spectrophotometer dr. maha al-sedik. principle: the solutions of many compounds have characteristic...
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Principle: The solutions of many compounds have
characteristic colors.
The intensity of such a color is 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.
Beer’ s law
• Beer’s Law talks about the relationship between color and
concentration.
• Beer’s Law states that the absorbance of light by a solution is
directly proportional to concentration or inversely proportional to
light transmitted within limits.
A α b x c
A : absorbance.Α : directly proportional .B : light path in cm .C : concentration of substance.
A=© x b x c
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
Question no 1 :
From the next standard curve, if the absorbance is
0.35 or 0.55 , the concentration will be ----------- or
-----------------------.
Question no 2 :
If the absorbance of standard is 0.5 , the absorbance
of unknown is o.4 , and the concentration of standard
is 50 ml / dl . Calculate the concentration of the
unknown .
Question no 2 :
If the absorbance of standard is 0.24 , the absorbance
of unknown is 0.3 , and the concentration of standard
is 120 ml / dl . Calculate the concentration of the
unknown .
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.
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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