2. A spectrophotometer is an instrument that
measures the amount of light absorbed by a
sample.
Spectrophotometer techniques are used to
measure the concentration of solutes in
solution by measuring the amount of the light
that is absorbed by the solution in a cuvette
placed in the spectrophotometer .
3. Lambert’s Law
• The proportion of light absorbed by a medium is
independent of the intensity of incident light.
• A sample which absorbs 75% (25% transmittance) of the
light will always absorb 75% of the light, no matter the
strength of the light source.
• Lambert’s law is expressed as I/Io=T
Where I = Intensity of transmitted light
Io = Intensity of the incident light
T = Transmittance
• This allows different spectrophotometers with different
light sources to produce comparable absorption readings
independent of the power of the light source.
4. Beer’s Law
• The absorbance of light is directly
proportional to both the concentration of the
absorbing medium and the thickness of the
medium.
• In Spectrophotometry the thickness of the
medium is called the pathlength.
5. The spectrophotometer technique is to measures light
intensity as a function of wavelength. It does this by:
1. diffracting the light beam into a spectrum of
wavelengths
2. direct it to an object
3. receiving the light reflected or returned from the
object
4. detecting the intensities with a charge-coupled device
5. displaying the results as a graph on the detector and
then the display device .
6.
7. 1)Measure the concentration of the solution
A spectrophotometer optically determines the
absorbance or transmission of characteristic
wavelengths of radiant energy (light) by a chemical
species in solution. Each molecule absorbs light at
certain wavelengths in a unique spectral pattern
because of the number and arrangement of its
characteristic functional groups, such as double bonds
between carbon atoms.
According to the Beer-Lambert law, the amount of light
absorbed at these wavelengths is directly proportional to
the concentration of the chemical
• species.
8. 2) Identify organic compounds by determining
the absorption maximum.
Spectrophotometers are used to identify organic
compounds by determining the absorption maxima
(which for most compounds and groups of
compounds have very distinct fingerprints (that's
what the absorption curves and peaks are called).
3) Used for color determination within the
spectral range
If one is working in the range of 380 to 700 nm, the
spectrophotometers can also be used for color
determination within this spectral range
9. Observed Color of
Compound
Color of Light
Absorbed
Approximate
Wavelength of Light
Absorbed
Green Red 700 nm
Blue-green Orange-red 600 nm
Violet Yellow 550 nm
Red-violet Yellow-green 530 nm
Red Green 500 nm
Orange Blue 450 nm
Yellow Violet 400 nm
10. 1)Light source
The function of the light source is to provide
a sufficient of light which is suitable for
marking a measurement. The light source
typically yields a high output of
polychromatic light over a wide range of
the spectrum.
11. Tungsten Lamp
• the most common light source used in
spectrophotometer.
• This lamp consists of a tungsten filament
enclosed in a glass envelope,
• Wavelength range of about 330 to 900 nm
• used for the visible region.
• It has long life about 1200h.
12. II) Hydrogen / Deuterium Lamps
• For the ultraviolet region
• hydrogen or deuterium lamps are frequently
used.
• range is 200 to 450 nm.
• Deuterium lamps are generally more stable
and has long life about 500h.
• This lamp generates continuous or
discontinuous spectral.
13. III) Xenon flash lamps
1)Their range between ( 190nm - 1000 nm)
2) Emit both UV and visible wavelengths
3) Long life
4) Do not heat up the instrument
5) Reduce warm up time
14. 2) Dispersion devices
a)Monochromator
Accepts polychromatic input light from a lamp
and outputs monochromatic light.
Monochromator consists of three parts:
I) Entrance slit
II) Exit slit
III) Dispersion device (includes prism, filter,
diffraction grating )
15. • 3)Focusing devices
Combinations of lenses, slits, and mirrors.
Variable slits also permit adjustments in the
total radiant energy reaching the detector.
