1. SPECTROPHOTOMETRY
PRACTICAL 213 PHC
INSTUMENTAL ANALYSIS

2. Instrumental Analysis
Analytical Chemistry
Volumetric Analysis
-Quantitative
Instrumental Analysis
- Qualitative

3. Principles of Spectrophotometry
There is interaction between electromagnetic radiation (light) and sample.
The amount of light absorbed is directly related to the concentration of the analyte.

4. Electromagnetic Radiation (EMR)
Energy

5. The Spectrophotometer
Instrument
The Spectrophotometer

6. Application qualitative : ( λ max)
The wavelength at which the maximum absorbance .
quantitative : Determination of conc. By
- Beer’s lambert law A = abc.
- Matching point.
- Calibration curve.

7. Qualitative
Absorbance spectrum A

8. Absorption spectrum
Absorption spectrum is obtained by plotting Absorbance (A) as a function of wavelength ().
It has characteristic shape with the  of maximum absorbance (max).
It is characteristic for each molecule according to its structure.
Therefore it is used for identification of a chemical substance
(qualitative analysis). Also max is used for quantitative measurement, in order to increase sensitivity and to minimize error of the analytical method.

9. -Bathochromic shift (red hift)
It is the shift of max to a longer wavelength due to substitution with certain functional groups (e.g. –OH and –NH2), change in the pH and the effect of the medium (solvent).
- Hypsochromic shift (blue shift)
It is the shift of max to a shorter wavelength due to removal
of conjugation by changing pH or polarity of the solvent.

10. - Hypochromic effect An increase in the intensity of absorption
- Hyperchromic effect
An increase in the intensity of absorption
- Hypochromic effect
It involves a decrease in the intensity of absorption

11. Changes in Absorption spectrum
Example 1): effect of conjugation on absorption spectrum
Increase in conjugation, increase absorbance of light to higher , bathochromic shift with hyperchromic effect.

12. Example 2) Effect of pH
The spectra of compounds containing (-OH) or (-NH2) groups are dependent on the pH of the medium.
-The U.V spectrum of phenol in acid medium, benzenoid form while in alkaline medium is the phenolate ion.
-The free pair of electrons of O2 increasing the delocalization of the -electrons, leading to the formation of conjugated system.
-So , electrons become more energetic and need less energy to be excited, therefore absorb longer  bathochromic shift ; red shift) with hyperchromic effect
Phenol

13. Absorption Spectra of Phenol and Effect of PH

14. Procedure :
Part 1:
1- Pipette 3 ml of phenol and transfer into 100 ml volumetric flask.
2- Add 10 ml of 0.1 N HCL.
3- Complete to the mark with distilled water and mix will.
4- Prepare a blank (10 ml HCL and complete to 100 with distilled water).
5- Fill quartz cell with blank and other with sample.
6- Measure the absorbance in the range (every 5 nm).
7- Draw absorbance spectrum and Determine λ max.
Because other compounds in a solution (or the solvent itself) may absorb the same wavelengths as the compound being analyzed, we compare the absorbance of our test solution to a reference blank. Ideally, the reference blank should contain everything found in the sample solution except the substance you are trying to analyze.

15. Part 2:
1- Pipette 3 ml of phenol and transfer into 100 ml volumetric flask.
2- Add 10 ml of 0.1 N NaOH.
3- Complete to the mark with distilled water and mix will.
4- Prepare a blank (10 ml NaOH and complete to 100 with distilled water).
5- Fill quartz cell with blank and other with sample.
6- Measure the absorbance in the range (every 5 nm).
7- Draw absorbance spectrum and Determine λ max.