Absorption Spectroscopy

Absorption Spectroscopy
Postlab Discussion
Chem 137.1 1st Sem AY 2019-2020
Jethro T. Masangkay
Instructor 7, Institute of Chemistry

Optical Spectrometry
Colorimeter – instrument for absorption measurements in which the human eye is the
detector
Photometer – uses an absorption or interference filters for wavelength selection in
conjunction with a suitable radiation transducer
Spectrometer – a spectroscopic instrument that uses a monochromator equipped with an
appropriate transducer; in organic chemistry, this is an instrument that measures
spectra
Spectrophotometer – spectrometers with phototransducers located at the exit slit; thus,
designed for measuring absorbance or transmittance
Transducer – a type of detector that converts a chemical or a physical signal (i.e. light
intensity, pH, mass, & temperature) to an electrical signal

Spectroscopy - general term used to describe
techniques based on the interactions of radiation
and matter
1. Energy is transferred between from the photon of EMR
and the analyte– absorption/emission
2. EMR undergoes a change in amplitude, phase angle,
polarization, or direction of propagation as a result of its
refraction, reflection, scattering, diffraction, or dispersion by
the sample – x-ray, refractometry

Absorption Spectroscopy
 the energy carried by a photon is absorbed by the analyte, promoting the
analyte from a lower-energy state to a higher-energy or excited state
 transition results from can be a change in electronic levels, vibrations, rotations,
etc.
Eo
E1
h Energy required of photon
to give this transition:
h = DE = E1 - Eo
(excited state)
(ground state)

Emission
 The release of a photon when an analyte returns to a lower-energy state from a
higher-energy state
photoluminescence - emission following absorption of a photon.
chemiluminescence - emission following a chemical reaction.
(excited state)
(ground state)

Beer’s Law: A = ebc
The amount of light absorbed (A) by a sample is dependent on the path length (b),
concentration of the sample (c) and a proportionality constant (e – molar absorptivity)
c
Absorbance is directly proportional to concentration Fe2+

Beer’s Law: A = ebc
Transmittance (T) = P/Po %Transmittance = %T = 100T
Absorbance (A) = -log T = log10 Po/P
No light absorbed-
% transmittance is 100%  absorbance is 0
All light absorbed-
% transmittance is 0%  absorbance is infinite

A = Absorbance = ebc = -log(%T/100)
Therefore, by measuring absorbance or percent transmittance at a given
concentration range, information related to the amount of sample (c)
present in the sample can be calculated for given e and l.
Note: law does not hold at high
concentrations, when A > 1

Spectroscopic Instrumentation
Basic Design:
Hitachi Instruments U-3010
Light Source, l selector, Sample cell holder, Detector, signal processor

Desired Properties of Components of spectrophotometer:
Light Source l Selector
Creates Proper l Narrow Bandpass (effective bandwidth):
Stable: Selects Desired l
Constant P (radiant power) Large Light Throughput:
Good Precision Increase P
Intense:
Increase P
Easier to See Absorbance
Sample Cell Holder Detector
Fixed Geometry: Stable (constant response over l range of interest)
Constant b highly sensitive to l of Interest
Transmits l of Interest good signal to noise ratio
little or no signal in absence of light

Light Sources
Two types:
1. Continuous Source
– Produces spectra over broad
range
Tungsten lamp
-provides visible spectrum; 400-1200
nm
Deuterium lamps
-provides ultra-violet spectrum; 190-
400 nm

2. Discontinuous or Discrete/Line Sources
– Produce only specific (discrete) wavelengths
– e.g. hollow cathode lamp (HCL) or an electrodeless discharge lamp
(EDL)
Light Sources
Hollow
Cathode
Lamp

Light Sources: UV/Vis (~ 200 – 800 nm):
1. Deuterium & Hydrogen Lamps (UV range)
- continuous source, broad range of frequencies
- based on electric excitation of H2 or D2 at Low pressure
40V ElectricArc
Electrode
Filament
D or H Gas2 2
Sealed QuartzTube
In presence of arc, some of the electrical energy is absorbed by D2 (or H2)
which results in the disassociation of the gas and release of light
D2 + Eelect  D*
2  D’ + D’’ + h (light produced)
Excited state

