUORESCENCE I. BACKGROUND. (VERSION 1.0) In this experiment, a solid unknown will be analyzed by use of infrared spectroscopy and molecular fluorescence. The unknown species will be identified from a list of possibilities by collecting the characteristic infrared fingerprint of the compound. A quantitative analysis will then be performed by use of fluorescence spectroscopy. Infrared Spectroscopy Infrared spectroscopy is based on the absorption of infrared energy by molecular species and the corresponding excitation of vibrational modes of the molecule. The mechanism of the absorption transition is based on the interaction of a bond dipole in the molecule with the electric field of the incident photon. The presence of multiple bond dipoles within a typical organic molecule, the possibility for several vibrational modes being associated with each dipole, and the coupling of vibrational and rotational transitions gives rise to a rich pattern of absorptions and a corresponding characteristic infrared fingerprint for a compound. Because a bond dipole requires a difference in electronegativity between the bonded atoms, bond dipoles are naturally associated with organic functional groups. The infrared spectral pattern is thus rich in information regarding the functional groups present in a molecule. The spectra of solid samples can be acquired in several ways. The solid material can be formed into a transparent disk by mixing it with an infrared-inactive material such as KBr and applying pressure. The resulting disk is analyzed with a conventional transmission measurement. The solid can be dissolved in a solvent and also analyzed with a transmission measurement. The drawback to this approach is the strong infrared absorbance of most common solvents. The solvent absorbance may effectively blank out entire regions of the spectrum. Those solvents with relatively good infrared transparency unfortunately also tend to be hazardous compounds (e.g., CS 2, CHCl 3, CCl 4, etc.). Handling and disposal issues thus arise when these solvents are used. Another possibility is to make a mull with a mineral oil (Nujol) and place the resulting material between two salt plates. A transmission measurement is then performed. The mineral oil obliterates the C-H stretching region between cm -1, but the fingerprint region is preserved. A third sampling approach, termed attenuated total reflectance (ATR), is depicted in Figure 1. In the figure, the infrared beam is propagated through a diamond waveguide by use of the principle of total internal reflection. Light launched into the waveguide reaches the interface between the waveguide material and the analyte in contact with it. The refractive indices are such that the light is reflected at the interface and returns to the waveguide. The light thus propagates through the waveguide as a series of reflective bounces back and forth at the interface. This is illustrated in Figure 1. Light exiting the waveguide is detected. When undergoing reflection, the electric field associated with the propagating photon penetrates a short distance into the analyte. This is termed the evanescence field of the photon. This penetration of the evanescence field allows interaction between the photon and analyte and can result in absorption if the vibrational and photon frequencies match. Thus, the spectral signature of the analyte is superimposed on the detected light. When a solid material is analyzed by ATR, an important requirement is good contact

2 REVISED Infrared Spectroscopy/Molecular Fluorescence, Page 2 Figure 1. Schematic of attenuated total reflectance with a diamond waveguide. between the sample and the waveguide. The short penetration depth (µm) of the evanescence field into the sample dictates that the sample must be pressed against the waveguide. The ATR accessory for use with solids often has a mechanism for applying pressure to the sample, thereby assuring good contact with the waveguide. Fluorescence Spectroscopy Fluorescence spectroscopy is a sensitive optical emission technique in which sample molecules are excited with a photon source. Those molecules that relax by radiant emission can be subsequently detected by measuring the intensity of that emission. The mechanism of this excitation and emission is illustrated in Figure 2. In the diagram, electronic excitation can occur from the ground state (S 0 ) to either of two excited states (S 1 and S 2 ). Each electronic state has as associated set of vibrational and rotational states. Following excitation, a variety of relaxation processes can occur to return the molecule to the preferred lower energy state. These processes can either be radiative (i.e., light-emitting) or non-radiative (i.e., non-lightemitting). Fluorescence is defined as radiative emission arising from a transition between a singlet excited electronic state and the ground state. The non-radiative processes are more efficient and are typically favored. Often, these processes are driven by collisions between the analyte molecule and the surrounding molecules of the solvent. For this reason, most molecules do not emit light upon relaxation from the excited electronic state. Instead, the energy is returned to the environment, typically causing the sample to increase in temperature. However, certain compounds under certain conditions (e.g., temperature, ph, etc.) do relax radiatively with enough probability that the photon emission can serve as the basis for an analytical measurement. In these cases, the fluorescence measurement is very sensitive because it is a zero-background method (i.e., one is observing the emission against a zero background as opposed to measuring a ratio of light intensities such as that required in a transmission experiment). Fluorescence can also be more selective than an absorption measurement because other potentially interfering constituents of the sample may not relax radiatively.

3 REVISED Infrared Spectroscopy/Molecular Fluorescence, Page 3 Figure 2. Schematic for radiative and non-radiative relaxation processes following absorption of a photon. Transmission experiments produce measurements of absorbance, which is linearly related to concentration through the Beer-Lambert law: A = -log 10 (P / P 0 ) = εbc (1) In Eq. 1, A is absorbance, P is the light power transmitted through the sample, P 0 is the light power incident on the sample, ε is the molar absorptivity, b is the optical path length, and C is analyte concentration. In the fluorescence experiment, F, the light power emitted by the sample is proportional to the light power absorbed: F = kφ F (P 0 P) (2) In Eq. 2, Φ F is termed the fluorescence quantum yield, defined as the fraction of excited molecules that undergo fluorescence. The constant, k, is the optical collection efficiency, defined as the fraction of emitted photons that are detected by the spectrometer. Solving Eq. 1 for P yields: P = P εbc (3) Combining Eqs. 2 and 3 yields:

