Lab 3
Spectrophotometry
Photometry:
- Photometry means “the measurement of light”
- Photometer & Spectrophotometer are instruments used for this type
of measutment, in which a photocell or photomultiplier tube is used to
detect the amount of light that passes through a colored solution from a
light source.
Basic Characteristics of Light:
- Light is a form of electromagnetic energy that travels in waves.
- The wavelength of light is the distance between two beaks of the light
wave, is inversely proportional with its energy.
- In the visible region the color of light is a function of its wave length
increasing from violet towards the red color.
- Objects that appear colored absorb light at particular wavelength and
reflect
the other parts of the visible spectrum resulting in many shades of color.
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Spectrophotometry :
 Is a method to measure how much a chemical substance absorbs
light by measuring the intensity of light as a beam of light passes
through sample solution.
 Each compound absorbs or transmits light over a certain range of
wavelength.
 This measurement can also be used to measure the amount of a
known chemical substance.
 Every chemical compound absorbs, transmits, or reflects light
(electromagnetic radiation) over a certain range of wavelength.
 Spectrophotometry is a measurement of how much a chemical
substance absorbs or transmits.
 Is an instrument that measures the amount of photons (the intensity
of light) absorbed after it passes through sample solution.
 With the spectrophotometer, the amount of a known chemical
substance (concentrations) can also be determined by measuring
the intensity of light detected.
Types of spectrophotometers :
Depending on the range of wavelength of light source, it can be
classified into two different types:
1. UV-visible spectrophotometer: uses light over the ultraviolet range
(185 - 400 nm) and visible range (400 - 700 nm) of
electromagnetic radiation spectrum.
2. IR spectrophotometer: uses light over the infrared range (700 15000 nm) of electromagnetic radiation spectrum.
Basic structure of spectrophotometers
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Spectrophotometer in general consists of two devices:
• Spectrometer: It produces a desired range of wavelength of light.
First a collimator (lens) transmits a straight beam of light (photons)
that passes through a monochromator (prism) to split it into several
component wavelengths (spectrum). Then a wavelength
selector (slit) transmits only the desired wavelengths .
• Photometer: After the desired range of wavelength of light passes
through the solution of a sample in cuvette, the photometer detects
the amount of photons that is absorbed and then sends a signal to a
galvanometer or a digital display .
Beer-Lambert Law:
Beer-Lambert Law (also known as Beer's Law) states that there is a linear
relationship between the absorbance and the concentration of a sample.
For this reason, Beer's Law can only be applied when there is a linear
relationship. Beer's Law is written as:
where A is the measure of absorbance (no units), ϵ is the molar extinction
coefficient or molar absorptivity (or absorption coefficient), l is the path
length, and c is the concentration. The molar extinction coefficient is
given as a constant and varies for each molecule. Since absorbance does
not carry any units, the units for ϵ must cancel out the units of length and
concentration. As a result, ϵ has the units: L·mol-1·cm-1. The path
length is measured in centimeters. Because a standard spectrometer uses a
cuvette that is 1 cm in width, l is always assumed to equal 1 cm. Since
absorption, ϵ, and path length are known, we can calculate the
concentration c of the sample.
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If the concentration of a solution is increased, the It will decrease and
then % T is decreased.
- The relationship between the concentration and %T is not linear, but if
the logarithm of the %T is plotted against the concentration, a straight
line is obtained .
-The term absorbance is used to represent – log % T
A = - log % T
= 1/ log % T
% T = It \ Io x 100 %
For x substance
For standard substance
Ax = a b c x
Ast = a b cst
From 1,2
Ax
Cx
Ast
Cst
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(1)
(2)
Cx = Ax . Cst
Ast
Glucose test (GOD)
Equipment:
 Automatic pipettes Tips
 Test tubes
 Glucose reagent
Reagents
TRIS buffer pH 7.5, phenol, Glucose oxidase, Peroxidases, 4 –
Aminophenazone
Principle
Glucose is oxidized by glucose-oxidase to gluconate and hydrogen
peroxide and gives pink color read at 500nm.
Sample
Serum free of hemolysis plasma.
Serum or plasma glucose is stable for at least 3 days at 2-8ºc.
Procedure
Blank
Standard
Stander
Sample
10µl
Sample
10 µl
Control
Reagent
Contro
l
1.0µl
1.0ml
1.0 ml
1.0 ml
Mix. Incubate 10 min. at 37ºc. or 30 min at room temperature.
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Measure the Absorbance at 505nm (490-550) against the blank. The
color is stable 60 min.
Calculation
Glucose (mg/dl) = (Abs. sample/Abs. standard)* standard conc.
Linearity
This method is linear up to 500mg/dl.
If the glucose concentration is greater than 500mg/dl. , dilute the sample
1:2 with saline solution and repeat the determination and multiply the
result by 2
Reference value
Serum: 70 -110mg/dl
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