2. Tungsten Filament Lamp (Vis – Near IR: 350- 2500 nm)
- continuous source, broad range of frequencies
- based on black body radiation

Wavelength Selectors:
- used to isolate wavelength of interest (monochromator or filters)
Ideal specs:
 high throughput of radiation – more photons can pass through thus signal
is strong and noise is minimized
Narrow effective bandwidth - provides a higher resolution

1. Monochromator
- separates frequencies () from polychromatic light.
- contains entrance and exit slit allowing only certain ls to be
selected and used.
-fixed wavelength or scanning
Dispersing Monochromator:
a) Prism: based on refraction of light and fact that different l’s
have different values of refraction index (hi) in a medium.
Recall Snell’s
law of refraction

b. Grating Monochromator
A grating is made by etching a highly
polished metal surface
Light is diffracted off the surface
The angle of diffraction is related to the
wavelength
A polychromatic radiation at the
entrance slit  monochromatic at the
exit slit
Typical bandpass : 20nm -0.5 nm for UV-vis instruments.
- decrease slit size  decrease effective bandwidth (good)
- less undesired l’s, but less intensity (bad)

Slits in the Monochromator:
- need to be carefully made, since they control the range of
ls emerging from the monochromator.
- typical slit widths are 0.01 nm to 2 mm and are often adjustable.
a) Bandpass of the monochromator (effective wavelength):
range of l’s transmitted at the half-height of
transmitted light band.
Typical bandpass :
20 nm to 0.5 nm for UV-vis instruments.
- decrease slit size  decrease bandpass (good)
- less undesired ls, but less intensity (bad)

Two l’s can be resolved by the
monochromator if they differ by
2 or more times the bandpass.
- l resolution is directly related to
slit size.

2. Filter
Absorption Filters
- remove undesired l’s by absorbing them
- typically made from colored glass or dye
suspended in gelatin between glass plates.
-wide range of l allowed through
- typical bandpass: 30-250 nm
- can combine filters with different l range
Effective bandwidth for two types of
filters and the result of combining
filters.

Interference Filters
- made up of thin layers of metal and dielectric (eq. CaF2)
material coated on both sides with a film that is thin
enough to transmit approximately half the radiation
striking it and reflect the other half
Bandpass can be 1-20nm

Sample Cell:
Must have windows that are transparent in the spectral region of interest
Visible region – silicate glass (low cost)
UV (and below 380 nm) - glass absorbs  fused silica or quarts
IR – material from halide salts or polymeric
Fingerprints, grease, etc – alter transmission characteristic of cell
Drying (esp by heating)– physical damage or change in pathlength

1) photovoltaic cell (Barrier-Layer Cell)
Process:
light of sufficiently high energy passes through the thin
transparent silver layer and hits selenium causing electrons to
be released which move across barrier toward silver layer
(electropositive) and collected at iron layer to neutralize
selenium layer.
- Current produced is proportional to photons hitting surface
- Maximum response at 550 nm (10% at 350-750 nm ~ same as
human eye).
Advantage: cheap, rugged, no external power source, good for portable instruments.
Disadvantage: not very sensitive, shows fatigue (decrease in response with continued
illumination)
Detectors:

a. Phototube
(Photoemission material)
Advantages: sensitive,
signal easily amplified.
• Converts the energy of an
incoming photon into a
current pulse
• Conversion is done on a
photo emissive surface by the
“photoelectric effect”
1)Human eye
2)Modern detectors - use sensitive transducer to convert a signal
consisting of photons into an easily measured electrical signal
Detectors:

b. Photomultiplier tube (PMT)
Process:
a) light hits cathode and e- emitted.
b) an emitted e- is attracted to electrode #1
(dynode 1), which is 90V more positive.
Causes several more e- to be emitted.
c) these e- are attracted to dynode 2, which is
90V more positive then dynode 1, emitting
more e-.
d) process continues until e- are collected at
anode after amplification at 9 dynodes.
e) overall voltage between anode and cathode
is 900V.
f) one photon produces 106 – 107 electrons.
g) current is amplified and measured
Advantages: very sensitive to low intensity, very fast response.
Disadvantages: need high voltage power supply, intense light damages

