5. Light source:
The function of the light source is to provide a sufficient light which is
suitable for marking a measurement.
Tungsten lamp:
The tungsten lamp is commonly used as a source of visible light.
Its wavelength range is 330-1000 nm.
It has a long life about 1200h.
6. Monochromator:
Accepts polychromatic input light from a lamp and outputs
monochromatic light
All monochromators contain the following component parts;
• An entrance slit
• A collimating lens
• A dispersing device
• A focusing lens
• An exit slit
7. Radiation enters the monochromator through entrance slit.
The beam is collimated and then strikes the dispersing element at an angle.
The beam is split into its component wavelength by the grating.
By moving the dispersing element, radiation of only a particular wavelength
leaves the monochromator through exit slit.
8. Dispersion devices:
Dispersion devices causes a different wavelength of light to be
dispersion at different angles monochromator used for function.
Types of dispersion devices:
• Prism
• Diffraction gratings
Prism:
Prism is used to isolate different wavelength.
Prism may be made of glass and quartz.
Glass prism used in visible range whereas quartz prism used in
ultraviolet spectrum.
9. Diffraction Grating:
• It is an optical component , which splits light into several beams
travelling in different directions.
Focusing devices:
• Combinations of lenses, slits and mirrors.
• Relay and focus light through the instrument.
mirrors
Type of rays Mirror material
X-rays ultraviolet aluminum
visible aluminum
10. Lenses
Cuvette:
Designed to hold sample for spectroscopic experiment.
Made of plastic, glass or optical grade quartz.
Glass cuvettes are only suitable for the visible region, whereas quartz may be
used in both the UV and visible region.
rays material
Ultraviolet Fused silica, sapphire
visible glass
11. Detectors:
Convert radiant energy into an electrical signal.
• The photocell and phototube are the simplest detectors, producing current
proportional to the intensity of light striking them.
Photomultiplier tube detector:
It consist of
Photo emissive cathode
Several dynodes
An anode
12. 1. A photon of radiation entering the tube strikes the cathode, causing emission of
several electrons.
2. These electrons are accelerated towards the first dynodes causing emission of
several electrons for each incident electrons.
3. These electrons are then accelerated toward the 2nd dynode, to produce more
electrons which are accelerated toward 3rd dynodes and so on.
4. Eventually the electrons are collected at the anode.
5. The resulting current is amplified and measured.
13.
14. Principle:
Beer lambert law:
Discovery:
Beer’s law was independently discovered by Pierre Bouguer in 1729,
Johann heinrich lambert in 1760 and August Beer in 1852.
Background:
Many compound absorb light. Beer’s law provide a scientist tool to
determine a solution concentration based on the level of light absorbed.
The more light a solution absorb ,the higher the solution
concentration. For this to work effectively the solution must be clear
meaning there can not be any precipitate in the liquid.
15. Statement:
“The amount of intensity, when passing through any sample decreases exponentially
with increase in thickness of the sample and the concentration of the medium. This
phenomenon of decrease in intensity with medium and concentration is called
Lambert-Beer law”.
16. Mathematical form:
A ∝ c
A ∝ l
A ∝ c * l
A = ε c l
ε = molar absorptivity
l = the sample “thickness”
c = the molar concentration of the solution
17. Transmittance:
• The ratio of the intensity of the transmitted light (I) to the intensity of the incident
light (Io) is called transmittance (T) .
T = I / Io
Because the intensity of the transmitted light (I) is never greater than the intensity of
the incident light (Io), transmittance (T) is always less than 1.
Percent transmittance:
Multiplies T by 100 to obtain the percent transmittance (%T), which ranges from 0
to 100%.
%T = T * 100
18. Absorbance:
Absorbance is the amount of light absorbed by a sample. It is calculated from T or
%T using the following equations:
A = - log10 T or A = log10 (1/T)
A = 2 - log10 %T
Relationship between transmittance and absorbance:
Transmittance and absorbance are inversely related. That is, the more a particular
wavelength of light is absorbed by a substance, the less it is transmitted. Moreover,
the inverse relationship between A and T is not linear, it is logarithmic.
19. • Two Sample Calculations of Absorbance from T and %T
• Calculation #1:
• if T = I / Io = 0.50
• then %T = T * 100 = 50
• and A = 2 - log10 %T = 2 - log10 50 = 2 - 1.69897 = 0.301
• Calculation #2:
• if T = I / Io = 0.999
• then %T = T * 100 = 99.9
• and A = 2 - log10 %T = 2 - log10 99.9 = 2 - 1.999 = 0.001
20. Absorbance spectrum:
• An absorbance spectrum is normally measured to determine the optimal
wavelength for measuring the absorbance of a given solution.
