Application of centrifugation Spectrophotometry

1. 1
Application of centrifugation
◘ 1- Velocity Sedimentation Centrifugation :-
♦ Centrifuges are used most often For Preparative-Scale Separation
procedures .
♦ this technique consist of :-
- Placing the sample in a tube or similar container
- Inserting the tube in the rotor
- Spinning the sample for a fixed period .
- The sample is removed and the two phases ( Pellet and supernatant )
may be separated by careful decantation .
♦ It separate particles ranging in size :
- From Coarse precipitates to cellular organelles .
- Heavy precipitates are sediment in Low-Speed Centrifuges .
- Lighter Organelles such as ribosomes require the High centrifugal
forces of an ultracentrifuge
♦ Further Characterization or analysis is usually carried out in the
individual phases .

2. 2
◘ 1- Fractional Centrifugation :
♦ Much of our current understanding of cell structure and function depends
on separation of subcellular components by centrifugation .
♦ This specific method of separation consists of successive centrifugation at
increasing rotor speeds .
♦ the rotor chamber must be kept at low temperature to maintain the native
structure and function of each cellular organelle ant its component
biomolecules .
♦ The Operation :-
- A high-speed centrifuge equipped with a
fixed angle rotor is the most appropriate
for the first two centrifugations at 600 xg
and 20,000 xg .
- After each centrifuge run , the supernatant
is poured into another centrifuge tube ,
which is then rotated at the next higher
speed .
- The final centrifugation at 100,000 xg to
sediment microsomes and ribosomes must be done in an ultracentrifuge .
- The 100,000 xg supernatant , the cytosol , is the soluble portion of the cell and
consists of soluble proteins and smaller molecules.

3. 3
Spectrophotometry
◘ The instruments that measure electromagnetic radiation have several
concepts and components in common .
◘ Photometric instruments measure light intensity without consideration of
wavelength .
◘ Most instruments today use :-
- filter (photometers)
- prisms , gratings ( spectrometers )
♠ to isolate or select a narrow range of the incident wavelength .
◘ Radiant energy that passes through an object will be partially reflected ,
absorbed , and transmitted .

4. 4
◘ Electromagnetic radiation : is described as photons of energy travelling
in waves .
◘ The relationship between wavelength and energy E
is described by Planck’s formula :
- Because the frequency of a wave is inversely
proportional to the wavelength .., the energy of
electromagnetic radiation is inversely
proportional to wavelength .
◘ Electromagnetic radiation includes a spectrum of energy from ( short-
wavelength , high energetic x-rays & γ-rays ) to ( long-wavelength Radio-
frequencies )
◘ Visible light falls in between , the color violet at 400 nm and red at 700
nm wavelengths being the approximate limits of the visible spectrum .

5. 5
♣ Absorption & Emission Spectra :
• Instruments measure either absorption or
emission of radiant energy to determine
concentration of atoms or molecules .
• The two phenomena , absorption and
emission , are closely related .
• For a ray of electromagnetic radiation to be absorbed , it must have the
same frequency as a rotational or vibrational frequency in the atom or
molecule that it strikes ( hits ) .
• levels of energy that are absorbed move in discrete steps and any
particular types of molecule or atom will absorb only certain energies and
not others .
• when energy is absorbed valence electrons move to an orbital with a
higher energy level .
• Following energy absorption , the
excited electron will fall back to the
ground state by emitting a discrete
amount of energy in the form of a
characteristic wavelength of radiant
energy .
• Absorption or emission of energy by atoms
results in a line spectrum .

6. 6
• Because of the relative complexity of molecules , they absorb or emit a
bank of energy over a large region .
• light emitted by incandescent solids ( tungsten or deuterium ) is in
continuum .

7. 7
◘ Principle :-
○ Beer’s Law :-
♦ Definition :
- The relationship
between absorption of light by a solution and the concentration of
that solution .
- States that the conc. Of the a substance is directly proportional to
the amount of light absorbed or inversely proportional to the
logarithm of the transmitted light .
♦ when a beam of monochromatic light entering a solution , some of the
light is absorbed and the remainder passes through , strikes a light detector .
- The detector convert the light to an electric signal .

8. 8
♦ Percent Transmittance ( % T ) :-
- Is the ratio of the radiant energy transmitted ( I ) divided by the
radiant energy incident on the sample ( Io )
- All light absorbed or blocked results in 0% (T) transmittance .
- A level of 100% T is obtained if no light is absorbed .
- In practice , the solvent without the constituent of interest is placed
in the light path , most of the light is transmitted , but a small
amount is absorbed by the solvent and cuvet or is reflected away
from the detector .
- The electrical readout of the instrument is set arbitrarily at 100% T,
while the light is passing through a “blank” or reference .
- The sample containing absorbing molecules to be measured is
placed in the light path .
- The difference in amount of light transmitted by the blank and that
transmitted by the sample is due only to the presence of the
compound being measured .

9. 9
- The % T measured as : the ratio of
the sample transmitted beam
divided by the blank transmitted
beam .
- %T = sample beam signal / blank
beam signal ×100
♦ Absorbance :-
• Is the amount of light absorbed .
• It cannot be measured directly by a spectrophotometer .
• It is mathematically derived from %T as follow :-
%T = ( I/Io) ×100
A = - log ( I/Io)
A = log ( 100%) – log %T
A = 2-log %T
- Where : ( Io) is incident light & ( I ) is Transmitted light .
• According to beer’s law :- Absorbance is directly proportional to
concentration .
A = ɛ*l*c
- Where ( ɛ ) is the molar absorptivity , the fraction of a specific
wavelength of light absorbed by a given type of molecule .
- ( l ) is the length of light path through the solution .
- ( c) is the concentration of absorbing molecules .

