FTIR spectroscopy measures how much infrared light of varying frequencies is absorbed by a sample. The sample is located after the interferometer in an FTIR setup. As the moving mirror in the interferometer modulates different wavelengths at different rates, this allows an array of wavelengths to be measured in a short time to see how the sample absorbs light across the infrared spectrum. In UV/visible spectroscopy, a sample is placed behind the dispersive element, usually a prism or diffraction grating. This separates light from the source into individual wavelengths, and the sample is placed after this point so it can absorb specific wavelengths that can identify compounds in the sample.

1. Sample Placement in
Spectroscopy

2. 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

3. Sample Location

4. Sample Location

5. 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

6. The Moving mirror causes the array
of wavelengths within the FTIR
spectrometer.
A. True
B. False
C. Neither
D. Both
E. This is not the answer

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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

14. Fluorescence Spectroscopy
 Emission at lower energy than absorption
 Greater selectivity but fluorescent yields vary for
different molecules
 Detection at right angles to excitation
 S/N is improved so sensitivity is better
 Fluorescent tags

17. 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

18. 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

19. Resources
 http://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/s
pectrpy/uv-vis/spectrum.htm
 http://en.wikipedia.org/wiki/Ultraviolet–
visible_spectroscopy
 http://en.wikipedia.org/wiki/Fourier_transform_infrared_spe
ctroscopy
 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