I am HAFIZ M WASEEM FROM mailsi vehari BSc in science college Multan Pakistan MSC university of education Lahore Pakistan i love Pakistan and my teachers

1. HAFIZ
MUHAM
MAD
WASEEM
BA BA G
LAHORE
Micrometry

2. Biological Techniques
UNIVESITY OF
EDUCATION
LAHORE

3. Introduction
Spectrophotometry?
 Every chemical substance
absorbs, transmits or reflects
light as it passes through it
 Spectrophotometry is branch
of spectroscopy that deals
with measurement of the
absorbed or transmitted light
by a substance
 It measures the concentration
of solutes in the solution by
measuring the amount of the
absorbed or transmitted light
as it passes through the
solution

4. Introduction
 White light is a mixture of all
visible colors (red, yellow,
green, blue and violet)
 When this light passes
through a substance some
radiations are absorbed while
others are allowed to pass
through or are reflected by
the substance
 If a substance does not absorb
any light it appears white (all
light is reflected)
 Similarly if a substance
absorbs all colors or transmits
none it appears black

5. Introduction
 A solution looks of a certain
color due to absorbance or
transmittance of some
radiations
 A solution appears of blue
color because it absorbs all of
the colors except blue
 It a technique for quantitative
analysis in various fields such
as chemistry, physics, material
and chemical engineering
 Spectrophotometer is an
instrument which measures
intensity of absorbed or
emitted radiations

6. Biological Techniques

7. Principle of Spectrophotometry
 A Spectrophotometer consists
of a spectrometer for
producing light of a specific
color and a photometer to
measure intensity of the light
 A solution is placed in a cuvette
between a spectrometer and a
photometer
 Photometer measures intensity
of absorbed or emitted light in
the form of a voltage signal as
it passes through the solution
 The signal changes as intensity
of absorbed or emitted light
changes

8. Principle of Spectrophotometry
 Spectrophotometry deals with
production, measurement and
interpretation of the spectra
arising from the interaction of
electromagnetic radiation with
the matter
 Beer-Lambert Law forms the
basis of the measurement
procedure
 It states that amount of the
light absorbed is directly
proportional to
• Concentration of the
solution
• Path length

9. Principle of Spectrophotometry
 Degree of absorption is
proportional to concentration
of the solution
 A standard curve is prepared
between concentration of the
solution and absorbance of
light
 It is usually in the form of a
straight line
 It is prepared by preparing
series of dilutions of a standard
solution of the analyte with
known concentration
 A blank is used to set zero
absorbance

10. Principle of Spectrophotometry

11. Principle of Spectrophotometry
 Absorbance of each of the
standards is read using the
spectrophotometer
 Concentration is plotted (x-
axis) versus absorbance of
the solutions (y-axis)
 Concentration of an unknown
solution is measured by
measuring its absorbance by
using the standard curve
 With same compound and
path length the absorbance is
directly proportional to
concentration of the solute in
the solution

12. Biological Techniques

13. Components of Spectrophotometry
Spectrophotometer
 It measures intensity of the
light absorbed as it passes
through the sample solution
 It has two main devices
 Spectrometer which consists of
• Light source
• Sample cell
• Monochromator
 Photometer which consists of
• Detector
• Optical system

14. Components of Spectrophotometry

15. Spectrometer
 It produces a desired range of
wavelengths of the light
depending on the nature of
biological sample to be
analyzed
 It may use
• Tungsten Filament Lamp
• Dueterium (D2) Arc Lamp
• Mercury and Xenon Arc
Lamps
• Silicon Carbide (SiC) Rod
• Laser
Components of Spectrophotometry

16. Monochromator
 It splits light into several
component wavelengths
 A wavelength selector (slit)
transmits only the desired
wavelengths to the sample
Sample cell
 Sample solution is placed in a
cuvette
Fused silica Cells (for visible
and UV)
Plastic and Glass Cells (visible)
NaCl and KBr Crystals (IR)
Components of Spectrophotometry

17. Photometer detector
 Produces an electric signal
proportional to intensity of
the radiations
• Phototube (UV)
• Photomultiplier tube
(UV-Vis)
• Thermocouple (IR)
Optical system
 Signal is amplified and made
available for direct display by
a signal processor and
measurement as it passes
through the sample
Components of Spectrophotometry

