Spectrophotometry colorimetry fluorometry FTIR Shrraddha Suman M. Sc. (Plant science) Khallikote university (unitary) Odisha

1. Submitted By:
SHRRADDHA SUMAN
PG 2nd YEAR (4th SEM)
ROLL NO.: PG20BO-08
EXAM ROLL NO.: 012004BO015
SPECTROPHOTOMET
RY
DEPARTMENT OF BOTANY
& BIOTECHNOLOGY

2. CONTENTS
• Introduction
• Spectrophotometry
 History
 Spectrophotometer
 Wavelength
 Beer Lambert Law
 Application
 Colorimetry
 Application
 Fluorometry
• Application
• FTIR
• Application
• Conclusion
• Reference

3. INTRODUCTION
• Spectroscopy is the study of the interaction between
electromagnetic radiation & matter.
• Nature of interaction: Absorption
Emission
Scattering
Spectroscopy
Qualitative
Quantitative

4. SPECTROPHOTOMETRY
• It is a branch of electromagnetic spectroscopy that deals with
measurement of intensity of light at selected wavelength.
• The basic principle is that each compound absorbs or transmits light
over a certain range of wavelength.
• It uses photometers known as spectrophotometers that can measure
the intensity of a light beam at different wavelength.
HISTORY
• Invented by Arnold O. Beckman in 1940.
• The spectrophotometer was created with the aid of his colleagues at his
company National Technical Laboratories.
Beckman Model DB Spectrophotometer (a
double beam model), 1960

5. SPECTROPHOTOMETER
• It is an instrument composed of two units, a spectrometer that produces
light of a definite wavelength and a photometer to measure the intensity
of the transmitted or absorbed light.
• It measures the amount of light absorbed, or the intensity of color at a
given wavelength.
• The intensity of color can be given a numerical value by comparing the
amount of light prior to passing it through the sample and after passing
through the sample.
• These quantitative measurements of light absorbed are the Transmittance
and the Absorbance.

7. WAVELENGTH
• It describes a position within a spectrum i.e. the distance between 2
peaks as the light travels in a wave-like manner.
• It is measured as nanometer.
Near Ultraviolet region 200-380nm
Visible region 380-750nm
Near Infrared region 750-2000nm

8. BEER LAMBERT LAW
• The two laws governing the absorption of radiation are known as
Lambert’s law and Beer’s law.
• LAMBERT’S LAW: It states that when a monochromatic light passes
through a medium , the intensity of transmitted light decreases
exponentially as the thickness of absorbing material increases.
• BEER’S LAW: It states that the intensity of transmitted monochromatic
light decreases exponentially as the concentration of the absorbing
material increases.
The relationship between concentration, length of the light path and the light
absorbed by a particular substance is expressed mathematically by:

9. APPLICATIONS
Detection of concentration of substances
Detection of impurities
Monitoring dissolved oxygen content in freshwater and marine ecosystems
Characterization of proteins
Detection of functional groups
Respiratory gas analysis in hospitals
Molecular weight determination of compounds

10. COLORIMETRY
• Colorimetry is a method for determining the concentration of
biochemical compounds.
• The basic principle involved is that when white light passes
through a coloured solution, some wavelength are absorbed
more than others.
• The earliest colorimeters relied on the human eye to match the
color of a solution with that of one of a series of coloured discs.
• The result obtained were too subjective and are not particularly
accurate.

11. COLORIMETER
• It is an apparatus that allows the absorbance of a solution at a particular
frequency (colour) of visible light to be determined.
• It helps to determine the concentration of a known solute, since it is proportional
to the absorbance; i.e. a more concentrated solution gives a higher absorbance
reading.
• Filter in the colorimeter is used to select the color of light which the solute
absorb the most, in order to maximize the accuracy of the experiment.
• Note: the color of absorbed light is the opposite of the color of the specimen.
COLORIMETER

12. Observed Color of
Compound
Color of Light Absorbed Approximate Wavelength
of Light Absorbed
Green Red 700 nm
Blue-green Orange-red 600 nm
Violet Yellow 550 nm
Red-violet Yellow-green 530 nm
Red Green 500 nm
Orange Blue 450 nm
Yellow Violet 400 nm

13. APPLICATIONS
• Colorimeters are used for a wide variety of applications in the
chemical and biological fields including:
APPLICATIONS
Analysis
of blood
Analysis
of water
Analysis of
soil
nutrients
Analysis
of
foodstuffs
Determination
of solution
concentration
Determination
of reaction
levels
Determination
of bacterial
crop growth

14. FLUOROMETRY
• It is defined as the measurement of
emitted fluorescent light.
• Fluorescence: A fluorescent compound
absorbs ultraviolet and visible light & the
molecule comes to an excited state. The
phenomenon of light emission during the
process of returning to the ground state
is called fluorescence.
PRINCIPLE
• A solution containing the molecules of
interest is irradiated with light of a
selected wavelength (excited) & is
absorbed returning to their original state
releasing their absorbed energy in the
form of radiant energy at longer
wavelength (emission).
• This energy falls on the sensitive photo
detector where it is converted to a signal
for feeding to a readout device.
Fluorometer

15. FLUOROMETER

16. APPLICATIONS
• Determination of uranium in salts used extensively in the field of nuclear
research.
• Estimation of traces of boron in steel by means of the complex formed
with benzene.
• Estimation of calcium by fluorometry with a calcium solution.
• Determination of Vitamin B (B1 thiamine and B2 riboflavin) in the food
samples like meat, cereals, etc.
• Fluorometry is employed to carry out both qualitative and quantitative
analyses for various aromatic compounds present in cigarette smoke, air-
pollutant, concentrates, and automobiles exhaust.

17. FOURIER TRANSFORM INFRARED
SPECTROMETER (FTIR)
• It is one of the instruments based on
infrared spectroscopy.
• It is the most modern type and preferred
over the other dispersive spectrometers.
• It is because of its high precision,
accuracy, speed, enhanced sensitivity,
ease of operation, and sample non
destructiveness.
• The fundamental of infrared spectroscopic
technology is on atomic vibrations of a
molecule that only absorbs specific
frequencies and energies of infrared
radiation.

18. Fourier transform infrared spectrometer

19. APPLICATION
• FTIR spectroscopy is used to quickly and definitively identify compounds
such as compounded plastics, blends, fillers, paints, rubbers, coatings,
resins, and adhesives.
• Other applications of FTIR includes:
Pharmaceutical research
Forensic investigations
Polymer analysis
Foods research
Quality assurance & control
Environmental & water quality analysis
Biochemical & biomedical research

20. CONCLUSION
• Spectrophotometric analysis is essential for determining biomolecule
concentration of a solution and is employed ubiquitously in
biochemistry and molecular biology.
• The future of spectrophotometry lies especially in the improvement of
pathological diagnostics, disease detection and general clinical
research as “UV-Vis spectroscopy enables safer, non-invasive
analysis of soft tissue, and can enhance accuracy and speed in
clinical diagnostics and medical research.”

21. REFERENCE
• Biotechniques, S.V.S. Rana
• Fluorometry; /www.sciencedirect.com/
• Spectrophotometry; https://microbenotes.com/
• Colorimetry; https://byjus.com/