spectrophotometer 2.pdf

Spectrophotometer 2

1. Presented by:
Dr. Mohammed Al-olfe
ةيلودلا ةيتاراملاا ةعماجلا
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2. The spectrophotometeris used in the laboratory for determining
the presenceor concentrationof a substance in a solution, thus
allowing a qualitative or quantitative analysis of the sample.
The word spectrophotometeris derived from the Latin word
spectrum, which means image, and the Greek word phosor
photos, which means light.
The spectrophotometeris one of the main diagnostic and research
instruments developed.
It uses the properties of lightand its interactionwith other
substances.
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3. How it is work:
Generally, light from a lamp with special characteristics is
guided through a device, which selects and separates a
determined wave length and makes it pass through a sample.
The light intensity leaving the sample is captured and
compared with that which passed through the sample.
Transmittance, which depends on factors such as the
substance concentration is calculated from this intensity ratio.
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4. Measurement Processes:
Most spectroscopic methods are differentiated , they can be classified into:
1)Absorption spectroscopy uses the range of the electromagnetic spectra in
which a substance absorbs, such as infrared, ultraviolet-visible and
microwave spectroscopy.
2)Emission spectroscopy uses the range of electromagnetic spectra in which a
substance radiates (emits). The substance first must absorb energy. This
energy can be from a variety of sources, which determines the name of the
subsequent emission, like luminescence. Molecular luminescence
techniques include spectrofluorimetry.
3)Scattering spectroscopy measures the amount of light that a substance
scatters at certain wavelengths, incident angles, and polarization angles.
Such that Raman spectroscopy.9/27/2021 4

5. As a basic principle, lightis considered to be a form of
electromagnetic energy. In space, it has a constant and universal
velocity[C] of approximately 3 x 10
8
m/s.
In any other medium (transparent) through which light passes, its
velocity will be slightly lower and can be calculated by the
following equation:
Where:
V
o
= Velocity at which light passes through the medium
n= Medium refraction index: whose value oscillates, in general, between 1.0
and 2.5.
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6. What is a wave?!
Harmonic
wave:
wavelength
frequency
or
wavelength
amplitude
velocity
frequency
wavelength
=
c
n
l
=
Frequency
(Hertz)
Velocity (300,000,000 meters/sec)
Wavelength (meters)
Propagation

7. The electromagnetic energy has a very wide range of wavelengths.
Some examples are shown in the following table:
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8. The electromagnetic energy has a very wide range of wavelengths.
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9. The electromagnetic energy has a very wide range of wavelengths.
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X-Ray UV Visible IR Microwave
200nm 400nm 800nm
WAVELENGTH(nm)

10. The table below shows the wavelength ranges used for carrying
out spectrophotometer tests.
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11. Upon passingor interactingthe lightwith diverse mediums, light undergoes a
series of phenomena. Among these are featured reflection, refraction,
diffraction, absorption, diffusion, polarizationand other phenomena measured
by various instruments and devices.
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12. The phenomenaon which spectrophotometryis based are mainly
absorptionand transmission.
In order to understand how, it is necessary to take Beer Lambert’s
lawinto account.
Beer Lambert’s Law. Also known as Beer’s law or Beer Lambert
Bouguer’slaw, it identifies the relationship between the
concentration of the sample and the intensity of light transmitted
through it.
With regard to the law mentioned, there are two implicit
concepts: transmittance[T]and absorbance[A].
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13. The transmittance [T]is the fractionof the incidental light of determined
wavelength passing through the sample.
Where:
I
t
= intensity of the transmitted radiation
I
o
= intensity of the incidental radiation
The percentage of transmittance [%T] can be expressed by the following
equation:
Absorbance [A] is related to transmittance [T]through the following equation:
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14. The concentration of light absorbing molecules in a sample is proportional to the
absorbance [A] of that sample. It is expressed mathematically as:
Where:
A= Absorbance measured
ε = Molécule absorbance coefficient [liters/moles/cm]
l = Distance of the trajectory traversed (path length) by the light in the
sample
c= Sample concentration [moles/liters]
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15. The graphs demonstrated how absorbance [A]and transmittance [T]vary as a
function of the concentration [C] according to Beer Lambert’s law.
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Transmittance graphAbsorbance graph

