Chem 445, Lab Section Modified 8-4-14 EXPERIMENT #2: Determination of Unknowns Using Spectroscopy

Fluorescence is one of the most common methods of spectroscopy used in biology and . In addition it is critical for determining fluorescent properties of any fluorescent molecule. In this lab, you will learn how to perform fluorescence spectroscopy on our Fluorometer and will use your data to better characterize the 3 unknown compounds from last week. You will need the information you obtained last week to determine λex.

READING ASSIGNMENT: Attached

Safety Information

Wear gloves when handling the unknown compounds. Avoid all contact with skin, eyes, and clothing. Also, these compounds are dyes and will stain your hands and all clothing and therefore must be handled with care.

EXPERIMENTAL NOTES:

Using the Fluorometer: You instructor will guide you through a preliminary run of the fluorometer. During this time you will want to record notes as to how to acquire the spectra as well obtaining the data from the computer.

Unknowns Identities:

Your group should have stock solutions for the following molecules from last week. Your job is to expand your data from last week to acquire λem from each molecule. You will need to use the most “red- shifted” λmax values from last week for the λem for the experiment.

Unknown Sample Preparation: For each sample you should have made ~100 µM and ~100 nM stock solutions (Vial 2 and Vial 3 which you should acquire from your instructor). Using a Pasteur Pipet, transfer the contents of Vial 3 to a fluorometer cuvette. Acquire the fluorescence spectrum for sample. If the sample is too concentrated (as shown by a huge fluorescent peak), dilute by a factor 10 with ethanol. If too dilute, add more of the 100 µM stock solution (Vial 2, ask your instructor how much should be added) to strengthen the signal.

Chem 445, Lab Section Modified 8-4-14 NOTES FOR WRITING YOUR LAB REPORT:

Typically in publications fluorescence data is reported in tabular form such as the following:

Note: λmax will be the values you determined last week and Fluor. λmax = λex maximum (what you figured out this week).

You’ll want to assign the data you have obtained with the chemical structures of fluorescein, Rhodamine B, and dihydroxyxanthone.

Chem 445, Lab Section Modified 8-4-14 Reading:

The measurement of fluorescence signals provides a sensitive method of monitoring the chemical/biochemical environment of a . Instruments have been designed to measure fluorescence intensity, spectrum, lifetime and polarization. Fluorescence intensity measurement allows the determination of the presence of and their concentrations. Fluorescence intensity measurement is used in numerous chemical/biochemical assays and experiments. The instrument designs for these assays are rather straightforward but are as varied as the applications.

Fluorometers are general-purpose instruments designed to measure fluorescence spectrum, polarization and/or lifetime. A typical fluorometer includes a light source, a specimen chamber with integrated optical components, and high sensitivity detectors (Figure 2). The most common light source for fluorometers are lamp sources, such as xenon arc lamps. These lamps provide a relatively uniform intensity over a broad spectral range from the to the near infrared. The optical paths of the excitation and the detection light paths are along the orthogonal axis (90o away from where light enters). The orthogonal arrangement ensures minimal leakage of excitation light into the detection side. High sensitivity photodetectors such as photomultipliers or chargecoupled device cameras are commonly used.

For spectral measurement, monochrometers or bandpass filters are placed in the excitation and emission light paths to select a specific spectral band. The excitation spectrum is defined as the fluorescent intensity measured as a function of excitation wavelength at a constant emission wavelength; the is the fluorescent intensity measured as a function of emission wavelength at a constant excitation wavelength. Fluorometers have also been designed to measure fluorescence lifetime. Given the typical lifetime of fluorophores, accurate lifetime measurement requires photodetectors and signal processing electronics with subnanosecond resolution.

Figure 1. A typical fluorometer design. LS is the light source, EXO is the excitation optical train, SC is the specimen chamber, EMO is the emission optical train, and DET is the optical detector. Both the excitation and emission optical trains contain beam-shaping and collimation optics. For wavelength-resolved measurements, spectral selection optical components such as monochrometers and filters are included in the EXO and EMO.