View
269
Download
5
Category
Preview:
Citation preview
UV/VISIBLE SPECTROSCOPY
UV/VISSpectroscopy
What does a UV/Vis Spectrometer
Measure?
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.
UV/VISSpectroscopy
UV/VISSpectroscopy
As the radiation is absorbed in the sample,the total intensity of radiation is reducedas it travels through the sample. This results in a non-linear relationship between transmittance and concentration.
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)
UV/VISSpectroscopy
UV/VISSpectroscopy
Within limits, the relationship between absorbance and concentration is linear.
Beer’s law or the Beer-Lambert law
A = bc
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)
UV/VISSpectroscopy
Increased interaction between the molecules can affect absorbance at high concentrations.
UV/VISSpectroscopy
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.
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.
UV/VISSpectroscopy
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.
UV/VISSpectroscopy
Quantification of Two Compounds
Spectra of Compound 1Spectra of Compound 2
Wavelength
Ab
sorb
ance
UV/VISSpectroscopy
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
UV/VISSpectroscopy
UV/VISSpectroscopy
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.
A = bc
Quantification of Two Compounds
Sample Absorptivity
at λ465 Absorptivity at
λ540 Compound a 11636 26579 Compound b 17949 2667
Absorbance of the mixture at λ465 = 0.870 at λ540 = 0.362
EXAMPLE
UV/VISSpectroscopy
Quantification of two compounds
Set up equations:
(1) 0.870 = 11636ca + 17949cb
(2) 0.362 = 26579ca + 2667cb
(3) 0.870 = 11636ca + 17949cb
(4) 2.436 = 178877ca + 17949cb
multiply equation (2) by 6.73, giving equation (4):
UV/VISSpectroscopy
Quantification of two compounds
Set up equations:
(3) 0.870 = 11,636ca + 17,949cb
(4) 2.436 = 178,877ca + 17,949cb
Subtract (3) from (4), giving equation (5):
(5) 1.566 = 167,240ca
UV/VISSpectroscopy
Quantification of Two Compounds (5) 1.566 = 16724ca
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) + 17949cb
0.870 = 0.109 + 17949cb
0.760 = 17949cb
cb = 4.24 x 10-5 moles/L = concentration of compound b
UV/VISSpectroscopy
Recommended