What is Spectroscopy?

• PART I : Basics of Spectroscopy & UV-Visible Spectroscopy
• PART II: CD Spectroscopy
• PART III: ESR Spectroscopy
• PART IV: FTIR Spectroscopy
• PART V: Fluorescence Spectroscopy

Spectroscopy: The study of molecular structure and dynamics through the absorption, emission and scattering of light.

Schematic of a conventional single-beam spectrophotometer

UV-Visible important points:

• Absorbance rises linearly with concentration.

• Transmittance decreases in a non-linear fashion.

• A=ecl

• Every instrument has a useful range for a particular analyte.

• Often, this range is determined experimentally.

• This is done by making a dilution series of the known solution.

• These dilutions are used to make a working curve.

Light Sources

A-) UV Spectrophotometer

1. Hydrogen Gas Lamp

2. Mercury Lamp

B-) Visible Spectrophotometer

1. Tungsten Lamp

C-) InfraRed (IR) Spectrophotometer

1. Carborundum (SIC)

Circular Dichroism (CD) is a form of spectroscopy based on the differential absorption of left- and right-handed circularly polarized light. It can be used to determine the structure of macromolecules.

What changes in light when it is absorbed by a molecule:

i. Intensity (amplitude)èDue to absorption
ii. Polarization èDue to absorption
iii. Velocity èDue to refractive index
iv. Wavelength èDue to refractive index

PLANE polarization + PLANE polarization = CIRCLE polarization

R-CIRCLE polarization + L-CIRCLE polarization = PLANE polarization

Important points:

• Components of the plane polarized light are absorbed by different “amounts” by different stereoisomers.

• Structural asymmetry è differences in the absorption of left-handed polarized light versus right-handed polarized light

Electron paramagnetic resonance (EPR) or electron spin resonance (ESR) spectroscopy is a technique for studying chemical species that have one or more unpaired electrons such as organic and inorganic free radicals or inorganic complexes possessing a transition metal ion.

In EPR study, we measure and interpret the energy differences between the atomic or molecular states.

• Spin gives a magnetic property to electron known as a magnetic moment.

• When an external magnetic field is supplied, the paramagnetic electrons can either orient in a direction parallel or antiparallel to the direction of the magnetic field.

• This creates two distinct energy levels for the unpaired electrons and measurements are taken as they are driven between the two levels.

Interactions of an unpaired electron with its environment influence the shape of an EPR spectral line. Line shapes can yield information about, for example, rates of chemical reactions.

Sensitivity of EPR depends on:

1-) Frequency

2-) Spectrometer Type

3-) Resonance Condition

4-) Sample Size

In FTIR Spectroscopy infrared (760-1000 nm) is passed through sample and Fourier transform equations are used in graphical interpretation.

No two unique molecular structures produce the same infrared spectrum.

IR & Raman Spectroscopies are used especially in:
-Pathology: Cancer, Neurological Disorders, Cardiology
-Clinical Chemistry: Reagent free quantitative analysis of
analytes in biological fluids (in vitro/in vivo)

Advantages:

Simultaneouity è Higher Signal/Noise Ratio

No Slit è Higher Signal/Noise Ratio

Ease of Addition & Substraction of Spectra

Disadvantages:

Measurement of Interferogram, not Spectra è Need to Fourier analysis

Noise at one part spreads to all spectra (so use middle ranges of IR)

1 single beam è Results more effected by atmospheric change

Fluorescence is a luminescence that is mostly found as an optical phenomenon in cold bodies, in which the molecular absorption of a photon triggers the emission of another photon with a longer wavelength. The energy difference between the absorbed and emitted photons ends up as molecular vibrations or heat. Usually the absorbed photon is in the ultraviolet range, and the emitted light is in the visible range, but this depends on the absorbance curve and Stokes shift of the particular fluorophore.

Fluorescence is named after the mineral fluorite, composed of calcium fluoride, which often exhibits this phenomenon.

Fluorescence lifetime is the average time that an electron spends in the excited state before a photon is emitted.

Measurement of the fluorescence from a large number of molecules, following a short pulse excitation, will show an exponential decay. (Intensity-time graph is exponential decay graph)

Photobleaching (fading): permanent loss of fluorescence due to photo-induced chemical modification of molecule

Quenching: Competing processes that induce non-radiative relaxation of excited-state electrons to the ground state

IMPORTANT POINTS:

• The fluorescence spectrum is shifted to longer wavelengths compared with the absorption spectrum

• Fluorescence spectroscopy allows in general more sensitive measurements than absorption spectroscopy

• Both the fluorescence spectrum and the lifetime are sensitive to the environment

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