Introduction to UV-Vis

Spectroscopy

Electromagnetic radiation
Electromagnetic (EM) radiation has both particle and wave
properties.

EM consists of particles called photons. The energy of a photon is

related to the wavelength of the EM radiation.
Energy of a photon = h = hc/
h = Plancks constant = 6.63 x1034 J s
= frequency (s 1 or Hz)
c = speed of light = 3.00 x 108 m/s
= wavelength ( m or nm or )
1 = 10-10 m; 1 nm = 10-9 m; 1 m = 10-6 m
As increases, the frequency decreases, and the energy of the
photon decreases.

The Electromagnetic Spectrum

Type

Wavelength

Interaction

Gamma

5x10-4 0.14 nm

Nuclear emission

X-ray

10-2 10 nm

Atomic ionization

UV

10 380 nm

Electronic transitions

Vis

380 780 nm

Electronic transitions

IR

0.78 100

Radio

meters

Bond interaction
Nuclear absorption

Molecular Absorption
IR

VIS

UV

E2

Absorption in the UV and VIS

regions involves promotion of
an electron from low-energy
state to high-energy state.

E1

IR absorption involves lower

energy vibrational and rotational
transitions associated with
bonds that hold molecules
together.
electronic excited states
vibrational states

E0

rotational states
electronic ground state

Atomic
absorption
results in line
spectra

Molecular
absorption
results in
band spectra

The Absorption Process and Beers Law

Incident radiation Po can be absorbed by the analyte resulting in a
transmitted radiation P, shown in (a) when the energy of the incident
beam matches one of the energy differences shown in (b).

(path length)

(incident
radiation)

(transmitted
radiation)

absorbing solution of
concentration c
(a)

(b)

Transmittance: T = P/Po
Absorbance: A = log Po/P = -log T
Beers Law: A = abc
where a is absorptivity, c in g/L
A = bc
where is molar absorptivity, c in mol/L

The Visible Spectrum

Region
absorbed, nm

Color of light
absorbed

Complementary
color
transmitted

400 435
435 480
480 490

Violet
Blue
Blue-green

Yellow-green
Yellow
Orange

490 500
500 560
560 580

Green-blue
Green
Yellow-green

Red
Purple
Violet

580 595
595 650
650 - 750

Yellow
Orange
Red

Blue
Blue-green
Green-blue

Color of a Solution

White light from a lamp or the sun strikes the solution of

Fe(SCN)2+. The fairly broad absorption spectrum shows a
maximum absorbance in the 460- to 500 nm range. The
complimentary red color is transmitted.

Question: A sample has a percent transmittance of 50%. What is its

absorbance?

Question: What is the %T for a sample if its absorbance is 1.27?

Question: A 5.00 104 M solution of an analyte is placed in a

sample cell with a pathlength of 1.00 cm. When measured at a
wavelength of 490 nm, the solutions absorbance is 0.338. What is
the analytes molar absorptivity at this wavelength?
Question: A solution of the analyte from Example 10.4 has an
absorbance of 0.228 in a 1.00-cm sample cell. What is the
analytes concentration?

Limitations to Beers Law

1. Real limitations: encountered only in relatively

concentrated solutions; refractive index may change
with concentration, hence changing molar
absorptivity.

Limitations to Beers Law

2. Apparent chemical deviation:
arises when an analyte
dissociates, associates, or
reacts with a solvent to
generate a product that has a
different absorption spectrum
from that of the analyte
HIn (color 1)
570 nm
(-) deviation

H+ + In- (color 2)
430 nm
(+) deviation

11

Limitations to Beers Law

3. Apparent instrumental deviations with polychromatic
radiation: law is adhered only with truly monochromatic
radiation; in practice, devices can only have a band of
wavelengths around the desired one. Measurements are
done at max (Band A) where Beers Law is followed.

