SPECTROPHOTOMETER
/COLORIMETER
Dr. Benish Zafar
LEARNING OBJECTIVES
At the end of this session, 2nd Year MBBS students will be able to:
• Define Spectrophotometer.
• Describe the principle of spectrophotometry.
• Discuss the terms Sample size, Incident light, transmitted light, transmittance
and optical density.
• Describe Lambert-Beers Law.
• Relate the function of spectrophotometer with that of estimating the
concentration of biomolecules in a fluid.
• Identify the applications of spectrophotometer.
• Differentiate between spectrophotometer and colourimeter.
SPECTROMETER
• Def: is an instrument which deals with the production, measurement, and
interpretation of spectra, arising from the interaction of electromagnetic
radiation with matter.
• EM Spectrum of energy: radio waves (λ>250 mm) to the gamma rays
(λ<0.1nm)
SPECTROSCOPY/
SPECTROCHEMICAL ANALYSIS
• The study of how the chemical compound interacts with different
wavelengths in a given region of electromagnetic radiation.
Spectroscopy deals with:
• Ultraviolet (180-380 nm)
• Visible light (380- 800 nm)
• Infrared (0.8 – 50 μm)
SPECTROPHOTOMETRY
• SPECTROPHOTOMETER: A device that is used to measure intensity of
light or amount of light of a specified wavelength that passes through (is
transmitted through)a sample of solution.
• SPECTROPHOTOMETRY a mean of measuring how densely coloured a
solution is in order to find the concentration of particles in a solution.
COMPONENTS OF
SPECTROPHOTOMETER
• Light source
• Collimator (lens)
• Monochromator (Prism + filter)
• Slit
• cuvette
• Photodetector
• Output display (of transmittance or optical density)
WORKING
• White light radiation source that passes through a MONOCHROMATOR
(prism or a diffraction grating that separates the white light into all colours
of the visible spectrum)
• After the light is separated, it passes through a FILTER (to block out
unwanted light, sometimes light of a different colour) and a SLIT (to
narrow the beam of light)
• Next the beam of light passes through the SAMPLE that is in the sample
holder (cuvette)
WORKING
• The light passes through the sample and the unabsorbed portion (reflected)
strikes a PHOTODETECTOR that produces an electrical signal which is
proportional to the intensity of the light.
• The signal is then converted to A READABLE OUTPUT (absorbance)
that is used in the analysis of the sample.
TERMINOLOGIES
• Incident light: the light which falls on the solution in the cuvette from the source. (Io)
• Transmitted light: the light which transmits or passes through the solution in the cuvette
(It).
• %Transmittance: the manner in which the spectrophotometer reports the amount of light
that passes through a sample.
• Absorbance (OPTICAL DENSITY): amount of light absorbed by a sample (the amount
of light that does not pass through or reflect off a sample)
• Sample Size: the amount of sample marked as T test tube used in the experiment of
estimation of a biomolecule.
BEER LAMBERT LAW
• Def: When a monochromatic light is passed through a solution containing
the absorbing substance, the intensity of transmitted light with path length
is indirectly proportional to the concentration of the solution and the
absorbed light.
CALIBRATION CURVE
It is a graph generated by measuring the absorbance of several solutions made
of stock standards marked as ‘S’ test tubes that contain known concentrations
of the analyte (biomolecule).
APPLICATIONS
• Quantitative analysis of • Identifying the dissolved oxygen
concentration of biomolecules. content in a body of water.
• Enzyme assay. • Analysis of respiratory gas in
• Identifying impurities in products hospitals.
• Organic compounds’ structure. • Functional group detection.
• Identifying the characteristics of a • Determining molecular weight in a
protein. particular compound.
• Identifying classes of compounds.
PROPERTIES OF
COLOURIMETER
• Both spectrophotometer and colourimeter are used to measure color-
absorbing properties of a particular substance.
• In colorimeter, the specific color absorbance is measured.
• Colorimeters have a set of colored filter or LED bulb that can emit a
particular color of light.
• When using a colorimeter, you have to choose the appropriate color filter .
• A solution of a particular color absorbs its own color the least.
COLOURIMETER SPECTROPHOTOMETER
• It only works with light in the • It has the ability to work with
visible part of the electromagnetic infrared, ultraviolet light and visible
spectrum. light.
• Colorimeter is cheap and mostly • spectrophotometer is more
used lab experiments and routine expensive and advanced than the
labs. colorimeter.
SUMMARY
• Spectrophotometer is a device used for measuring the intensity of light given
off by a solution sample.
• It measures the wavelength of visible light of EM spectrum.
• Beer lamberts law describes the relationship between the intensity of
transmitted and absorbed light of solution with that of the concentration of
solution.
REFERENCES
• [Link]
• Spectrophotometer – Principle, Types, Uses and Applications -
[Link]