Signal Processors and Read-out
-devices that amplify the electric signal from the detector
-may also perform mathematical operations on the signal
as a differentiation, integration or conversion to a
logarithm
-digital meters, scales of potentiometers, recorders,
computers

Single vs. Double Beam Instruments:
- To determine absorbance both Po and P must be measured.
Main difference between single and double beam spectrophotometer:
process of measuring the Po and P
Absorbance (A) = -log T = log10 Po/P

Process for a Single Beam Spectrometer:
a) Po is measured with a blank and spectrophotometer is
adjusted to read 100%T or 0% A which is set by
blocking the light beam (P=0)
Schematic of Spectronic 20
Advantages: cheap, rugged.
Disadvantages: must readjust 100%T at every l and periodically check for drift, cell is
round so pathlength can vary if the cell is not aligned constantly (the same) each time

Double-beam instrument with beams separated
in space
• Radiation from the wavelength selector is split in two beams that
simultaneously pass through the reference and sample cells before
striking two matched photo detectors

Double-beam instrument with beams
separated in time
• Radiation from the 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.
Advantages: l scanning, little drift – only one PMT.
Disadvantages: more complex and expensive

Common Problems in UV/Vis Spectroscopy
a) the sample/matrix is a not pure
- blank samples often contain multiple absorbing species.
- the absorbance is the sum of all the individual absorbances
A= A1 + A2 +A3 + … = e1bc + e2bc + e3bc …
- substances in both the blank and sample which absorb can be
“blanked out” in both double and single beam spectrometers

If blank absorbance is too high:
- use a different l where the analyte absorbs more relative to the interference
- use a different method of separation
If the blank absorbance is high, Po will decrease too much, the response
will be slow and the result is inaccurate
l scan of substance
Large blank
absorbance

b. Instrumental Limitations – Stray Light
A = -log P/Po = log Po/P
 When stray light (Ps) is present, the absorbance observed (Aapparent) is
the sum of the real (Areal) and stray absorbance (Astray)
Aapp = Areal + Astray = log (Po + Ps)
P + Ps
 As the analyte concentration increases ([analyte]↑), the intensity of
light exiting the absorbance cell decreases (P↓)

• Result – non-linear
absorption (analyte vs.
conc.) as a function of
analyte concentration

C A (actual) A(expected)
10-6M 0.0075 0.0075
10-5M 0.074 0.075
10-4M 0.068 0.75
10-3M 5.3 7.5 0
1
2
3
4
5
6
7
8
2 10-4
4 10-4
6 10-4
8 10-4
1 10-3
Concentration (M)
Negative Deviation
A = bce
The results are the same for more l’s of light. The situation is worse for greater
differences in e’s and the instrument has a broad effective bandwidth.
Let us say that exactly 2 wavelengths of light were entering the sample
l = 254 nm e254 =10,000
l = 255 nm e255 = 5,000
let Po = 1 at both l’s
What happens to the Beer’s law plot as c increases?
c) Instrumental Limitations - polychromatic light (more than a single l)
since all instruments have a finite bandpass, a range of l’s are sent through the sample
e may be different for each l

HIn
Ka
H+ + In-
Red, l =600nm colorless
phenolphthalein:
If solution is buffered, then pH is constant and [HIn] is related to absorbance.
d) Chemical Deviations from Beer’s Law:
- Molar absorptivity change in solutions with concentrations > 0.01M
 due to molecular interactions
 Beer’s law assumes species are independent
- association, dissociation, precipitation or reaction of analyte
 recall A = abc ( c= the concentration of the absorbing species)
if dissociation happens:

CHIn [HIn] [In-] [HIn]/[In-]
10-5 8.5x10-7 9.2x10-6 0.0924
10-4 3.8x10-5 6.2x10-5 0.613
10-3 7.3x10-4 2.7x10-4 2.70
But, if unbuffered, equilibrium will shift depending on total analyte concentration
example: Ka = 10-4
HIn
Ka
H+ + In-
C
HIn
Expected
Actual

Isosbestic point
At the isosbestic point in spectra:
A = eb([HIn] + [In-])

e) Non-constant b
- worse for round cuvettes
- use parallel cuvettes to cancel pathlength differences
P0
A = log10 Po/P = ebc
P1
B1
B2
P2

f) Instrument noise
noise - short term baseline fluctuations, which decrease the precision of the analysis
1) 0%T noise
- noise when light beam is blocked
- seldom important
- typically around 0.01%T
2) Readout Precision
- especially with a meter
- typically around 0.5%T  1-3% error in concentration
3) Shot Noise
- occurs when e- transfers across a junction (like the space between
cathode & anode in PMT).
- causes random fluctuations in current since individual e- arrive at
random times
- increases with increase current (%T). Especially bad above 95%T.

