UV-visible spectroscopy

How They Work

What is Spectroscopy?
The study of molecular structure and
dynamics through the absorption,
emission and scattering of light.
What is Light?
According to
Maxwell, light is an
electromagnetic field
characterized by a
frequency f, velocity
v, and wavelength .
Light obeys the
relationship

f = v / .
The Electromagnetic
Spectrum
v = c / E = hv
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
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
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
Prism - spray out the spectrum and choose the certain
wavelength (l) that you want by moving the slit.
Polychromatic
Ray
Infrared
Red
Orange
Yellow
Green
Blue
Violet
Ultraviolet
monochromatic
Ray
SLIT
PRISM
Polychromatic Ray Monochromatic Ray
Photomultiplier Tube
Detector
Anode
High sensitivity at
low light levels
Cathode material
determines spectral
sensitivity
Good signal/noise
Shock sensitive
The Photodiode Detector
Wide dynamic range
Very good
signal/noise at high
light levels
Solid-state device
Schematic Diagram of a
Photodiode Array
Same characteristics
as 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
Definition of Resolution
Spectral resolution is a measure of the ability of an instrument to
differentiate between two adjacent wavelengths
Instrumental Spectral
Bandwidth
The SBW is defined as the width, at half the maximum intensity, of
the band of light leaving the monochromator
Natural Spectral
Bandwidth
The NBW is the width of the sample absorption band at half the
absorption maximum
Transmission
Characteristics of Cell
Materials
Note that all materials exhibit at least approximately 10% loss in
transmittance at all wavelengths
Cells
UV Spectrophotometer
Quartz (crystalline silica)
Visible Spectrophotometer
Glass
IR Spectrophotometer
NaCl
Open-topped rectangular standard cell (a)
and apertured cell (b) for limited sample volume
Cell Types I
Cell Types II
Micro cell (a) for very small volumes and flow-through cell (b)
for automated applications
Transmittance and
Concentration
The Bouguer-Lambert Law
Pathlength Const
e I I T

= =
0
/
Transmittance and Path
Length: Beers Law
ion Concentrat Const
e I I T

= =
0
/
Concentration
The Beer-Bouguer-
Lambert Law
( ) ( ) c b I I I I T A = = = = c / log / log log
0 0
BEER LAMBERT LAW
Glass cell filled with
concentration of solution (C)
I
I
Light
0
As the cell thickness increases, the intensity of I
(transmitted intensity of light ) decreases.

R- Transmittance
R = I
0
- original light intensity
I- transmitted light intensity

% Transmittance = 100 x

Absorbance (A) or optical density (OD) = Log

= Log = 2 - Log%T

Log is proportional to C (concentration of solution)
and is also proportional to L (length of light path
through the solution).
I
I
0
I
I
0
I
0
I

1

T

I
I
0
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
E =
Absorbance x Liter
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)

E =
Liter
Cm x Mole

L = Cm
C = Moles/Liter


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






A = ECL = (
Liter
Cm x Mole
) x
Mole
Liter
x Cm
K=
Liter
Cm Gram
A = KLC = (
Liter
Cm x Gram
Gram
Liter
x Cm
) x
STEPS IN DEVELOPING A
SPECTROPHOTOMETRIC
ANALYTICAL METHOD
1. Run the sample for
spectrum
2. Obtain a monochromatic
wavelength for the
maximum absorption
wavelength.
3. Calculate the concentration
of your sample using Beer
Lambert Equation: A = KCL

Wavelength (nm)
Absorbance
0. 0
2. 0
200
250 300
350
400 450
Slope of Standard Curve =
A
A
A
C
1 2 3
4
5
1.0
0.5
Concentration (mg/ml)
Absorbance at 280 nm
There is some A vs. C where graph is linear.
NEVER extrapolate beyond point known where
becomes non-linear.
SPECTROMETRIC ANALYSIS USING
STANDARD CURVE
1
2
3
4
0. 4
0. 8
1. 2
Absorbance at 540 nm
Conc entration (g/l) glucose
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 +