The document provides a comprehensive overview of spectrophotometry, detailing its principles, applications, and key components. It discusses the laws governing light absorption, types of spectrophotometers, and the significance of calibration curves. Additionally, the document highlights the wide-ranging applications of spectrophotometry in various scientific fields, emphasizing its importance in analytical chemistry.

1. SPECTROPHOTOMETRY
Dr. S.A. Ojedokun
Department of Chemical Pathology
LTH Ogbomoso
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2. Outline
• Introduction
• Light spectroscopy
• Principles of light Absorption
• Principles of Spectrophotometry
• Classifications/Types
• Interferences
• Quality assurance
• Applications
• Conclusion
• References
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3. Introduction
• Spectrophotometry is a method that hinges on the quantitative
analysis of molecules depending on how much light is absorbed by
coloured compounds.
• Important features of spectrophotometers are spectral bandwidth
(the range of colours it can transmit through the test sample), the
percentage of sample transmission, the logarithmic range of sample
absorption, and sometimes a percentage of reflectance
measurement.
• Photometry is the measurement of the amount of luminous light
(luminous intensity) falling on a surface from a source.
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4. Intro cont’d
• Spectrophotometry is a scientific analytical technique based on the
absorption of light by a solution at a particular wavelength with
relevant properties of the solution., e.g., concentration.
• Spectrophotometry depends on the light-absorbing properties of
substances or a derivative of the substance being analyzed.
• An instrument for measuring the intensity of light in a part of the
spectrum, as transmitted or emitted by particular substances is a
spectrophotometer
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5. Light spectroscopy
• Light is a form of energy propagating into space at a very high speed.
• As an electromagnetic wave travelling into space – it is radiant energy.
• The energy of light oscillates periodically between a minimum and a
maximum as a function of time – like a wave.
• The distance between two maxima or two minima, respectively of the
electromagnetic wave is defined as the wavelength, given in
nanometers (nm).
• Light behaves like discrete energy packets called photons whose
energy is inversely proportional to the wavelength
• The shorter the wavelength, the higher the energy
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6. Light spectroscopy cont’d
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7. Light spectroscopy cont’d
• Thus, the different components of light are characterised by a specific
wavelength.
• The sum of all components i.e. of all wavelengths, is called a
spectrum.
• More specifically, a spectrum represents a distribution of radiant
energy.
• For instance, the electromagnetic spectrum of visible light ranges
from approximately 390nm up to approximately 780 nm with
different colours.
• Each colour has a specific wavelength, e.g. red light has a wavelength
of 660 nm, while green light has a wavelength of 520 nm.
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8. 8

9. Light spectroscopy cont’d
• Optical spectroscopy is based on the interaction of light with matter.
• The light which is not absorbed by the object is reflected and can be
seen by the eye
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10. Principles of Light Absorption
10
Properties of Light

11. Beer’s Law
• The absorbance of light is directly proportional to both the
concentration of the absorbing medium and the thickness of the
medium.
• In Spectrophotometry the thickness of the medium is called the path
length.
• Beer’s law allows the measurement of samples of differing pathlength
and compares the results directly with each other.
• In basic terms: Absorbance = Concentration × Pathlength
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12. 12

13. Lambert’s Law
• The proportion of light absorbed by a medium is independent of the
intensity of incident light.
• A sample which absorbs 75% (25% transmittance) of the light will always
absorb 75% of the light, no matter the strength of the light source.
• Lambert’s law is expressed as I/Io=T
Where I = Intensity of transmitted light
Io = Intensity of the incident light
T = Transmittance
• This allows different spectrophotometers with different light sources to
produce comparable absorption readings independent of the power of the
light source.
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14. Beer-Lambert law
• When light, passes through a transparent cuvette filled with sample
solution, the light intensity is attenuated proportionally to the sample
concentration. In other words, a highly concentrated sample solution
will absorb more light.
• In addition, the attenuation is also proportional to the length of the
cuvette; a longer cuvette will lead to a higher absorption of light.
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15. • Mathematically,
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16. This relationship is called the Lambert-Beer law where:
1. The sample concentration is c.
2. The path length, d of the cuvette.
3. The extinction coefficient ε (epsilon) is a sample-specific constant
describing how much the sample is absorbing at a given wavelength
• When the path length is 1 cm and the concentration is 1% w/v, the
extinction coefficient is called specific absorbance (E )
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17. • The Lambert-Beer law allows for the determination of the sample
concentration from the measured absorbance value. If the extinction
coefficient ε and the path length d are known, then concentration c
can be calculated from absorbance A as given below:
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18. Beer’s law holds if the following conditions are met
• Incident radiation on the substance of interest is monochromatic.
• The solvent absorption is insignificant compared with the solute
absorbance.
• The solute concentration is within given limits.
• An optical interference is not present.
• A chemical reaction does not occur between the molecules of interest
and another solute or solvent molecule.
• The sides of the cell are parallel
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19. Calibration curve/ standard curve
• A solution of known concentration is prepared.
• Serial dilutions up to about 5 solutions of different concentrations.
• Spectrophotometric absorbance is set at zero using a blank.
• Measurement of absorbances of the solutions.
• Plotting of absorbances(y axis) against concentrations (x axis).
• Determination of the concentration of unknown solution using the
graph.
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20. • Calibration curve for glucose assay
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21. Principles of Spectrophotometry
The spectrophotometry technique is used to measure light intensity as
a function of wavelength using a spectrophotometer.
And this is achieved through:
1. Diffraction of the light beam into a spectrum of wavelengths
2. Directing the diffracted light onto an object
3. Reception of the light reflected or returned from the object
4. Detecting the intensities with a charge-coupled device
5. Displaying the results as a graph on the detector and then the
display device 21

