Chapter 6
Spectroscopic method-
UV/Vis
Energy diagram
Components of Spectrophotometer
Sample container (Cuvette)
For Visible and UV spectroscopy, a liquid sample is usually
contained in a cell called a cuvette.
Glass is suitable for visible but not for UV spectroscopy because it
absorbs UV radiation.
Quartz can be used in UV as well as in visible
spectroscopy Long 1 cm pathlength cu
pathlength
Opaque
Face
Transparent
Face
1 cm 1 cm
Short
Holding capacity 1.5 mL to 3.5 mL
pathlength (b)
Absorbance Spectra
Visible absorbance spectrum for cranberry juice
(anthocyanin dyes )
Transmittance and Absorbance
Principles
Beer’s Law
The amount of light absorbed is proportional
to the concentration of the absorbing
substance
100% light 40% light 100% light 70% light
60% light absorbed 30% light absorbed
Rate of Absorption
Lambert ’s Law
The amount of light absorbed is proportional to the
thickness (length) of the absorbing material
(Cuvette)
100% light 40% light 100% light 70% light
Larger path length
Smaller path length
60% light absorbed 30% light absorbed
Rate of Absorption
Absorbance and Concentration:
Lambert-Beer’s Law
a is the analyte’s absorptivity [cm–[Link]–1]
If the concentration is expressed in molarity:
a: molar absorptivity, , [cm–1.M–1), the Beer’s Law is
rewritten:
Lambert-Beer’s Law and
Multicomponent Samples
For a two-component mixture of analyte’s X and Y, the total
absorbance, Atot, is
Generalizing, the absorbance for a mixture of n components,
Amix, is:
Applications
The applications of UV/Vis Spectrometer are quite
vast.
In analytical chemistry: quantitative determination
of different analytes, such as metal ions,
highly conjugated organic compounds, and
biological macromolecules.
Spectroscopic analysis is commonly carried out in
solutions but solids and gases may also be studied.
*** if an analyte does not absorb UV/Vis radiation—
(or too weak): react it with another species that is
strongly absorbing (EX: Fe2+ with o-phenanthroline)
Q&A
Q&A
The concentration of Cu2+ in a sample is determined by reacting
it with the ligand cuprizone and measuring its absorbance at 606
nm in a 1.00-cm cell. When a 5.00-mL sample is treated with
cuprizone and diluted to 10.00 mL, the resulting solution has an
absorbance of 0.118. A second 5.00-mL sample is mixed with
1.00 mL of a 20.00 mg/L standard of Cu2+, treated with cuprizone
and diluted to 10.00 mL, giving an absorbance of 0.162. Report
the mg Cu2+/L in the sample. (5.4)
Q&A
The absorbance spectra for Cr3+ and Co2+ overlap significantly.
To determine the concentration of these analytes in a mixture, its
absorbance is measured at 400 nm and at 505 nm, yielding
values of 0.336 and 0.187, respectively. The individual molar
absorptivities (M–[Link]–1) for Cr3+ are 15.2 at 400 nm and 0.533 at
505 nm; the values for Co2+ are 5.60 at 400 nm and 5.07 at 505
nm.