4) Flicker Noise
- noise from the lamp due to intensity changes
- important at high transmittances.
5) Cell positioning uncertainty
- not really noise, but affects precision
- minor imperfections, scratches or dirt change %T
- may be the major cause of imprecision
Taken together, these noise sources indicate that the intermediate
absorbance and transmittance ranges should be used.
- at low %T, noise and readout precision are important
- at high %T, shot and flicker noise are large.
Keep A in range of 0.2 – 0.8 absorbance units

Equipment Management
For a reliable results, all aspects of analytical work must be controlled,
and potential errors are controlled by carrying out preventive maintenance
and calibration procedures.
Who carries out the calibration?
personnel of the laboratory, external personnel, formed, authorized
Frequency of calibration?
depends on the use and conservation
Which equipments?
All the ones that intervene in the results

UV-Vis instruments
1. wavelength accuracy and repeatability
Deuterium lamp - sharp emmission lines at 656.1nm and 486.0 nm

UV-Vis instruments
1. wavelength accuracy and repeatability
use of filters:
Holmium oxide filter Didymium filters
Use of solutions: dilute KMnO4 : λmax at 526nm and 546 nm
**deviation of the wavelength
reading at an absorption band or
emission band from the known
wavelength --- causes errors in
the qualitative and quantitative
results

UV-Vis instruments
2. photometric accuracy and repeatability
-how accurately an instrument measures absorbance
Examples:
Potassium Dichromate Liquid Filter
Neutral Density Solid Filter
Didymium Glass Solid Filter

UV-Vis instruments
3. Stray Light – commercially available standards
ex. NIST – National Institute for Standards and Technology
use of filter – absorbs all light of the λ at which the measurement is
made and transmits higher and lower λ’s
Procedure:
1. Measure reference/balance with nothing in the sample area (that is, on air).
2. Insert cell with appropriate test solution and measure transmittance at the test
wavelength. This is the amount of stray light.

UV-Vis instruments
3. Stray Light

UV-Vis instruments
4. Baseline Stability
Most UV-Vis spectrophotometers have dual light sources:
UV range - deuterium lamp
Vis range - tungsten lamp
The intensity of the radiation coming from the light sources is not constant over the
whole UV-Visible range. The response of the detector also varies over the spectral
range. A flat baseline demonstrates the ability of the instrument to normalize the
output of the lamp and detector responses.

Applications:
A) Determination of Molar Absorptivities (e) in UV-Vis Range:
Name Structure lmax e
but-1-en-3-yne 219 7,600
cyclohex-2-enone 225 10,300
toluene 206 7,000
3,4-dimethylpent-3-en-2-one 246 5,300

-For Compounds with Multiple Chromophores:
If greater then one single bond apart
- at constant l, e are additive
CH3CH2CH2CH=CH2 lmax= 184 emax = ~10,000
CH2=CHCH2CH2CH=CH2 lmax=185 emax = ~20,000
If conjugated
- shifts to higher l’s (red shift)
H2C=CHCH=CH2 lmax=217 emax = ~21,000

Applications:
B) Determination of Absorbing Species in UV/Vis
1)Electronic transitions involving organic compounds, inorganic compounds,
complexes, etc.
Basic process:
M + h  M*
10-8 – 10-9s
M*  M + heat
(or fluorescence, light, or phosphorescence)
or
10-8 – 10-9s
M*  N
(new species, photochemical reaction)

2) Absorption occurs with bonding electrons.
- E(l) required differs with type of bonding electron.
- UV-Vis absorption gives some information on bonding electrons (functional
groups in a compound
- Most organic spectra are complex
 electronic and vibration transitions superimposed
 absorption bands usually broad
 detailed theoretical analysis not possible, but semi-quantitative or qualitative
analysis of types of bonds is possible
 effects of solvent & molecular details complicate comparison

