Saleem Siddiqui (Sharda University) 1

SPECTROPHOTOMETER

Light is electromegnatic radiation that is transmitted through space at enormous

velocities. It travels in the form of transverse waves. The wavelength (λ) of a beam of
radiation is the linear distance between successive maxima or minima of a wave.
The electromagnetic spectrum of light covers an immense range of wavelengths or
energies. The, visible light, which the human eyes can perceive, lies only between 400-700
nm. The modern instruments can, however, permit measurements at both shorter wavelength
(ultraviolet [UV]) and longer wavelength (infrared [IR]) portions of the spectrum.
When a beam of radiant energy impinges upon a substance, several things may
happen to it. (1) It may pass through the matter with little absorption taking place and,
therefore, little loss of energy. (2) The direction of propagation of the beam may be altered by
reflection, refraction, or diffraction. Scattering by particulate suspended matter must also be
included. (3) The radiant energy may be absorbed entirely or in part. The absorption involves
a transfer of energy to the medium and the absorption process is a specific phenomenon
related to characteristic molecular structures.

The wavelength of electromagnetic radiation that is absorbed or emitted by the atom

or molecules of a substance is characteristic of the atomic and molecular structure of that
substance. A number of different measures are used to characterize these wavelengths. The
amount of radiation that is taken up or given off by the atom or molecules are proportional to
the amount of substance present.
Spectrophotometry involves the use of a spectrophotometer. A spectrophotometer is a
photometer (a device for measuring light intensity) that can measure intensity as a function of
the color, or more specifically, the wavelength of light.

UV-Visible Spectrophotometer (Absorption Spectrophotometer)

When white light passes through a solution certain wavelengths are absorbed and
unabsorbed wavelengths are transmitted. This is called light transmittance or absorption. The
colour of the substance visible to the eye is the light that is transmitted by the solution.

Absorption laws
When a beam of monochromatic light passes through a solution that absorbs radiation,
a certain amount of the light is absorbed by the solution. The quantity of light absorbed is
well defined and follows definite physical laws. For example, if the power (also referred as
intensity, I) of a beam of monochromatic radiation that falls on a solution is Po, then the
intensity of the radiation emerging from the solution is P, not Po. The ratio P to Po is known
as the transmittance (T), generally expressed as % T
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Figure 1. Absorption of light

Absorbance (A) of a solution is defined as

A= -log10T = log Po/P

Lambert’s law
The proportion of monochromatic light absorbed by a homogeneous medium is
proportional to the path length (parallel surfaces tangential to incident and transmitted light
beam). Transmittance decreases exponentially with increasing path length.

A = ab

Where a is proportionality constant called absorptivity of the liquid and b is the

optical path length.

Beer’s law
Beer applied Lambert’s findings to spectrophotometry. This law states that the amount
of light absorbed is proportional to the number of molecules in the absorbing substance
through which the light passes. Therefore, the concentration of a substance in solution is
directly proportional to the amount of light absorbed.

A = ac

Where a is the absorptivity of the liquid and c is the concentration of the solution.

The combination of these laws, known as the Beer-Lambert law, is the basic law of
spectrophotometry and can be expressed as

A = abc = -log10T = log Io/I

When the concentration c is expressed in moles per litre and b is in centimeters, the
proportionality constant a is refered as molar absoptivity (ε). Thus
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A=εbc
-1 -1
where ε has the units of L cm mol .

Beer-Lambert’s law, which is usually found to hold at low concentrations, is the basic
principle in absorption spectrophotometry, such as colorimetry, absorption spectrometry and
uv-visible spectrometry.
Beer-Lambert’s law is better applicable for absorption of that wavelength of light
which is absorbed more. Therefore, first the characteristic absorption spectrum of the test
substance has to be determined.

Absorbance Spectrum
The extent to which a sample absorbs light depends strongly upon the wavelength of
light. For this reason, spectrophotometry is performed using monochromatic light.
Monochromatic light is light in which all photons have the same wavelength. In analyzing a
new sample, a chemist first determines the sample's absorbance spectrum.
The absorbance spectrum shows how the absorbance of light depends upon the
wavelength of the light. The spectrum itself is a plot of absorbance vs wavelength and is
characterized by the wavelength (max) at which the absorbance is the greatest. The value of
max is important for several reasons. This wavelength is characteristic of each compound and
provides information on the electronic structure of the analyte. In order to obtain the highest
sensitivity and to minimize deviations from Lambert-Beer's Law, analytical measurements are
made using light with a wavelength of max. This wavelength provides maximum sensitivity to
the measurements.

