Principle, Beers law, types, instrumentation, difference between colourimeter and spectrophotometer.

1. Fundamentals of
Spectrophotometer
By
Muhammad Asif Shaheen
Lecturer KEMU Lahore.

2. Fundamentals of Spectrophotometer
Introduction
Spectrophotometer
 An instrument that measures the amount of photons (the intensity of light)
absorbed after it passes through sample at specific wavelength.

3. Fundamentals of Spectrophotometry
• Almost all of the energy available at Earth’s surface comes from the sun. The sun
gets its energy from the process of nuclear fusion.
• This energy eventually makes its way to the outer regions of the sun and is radiated
or emitted away in the form of energy, known as electromagnetic radiation.
• A particle of electromagnetic radiation is known as a photon.
• Electromagnetic radiation, also known as radiant energy (or radiation), is spread in
the form of electromagnetic waves.

4. • Electromagnetic waves are waves that can cause charged particles (such as
electrons) to move up and down. These waves have both electrical and magnetic
properties and can travel through gases, liquids, solids, and through empty space (or a
vacuum) at nearly 300,000 kilometers per second (the speed of light).
• Electromagnetic waves are characterized by wavelength and frequency.
• The wavelength is the distance between two wave crests or troughs. The highest point
of a wave is called the crest, and the lowest point of a wave is called the trough.
• Frequency is expressed in hertz (Hz) and refers to the number of wavelengths that
pass a fixed point in 1 second. The shorter the wavelength is, the higher its frequency
will be. The reverse is also true. For example, radio waves have the longest wavelength
and the lowest frequency.

6. • The electromagnetic spectrum is the term used by scientists to describe the entire
range of light that exists. From radio waves to gamma rays, most of the light in the
universe is, in fact, invisible to us! Light is a wave of alternating electric and magnetic
fields

9. Fundamentals of Spectrophotometry
Properties of Light
1.) Particles and Waves
 Light waves consist of perpendicular, oscillating electric and magnetic fields
 Parameters used to describe light
- amplitude (A): height of wave’s electric vector
- Wavelength (λ): distance (nm, cm, m) from peak to peak
- Frequency (ν): number of complete oscillations that the waves makes each
second
 Hertz (Hz): unit of frequency/second-1
(s-1
)
 1 megahertz (MHz) = 106
Hz

10. Fundamentals of Spectrophotometry
Properties of Light
1.) Particles and Waves
 Parameters used to describe light
- Energy (E): the energy of one particle of light (photon) is proportional to its
frequency
νhE =
where: E = photon energy (Joules)
ν = frequency (sec-1
)
h = Planck’s constant (6.626x10-34
J-s)
As frequency (ν) increases, energy (E) of light increases

11. Fundamentals of Spectrophotometry
Properties of Light
1.) Particles and Waves
 Relationship between Frequency and Wavelength
 Relationship between Energy and Wavelength
λν /c=
where: c = speed of light (3.0x10810
cm/s in vacuum))
ν = frequency (sec-1
)
λ = wavelength (cm)
λ
hc
E =
As frequency (λ) decreases, energy (E) of light increases

12. Fundamentals of Spectrophotometry
Absorption of Light
1.) Colors of Visible Light
 Many Types of Chemicals Absorb Various Forms of Light
 Light is made up of wavelengths of light, and each wavelength is a particular
colour. The colour we see is a result of which wavelengths are reflected back
to our eyes.
 White light is actually made of all of the colours of the rainbow because it
contains all wavelengths, and it is described as polychromatic light. Light from
a torch or the Sun is a good example of this.
 Light from a laser is monochromatic, which means it only produces one colour.

14. Fundamentals of Spectrophotometry
Absorption of Light
2.) Colors of object
 Objects appear different colours because they absorb some colours (wavelengths) and
reflected or transmit other colours. The colours we see are the wavelengths that are
reflected or transmitted.
 For example, a red shirt looks red because the dye molecules in the fabric have
absorbed the wavelengths of light from the violet/blue end of the spectrum.

