The document provides an overview of UV-visible molecular absorption spectrophotometry, discussing its theory, instrumentation, advantages, limitations, and applications. Key concepts include the Beer-Lambert law, sources of radiation, types of detectors, and the construction of spectrophotometric methods to analyze samples at trace levels. Specific applications are mentioned, including the determination of proteins, amino acids, and various metals.

1. UV-Visible Molecular
Absorption Spectrophotometry
Theory, Instrumentation, Merits,
Limitations, Applications
By
Ch.Krishna
Analytical laboratory
EPTRI, Hyderabad

2. Property of matter is used
Gravimetry - Weight
Volumetry – precipitation, redox
complexometric
Light - matter - Absorb
Reflect
Transmits

3. Why spectrophotometry ?
A need to to analyse at trace quantities in
the samples
Gravimetry and volumetry fails at ppm and
lower levels

4. Electromagnetic Spectrum
Spectral Distribution of Radiant Energy
Wave Number (cycles/cm)
X -Ra y UV V isib le IR M icro wa v e
2 0 0 n m 4 0 0 n m 8 0 0 n m
W AV E L E NGT H(n m )

6. V = Wave Number (cm-1)
l = Wave Length
C = Velocity of Radiation (constant) = 3 x 1010
cm/sec.
u = Frequency of Radiation (cycles/sec)
The energy of photon:
h (Planck's constant) = 6.62 x 10-27 (Ergsec)
V =

C 


E = h = h
C

 C
= 
 C =
u

8. LAMBERT LAW
When a monochromatic light is allowed to pass
through a transparent medium , the rate of
decrease of intensity with the thickness of the
medium is directly proportional to the intensity of
the incident light .
dIo / dt α Io
dIo / dt = kt
Io - Intensity of Incident light
t - Thicknesss of the medium
It = Io e-kt

9. BEERS LAW
The intensity of a beam of monochromatic
light decreases exponentially with the
increase in the concentration of the absorbing
substance arithmetically
It = Io e-kc
c - concentration
= Io e-kc

10. It = Io e-kct
A = Log Io/It = kct
A = Absorbance
c = Concentration moles / litre
t = thicknesss in cm
k = ε = Molar absorption coefficient ( lit mole-1 cm -1)
Beer-Lamberts Law

13. Limitations of the Beer-Lambert law
The linearity of the Beer-Lambert law is limited by
chemical and instrumental factors. Causes of
nonlinearity include:
deviations in absorptivity coefficients at high
concentrations (>0.01M) due to electrostatic
interactions between molecules in close proximity
scattering of light due to particulates in the sample
fluoresecence or phosphorescence of the sample

14. changes in refractive index at high analyte
concentration
shifts in chemical equilibria as a function of
concentration
non-monochromatic radiation, deviations can be
minimized by using a relatively flat part of the
absorption spectrum such as the maximum of an
absorption band
stray light

15. T - Transmittance
T = I0 - original light intensity
I- transmitted light intensity
% Transmittance = 100 x
Absorbance (A) or optical density (OD) = Log
= Log = 2 - Log%T
Log is proportional to C (concentration of solution) and is
also proportional to L (length of light path
through the solution).
I
I0
I
I0
I0
I
1
T
I
I0

17. STEPS IN DEVELOPING A SPECTROPHOTOMETRIC
ANALYTICAL METHOD
1. Run the sample for spectrum
2. Obtain a monochromatic
wavelength for the maximum
absorption wavelength.
3. Calculate the concentration
of your sample using Beer
Lambert Equation: A = KCL

18. Wavelenth Vs Absorbance

19. Block diagram of UV-visible Spectrophotometer

21. Instruments for measuring the absorption of U.V.
or visible radiation are made up of the following
components:
Sources (UV and visible)
Wavelength selector (monochromator)
Sample containers
Detector
Signal processor and readout

22. Sources of UV radiation
The electrical excitation of deuterium or hydrogen at low
pressure produces a continuous UV spectrum Both
deuterium and hydrogen lamps emit radiation in the
range 160 - 375 nm.
Quartz windows must be used in these lamps, and quartz
cuvettes must be used, because glass absorbs radiation of
wavelengths less than 350 nm.

23. Sources of visible radiation
The tungsten filament lamp is commonly employed as
a source of visible light. This type of lamp is used in
the wavelength range of 350 - 2500 nm. The energy
emitted by a tungsten filament lamp is proportional to
the fourth power of the operating voltage. This means
that for the energy output to be stable, the voltage to
the lamp must be very stable indeed. Electronic voltage
regulators or constant-voltage transformers are used to
ensure this stability.

24. Czerney-Turner grating monochromator

25. Cuvettes
The containers for the sample and
reference solution must be transparent
to the radiation which will pass through
them. Quartz or fused silica cuvettes are
required for spectroscopy in the UV
region. These cells are also transparent
in the visible region.

26. Detectors
The photomultiplier tube is a commonly used detector in UV-Vis
spectroscopy.
photoemissive cathode (a cathode which emits electrons when
struck by photons of radiation),
dynodes (which emit several electrons for each electron striking
them) and an anode.
Each original photon produces 10-6 TO 10-7 electrons. The
resulting current is amplified and measured.
Photomultipliers are very sensitive to UV and visible radiation.
They have fast response times. Intense light damages
photomultipliers; they are limited to measuring low power
radiation.

27. Cross section of a photomultiplier tube

28. Advantages
Very simple to handle
Determine elemental concentration at ppm levels
It has good precision when compared to flame techniques
Non-metals like S, P, F can be determined easily
Some refractory elements like Th, Zr, ant Ti can be determined at
ppm levels

29. Limitations
Long sample preparation time
Requires separation of interfering ion
one or two elements can be determined at a time
Error is more at high and low absorbance range
Self at high concentrations absorption is not linear

32. Slope of Standard Curve =
 A
C
1 2 3 4 5
1.0
0.5
Concentration (mg/ml)
Absorbance at 280 nm
There is some A vs. C where graph is linear.
NEVER extrapolate beyond point known where
becomes non-linear.

33. Element Reagents λ(max)
Ti Hydrogen
peroxide
440nm
P Ammonium
molybdate and
Ammonium
monovanadate
430nm
Si Ammonium
molybdate and
reducing agent
650nm
Th Arsenazo III 660nm
Some Application in Geological materials

34. CHEMICAL STRUCTURE & UV
ABSORPTION
Chromophoric Group ---- The groupings of the
molecules which contain the electronic system which
is giving rise to absorption in the ultra-violet region.

35. CHROMOPHORIC STRUCTURE
Group Structure nm
Carbonyl > C = O 280
Azo -N = N- 262
Nitro -N=O 270
Thioketone -C =S 330
Nitrite -NO2 230
Conjugated Diene -C=C-C=C- 233
Conjugated Triene -C=C-C=C-C=C- 268
Conjugated Tetraene -C=C-C=C-C=C-C=C-315
Benzene 261

36. UV SPECTROMETER APPLICATION
Protein
Amino Acids (aromatic)
Pantothenic Acid
Glucose Determination
Enzyme Activity (Hexokinase)

37. VISIBLE SPECTROPHOTOMETER APPLICATION
Niacin
Pyridoxine
Vitamin B12
Metal Determination (Fe)
Fat-quality Determination (TBA)
Enzyme Activity (glucose oxidase)

38. Thank you