Spectrophotometric analysis is a technique to measure the concentration of solute solution by measuring the amount of light absorbed by solution. Absorption can be calculated in terms of transmittance by using Beer's Lambert law.

1. Spectrophotometer
BCH-503

2. Spectrophotometric Analysis
• Spectrophotometric techniques are used to measure
the concentration of solutes in solution by measuring the
amount of light that is absorbed by the solution in a
cuvette placed in the spectrophotometer.
• The spectrophotometer can measure the amount of
light or electromagnetic radiation (of certain frequency)
transmitted or absored by the solution.

3. Absorption of Radiant Energy
Wave-particle Nature of Radiant energy:
Light and other forms of radiant energy have a dual nature (wave
and particle)
Electromagnetic radiation is a type of energy that is transmitted
through space as a transverse wave at enormous velocity.
It takes numerous forms known as electromagnetic spectrum.
The electromagnetic spectrum include gamma ray, X-ray,
ultraviolet (UV), visible, infrared (IR), microwave and radio-wave
radiation.

4. Wave motion of light
Wave Properties
The wave is described either in terms of its wavelength (l),the distance
between successive maxima or minima of a wav(nm), or in terms of the
frequency(n), the number of oscillation of the field per second.
The velocity of light, c, is given by the equation:
C= n l

5. Particle properties
Electromagnetic radiation of light can be viewed as a stream of
discrete wave packets of distinct particles called photons.
The energy E of photon depends upon the frequency of the
radiation
E = hn
Therefore:
h = Planck’s constant (6.626 x 10-34 J s)
n = frequency of the radiation (most common units = cm-1
Energy is inversely proportional to wavelength

6. • The ultraviolet region extends from about 10 to 380 nm
•The most analytically useful region is from 200 to 380
nm, called the near- ultraviolet region or quartz UV region.
• The visible (VIS) region extends from about 380 to
780nm.
•The infrared (IR) region extends from about 0.78μm to
300 μm.
•The near-infrared (IR) region extends from about 0.80μm
to 2.5 μm.
•The far-infrared (IR) region extends from about 2.5μm to
16 μm.
The electromagnetic spectrum

7. Regions of Electromagnetic Spectrum-the “colour” of light

8. The absorption process
How does matter absorb radiation
When polychromatic light (white light), which contains the
whole spectrum of wavelengths in visible region, is passed
though an object will absorb certain of the wavelengths, leaving
the unabsorbed wavelengths to be transmitted. These residual
transmitted wavelengths will be seen as a color. This color is
complementary to the absorbed colors.

9. Absorption is a process in which chemical species (atom, ion or molecule)
in a transparent medium selectively attenuate certain frequencies of EMR.
Absorption spectrum is a plot of the amount of light absorbed by
a sample as a function of wavelength.
At room temperature most substance are in their lowest energy or
ground state. When an atom, molecule or ion absorbs EMR it is
promoted to higher energy states or excited states.
The excited state is a transition one and the species soon looses
the energy it gained and returns to its ground state by relaxation
process either as heat of collision or sometimes emits radiation of
specific wavelength.

10. Ground state
First Excitation state
Second Excitation state
E2
E1
E0
hv1
hv2

11. • When a molecule interacts with photons of UV or VIS
radiation excitation of electrons takes place to higher
electronic energy level at any of its vibrational level.
• Eex-Eg= hn of the photon absorbed.
• UV / VIS radiation cause electronic transition which is
accompanied by vibrational and rotational.
• If the compound subjected to IR radiation vibrational and
rotational transitions in ground state occure.
• Rotational transitions alone can be brought about by microwave.

12. • Ultraviolt and visible radiations have sufficient energy to cause
transitions of the outermost or valence electrons.
• If large amount of energy is absorbed by certain substance, bonds may
be ruptured and new compounds are formed photolysis.
This may occur upon absorption of far Ultraviolt as its energy is
sufficiently high to exceed the energy of formation of certain bonds.
• The total energy of a molecule is given by
Etotal=Eelectronic+Evibrational +Erotational

13. Two methods to detect molecules:
Indicator – Quickest method.
Indicator solutions change colors when a
molecule of interest is present.
Allows scientist to detect colorless
molecules in a solution.
Examples: Bradford protein reagent
Diphenylamine (DPA) or
Biuret (NaOH+CuSO4).
What kind of data is this?

