Introduction to UV-Visible spectrophotometry

Content
●
Fundamental concepts of spectrophotometry.
●
Absorbance and Transmittance
●
Beer's Law and quantitative analysis
●
UV-Vis spectrum and qualitative analysis
●
Chemical and instrumental deviations from Beer's law.
●
Electronic model of UV-Vis Absorption
●
Absorption of organic and inorganic compounds in the ultraviolet and
visible region.
●
Components and instrumentation used in UV-Vis spectrophotometry.
●
Qualitative and quantitative determination of substances of interest.
 Content
●
Fundamental concepts of spectrophotometry.
●
Absorbance and Transmittance
●
Beer's Law and quantitative analysis
●
UV-Vis spectrum and qualitative analysis
●
Chemical and instrumental deviations from Beer's law.
●
Electronic model of UV-Vis Absorption
●
Absorption of organic and inorganic compounds in the ultraviolet and
visible region.
●
Components and instrumentation used in UV-Vis spectrophotometry.
●
Qualitative and quantitative determination of substances of interest.
Fundamental concepts of spectrophotometry.
Fundamental concepts of spectrophotometry.
 Fundamental concepts of spectrophotometry.
Many properties of electromagnetic radiation are adequately explained with a
classical sine wave model that uses parameters such as wavelength, frequency,
speed and amplitude. Magnetic radiation propagates in a vacuum
 Fundamental concepts of spectrophotometry.
A wave consists of successive troughs (Min) and crests (Max). The distance between two
adjacent crests or troughs is called the wavelength. Waves of the electromagnetic
spectrum vary in size, from very long radio waves the size of buildings to very short gamma
rays smaller than atom nuclei. Frequency is inversely proportional to wavelength, according
to the equation:

When the waves cross boundaries between different media, their speeds change but their
frequencies remain constant. In vacuum, the velocity is 2,99 x 10-8 m/s.
 Fundamental concepts of spectrophotometry.
Mathematical description of a wave

Electric Phase
field angle
Fundamental concepts of spectrophotometry.
Types of interaction of radiation with matter
 Fundamental concepts of spectrophotometry.
Interaction when the radiation is in the UV-Vis range
 Absorbance and Transmittance
Absorption of electromagnetic radiation is the process in wich matter (typically electrons
bound in atoms) takes up a photon's energy and so transforms electromagnetic energy into
internal energy of the absorber.
 Absorbance and Transmittance
The absorbance of an object quantifies how much of the incident light is absorbed by it
(instead of being reflected or refracted). This may be related to other properties of the object
through the Beer–Lambert law. Precise measurements of the absorbance at many
wavelengths allow the identification of a substance via absorption spectroscopy, where a
sample is illuminated from one side, and the intensity of the light that exits from the sample in
every direction is measured. A few examples of absorption are ultraviolet–visible
spectroscopy, infrared spectroscopy, and X-ray absorption spectroscopy.

If the concentration is expressed in molarity, the

absorptivity is named molar absorptivity (L M-1 cm-1)
Absorbance and Transmittance
FT-IR

X-Ray
UV

Visible
 Absorbance and Tramitance
Transmittance of a material is its effectiveness in transmitting radiant energy. It is the
fraction of incident electromagnetic power that is transmitted through a sample, in contrast
to the transmission coefficient, which is the ratio of the transmitted to incident electric field
Absorbance and Tramitance
 Beer's Law and quantitative analysis
Two solutions of the H-In (Ka = 1,42 x 10-5) base-acid indicator are prepared by diluting
solutions of this compound to the concentration of 1,02 x 10-4 M with equal volumes of 0.2 M
NaOH and 0.2 M HCl. When the absorbance is measured in a 1 cm cell, obtain the following
data at 430 nm and 570 nm:

Reactant A (430 nm) A (570 nm)

0,2 M NaOH 1,051 0,049
0,2 M HCl 0,032 0,363
Deduct the absorbance data at the two wavelengths for the non-buffered solutions that have
indicator concentrates within the range of 2 x 10-5 to 1,6 x 10-4 M.

