Spectrophotometer uses. Biophysics. Biochemistry. Red blood cell hemolysis. Studying concentrations of solutions

1. ABSORPTION
SPECTROPHOTOMETRY
BY: KAREEM JELILAT T.

2. BASED ON:
❖ Determination of concentration of solutions
❖ Studying acid hemolysis of erythrocytes
Of particular significance is:
The Lambert- Beer Law

3. Different methods have been developed for measuring the concentrations of substances
within the body fluids, but a commonly used method is the Spectrophotometry method.
This method works on the principle that electromagnetic radiation of a particular
frequency is passed through the studied sample, and the transmitted radiation is then
analysed in terms of the intensity at different frequencies.
In normal circumstances, atoms and molecules exist in their ground state, but when they
move to a higher energy level under the influence of an electromagnetic radiation (source
of energy), there will be a relationship between the absorbed radiation and the transition
created by the jump; this relationship is termed the absorption spectrum and is represented
graphically by the energy absorbed against the wavelength.
LAMBERT’S LAW
On passing through a solution, the intensity of radiation attenuates and this intensity is
denoted by I0 and the intensity of transmitted radiation by I.
The light intensity however decreases exponentially with the increase in medium
thickness x and this is expressed by:
I=I0e-x/𝛅 where e is 2.71 and 𝛅 is the layer thickness.

4. The attenuation or extinction coefficient is given by: μ=1/𝜹 and its unit of measure is
m-1 or cm-1.
Extinction is the absorbance of light by 1cm thick of a solution having a concentration
of 1mol/L. It is a unitless parameter however and its formula of derivation is: E=I0/I
Another way to represent the Lambert’s law is by using the transmittance:
T=I/I0 x 100%
BEER’S LAW
This is valid for solutions whose attenuation of light is related tp the molar
concentration of the absorbing medium and the thickness through which the light is
transmitted and the equation that explains this statement is: μ=εc ⇒ I= I010-εcx
The combination of both laws gives us the Lambert- Beer law which states that ‘if a
solution contains a solute that absorbs light, then the more concentrated the solution is,
the more the light that is transmitted through it’.
E= log I0/I= εcx where ε is the molar extinction coefficient and varies with the
❖ The nature of the absorbing solute

5. ❖ Nature of the solvent
❖ Wavelength of light
Thus, the Lambert- Beer law is only valid for monochromatic light (light of one
frequency).
SPECTROPHOTOMETRY
It works on the basis of the law and is used to measure the concentration of any
sample of body fluids by comparing its absorbance or transmittance with that of a
standard concentration.
The modern spectrophotometer consists of:
● A source of radiation
● A monochromator or filter
● A transparent cuvette containing the sample
● A light detector (transforms light energy into electrical energy)
● An electrical signal amplifier
● A measuring device to record the value of extinction

6. EXPERIMENTAL PROCEDURE
Two transparent cuvettes are filled with distilled water and methyl blue solution
respectively. The spectrophotometer must have been set to a certain wavelength about
10minutes before the commencement of the experiment.
The cuvette containing the methyl blue solution is placed on the outer
compartment of the device while tat filled with distilled water in the inner
compartment.
The potentiometer is adjusted according to the sensitivity of the solution with the
aid of the fine adjustment knobs.
The extinction and transmittance can then be read from the scale with the latter
having its values in percentages. The graph of both parameters against the
concentration can then be plotted.
The concentration of unknown solution can be obtained from the graph afterwards
simply using the slope of the graph to compute its value.

7. ACID HEMOLYSIS OF ERYTHROCYTES
Under physiologic conditions,slightly less than 1% of RBCs are destroyed each day and
are replaced by new ones
The important geometric parameters of a RBC is its area and volume, and as it ages, it
loses water and its surface area diminishes thus causing cell deformity.
The young and old cells can be separated by centrifugation whilst the heavy old cells have
the lower diameter and surface area.
Hemolysis is a premature destruction of RBC and can either be within the circulation-
intravascular or extravascular.
HCl is a potential hemolytic agent of RBC and it hemolyzes in three stages: penetration of

8. ● The extinction of RBC suspension follows and this decreases over time but
NEVER reaches zero
● This is due to the red colour of the chlorematin and its light absorbing
characteristic formed from the reaction between Hb and HCl.
● The old cells undergo hemolysis first followed by the young cells
● The cessation of the decreasing extinction signals the hemolysis of the sample.
Partial hemolysis
Is the difference between two consecutive readings of the extinction and ut determines
the percentage of hemolyzed RBC within a specific time interval
The curve obtained is the erythrogram and in a case where acd is used as the hemolytic
agent, it is called an acid erythrogram.

9. EXPERIMENTAL PROCEDURE
The spectrophotometer is also set to a certain wavelength (555 nm) 10minutes
before the experiment.
Distilled water is put in one of the cuvettes and erythrocyte solution in the
other. A measurable quantity of HCl should be added to the cuvette containing
the erythrocyte immediately.
The extinction is read after every 30 seconds, and then the partial hemolysis
in percentage can be plotted against the time in seconds on an acid
erythrogram.
In order to calculate the partial hemolysis of a sample in percentage, the
equation below should be used:
△E=△E%/△E100% x 100%
where △E% is the absorbance or extinction and △E100% is the difference
between the first and last extinction.

10. Best of Luck in the Colloquium and Final Exam!!!