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Spectrophotometer
Spectrophotometer
A device to measure how much a chemical
substance absorbs light by measuring the intensity
of light as a beam of light passes through sample
solution.
The basic principle is that each compound
absorbs or transmits light over a certain range of
wavelength.
This measurement can also be used to measure
the amount of a known chemical substance.
The most useful devices of quantitative analysis in
various fields such as chemistry, physics,
biochemistry, material and chemical engineering
and clinical applications.
Common terms
Spectroscopy
The science of studying the interaction between matter and radiated
energy
Spectrometry
Method used to acquire a quantitative measurement of the spectrum.
Spectrum
Band of colours, as seen in a rainbow, produced by separation of the
components of light by their different degrees of refraction according to
wavelength. (refer figure 1 & figure 2)
Photometry
the science of the measurement of light, in terms of its perceived
brightness to the human eye.
Spectrophotometer
Instrument for measuring the intensity of light in a part of the spectrum,
especially as transmitted or emitted by particular substances.
Spectrum
Band of colours, as seen in a rainbow, produced by
separation of the components of light by their different
degrees of refraction according to wavelength.
Figure 1
Wavelength
The distance between successive crests of a wave, especially
points in a sound wave or electromagnetic wave.
Figure 2
Spectrometry & Photometry
Spectrometry Photometry
Spectrometer: It produces a desired range of wavelength Photometer: After the desired range of
wavelength of light passes through the
of light. First a collimator (lens) transmits a straight beam of solution of a sample in cuvette, the
light (photons) that passes through a monochromator photometer detects the amount of
(prism) to split it into several component wavelengths photons that is absorbed and then
(spectrum). Then a wavelength selector (slit) transmits only sends a signal to a galvanometer or a
the desired wavelengths digital display
Prism
• Visible Spectrophotometer
Glass
Colour intensity
many solutions have distinctive colours
absorb/transmit different wavelengths of visible light
Figure 3
Electromagnetic Spectrum
400 700
nm nm
UV-Vis and IR
Spectrophotometer
Depending on the range of wavelength of light
source, it can be classified into two different
types:
A) % Transmittance
B) Transmittance
C) Nanometer
Question & Answer
General procedure:
sample is placed into cuvette a cuvette
light of selected wavelength (λmax)
is passed through sample
instrument measures the amount of light
absorbed by the sample
Reference blank
contains everything found in the sample
solution, except the substance being
analyzed
calibrates the absorbance reading to
A=0.000
spectrophotometer
• Io = Original intensity of light
• It = Intensity of transmitted light
2. Absorbance, A = -log(%T)
Absorbance and
wavelength
Choosing a Wavelength
use wavelength at which most light is absorbed (λmax)
λmax
equation of line: y = mx + b
A = εLc
ε=?
l =?
A =?
c= A/ εl
Calculation 1
Guanosine has a maximum absorbance of 275
nm. ϵ275 = 8400 L mol-1 cm-1, and the path length
is 1 cm. Using a spectrophotometer, you find the
that A275=0.70. What is the concentration of
guanosine?
Answer:
ϵ275 =?
A275=?
I=?
c=?
Lambert law =
Calculation 2
Answer
Uses of Spectrophotometry
Absorption Spectra
Kinetic Assays
Measure quantity
Some bio-molecules have properties
which allow direct measurement
proteins have aromatic amino acids
(280nm)
Nucleic acids have unsaturated ring
structures (260nm)
Protein
Amino Acids (aromatic)
Pantothenic Acid
Glucose Determination
Enzyme Activity (Hexokinase)
GeneRay UV Photometer
The GeneRay UV Photometer is
a powerful tool for the
molecular biologist enabling
rapid
UV purity determination and
quantification of single-
stranded and double-stranded
DNA, ssRNA and
oligonucleotides,
as well as bacterial growth
rates.
Results can be obtained in
µg/ml µg/µl or pmol/ml
Measures at 260, 280 and 600
nm
No warm-up time
Automatic calculation of
concentrations
Virtually unlimited life time of
lamp
VISIBLE SPECTROPHOTOMETER APPLICATION
• 340 nm to 700 nm
• Tungsten Lamp
• Material detection like
Vitamins like Niacin (Vit B3), Vit B12 &
Pyridoxine (Vit B6)
Metal Determination (Fe)
Fat-quality Determination (TBA)
Enzyme Activity (glucose oxidase)
Common spectrophotometer
Precautions When Handling a
Spectrophotometer
Calibrate the machine with the blank. Place the blank into
the cuvette holder and shut the lid.
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