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UV-Visible Spectroscopy

By
Asmara Aslam

Session: 2016-2018
Registration#:

Department of Chemistry,
The National College Toba Tek Singh.
Affiliated
Govt. College University Faisalabad, Faisalabad
Introduction: Spectroscopy

Spectrum is the distribution of color into


various regions on the basis of wavelength
Fortunately, certain molecules absorb light in a
characteristic way. Over the years, this has
helped us identify and quantify biological
molecules.
Absorption: Physical Basis

Absorption occurs when the energy


contained in a photon is absorbed by an
electron resulting in a transition to an
excited state and electron energy levels are
Since photon
quantized, we can only get specific allowed
transitions
E=h (h = 6.626*10-34 Js)

~ 400 - ~ 115
700 nm nm
~ 200 – 400
nm
~ 150-
250 nm
http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/uvvisab1.htm
Absorption: Lineshape

h
  *

So, our absorption


spectrum should
probably look like this:
But they don’t…
Absorption: Intensity

The absorption efficiency of an analyte is


affected
Theby:
nature of the analyte
The number of available microstates
The solvent (sortof)
The absorption efficiency of an analyte
generally not affected by:
Other (low conc.) solutes
Temperature (within reason)
Concentration
This makes absorption spectroscopy one of
the few bioanalytical methods where the
signal intensity is directly proportional to the
concentration
Absorption: The Beer-Lambert Law

The Beer-Lambert law sortof has the wrong


name…
A   log( I1 / I 0 )  cl
Extinctio
n Concentrat
coefficie ion
nt Path
length
Pierre Johan
Bouguer Lambert
(1698-
Astronomer: (1728-
Mathematician,
1758)is
Light first1777)
to prove
diminished as that  is
it passes irrational. No
through the absorption
August Beer
atmosphere. (1825-1863): Added
coefficient.
absorption co-efficient and related
to conc. in solution.
UV/Visible Spectroscopy:
Instrumentation
In absorption spectroscopy, we measure  as
a function of wavelength 

The instrument we use to do this is called a


UV/visible Spectrophotometer

The Major Components Are:

A light source
A monochromator
A Sample
Compartment
A detector
UV/Visible Spectroscopy: Light
Sources
Xenon, Mercury/Xenon

Flash Arc-Lamps –
light generated
from Xe plasma
Pure Xenon has very wide emission spectrum
~200 – 1200 nm
Xenon/Mercury is blue shifted for more power
in the UV region, used more often for
sterilization
UV/Visible Spectroscopy: Light
Sources
Deuterium

D2 gas is discharged by contact with a high


voltage tungsten cathode
Continuous spectrum from ~150 nm - ~370 nm
Usually used in conjunction with a
tungsten/halogen source, which handles the
visible spectrum
Monochromators

The light sources we use produce continuous


emission spectra. But we need single
wavelengths, so…
This is called the
Czerny-Turner setup
B and C set up a
beam that is at
infinite focus
D is either a prism or
a diffraction grating
(almost always the
latter
E these one
refocuses dayswavelength
on slit F wavelengths can be focused on F
Different
by rotating D or E (it’s almost always D).
Monochromators: Defraction
Gratings
Diffraction gratings are a surface with closely
spaced parallel lines

distance between
slits diffraction
wavelength!
order
a sin( )  n
These can be semi-transparent gratings or
ridged mirrorsthough a slit (or bouncing
After passing
off a ridge) the angle at which the light
leaves is given by
Sample Compartments/Holders

These days, sample compartments are


designed to accept accessories…

The sample itself is held in a cuvette, usually


plastic or quartz:
The detector

Silicon diode:
Basically a solar cell – light ionizes n-doped
(phosphate) silicon, placing the electrons
in the conduction band (i.e. having a
voltage).

Wide wavelength range, less sensitivity


Photomultiplier

In photomultipliers, light hits a photocathode,


releasing a small number of electrons, which
are then made to collide with a series of
dynodes, each more positive than the last

Each collision produces more and more


activated electrons
Sensitive, but noisy. Pretty much needed for
low energy (IR) photons
The Whole Instrument

Sample
Detector Czerny-Turner

Light
Source
  APPLICATIONS:
Detection of Impurities
UV absorption spectroscopy is one of the best methods for determination of
impurities in organic molecules. Additional peaks can be observed due to
impurities in the sample and it can be compared with that of standard raw
material. By also measuring the absorbance at specific wavelength, the
impurities can be detected.
Benzene appears as a common impurity in cyclohexane. Its presence can be
easily detected by its absorption at 255 nm.

Structure elucidation of organic compounds.


UV spectroscopy is useful in the structure elucidation of organic molecules,
the presence or absence of unsaturation, the presence of hetero atoms.
From the location of peaks and combination of peaks, it can be concluded
that whether the compound is saturated or unsaturated, hetero atoms are
present or not etc.
Quantitative analysis
UV absorption spectroscopy can be used for the quantitative determination of
compounds that absorb UV radiation. This determination is based on Beer’s
law which is as follows.
A = log I0 / It = log 1/ T = – log T = abc = εbc
Where ε is extinction co-efficient, c is concentration, and b is the length of
the cell that is used in UV spectrophotometer.

Qualitative analysis
UVabsorption spectroscopy can characterize those types of compounds which
absorbs UV radiation. Identification is done by comparing the absorption
spectrum with the spectra of known compounds.
UV absorption spectroscopy is generally used for characterizing aromatic
compounds and aromatic olefins
Dissociation constants of acids and bases.
PH = PKa + log [A-] / [HA]
From the above equation, the PKa value can be calculated if the ratio of [A-] / [HA] is
known at a particular PH. and the ratio of [A-] / [HA] can be determined
spectrophotometrically from the graph plotted between absorbance and wavelength at
different PH values.

Chemical kinetics
Kinetics of reaction can also be studied using UV spectroscopy. The UV radiation is
passed through the reaction cell and the absorbance changes can be observed.

As HPLC detector
A UV/Vis spectrophotometer may be used as a detector for HPLC. The presence of an
analyte gives a response which can be assumed to be proportional to the concentration.
For more accurate results, the instrument's response to the analyte in the unknown
should be compared with the response to a standard; as in the case of calibration curve.

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