4 REVISED Infrared Spectroscopy/Molecular Fluorescence, Page 4 F = kφ F P 0 (1-10 -εbc ) (4) The expression in Eq. 4 defines the relationship between measured fluorescence intensity and analyte concentration. Clearly, this relationship is nonlinear. At dilute concentrations, however, the functional relationship between F and C becomes approximately linear. One component of the experimental design in fluorescence is to define a working range of concentration that yields a response that is linear enough to allow use of standard linear calibration methods based on least-squares analysis. Another issue in implementing a successful analysis based on fluorescence measurements is the control of experimental parameters such as ph. Changing the ph can change the probability of radiative emission through the creation of different resonance structures of the molecule. Consequently, optimization of ph is an important part of the experimental design. II. REAGENTS. The following solutions will be provided: ph 7 buffer (0.1 M NaH 2 PO 4 + NaOH to ph 7) ph 10 buffer (0.1 M H 3 BO 3 + NaOH to ph 10) ph 2 buffer (0.05 M H 2 SO 4 + NH 4 OH to ph 2) III. PROCEDURE. First Laboratory Period The goal of the work in the first laboratory period is to identify the unknown and to establish optimal conditions for the fluorescence analysis of each compound. Groups 1 and 2 should begin with infrared spectroscopy, while Groups 3 and 4 should begin with fluorescence spectroscopy. Infrared Spectroscopy Your group will be given an unknown and will be assigned one standard for which you will acquire infrared spectra. The instructor will demonstrate the operation of the infrared spectrometer and provide instruction in the sample preparation methods available. Collect spectra of the unknown and the standard. Provide copies of the spectra of the standard to the other groups. Fluorescence Spectroscopy A. Prepare 10 ppm solutions of your assigned standard in 100 ml volumetric flasks. Make three separate solutions by diluting with the ph 2, ph 7, and ph 10 buffers. For each solution, determine the peak excitation wavelength and record an emission spectrum based on the selected excitation wavelength. Save the recorded spectra in text files. In each case, use the corresponding buffer as the spectroscopic blank (i.e., the measurement of the solution diluted with ph 2 buffer uses ph 2 buffer as the blank). It is possible that your standard will fluoresce strongly enough for the emission intensity to be off-scale at 10 ppm. If this is the case, proceed to part B. B. With each solution, perform serial dilutions by a factor of 4 by taking a 25 ml aliquot of the

5 REVISED Infrared Spectroscopy/Molecular Fluorescence, Page 5 solution with a volumetric pipette and diluting to 100 ml in a volumetric flask. Using the same excitation wavelength determined previously, record emission spectra of each solution. Continue this 1:4 serial dilution until the emission spectra are no longer distinguishable from noise. Second Laboratory Period -- End of First Laboratory Period -- Table 1. Optimal Fluorescence Conditions Compound Optimal ph Excitation λ (nm) Emission λ (nm) Working Range (ppm) Quinine sulfate Salicylic acid Salicylamide A. On the basis of the collected infrared spectra of the standards, identify your unknown. B. Using a pure sample of your unknown compound, prepare a 10 ppm solution in a 1 L volumetric flask. Dilute with water. If the compound will not dissolve, the ph may need to be adjusted. Using 100 ml volumetric flasks, perform serial dilutions of this solution as needed in order to produce four standards that span the working range of your unknown as defined in Table 1 above. Perform all dilutions with water until the final solution. Dilute that solution with the appropriate ph buffer as defined in Table 1. C. Set the excitation monochromator to the optimal excitation wavelength for your compound. Establish a 20 nm scanning window centered on the optimal emission wavelength. Scan the emission spectral window. Make two replicate measurements of each standard without refilling the sample cell. Save the recorded spectra in text files so that they can be exported into Excel. Note the maximum and minimum fluorescence intensities across the four standards. D. Prepare a solution of your unknown that produces a fluorescence signal that falls in the middle of the range spanned by your standards. Begin by using the same procedure used to produce the high concentration standard. Start with 0.01 g of the unknown diluted to 1 L in a volumetric flask. As before, perform dilutions with water until the final solution that is to be measured. Dilute that solution with the appropriate ph buffer. Measure the fluorescence and adjust the dilution scheme as needed. When the optimized solution is found, make two replicate measurements. Save the recorded spectra in text files IV. CALCULATIONS. -- End of Second Laboratory Period -- A. Draw the chemical structure of your unknown. Use the data in the provided table of infrared group frequencies to assign at least three major bands in the infrared spectrum of your unknown to a specific vibrational mode (e.g., C-O stretch, etc.).

6 REVISED Infrared Spectroscopy/Molecular Fluorescence, Page 6 B. Compute the exact concentration for each of your calibration standards. C. For the calibration data, your measured emission spectra consist of recorded emission intensities across the 20 nm scanning window. Import these traces into Excel and compute the maximum intensity within the scanning window (use the max function in Excel). This value will be used as fluorescence intensity in subsequent calculations and plots. Note that fluorescence is measured in arbitrary units. D. For the measurement of the calibration standards, use your replicate measurements to tabulate the mean, standard deviation, and 95% confidence interval for the fluorescence intensities. E. Construct a plot (with error bars) of fluorescence intensity vs. analyte concentration (mg/l) for the calibration data. Use least-squares procedures to determine the calibration model. Report the slope, intercept, the value of r 2 and the standard error of estimate for your calibration model. F. Using your calibration model, compute the concentration of your unknown as measured and work backward through your dilution procedure to calculate the weight percent of the analyte in the original solid sample. Predict each replicate measurement separately and then compute an average, standard deviation, and 95% confidence interval.