1. chapter 2
Quantitative

2. Spectroscopy – Light Properties

3. Spectrophotometer
Model of light interaction with matrix and how spectrophotometer works

4. Spectroscopy – Quantification Terms
Transmittance (T) – the fraction of incident
radiation transmitted by the medium:
T = I/I0
Absorbance (A) – the amount of incident
radiation absorb by the medium and
expressed by:
A = log(1/T) = - logT = log P0/P

5. BEER LAMBERT LAW
As the cell thickness increases, the intensity of I (transmitted intensity of light ) decreases.

6. T- Transmittance
T = I0 - Original light intensity
I- Transmitted light intensity
 % Transmittance = 100 x
T =
Absorbance (A) = Log
A = 2 – Log %T
%T= antilog (2 – A ) I I0 I I0 %T
100
100 %T

7. Spectroscopy - Quantification
Beer’s Law (Beer – Bourguer – Lambert
Law)
Radiation energy decays exponentially
T = P/P0 = 10-kb
Where:
k – constant
T – the transmittance – the fraction of
transmitted radiant energy
b – the pathlength of the medium

8. A  CL = KCL by definition and it is called the Beer Lambert Law.
A = KCL
K = Specific Extinction Coefficient g of solute per liter of solution
 A = ECL
E = Molar Extinction Coefficient Extinction Coefficient of a solution containing 1g molecule of solute per 1 liter of solution

9. Beer Law Chart

10. E differs from K (Specific extinction Coefficient) by a factor of molecular weight.
UNITS
  A = ECL
A = No unit (numerical number only)

11. L = Cm
C = Moles/Liter
A = KCL
A = No unit C = Gram/Liter L = Cm K=
Liter
Cm  Gram

12. STEPS IN DEVELOPING A SPECTROPHOTOMETRIC ANALYTICAL METHOD
Run the sample for spectrum
2. Obtain a monochromatic wavelength for the maximum absorption wavelength.
3. Calculate the concentration of your sample using Beer Lambert Equation: A = KCL

13. SPECTROPHOTOMETR READINGS

14. ULTRAVIOLET SPECTRUM

15. There is some A vs. C where graph is linear.
NEVER extrapolate beyond point known where becomes non-linear.

16. SPECTROMETRIC ANALYSIS USING STANDARD CURVE
Avoid very high or low absorbencies when drawing a standard curve. The best results are obtained with 0.1 < A < 1. Plot the Absorbance vs. Concentration to get a straight line

17. CELLS UV Spectrophotometer Quartz (crystalline silica)
 Visible Spectrophotometer
Glass
 IR Spectrophotometer
NaCl

18. LIGHT SOURCES UV Spectrophotometer 1. Hydrogen Gas Lamp
2. Mercury Lamp
Visible Spectrophotometer
1. Tungsten Lamp
IR Spectrophotometer
1. Carborundum (SIC)

19. UV SPECTROMETER APPLICATION
Protein
Amino Acids (aromatic)
Pantothenic Acid
Glucose Determination
Enzyme Activity (Hexokinase)

20. VISIBLE SPECTROPHOTOMETER APPLICATION
Niacin
Pyridoxine
Vitamin B12
Metal Determination (Fe)
Fat-quality Determination (TBA)
Enzyme Activity (glucose oxidase)

21. EXAMPLES
Calculate the Molar Extinction Coefficient ε at 351 nm for aquocobalamin in 0.1 M phosphate buffer. pH = 7.0 from the following data which were obtained in 1 Cm cell.
Solution C x 105 M Io I A B
 2. The molar extinction coefficient (ε) of compound riboflavin is 3 x 103 Liter/Cm x Mole. If the absorbance reading (A) at 350 nm is 0.9 using a cell of 1 Cm, what is the concentration of compound riboflavin in sample?
3. The concentration of compound Y was 2 x moles/liter and the absorption of the solution at nm using 1 Cm quartz cell was What is the molar extinction coefficient of compound Y?

22. 3. Complete the following table.
 [X](M) Absorbance Transmittance(%) ε(L/mole-cm) L(cm)  
2.5 x
4.0 x
2.0 x
  [X](M) = Concentration in Mole/L
4. The molar absorptivity of a pigment (molecular weight 300) is 30,000 at 550 nm What is the absorptivity in L/g-cm.!
5. The iron complex of o-phenanthroline (Molecular weight 236) has molar absorptivity of 10,000 at 525 nm. If the absorbance of 0.01 is the lowest detectable signal, what concentration in part per million can be detected in a 1-cm cell?

23. A = 0. 01 ε= 10000L / mole x cm L = 1cm A = ECL 0
A = 0.01 ε= 10000L / mole x cm L = 1cm A = ECL 0.01= 10000L/mole X Cm X C (Concentration) x 1Cm C = mole / Liter C = X mole / Liter = X mole (236 g/mole) / Liter (1000 Cm3) x PPM (10-6 g/Cm3) = X mole (236 g / mole) / Liter x 1 Liter / 1000 Cm3 x ( PPM) g / Cm3) =x PPM PPM = 1ug / Cm3 1ug = 10-6 g

24. Spectroscopy –Multimolecules Effect

25. Spectroscopy -Mixture
Multi-component mixture:
AT = A1 + A2 +A3 + An
AT = ε1bc ε2bc ε3bc3 + εnbcn
Where: 1,2,3,….,n – refer to absorbing
components

26. EXAMPLES
Potassium dichromate and potassium permanganate in 1 M H2SO4 has absorbance spectra overlap (overlab). K2Cr2O7 have maximum absorbance at 440 nm and KMnO4 545 nm. Mixture of both substances have been analyzed by measuring the absorbance of solution at the second wavelength with the following results: A 440 = 0.405, A545 = cm cell used. Absorbance K2Cr2O7 pure solution (1 x M) and KMnO4 (2 x 10-4 M) in 1 M H2SO4 by using the same cell are as follows: A Cr, = , A Cr, 545th = 0.009, A Mn, 440 = 0.019, A Mn, 545 = Calculate the concentration of dichromate and permanganate in the sample solution?

27. Spectroscopy – Beer’s Law Limitations
Deviations from the direct
proportionality (b=const)
Instrumental Deviations
Chemical Deviations

28. Beer’s Law Limitations
Deviations from the direct proportionality (b = const)
Valid at concentration usually below
0.01M
Molecules interference – distance between molecules affects charge distribution of molecules (ions)
Effect of refractive index –concentration affects refractive index ε is affected (low, less important)

29. Spectroscopy - Deviations

30. Beer’s Law Limitations
Chemical Deviations
Association, dissociation and reaction with solvent and other molecules

31. Beer’s Law Limitations
Instrumental Deviations
monochromatic radiation – quality of monochromator and control of bandwidth and slit
Instrumental noise – accuracy of measurement of transmittance – quality of detector