3. WHAT IS A SPECTROPHOTOMETER?
• A spectrophotometer is a device that precisely measures electromagnetic energy
at specific wavelengths of lights. It uses the characteristics of light and energy
to identify colours and determine how much of each colour is present
in a ray of light. The two main components of a spectrophotometer are a
spectrometer and a photometer.
• Spectrometer: The spectrometer uses a lens to send a straight beam of light to
a prism, which splits it up into its individual wavelengths. Then, a wavelength
selector filters out just the specified wavelengths and sends it toward the
photometer.
• Photometer: The photometer is the part of the device that detects the number
of photons that are absorbed, sending that measurement to a digital display.
5. • It is similar to calorimeter except that it uses prism or
diffraction grating to produce monochromatic light. It can be
operated in UV (Ultraviolet) region, Visible spectrum as well as
IR (Infrared) region of the electromagnetic spectrum.
• Absorption of light – Light falling on a coloured solution is
either absorbed or transmitted. A coloured solution absorbs all
the colours of white light and selectively transmits only one
colour. This is its own colour.
7. Like all tools, this one has its benefits and drawbacks. Here are some of the advantages of
a spectrophotometer:
•Displays clear, definitive results: With a definite colour measurement, differences in
viewing circumstances won’t influence the colour you work with. For instance, variations
in computer screen settings and lighting can make a colour appear different than
intended. Even differences in human perception, such as colour blindness and eye fatigue,
can result in many different colour interpretations. With a specific measurement, this issue
goes away.
•Quantifies a qualitative characteristic: Since a colour sample is precisely defined, you can
say goodbye to subjective differences in how people interpret colours. “Maroon” might be
an entirely different colour to two different people, but they can’t argue with a specific
colour measurement. This improves communication and makes it easier to discuss your
work.
Advantages of Spectrophotometer
8. • Creates consistency: For many applications and brands, consistent colour is a
vital part of success. A car manufacturer needs to make sure every piece of
the body of the car is the same colour, and brands need to make sure that all
of their marketing maintains consistent colours across mediums. In print and
on a screen, it must all look the same. Precise measurements can help them
achieve that.
• Provides incredibly precise measurements: The human eye is surprisingly
good at noticing colour differences. With precise measurements, you can
confidently keep this issue to a minimum.
• Offers fast operation: Spectrophotometers can gather results in just a few
seconds and some models are designed for mass quantities.
• Comes in various configurations: These tools come in different models for
different samples and environments and offer portability, infrared detection
and scans of irregular samples.
9. Some of the disadvantages of a spectrophotometer include the following:
•It has a somewhat steep learning curve on operating the instrument and
interpreting data. Some training may be necessary to fully understand the
data provided, though many tasks are easier than others.
•Not every spectrophotometer can measure all types of samples. You may
need a model that’s configured to your needs.
DISADVANTAGES OF SPECTROPHOTOMETER
10. Applications of Spectrophotometer
Research, product development, quality control and diagnoses can all benefit from the data that a
spectrophotometer provides. Here are a few specific examples of how these powerful tools are used:
•Beverages: Color can indicate quality in many beverages from soft drinks and juices to spirits and beer, and
consistent color is critical to inspire confidence in customers.
•Pharmaceuticals: The color of a pill is an integral part of identification. It may not affect its functioning, but it tells
people what they’re using. Other pharmaceutical products, like liquid ingredients, have strict standards to meet,
some of which involve its color and transparency. Spectrophotometry helps ensure brand colors and identify
counterfeit medications.
•Building products: If a contractor builds a house with mismatched pieces of vinyl siding, the client isn’t likely to be
happy with the results. When appearance is involved, color is critical. Many materials also reflect property
changes in their color. Anodized metal, for instance, uses a layer of natural oxide to increase its resistance and
adhesive properties. Color analysis can confirm that each piece produced reflects these improvements.
•Chemicals: Chemicals must be clean, consistent in color and free of contaminants to ensure proper functionality
and that your customer trusts them. Color is a key part of classifying many chemical products and identifying their
composition.
•Food: Food production uses spectrophotometry in many ways. From evaluating the ripeness of fruits to identifying
the appropriate baking contrast of breads and buns, color analysis lends itself to plenty of food-based applications.
11. Spectrophotometer is based on the photometric technique which states that “when a beam of
incident light of intensity Iₒ passes through a solution, a part of the incident light is reflected
(Ir), a part is absorbed (Ia) and rest of the light is transmitted (It)”
Thus,
Iₒ = Ir + Ia + It
⇒ In photometers (colorimeter & spectrophotometer), (Ir) is eliminated because the
measurement of (Iₒ) and It is sufficient to determine the (Ia). For this purpose, the amount
of light reflected (Ir) is kept constant by using cells that have identical properties. (Iₒ) & (It)
is then measured.