Chromophore Example Solvent lmax (nm) emax Type of
transition
Alkene n-Heptane 177 13,000 pp*
Alkyne n-Heptane 178
196
225
10,000
2,000
160
pp*
_
_
Carbonyl n-Hexane
n-Hexane
186
280
180
293
1,000
16
Large
12
ns*
np*
ns*
np*
Carboxyl Ethanol 204 41 np*
Amido Water 214 60 np*
Azo Ethanol 339 5 np*
Nitro CH3NO2
Isooctane 280 22 np*
Nitroso C4H9NO Ethyl ether 300
665
100
20
_
np*
Nitrate C2H5ONO2
Dioxane 270 12 np*
C6H13HC CH2
C5H11C C CH3
CH3CCH3
O
CH3CH
O
CH3COH
O
CH3CNH2
O
H3CN NCH3
Absorption Characteristics of Some Common Chromophores

C) Qualitative Analysis
1) Limited since few resolved peaks
- unambiguous identification not usually possible.
2) Solvent can affect position and shape of curve.
- polar solvents broaden out peaks, eliminates fine structure.
Loss of fine structure for acetaldehyde
when transferred to a solvent from gas
phase
Also need to consider
absorbance of solvent

Loss of fine structure for 1,2,4,5-
tetrazine as solvent polarity increases
(a) Vapor
(b) Hexane solution
(c) Aqueous
Solvent can affect position and shape of curve
- polar solvents broaden out peaks, eliminates fine structure
Solvent can also absorb in UV-Vis spectrum.

C) Quantitative Analysis (Beer’s Law)
Calibration curve
 for analysis wherein analytical signal is proportional to the quantity of analyte
 limited to linear range of the curve
Dynamic range - concentration range over which there is a measurable response to analyte

C) Quantitative Analysis (Beer’s Law)
Strategies:
a) absorbing species
- detect both organic and inorganic compounds containing any of these species
(all the previous examples)

b) non- absorbing species
- react with reagent that forms colored product
- can also use for absorbing species to lower limit of detection
- items to consider: l, pH, temperature, ionic strength
- prepare standard curve (match standards and samples as much as possible)
reagent
(colorless)
Complex
(red)
Non-absorbing
Species (colorless)
As Fe3+ continues to bind protein
red color and absorbance increases.
When all the protein is bound to Fe3+,
no further increase in absorbance.

c. Standard Addition Method (spiking the sample)
- used for analytes in a complex matrix where interferences are likely to
occur: i.e. blood, sediment, human serum, etc..
- Method:
(1) Prepare several identical aliquots, Vx, of the unknown sample.
(2) Add a variable volume, Vs, of a standard solution of known
concentration, cs, to each unknown aliquot.
Note: This method assumes a linear relationship between instrument response and sample concentration.

(3) Dilute each solution to an equal volume, Vt.
(4) Make instrumental measurements of each sample to get an
instrument response, IR.

Where:
S = signal or instrument response
k = proportionality constant
Vs = volume of standard added
cs = concentration of the standard
Vx = volume of the sample aliquot
cx = concentration of the sample
Vt = total volume of diluted solutions
Vs
InstrumentResponse(S)
m = D y/ D x
b = y-intercept
(V s ) 0
t
xx
t
ss
V
ckV
V
ckV
S =
bmVS s =
x
s
x
mV
bc
c =
VsSS
cVS
C ss
x
)( 12
1

=
(5) Calculate unknown concentration, cx, from the following equation.

d) Analysis of Mixtures
- use two different l’s with different e’s
A1 = e1
MbcM + e1
NbcN (l1)
A2 = e2
MbcM + e2
NbcN (l2)

II. Elemental Analysis - Atomic Spectroscopy
A) Introduction
Based on the breakdown of a sample into atoms, followed by the measurement of the
atom’s absorption or emission of light.
i. deals with absorbance or emission of atoms or elemental ions
rather then molecules
ii. Provides information on elemental composition of sample or compound
- UV/Vis, IR, Raman gives molecular functional group information, but not
elemental information.
iii. Basic process the same as in UV/Vis, fluorescence etc. for molecules
Eo
E1
h
Absorbance
E0
E2
AES - radiative
emission
Emission

iv. Differences from Molecular Spectroscopy
- no vibration levels  much sharper absorbance, fluorescence, emission
bands
- position of bands are well-defined and characteristic of a given element
thus qualitative analysis is easier compared to molecular (UV/VIS)
spectroscopy
Examples:
carbon
oxygen
nitrogen

