Fluorimetry Questions

Because vibrational and rotational transitions can occur as well Because after you have excite absorption, it falls to the ground excited state. When it falls back to the ground stage, it is at a longer wavelength.

Explain why emission occurs at a longer wavelength (lower energy) than absorption.

Allows us to evaluate the efficiency of the extraction process since the amount of quinine in this solution is known --> percent recovery or extraction efficiency (So that you don't have straight kind of metal sitting on the background because there are metal everywhere.

What is the purpose of the control solution?

The extraction process used to separate the quinine from the urine exposes the sample to multiple reagents that could have resulted in contamination. The blank allows us to correct for this To identify whether there is any metals which are straight metal in there.

What is the purpose of the extraction blank?

Either increase the amplitude of the signal or reduce the amplitude of the signal so that it fits.

What should you do if the excitation scan flat-lines or goes above 3x106 photons/sec?

subtract the blank concentration from the concentrations determined for the other extracted sample.

What should you do if you observe significant emission intensity at the quinine emission wavelength in the extraction blank or acid blank?

UV-vis and Fluorescence are both electronic transitions as oppose to IR, which is vibrational.

What energy level transitions are involved in UV-vis absorbance/fluorescence as opposed to IR?

Fluorimeter

What instrument will you use to measure the absorbance of quinine?

1.49 g/cm^3

What is the density of chloroform?

1 g/cm^3

What is the density of water?

Phosphorescence is an emission of a photon during a transition between states with different spin quantum numbers and fluorescence is an emission of a photon during a transition between states with the same spin quantum numbers

What is the difference between phosphorescence and fluorescence

The excitation monochromator selects an excitation wavelength and the emission monochromator observes luminescence. The first monochromator separate the light that is going to be absorbed and the second monochromator separates the light that had been emitted as fluorescence.

What is the function of each monochromator?

the organic (chloroform) phase will be the bottom layer since it has a higher density.

Chloroform and water are immiscible. If combined in a container, where will the organic (chloroform) phase be located (top or bottom layer)?

Light source, excitation monochromator, emission monochromator, detector and cuvette containing sample.

Describe the components of a typical fluorimeter.

In absorption atoms absorb part of the light from the source (UV-Vis and Flame AA) and in emission, photons are emitted from an atom that moved to a ground state from a higher excited state (Fluorescence spectroscopy)

Differentiate between absorption and emission

Excitation spectrum come from atoms releasing photons at specific wavelengths while absorbance spectrum measures the light wavelength that was absorbed by the atom to excite the electrons Because you have two monochromator in fluorescence, you can either be measuring the absorbance directly or the excitation directly, however if you are doing fluorescence, you have a choice. You can either be diling the excitation stores and varying it while holding the emission spectrum constant OR you can hold the excitation constant (typical) and vary the emission spectrum so that you can watch the various vibrational modes as they come up.

Differentiate between an excitation spectrum and an absorbance spectrum.

Emission spectrum is when you hold the excitation wavelength fixed and scan through the emitted radiation Excitation spectrum is measured by varying the excitation wavelength and measuring emitted light at one particular wavelength

Differentiate between the emission spectrum and the excitation spectrum.

creates an electric current proportional to irradiance in order to create an extremely sensitive light detector You have your photon on a surface of metal that eject electrons following the photoelectric effect and from there either accelerate into a series of dynodes that amplify the signal to generate a measurable current that is turn into a voltage, that is put into your computer.

Explain how a photomultiplier tube detector works.

2

How many monochromators are there in this instrument?

Flush down the toilet

Where should you dispose of your urine waste?

helps to prevent the scattered light from reaching the detector. The smaller scattering happen at a 90 degree angle.

Why is the detector is typically set at a 90 degree angle from the excitation beam path?