SPECTROPHOTOMETERDr. Maha Al-Sedik

                                                           

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

As /bs x cs=Au /bu x cu

B ….light path in cm is constant

As / cs = Au / cu

cu = Au x cs / As

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.

Components of spectrophotometer:

Question?

Cleaning of the cuvet: Cleaned in solution of

concentrated HCL : water : ethanol = 1 :3 :4

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.

THANK YOU