1. UV/Visible Spectroscopy

2. UV/Vis Spectrometer Measure?
Spectroscopy
What does a
UV/Vis Spectrometer Measure?

3. UV/VIS Spectroscopy T = P/Po
Transmittance (T) is defined as the amount of light passing through the sample solution (P) divided by the amount of incident radiation (Po)
T = P/Po
In practice Po is estimated by a “blank”
which accounts for reflection and
scattering losses.

4. UV/VIS Spectroscopy As the radiation is absorbed in the sample,
the total intensity of radiation is reduced
as it travels through the sample. This results in a non-linear relationship between transmittance and concentration.

5. UV/VIS Spectroscopy A = log10(1/T) Absorbance (A) is based
on the amount of light
absorbed by the solution
and is defined as the log
of the inverse of the
transmittance.
A = log10(1/T)

6. UV/VIS
Spectroscopy
Within limits, the relationship between absorbance and concentration is linear.

7. A = bc UV/VIS Spectroscopy Beer’s law or the Beer-Lambert law
 is the molar absorptivity with units of (L/mol-cm)
b is the path length of the sample ie. the inside cross section of the sample cuvette (cm)
c is the concentration of the compound in solution in (mol/L)

8. UV/VIS Spectroscopy Increased interaction between the molecules
can affect absorbance at high concentrations.
Electrolytes can also alter the molar absorptivity
of the analyte by electostatic interactions.
Molar absorptivity is also altered by the refractive index of the solution.

9. UV/VIS Spectroscopy Hindicator H+ + Indicator-
When an analyte associates or dissociates in solution producing products with different absorption spectra, deviations from Beer’s law can be observed if the equilibrium shifts at different concentrations.
Hindicator H+ + Indicator-

10. UV/VIS Spectroscopy Other causes of deviation from Beer’s law:
Radiation is not monochromatic
Stray radiation
Absorbance readings less than 0.10 and higher than 1.5 can contain significant error and should be avoided.

11. UV/VIS Spectroscopy Quantification of Two Compounds Absorbance
Spectra of Compound 1
Absorbance
Spectra of Compound 2
Wavelength

12. At wavelength 1 A1 = ea1ca + eb1cb At wavelength 2 A2 = ea2ca + eb2cb
UV/VIS
Spectroscopy
Quantification of Two Compounds
The method to determine the concentrations of two compounds (a & b) in a mixture involves the simultaneously solution of the following two equations:
At wavelength 1 A1 = ea1ca + eb1cb
At wavelength 2 A2 = ea2ca + eb2cb

13. UV/VIS Spectroscopy Quantification of Two Compounds
A = bc
Quantification of Two Compounds
At wavelength 1 A1 = ea1ca + eb1cb
At wavelength 2 A2 = ea2ca + eb2cb
Where ea1, eb1, ea2, eb2 are the molar absorptivities for the two compounds at the two wavelengths which are determined from standard solutions -
and ca and cb are the concentrations of the two unknown compounds.

14. UV/VIS
Spectroscopy
Quantification of Two Compounds
EXAMPLE

15. multiply equation (2) by 6.73, giving equation (4):
UV/VIS
Spectroscopy
Quantification of two compounds
Set up equations:
(1) = 11636ca cb
(2) = 26579ca cb
(3) = 11636ca cb
(4) = ca cb
multiply equation (2) by 6.73, giving equation (4):

16. UV/VIS Spectroscopy Quantification of two compounds Set up equations:
(3) = 11,636ca ,949cb
(4) = 178,877ca ,949cb
Subtract (3) from (4), giving equation (5):
(5) = 167,240ca

17. UV/VIS Spectroscopy Quantification of Two Compounds
Solve for ca:
(6) ca = 9.36 x 10-6 moles/L = concentration of compound a
Substitute value from (6) into equation (1) and solve for cb:
0.870 = 11636(9.36 x 10-6) cb
0.870 = cb
0.760 = 17949cb
cb = 4.24 x 10-5 moles/L = concentration of compound b