This document discusses molecular spectroscopy and spectrophotometry. It describes how a spectrophotometer works by measuring the intensity of light passing through a sample as a function of wavelength. Key components of a spectrophotometer include a light source, monochromator, cuvette containing the sample, and detector. The document also explains Beer's and Lambert's laws which relate absorbance to characteristics of the sample like concentration and path length. Colorimeters are also covered as devices that can determine concentration by measuring absorbance at specific wavelengths based on the Beer-Lambert law.

1. Molecular
Spectroscopy
BY,
TAHIRA KHALID
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2.  Spectroscopy
 Construction Of Spectrophotometer
 Instrumentations Deviations
 Absorbance
 Absorption Laws
 Colorimeter
 Construction Of Colorimeter
 Other Instruments
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3. Spectrophotometry involves the use of a
spectrophotometer. A spectrophotometer
is a photometer that can measure intensity
as a function of the light source
wavelength. Important features of
spectrophotometers are spectral
bandwidth and linear range of absorption
or reflectance measurement.
A spectrophotometer is commonly
used for the measurement of transmittance
or reflectance of solutions, transparent or
opaque solids, such as polished glass, or
gases. However they can also be designed
to measure the diffusivity on any of the
listed light ranges that usually cover around
200 nm - 2500 nm using different controls
and calibrations. Within these ranges of
light, calibrations are needed on the
machine using standards that vary in type
depending on the wavelength of
the photometric determination.
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4. SINGLE BEAM SPECTROPHOTOMETER
DOUBLE BEAM SPECTROPHOTMETER

5. SOURCE MONOCHROMATOR CUVETTE DETECTOR
Source must be:
• Continuous
• Stable
• Intense
Cuvette
must be
made up
of silica or
quartz
More the
grooves present
in the
monochromator,
efficient will the
monochromator
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6.  STRAY DEVIATION:
 Some external source is entering like sunlight
 VOLTAGE FLUXUATION:
 Voltage is varying continuously which effects the sensitivity of the spectrophotometer
 CHANGE IN SECSITIVITY OF DETECTOR:
 With the passage of time sensitivity of the spectrophotometer decreases
 POLYCHROMATIC RADIATION:
 Light passing through the sample is polychromatic because the monochromator is not
working properly
 UNEXPECTED CHEMICAL REACTION:
 The given sample is dissociated after some time
 Concentration of the given sample is changed
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7. 7
For each wavelength of light passing through the spectrometer, the intensity of the light passing through
the reference cell is measured i.e. Io
The intensity of the light passing through the sample cell is also measured for that wavelength - given the
symbol, I.
If I is less than Io, then obviously the sample has absorbed some of the light. A simple bit of maths is then
done in the computer to convert this into something called the absorbance of the sample - given the
symbol, A.

8.  LAMBERT’S LAW:
According to this law
“ Absorbance is directly proportional to the breadth of the cuvette
i.e. A α b
 BEER’S LAWS:
According to this law
“ Absorbance is directly proportional to the concentration of the sample
i.e. A α C
By combining both laws we get
A α bC
A= ε bC
Where ε is the molar absorptivity
where “l” is the breadth of the cuvette
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9. A Colorimeter is a device used
in colorimetry. In scientific fields the
word generally refers to the device
that measures the absorbance of
particular wavelengths of light by a
specific solution. This device is most
commonly used to determine
the concentration of a
known solute in a given solution by
the application of the Beer-Lambert
law, which states that the
concentration of a solute is
proportional to the absorbance.

10. The essential parts of a colorimeter are:
 A light source (often an ordinary low-voltage filament lamp)
 An adjustable aperture
 A set of colored filters
 A cuvette to hold the working solution
 A detector (usually a photoresistor) to measure the transmitted light
 A meter to display the output from the detector
 In addition, there may be:
 A voltage regulator ,to protect the instrument from fluctuations in main voltage.
 A second light path, cuvette and detector. This enables comparison between the working
solution and a "blank", consisting of pure solvent, to improve accuracy.
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11. Filters
Changeable optics filters are used
in the colorimeter to select the
wavelength of light which the
solute absorbs the most, in order
to maximize accuracy. The usual
wavelength range is from 400 to
700 nanometers (nm). If it is
necessary to operate in
the ultraviolet range (below
400 nm) then some modifications
to the colorimeter are needed. In
modern colorimeters the filament
lamp and filters may be replaced
by several light-emitting diodes of
different colors.
Cuvettes
In a manual colorimeter the
cuvettes are inserted and
removed by hand. An automated
colorimeter (as used in
an AutoAnalyzer) is fitted with
a flawcell through which solution
flows continuously.
Output
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The output from a colorimeter
may be displayed by an
analogue or digital meter and
may be shown
as transmittance (a linear scale
from 0-100%) or
as absorbance (a logarithmic
scale from zero to infinity). The
useful range of the absorbance
scale is from 0-2 but it is desirable
to keep within the range 0-1
because, above 1, the results
become unreliable due to
scattering of light.