18. Biological Techniques

19. Types of Spectrophotometry
Single beam
 It utilizes single beam of light
that passes through the sample
holder
 It is standardized by placing a
reference in the sample holder
and the resulting value is
subtracted from the sample
measurements
 Suitable for applications in the
wavelength range from 190 to
1100 nm
 Nucleic acids and proteins are
measured by this type
 It is cheaper and simple

20. Types of Spectrophotometry
Double beam
 Light source is split into two
separate beams before it
passes through the sample
 One beam is used for the
reference and the other passes
through the sample
 Two detectors measure the
sample and reference beams at
one time
 This is advantageous because
the reference reading and
sample reading can be taken at
the same time
 It has high speed and reliability

21. Types of Spectrophotometry

22. Types of Spectrophotometry
Visible spectrophotometer
 It uses light of visible range
(400 - 700 nm)
 Uses a prism to narrow down a
certain range of wavelength so
that particular beam of light is
passed through a sample
 Visible region
spectrophotometers vary in
accuracy
 Some have CCD detectors with
enough pixels to take reading
every 10nm while others can
take several reading per
nanometer

23. Types of Spectrophotometry
 It uses light over the ultraviolet
range (185 - 400 nm)
 UV is most commonly used for
liquids but can also be used for
gases and even solids
 It obeys Beer Lambert law
which states that amount of
the light absorbed is directly
proportional to Concentration
of the solution
 Samples are placed in a cuvette
usually 1cm in width
 These can be made of plastic,
glass or quartz
UV spectrophotometer

24. Types of Spectrophotometry
Atomic absorption
 Uses a nebulizing chamber to
create a sample mist
 Flame is used to desolvate
and atomize the sample
 But the excitation of the
analyte atoms is done by
lamps shining through the
flame at various wavelengths
for each type of analyte
 Amount of light absorbed
after going through the flame
determines the amount of
analyte in the sample
 Used in research and industry

25. Biological Techniques

26. In Medicine
 Used in measuring the growth
of micro organisms like
bacteria in microbiological
preparations
 Used to measure
concentration of enzymes,
hormones, etc.
 Role played by
spectrophotometric methods
in research on vitamins,
hormones, enzymes, etc. has
been spectacular
 Used intensively in toxicologic
investigations
Applications of Spectrophotometry

27. Estimation of proteins
 Since proteins absorb light at
a specific wavelength, direct
UV measurement at 280 nm
yields highly reproducible
measurements
 Absorption is affected by
composition of amino acids
like tyrosine and tryptophan
 Structure of proteins also
influence the UV absorbance
by aromatic side chains
 It produces quick results since
the sample does not need to
be processed
Applications of Spectrophotometry

28. Applications of Spectrophotometry
In Forensics
 Used in identifying polymer
degradation in various types
of criminal investigations
 Used to measure blood alcohol
contents of the addict
 Estimation of glucose and
other parameters like
vitamins, hormones, enzymes,
etc. which are important in
deciding health conditions
 Used in toxicological
investigations to determine
causes of death in various
types of cases

29. In Agriculture
 Soil quality is an important
part of agriculture and
sustainability
 Soil quality depends on
quantitative analysis and the
careful balance of nutrients in
the soil
 This promote better growing
conditions and thus enhanced
productivity
 Spectrophotometers are used
to monitor nitrogen and
phosphorus levels in the
fertilizers and soil
Applications of Spectrophotometry

30. Dissolved organic carbon
measurement
 DOC in marine and
freshwater systems is one of
the greatest reservoirs of
organic matter on earth
 This accounts for the same
amount of carbon as the
atmosphere and up to 20% of
all organic carbon
 It is important to measure
DOC for quality of life
 DOC is also extremely
important in the transport
of metals in aquatic systems
Applications of Spectrophotometry

31. Biomedical engineering
 Biomedical optics are
important in the future of
health care industry
 It provides a safe, non-
invasive and non-destructive
method of analysis for a
variety of medical needs
 Instrumentation is durable
and portable
 It involves the monitoring of
blood oxygen levels and
hemoglobin saturation
 It can be used in all of the
fields related to health
Applications of Spectrophotometry