14. Two methods to detect molecules:
• Spectrophotometer (Spec)
–An instrument that measures the
amount of light that passes through (is
transmitted through) a sample.

15. Spectrophotometer cont…
–Uses a type of light to detect
molecules in a solution
–Light is a type of energy, and the
energy is reported as wavelengths,
in nanometers (nm).

16. Two different types of Spectrophotometer:
Ultraviolet (UV) Spectrophotometers.
Uses ultraviolet light of wave lengths from
200 nm to 350 nm.
Visible (VIS) Light Spectrum
Spectrophotometers.
Uses visible light (white light) of wave
lengths from 350 nm to 700 nm.

17. The visible light spectrum
R O Y G B I V

18. Spectrophotometer cont…
• Shines a beam of light on a sample.
• The molecules in the sample interact with
the light waves in of 3 ways:
–Absorb the energy
–Reflect the energy
–Transmit the energy between and
through the atoms and molecules of the
sample.

19. How a spectrophotometer works:
Consider blue molecules, all the
wavelengths of light are absorbed, except
for the blue ones.
The blue wavelengths are transmitted or
reflected off the molecules. If these blue
wavelengths hit a detector (such as in the
spectrophotometer or the nerve cells in
your eye), they appear blue.

20. How a spectrophotometer works:
• Molecules are whatever color of
light that they do not absorb.
• Green molecules appear green
because they absorb most
wavelengths of visible light, except
the green wavelengths.

21. Spectrophotometer cont…
The spectrophotometer measures the
amount of light transmitted through
the sample (Transmittance).
By using an equation (Beers law), it
converts the transmittance data to an
absorbance value.
What kind of data is this?

22. Spectrophotometer cont…
The concentration of an unknown
sample can be determined by comparing
the absorbance data to standards of
known concentration.
The data generated with the set of
known standards is called a standard
curve.

23. Parts of a spectrophotometer
• Inner parts
–Lamp
–Prism or grating that direct light of a
specific wavelength.

24. VIS Spec vs. UV spec
Visible spectrophotometer
Contains a tungsten lamp that produces
white light.
Ultraviolet spectrophotometer
Contains a deuterium lamp that
produces light in the UV light part of the
spectrum.

25. Parts of a Spectrophotometer
• Outer parts:
Sample Holder
Display
Knobs or buttons used to calibrate the
spec to measure the designated molecule.
Wavelength
Selection

27. How a spectrophotometer works:
Visible Spectrophotometer
White light hits the prism or grating, it is
split into the colors of the rainbow
(Visible Spectrum).
The wavelength knob rotates the
prism/grating, directing different color
of light toward the sample.

28. How a spectrophotometer works:
• The wavelength of light produced by the
tungsten lamp range from about 350 nm
(Violet light) to 700 nm (red light).
• The molecules in the sample either
absorb or Transmit the light energy of
one wavelength or another.

29. How a spectrophotometer works:
• The detector measures the amount of
light being transmitted by the sample
and reports that value directly (%
transmittance) or converts it to the
amount of light absorbed in absorbance
units (au) using Beers Law.
A = 2 – log10%T

32. Absorption Spectrum
• After collecting data for your
concentration an absorption
spectrum graph is created.
• These can be used when attempting
to identify unknown substances (e.g.
CSI)

33. Example of absorption spectrum of
Chlorophyll a & b, and Carotenoids

34. The relationship of concentration in a
solution:
The concentration of molecules in a
solution affects the solution’s
absorbance.
If there are more molecules in one
solution than in another, than there are
more molecules to absorb the light.

35. Applications of a spectrophotometer
• Determines the presence and
concentrations of samples.
• Determines the purity of a sample.
• Look at the change of samples over
time.

37. OVERVIEW OF QUANTITIVE
SPECTROPHOTOMETRY
A. Measure the absorbance of standards
containing known concentrations of the analyte
B. Plot a standard curve with absorbance on the X
axis and analyte concentration on the Y axis
C. Measure the absorbance of the unknown(s)
D. Determine the concentration of material of
interest in the unknowns based on the standard
curve