dr. maha al-sedik. objectives: electromagnetic radiation white light beer’ s law ...
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Biochemical instrumental analysis-1
Dr. Maha Al-Sedik
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Objectives:
Electromagnetic Radiation
White light
Beer’ s law
Spectrophotometer
Components of spectrophotometer
Types of spectrophotometer
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Light is the most important example of
electromagnetic radiation.
Light moves in waves.
different types of light have different wavelengths.
Wave length: distance between 2 peaks and is
expressed by nanometers.
Electromagnetic Radiation
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Do you think (a) or (b) has longer wave length and lowest amplitude?
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The human eye responds to radiant energy
between 380 and 750.
Sun light is mixture of radiant energy of
different wavelengths that eye recognize it as
white.
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White light
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Colors & WavelengthsCOLOR WAVELENGTH (λ in nm)
Ultraviolet < 380
Violet 380 – 435
Blue 436 – 480
Greenish-blue 481 – 490
Bluish-green 491 – 500
Green 501 – 560
Yellowish-green 561 – 580
Yellow 581 – 595
Orange 596 – 650
Red 651 – 780
Near Infrared > 780
Vis
ible
Lig
ht
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Each object when exposed to light , It absorbs some of lights and transmit some others.
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Q- When do we see solution with green light?
A- if it transmits light maximally between 500 -560 ( green wave length) and absorbs light at other wave length.
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All colors absorbed except red color
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Beer’ s law
Beer’s Law talks about the relationship between
color and concentration.
Beer’s Law states that the concentration of
substance in solution is directly proportional to
the light absorbed by the solution or inversely
proportional to light transmitted within limits.
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A α b x c
A : absorbance.α : directly proportional .b : light path in cm .c : concentration of substance.© : constant
A=© x b x
c
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If we suppose that I have two solution one with known concentration we will name it standard (s) . The second with unknown concentration and we will name it unknown (u).
As = © x bs x csAu = © x bu x cu
©= As /bs x cs©= Au /bu x cu
As /bs x cs=Au /bu x cu
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As /bs x cs=Au /bu x cu
B ….light path in cm is constant
As / cs = Au / cu
cu = Au x cs / As
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Beer’ s law is followed only if the following
conditions are met :
The light must be monochromatic.
The solute concentration is within given limits.
A chemical reaction does not occur between the
molecule of interest and another solute.
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Spectrophotometer
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Spectrophotometric techniques
Photometry: measurement of intensity of
light.
Spectrophotometry: measurement of
intensity of light at selected wave length.
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Principle:
The solutions of many compounds have
characteristic colors.
The intensity of a color is directly proportional to the
concentration of the compound.
Light is absorbed by dissolved substances.
The concentration of the substance is directly
proportional to the amount of light absorbed.
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Q- Is spectrophotometer quantitative or
qualitative technique ?
A- Both
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Components of spectrophotometer:
Light source
Monochromator
Filter
Cuvet
Photo detector
Signal processor
Record
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Components of spectrophotometer:
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1-Light source:
Tungsten Filament Lamps: the most common
source of visible radiation.
Deuterium Lamps: also may be used.
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2-Monochromator:
Used as a filter: the monochromator will select a
narrow portion of the spectrum of a given source.
Monochromator may be filters or prism and grating.
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3-Cuvet:
It may be Glass or plastic.
It may be round, square or
rectangular.
It must be clean and clear.
The most popular is 1 cm light
path.
Cleaned in solution of
concentrated HCL : water :
ethanol = 1 :3 :4
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3- Photodetector:
Photodetector transforms light to electrical
impulses.
When light strikes the photodetector , electrons are
exited in the semiconducting material .
The electricity is directly proportional to the
intensity of light.
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4- SIGNAL PROCESSOR:
Digital signals from the blank are subtracted from
standered and uknown
5- RECORD:
Gives the net results on the monitor.
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General rules and precautions in spectrophotometer:
A blank should be used for each set of determinations.
Cuvet should be clean, clear and dry.
For each size of cuvet there is a specified minimum
volume.
The solution must be free of air.
Cuvet must be put with the same side facing light
every time.
Cuvet must be covered before each reading.
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Types of spectrophotometer:
Single beam spectrophotometer.
Double beam spectrophotometer.
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http://www.youtube.com/watch?v=pxC6F7bK8CU
http://www.youtube.com/watch?v=QdufRwbkeKo
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THANK YOU