Prepared by: Nabhan & Mohanned

Supervisor : Eng shehdeh zahdeh

Background
Beer-Lambert law
The Spectrophotometer
Basic components
Types of Spectrophotometers
Operation
Maintenance
Applications of Spectrophotometer



CONTENTS:-
Background :
At natural state, most of the atoms, molecules and electrons are in
the lowest energy level called ground state.
To transits from lower energy level to highest the electron need
promotion as light and its called energy transition.
When a chemical absorbs light, it goes from a low energy state
(ground state) to a higher energy state (excited state)






Only photons with energies exactly equal to the energy difference
between the two electron states will be absorbed
Since different chemicals have different electron shells which are
filled, they will each absorb their own particular type of light
When transitions occurs, the wavelength and
energy decreases, and increases of frequency.
The Light waves consist of perpendicular,
oscillating electric and magnetic fields
Electro- magnetic waves and described by
1. amplitude(A),
2. wavelength(),
3. frequency(F).

For the light the freq. increases, energy
increases and wavelength decreases through




Background :
E = h = h
C

u
C
=

u
C = u
Visible light is only a
small portion of the
entire electromagnetic
spectrum
it includes the colors
commonly observed
(red, yellow, green, blue
and violet).
The visible spectrum
consists of electro-
magnetic radiation
whose wavelengths
range from 380nm to
nearly 760nm.
Background :
(nm) Region Color Observed
< 380 Ultraviolet Not visible
380-440 Visible Violet
440-500 Visible Blue
500-580 Visible Green
580-600 Visible Yellow
600-620 Visible Orange
620-750 Visible Red
750-2000 Short IR Not visible
Background :
Why do some substances appear colored?
When light passes through a substance, certain
energies (or colors) of the light are
1. Absorbed
2. Other colors allowed to pass
3. Other are reflected.

If the substance does not absorb any light, it appears
white (all light is reflected). colorless (all light is
transmitted).
A solution appears a certain color due to the
absorbance and transmittance of visible light. For
example, a blue solution appears blue because it is
absorbing all of the colors except blue.

Background :
Lamberts Law of Absorption:
Lambert described how intensity changes with
distance in an absorbing medium.
The intensity I
0
of light beam decreases exponentially
as it passes though a uniform absorbing medium.






q q q q q q A = A + ) ( ) ( ) ( ) ( I I I
q
q q A +
q

0
I
source
) (q I detector
q
q

= = e I I I
0
) (
q
q
q
d
I
dI
=
) (
) (
Beer-Lambert law:
Lamberts Law of Absorption (base 10)

Typically base 10 is used in photometry
Beer-Lambert law:
q q k
I e I I

= = 10
0 0
10 ln = k
q q k
e
I
I

= = 10
0
k is the path length over
which the intensity is
attenuated to 1/10.
q k
I
I

=10
0
Beers Law
Beer found that Lamberts linear decay constant
k for a solution of an absorbing substance is
linearly related to its concentration c by a
constant.
the absorptivity , a characteristic of the
absorbing substance.
Restatement: The linear decay constant k is
linear in concentration c with a constant of
proportionality .

c k c =
Beer-Lambert law:
Photometric Quantities
In photometry we measure the intensity of light and characterize its
change by substance.
This change is typically expresses as percent transmittance or
absorbance.

Transmittance (T)


Absorbance (A)
0
I
I
T =
T
I
I
A log log
0
=
|
|
.
|

\
|
=
usually given in percent
by convention, base 10 logs are used
Beer-Lambert law:
Absorbance and the extinction coefficient
Absorbance is useful since it can be summed
for layers of different materials

A = A + A + A +
A = C x + C x + C x +

A specialized device to measure the intensity
of light as a function of wavelength is the
spectrophotometer.
tot 1 2 3
tot
1 1 1 2 2 2 3 3 3
one of the basic medical laboratory
instruments uses to measure light intensity
as a function of wave length().
Measures absorbance as a function of
wavelength
The device important
for determining the
unknown substances
and for calculating
the concentration of
known substances.
The Camspec M550 Double Beam Scanning UV/Vis Spectrophotometer
The Spectrophotometer
, monochromator , light source : Components
. optical system & detector , sample cell
monochromator
sample cell
detector
light source
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optical system
The Spectrophotometer
Basic components:
1- Light Source: provides the light to be passed
through the sample.
a source must generate a beam of radiation
that is sufficiently powerful for easy
detection and measurement.
- Hollow Cathode Lamps (HCL)
- tungsten Lamp: visible light .
- Hydrogen discharge: ultraviolet Light.

its output power should be stable for reasonable periods.
2- monochromator: used to select a given wavelength of
the light from the light source.
mono single.
chroma color.
ator denoting an agent

There exists many techniques for that
Diffraction gratings
Prisms
Collimation
Stray light
Wavelength range
Double monochromator
Basic components:
2- monochromator:
Diffraction gratings


To obtain of specific wavelength:
1. entrance slit.
2. concave mirror or lens.
3. a prism or grating.
4. focal plane.
5. exit slit.
Basic components:
2- monochromator:
Prisms spray out the spectrum and choose the certain
wavelength that you want by slit.
Basic components:
3- Sample Cell:
A container that contains a sample is usually called
"cell"
has fixed length & volume.
usually round or square cuvette.
made of material that does not absorb light in the
wavelength range.
two types are available:-
- Glass visible region.
- Quartz ultraviolet.

.
Basic components:
4- Detector:
to convert the radiant energy to a
measurable signal; and to a readout device
Detector is a device that indicates the
existence of some physical phenomenon.
The term transducer is used to indicate the
type of detector that converts quantities,
such as light intensity, into such electrical
signals that can be subsequently amplified,
manipulated, and finally converted into
numbers.

Basic components:
Basic components:
Ideal detector : high sensitivity.
high signal/noise.
fast response time.
constant response for s.
responds to low levels of energy.

Single-Beam Instrument: -
sample and blank are alternatively measured in same sample
chamber.
use a single-wavelength light source, such as a light
emitting diode (LED), a sample container, and a photodiode
detector.
can utilize a fixed wavelength light source or a continuous source.
and offers a small and inexpensive device configuration



Types of Spectrophotometers:
2-Double-Beam Instrument:
A double beam system has two dedicated
positions for the sample and the blank
Continuously compares sample and blank.
The change of the light source can be
corrected, and it is possible to measure the
system with stability.


Types of Spectrophotometers:
Operation
Periodically maintenance:
Change the light source.
Clean lenses, mirrors and light paths
periodically
Keep lenses and mirror away from touch
and dust. Affect the readings
Be careful when dealing with mirrors and
lenses and clean them with soft material
Maintenance:

Basic faults:
Damage of light source, detector.
Illogical measurements-dusty of
mirrors and lenses
ICs damaged check each ICs
individually
Maintenance:

Safety notes:
Keep U-light away from human
eyes
There exist high voltage be
aware when removing the cover
Remove the power supply when
change device kits


Maintenance:
Applications of Spectrophotometer
Spectroscopy
Chemical Analysis: concentration ,trace
analysis, pH and remote monitoring
Geology.
Astronomy.
Particle size.
Thin film characterization
Color matching
Optics