To observe atomic spectra, sample should be broken down into its
constituent atoms.
Basic steps:
a) nebulization – formation of aerosol from liquid sample (by spraying thru thin nozzle or
passing over vibrating crystal)
b) desolvation - heat droplets causes solvent evaporation leaving analyte and other
matrix compounds
c) volatilization – convertion of solid analyte/matrix particles into gas phase
d) atomization/dissociation – break-up molecules in gas phase into atoms.
e) excitation – transition to higher E levels (spectra measurement)
E) Sample Atomization – expose sample to flame or high-temperature

Atomic Absorption Spectroscopy (AAS)
– commonly used for elemental analysis
– sample is exposed to flame or high-temperature
– characteristics of flame impact use of atomic absorption spectroscopy
Flame AAS:
• simplest atomization of gas/solution/solid
• laminar flow burner - stable "sheet" of flame
• flame atomization best for reproducibility (precision) (<1%)
• relatively insensitive - incomplete volatilization, short time in flame

1)Atomizer - Laminar Flow Burner
- adjust fuel/oxidant mixture for optimum excitation of desired
compounds
- different mixes give different temperatures.
Main advantage:
- good reproducibility
disadvantages:
- small amount of time that sample is
in light path (~10-4 s)
- needs lots of sample

Different mixes and flow rates give different temperature profile in flame
- gives different degrees of excitation of compounds in path of light source
Temperature varies significantly across
flame – need to focus on part of the flame

 selection of right region in flame important for optimal performance
a) primary combustion zone – blue inner cone
- not in thermal equilibrium and not used
b) interconal region
- region of highest temperature (rich in free atoms)
- often used in spectroscopy
c) outer cone
- cooler region
- rich in O2 (due to surrounding air)
- gives metal oxide formation
Not in thermal equilibrium
and not used for
spectroscopy
Primary region
for spectroscopy

b) Light source
-need light source with a narrow bandwidth for light output
- AA lines are remarkably narrow (0.002 to 0.005 nm)
Problem: Line Broadening

Line Broadening:
1. Doppler effect - an atom moving toward the radiation source experiences
more oscillations of the electromagnetic wave in a given time period than
one moving away from the source
- emitted or absorbed wavelength changes as a result of
atom movement relative to detector
- wavelength decreases if motion is toward receiver
- wavelength increases if motion is away from receiver

Line Broadening:
2. Pressure broadening - The collision of other particles with the emitting particle
interrupts the emission process, and thus shortening the characteristic time for the
process  increases the uncertainty in the energy emitted
Effect: worse at high pressures
• For high pressure Xe lamps (>10,000 torr) turns lines into a broad band

Solution: use light source that has line emission in range of interest
- hollow cathode lamp (HCL)
Hollow Cathode
Lamp
Coated with element
to be analyzed
Process: use element to detect element
1. ionizes inert gas to high potential (300V)
Ar  Ar+ + e-
2. Ar+ go to “-” cathode & hit surfaces
3. As Ar+ ions hit cathode, some of deposited element is excited and
dislodged into gas phase (sputtering)
4. excited element relaxes to ground state and emits characteristic radiation
- advantage: sharp lines specific for element of interest
- disadvantage: can be expensive, need to use different lamp for each element tested.

Continuous Light source
Typical problem:
- have right l, but also lots of interfering l ’s
(non-monochromatic light)
- hard to see decrease in signal when atoms
absorb in a small bandwidth
- with large amount of elements  bad
sensitivity

Spectral Interferences Due to Matrix :
-problem if absorbance spectra overlap since molecular spectrum is much
broader with a greater net absorbance
- need a way of subtracting these factors out
Matrix - everything in a sample
other than analyte. Ideally, the
matrix decomposes and
vaporizes during the charring
step.

