Spectroscopy

Spectroscopy

—  Spectroscopy is derived from two words a Latin word specere which means “ to look at” and a Greek word skopia means “ to see”

—  The study of the interaction between matter and electromagnetic radiation is known as spectroscopy.

—  The study of the emission and absorption of light and other radiations by matter is known as spectroscopy.

—  Spectroscopy were famous for visible spectrum traditionally, however UV, X-ray, and gamma spectroscopy are also very applicable as an analytical tool.

—  The data obtained from spectroscopy is usually presented as spectrum.

—  Spectroscopy can involve any interaction between light and matter, including absorption, emission, scattering, etc.

—  When a beam of electromagnetic radiation passes through a sample, the photons interact with the sample.

—  As a result of the interaction of photons with the sample they are either absorbed, reflected, refracted, etc.

—  Absorbed radiation affects the electrons and chemical bonds in a sample.

—  In some cases, the absorbed radiation leads to the emission of lower- energy photons.

—  Spectroscopy reveals the effect of incident radiation upon the sample.

—  Spectrum obtained as a result of emission and absorption are used to collect information about the sample material.

UV Visible Spectroscopy

—  Analytical technique that measures the amount of discrete wavelength of light in UV-visible range that are absorbed by or transmitted through a sample in comparison to a reference or blank sample.

—  UV range 100 to 400nm

—  Deep UV range 100 to 200nm

—  Visible range 400 to 700nm

Principal

—  Molecules containing bonding and non bonding electrons can absorb energy in the form of UV or Visible light and gets excited to higher molecular orbitals.

—  Working of UV Visible spectroscopy is based on Beer-Lambert law which states that..

—  When a beam of monochromatic light is passed through a solution of an absorbing substance,

—  the rate of decrease of intensity of radiation with thickness of the absorbing solution is proportional to the incident radiation as well as the concentration of the solution.

—  The expression of Beer-Lambert law is

—   A = log (I0/I) = Ecl

—  Where, A = absorbance

—  I0 = intensity of light incident upon sample cell

—  I = intensity of light leaving sample cell

—  C = molar concentration of solute

—   L = length of sample cell (cm.)

—  E = molar absorptivity

—  More the concentration of solution the higher will be the absorbance due to the increased interaction of molecules.

—  Similarly longer the path length the more molecules will interact and hence more will be the absorbance

Instrumentation

—  Light source

—  Mostly used light sources are tungsten filament lamps and Hydrogen-Deuterium lamps

—  Monochromator

—  It is mostly consist of a prism and a slit and select particular wavelength of light.

—  Sample and Reference Cell

—  They are made up of quarts and are called cuvetts

—  Detector

—  Photocells working as detector and it measures the light transmitted from the sample

—  Recording Device

—  A computer system

Application

—  It is used in qualitative and quantitative determination of different analytes such as metal ions, organic compounds and bilogical macro and micro molecules.

—  Used in polymer analysis.

—  Determination of functional groups.

—  Organic compounds structural elucidation

—  Quantitative determination of pharmaceutical substances.

—  Identification and quantitative determination of poly nuclear aromatic compounds.

—  Determination of impurities.

—  As HPLC detector

FTIR
Fourier Transform Infrared Spectroscopy

What is FTIR

Introduction

—  FTIR spectroscopy is an analytical technique, which is used for identification of Organic, Polymeric, Inorganic compounds in Pharmaceutical industry, Petrochemical engineering, And Food industries.

—  FTIR uses Infrared light  to scan test sample and observe chemical properties. It works on the fingerprint of molecules, which is a great tool for us in chemical identification.

—  This tiny beautiful optical piece was invented by Albert Abraham Michelson, also received NOBLE prize in 1907.

—  The range of infrared region is 12800 ~ 10 cm^-1 near-infrared region (12800 ~ 4000 cm^-1), mid-infrared region (4000 ~ 200 cm^-1) and far-infrared region (50 ~ 1000 cm^-1)

Instrumentation

—  Input

—  Mirror 1

—  Mirror 2

—  Beam Splitter

—  Output (Detector)

How Ftir works

—  Ftir is based on the Michelson interferometer Experimental setup as shown in previous slide

—  The interferometer consist of Source, beam splitter, fixed mirror, movable mirror(translates back and forth very precisely), and output.

—  The beam splitter is made of special material which transmits half radiation and reflects half radiation.

—  The radiation from the source strikes beam splitter which is divided into two beams, one beam transmits to fixed mirror and 2^nd beam reflects back to beam splitter.

—  Again half radiation transmits and half reflects back to beam splitter.

—  This transmission and reflection results in one beam passes to output (detector) and the other beam back to the source.

Principle of Ftir

—  The basic principle of Ftir is to identify the functional group by providing or striking the material with some energy packets (in the form of IR light/Radiation).

—  The functional group in a material attached with each other with specific bonds. And these bonds will produce some stretching, wagging and vibrational movement when struck by IR source.

—  Now when IR radiation passes through a molecule, some radiation will be absorbed and some radiation passes through. The absorbed radiation is converted into that movement   ( Rotational, Vibrational) by sample molecule, representing the molecular fingerprint (Functional Group) of sample.

—  The resulting signal on detector produce a spectrum which is shown on our monitor of computer attached to Ftir.

