# Lab 4: Molecular Fluorescence

When a molecule absorbs a photon in the ultraviolet or visible (UV VIS) region (180 - 780 nm), an electronic transition occurs within the molecule. This transition involves moving an electron from the singlet ground state to a singlet excited state.

### Theory

An excited molecule will undergo de-excitation to regain its ground state electronic configuration. De-excitation of the molecule can occur in three distinct ways: by collisional deactivation (external conversion), fluorescence, or phosphorescence (Figure 4.1). The first relaxation mechanism, collisional deactivation, occurs when the excited molecule transfers its excess energy to molecules with which it collides without photon emission. The second mechanism, fluorescence, involves the electron returning from the excited singlet state to the ground state accompanied by the emission of a photon of lower energy (longer wavelength) than the absorbed photon; the energy loss is due to vibrational relaxation while in the excited state. When fluorescence is favored, it occurs within about 10-8 seconds after absorption (picoseconds are 10-9 seconds).

Figure 4.0: UCD Fluorometer

Phosphorescence, the third route for de-excitation, occurs when the excited electron enters the lowest triplet state from the excited singlet state by intersystem crossing, and subsequently emits a photon in returning from the lowest triplet state to the singlet ground state. These singlet-triplet and triplet-singlet transitions involve electron spin reversal, which is a low probability occurrence. Thus, the time between absorption and phosphorescence can be from 10-2 seconds to several minutes.

Figure 4.1: Pathways for production and de-excitation of an excited state.

Since our interest involves fluorescence, a typical experimental arrangement is shown in Figure 4.2. This is used in either of two distinct ways. The first, which holds the excitation monochrometer M1 fixed, and varies the emission monochrometer M2, yields an emission spectrum (the wavelength distribution of light emitted by the excited singlet state). Alternately, M2 can be fixed and M1 varied; this procedure produces an excitation spectrum (a plot of fluorescence intensity as a function of excitation wavelength). Often, the excitation spectrum of a pure compound has exactly the same profile as the absorption spectrum.

Figure 4.2: Schematic Diagram of Fluorometer. M1 = excitation monochrometer, M2 emission monochrometer, Photo Multiplier Tube detector.

Coupling the above techniques with a relationship between fluorescence intensity and concentration would be exceptionally useful. Such a relationship can be derived from Beer's Law, which states that the fraction of light intensity transmitted by a sample is

$\dfrac{I}{I_o} = 10^{-\epsilon b c} \label{1}$

where

• $$I$$ is the transmitted light intensity,
• $$I_o$$ is the incident intensity,
• $$ε$$ is the molar absorptivity at a given wavelength in units of L*mol-1*cm-1,
• $$b$$ is the cell path in centimeters, and
• $$c$$ is the concentration in moles per liter.

The fraction of absorbed light is: $1 - \dfrac{I}{I_o} = 1 - 10^{-\epsilon b c} \label{2}$

from which it follows that the absolute amount of absorbed light is equal to: $I_o - I = I_o - I_o \left(10^{-\epsilon b c} \right) \label{3}$

The fluorescence intensity, F, is proportional to the amount of light absorbed and fluorescence quantum yield, Φ. Thus, $F = kI_o \phi \left[1-\left(10^{-\epsilon bc} \right) \right] \label{4}$

where $$k$$ is a proportionality constant. If dilute solutions are used, so that less than 2% of the excitation energy is absorbed, the exponential term in Equation $$\ref{4}$$ can be approximated by the first two terms in the corresponding Taylor series expansion

$e^x \approx 1 + x$

Then,  $F = kI_o \phi \epsilon bc \label{5}$

hence fluorescence intensity is proportional to concentration:

$F \propto c$

### Experimental

This experiment involves the analysis of vitamin B2, riboflavin, by measuring its native fluorescence. Riboflavin (Figure 4.3) is a common water-soluble vitamin found in eggs, milk, and other foods, that strongly fluoresces and is very sensitive to light. Its two predominant irradiation decomposition products are lumichrome and lumiflavin, which are themselves highly fluorescent.

Figure 4.3: Riboflavin (A) and the two most predominant irradiation decomposition products, lumiflavin (B) and lumichrome (C).

The analysis method requires working with riboflavin samples at concentrations well below the part per million (ppm) level. Therefore, it is extremely important that the utmost care be exercised in cleaning and handling all of the glassware and solutions during this experiment.

In the first lab period the standard and unknown solutions mentioned below should be prepared. Later during this period you should consult the T.A. about operating the UV VIS spectrophotometer as well as the fluorescence spectrometer. You should also obtain the absorption spectra of riboflavin and perform a practice run on the fluorescence spectrometer.

The second lab period should be used to complete the experiment and Part II of the unknown analysis.

