8 Spectrophotometric Methods
8 Spectrophotometric Methods
8 Spectrophotometric Methods
SPECTROPHOTOMETRIC
Descriptors
1.Introduction and generalities
2. Molecular spectrophotometry
4.Instrumentation and analytical applications
Spectroscopic optical methods
This is the generic name for a very large number of instrumental
methods that use instrumental techniques in which an optical signal
is generated whose foundation is based on the interaction of
electromagnetic radiation with the analyte.
When electromagnetic radiation interacts with the sample containing
the analyte, different phenomena can arise, among which the
absorption and emission of light are the most relevant and give rise to
spectrophotometric methods of absorption and (or) emission.
The methods can be molecular or atomic absorption, depending on
whether the analyte is in a molecular or atomic state.
There are also fluorescent methods, in which there is absorption
preceded by emission
All of these methods are also called spectroscopic, because they are
based on the absorption or emission spectra of the analyte.
The electromagnetic spectrum
Given the width of lines and spectral frequencies it contains, the
electromagnetic spectrum, the spectrum that results from the
interaction of matter-radiant energy, can be very complex and cover wide
areas: Vis-Uv, IR, Microwaves...etc.
Photon energy = hv
v=c
C- light speed
Atomic Molecular
Absorption Absorption
uv
visibl
e
G
O
l CONTINUOUS
SPECTRUM
LINE SPECTRUM
Molecular absorption spectrophotometry
It is useful for both qualitative and quantitative analysis
Electronic
transactions Interactions
link
UV A NA Visible nearby N |R | Yo
UJ\W“NJ Spectrum
By varying the wavelength within each area of the electromagnetic spectrum, the
absorption spectrum of a substance in each region can be obtained.
Qualitative information:
It is obtained by comparing the spectrum of the sample with that of a
reference material.
This information is more relevant in IR spectroscopy and less in the case of
Vis-UV spectroscopy.
absorbance
total At =
measureme T, l
nt
to the
Q
= Transmittance
P
0
(T)
MOLAR ABSORPTIVENESS
DEPENDS ON:
FEATURES OF THE
SOLVENT
ABSORBENT SPECIES
WORKING WAVELENGTH
UNITS
L /mol.cm
REAL
APPARENTS
Most species comply with the law in a certain concentration range. Outside of it,
they experience positive or negative deviations. This is well observed in the
calibration:
Vis-UV Spectroscopy:
IR Spectroscopy:
Molecular fluorescence:
(issue)
detector reader
RADIANT ENERGY SOURCE
KEEP GOING
STABLE
Lamp of
In molecular absorption, the sample must be irradiated with light energy from a
source. Depending on the work area, the properties of the source are different.
The sources must be uniform in intensity.
deuterium lamp
(UV)
tungsten source (W)
D2 * electrical energy
Used in VIS and near IR
produces light in ranges of 350-2200 nm D,* —.D,hv
160-380nm
VISIBLE SPECTRUM AND COLORS
COMPLEMENTARY
display complementary
l (nm) color l (nm) color
380-420 violet 520 -550 yellow green
420 -440 blue violet 550 -580 yellow
440 -470 blue 580 -620 orange
470 - 500 green Blue 620 -680 red
500 -520 green 680 -780 purple
520 -550 yellow green 380 -420 violet
550 -580 yellow 420 -440 blue violet
580 -620 orange 440 -470 blue
620 -680 red 470 - 500 green Blue
680 -780 purple 500 -520 green
A SOLUTION APPEARS BLUE IN COLOR WHEN IT IS
ILLUMINATED WITH POLYCHROMATIC LIGHT BECAUSE IT
ABSORBS ALL COLORS EXCEPT BLUE, WHICH IS THE
COLOR IT ALLOWS TO PASS
WAVELENGTH SELECTOR
CHARACTERISTICS
MAXIMUM TRANSMISSION WAVELENGTH
PERCENTAGE TRANSMITANCE AT MAXIMUM
EFFECTIVE BANDWIDTH
DESIGNS
CUT FILTERS
ABSORPTION FILTERS
INTERFERENCE FILTERS
* PRISM MONOCHROMATORS
* NETWORK MONOCHROMATORS
WAVELENGTH SELECTION
WITH MONOCHROMATORS
BASIC COMPONENTS
COLLIMATING MIRROR
with
diffraction
grating
with prism
BUCKETS
G
O
PHOTOTUB
In IR spectroscopy, a Fourier transform
processor (FTIR) can be coupled to the detector. E
DETECTOR REQUIREMENTS
They are based on the photoelectric effect, in which the incidence of a photonic
beam on a metal is capable of generating electrical energy.
In the case of
photomultipliers, the signal is amplified through the use of dynodes in series.
DETECTOR
PHOTOMULTIPLIER TUBE
array photodiode
Made up of a series of photodiodes spaced at regular
intervals within an integrated circuit chip
It can measure multiple wavelengths at the same time.
They may be :
1 Single beam or simple beam
• The beam can be unfolded at regular intervals with the help of a chopper
• Half of the light passes through the sample and the other a “target”
• The measured absorbance takes into account the sample/blank absorption ratio and
allows correction of deviations in the detector and the output source.
• The chopper change speed is a limitation on what the instrument can select.
3 IR Spectrophotometers
They are of two types:
1) Dispersive (they rely on a monochromating and scanning system to produce
the spectrum).
2) Fourier transform (they use a combination of constructive and destructive
interference along with the transform to produce the spectrum).
Dispersive
Basis:
At a fixed time many l are passed through the cell and the signal is recorded.
With each sweep, any l can traverse the cell many times. The interferogram is
obtained like this:
Analytical applications
ANALYTICAL APPLICATIONS
GOOD PRECISION
OPERATIONAL EASE