NMR
Nuclear Magnetic Resonance Spectroscopy

—  Nuclear magnetic resonance is the most powerful physicochemical tool to determine the organic structures of molecules.

—  NMR Spectroscopy is the study of molecular structure determination by recording the interaction of radiofrequency (Rf) electromagnetic radiations with the nuclei of molecules placed in a strong magnetic field.

Basis of NMR

—  NMR is used in determination of molecular structures as well as the content and purity os samples.

—  The most widely used method of NMR is Proton (1H) NMR in analytical chemistry.

—   The protons present in the molecule will behave differently depending on the surrounding chemical environment, making it possible to elucidate their structure.

NMR Principal

—  As per the NMR principal most of the nuclei are exhibiting spin and all the nuclei are charged electrically.

—  A nucleus with an odd atomic number or an odd mass number has a nuclear spin.

—  The spinning charged nucleus generates a magnetic field.

—  When placed in an external field these spinning protons act like bar magnets.

—  When an external magnetic field is applied energy transfer from base energy state to higher energy state occur.

—  The magnetic fields of the spinning nuclei will align either with the external field, or against the field.

—  The wavelength at which energy transfer occur coincide with the radio waves.

—  Emission of energy at the same frequency occur when the spin comes back to the base state.

—  Processing of NMR spectrum for the concerned nuclei is measured by the signals that matches this transfer.

—  The number of signals shows how many different kinds of protons are present.

—  The location of the signals shows how shielded or deshielded the proton is.

—  The intensity of the signal shows the number of protons of that type.

—   Signal splitting shows the number of protons on adjacent atoms.

Working of NMR

—  1^st of all sample is placed within magnetic field.

—  The nuclei sample is excited with the help of radio waves within the magnetic field to generate NMR signals.

—  A detector sensitive to radio waves is used to detect these signals.

—  The intramolecular magnetic filed surrounding the molecules changes the resonance frequency of the atoms in the molecule.

—  These changes in the resonance frequency of atom gives detain about the functional groups and structure of the molecule.

—  Reaction state, structure of molecule, chemical environment and dynamics of a molecule are determined in this way by applying this technique.

Chemical Shift

—  A nuclei that exhibit charge when do spinning it generates magnetic field, this magnetic field results magnetic moment.

—  This magnetic moment is directly proportional to the spin.

—  When an external magnetic field is applied it results in two spin states

—  Up spin

—  Down spin

—  Among these up spin and down spin one aligns with the external magnetic filed and the other one opposes it.

—  Difference between the resonant frequency of the spinning charged nuclei i.e proton and the signals of the reference molecule characterize the chemical shift.

—  Magnetic shielding

—  If all protons absorbed the same amount of energy in a given magnetic field, not much information could be obtained.

—  But protons are surrounded by electrons that shield them from the external field.

—  Circulating electrons create an induced magnetic field that opposes the external magnetic field.

—  Magnetic field strength must be increased for a shielded proton to flip at the same frequency.

—  Nuclear magnetic resonance chemical change is one of the most important properties usable for molecular structure determination.

—   1H and 13C are the most widely used nuclei that are detected by NMR

—  Beside the above, 15N (nitrogen 15), 19F (fluorine 19), are also used.

—  Chemical shift is measured in (ppm)parts per million.

—  Chemical shift δ = νsample - ν reference

—                                                 ν _reference

—  V sample = absoulte frequency of sample

—  V reference = absoulte frequency reference

—  Magnetic field B₀ would be same for both reference and sample

—  numerator is usually expressed in hertz

—   and the denominator in megahertz 

—  Chemical Shift δ is expressed in ppm

—  The detected frequencies (in Hz) for ¹H, ¹³C, and ²⁹Si nuclei are usually referenced against TMS (tetramethylsilane), TSP (Trimethylsilylpropanoic acid), or DSS (Sodium trimethylsilylpropanesulfonate).

—  TMS, TSP and DSS have a chemical shift of zero if chosen as the reference.

—  NMR signal observed at a frequency 300 Hz higher than the signal from TMS, where the TMS resonance frequency is 300 MHz, has a chemical shift of:

—   

—  δ =    300Hz        =   1 x 10^-6  = 1ppm

—           300 x 10⁶

Instrumentation

—  Sample holder: Sample holder is 8.5 cm long and 0.3 cm in diameter glass tube.

—  Magnetic coils : Magnetic coil generates magnetic field whenever current flows through it.

—  Permanent magnet : Permanent magnet provides a homogenous magnetic field at 60 – 100 MHZ.

—  Sweep generator: It modifies the strength of the magnetic field which is already applied.

—  Radiofrequency transmitter: It generates a powerful and short pulse of the radio waves.

—  Radiofrequency: It supports detecting receiver radio frequencies.

—  RF detector: It determines unabsorbed radio frequencies.

—  Recorder: It records the NMR signals which are received by the RF detector.

—  Readout system: Computer system records the data.

Applications

—  NMR is used in the structural determination of organic and inorganic compounds.

—  Microstructure determination of polymer chain.

—  Determination of physical and chemical properties of atoms.

—  In medicine it is used in MRI, Tumors, tissue perfusion studies and angiography.

 

Presented By

Muhammad Atif

PhD Scholar Pharmaceutical Sciences AWKUM

Manager QA Decent Pharma Islamabad Pakistan.