Limitations to Beers Law

4. Instrumental deviations
in the presence of stray
radiation: absorbance
measurements is usually
contaminated with small
amounts of stray radiation,
Ps, (result of scattering
phenomena off the surfaces
of prisms, lenses, filters,
and windows) due to
instrumental imperfections.
The observed absorbance
A is given by:

Po Ps
A' log
P Ps

Instruments for Optical Spectrometry

Optical Materials

Sample Cells

Continuum Sources for Optical Spectroscopy

Wavelength Selectors
In absorption spectrometry, a single
wavelength is desired at any given
time.
The purpose of a wavelength
selector (monochromator or filter) is
to allow only a specific wavelength
to reach the detector at any given
time; however, a single wavelength
cannot be obtained whatever the
source is.
The effective bandwidth of a
wavelength selector is the width of
the band of radiation in wavelength
units at half-peak height.

Monochromators
A grating consists of a
polished surface with a
series of lines etched into
it that is used to disperse
polychromatic radiation by
diffraction.
A prism is a transparent,
prism-shaped solid that
disperses polychromatic
radiation into its
component wavelengths
by refraction.
Most modern instruments
use gratings, while older
instruments use prisms.

Filters
Filters operate by absorbing all but a restricted band of radiation from a
continuum source.
Interference filters are used
with UV and visible
radiation. They rely on
optical interference to
provide a band of radiation,
typically 5 to 20 nm.
Absorption filters are limited
in use to the visible region;
usually consist of a colored
glass plate that removes
part of the incident radiation
by absorption to give a
bandwith of about 30 to 250
nm.

Detectors for Absorption Spectroscopy

A detector is a device that responds to some characteristic of the system
under observation and converts that response into a measurable signal.

Signal Processors and Readout

Signal processors
Devices that amplify the electrical signal from the detector
May alter the signal from ac to dc (or the reverse), change the
phase of the signal and filter it
May also perform mathematical operations on the signal as
differentiation, integration or conversion to a logarithm.
Readout devices in modern instruments
Digital meters
Scales of potentiometers
Recorders
Cathode-ray tubes
Monitors of microcomputers

Single-beam UV/Vis Instrument

Radiation from the wavelength selector (filter or monochromator)

passes through either the reference cell or the sample cell before
striking the photodetector.

Double-beam-in-space UV/Vis Instrument

Radiation from the wavelength selector (filter or monochromator) is

split into two beams that simultaneously pass through the reference
and sample cells before striking two matched photodetectors.

Double-beam-in-time UV/Vis Instrument

Radiation from the wavelength selector (filter or monochromator) is

alternately sent through reference and sample cells before striking a
single photodetector. Only a matter of milliseconds separates the beams
as they pass through the two cells.

Quantitative Analysis
Calibration Method: standards for photometric or a
spectrophotometric analysis should approximate as closely as
possible the overall composition of the actual samples, and should
encompass a reasonable range of the analyte concentrations
Standard-Addition Method: involves adding one or more increments
of a standard solution to sample aliquots of the same size; helpful in
counteracting matrix effects

The determination of Fe in an industrial waste

stream was carried out by the o-phenanthroline
Method. Using the data in the following table,
determine the mg Fe/L in the waste stream.

Quantitative Analysis
Analysis of Mixtures
The total absorbance of a
solution at any given
wavelength is equal to the
sum of the absorbances of
the individual components in
the solution.

A1 =

M1bcM

N1bcN

A2 =

M2bcM

N2bcN

Quantitative Analysis
Example. The absorption spectra of two colored
substances A and B are determined and the following
data obtained using 1.00-cm cell:
Solution

Molarity

A alone

5.0 x 10

B alone

2.0 x 10

A+ B

Unknown

A450 nm

A750 nm

0.800

0.100

0.100

0.600

0.600

1.000

Calculate the concns of A and B in the unknown solution.

Spectrophotometric Titrations
The application of absorption measurements requires that
one or more of the reactants or products absorb radiation or
that an absorbing indicator be added to the analyte solution.

Test Yourself
SWHC 8ed #24-18, 24-21, 24-24 pp740-741