COLORIMETRY & SPECTROPHOTOMETR
- Many biochemical experiments involve the measurements of compound or group of
compounds present in a complex mixture.
- The most widely used method for determining the concentration of biochemical
compounds is colorimetry, which makes use of the property that when white light passes
through a colored solution, some wavelength are absorbed more than others.
- Many compounds are not themselves colored but can be made to absorb light in visible
region by reaction with suitable reagents.
- These reactions are fairly specific and in most cases very sensitive, so that quantities of
material in the region of mM / L concentrations can be measured.
- The big advantage of is that complete isolation of compound is not necessary and the
constituents of a complex mixture such as blood can be determined after little treatment.
- The depth of the color is proportional to the concentration of the compound being
measured, while the amount of light is proportional to the intensity of the color and hence
the concentration.
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Organized by: Sharifa Al-Ghamdi& Huda Al-Shaibi
Measurement of Extinction:
- The earliest colorimeters relied on the human eye to match the color of a solution with
that of one of a series of colored discs. The results obtained were too subjective and not
particularly accurate.
The Colorimeter:
- Colorimeter is generally any tool that characterizes colour samples to provide an
objective measure of colour characteristics. In chemistry, the colorimeter is an apparatus
that allows the absorbance of a solution at a particular frequency (colour) of visual light to
be determined. Colorimeters hence make it possible to determine the concentration of a
known solute, since it is proportional to the absorbance.
- Different chemical substances absorb varying frequencies of the visible spectrum.
Colorimeters rely on the principle that the absorbance of a substance is proportional to its
concentration i.e., a more concentrated solution gives a higher absorbance reading.
- Filter in the colorimeter is used to select the color of light which the solute absorbs the
most, in order to maximize the accuracy of the experiment. Note that the colour of the
absorbed light is the 'opposite' of the colour of the specimen, so a blue filter would be
appropriate for an orange substance. Sensors measure the amount of light which has
passed through the solution, compared to the amount entering, and a display reads the
amount absorbed.
- A quantitative reading for the concentration of a substance can be found by making up a
series of solutions of known concentration of the chemical under study, and plotting a
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Organized by: Sharifa Al-Ghamdi& Huda Al-Shaibi
graph of absorbance against concentration. By reading off the absorbance of the specimen
substance on the graph, a value for its concentration is found.
How colorimeter works?
1- White light from a tungsten lamp passes through a slit, then a condenser lens, to give a
parallel beam which falls on the solution under investigation contained in an absorption
cell or cuvette. The cell is made of glass with the sides facing the beam cut parallel to each
other.
2- Beyond the absorption cell is the filter, which is selected to allow maximum transmission
of the color absorbed. If a blue solution is under examination, then red is absorbed and a
red filter is selected.
NOTE: The color of the filter is complementary to the solution.
3- The light then falls on to a photocell which generates an electrical current in direct
proportion to the intensity of light falling on it.
4- This small electrical signal is increased by the amplifier which passes to a galvanometer
of digital readout to give absorbance reading directly.
-Among the simplest and most common colorimeters are the Spectronic 20 and Spectronic
21. They are commonly called the Spec 20 and Spec 21.
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Organized by: Sharifa Al-Ghamdi& Huda Al-Shaibi
Light source
slit condenser
cuvette
filter
photocell
galvanometer
Lens
The Spectrophotometer:
- Is a sophisticated type of colorimeter where monochromatic light is provided by prism.
The band with of the light passed by a filter is quite board, so that it may be difficult to
distinguish between two components of closely related absorption with a colorimeter. A
spectrophotometer is then needed.
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- All types require a Blank: which is a solution that contains the entire reagents except the
substance to be measured. It is used to adjust the device to zero.
Here is a summary of the steps of operation of a Spec 20 & spectrophotometer:
1- Power >>>>>>>>Turn on power.
2-Warmup>>>>>>Allow about 5 minutes when first turned on.
3-Wavelength>>>>>Select appropriate wavelength.
4- Zero>>>>>With sample holder empty and closed, adjust meter needle to 0%T (or
infinite A) using zero control knob.
5- Blank >>>>Fill tube half full with water. Place in sample holder and close cover. Adjust
meter needle to 100%T (or 0 A) using light control knob.
6- Standard>>>>Measure absorbance (or %T) of known solution. Fill tube half full with
sample of known concentration. Place in sample holder and close cover. Read absorbance
value (or %T) from meter. Repeat this step if making a calibration curve or verifying
proportionality (Beer's Law).
7- Sample>>>>>Measure absorbance (or %T) of solution with unknown concentration as
in previous step.
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Types of spectrophotometer:
1- Visible spectrophotometer.
2- Ultraviolet (UV) spectrophotometer.
References:
www.wikipedia.org
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Organized by: Sharifa Al-Ghamdi& Huda Al-Shaibi
COLORIMETRIC DETERMINATION OF
VITAMIN B-12
- Vitamin B12 is water soluble vitamin.
- RDA= 2.4 µg /day for adult.
- It is found only in animal sources.
Functions:
1- Aids folic acid in synthesis of heme.
2- It prevents anemia.
3- Required for protein digestion and absorption.
Method:
- Prepare series of tubes according to the following table
Tube
B
1
2
3
4
5
UNK
Standard
-
1ml
2ml
3ml
4ml
5ml
-
Unk
-
-
-
-
-
-
5ml
H2 O
5ml
4ml
2ml
2ml
1
-
vitamin
concentration
- Vortex.
- Read the absorbance at 555nm.
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RESULTS & LAB REPORT
- You are supplied with the sample of unk vitamin.
- Plot the standard curve to calculate the concentration of unk.
- Calculate the concentration by using the equation>>>>
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Cunk = Aunk
A std
X C std
Organized by: Sharifa Al-Ghamdi& Huda Al-Shaibi