UV-visible spectroscopy

Laida Neti Mulyani, M.Si

 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.
The Electromagnetic Spectrum

E = hn n=c/l
 Spectroscopy
Spectral Distribution of Radiant Energy

Wave Number (cycles/cm)

X-Ray UV Visible IR Microwave

200nm 400nm 800nm

WAVELENGTH(nm)
Transmission and Color

The human eye sees the complementary color to that which is

absorbed
Absorbance and Complementary Colors
 UV and Visible Spectroscopy

In structure determination : UV-VIS

spectroscopy is used to detect the presence of
chromophores like dienes, aromatics, polyenes,
and conjugated ketones, etc.
 Electronic transitions

There are three types of electronic transition

which can be considered;
Transitions involving p, s, and n electrons
Transitions involving charge-transfer
electrons
Transitions involving d and f electrons
Absorbing species containing p,
s, and n electrons
Absorption of ultraviolet and visible
radiation in organic molecules is restricted
to certain functional groups
(chromophores) that contain valence
electrons of low excitation energy.
N
O
 Vacuum UV or Far UV
(<190 nm )

UV/VIS
 Transitions

An electron in a bonding s orbital is excited to

the corresponding antibonding orbital. The
energy required is large. For example, methane
(which has only C-H bonds, and can only
undergo transitions) shows an
absorbance maximum at 125 nm. Absorption
maxima due to transitions are not seen
in typical UV-VIS spectra (200 - 700 nm)
 n Transitions
Saturated compounds containing atoms with
lone pairs (non-bonding electrons) are capable
of n transitions. These transitions
usually need less energy than
transitions. They can be initiated by light
whose wavelength is in the range 150 - 250 nm.
The number of organic functional groups with
n peaks in the UV region is small.
 n and Transitions
Most absorption spectroscopy of organic
compounds is based on transitions of n or
electrons to the excited state.
These transitions fall in an experimentally
convenient region of the spectrum (200 - 700
nm). These transitions need an unsaturated
group in the molecule to provide the
electrons.
Chromophore Excitation lmax, nm Solvent

C=C * 171 hexane

n* 290 hexane
C=O
* 180 hexane

n* 275 ethanol
N=O
* 200 ethanol

C-X n* 205 hexane

X=Br, I n* 255 hexane
 Terms describing UV absorptions

1. Chromophores: functional groups that give

electronic transitions.
2. Auxochromes: substituents with unshared pair e's like
OH, NH, SH ..., when attached to chromophore they
generally move the absorption max. to longer .
3. Bathochromic shift: shift to longer , also called red
shift.
4. Hysochromic shift: shift to shorter , also called blue
shift.
5. Hyperchromism: increase in of a band.
6. Hypochromism: decrease in of a band.

 Light Sources

UV Spectrophotometer
1. Hydrogen Gas Lamp
2. Mercury Lamp
Visible Spectrophotometer
1. Tungsten Lamp
InfraRed (IR) Spectrophotometer
1. Carborundum (SIC)
 Dispersion Devices

Non-linear dispersion
Temperature sensitive

Linear Dispersion
Different orders
 Dispersion of polychromatic light
with a prism

Infrared
monochromatic
Ray
Red
Orange
Yellow SLIT
Polychromatic PRISM
Green
Ray Blue
Violet

Ultraviolet

Polychromatic Ray Monochromatic Ray

Prism - spray out the spectrum and choose the certain

wavelength (l) that you want by moving the slit.
 Photomultiplier Tube Detector

High sensitivity at
low light levels
Cathode material
determines spectral
sensitivity
Good signal/noise
Shock sensitive

Anode
 The Photodiode Detector

Wide dynamic range

Very good
signal/noise at high
light levels
Solid-state device
 Schematic Diagram of a Photodiode
Array

Same characteristicsas photodiodes

Solid-state device
Fast read-out cycles
Conventional Spectrophotometer

Schematic of a conventional single-beam

spectrophotometer
Conventional Spectrophotometer

Optical system of a double-beam spectrophotometer

Conventional Spectrophotometer

Optical system of a split-beam spectrophotometer

Two-Component Mixture

Example of a two-component mixture with little spectral

overlap
Two-Component Mixture

Example of a two-component mixture with significant

spectral overlap
 Influence of 10% Random Error

Influence on the calculated concentrations

Little spectral overlap: 10% Error
Significant spectral overlap: Depends on similarity, can be much higher (e.g. 100%)
Absorption Spectra of Hemoglobin
Derivatives
 Definition of Resolution