16. Absorption Spectra:
Absorption
Spectra Plot of
Absorbance vs.
wavelength
called
absorption
spectrum.

17. Emission Spectra:
Emission
Spectra Plot of
emission
intensity vs.
wavelength
called
emission
spectrum.

18. There are two major classes of devices: single beam and double beam.
1)A double beam spectrophotometer compares the light intensity
between two light paths, one path containing a reference sample and
the other the test sample.
2)A single beam spectrophotometer measures the relative light intensity
of the beam before and after a test sample is inserted.
Although comparison measurements from double beam instruments are easier
and more stable, single beam instruments can have a larger dynamic range and
are optically simpler and more compact.
The most common spectrophotometers are used in the UVand visible regions
of the spectrum, and some of these instruments also operate into the near-
infraredregion as well.
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24. The most important components of the basic spectrophotometer,
not covering novel technology incorporated by manufacturers in
advanced models are the following:
1)The light source
2)The monochromator
3)Filters
4)The sample carrier
5)The detector system
6)The reading system
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25. Light Source:
Depending on the type of spectrophotometer, the light source can be a tungsten
lamp for visible light (400 –800 nm) or a deuterium arc lamp for ultraviolet light
(200 –400 nm).
Some manufacturers have designed spectrophotometers with long lasting xenon
intermittent lampsemitting light in the visibleand ultraviolet ranges.
The lamp(s) come factory-assembled on a base that ensures a fixed position, to
maintain optical adjustment and focus when operating or when replacing the
bulb.
The typical radiating energy emitted from a tungsten lamp is between 2600and
3000°K (Kelvin degrees).
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26. Filter:
Filtersabsorb light of different wavelengths, allowing only
light with a narrow range of wavelengths (bandwidth) to
bass through.
Filtershave a bandwidth of about 2 nm.
Filtersmay be fixedor removable.
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27. Monochromator:
The monochomatoris a set of elements used to disperse white light into waves
of different wavelengths, one of which is used in the sample reading.
In general, it has an entry creviceor groovewhich limits the light radiation
produced by the source and confines it to a determined area;
a set of mirrors for transmitting light through the optic system; an element for
separating the light radiation wavelengths (which may be a prism or a diffraction
(or transmission) grating); and an exit opening for selecting the wavelength
required to illuminate the sample.
Diffraction gratings have the advantage of eliminating the non-linear dispersion
and being insensitive to changes in temperature.
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28. Monochromator:
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29. Simple holder:
This device holds the sample(s)to be analyzed.
There are various sample holder types to accommodate different
spectrophotometer models and sample volumes: these come as cuvettes,
microcells, microplates, test tubes and continuous flow cells, etc.
In conventional spectrophotometers, the holder is a cell or cuvette of rectangular
shape.
Cuvettesare made of glass to read in the range of 340to 1000 nm and others of
silicato read in the visible range of 220to 340 nm.
There are also cuvettesand other sample holder types (e.g. microplates) in plastic
such as styreneor polystyrenewhich are disposable.
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30. Detector System:
The detection system can be designed with photocells, phototubes, photodiodes
or photomultipliers.
This dependson the rangesof wavelength, the sensitivityand the required speed
of response.
The detection system receives light from the sampleand convertsit into an
electrical signal proportionalto the energy received.
This electrical signal can be processedand amplifiedto be interpretedby the
reading system.
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31. Detector System:
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32. Reading System:
The signal which leavesthe detectorgoes through various transformations. It is
amplifiedand transformed until its intensitybecomes a proportional
transmittance/absorbancepercentage.
There are analogous reading systems (displaying results on a reading scale) or
digital ones (showing results on a screen).
Analogous indicators traditionally bear the name meters. Their exactitude
depends among other factors, on the length and the number of divisions of the
scale (the more divisions, the more exact it is). Their main disadvantage is that
they can be incorrectly read, due to the operators’ fatigue or errors identifying
scales when there are several.
Digital indicators usually show results on a screen as illuminated alpha numerals.
This makes reading errors less likely.
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33. Maintenance includes:
Calibration adjustment and replacement of light source
bulbs and photo detectors.
Mechanically rotating assembles (mirrors, diffraction
grating) will occasionally malfunction.
The electronics is very reliable.
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37. Colorimeteror filter photometer is an optical electronic
device that measures the color concentration of a
substance in solution (following the reaction between the
original substance and a reagent).
Optical color filters are used to select a narrow wavelength.
The results are displayed in percent optical color
transmittance or absorption to indicate hemoglobin
concentration.
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39. Light passes through an optical color filter, is focused by
lenses on the reference and sample cuvettesand falls on
the reference and sample photodetectors.
The difference in voltage between the two detectors is
increased by a dc amplifier and applied to a meter.
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40. A calibration procedure is as follows:
1)Ground the amplifier input (V
1) and adjust potentiometer (R
a)
for 0 volt
2)Remove the ground and place reference concentration in
cuvettes1 an 2
3)Adjust potentiometer R
1for 0 V
4)Leave the reference concentration in cuvette1 and replace
cuvette2 with a cuvettecontaining the sample
5)Read the unbalanced voltage on the meter in percent
transmittance or absorbance units.
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43. The flame photometer measures the color intensity of
flame that supported by oxygen and specific substance.
The basic schematic shows that a reference gas containing
a lithium salt causes a red color to shine on the reference
photo detector through the reference optical filter.
A yellow or violetlight from sample sodiumor potassium
falls on the sample photo detector.
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Calibration:
Basically, the flame photometer is calibrated in
a manner similar to the colorimeter
Continuous calibration can be accomplished by
inspiration of air and lithium.
the output is read in units of sodium or
potassium concentration.

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Maintenance:
Maintenance includes calibration, adjustment of
bulbs and photo detectors.
Aspiration devices and flame chambers
occasionally require cleaning.
Electronic failures are usually infrequent.

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