SPECTROPHOTOMETRY

Objectives
 To identify and explain spectroscopy or
spectrophotometry
 To illustrate the EM spectrum
 To identify the specific components of
spectrophotometer
 Explain the principle involved in analyzing
organic substances based on their
chromophores.
 To discuss the applications of
spectrophotometry
REFERENCES
 Molecular spectroscopy . www.scribd.com
 Pharmaceutical Analysis. A textbook for
Pharmacy Students and Pharmaceutical
Chemists. By : David G. Watson
Spectroscopy

• Study of the Interaction of Electromagnetic

 Radiation (Energy) and Matter
• When energy is applied to matter it can be
absorbed, emitted, cause a chemical change
(reaction), or be transmitted.
• Electromagnetic Spectrum:-
 Cosmic g (Gamma) X-Ray
 Ultraviolet Visible Infrared
 Microwave Radio
 LIGHT SOURCE – deuterium lamp UV = 190-
350 nm
- quartz halogen/tungsten =
Vis region = 350 nm – 900nm

 Monochromator – disperses light into

constituents.
 Optics – designed to split the beam so that it
passes to 2 compartments.
Spectroscopy Types:
1. Ultraviolet Spectroscopy (UV) – Electronic Energy
States
• Use –Conjugated Molecules; Carbonyl Group, Nitro
Group
2. Infrared Spectroscopy (IR) – Vibrational Energy States
• Use – Functional Groups; Compound Structure
3. Nuclear Magnetic Resonance (NMR) – Nuclear Spin
States
• Use – The number, type, and relative position of protons
(Hydrogen nuclei) and Carbon-13 nuclei
4. Mass Spectrometry (MS) – Hi-Energy Electron
Bombardment
• Use – Molecular Weight, Presence of Nitrogen, Halogens
5. AES AND AAS
Terms describing UV absorptions

 1. Chromophores: functional groups that give electronic

transitions.
 2. Auxochromes: substituents with unshared pair e's
like OH,NH, SH ..., when attached to π
chromophore they generally
move the absorption max. to longer λ.
 3. Bathochromic shift: shift to longer λ, also called red
shift.
 4. Hysochromic shift: shift to shorter λ, also called blue
shift.
 5. Hyperchromism: increase in ε of a band.
 6. Hypochromism: decrease in ε of a band.
UV Spectroscopy
Chromophores
 A. Definition:-
• Remember the electrons present in organic
molecules are involved in covalent bonds or lone pairs of electrons
on atoms such as O or N
• Since similar functional groups will have electrons
capable of discrete classes of transitions, the characteristic energy
of these energies is more representative of the functional group than
the electrons themselves
• A functional group capable of having characteristic electronic
transitions is called a chromophore (color loving)
• Structural or electronic changes in the chromophore can be
quantified and used to predict shifts in the observed
electronic transitions
APPLICATION IN
PHARMACEUTICAL ANALYSIS
 Analysis of inorganic
substances
 AAS – Metals
 FTIR – functional groups
 Analysis of drugs –
furosemide, cyclizine
lactate and penicillin,
adrenaline in lidocaine.
USE OF UV-VIS TO COMPUTE
FOR pKa values
 pH are UV dependent
 Ex. Phenylepinephrine = phenolic group @ 292
nm.
 FORMULA
 pKa = pH + log Ai –A
A-Au
A = absrobance of the buffer
Ai = absorbance of fully ionised specie
Au = absorbance of unionised
EXAMPLE
 The absorbance of a fixed concentration of
phenylepinephrine at 292 m is found to be
1.224 in 0.1 M NaOH and 0.02 in 0.1 M
HCl. Its absorbance in buffer at pH 8.5 is
found to be 0.349. Calculate the pKa
value of its acidic phenolic group.