16. Fundamentals of Spectrophotometry
Absorption of Light
3.) Beer’s Lambert's Law
 Beer’s law states that the concentration of a substance is directly proportional
to the amount of light absorbed or inversely proportional to the logarithm of the
transmitted light.
 Lambert's law stated that absorbance of a material sample is directly
proportional to its thickness (path length)
 Light of a particular wavelength enters the ‘sample’.
 Light scatters from particles in solution, reducing light transmission
 Light is absorbed by molecules/particles reducing light transmission
 The relative amount of light absorbed (A) through a sample is dependent on:
- distance the light must pass through the sample (cell path length - b)
- amount of absorbing chemicals in the sample (analyte concentration – c)
- ability of the sample to absorb light (molar absorptivity - α

18. Fundamentals of Spectrophotometry
Absorption of Light
3.) Beer’s Law
 The relative amount of light passing through the sample is known as the
transmittance (T)
oI
I
T =






×=
oI
I
T 100%Percent transmittance

19. Fundamentals of Spectrophotometry
Absorption of Light
3.) Beer’s Law
 Absorbance (A) is the relative amount of light absorbed by the sample and is
related to transmittance (T)
 It cannot be measured directly by a spectrophotometer but rather is
mathematically derived from % T
Absorbance is sometimes called optical density (OD)
)(loglog T
I
I
A
o
−=





−=

20. Fundamentals of Spectrophotometry
Absorption of Light
3.) Beer’s Law
 Absorbance is useful since it is directly related to the analyte concentration, cell
pathlength and molar absorptivity.
 This relationship is known as Beer’s Law
abcA =
where: A = absorbance (no units)
α= molar absorptivity (L/mole-cm)
b = cell pathlength (cm)
c = concentration of analyte (mol/L)
Beer’s Law allows compounds
to be quantified by their ability
to absorb light, Relates directly
to concentration (c)

21. Fundamentals of Spectrophotometry
Absorption of Light
3.) Beer’s Law
 Absorptivity depends on molecular structure and the way in which the
absorbing molecules react with different energies. For any particular molecular
type, absorptivity changes as wavelength of radiation changes. The
amount of light absorbed at a particular wavelength depends on the molecular
and ion types present and may vary with concentration, pH, or temperature.
 b is the length of light path (1 cm) through the solution.
 Because the path length and molar absorptivity are
constant for a given wavelength
cA ∝

23. Fundamentals of Spectrophotometry
Spectrophotometer
1.) Basic Design
 An instrument used to make absorbance or transmittance measurements is
known as a spectrophotometer

24. Fundamentals of Spectrophotometry
Spectrophotometer
1.) Basic Design
 Light Source:
 The light source typically yields a high output of polychromatic light over a
wide range of the spectrum
Low pressure of iodine or bromin
(vacuum) to increase life of lamp up to
2000 to 5000 hours
Tungsten Filament

25. • Types of light sources used in spectrophotometers include:
• Incandescent lamps and lasers
• Incandescent Lamps:
• Halogen Lamp
• Also known as tungsten or quartz lamp, and the wavelength range of halogen lamp is
in the visible light region, which is in the range of 320nm to 700 nm. If the instrument
is equipped with a halogen lamp only, it means the instrument can only measure
visible light. General halogen lamp life is about 2000 hours, or more
• LED Lamp
• Produce a single wavelength of light, thus, LED lamp does not require a
monochromator. Its life is very long. LED light source has little variation in bandwidth,
and it’s stable. LED lamp is a low-cost light source.

26. • Deuterium Lamp
• A continuous spectrum in the ultraviolet region is produced by electrical excitation of
deuterium at low pressure. It is Also known as D2 lamp, its wavelength range is from
of 190nm - 370nm. Because of its high temperature behavior, the normal glass
housing is not suitable, but requires quartz, or other materials. Life time of a typical
deuterium lamp is about 1000 hours. An UV / Vis spectrophotometer, will design a
deuterium lamp with a halogen lamps, in order to cover the entire UV and visible light
wavelength.
• Xenon Lamp
• Xenon lamp provides high energy light source, and it can reach a steady state in a
short time period. Its light covers the entire UV and visible wavelength range, from
190nm to 1100nm. An xenon lamp flashes in a frequency of 80Hz, so that the life
time is longer than deuterium lamp or halogen lamp. However, the cost of a xenon
lamp is higher.
• Hydrogen Gas Lamp and Mercury Lamp are commonly used in UV absorption
measurements as well as visible light.
• Globar (silicon carbide rod): Infra-Red Radiation at wavelengths: 1200 - 40000 nm
NiChrome wire (750 nm to 20000 nm) for IR Region.

