SPECTROPHOTOMETRY The ranges of the wavelength of radiant the practice of
radiation of single energy of importance in
PHYSICOCHEMICAL AND INSTRUMENTAL METHODS USEDIN spectrophotometry are:
PHARMACEUTICAL ANALYSIS UV = 200 – 380 nm
Visible = 380 – 780 nm
LIGHT Near IR = 780 – 3000 nm
is electromagnetic radiation within a certain portion of Medium = 3.0 – 15 um
the electromagnetic spectrum. The word usually refers Far IR = 15 – 300 um
to visible light, which is visible to the human eye and is
responsible for the sense of sight.
FREQUENCY + ENERGY AND WAVELENGTH – INVERSE
PROPORTIONAL
GROUP FREQUENCY REGION
The region from 3 to 8 um, because the absorption
peaks which appear in this region are due to functional
groups (carbonyl, amine, hydroxyl, etc.) formed in the
organic compounds.
FINGERPRINT REGION
The region from 8 to 15 um because this region gives a
spectrum of the molecule as a whole.
BEER'S LAW
states that the power of a transmitted radiant beam
decreases exponentially as the concentration of the
solution containing the absorbing chemical species
increases arithmetically.
LAMBERT OR BOUGER'S LAW
states that the power of a transmitted radiant beam
decreases exponentially as the thickness of the solution
containing the absorbing chemical species increases
arithmetically.
BEER-LAMBERT OR BEER-BOUGER'S LAW
is a combination of the above law and relates the power
of the incident and the transmitted radiant beam to the
thickness and concentration of the solution containing
absorbing chemical species.
BEER'S PLOT
The concentration could also be determined by the
following mathematical relationship :
ELECTROMAGNETIC SPECTRUM
Is the term to define the complete system of energy
propagated in waveform. Energy in this nature is
referred to as radiant energy and appears in such
apparently different forms as sunlight, color, radiowaves, TRANSMITTANCE
etc. the ratio of the amount of light transmitted to the
amount of light that initially fell on the surface.
RADIANT ENERGY
refers to energy in the uv, visible, and ir regions of the ABSORBANCE
electromagnetic spectrum. the negative logarithm of transmittance.
SPECTROMETRY- Defined as a method of analysis that deals ABSORBED ENERGY
with the measurement of spectra. The molecule absorbs energy that requires to elevate to a
SPECTROSCOPE: Measures the light higher level
emitted/transmitted Initial energy state – ground state to higher energy
SPECTROPHOTOMETER: Measures the light state
transmitted – qualitative and quantitative Energy can be transferred to the following forms:
ABSORPTIMETRIC: Analytical methods based on Electronic (UV)
the absorption of radiant energy Associated with the overall motion of
COLORIMETRIC: Type of absorptiometry utilize the electrons around the nuclei
visible region of spectrum Vibrational (IR)
Associated with the motion of atoms within
COLORIMETRY- a branch of spectrophotometry in which the the molecule
absorption measurement is made in the visible region of the Rotational (Microwave)
spectrum Associated with the overall rotation of the
molecule
Translational (not quantized)
Associated with the motion of the molecule
as a whole
SPECTROPHOTOMETRY- a branch of spectrometry which
embraces the measurementof the absorption by chemical
species of radiant energy of definite and narrow wavelength
approximating monochromatic radiation.
ELECTRONIC TRANSITIONS (UV)
Involves the transfer of electrons between different
electronic state -- > ground state to the excited state
When UV is absorbed, an electron is raised from the
ground state to the excited state, loses its vibrational
energy, decays to the ground state, emitting UV or
visible light -- flourescence
When the transition back to the ground state is delayed
it results in phosphorescence.
Monochromatic radiation - is radiation of a single VIBRATIONAL (IR)
wavelength. Two general regions under IR
Monochromatic radiation is obtained by using prism or Group frequency - region of 3.0 to 8.0 μm, wave
diffraction grating. number of 4000-1300cm-1
vibrational bands are associated with specific
PRINCIPLES structural or functional groups
Every substance absorbs or wavelengths of radiant Fingerprint region - 8.0 to 15.0 μm, wave number
energy wavelengths of 1300 - 400cm-1
Intensity of the transmitted radiant energy is a function vibrational modes depend strongly on the
of the concentration rest of the molecule
Chromophore – functional group which absorbs radiant SPECTROPHOTOMETER
energy in the UV or vis regions These instruments may either be manually operated or
Ethylene automatic recording instruments.
Acetylene Instruments have
Ketones radiant power source
Organic acids radiant energy dispersing device
Aldehydes a sample compartment
Azomethines the associated electronics which permit the
measurement of
radiant power transmitted by the sample.
TERMS & SYMBOLS APPLICABLE TO
SPECTROPHOTOMETRICANALYSIS:
Transmittance (T). The ratio of the radiant power
transmitted by the solution to the radiant power
transmitted by the blank (solvent).
T = P (Solution)/P (Blank)
Absorbance (A). Also called optical density, absorbancy,
extinction coefficient.
A = Log 1/T or A = Log (P (Blank)/ P(Solution)
Concentration (C). Expressed in grams of solute per liter
of solution.
Absorptivity (a). The value obtained by dividing the
absorbance (A) by the product of concentration,
expressed in grams per liter, and the path length,
expressed in centimeters.
Molar Absorptivity (ͼ). The value obtained by dividing
the absorbance (A) by the product of concentration of
the solution, expressed in moles per liter, and the path
length, expressed in centimters.
Absorption Spectrum. The graph obtained when
absorbance or any function of absorbance is plotted
against wavelength.
