METHODS USED FOR
OFFICIAL ASSAY
General Methods
■General Methods are the “classical methods”.
Types:
A. Titrimetry
B. Gravimetry
Instrumental Methods
■Based on the utilization of an instrument to
measure physicochemical properties.
Types of Instrumental Methods:
A. Spectrometric
B. Chromatographic
C. Electrometric
SPECTROMETRIC
METHODS
SPECTROSCOPY
■A branch of science that deals with the study of
interaction of electromagnetic radiation with
matter or sample substances.
■Based on the absorption or emission of
electromagnetic (EM) radiation as a result of
interaction with matter.
■Instrument: SPECTROPHOTOMETER
PLAYS AN IMPORTANT ROLE IN SPECTROSCOPY
Chromophore –functional group that absorbs maximum radiation in the
UV or visible regions
Auxochrome – functional group which does not give rise to an
absorption band by itself, but upon being attached to a chromophore
Electromagnetic radiation – energy propagated in a wave form
Frequency – number of complete cycles that pass a single point per
second
Wavelength – length of one complete wave or cycle
■ Spectrophotometer – a device with a light source, prism, filter and a
sensor of energy
CHROMOPHORE
■ Functional group that shows a characteristic absorption
of EMR in the UV or visible region.
■ From the Greek word. Chroma = “color” & phoros =
“bearer
■ Responsible for the change in color of molecules
Examples: (EMO KA)
Ethylene, Methylene, Organic acids, Ketones,
Aldehydes
AUXOCHROME
■A functional group that, when attached to
chromophore alters both wavelength and
intensity of absorption, leading to an enhanced or
modified color.
Example:
Hydroxyl group (-OH)
ABSORPTION AND INTENSITY SHIFTS
There exist four types of shifts
corresponding to auxochromes:
■ Bathochromic shift (redshift)
– towards a longer wavelength
■ Hypsochromic shift (blueshift)
– towards a shorter wavelength
■ Hyperchromic shift – towards
a higher absorbance
■ Hypochromic shift – towards a
lower absorbance
ELECTROMAGNETIC RADIATION
(EMR)
■ The electromagnetic radiation refers to the emission and
propagation of energy through space in the form of
electromagnetic waves or particles.
PARTS OF WAVES
Crest: The highest point or peak of a wave.
1. Trough: The lowest point or dip of a wave.
2. Amplitude (A) : The maximum displacement of a point on the
wave from its undisturbed position, often measured from the
midpoint to the crest or trough.
3. Wavelength (λ) : The distance between two successive points in a
wave, such as from one crest to the next or one trough to the next.
4. Wavenumber (k) –the number of cycles per unit distance
5. Frequency (f) : The number of complete cycles of a wave passing
a point in a given time.
6. Period (T) : The time it takes for one complete cycle of a wave to
pass a given point.
PARTS OF WAVES
Formula:
K= cycles/unit distance
Or
K= 1/λ
PARTS OF WAVES
Formula:
K= cycles/unit distance
Or
K= 1/λ
PARTS OF WAVES
Formula:
(T)- Period Unit of Frequency = Hz
(f)- Frequency • Hz= cycle/s
(v)- Velocity Unit of Wave Period = s
(λ)- Wavelength
There is an inverse relationship between wavelength and frequency.
• The higher the frequency, the shorter the wavelength
• The longer the wavelength, the lower the frequency
PARTS OF WAVES
Formula:
(T)- Period Unit of Frequency = Hz
(f)- Frequency • Hz= cycle/s
(v)- Velocity Unit of Wave Period = s
(λ)- Wavelength
PLANCK’S EQUATION
■ The energy of EMR is given by this equation:
■ Formulas:
Energy is directly proportional to frequency
• Energy is inversely proportional to wavelength
ELECTROMAGNETIC SPECTRUM
■ The electromagnetic spectrum is the division of
electromagnetic radiation based on the energy,
frequency, or wavelength of a photon.
ELECTROMAGNETIC SPECTRUM
WAVELENGTH RANGE OF EACH REGION
FUNDAMENTAL LAWS OF SPECTROMETRY
1. Beer’s Law
■ Transmittance decreases exponentially as the concentration of the solution
increases arithmetically
2. Lambert’s Law/Bouger’s Law
■ Transmittance decreases exponentially as the thickness of the solution
increases arithmetically
3. Beer-Lambert Law
■ combination of Beer’s Law & Lambert’s/Bouger’s Law
■ Absorbance is directly proportional to the path length and concentration
INSTRUMENT: SPECTROPHOTOMETER
COMPONENTS OF SPECTROPHOTOMETER
1. Source of radiation- Generates a beam of radiation 4. Radiation Detector- Converts
a. Continuum sources (ex: deuterium lamp, radiant energy into a usable
tungsten lamp, Nernst glower) electrical signal
b. Line sources (ex: hollow cathode) a. Phototubes
b. Photomultipliers
2. Wavelength Selector- Isolates a restricted region of the c. Photodiodes – more
spectrum sensitive
c. Filters – dedicated to a single band of wavelength
d. Monochromators – designed for spectral scanning 5. Signal processor and readout-
(ex: prisms, gratings) Displays the transduced electrical
signal into numbers
3. Sample cell- Holds the sample to be analyzed Ex: charge transfer devices
e. Quartz cuvette – UV or visible region
f. Silicate glass or plastic cell – visible region
g. NaCl or KBr plates – IR region
ANALYTICAL METHODS OF SPECTROSCOPY
1. FLAME 2. MASS 3. INFRARED
SPECTROSCOPY SPECTROMETER SPECTROSCOPY
■ Same with Flame test ■ Bombardment of Absorption of energy in the
molecules w/ stream of IR region is associated
■ For elemental Analysis
electrons causing with vibrational transitions
■ Methods: cracking & of atoms w/in molecules
– Flame emission fragmentation
■ Structure determination
for groups I & II ■ Determine MW, protein
– Atomic structure, struct ID
Absorption (AAS)
important analysis
of trace minerals in
multivitamins
preparations.
ANALYTICAL METHODS OF SPECTROSCOPY
4. NUCLEAR MAGNETIC 5. COLORIMETRY 7. TURBIDIMETRY
RESONANCE ■ Branch that deals with ■ Measurement of
■ Transition between the absorption measurement transmitted light in a
energy level that may be made in visible suspension
generated by radiant spectrum.
■ Application: Antibiotics
energy in the molecule that ■ Uses filter instead of
& Vitamins
are placed in magnetic prism.
field.
■ Analysis of colored 8. FLUOROMETRY
■ Rotation of spinning compounds.
nucleus when magnetic ■ Measurement of
6. NEPHELOMETRY excess energy lost by
field is applied
■ Measurement of emission
■ Determine 3D structure/ (fluorescence)
reflected light/ light
identity studies, molecular
scattered in a Application: Vit. B1, B2,
configuration
suspension Phosphorous