Applied biochemistry
Presented by
D/Ahmed Abd El Ghaffar
D/Ahmed Abd El Ghaffar 1
Others Urine
Body fluid
examination Semen Blood
CSF
Site of blood collection in different animals
Techniques in collection
of blood samples
1. Shaving or clipping of hair at the site
of collection
2. Sterlize the site with 70% alcohol
3. Tie tourniquet above site of collection
(at least 10 cm )
4. Insert the needle in 30ᵒ inside the vein
5. Before removal of needle remove the
tourniquet first to avoid blood leakage
Types of blood
collecting tubes
What is the Light?
Light is the visible portion of EMR
Any solution containing a compound that absorb light in
range of 380-750 nm will appear colored to the eye
Incident
light (I0) Eye
Transmitted
Absorbed light
light (I)
is a quantitative method to measure how much a
chemical substance absorbs EMR using an
instrument called “Spectrophotometer”.
Spectrophotometer
an instrument which can measure the amount of the
light absorbed by the sample at any selected
wavelength.
Spectrophotometer
A spectrophotometer consists of two instruments:
1) a spectrometer for producing light of any selected color (wavelength)
2) a photometer for measuring the intensity of light.
Spectrophotometer
The instrument is arranged so that liquid in a cuvette can be placed
between the Spectrometer beam and the Photometer
1-LIGHT SOURCE
Types of spectrophotometer
1 UV-visible spectrophotometer:
uses light over the ultraviolet range (185 - 400 nm) and
visible range (400 - 700 nm) of EMR spectrum.
2 IR spectrophotometer:
uses light over the infrared range (700 - 15000 nm) of
EMR spectrum.
Spectrophotometer
1-LIGHT SOURCE
UV Spectrophotometer
1 Hydrogen/ Deuterium Lamp (200-450nm)
2 xenon Lamp (185-2000 nm) for UV and IR spectrophotometer.
Visible Spectrophotometer
1 Tungsten Lamp (330-2500 nm)
Spectrophotometer
1-LIGHT SOURCE
Spectrophotometer
2-MONOCHROMATOR
For selection of certain wavelength and achieved using either:
1-Prism
2-Diffraction Grating
3-Filters
Spectrophotometer
2-MONOCHROMATOR
Split white light to color of rainbow with
several wavelengths Select one color with specific wavelength
Prism Diffraction Grating Filters
Spectrophotometer
3- SAMPLE HOLDER
(CUVETTE)
Visible light UVR
Plastic Glass Quartz
Spectrophotometer
4- PHOTO DETECTOR
Photocell
convert photons into
electrical signal (produce
current proportional to the
intensity of light)
Spectrophotometer
5- Recorder ( Read out device)
The data from detector are
displayed by the read out
device(G alvanometer) which
may be :
A- Anal oge
B- Digi tal
Colorimeter
Colorimeter is an instrument
Used to measure the conc. of colored solutions.
Using visible range (400-800 nm) of EMR
Difference between
Colorimeter and Spectrophotometer
Point of comparison Colorimeter Spectrophotometer
LIGHT SOURCE: Tungsten lamp Tungsten, Xenon, and Deuterium
LED bulb lamp.
Fixed wavelength in the visible range Wide Wavelength Range (visible, UV
and IR)
WAVE LENGTH SELECTOR Color filter monochromator
USE Limited parameters according to the Numerous parameters
fixed wavelength range
SAMPLE coloured Coloured and colourless
COST Cost effective Expensive.
Sample Quantification
(Calculation of Concentration)
There’re 2 methods for sample quantification
Standard Curve
Beer-Lambert Law
Beer-Lambert Law
The concentration of a substance in a solution is directly proportional
to the absorbance (A) of the solution
Aα C
When monochromatic light passes through a solution , the intensity
of light absorbed increases exponentially with increasing path length
Aα L
SO
A α CL
Beer-Lambert Law
HOW to use beer lambert law for calculation of UK conc ?
Standard
A specimen c specimen
=
A standard c standard
C specimen = A specimen x C standard
A standard
Why must you use a blank solution?
It is similar to the sample in every thing
except the measured compound is
absent from the solvent
It is used to adjust the zero of the
spectrophotometer and eliminate any
interference .
STEPS
1. The instrument must be warmed
for 15 min.
2. Set a monochromatic wavelength for
the maximum absorption wavelength.
3-Wipe the tube
containing the blank
solution with a lab wipe
4-place it into
sample holder .
Close cover
32
5 set Zero absorbance
6 Remove blank tube,
wipe off the cuvette of
sample and insert it
7 Read and record the
absorbance (A) or optical
density(OD).
8 Calculate the concentration of
The sample
The absorbance of sample is A=0.98
What will be the concentration ?
Absorbance of standard Concentration of standard
0.157 0.2 M
C specimen = A specimen x C standard
A standard