1. SPECTROPHOTOMETER Dr. Maha Al-Sedik

4. 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.

5. 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.

7. A α b x c A : absorbance. Α : directly proportional. B : light path in cm. C : concentration of substance. A =© x b x c

8.  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 cs Au = © x bu x cu ©= As /bs x cs ©= Au /bu x cu As /bs x cs=Au /bu x cu

9. B ….light path in cm is constant As / cs = Au / cu cu = Au x cs / As

10. Question no 1 : From the next standard curve, if the absorbance is 0.35 or 0.55, the concentration will be ----------- or ----- ------------------.

12. 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.

13. 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.

14. 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.

15. Components of spectrophotometer: Question?

16. Cleaning of the cuvet: Cleaned in solution of concentrated HCL : water : ethanol = 1 :3 :4

17. 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.