Methods for Correction:
1) Two-line method
- a reference line from the source (from an impurity in cathode or any emission
line) is selected where this line should have the following properties:
1. Very close to analyte line
2. Not absorbed by analyte
- A & e are approximately constant if two ls are close
- comparing Al1, Al2 allows correction for absorbance for molecular species
Al1 (atom&molecule) – Al2 (molecule) = A (atom)
If such a line exists, since the reference line is not absorbed by the analyte, its
intensity should remain constant throughout analysis. However, if its intensity
decreases, this will be an indication of absorbance or scattering by matrix
species.
Problem: Difficult to get useful second l with desired characteristics

2) Continuous source method
- alternatively place light from HCL or a continuous source D2 lamp thru flame
- HCL  absorbance of atoms + molecules
- D2  absorbance of molecules
advantage:
-available in most instruments
-easy to do
disadvantage:
-difficult to perfectly match lamps (can give + or – errors)

3) Zeeman Effect
-Effect of splitting the spectral line into several components in the presence of
static magnetic field
-sum of split absorbance lines  original line
An alternating magnetic field is applied at the atomizer (graphite furnace) to
split the absorption line into three components, the π component, which
remains at the same position as the original absorption line, and two σ
components, which are moved to higher and lower wavelengths,
respectively. Total absorption is measured without magnetic field and
background absorption with the magnetic field on.

Chemical Interference:
1) Formation of Compounds of Low Volatility
- Anions + Cations  Salt
Ca2+ +SO4
2-  CaSO4 (s)
- Decreases the amount of analyte atomized  decreases the absorbance
signal
- Avoid by:
> increase temperature of flame (increase atom production)
> add “releasing agents” – other items that bind to interfering
ions (Sr or La)
eg. For Ca2+ detection add Sr2+
Sr2+ + SO4
2-  SrSO4 (s)
increases Ca atoms and Ca absorbance
> add “protecting agents” – bind to analyte but are volatile
eg. For Ca2+ detection add EDTA4-
Ca2+ + EDTA4-  CaEDTA2-  Ca atoms

Chemical Interference:
2) Formation of Oxides/Hydroxides
M + O  MO
M + 2OH M(OH)2
- Avoid by:
> increase temperature of flame (increase atom production)
> use less oxidant
non-volatile & intense molecular absorbance

3) Ionization
M  M+ + e-
- Avoid by:
> lower temperature
> add ionization suppressor like CsCl – creates high concentration of e-
suppresses M+ by shifting equilibrium.

Atomic Emission Spectroscopy (AES) – similar to AA with flame used for
atomization and excitation of the sample for
light production
1) Atomic Processes
heat
Degree of Excitation Depends on Boltzmann Distribution:
N1 and No – are the number of atoms in excited and ground states
k – Boltzmann constant (1.28x10-23 J/K)
T – temperature
DE – energy difference between ground and excited states
P1 and Po – number of states having equal energy at each quantum level
Increase Temperature  increase in N1/No (more excited atoms)

Need a good temperature control to get reproducible signal
eg. For Na, temperature difference of 10o C 2500  2510
results in a 4% change in N1/No
2) Comparison of AA and AES Applications
Flame Emission More
Sensitive
Sensitivity About the
Same
Flame Absorption
More Sensitive
Al, Ba, Ca, Eu, Ga, Ho,
In, K, La, Li, Lu, Na,
Nd, Pr,Rb, Re, Ru, Sm,
Sr, Tb, Tl, Tm, W, Yb
Cr, Cu, Dy, Er, Gd, Ge,
Mn, Mo, Nb, Pd, Rh,
Sc, Ta, Ti, V, Y, Zr
Ag, As, Au, B, Be, Bi,
Cd, Co, Fe, Hg, Ir, Mg,
Ni, Pb, Pt, Sb, Se, Si,
Sn, Te, Zn
Comparison of Detection Limit
AES – simultaneous emission from multiple species

3) Instrumentation
- Similar to AA, but no need for external light source (HCL) or chopper
> flame acts as sample cell & light source
Source Temperature (oC)
Flame 1700-3150
Plasma 4,000-6,000
Arc/Spark 4,000-5,000/40,00
Atomization Sources:

