uv spectrophotometery

1. Molecular Spectroscopy
Visible and Ultraviolet Spectroscopy
－UV/VIS Spectroscopy
－UV/VIS Spectrometer
－Application for Quantitative Analysis

2. U V Spectrophotometer
n 2014
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3.  Ultraviolet: 190~400nm
 Violet: 400 - 420 nm
 Indigo: 420 - 440 nm
 Blue: 440 - 490 nm
 Green: 490 - 570 nm
 Yellow: 570 - 585 nm
 Orange: 585 - 620 nm
 Red: 620 - 780 nm

4. Electronic Spectroscopy
 Ultraviolet (UV) and visible (VIS) spectroscopy
 This is the earliest method of molecular
spectroscopy.
 A phenomenon of interaction of molecules with
ultraviolet and visible lights.
 Absorption of photon results in electronic
transition of a molecule, and electrons are
promoted from ground state to higher
electronic states.

5. Quantitative Analysis
Beer’s Law
A=ebc
e: the molar absorptivity (L mol-1 cm-1)
b: the path length of the sample
c :the concentration of the compound in
solution, expressed in mol L-1

6. Transmittance
I0 I
b
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.
2
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log(
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log(
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2
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ln(
0
0
0
0
0
0
0
0
k
bc
A
T
I
I
I
I
kbc
I
I
db
kc
I
dI
kcdb
I
dI
I
I
T
I
I
b
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e
e

7. UV Spectroscopy
Instrumentation and Spectra
A. Instrumentation :
1. The construction of a traditional UV-VIS spectrometer is very similar
to an IR, as similar functions – sample handling, irradiation, detection
and output are required
2. Here is a simple schematic that covers most modern UV
spectrometers:
sample
reference
detector
I0
I0 I0
I
log(I0/I) = A
200 700
l, nm
monochromator/
beam splitter optics
UV-VIS sources

8. Components of a Spectrophotometer
Light Source
 Deuterium Lamps－a truly continuous
spectrum in the ultraviolet region is
produced by electrical excitation of
deuterium at low pressure.
(160nm~375nm)
 Tungsten Filament Lamps－the most
common source of visible and near
infrared radiation.

9. Components of a Spectrophotometer
Monochromator
 Used as a filter: the monochromator will
select a narrow portion of the spectrum
(the bandpass) of a given source
 Used in analysis: the monochromator will
sequentially select for the detector to
record the different components (spectrum)
of any source or sample emitting light.

10. Detector
Photomultiplier

11. Principle of Photomultiplier
Detector
 The type is commonly used.
 The detector consists of a photo emissive
cathode coupled with a series of electron-
multiplying dynode stages, and usually called
a photomultiplier.
 The primary electrons ejected from the photo-
cathode are accelerated by an electric field so
as to strike a small area on the first dynode.

12. Principle of Photomultiplier
Detector
 The impinging electrons strike with enough
energy to eject two to five secondary electrons,
which are accelerated to the second dynode to
eject still more electrons.
 A photomultiplier may have 9 to 16 stages,
and overall gain of 106~109 electrons per
incident photon.

13. Single and Double Beam
Spectrometer
 Single-Beam: There is only one light beam or
optical path from the source through to the
detector.
 Double-Beam: The light from the source, after
passing through the monochromator, is split
into two separate beams-one for the sample
and the other for the reference.

14. 14
Instrumentation – Sample Handling
• Virtually all UV spectra are recorded
solution-phase
• Cells can be made of plastic, glass or
quartz
Only quartz is transparent in the full 200-
700 nm range; plastic and glass are only
suitable for visible spectra.
A typical sample cell (commonly called a cuvet):

15.  Steps in carrying out a colorimetric analysis.
 Choose the wavelength of maximum absorbance.
 Prepare a calibration curve using known quantities of
the complex measured at this wavelength.
 Measure the absorbance of your unknown sample.
 Calculate the concentration from the equation of the
best fit line.
UV / visible Spectroscopy

16. Standard Addition Method
 Standard addition must be used whenever
the matrix of a sample changes the
analytical sensitivity of the method. In
other words, the slope of the working
curve for standards made with distilled
water is different from the same working
curve.

17. Preparation of the Standards
The concentration and volume of the stock solution
added should be chosen to increase the
concentration of the unknown by about 30% in
each succeeding flask.

18. Preparation of the Sample
Sampling
Homogenization (By Grinding/ Blender)
Digestion
Wet Oxidation
Dry Ashing
Microwave
Isolation
Colour development by suitable reagent
Dilution to different volume
Preparation of Standard & read absorbance

19. External Standard and the
Calibration Curve

20.  Clarity of the solution
 The solution must be free of precipitates.
 Turbidity scatters and absorbs light.
 High sensitivity
 It is desirable that the colour reaction be highly
sensitive.
 i.e. e is very large.
UV / visible Spectroscopy

21.  Advantages of colorimetric analysis.
 Better at low concentrations than titrimetric
or gravimetric analysis.
 Can be applied under conditions where there
are no satisfactory titrimetric or gravimetric
procedures.
 Very rapid once a calibration curve as been
obtained.
UV / visible Spectroscopy

22. UV / visible Spectroscopy
 Six criteria for a successful analysis
 Specificity of the colour reaction
 Proportionality between colour and
concentration
 Stability of the colour
 Reproducibility
 Clarity of the solution
 High sensitivity.

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