Visible Spectrophotometry and Colorimetry.pptx Color comparison was one of the oldest methods used for quantitative estimation of elements and substances. The variation of a color of a system with change in concentration of some components forms the basis of colorimetric analysis. The color is usually due to the formation of a colored compound by the addition of an appropriate reagent at specific condition or it may be inherent in the desired constituent itself. The intensity of the color may then be compared with that obtained by treating a known amount of the substance in the same manner. Colorimetry is concerned with the determination of the concentration of a substance by measurement of the relative absorption of light with respect to a known concentration of a substance. In visual colorimetry, natural or artificial white light.

1. VISIBLE SPECTROPHOTOMETRY AND
COLORIMETRY
BY
DR. NEETU SONI ,SHUATS, PRAYAGRAJ

2. Instrumental visible spectroscopy is a
logical extension of the color tests and
visual observations. It is a sensitive,
accurate method of measuring the
colors or mixtures of colors that our
eyes perceive.

3. Spectrophotometry is the quantitative measurement
of how much a chemical substance absorbs light by
passing a beam of light through the sample using a
spectrophotometer. By measuring the intensity of
light detected, this method can be used to determine
the concentration of solute in the sample.

5. The visible light spectrum is the segment of
the electromagnetic spectrum that the
human eye can view. More simply, this range
of wavelengths is called visible light.
Typically, the human eye can detect
wavelengths from 380 to 700 nanometers.

6. Spectrophotometer
The spectrophotometer refers to an
instrument that measures the absorbance of
the test sample at a specific wavelength by
measuring the amount of light transmitted by
the sample. This device contains several
components like a light source, collimator,
monochromator, cuvette, light detector, and
digital meter.

7. Colorimetry is a scientific technique that
is used to determine the concentration of
colored compounds in solutions by the
application of the Beer–Lambert law,
which states that the concentration of a
solute is proportional to the absorbance.

9. Theory of spectrophotometry and
colorimetry
When light (monochromatic or heterogeneous) is
incident upon a homogeneous medium, a part of the
incident light is reflected, a part is absorbed by the
medium, and the remainder is transmitted as such.
Color comparison was one of the oldest methods
used for quantitative estimation of elements and
substances. The variation of a color of a system
with change in concentration of some components
forms the basis of colorimetric analysis.

10. In visual colorimetry, natural or artificial white light
(the whole range of the visible radiation is from
400-760 nm) is generally used as a light source
and determinations are usually made with a simple
instrument termed as colorimeter or color
comparator.

11. Lambert’s and Beer’s law.
When light (monochromatic or heterogeneous) is
incident upon a homogeneous medium, a part of the
incident light is reflected, a part is absorbed by the
medium, and the remainder is transmitted as such. If I0
denotes the intensity of the incident light, It, the
transmitted light, Ir the reflected light and Ia the
absorbed light, then it may be written:
I0 = Ia + It + Ir
Ir is very small then
I0 = Ia + It

12. Lambert’s law: When a monochromatic light passes through a
transparent medium, the rate of decrease in intensity (I) with thickness
(l) of the medium is proportional to the intensity of light.
Mathematically, –dI/dl∞I
On deduction it stands to It = I0 × e –kl (k is a proportionality constant)
On changing the above equation from natural to common logarithm,
It = I0 × 10–0.4343kl or
It = I0 × 10–Kl (K is called absorption coefficient)
The absorption coefficient is generally defined as the reciprocal of
the thickness (l cm.) required to reduce 1/10th of its intensity.
It/I0 = 0.1 = 10–Kl
or
K × l = 1
or
K = 1/l So unit of K is cm–1

13. The ratio (It/I0) is termed as Transmittance (T)
The ratio (I0/It) is termed as Opacity
log(I0/It)is termed as the Absorbance (A) of the medium or
Optical density (OD).