21. Calibration graph:
To determine the concentration of absorbing compound by construction of the calibration graph.
Prepare a series of standards of known concentration of the absorbing compound ,measure
absorbance for each of them and plot the absorbance values against their concentration.
measure absorbance of the “unknown” sample, its concentration can then be read from
calibration graph.
22. WORKING:
SET WAVELENGTH
To set the wavelength correctly, view the dial from directly above.
Otherwise, reading of the dial may be wrong. Such 'parallax' errors
occur if line of sight is NOT PERPENDICULAR to the face of the dial.
FILLING VOLUME
When pouring a liquid into the cuvette, the solution must fill the cuvette
to a sufficient height so that the internal light beam passes through the
solution in the cuvette, and not through air.
The Spec 21 cuvettes have a horizontal index mark to show the
minimum required filling volume.
23. CLEAN WITH KIMWIPES
Wipe the cuvette first with a Kimwipe and then with a dry tissue.
After cleaning the cuvettes, handle cuvette by their tops. Don't touch
the lower portion of the glass.
AIR BUBBLES
Even after cleaning the cuvette errors may still occur in a reading if air
bubbles are present in the solution.
Before reading a sample of even a blank, must REMOVING AIR
BUBBLES
Removing air bubbles can be done by tapping the bottom of the
cuvette to dislodge the bubbles.
Remove all air bubbles.
24. SAMPLE HOLDER
Once the sample or blank is free from bubbles and in a clean cuvette,
it can be inserted into the sample holder.
The sample holder is located on the left, top surface of the Spec 21. It
is fitted with a cover, which must be closed before taking readings.
INSERTING A CUVETTE
When inserting a cuvette into the sample chamber, GENTLY push the
cuvette into its position. Hard pushing could damage the instrument.
25. ADJUSTING THE BLANK
The blank solution is used to calibrate the instrument so that the
internal light beam passes through the cuvette to the light-sensing
device. This is indicated by a dial reading of 100% Transmittance or
zero Absorbance.
REMOVE CUVETTE AFTER READING
Always remove the cuvette from the sample holder as soon as the
necessary adjustment or reading has been completed. Leaving the
cuvette in the sample holder for an extended period can damage the
light-sensing device.
26. MATCHING CUVETTES
To reduce reading errors due to imperfections or scratches on the
cuvettes, the sample and blank cuvettes should be matched to one
another.
This entails finding two cuvettes, which give exactly the same reading
when containing the same solution.
An alternative to using matched cuvettes is to read all solutions, both
blanks and samples, in a single cuvette. This is cumbersome, but it
assures accuracy.
If the second cuvette reads within 1% of the blank cuvette, then they
may be considered as matching.
27. LEARNING TO READ THE METER
The meter simultaneously indicates Absorbance (the amount of light
absorbed by the sample) on the lower scale and Percent Transmittance
(the portion of light passing through the sample) on the top scale.
HOW TO READ THE METER
The top scale (%T), which is divided into increments of constant size,
must be read from left to right. This is an easy scale to read.
The bottom absorbance scale has increments between tick marks,
which vary across the scale, making for some difficult readings. Also,
the scale is read from right to left, the opposite of the %T scale.
28. PRACTICE TAKING ABSORBANCE READINGS
Select one of the three concentrations of CuSO4 to work with.
The Spec 21 must first be adjusted to 100% T using the blank cuvette.
Verify that the Spec 21 is set to 100% T when the blank cuvette is
inserted properly into the chamber.
Now prepare the sample cuvette. Pour the water out of the second,
matched cuvette, rinse it with the selected blue solution several times,
fill it to above the mark, wipe the outside clean, remove any bubbles,
gently insert it into the sample chamber, and close the cover.
29. CHANGE THE WAVELENGTH
Change the wavelength and read the values for Absorbance and %T
again for the same sample of copper sulfate. Use the same cuvette to
take the reading.
However -- and this is important to remember -- every time the
wavelength is changed, readjust the Spec 21 using the blank solution.
CHECK ABSORBANCE AT ALL WAVELENGTHS
Determine the absorbance and %T of the sample at all three suggested
wavelengths, checking the accuracy of all the readings.
