This document discusses spectrophotometry and spectrophotometers. It begins by defining spectrophotometry as a method to quantitatively measure the absorption or transmission of a material as a function of wavelength. It then describes the basic components and mechanisms of a spectrophotometer, including that it measures the amount of light absorbed by a sample. The document also covers the different types of spectrophotometers based on their light source or beam structure, and applications such as concentration measurement and detection of impurities. Finally, it introduces Beer's law which states absorbance is directly proportional to concentration.
A spectrophotometer is an instrument containing a monochromator, a device which produces a light beam containing wavelengths in a narrow band around a selected wavelength, and a means of measuring the ratio of that beam's intensity as it enters and leaves a cuvette 99 This describes a single-beam photometer.
A spectrophotometer is an instrument containing a monochromator, a device which produces a light beam containing wavelengths in a narrow band around a selected wavelength, and a means of measuring the ratio of that beam's intensity as it enters and leaves a cuvette 99 This describes a single-beam photometer.
It is the most common analytical technique used in biochemical estimation in clinical laboratory.
It involves the quantitative estimation of color.
A substance to be estimated colorimetrically, must be colored or it should be capable of forming chromogens (colored complexes) through the addition of reagents.
A spectrophotometer is an instrument that measures the amount of light absorbed by a sample. Spectrophotometer techniques are used to measure the concentration of solutes in solution by measuring the amount of the light that is absorbed by the solution in a cuvette placed in the spectrophotometer .
Ultraviolet-visible (UV-Vis) spectrophotometry is a technique used to measure light absorbance across the ultraviolet and visible ranges of the electromagnetic spectrum. When incident light strikes matter it can either be absorbed, reflected, or transmitted. The absorbance of radiation in the UV-Vis range causes atomic excitation, which refers to the transition of molecules from a low-energy ground state to an excited state.
It is the most common analytical technique used in biochemical estimation in clinical laboratory.
It involves the quantitative estimation of color.
A substance to be estimated colorimetrically, must be colored or it should be capable of forming chromogens (colored complexes) through the addition of reagents.
A spectrophotometer is an instrument that measures the amount of light absorbed by a sample. Spectrophotometer techniques are used to measure the concentration of solutes in solution by measuring the amount of the light that is absorbed by the solution in a cuvette placed in the spectrophotometer .
Ultraviolet-visible (UV-Vis) spectrophotometry is a technique used to measure light absorbance across the ultraviolet and visible ranges of the electromagnetic spectrum. When incident light strikes matter it can either be absorbed, reflected, or transmitted. The absorbance of radiation in the UV-Vis range causes atomic excitation, which refers to the transition of molecules from a low-energy ground state to an excited state.
Instrumentation of uv visible spectrophotometerTalha Liaqat
A spectrophotometer is an apparatus for measuring the intensity of light in a part of the spectrum, especially as transmitted or emitted by particular substances. The instrumentation of the Spectrophotometer is described in this presentation.
Fourier Transform Infrared Spectroscopy-:A type of infrared spectroscopy.It is method of obtaining an infrared spectrum by measuring interferogram and then performimg a Fourier Transform upon the interferogram to obtain the spectrum.
Spectrophotometer techniques are mostly used to measure the concentration of solutes in solution by measuring the amount of light that is absorbed by the solution in a cuvette placed in the spectrophotometer.
Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region. Ultraviolet-Visible (UV-VIS) Spectroscopy is an analytical method that can measure the analyte quantity depending on the amount of light received by the analyte.
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3. INTRODUCTION
• Spectrophotometer is an instrument which
measures light absorption as a function of
wavelength in the UV as well as visible
regions.
• It also follows essentially the laws of light
absorption viz the beer-lambert’s law.
• Unlike colorimeters in spectrophotometers
the compound can be measured at precise
wavelength.
4. SPECTROPHOTOMETRY
• A method in which the absorption or
transmission properties of a material is
quantitatively measured as a function of
wavelength.
• The basic principle behind this method is
that:
“Each compound absorbs or transmits light
over a certain range of wavelength”
5.
6. • A spectrophotometer is an instrument that
measures
• the amount of photons absorbed by a sample
after it is passed through its solution.
• With the spectrophotometer , the amount of
a known chemical substance can be
determined by measuring the absorbance.
7. • Spectrophotometer can be classified into two
different types :
1) SINGLE BEAM SPECTROMETER
• To measure the intensity of the incident light
the sample must be removed so that the
reference can be placed each time. This type
of spectrometer is usually less expensive and
less complicated.
8. 2) DOUBLE BEAM SPECTOMETER
• In this type, before it reaches the sample, the
light source is split into two separate beams.
From these one passes through the sample
and second one is used for reference. This
gives an advantage because the reference
reading and sample reading can take place at
the same time.
9.
10. • Based on the wavelength of light used it can be
classified into :
1) VISIBALE SPECTROMETER
• Uses visible range (400 – 700nm) of electromagnetic
radiation spectrum. Visible spectrophotometers vary in
accuracy. Plastic and glass cuvettes can be used for
visible light spectroscopy.
2) UV SPECTROMETER
• Uses light over the UV range (180 - 400 nm).UV
spectroscopy is used for fluids, and even solids.
Cuvettes, only made of quartz, are used for placing the
samples.
3) IR SPECTROMETER
• Uses light over infra red range (700 -15000) of
electromagnetic radiation spectra.
11. DEVICE AND MECHANISM
• The spectrophotometer, in general, consists of
two devices. They are the following :
1. SPECTROMETER :
• A device that produce, typically disperse and
measure the light.
2. PHOTOMETER
• Indicates the photoelectric dictator that
measures the light.
12. • The photometer detects the light absorbed
by the sample as the light from the slit is
passed through the solution. And then it
sends signal to the galvanometer or digital
display.
13. • The spectrometer consists of the following parts :
1. LIGHT SOURCE :- it produce a desired range of
wavelength of light.
2. COLIMATOR :- transmits a straight beam of light.
3. MONOCHROMATOR :- split the light into its
component wavelength
4. WAVELENGTH SELECTORE :- transmits only the
desired wavelength.
5. CUVETTES :- The optically transparent cells
(cuvett) are made up of glass,plastic,silica or
quartz,glass and plastic absorb UV light below 310
nm.
14. 6. Photocell and photo-multipller tubes:-
‘a photocell is a photoelectric device
which converts light energy into electrical
energy, which is then amplified, detected and
recorded.’’
15. APPLICATION
• 1. Concentration measurement.
• 2. Detection of impurities.
• 3. Chemical kinetics
• 4. Detection of functional group
• 5. Molecular weight determination
16. BEER- LAMBERT LAW
• It states that the absorbance of light by a
material in a solution is directly proportional
to its concentration in that solution.
A = ϵlc
Where,
A – absorbance
ϵ - molar absorptivity
l – length of solution
c – concentration