27. • Laser Sources:
• Acronym for: light amplification by stimulated emission of radiation
• These devices transform light of various frequencies into an extremely intense,
focused, and nearly non-divergent beam of monochromatic light
• Used when high intensity line source is required
• Cost as varied as possible wavelength range: from two-dollar pointers to million-dollar
devices
• Properties of laser sources include
• Spatial coherence: Wave fronts of all the photons are “launched in unison,” all
moving in step with the others (coherent).
• Strong, concentrated, tight beam (directional).
• Laser light is typically monochromatic – emitting only one wavelength

28. Coherent Laser light

29. Fundamentals of Spectrophotometry
Spectrophotometer
1.) Basic Design
 Wavelength Selector (monochromator
 Spectral Isolation
 A system for isolating radiant energy of a desired wavelength and excluding
that of other wavelength is called spectral isolation and it is achvied with the
help of Monochromator.
 Monochromator consists of these parts:
 Entrance slit –
 Collimating lens or mirror –
 Dispersion element: A special plate with hundreds of parallel grooved lines.
The grooved lines act to separate the white light into the visible light
spectrum
Devices used for spectral isolation include:
Filters,
Prisms
Diffraction gratings.

30. • Filters:
• In applications requiring control of light transmissions, optical filters can
serve as an effective means of selectively blocking or transmitting light
based on certain properties, such as wavelengths or intensity
Colored Optical Filters
• An interference filter or dichroic filter is an optical filter that reflects
one or more spectral bands or lines and transmits others, while
maintaining a nearly zero coefficient of absorption for all wavelengths of
interest.
• Absorption filters, commonly manufactured from dyed glass or
pigmented gelatin resins. These compounds absorb some wavelengths of
light while transmitting others

31. • Filters ranges:
• A) Long-Pass Filter: A long-pass configuration transmits longer
wavelengths above a specified range while attenuating shorter
wavelengths.
• B) Short-Pass Filter: A short-pass configuration transmits shorter
wavelengths over an active range while attenuating longer wavelengths.
• B) Bandpass Filter: Short-pass and long-pass filters can be combined to
form a bandpass filter, which features lower transmittance values and
rejects any wavelengths outside a predetermined interval. The size of the
interval depends on the number of filter layers used in the device.

32. • Filters:
• Thin-Film Optical Filters
• Although colored-glass filters can be configured in a wide variety of forms, their
transmission curves normally cannot be customized to fit a specific application.
Thin-film optical filters, however, can be designed to meet nearly any type of
transmission curve, making them more effective for certain applications. In
addition to standard light control systems, thin-film filters can be used to transmit
or block laser light, which may contain extra wavelengths than those in the main
laser line.
• Neutral-Density Filters
• A neutral-density filter is typically used to attenuate light at all wavelengths
equally.

34. • Prism
• The prism is another type of monochromator. A narrow beam of light focused on
a prism is refracted as it enters the more dense glass. Short wavelengths are
refracted more than long wavelengths, resulting in dispersion of white light into a
continuous spectrum. The prism can be rotated, allowing only the desired
wavelength to pass through an exit slit

35. • Diffraction gratings
• A diffraction grating consists of many parallel grooves (15,000 or 30,000 per inch)
etched onto a polished surface (Thin layer of aluminum-cupper alloy on flat glass
surface)..
• Diffraction, the separation of light into component wavelengths, is based on the
principle that wavelengths bend as they pass a sharp corner.
• The degree of bending depends on the wavelength. As the wavelengths move past
the corners, wave fronts are formed.

36. Fundamentals of Spectrophotometry
Spectrophotometer
1.) Basic Design
 Sample Cell: sample container of fixed length (b).
- Usually square cuvet
- Made of material that does not absorb light in the wavelength
range of interest
1. Glass – visible region
2. Quartz – ultraviolet
3. NaCl, KBr – Infrared region

37. Fundamentals of Spectrophotometry
Spectrophotometer
1.) Basic Design
 Sample Cell:
 A cuvette (French: cuvette = "little vessel") is a small tube
of circular or square cross section, sealed at one end, and
designed to hold samples for spectroscopic experiments.