Figure 2. Absorption spectrum. A graph of absorbance vs. wavelength for a hypothetical compound. The Amax
for this compound is about 500 nm and should be used for the study.

Standard Calibration Curves

Calibration curves are made experimentally by preparing a series of standard solutions
of analyte, each with a known concentration. The absorbance of each solution is then
measured and a curve relating the experimentally determined absorbance to the concentration
of the solution is prepared. The typical calibration curve in prepared by plotting absorbance
(A) against the concentration (c) of the solution. The measurements are made only in that
range where the curve is linear.
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Figure 3. The calibration or standard curve.

INSTRUMENT COMPONENTS
All spectroscopic instruments contain the same basic components: a sourceof light
radiations. an attenuating device, a monochromator, a cell to hold the sample, a detector, an
amplifier and a meter or recorder to display the signal.

A. Single beam UV-Visible spectrometer

Figure 4. Single beam spectrophotometer

Light Source/Lamps
In the visible region, a tungsten lamp is a satisfactory radiation source. Only about 15% of
the energy from a tungsten lamp is in the visible region, yet can provide sufficient energy for
most measurements, in that region. Only a small percentage of the radiation from a tungsten
lamp is ultraviolet radiation. The most common sources for the UV region are the halogen
discharge lamp, the mercury vapor lamp, and the deuterium (D2) discharge lamp. Hydrogen
or deuterium lamps, which provide a continuous spectrum in the UV region, are most often
used. A deuterium lamp is more stable and has a longer life than a hydrogen lamp. Frequently
these lamps operate for 2000 to 5000 hours before replacement is necessary.

Cuvettes
The cuvettes/cells may be either round or square. For measurements in the visible
region, cells are usually made of glass. However, glass is not satisfactory in the UV region
because it absorbs most of the radiation if below 360 nm. Two good materials for the UV
region are quartz and fused silica. Fused silica transmits down to 185 nm, whereas quartz
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transmits only to 200 nm.

Round cells should always be placed in the instrument in the same direction every
time. This is necessary because round cells are seldom uniform in diameter and the glass
thickness varies.
Cells also should be filled nearly full. If they are filled only a little past the light path,
reflection and stray-light errors may occur.

Monochromators
A monochromator is a device that will pass a very narrow band of wavelengths of
radiations. An entrance and/or exit slit may be associated with these components. In practice,
however, monochromators allow a narrow range of wavelengths to reach the specimen. The
general monochromators been used are coloured glass, glass prism and gratings. Gratings are
most effective.

Detectors
The main type of detector for the UV and visible regions is the photocell or phototube.
The incident radiation strikes the cathode, which is coated with a mixture of cesium oxide,
silver oxide, and silver. It is connected to a Recorder or a Read out device (an ammeter)

Limitations of a UV-Visible spectrophotometry

1. Only coloured substances (by itself are coloured or that can be converted to coloured
compound by some chemical reaction) can be analysed by visible spectrophotometers.
However, UV-spectrophotometer can be used for colourless compounds (proteins and
aminoacids).

2. It is applicable only to clear solutions that are not turbid.

3. It is not applicable at high concentrations that lead to crystallization or polymerization.

Recommended only in the range of 20-80% transmission range.
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Complementary hue

The colour of the substance visible to the eye is the light that is transmitted by the
solution. This colour is complementary to the absorbed colours. The complementary colours
(hue) for various coloured substances is given below.

Wavelength region (nm) Transmitted colour Complementary hue

< 380 Ultraviolet
380-435 Violet Yellowish green
435-480 Blue Yellow
480-490 Greenish blue Orange
490-500 Bluish green Red
500-560 Green Purple
560-580 Yellowish green Violet
580-595 Yellow Blue
595-605 Orange Bluish green
605-780 Red Greenish blue
> 780 Near Infra-red

Some of the major applications of spectrophotometers include the following:

 Detection of concentration of various nutrients and biomolecules in food.

 Detection of anti-nutritional compounds in food.
 Detection of impurities and aflatoxins.
 Identification of pigments and browning of product during storage or processing.
 Monitoring deteriorative changes in food (oil rancidity, peroxide value, etc.) during
storage.
 Determination of antioxidant activity in food.
 Characterization of proteins, fats, carbohydrates, etc.
 Quantification of active ingredients in a neutraceuticals.
 In monitoring and quality control of end product.