⇒ The mathematical relationship between the amount of light absorbed and the concentration of
the substance can be shown by the two fundamental laws of photometry on which the
Spectrophotometer is based.
PRINCIPLE OF SPECTROPHOTOMETER
13. Beer’s Law
This law states that the amount of light absorbed is
directly proportional to the concentration of the
solute in the solution.
Log10 I0/It = asc
where,
as = Absorbency index
c = Concentration of Solution
14. Lambert’s Law
The Lambert’s law states that the amount of light absorbed
is directly proportional to the length and thickness of the
solution under analysis.
A = log10 I0/It = asb
Where,
A = Absorbance of test
as = Absorbance of standard
b = length / thickness of the solution
15. The mathematical representation of the combined form of Beer-
Lambert’s law is as follows:
Log10 I0 / It = asbc
If b is kept constant by applying Cuvette or standard cell then,
Log10 I0/It = asc
The absorbency index as is defined as
as = A/cl
Where,
c = concentration of the absorbing material (in gm/liter).
l = distance traveled by the light in solution (in cm).
16. In simplified form,
The working principle of the Spectrophotometer is based on Beer-
Lambert’s law which states that the amount of light absorbed by a
colour solution is directly proportional to the concentration of the
solution and the length of a light path through the solution.
A cl
∝
Where,
A = Absorbance / Optical density of solution
c = Concentration of solution
l = Path length
or, A = cl
∈
∈ = Absorption coefficient
17. A = cl
∈
For two solutions i.e. Test and standard,
∈ = Constant
l = Constant (using the same Cuvette or Standard cell)
AT = CT ….. (i)
AS = CS ….. (ii)
From (i) & (ii),
AT × CS = AS × CT
CT = (AT/AS) × CS
Where,
CT = Concentration of the Test solution
AT = Absorbance/ Optical density of the test solution
CS = Concentration of the standard
AS = Absorbance / Optical density of the standard
18. A spectrophotometer consists of four general parts; light
source, an optical system (monochromator), sample
holder, and detector (photometer).
Light source: Any spectrophotometer requires light of
various wavelengths. Commonly tungsten lamp provides a
visible spectrum of light in a spectrophotometer.
Likewise, hydrogen and deuterium lamps provide
ultraviolet radiation, and Nernst filament provide IR
(infrared) radiation.
Parts of Spectrophotometer
19. Optical system (monochromator): An optical system of spectrophotometer
consists of the following parts:
•Lenses: It collects the radiation from the source and directs it into the slit.
•Entrance slit: It provides a narrow image of the radiation.
•Collimator lens: It depicts the light from the entrance slit parallel.
•Exit slit: It selects the desired spectrum of the light-emitting from the exit slit.
•Dispersive device: A prism and grating function as dispersive devices. These
help in dispersing the incident ray of light. The action of the prism depends on
the refraction of light. Quartz or fused silica prism is a must for ultra-violet
spectrum below 350 nm. A diffraction grating consists of many parallel lines
ruled at highly close intervals, likely 15000/30000 lines per inch on a highly
polished surface like aluminum. The recommended ruling number is from 20
grooves/mm in infrared to as many as 3600 or more grooves/mm for the visible
and ultraviolet rays. The grating is better than a prism.
20. Sample holder
The sample solution is placed in the cuvette ( glass
tubes) directly before the detector and after the
dispersive device. Cuvette varies from test tubes
because it has uniform thickness and optical path
length. Usually, the cuvette is made up of glass or
quartz.
Detector (photometer)
After the desired light passes through the sample
solution in the cuvette, the photometer detects the
photons and gives the signals to the
galvanometer for digital display.
21. Working Mechanism of
Spectrophotometer
When using a Spectrophotometer, it requires being calibrated first which is
done by using the standard solutions of the known concentration of the solute
that has to be determined in the test solution. For this, the standard solutions
are filled in the Cuvettes and placed in the Cuvette holder in the
spectrophotometer that is similar to the colorimeter.
There is a ray of light with a certain wavelength that is specific for the assay is
directed towards the solution. Before reaching the solution the ray of light
passes through a series of the diffraction grating, prism, and mirrors. These
mirrors are used for navigation of the light in the spectrophotometer and the
prism splits the beam of light into different wavelength and the diffraction
grating allows the required wavelength to pass through it and reaches the
Cuvette containing the standard or Test solutions. It analyzes the reflected light
and compares with a predetermined standard solution.