Flame Source:
- used mostly for alkali metals
> easily excited even at low temperatures
- Na, K
- need internal standard (Cs usually) to correct for variations flame
Advantages
- cheap
Disadvantage
- not high enough temperature to extend to many other elements

Sample Problem 1
Manganese in steel is determined colorimetrically by oxidizing it to permanganate. The steel is
first dissolved in nitric acid, forming a nearly colorless manganese (II) aquo ion:
Mn(s) + 2 H+ + 6 H2O  Mn(H2O)6
2+ + H2(g)
The aquo ion is fairly stable and does not readily form intense colored complex ions, hence it is
oxidized to the permanganate ion by oxidation with periodate:
2 Mn2+ + 5 IO4
- + 3 H2O  2 MnO4
- + 5 IO3
- + 6 H+
KMnO4 conc, M Absorbance
0 0.000
0.50 × 10-5 0.101
1.00 × 10-5 0.202
2.00 × 10-5 0.405
3.00 × 10-5 0.606
4.00 × 10-5 0.809
Unknown sample 0.725
The absorbance of the permanganate ion is then measured at 540 nm.
Suppose a 0.2000 g sample of a metal alloy was dissolved in an acid and diluted to exactly
200.0 mL. A 25.00 aliquot was obtained from this solution and was added with periodate, and
boiled for several minutes. After cooling, the resulting solution was diluted to 100.0 mL. The
absorbance of this final solution was measured together with the KMnO4 standard solutions in a
1-cm cell at 504 nm. The results are given in the table. (MM of KMnO4 = 158.03, MM of Mn =
54.94)
1. What is the type of calibration used?
2. Calculate the molar absorptivity of KMnO4 at 504 nm.
3. Calculate the % (w/w) Mn in the metal alloy sample

Sample Problem 2
2.1 A spectrophotometric method for the quantitative determination of the
concentration of Pb2+ in blood yields an Ssamp of 0.193 for a 1.00-mL
sample of blood that has been diluted to 5.00 mL. A second 1.00-mL
sample is spiked with 1.00L of 1560-ppb Pb2+ standard and diluted to
5.00 mL, yielding an Sspike of 0.419. Determine the concentration of Pb2+ in
the original sample of blood.
2.2 A spectrophotometric method for the quantitative determination of the
concentration of Pb2+ in blood yields an Ssamp of 0.193 for a 5.00-mL
sample of blood. After spiking the blood sample with 5.00-mL of a 1560-
ppb Pb2+ standard, an Sspike of 1.546. Determine the concentration of Pb2+
in the original sample of blood.

Sample Problem 3
Ten-milliliter aliquots of a natural water sample were pipetted into 50.00 mL
volumetric flasks. Exactly 0.00, 5.00, 10.00, 15.00, and 20.00 mL of a standard
solution containing 11.1 ppm of Fe3+ were added to each, followed by an excess
of thiocyanate ion to give the red complex Fe(SCN)2+ . After dilution to volume,
absorbances for the five solutions, measured with a photometer equipped with a
green filter, were found to be 0.240, 0.437, 0.621, 0.809, and 1.009, respectively
(0.982-cm cells).
1. What was the concentration of Fe3+ in the water sample?
2. Calculate the standard deviation of the slope, the intercept, and the
concentration of Fe.

Sample Problem 4
In a preliminary experiment, a solution containing 0.0837 M X and 0.0666 M S
gave peak areas of Ax=423 and As=347. To analyze the unknown, 10.0 mL of
0.146 M S were added to 10.0 mL of unknown and the mixture was diluted to 25.0
mL in a vol. flask. The mixture gave the chromatogram for which Ax=553 and
As=582. Find the concentration of X in the unknown.

Sample Problem 4
Infrared spectra are customarily recorded on a transmittance scale so that weak
and sstrong bands can be displayed on the same scale. The absorption
corresponds to a downward peak on this scale. The spectra were recorded from a
0.0100 M solution of each, in cells with 0.00500 cm path lengths. A mixture of A
and B in a 0.00500 cm cell gave a transmittance of 34.0% at 2022 cm-1 and
38.3% at 1.993 cm-1. Find the concentration of A and B.
Wavenumber Pure A Pure B
2022 cm-1 31.0% 97.4%
1993 cm-1 79.7% 20.0%

Absorption Spectroscopy

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