This document discusses various spectroscopic methods used for official assay. It describes instrumental methods like spectrometric and chromatographic techniques that use instruments to measure physicochemical properties. Spectrometric methods are discussed in detail, including how spectroscopy works via absorption or emission of electromagnetic radiation when interacting with matter. Key components of a spectrophotometer and analytical spectroscopic techniques are also outlined, such as atomic absorption spectroscopy, infrared spectroscopy, and nuclear magnetic resonance spectroscopy.

1. METHODS USED FOR
OFFICIAL ASSAY

2. General Methods
■ General Methods are the “classical methods”.
Types:
A. Titrimetry
B. Gravimetry

3. Instrumental Methods
■ Based on the utilization of an instrument to
measure physicochemical properties.
Types of Instrumental Methods:
A. Spectrometric
B. Chromatographic
C. Electrometric

4. SPECTROMETRIC
METHODS

5. 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

6. 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

7. 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

8. 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)

9. 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

11. ELECTROMAGNETIC RADIATION
(EMR)
■ The electromagnetic radiation refers to the emission and
propagation of energy through space in the form of
electromagnetic waves or particles.

12. PARTS OF WAVES
1. Crest: The highest point or peak of a wave.
2. Trough: The lowest point or dip of a wave.
3. 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.
4. 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.
5. Wavenumber (k) –the number of cycles per unit distance
6. Frequency (f) : The number of complete cycles of a wave passing
a point in a given time.
7. Period (T) : The time it takes for one complete cycle of a wave to
pass a given point.

13. PARTS OF WAVES
Formula:
K= cycles/unit distance
Or
K= 1/λ

14. PARTS OF WAVES
Formula:
K= cycles/unit distance
Or
K= 1/λ

15. PARTS OF WAVES
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
Formula:
(T)- Period
(f)- Frequency
(v)- Velocity
(λ)- Wavelength
Unit of Frequency = Hz
• Hz= cycle/s
Unit of Wave Period = s

16. PARTS OF WAVES
Formula:
(T)- Period
(f)- Frequency
(v)- Velocity
(λ)- Wavelength
Unit of Frequency = Hz
• Hz= cycle/s
Unit of Wave Period = s

17. 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

18. ELECTROMAGNETIC SPECTRUM
■ The electromagnetic spectrum is the division of
electromagnetic radiation based on the energy,
frequency, or wavelength of a photon.

19. ELECTROMAGNETIC SPECTRUM

20. WAVELENGTH RANGE OF EACH
REGION

21. 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

22. INSTRUMENT:
SPECTROPHOTOMETER

23. COMPONENTS OF
SPECTROPHOTOMETER
4. Radiation Detector- Converts
radiant energy into a usable
electrical signal
a. Phototubes
b. Photomultipliers
c. Photodiodes – more
sensitive
5. Signal processor and readout-
Displays the transduced electrical
signal into numbers
Ex: charge transfer devices
1. Source of radiation- Generates a beam of radiation
a. Continuum sources (ex: deuterium lamp,
tungsten lamp, Nernst glower)
b. Line sources (ex: hollow cathode)
2. Wavelength Selector- Isolates a restricted region of the
spectrum
a. Filters – dedicated to a single band of wavelength
b. Monochromators – designed for spectral scanning
(ex: prisms, gratings)
3. Sample cell- Holds the sample to be analyzed
a. Quartz cuvette – UV or visible region
b. Silicate glass or plastic cell – visible region
c. NaCl or KBr plates – IR region

24. ANALYTICAL METHODS OF
SPECTROSCOPY
1. FLAME
SPECTROSCOPY
■ Same with Flame test
■ For elemental Analysis
■ Methods:
– Flame emission
for groups I & II
– Atomic
Absorption (AAS)
important analysis
of trace minerals in
multivitamins
preparations.
2. MASS
SPECTROMETER
■ Bombardment of
molecules w/ stream of
electrons causing
cracking &
fragmentation
■ Determine MW, protein
structure, struct ID
3. INFRARED
SPECTROSCOPY
■ Absorption of energy in
the IR region is
associated with
vibrational transitions
of atoms w/in
molecules
■ Structure determination

25. ANALYTICAL METHODS OF
SPECTROSCOPY
4. NUCLEAR MAGNETIC
RESONANCE
■ Transition between the
energy level that may be
generated by radiant
energy in the molecule that
are placed in magnetic
field.
■ Rotation of spinning
nucleus when magnetic
field is applied
■ Determine 3D structure/
identity studies, molecular
configuration
5. COLORIMETRY
■ Branch that deals with
absorption
measurement made in
visible spectrum.
■ Uses filter instead of
prism.
■ Analysis of colored
compounds.
6. NEPHELOMETRY
■ Measurement of
reflected light/ light
scattered in a
suspension
7. TURBIDIMETRY
■ Measurement of
transmitted light in a
suspension
■ Application: Antibiotics
& Vitamins
8. FLUOROMETRY
■ Measurement of
excess energy lost by
emission
(fluorescence)
Application: Vit. B1, B2,
Phosphorous