22. When the monochromatic light (light of one wavelength) reaches the Cuvette
some of the light is reflected, some part of the light is absorbed by the solution
and the remaining part is transmitted through the solution which falls on the
photodetector system. The photodetector system measures the intensity of
transmitted light and converts it into the electrical signals that are sent to the
galvanometer.
The galvanometer measures the electrical signals and displays it in the digital
form. That digital representation of the electrical signals is the absorbance or
optical density of the solution analyzed.
If the absorption of the solution is higher then there will be more light absorbed
by the solution and if the absorption of the solution is low, then more lights will
be transmitted through the solution which affects the galvanometer reading
and corresponds to the concentration of the solute in the solution. By putting all
the values in the formula given in the below section one can easily determine
the concentration of the solution.
26. Single Beam Spectrophotometer
Single beam spectrophotometer is an analytical instrument in
which all the light waves coming from the light source passes
through the sample.
Therefore, the measurements are taken as the intensity of light
before and after the light pass through the sample.
These single beam spectrophotometers are more compact and
optically simpler than double beam spectrophotometers. And
these instruments are less expensive.
28. Double Beam Spectrophotometer
The light coming from the monochromator splits into two
beams.
One falls on the reference sample and another on the test sample.
The measurements taken from double beam spectrophotometers
are highly reproducible because electronic and mechanical effects
on both sample and reference beams are equal.
30. UV Spectrophotometer
• Specialized quartz is used in this type instead of normal glass
cuvette, and the light source used is either hydrogen or
deuterium lamps.
• Hydrogen lamp emits various continuous or discontinuous
spectral UV-light, which ranges between 200-450nm.
• It estimates the OD or transmittance for fluids and even
solutions.
32. Visible Spectrophotometer
• Either glass or plastic cuvettes can be used.
• Light source: tungsten halogen lamp.
• Tungsten lamps contain tungsten filament, which emits light of
visible spectrum ranging between 330-900nm.
• The life of the lamp is 1200h.
• This type can estimate the color change intensity according to
the change in concentration of moderately diluted solutions.
33. Infrared Spectrophotometer
• Conductive device: Nernst glowers.
• They have a long life.
• It helps in studying different vibrations generated by different
molecules at a specific wavelength.
• Near and mid-IR-rays cause rotational and harmonic
vibrations.
35. Nuclear magnetic resonance
spectrophotometer
NMR uses low energy radiation from the radio frequency (RF)
region of the electromagnetic spectrum. Only certain nuclei are
NMR active. Only nuclei containing odd mass numbers or odd
atomic numbers will give NMR signals. These nuclei possess a
property known as nuclear spin.
Gives entire structural information of the entire molecule as
well as the dynamic information of organic reactions.
Used to determine the structure of organic compounds
37. Atomic absorption
spectrophotometer
• Water in the sample is allowed to dissociate into ions after
subjecting it to evaporation using a flame.
• This dissociation leads to changes in the intensity of light as
observed by the detector.
• It helps in estimating the unknown concentration of the sample
• High in precision.
• Application in toxicology, testing of environmental samples, and
quality control in laboratories.
39. Mercury
spectrophotometer/analyzer
Mercury Analyzer is a portable multifunctional
atomic absorption spectrometer with Zeeman
background correction, which eliminates the effect
of interfering impurities.
It measures the trace amount of mercury in water.
41. Fluorometers
A fluorimeter measures the fluorescence or light
emitted by different fluorescing objects.
Fluorescence occurs when light of specific
wavelength hits and excites electrons in a sample,
and the electrons in that sample instantly emit light of
a different wavelength.
43. Spectroscopy vs Spectrophotometry
• Spectrophotometry may seem similar to spectroscopy.
• However, there is a vital difference between spectroscopy vs.
spectrophotometry. Spectroscopy is used to analyze matter based on the
wavelengths produced in the spectrum, while spectrophotometry analyzes
matter based on the intensity of light in each wavelength involved in
light’s interaction with matter (absorbance, reflectance, transmittance).
• Thus, spectrophotometry is based on spectroscopy and can be called an
application of the latter.
• Spectrophotometry applications are useful to measure the absorbance,
reflectance, and transmission of light by gases, liquids, and solids.
44. Precautions:
•The spectrophotometer should be turned on 10 to 15 minutes before
use.
•The device should be calibrated each time.
•The selected wavelength should be the maximum wavelength that the
solution can absorb.
•The sample used should not contain any substance which can
dissociate, react or change during the measurement.
•Since the absorbance depends on the concentration, sample
preparation should be within the acceptable concentration range.