16. • Optical Materials
• 1)Mirrors
Type of rays Mirror material
• X-rays – Ultraviolet(UV) Aluminum
• Visible - Aluminum
• Near infrared - Gold
• Infrared (IR) - Copper or Gold
18. • 4)Absorption cells(Cuvettes)
A cuvette is a kind of cell (usually a small square
tube) sealed at one end,
made of Plastic, glass or optical grade quartz and
designed to hold samples for spectroscopic
experiments.
For measurements in the visible region, cuvettes of
optical glass are sufficient; however, optical glass
absorbs light below 350 nm , and more expensive
quartz or fused silica must be used for these
wavelengths
19. 5)Detectors
Any photosensitive device can be used as a
detector of radiant energy .The photocell and
phototube are the simplest photodetectors,
producing current proportional to the
intensity of the light striking them .
20. 6)Display devices
The data from a detector are displayed by a
readout device, such as an analog meter, a
light beam reflected on a scale, or a digital
display , Or liquid crystal display(LCD) .The
output can also be transmitted to a computer
or printer.
21. Types
There are two classes of spectrophotometers:
1)Single beam
The single beam spectrophotometer was the first
invented, and all the light passes through the
sample. In this case, to measure the intensity of
the incident light, the sample must be removed
so all the light can pass through. This type is
cheaper because there are less parts and the
system is less complicated.
22. The advantages of the single beam design are low
cost, high throughput, and hence high Sensitivity
, because the optical system is simple. The
disadvantage is that an appreciable amount of
Time elapses between taking the reference (I)
and Making the sample measurement (Io) so that
there can be problems with drift. This was
certainly true of Early designs but modern
instruments have better electronics and more
stable lamps, so stability with single beam
instruments is now more than adequate for the
vast majority of application.
23.
24. 2)Double beam
* The double beam instrument design aims to eliminate
drift by measuring blank and sample virtually
simultaneously. A "chopper" alternately transmits and
reflects the light beam so that it travels down the blank
and the sample optical paths to a single detector..
* To measure a spectrum with a double beam instrument
the two cuvettes, both containing solvent are place in
the sample and reference positions and a "balance”
measurement is made. This is the difference between
the two optical paths and is subtracted from all
subsequent measurement. The sample is then placed
in the sample cuvette and the spectrum is measured. I
and Io are measured virtually simultaneously as
described above.
25. The advantage of the double beam design is
high stability because reference and sample
are measured virtually at the same moment in
time.
The disadvantages are higher cost, lower
sensitivity because throughput of light is
poorer because of the more complex optics
and lower reliability because of the greater
complexity.
26. Applications
• Nucleic acid estimation:
spectrophotometry can be used to estimate DNA or
RNA concentration and to analyze the purity of the
preparation. Typical wavelengths for measurement are 260
nm and 280 nm.
Purines and pyrimidines in nucleic acids naturally
absorb light at 260 nm. For pure samples it is well
documented that for a pathlength of 10 mm, an absorption
of 1A unit is equal to a concentration of 50 µg/ml DNA and
40 µg/ml for RNA. For oligonucleotides the concentration is
around 33 µg/ml but this may vary with length and base
sequence.
So for DNA: Concentration (µg/ml) = Abs 260 × 50.
27. Direct UV measurement:
A260/A280 Ratio
The most common purity check for DNA and RNA is
the A260/A280 ratio
Any protein contamination will have maximum
absorption at 280 nm
For DNA the result of dividing the 260 nm
absorption by the 280 nm needs to be greater or
equal to 1.8 to indicate a good level of purity in
the sample. For RNA samples this reading should
be 2.0 or above. Results lower than this are
indicative of impurities in the sample
28. A260/A230 Ratio
• An increase in absorbance at 230 nm can also
indicate contamination, which may in turn affect
the 260 nm reading for DNA and RNA. A number
of substances absorb at 230 nm, as this is the
region of absorbance of peptide bonds and
aromatic side chains
• Phenol contamination also increases a sample’s
absorption at 280 nm and therefore can be
identified through a lower A260/A280 ratio. An
A260/A230 ratio of 2 or above is indicative of a
pure sample.
