Sample Placement in Spectroscopy

FTIR

Fourier transform infrared spectroscopy shines a beam containing many frequencies of light at once, and measures how much of that beam is absorbed by the sample

Solves problems of IR difficult mechanics hard to transport limited resolution slow and inaccurate analysis stray light limited sensitivity lack of reproducibility

Sample Location

Sample Location

Reasons for Sample Locations

The sample is located after the interferometer

The moving mirrors reflect an array of frequencies

As this mirror moves, each wavelength of light in the beam is periodically blocked or transmitted by the interferometer, acting similar to a monochromator, due to wave interference

Different wavelengths are modulated at different rates by the moving mirror

This set up for FTIR is used to see how the sample absorbs at an array of wavelengths in a short period of time

The Moving mirror causes the array of wavelengths within the FTIR spectrometer.

A. True B. FalseC. NeitherD. BothE. This is not the answer

Infrared Spectroscopy

Pass a beam of infrared light through the sample.

When the frequency of the IR is the same as the vibrational frequency of a bond, absorption occurs.

Examination of the transmitted light reveals how much energy was absorbed at each frequency (or wavelength)

This can lead to identification/study of different molecules

Sample location

The sample is placed in line with a reference for comparison

Sample placed before the chopper/splitter The chopper/splitter alternates which beam (sample or reference) enters

the monochromator and detector This is done to prevent fluctuations in the output of the source affecting

the data and allows the effects of the solvent of the sample to be cancelled out

The sample is placed before the monochromator so that it can adjust which wavelength range is detected by the IR detector

UV/Visible Spectroscopy

Utilizes light in the UV/Visible region to measure the absorption

Measures transition of non-binding electrons from ground state to excited state as opposed to fluorescence, which measures excited to ground state

UV/Visible Spectroscopy A beam of light from a visible

and/or UV light source is separated into its component wavelengths by a prism or diffraction grating.

Each wavelength is split into two equal intensity beams by a half-mirrored device.

One beam passes through the sample and the other through the reference or blank.

The intensities of these light beams are then measured by electronic detectors and compared.

Sample is placed behind the dispersive element

Why the sample is located behind the dispersive element

UV/visible spectroscopy is used in quantitative determinations of solutions especially transition metal ions, conjugated organic compounds, and biological macromolecules

These samples used in UV/visible spectroscopy only absorb at specific wavelengths in the UV/visible range of 200-800 nm

A single wavelength is needed to identify the compound within the sample, so the sample must be behind the monochromator

For Example:NADH absorbs at 340 nm

NADH is broken down into NAD+ to allow many reactions to occur

Utilizing Beer’s Law the rate of reaction (decrease in NADH concentration) can be determined by the decrease in absorbance as the reaction

One can determine whether an enzyme, such as MDH above, used in this kinetic assay was successful or not depending on the rate of reaction

Where is the sample in UV/Visible Spectroscopy located?

A. After the monochromator

B. Before the monochromator

C. Right in front of the deuterium lamp

D. You don’t need a sample in UV/Visible spectroscopy

E. This one is wrong

Fluorescence Spectroscopy

Emission at lower energy than absorption

Greater selectivity but fluorescent yields vary for different molecules

Detection at right angles to excitationS/N is improved so sensitivity is better

Fluorescent tags

In fluorescence spectroscopy…

A) We can select for wavelength by having the excitation source pass through a filter or monochromator

B) We want the detection at ~180° to the excitation for an improved signal/noise ratio

C) Emission occurs at an equal energy to absorption

D) All of the above

Fluorescence within Chromatography

The use of an excitation monochromator can be avoided using a laser because it emits light at a very narrow wavelength interval

Disadvantage: Cannot drastically change the wavelength

Resources

http://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/uv-vis/spectrum.htm

http://en.wikipedia.org/wiki/Ultraviolet–visible_spectroscopy

http://en.wikipedia.org/wiki/Fourier_transform_infrared_spectroscopy

http://david-bender.co.uk/metonline/CHO/shuttles/shuttles9.htm

https://www.princeton.edu/~achaney/tmve/wiki100k/docs/Ultraviolet-visible_spectroscopy.html

http://en.wikipedia.org/wiki/Infrared_spectroscopy