38. Spectrophotometer
1.) Basic Design
Photo Detector:
 Measures the amount of light passing through the sample.
 Usually works by converting light signal into electrical signal
 The least expensive of the devices is known as a barrier-layer cell, or
photocell.
 The photocell is composed of a film of light-sensitive material,
frequently selenium, on a plate of iron. Over the light-sensitive material
is a thin, transparent layer of silver. When exposed to light, electrons
in the light-sensitive material are excited and released to flow to the
highly conductive silver in comparison with the silver, a moderate
resistance opposes the electron flow toward the iron, forming a
hypothetical barrier to flow in that direction. Consequently, this cell
generates its own electromotive force, which can be measured. The
produced current is proportional to incident radiation.
Fundamentals of Spectrophotometry

41. Spectrophotometer
1.) Basic Design
Phototube:
 A phototube is similar to a barrier-layer
cell in that it has photosensitive material that gives off
electrons when light energy strikes it.
 It differs in that an outside voltage is required for operation.
 Phototubes contain a negatively charged cathode and a positively
charged anode enclosed in a glass case.
 The cathode is composed of a material (e.g., rubidium or lithium) that
acts as a resistor in the dark but emits electrons when
exposed to light.
 The emitted electrons jump over to the positively charged anode,
where they are collected and return through an external, measurable
circuit. The cathode usually has a large surface area.
Fundamentals of Spectrophotometry

43. Spectrophotometer
1.) Basic Design
Phototube:
 The third major type of light detector is the photomultiplier (PM) tube, which
detects and amplifies radiant energy.
 incident light strikes the coated cathode, emitting electrons. The electrons are
attracted to a series of anodes, known as dynodes, each having a successively
higher positive voltage These dynodes are of a material that gives off many
secondary electrons when hit by single electrons. Initial electron emission at
the cathode triggers a multiple cascade of electrons within the PM tube itself.
Because of this amplification, the PM tube is 200 times more sensitive than the
phototube..
Fundamentals of Spectrophotometry

44.  PM tubes are used in instruments designed to be extremely sensitive to very low
light levels and light flashes of very short duration.
 The accumulation of electrons striking the anode produces a current signal,
measured in amperes, that is proportional to the initial intensity of the light. The
analog signal is converted first to a voltage and then to a digital signal through
the use of an analog to- digital (A/D) converter. Digital signals are processed
electronically to produce absorbance readings

45. • Readout device.
• In the past nearly all spectrophotometer used ammeters or galvanometers. Newer
digital devices and printers have now replaced these, and many instruments relay
their electrical output directly to computer circuits where calculations are performed,
allowing direct reporting of sample concentration.
• Microprocessor and recorders

46. Fundamentals of Spectrophotometry
Spectrophotometer
2.) Types of Spectrophotometers
 Single-Beam Instrument: sample and blank are alternatively
measured in same sample chamber.

47. Fundamentals of Spectrophotometry
Spectrophotometer
2.) Types of Spectrophotometers
 Double-Beam Instrument
- Continuously compares sample and blank
- Automatically corrects for changes in
electronic signal or light intensity of source

49. Single Beam Spectrophotometer
Advantages of Single Beam
• Single beam instruments are less
expensive
• High energy throughput due to
non-splitting of source beam
results in high sensitivity of
detection
Disadvantages
• Instability due to lack of
compensation for disturbances like
electronic circuit fluctuations,
voltage fluctuations, mechanical
component’s instability or drift in
energy of light sources. Such drifts
result in abnormal fluctuations in
the results.

50. Advantages of Double Beam
• Modern improvements in optics
permit high level of automation
and offer the same or even better
level of detection as compared to
earlier single beam systems.
Instability factors due to lamp drift,
stray light, voltage fluctuations do
not affect the measurement in
real-time.
• Little or no lamp warm up time is
required. This not only improves
throughput of results but also
conserves lamp life
Disadvantages
• The cost factor is more than offset
by the advantages offered by
modern double beam systems
Double Beam Spectrophotometer

51. Deference between Colorimeter and Spectrophotometers
Colorimeters
• . A colorimeter is generally any tool that
characterizes color samples to provide an
objective measure of color characteristics
• Colorimeters use a coloured light beam to
measure sample concentration.
• Colorimeter uses a tristimulus absorption
filter to isolates a broad band of
wavelengths. It is generally rugged and less
complex instrument than a
spectrophotometer.
• In colorimetry, coloured light passes through
an optical filter to produce a single band of
wavelengths.
• Both of these techniques use the Beer-
Lambert Law to determine concentration.
The difference is that colorimeters measure
the absorbency of light in a sample
Spectrophotometers
• A spectrophotometer is a photometer that
can measure intensity as a the wavelength
of light.
• Spectrophotometers use a white light that is
passed through a slit and filter to analyse
samples.
• Spectrophotometer uses a interference filter
or a grating and prism to isolate a narrow
band of wavelengths. It is a more complex
instrument than a colorimeter.
• spectrophotometers, the white light is
passed through a special filter that
disperses the light into many bands of
wavelengths.
• spectrophotometers measure the amount of
light that passes through it.

52. Colorimeter