10. 10
♦ Absorptivity :
• Depends on molecular structure and the way in which the absorbing
molecules react with different energies .
• For any particular molecular type , absorptivity changes as wavelength of
radiation changes .
• The amount of light absorbed at a particular wavelength depends on the
molecular and ion types present and may vary with , PH and temperature.
• because the path length and molar absorptivity are constant for a given
wavelength , C .
♦ Unknown concentration are determined
from a calibration curve that plots
absorbance at a specific wavelength
versus concentration for standards of
known concentration .
• For calibration curves that are linear and
have a zero Y-intercept , unknown
concentration can be determined from a
single calibrator .
• Beer’s Law deviations :
- Not all calibration curves result in straight lines .
- Deviation from linearity are typically observed at
high absorbance .
- The stray light within an instrument will
ultimately limit the maximum absorbance that a
spectrophotometer can achieve , typically 2.0 absorbance units .

11. 11
♦ Beer’s law Examples :
%T = ( I/Io) ×100
A = - log ( I/Io)
A = log ( 100%) – log %T
A = 2-log %T
A = ɛ*l*c
(ε ): (Greek letter, epsilon) is the molar absorptivity of the solute with units of M-1
cm-1
(or ( L mol-1
cm-1
or mol-1
dm3
cm-1
)
(l) is the path length of the light through the solution in units of cm.
(C) is the concentration of the solution in mol L-1
(or mol dm-3
or M )
◘ Using a cuvette with a length of 1 cm, you measured the absorbance of a solution with a
concentration of 0.05 mol/L. The absorbance at a wavelength of 280 nm was 1.5. What is
the molar absorptivity of this solution?
 ɛ280 = A/lc = 1.5/(1 x 0.05) = 30 L mol-1
cm-1
◘ A solution of Tryptophan has an absorbance at 280 nm of 0.54 in a 0.5 cm length cuvette.
Given the absorbance coefficient of Tryptophan is 6.4 × 103
LMol-1
cm-1
. What is the
concentration of solution?
Solution:
As : ε = A / l c
l = 0.5 cm
A= 0.54
ε = 6.4 × 103
LMol-1
cm-1
C=?
So c = A/ε l
= 0.54 / 6.4 × 103
× 0.5
Answer = 0.000168 M
◘ A solution of thickness 2 cm transmits 40% incident light. Calculate the concentration of the
solution, given that ε = 6000 dm3
/mol/cm.
Solution:
A = 2 - log %T = 2 – log 40 = 2 – 1.6020 = 0.398
A = ε l c
l= 2cm
ε = 6000 dm3
/mol/cm
A=0.398
c=?
So c = A/ ε l = 0.398/ 6000 × 2
Answer = 3.316 X 10 – 5
mol / dm3

12. 12
◘ A solution shows a transmittance of 20%, when taken in a cell of 2.5 cm
thickness. Calculate its concentration, if the molar absorption coefficient is
12000 dm3
/mol/cm.
Solution:
A = 2 – log %T = 2 - log 20 = 2 – 1.301 = 0.698
A = ε l c
l= 2.5 cm
ε = 12000 dm3
/mol/cm
A=0.698
c=?
So c = A/ ε l
= 0.698/ 12000 × 2.5
Answer = 2.33 X 10 – 5
mol / dm3
◘
Calculate the molar absorptivity of a 1 x 10-4
M solution, which has an
absorbance of 0.20, when the path length is 2.5 cm.
Solution:
A = ε l c
l= 2.5 cm
A= 0.20
C= 1 x 10 – 4
M
ε =?
So ε = A / l c
= 0.20/2.5 ×1×10-4
Answer = 800 dm3
/mol/cm
◘ Calculate the molar absorptivity of a 0.5 x 10-3
M solution, which has an
absorbance of 0.17, when the path length is 1.3 cm.
Solution:
A = ε l c
l= 1.3 cm
A= 0.17
C= 0.5 x 10-3
M
ε =?
So ε = A / l c
= 0.17/ 1.3 × 0.5 x 10-3
Answer = 261.53 dm3/mol/cm.

13. 13
◘ A CaCO3 solution shows a transmittance of 90%, when taken in a cell of 1.9 cm thickness.
Calculate its concentration, if the molar absorption coefficient is 9000 dm3
/mol/cm.
Solution:
A = 2 - log %T = 2 - log 90 = 2 – 1.954 = 0.045
A = ε l c
l= 1.9 cm
ε = 9000 dm3/mol/cm
A=0.045
c=?
So c = A/ ε l
= 0.045/ 9000 × 1.9
Answer = 2.631 × 10 -6
mol / dm3
◘ A 1.00 × 10–4 M solution of an analyte is placed in a sample cell with a path length of 1.00
cm. When measured at a wavelength of 350 nm, the solution’s absorbance is 0.139. What is
the analyte’s molar absorptivity at this wavelength?
l = 1.00 cm
c = 1.00 × 10–4
M
A=0.139
ε =?
So ε = A / l c
= 0.139/ 1.0 × 1.00 x 10-4
Answer = 1390 cm−1
M−1
Spectrophotometry