Spectral resolution is a measure of the ability of an instrument to

differentiate between two adjacent wavelengths
Cells

UV Spectrophotometer
Quartz (crystalline silica)
Visible Spectrophotometer
Glass
IR Spectrophotometer
NaCl
 Cell Types I

Open-topped rectangular standard cell (a)

and apertured cell (b) for limited sample volume
 Cell Types II

Micro cell (a) for very small volumes and flow-through cell (b)
for automated applications
The Beer-Bouguer-Lambert Law

A log T logI / I 0 logI 0 / I b c

 BEER LAMBERT LAW

Light
I0 I

Glass cell filled with

concentration of solution (C)

As the cell thickness increases, the intensity of I

(transmitted intensity of light ) decreases.
 R- Transmittance
I
R= I0 - original light intensity
I0
I- transmitted light intensity

% Transmittance = 100 x I
I0
1
Absorbance (A) or optical density (OD) = Log
T
I0
= Log = 2 - Log%T
I
I
Log is proportional to C (concentration of solution)
and is I0 also proportional to L (length of light path
through the solution).
A CL = KCL by definition and it is called
the Beer Lambert Law.
A = KCL
K = Specific Extinction Coefficient ---- 1 g of
solute per liter of solution

A = ECL
E = Molar Extinction Coefficient ----
Extinction Coefficient of a solution containing
1g molecule of solute per 1 liter of solution
 Absorbance x Liter
E =
Moles x cm

E differs from K (Specific extinction Coefficient) by

a factor of molecular weight.

UNITS
A = ECL
A = No unit (numerical number only)

Liter
E =
Cm x Mole
 L = Cm
C = Moles/Liter
Liter Mole
A = ECL = ( )x x Cm
Cm x Mole Liter

A = KCL
A = No unit C = Gram/Liter L = Cm

Liter
K=
Cm Gram

Liter Gram
A = KLC = ( )x x Cm
Cm x Gram Liter
 STEPS IN DEVELOPING A
SPECTROPHOTOMETRIC
ANALYTICAL METHOD

1. Run the sample for

spectrum Absorbance

2. Obtain a monochromatic 2.0

wavelength for the
maximum absorption
wavelength. 0.0

200 250 300 350 400 450

3. Calculate the concentration Wavelength (nm)
of your sample using Beer
Lambert Equation: A = KCL
 A
Slope of Standard Curve =
C
Absorbance at 280 nm

1.0

0.5

1 2 3 4 5
Concentration (mg/ml)

There is some A vs. C where graph is linear.

NEVER extrapolate beyond point known where
becomes non-linear.
 SPECTROMETRIC ANALYSIS USING
STANDARD CURVE
Absorbance at 540 nm
1.2

0.8

0.4

1 2 3 4
Concentration (g/l) glucos e
Avoid very high or low absorbencies when drawing a
standard curve. The best results are obtained with 0.1 < A
< 1. Plot the Absorbance vs. Concentration to get a
straight line
 Every instrument has a useful range for a
particular analyte.
Often, you must determine that range
experimentally.
This is done by making a dilution series of
the known solution.
These dilutions are used to make a
working curve.
Make a dilution series of a known quantity
of analyte and measure the Absorbance.
Plot concentrations v. Absorbance.
What concentration do you think the
unknown sample is?
In this graph, values above A=1.0 are not linear. If we
use readings above A=1.0, graph isnt accurate.
The best range of this spectrophotometer is A=0.1 to
A=1.0, because of lower errors. A=0.4 is best.
Relating Absorbance and Transmittance

Absorbance rises linearly with

concentration. Absorbance is
measured in units.
Transmittance decreases in a non-
linear fashion.
Transmittance is measured as a %.
Absorbance = log10
(100/% transmittance)
 Precision and Accuracy

Precision Precision + Precision Precision +

Accuracy Accuracy Accuracy + Accuracy +