SPECTROSCOPIC METHOD

OF ANALYSIS
At the end of this discussion, the students should be able to:
• Define spectroscopy and spectrophotometer
• Explain what is electromagnetic radiation and its association with
spectroscopy
• Know and differentiate the different types of spectrophotometer
• Describe the specific functions of the different parts of spectrophotometer
• Apply Beer’s law to determine the concentration of the unknown analyte
Spectroscopy is the study of how matter absorbs and emits light and other
radiation. It is the process of separating electromagnetic radiation into its
constituent wavelengths similar to how a prism splits light into a rainbow of
colors.
Electromagnetic radiation, is a form of energy whose behavior is described by
the properties of both waves and particles. The optical properties of
electromagnetic radiation, such as diffraction, are explained best by describing
light as a wave.
Electromagnetic radiation is made up of oscillating electric and magnetic fields
that travel in a linear path at a constant speed.
• Vacuum – Electromagnetic radiation travels at the speed of light c=________
The electric and magnetic fields oscillate perpendicularly to each other and the
wave's propagation direction.
ENERGY:
➢ Electromagnetic waves
➢ Characterized by _______ and _______
WAVELENGTH:
➢ Distance between two successive _______ and expressed in _______
NOMINAL WAVELENGTH

*Significant errors in absorbance values can be caused by even small wavelength alterations
PLANCK’S FORMULA:
E = hv
Where:
E – energy of photon in Joules or eV
h – 6.626 x10 ^-34 erg sec
V - frequency
• FREQUENCY
• WAVE FREQUENCY IS INVERSELY PROPORTIONAL TO WAVELENGTH
• WAVELENGTH IS INVERSELY PROPORTIONAL TO FREQUENCY AND ENERGY
• Moving particles - ________
• Moving electromagnetic disturbances - ________

• Length of electromagnetic wave

• Physical distance between two crests
• Meters, centimeters, NANOMETERS
• number of the moving electromagnetic waves that pass a fixed point in 1
sec
• SYMBOL: lowercase Greek letter nu, ν
• Cycles per second, or hertz (Hz)
• ELECTROMAGNETIC SPECTRUM OF LIGHT IS BROAD
• VISIBLE REGION
• Radio and television waves:
➢ Very low energy
➢ Do no harm
• Microwaves:
➢ Low energy
➢ Can be dangerous
• Infrared region
➢ Wavelengths are shorter and have high energy than televisions and
microwave
➢ No harm
• Ultraviolet region
➢ Shorter wavelengths
➢ Known to cause harm
• X-Ray
• Gamma rays
• VISIBLE SPECTRUM: 400-700 nm
• ULTRAVIOLET REGION: <400 nm
• INFRARED REGION: >700nm
• Some wavelengths of visible light are absorbed, which causes an object to
display color.

❑ ULTRAVIOLET-VISIBLE (UV-VIS) SPECTROPHOTOMETRY

❑ INFRARED (IR) SPECTROPHOTOMETRY
❑ ATOMIC SPECTROSCOPY
• A plot of the amount of light absorbed by a sample against the wavelength of
the light is called an absorption spectrum.

• Plotting the amount of light transmitted by a sample rather than the light
absorbed.
• The intensity of light striking the light sensor when a blank solution is held
in the path of the light. Symbol I(0)
• Blank solution - Solution that includes every chemical species found in the
standards and measurement samples—aside from the analyte species.
• Absorbance then is a parameter that increases linearly with concentration
and is important for quantitative analysis.

FORMULA:
A= 2- log%T
 • Involves the measurement of light transmitted by a solution to determine the
concentration of the light-absorbing substances in the solution.

• Simplest type
• One measurement at a time at one specified wavelength

• Splits monochromatic light into two components

• Additional beam
a) Double-beam in space – 2 photodetectors
b) Double-beam in time – 1 photodetector
❖ chopper

1. Stable source of radiant energy

2. Filter
3. Sample holder
4. Radiation detector
5. Signal processor
6. Readout device
I. Light/Radiant Source
✓ Polychromatic light and sufficient radiant energy or power
✓ Linear

• Continuum source: emits radiation that changes in intensity.

Examples:
✓ Tungsten, deuterium, xenon lamps
• Line source: emits limited radiation and wavelength
Examples:
✓ Mercury and sodium vapor lamps, hollow cathode lamp
✓ LASER
✓ Range
✓ Spectral distribution
✓ Source of radiant production
✓ Stability of the radiant energy
✓ Temperature

ALTERNATIVES FOR TUNGSTEN LIGHT BULB

✓ Mercury arc
✓ Deuterium lamp
✓ Hydrogen lamp
✓ Xenon lamp
✓ Merst glower
✓ Lobar (silicone carbide)
II. ENTRANCE SLIT
✓ Minimizes unwanted or stray light
✓ Stray light: any wavelengths outside the band transmitted by the
monochromator
III. MONOCHROMATOR
✓ ISOLATES specific wavelength of light
❑ PRISMS: Wedge-shaped pieces of glass, quartz, or sodium chloride
❑ DIFFRACTION GRATINGS
✓ Most commonly used
✓ Better resolution than prism
✓ Holographic gratings
❑ FILTERS
✓ Simple, least expensive, not precise, but useful monochromators
✓ These are made by placing semi-transparent silver films on both sides of a
dielectric (magnesium fluoride)
✓ Wide band of radiant energy and have a low transmittance of the selected
wavelength
IV. EXIT SLIT
✓ Controls the width of the light beam
✓ Bandpass
✓ Accurate absorbance measurement – bandpass <1/5 the natural bandpass
of the spectrophotometer
✓ Spectral purity of the spectrophotometer is reflected by the bandpass
V. CUVETTE
✓ Sample cell
✓ Holds the solution
KINDS OF CUVETTES
✓ Alumina silica glass
✓ Quartz/plastic
✓ Borosilicate glass
✓ Soft glass
VI. PHOTODETECTOR
✓ Detects and converts transmitted light into photoelectric energy
✓ Detects the amount of light

Kinds of Photodetectors
➢ BARRIER CELL
✓ Simplest, least expensive
✓ Basic photodetector – visible region
➢ PHOTOTUBE
✓ Cathode and anode
➢ PHOTOMULTIPLIER
✓ Most sensitive photodetector
✓ Should never be exposed to room light
➢ PHOTODIODE
VII. METER OR READOUT DEVICE
✓ Displays the output of the detection system

✓ Contaminating Substance
✓ QUALITATIVE ANALYSIS – Incorrect absorption spectrum
✓ QUANTITATIVE ANALYSIS – Higher absorbance
• SAFETY and CLEANLINESS
• CALIBRATION
✓ Concentration of the unknown substance is directly proportional to the
absorbed light and inversely proportional to the amount of light transmitted

Absorbance:
Amount of light absorbed

Formula:
A= abc = 2 – log%T
Where:
A = absorbance
a = molar absorptivity
b = length of light through the solution
c = concentration of absorbing molecules
Unknown solution: = Au/As x Cs
• Harvey, D. T. (1999). Modern Analytical chemistry.
http://dspace.fudutsinma.edu.ng/xmlui/handle/123456789/2059
• Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2013). Clinical chemistry: Principles, Techniques, and
Correlations. Lippincott Williams & Wilkins.
• Rodriguez, M. T. (2023). Review Handbook in Clinical Chemistry. C & E Publishing, Inc.