Tip:
1) Calculate the molar molar absorptivity for H-In and In- at every absorbance.
2) Calculate the absorbance for In- and H-In as a sum of both absorbances AT = AHIn + AIn-

.
Beer's Law and quantitative analysis
 Beer's Law and quantitative analysis

Interpolation Standard addition

 Content
●
Fundamental concepts of spectrophotometry.
●
Absorbance and Transmittance
●
Beer's Law and quantitative analysis
●
UV-Vis spectrum and qualitative analysis
●
Chemical and instrumental deviations from Beer's law.
●
Electronic model of UV-Vis Absorption
●
Absorption of organic and inorganic compounds in the ultraviolet and
visible region.
●
Components and instrumentation used in UV-Vis spectrophotometry.
●
Qualitative and quantitative determination of substances of interest.
 Chemical and instrumental deviations from Beer's law

Some deviations in linearity are connected with changes in absorptivity with wavelength and the
specificity of the instrument in being able to isolate the smallest increment of absorbing
wavelengths. If the increment of wavelengths measured by the instrument is small and the
molar absorptivity of the species being observed is near the maximum then any change in the
molar absorptivity in this region is small and Beer's Law is obeyed
 Chemical and instrumental deviations from Beer's law

If the concentration of the species being observed increases there is a greater probability in
interactions between the absorbing molecules, and a change (usually downward) in the
absorbance. Any interaction likely interferes with the electronic band structure in the absorbing
species and and energy absorption levels change with a resulting exhibition of a non-linear
perturbation of Beer's Law.
 Content
●
Fundamental concepts of spectrophotometry.
●
Absorbance and Transmittance
●
Beer's Law and quantitative analysis
●
UV-Vis spectrum and qualitative analysis
●
Chemical and instrumental deviations from Beer's law.
●
Electronic model of UV-Vis Absorption
●
Absorption of organic and inorganic compounds in the ultraviolet
and visible region.
●
Components and instrumentation used in UV-Vis spectrophotometry.
●
Qualitative and quantitative determination of substances of interest.
 Content
●
Fundamental concepts of spectrophotometry.
●
Absorbance and Transmittance
●
Beer's Law and quantitative analysis
●
UV-Vis spectrum and qualitative analysis
●
Chemical and instrumental deviations from Beer's law.
●
Electronic model of UV-Vis Absorption
●
Absorption of organic and inorganic compounds in the ultraviolet
and visible region.
●
Components and instrumentation used in UV-Vis spectrophotometry.
●
Qualitative and quantitative determination of substances of interest.
 UV-Vis spectrum and qualitative analysis
UV-Vis spectroscopy has limited applications in the qualitative analysis, because in these
spectra there is a relatively low number of maxima and minima. Consequently, the
identification of compounds is usually impossible. Example: Ligand effect
 UV-Vis spectrum and qualitative analysis
Solvent selection: When choosing a
solvent, you should consider not only
its transparency but also its possible
effects on the absorbent system.
Example: polar solvents such as
water tend to obliterate the fine
spectral structure produced by biblical
effects.
 Absorption of inorganic compounds in the ultraviolet and
visible region
A transition metal complex is made up of a centrally positioned transition metal atom or ion
surrounded by, and bonded to, a group of other ions or neutral molecules (these are known as
‘ligands’).

Neutral complex: [Pt(NH3)2Cl4]

Cationic complex: [Ir(pyridine)4Cl2]Cl
Anionic complex: K2[PtCl6]
Absorption of inorganic compounds in the ultraviolet and
visible region
Absorption of organic compounds in the ultraviolet and
visible region
Absorption of organic compounds in the ultraviolet and
visible region
 Absorption of organic compounds in the ultraviolet and
visible region
Molecule wavelength of maximum absorption
(nm)
Ethene 171
buta-1,3-diene 217
hexa-1,3,5-triene 258

https://www.youtube.com/watch?v=wxrAELeXlek
 Qualitative and quantitative determination of substances of
interest: mixtures
The content of chromium and manganese in a sample of 0.246 g of steel was analyzed,
dissolving it in acid and diluting it in 250 mL. A 50 mL aliquot was treated with potassium
persulfate in which, in the presence of catalytic amounts of the Ag+ ion, it converts chromium
and manganese to Cr2O72- and MnO4-. The resulting solution was diluted to 100 mL. It was
found that the absorbance of this solution, read in 1cm cells, was 0.932 at 440 nm and
0.778 at 545 nm respectively. Calculate the percentage of manganese and chromium in the
sample.

Wavelenght Ɛ (Cr2O72-) Ɛ (MnO4-)

(nm) (L/mol.cm) (L/mol.cm)
440 369 95
545 11 2350
Qualitative and quantitative determination of substances of
interest: mixtures