22. Basic components of spectrophotometer
• Light source
• Monochromator
• Cuvette or sample cell
• Photo detector
• Readout device
• Recorder
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23. 23
Basic components of a spectrophotometer

24. Lamp source
1. Incandescent lamps
UV spectrum
• Deuterium-discharge lamp
• Mercury arc lamp
• Xenon arc lamp
• Hydrogen lamp
Visible and near infrared region
• Tungsten halogen lamp containing
• iodine or bromine
2. Laser (Light amplification by stimulated emission of radiation)
• This is a device used in spectrophotometry, which transform light of various frequencies into an extremely
intense, focused, and nearly non divergent beam of monochromatic light. 24

25. Important factors for a light source
• Range
• Spectral distribution within the range
• Stability of radiant energy
• Source of radiant production
• Temperature
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26. Monochromator
• Necessary to isolate a desired wavelength of light and exclude other
wavelengths
• Wavelength isolation is a function of the type of device used and the
width of the entrance and exit slits.
• Devices used to obtain monochromatic light include
• Filters (colored glass and interference)
• Prisms
• Diffraction gratings
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27. Colored glass filters
• Least expensive
• Simple, although not always precise
• Usually pass a relatively wide band of radiant energy
Interference filters
• Produces a monochromatic light based on the principle of constructive
interference of waves
Prism
• A narrow beam of light focused on a prism is refracted as it enters the denser
glass.
• The prism can be rotated, allowing only the desired wavelength to pass through
an exit slit.
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28. Diffraction gratings
• Most commonly used as monochromators.
• 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
• Diffraction grating consists of many parallel grooves (15,000-30,000
per inch) etched onto a polished surface.
• Because the multiple spectra have a tendency to cause stray light
problems, accessory filters are used.
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29. Cuvette/sample cell
• Are small vessels used to hold liquid samples to be analysed in the
light path of the spectrophotometer.
• Can be round or square
• Square cuvettes have advantages over round cuvettes in that there is
less error from lens effects, orientation and refraction.
• It can be made of Glass (Visible range), or quartz(UV & Visible range)
• Light path must be kept constant
• Cuvettes with scratched optical surfaces should be discarded as they
scatter light.
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30. Photodetector
• Detects transmitted radiant energy and converts it into an equivalent
amount of electricity
Types include:
• Photocell/Barrier layer cell
• Phototube
• Photomultiplier tube
• Photodiode
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31. Photocells/Barrier layer cell:
• Least expensive and durable
• Composed of a film of light sensitive materials: Selenium on a plate of iron
covered by a thin transparent layer of silver
• When exposed to light, electrons in the light sensitive materials are excited
and released to flow through highly conductive silver
• Resistance prevents ions from flowing in the opposite direction towards
the iron thus forming a barrier.
• EMF is generated from the resistance which can be measured.
• It is temperature sensitive and becomes non linear at very high or very low
levels of illumination.
• Output is not easily amplified so it is used in instruments with illumination
levels such that there is no need to amplify the signals.
• Light-sensitive
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32. Phototube
• Similar to photocell but needs an outside voltage to operate it.
• Contains cathode and anode enclosed in a glass case
• Cathode: made up of lithium or rubidium 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
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33. Photomultiplier(pm) tube
• Detects and amplifies radiant energy, hence more sensitive than the
phototube.
• 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 a single electron
• Initial electron emission at the cathode triggers a multiple cascade of
electrons within the PM tube
• The accumulation of light striking the anode produces a current signal
measured in amperes
• They are used in instruments designed to be extremely sensitive to very
low light levels and light flashes of very short duration
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34. Photodiode
• In a photodiode, absorption of radiant energy by a reverse-biased pn-
junction diode (pn, positive-negative) produces a photocurrent that is
proportional to the incident radiant power.
• Photodiode array (PDA) detectors are available in integrated circuits
containing 256 to 2,048 photodiodes in a linear arrangement
• Each photodiode responds to a specific wavelength,
• Its excellent linearity (6–7 decades of radiant power), speed, and
small size make them useful in applications where light levels are
adequate
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35. Readout devices
Analog
• Uses deflector pin on a meter
• Zero error is common
• Parallax error
• Easily affected by current/light voltage
• No longer popular
Digital
• Now common with newer spec
• Limit zero error
• No parallax error
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36. Classifications
A. Electromagnetic forms
 UV, ViS, & IR
B. Geometry designs
 Scanning & Array
C. Optical pathways
 Single & double beam
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37. A. Based on electromagnetic form
1. UV spectrophotometry
• Uses light over the UV range (180-
400nm)
• A prism of suitable material and
geometry will provide a continuous
spectrum in which the component
wavelengths are separated in space
• In addition to prisms, diffraction
gratings are also employed for
producing monochromatic light
• Quartz cuvettes used to hold
samples
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38. 2. ViS Spectrophotometry
• Uses the visible range (~400-700nm) of the electromagnetic radiation
spectrum
• Plastic or glass cuvettes can be used for visible spectrophotometry
3. Infrared spectroscopy (IR)
Infrared spectrum refers to a spectrum greater than 760nm, which
is the most commonly used spectral region of organic compounds,
and can analyze a variety of conditions (gas, liquid, solid) of the sample.
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B. Based on Geometry designs
1. Scanning spectrophotometer
• The working principle of a conventional scanning spectrophotometer
is based on the measurement of the transmittance value at each
single wavelength.
• The transmittance at this specific wavelength is recorded. The whole
spectrum is obtained by continuously changing the wavelength of the
light (i.e. scanning) incoming onto the sample solution by rotating the
grating