#### Stock Solutions

First obtain the 100 ppm stock standard solution from the T.A., then allow the solution to equilibrate to room temperature for consistent volume measurements. Deliver 1 mL of the stock solution to a 10 mL volumetric flask and dilute using deionized water. This is your 10 ppm solution. Using a volumetric pipet, deliver 1.00 mL of the 10 ppm solution to a 100 mL volumetric flask, and dilute using deionized water. This is your 100 part per billion (ppb) stock solution. Fill in the table below, with the correct amounts of 100 ppb riboflavin to make the remainder of your standards. Check the table with your TA before you begin the experiment. Make certain that your glassware is clean and rinsed with deionized water.

Table 4.1: Standard Riboflavin Solutions
Solution # mL of 100 ppb sol. Dilute to (mL) Rib. Conc (ppb)
2   50 2
3   50 10
4   50 20
5   50 40
6   50 50

#### Unknown Solution

The dry milk sample should be reconstituted as indicated on the jar. Record your work (this is a quantitative experiment). The milk samples require special treatment in order to remove interfering fats and proteins, which would otherwise make analysis difficult or impossible. The procedure to accomplish this is outlined below.

To a 25 mL milk sample add exactly 75 mL of a solution consisting of equal parts 3M HAc and 3M NaCl. Do not use a volumetric flask for this. It is too difficult to stir the solution. Use your 25 mL pipet and a 250 or 400 mL beaker. Stir for 20 min and filter using a Buchner funnel and the vacuum line. Make sure you have a trap between the filtering flask and the house vacuum line. Take a 5 mL aliquot of the filtrate and dilute to 100 mL with deionized water in a volumetric flask. This solution will be used for your analytical measurements.

### Instrumental

#### Part 1: Approximate Excitation Wavelength

The instrument you'll be using is the Varian Eclipse Fluorescence Spectrometer which is computer-controlled through software running on Windows XP. Detailed operating instructions for the Eclipse follow.

The first step in conducting the analysis is to determine the optimum excitation wavelength for riboflavin. When no prior information is available, as in this case, the absorption spectrum can be used to approximate the best excitation wavelength.

Use the 10 ppm solution to obtain an absorption spectrum of riboflavin from 280 to 600 nm using an Agilent HP8453 UV-Vis spectrometer. Use distilled water as your reference solution. If you do not recall how to acquire an absorbance spectrum, check with your TA for instructions. The wavelength of maximum absorbance should be used as an initial excitation wavelength. There may be more than one absorbance peak.

#### Part 2: Actual Excitation and Emission Maxima

In this part of the experiment you will determine optimum instrumental parameters. Using the approximate excitation wavelengths determined above from the UV-Vis absorbance spectrum, obtain two emission spectra. The emission maximum observed in the two emission spectra will then be used to collect an excitation spectrum to "refine" the choice of excitation wavelength. You will see that the fluorescence excitation spectrum closely follows the absorbance spectrum.

You should collect the following spectra: (1) two emission spectra with the two approximate excitation wavelengths determined in part 1 of the 50 ppb standard solution, and (2) one excitation spectra of the 50 ppb standard and the milk sample you prepared.

Using the Varian Eclipse Fluorometer

1. Power up the Eclipse with the switch on the front panel, if necessary. The dim light on the front will become green when ready.
2. If the computer is not logged in, log into "Fluorimeter" by clicking on its icon.
3. Double click on the Scan icon on the desktop THEN Click on the Setup button.
4. Set the excitation wavelength to either of the absorption maxima in the UV-Vis spectrum
5. Set the scan limits. The starting wavelength for the emission spectrum should be 30nm above the excitation wavelength to avoid Rayleigh scattering. The upper wavelength should be 700nm.
6. The scan speed should be set to Slow (120 nm/minute) and both slits should be set to 10nm.
7. Click on the Reports tab. Set the Threshold value to 5. Click the OK button.
8. Click on the green traffic light icon to start the scan.
9. A window will appear where you can name your sample. Put in your desired name and click OK.
10. When the scan is complete, print the spectrum by clicking on the Print button.
11. Obtain excitation spectrum by repeating the above steps, start by clicking the Setup button. Click on the box that says excitation.
12. Set the emission wavelength to the value you determined above.
13. Record the excitation spectrum from 280nm to about 30nm below the emission wavelength.
14. Collect excitation spectra under the identical conditions for the milk sample you prepared.

#### Standard Curve

At two excitation wavelengths (both ~370 and ~440 nm) you determine the exact wavelengths to use.

1. Double click the Simple Reads icon on the desktop.
2. Click on the Setup button, and set the emission wavelength and excitation wavelength in the window.
3. Set the excitation and emission slits at 10 nm, and the Average Time at 5 seconds.
4. Click on Read to begin data acquisition.
5. Change the excitation wavelength and repeat the procedure.
6. Do this for all your standards, and repeat three times for the unknown sample.
7. Save the data on a flash drive or a floppy disk using File…Save as… and choose the Rich Text Format with the extension .rtf, for later import into Excel. You may also want to save the data on the computer hard drive (C:\Documents and Setting\Fluorimeter\Chem115-F09 ) or print the data out.
8. When you are done with the fluorimeter, exit the programs, switch out the instrument, and remember to sign the logbook.