Transcript Colorimeters

Colorimeters
Definition
colorimeter is an instrument which compares the
amount of light getting through a solution with the
amount which can get through a sample of pure
solvent.
A
 Substances
absorb light for a variety of reasons. Pigments
absorb light at different wavelengths. A cloudy solution will simply
scatter/block the passage of light (sometimes a colorimeter is
used to monitor the growth of a bacterial or yeast culture).
 The % transmission or the % absorbance is recorded (you can
use either).
 It is possible to change the color of the light that is used by using
filters in the simplest equipment or an "optical wedge" .
How the Colorimeter Works
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Light from a LED
light source passes
through a Cuvette
containing a solution
sample,
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Some of the incoming
light is absorbed by the
solution.
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As a result, light of a
lower intensity strikes
a photodiode.
Construction
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The essential parts of a
colorimeter are:
 a light source, which is usually
an ordinary filament lamp
 an aperture which can be
adjusted
 a detector which measures the
light which has passed
through the solution
 a set of filters in different
colors
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filters are used to select the
wavelength of light which the
solution absorbs the most.
Solutions are usually
placed in glass or plastic
cuvettes.
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Wavelength selection,
Printer button
Concentration factor adjustment,
UV mode selector (Deuterium
lamp)
Readout
Sample compartment
Zero control (100% T),
Sensitivity switch.
TRANSMITTANCE
Transmittance “T”
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The amount of light that passes through a solution is
known as transmittance T.
Transmittance can be expressed as the ratio of the
intensity of the transmitted light It to the initial intensity
of the light beam Io
 The transmittance formula is:
T = It /Io
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The Colorimeter produces an output voltage which
varies in a linear way with transmittance, allowing a
computer, calculator, or handheld to monitor
transmittance data for a solution.
Absorbance “A”
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The reciprocal of transmittance of the sample varies
logarithmically (base ten) with the product of three factors:
ε, the molar absorptivity of the solution, b the cell or cuvette width,
and C the molar concentration
A = log(1/T) = ε b C
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The relationship between these two variables is inverse and
logarithmic (base 10). It can be expressed as
A = log(1/T)
T&A
T&A
RELATION BETWEEN
ABSORBANCE AND
CONCENTRATION
 BEER’S LAW
Beer’s law
Mathematical statement of Beer’s law
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For a given solution contained in a cuvette with a
constant cell width, the Absorbance is proportional to the
concentration:
A=εC b=kC
This equation shows absorbance to be related directly to
concentration and represents a mathematical statement of Beer’s
law.
b is cuvette path length
C is concentration of absorbing substance
ε is absorptivity (~ substance)
In many chemical and biological experiments this law is
assumed for making calibration line for determining
concentration.
Beer’s Law Curve
Determination of C of an unknown substance
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To obtain a Beer’s law curve,
several standards (solutions of
known concentration) are prepared
and their absorbance values are
determined using a Colorimeter.
A graph of absorbance vs.
concentration is then plotted.
A solution of unknown
concentration is placed in the
colorimeter and its absorbance
measured.
When the absorbance of this
solution is interpolated on the
Beer’s law curve, its concentration
is determined on the horizontal
axis.
Alternatively, its concentration may
be found using the slope of the
Beer’s law curve.
Concentration of unknown solution
Cu
Cu= Cs Au/As
Cu is unknown concentration
Au is unknown absorbance
Cs standard concentration
As is standard absorbance
Ranges of A and T for the colorimeter
during calibration

For best results our laboratory testing of the
colorimeter indicates that absorbance or
transmittance values should fall with these
ranges:
Transmittance (T) 0.28 - 0.90
Absorbance (A) 0.050 - 0.550
To get sufficiently reliable results calibration solutions should
be used which lie in the flat part of the curve dT% = 1%
(i.e. 1% accuracy of the transmission).
The measurement error in percent in the concentration (or absorbance) dc/c
as function of the transmission T for different accuracy's (dT%) of the
transmittance range.
Determining of the wavelength
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You can select three LED light colors:
red (635 nm), green (565 nm) or blue (470 nm).
There are several ways you can decide which of three
wavelengths to use:
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Method 1.
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Look at the color of the solution. (Remember that the color of
solution is the color of light which is not absorbed).
use a different color of light that will be absorbed
For example: with a blue CuSO4 solution, use the red LED
(635 nm).
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Method 2.
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Place a cuvette containing the solution in the colorimeter.
And check to see which of three wavelengths yields the
highest absorbance (low transmittance).
Method 3.
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Directions for most colorimetry experiments express a
recommended wavelength. Use the closest of the three
wavelengths on the colorimeter. Even if the LED wavelength
is somewhat different, a Beer's law curve can usually be
obtained at almost any wavelength around the recommended
wavelength.
Basic Colorimeter Schematic
Electronics
Calibration
Example
Calculations
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Automatic CAL
….each time before use
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After powering up the Colorimeter and waiting for a 5 minute
warm up,
press to select a wavelength.
Open the lid, insert a cuvette filled about 3/4 full of distilled
water, and close the lid.
Make sure that one clear side of the cuvette is lined up with the
arrow at the top of the cuvette slot.
Press the CAL button and hold it until the red LED begins to
flash and then release the CAL button.
When the LED stops flashing, the calibration is complete.
Detector
Silicon Photo-diode
Filter options
Interchangeable glass
or interference type
filter
420 nm, 470 nm, 510
nm, 530 nm, 620nm,
600 nm
Light source
Tungsten lamp 3.0 V
0.5 A
Display
LCD
Cuvette
Round 10 mm ID, 12
mm OD 105mm L
Square 10 mm x 10 mm
x 45 mm
Wavelength range
420 nm - 660 nm
Photometric Range
Transmittance
10 ~ 100%
Absorbance
0.01 - 1 ABS
Power
NiCd battery or line
adapter
Dimensions
150 mm x 80 mm x 50
mm
Weight
~ 1 lb
Specifications for the
PC-10 handy photo
colorimeter
Maintenance
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Calibration
Adjustment
Replacement of burned-out lamp and photo
detector
Electronic problems are rare