PHOTOMETRIC CALIBRATION PROCEDURE
1. 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
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
THE COLORIMETER 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. And this what Beer’s states.
LIGHT AFFECTED BY SAMPLE CONCENTRATION
BEER’S AND LAMBERT’S LAW Beer’s law states that the concentration of a substance is
directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light
A = 2- log %TA ~C
Lambert’s law states that the rate of decrease in intensity with the thickness of the medium is proportional to the intensity of light
Beer-Lambert law gives
T= e -kct
Finally givesConcentration of Test = (absorb of Test/absorb of
STD)x concentration of STD This law represents the basic for colorimetric and
spectrophotometric measurements
COMPONENTSComponents:- Light source : The most important factors
for a light source are range, spectral distribution within the range, the source of radiant production, stability of the radiant energy, and temperature
- Monochromators : 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
COMPONENTS- Sample cell- Photodetectors : could be barrier-layer
cell, phototube, photomultiplier tube, or photodiode. The light then falls on to a photocell which generates an electrical current in direct proportion to the intensity of light falling on it. This small electrical signal is increased by the amplifier which passes to a galvanometer of digital readout to give absorbance reading directly
CALIBRATION CURVES Calibration curves used to help in
determining concentrations when for instance analyzing many samples. This is done by plotting absorbance at a specific wavelength versus concentration for standards of known concentration
Each method reads within a specific range, below and above this range the reading will not be convenient ,and sample then must be treated before measurement. Deviations from linearity are typically observed at high absorbances
FILTER SELECTION The filter chosen is usually
complementary to the solution colour to be measured
Solution colour Usual filter
BlueBluish-greenPurpleRed YellowYellowish-green
YellowRedGreenBluish-greenBlueViolet
FILTER SELECTION We should measure the absorbance of
the coloured reaction throughout the visible spectrum and then deciding the wavelength of filter which gives the highest absorbance
Once the correct wavelength has been chosen to give maximum absorption, the selectivity is determined by taking further readings at various wavelengths with two different concentrations of the same solution
SCIENCE IS AMAZING
SPECTROPHOTOMETERS
SPECTROPHOTOMETERS Is a sophisticated type of
colorimeter where monochromatic light is provided by prism or diffraction grating
The band width 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
How to calibrate colorimeters/spectrophotometers
Think about (Source of errors
in spectrophotomet
ric measurements)
ATOMIC ABSORPTION SPECTROPHOTOMERS The analyzed sample must contain the reduced metal in the
atomic vaporized state. Commonly, this is done by using the heat of a flame to break the chemical bonds and form free, unexcited atoms
The sample, in solution, is aspirated as a spray into a chamber, where it is mixed with air and fuel. This mixture passes through baffles, where large drops fall and are drained off. Only fine droplets reach the flame
Light from the hollow-cathode lamp passes through the sample of ground-state atoms in the flame. The amount of light absorbed is proportional to the concentration
When a ground-state atom absorbs light energy, an excited atom is produced. The excited atom then returns to the ground state, emitting light of the same energy as it absorbed
Flameless atomic absorption requires an instrument modification that uses an electric furnace to break chemical bonds (electrothermal atomization)
Atomic absorption spectrophotometry is sensitive, accurate, specific and Precise
It is routinely used to measure concentration of trace metals that are not easily excited
It is generally more sensitive than flame emission because the vast majority of atoms produced in the usual propane or airacetylene flame remain in the ground state available for light absorption
2. DENSITOMETRY Densitometry is the quantitative
measurement of optical density in light-sensitive materials, such as photographic paper or photographic film, due to exposure to light. Optical density is a result of the darkness of a developed picture and can be expressed absolutely as the number of dark spots (i.e., silver grains in developed films) in a given area, but usually it is a relative value, expressed in a scale
The densitometer is used to detect and differentiate electrophoresis patterns
TURBIDIMETRY Turbidimetric measurements are made
with a spectrophotometer to determine concentration of particulate matter in a sample. The amount of light blocked by a suspension of particles depends not only on concentration but also on size
NEPHELOMETRY Nephelometry is similar, except that
light scattered by the small particles is measured at an angle to the beam incident on the sample holder
FLAME PHOTOMETRY A sample aspirated into a flame,
producing atoms in an excited state, which are capable of emitting light of a specific wavelength depending on the element of interest
The intensity of the emitted light can be correlated to the quantity of ion present in the sample
FLUOROMETRY The source emits short-wavelength high-energy
excitation light. A mechanical attenuator controls light intensity
The primary filter, placed between the radiation source and the sample, selects the wavelength that is best absorbed by the solution to be measured
The fluorescing sample in the cuvet emits radiant energy in all directions
The photodetector must be placed at a 90° angle from the initial light source. This eliminates any interference from the initial light source and detects only fluorescent light
The electrical output of the photodetector is proportional to the intensity of fluorescent energy
FLUOROMETRY
FLUOROMETRY Definition of fluorescence : Certain molecules absorb light and a
given frequency, and then re-emit that light at a different and longer frequency
Advantages of fluorescence:Very specific and sensitive
Disadvantages of fluorescence: Few molecules fluoresce and very susceptible to pH and temperature changes
CHEMILUMINESCENCE A chemical reaction that emits energy in
the form of light. When used in combination with immunoassay technology, the light produced by the reaction indicates the amount of analyte in a sample
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