14. Beer’s law: When a beam of monochromatic light is allowed to pass
through a solution, the intensity of the light beam decreases
exponentially with the increase in concentration as well as thickness
of the solution. The equation becomes:
It = I0 × 10–a.c.l (a is called molar absorption coefficient)
The value of ‘a’ depends on the unit of concentration. If c is expressed in
gm-moles/litre, l in cm, then a is replaced by ε and is termed as molar
extinction coefficient.
The equation becomes: It = I0 × 10–ε.c.l
A = ε.c.l
or, ε = A/c.l
The unit of ε thus becomes litre gm-moles–1cm–1 Absorbance per unit
path length (l) per unit concentration (c) is known as specific extinction
coefficient

15. Instrumentation
All spectrophotomete, Photometers and Colorimeters have the following
basic components.
 Source of continuous radiation over the wavelengths of
interest.
 A Filter or Monochromator for selecting a narrow band of
wavelengths from the source spectrum.
Sample cell A container for the sample.
A Detector for converting radiant energy into electrical
energy, and
 A Read out system device to read out the response of the
detector.

16. The radiation source
Tungsten filament (300–2500 nm)
.
Filter
It can transmit or reflect a specified range of wavelength.
Filters are classified into three
types
Long wave pass filters,
Short wave pass filters
 Band pass filters

17. Types of filter from the viewpoint of function
(1) Absorption filter : a filter which absorbs a specific
range of wavelengths, for examples, colored filter
glass and gelatin filter.
(2) Interference filter : a filter which transmits a specific
range of wavelengths by utilizing the interference effect of
a thin film.

18. Prisms
A prism disperses polychromatic light from the source
into its constituent wavelengths by virtue of its ability to
reflect different wavelengths to a different extent
Two types of Prisms are usually employed in commercial
instruments. Namely, 600 cornu quartz prism and
300 Littrow Prism.

19. Grating
Gratings are often used in the monochromators of
spectrophotometers operating ultraviolet, visible and infrared
regions.

20. Monochromator
(1) entrance slit, (2) collimating mirror (to form a parallel beam after the slit),
(3) diffraction grating (dispersive element), (4) camera mirror (focuses light from
the dispersive element onto the exit slit), and (5) exit slit.

21. Detectors
Detectors are instruments that measure the amount of
ultraviolet or visible light absorbed by the sample
molecules.
In order to detect radiation three types of photosensitive
devices are
Photovoltaic cell
Phototubes
Photomultiplier

22. Photovoltaic cell
The Photovoltaic cell is the semiconductor device that converts the light
into electrical energy. The voltage induces by the PV cell depends on the
intensity of light incident on it.

23. Phototubes
Phototube is also known as a photoelectric cell.
It is filled with gas under low pressure.
It contains a light-sensitive cathode and anode inside an evacuated
quartz envelope.
A photon entering the tube strikes the cathode and leads to the ejection
of an electron.
 Electrons strikes the anode and results in the flow of current. The
resulting current is of low intensity and requires amplification.
The response in the phototube depends on the wavelength of incident
light.

24. Phototubes

25. Photomultiplier tube is used in UV-Vis spectrophotometers.
 It has an anode, cathode, and many dynodes.
Photon when entering the tube, strikes the cathode, resulting in
the emission of electrons.
The emitted electrons are accelerated towards the first dynode,
An electron striking the first dynode will result in the production of
several electrons.
The process repeats itself continuously from one dynode to
another and after passing to 10 dynodes.
Photomultiplier Tube

28. A colorimeter is an instrument that compares the amount of
light getting through a solution with the amount that can get
through a sample of pure solvent.
Visual comparators
•Multiple standard method
•Duplication Method
•Dilution methods
•Balancing method
Colorimeter

29. Dubosq colorimeter

31. Application of colorimetry and
spectrophotometry:
(i) Determination of molar composition of complexes.
(ii) Determination of instability constants of complexes.
(iii) Determination of pK value of an indicator.
(iv) Elucidation of structures of organic as well as
inorganic compounds.
(v) Determination of molecular weight of colored
substances.