40. Scanning spectrophotometer
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2. Array spectrophotometer
• In this configuration, the sample in the cuvette simultaneously absorbs
different wavelengths of light. The transmitted light is then diffracted by a
reflection grating located after the cuvette
• Subsequently, the diffracted light of various wavelengths is directed onto
the detector.
• The detector, with its long array of photosensitive, semiconductor
material, allows for simultaneous measurement of all wavelengths of the
transmitted light beam.
• This design is also known as "reverse optics"

42. Array spectrophotometer
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C. Based on optical paths
1. Single beam configuration
The light beam is directly guided through the sample onto the
detector. A cuvette containing only solvent has to be measured
first to determine the blank value. After measuring the blank
value, the solvent cuvette is replaced by a cuvette containing the
sample to measure the absorption spectrum of the sample.

44. 2. Double-beam configuration
In a double-beam configuration, the light beam is split into a
reference and a sample beam.
a) Simultaneous in time: The light beam of the lamp is split into two
beams of equal intensities. Each beam passes through a different
cuvette; one is the reference cuvette, whereas the second cuvette
contains the sample solution. The intensities of both beams are
measured simultaneously by two detectors.
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45. Simultaneous-in-time
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b) Alternating in time: This configuration is achieved by directing
the light path with an optical chopper (OC), which is a rotating
sectional mirror. The light is directed alternately through a
sample and a reference cell. A unique detector measures both
light beams one after the other.

47. Interference
• Interference is phenomenon that leads to changes in intensity of the
signal in spectrophotometry.
Types of interference
• Optical interference is a phenomenon in which two wavelengths
superimpose to form a greater or lower wavelengths.
• Chemical interference arises out of the reaction between different
interferents and the analyte.
• Physical interference are due to physical properties of the sample e.g.
impurities in the solution
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48. Quality assurance
Wavelength accuracy
• Checked using standard absorbing solutions or filters with maximal
absorbance of known wavelength.
Stray light
• Refers to any wavelength outside the band transmitted by the
monochromator. Most common causes are light reflection from scratches
on optical surfaces or dust particles in the light path. Stray light is detected
and eliminated by using cutoff filters.
Linearity
• Refers to the difference between the actually measured value and the
value derived from the equation. It is checked using coloured solutions of
different concentrations labelled with expected absorbances.
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49. Applications of Spectrophotometry
• Chemical reactions
• End point reaction
• Kinetics reaction
• Fixed time
• Two-point absorbance
• Fixed wavelength
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50. • Bio applications
• Use in clinical laboratory analysis
• Dissolution/ in vitro releases assay of drugs
• Quantification of DNA, RNA and proteins
• Dye, ink and paint industries
• Heavy metal and organic matter from environmental/agricultural
samples
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51. • Other applications
• Quantifying concentrations of compounds.
• Determining the structure of a compound.
• Finding functional groups in chemicals.
• Determining the molecular weight of compounds.
• Determining the composition of materials
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52. Conclusion
• The use of spectrophotometers spans various scientific fields,
such as physics, materials
science, chemistry, biochemistry, chemical engineering,
and molecular biology semiconductors, laser and optical
manufacturing, printing and forensic examination, as well as in
laboratories for the study of chemical substances.
• Spectrophotometry continues to enjoy wide popularity due to the
common availability of its instrumentation and simplicity of
procedures, as well as speed, precision and accuracy of its technique.
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53. References
• Tietz fundamentals of clinical chemistry, 2008 fifth edition chapter 4
pg 63-80.
• Cosimo A. De Caro, Haller Claudia. UV/VIS Spectrophotometry -
Fundamentals and Applications. 2015
https://www.researchgate.net/publication/321017142
• The principles of use of a spectrophotometer and its application in
the measurement of dental shades Chapter 11 Spectrophotometer
• Mass spectrometry
https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/
massspec/masspec1.htm
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Thank you