ATOMIC ABSORPTION

SPECTROSCOPY
Flame Emission and Atomic Absorption Spectroscopy

□ In these methods the solution sample is aspirated into

a flame that is hot enough to break the molecules
into their atomic states.

□ The concentration of the analyte in the flame may

be measured by either its absorption or emission of
the radiation.

□ The absorption mode is known as atomic absorption

spectroscopy (AAS) whereas emission mode as
flame emission Spectroscopy (FES)
Upper Diagram shows FES
while the lower one shows
AAS. In FES the flame also
provides the excitation, but
in AAS it provides only for
the atomization .
 Atomic Absorption (AA) spectroscopy.
Uses
:
• Atomic absorption spectroscopy is a
quantitative method of analysis that is
applicable to many metals.
• examples include:
Al in blood serum
Ca in blood serum, plants, soil,
water Cu in alloys
Cr in sea
water Fe in
plants
• Only a drop of sample needed
• The metals need not be removed from
other components (AA is a highly
selective technique)
• Sensitive in the ppm range (even ppb with
the right equipment)
Elements that are highlighted in pink
are detectable by AAS
 The principles of AA.

• When metals are exposed to heat, they

absorb light.
• Each metal absorbs light at a
characteristic frequency. For
example:

Metal Zn Fe Cu Ca Na
λ 21 24 32 42 589
(nm) 4 8 5 3
□ The metal vapor absorbs energy from
an external light source, and electrons
jump from the ground to the excited
states.

□ The ratio of the transmitted to

incident light energy is directly
proportional to the concentration of
metal atoms present.

□ A calibration curve can thus be

constructed [Concentration (ppm) vs.
Absorbance]
 The components of the AA spectrometer: fuel,
atomizer, monochromatic light source, monochromatic
detector, read out.
 the AAS instrument

:
 The simple diagram for the AAS
4. The element in the sample
will absorb some of the
light,
thus reducing its intensity

5. The
3. A beam of UV light monochromator
will be focused on the isolates the line of
sample interest

1. We set the
instrument at
certain wavelength 2. The
suitable for a certain element in
element the sample 6. The detector
will be measures the change
atomized by in intensity
heat

7. A computer data
system converts the
change in intensity
into an
absorbance
 Sample
Compartment

Light Source Detector

• The source of light is a lamp whose
cathode is composed of the element
being measured.
• Each analyzed element requires a different
lamp.
• For example, a hollow cathode
lamp for Aluminum (Al) is shown
below
The light source in AAS is the hollow cathode
lamp (HCL). The cathode is constructed of the
metal the analysis.
• The cathode lamps are
stored in a
compartment inside the
AA spectrometer. The
specific lamp needed
for a given metal
analysis is rotated into
position for a specific
experiment.
• The sample is made up, typically in water
• A flame is created, usually using
ethyne & oxygen (fuel)
• The flowing fuel and air mixture
provides the aspiration action drawing
the solution sample into the flame.
• The flame gases flowing into the burner
create a suction that pulls the liquid into
the small tube from the sample container.
This liquid is transferred to the flame
where the sample is atomized. The metal
atoms then absorb light from the source
(cathode lamp).
□ Three steps are involved in
turning a liquid sample into an
atomic gas:
1. Desolvation: the liquid solvent is
evaporated, and the dry sample
remains.
2. Vaporisation: the solid sample
vaporises to a gas.
3. Volatilization: the compounds
making up the sample are broken
into free atoms.
The atomizer-burner
 Light beam

Sample is
vaporized
in the
flame.

Aspirator
tube sucks the
sample into the
flame in the
sample
compartment.
The sample may also be atomized in AAS using
the electrically heated graphite furnace
□ The advantage of the Graphite
Furnace for atomization in AAS lies in
the smaller sample (microliter range)
required and the
possibility programming the heating
cycle to destroy organic materials
before the atomization occurs.

□ In the flame AAS the sample is

continuously flowing into the flame
and the flame thus dilutes the
concentration of atomized
atoms, lowering the sensitivity.
• The light passes through a
monochromater (a device used to select
a particular wavelength of light for
observation)
• The intensity of the light is fairly low, so
a photomultiplier tube (PMT) is used to
boost the signal intensity
• A detector (a special type of transducer)
is used to generate voltage from the
impingement of electrons generated by
the photomultiplier tube
A typical photomultiplier tube
• The read out specified
by the user is
displayed on the
computer screen for
each sample
measured.
The
resulting
data can
be
presented
in a variety
of ways,
but
typically a
print out is
made.
Signal
 Determine the concentration of a solution from a
calibration curve.

• AA can be used to identify the presence

of an element (qualitative analysis), or the
concentration of a metal (quantitative
analysis)
• Quantitative analysis can be
achieved by measuring the
absorbance of a series of solutions
of known concentration.
• A calibration curve and the equation for
the line can be used to determine an
unknown concentration based on its
absorbance.
 Sample Problem:
Lead is extracted from a sample of blood and analyzed at 283 nm and gave
an absorbance of 0.340 in an AA spectrometer. Using the data provided,
graph a calibration curve and find the concentration of lead ions in the blood
sample.

• The data provided

in the problem
appears in the
upper left hand
corner of this MS
EXCEL worksheet.
• The graph was
used to calculate
the best fit line.
• The equation
was then used to
calculate the
concentration of
Pb
(II) ions with an
absorbance of
0.340.
• The result,
0.357 ppm, is
displayed
above the
graph.
Applications:

1. This is the widely used technique

for quantitative determination of
metals in biological, environmental,
pharmaceutical samples.
2. It utilizes Beer-Lambert Law

A = εbc
3. A calibration curve and the equation for
the line can be used to determine an
unknown concentration based on its
absorbance.
Limitation:

1. Provides no information on the

chemical form of the metal.
2. Sample must be in solution or at least
volatile.
3. Individual source lamp is needed for
each element, since each metal has
its own characteristic absorption.
4. Limited to metal or metalloids.
5. Destructive technique.
Mass Spectrometry
Mass Spectrometer

Chapter 12 2
 Mass Spectrometry
• Molecular weight can be obtained
from a very small sample.
• It does not involve the
absorption or emission of light.
• A beam of high-energy electrons
breaks the molecule apart.
• The masses of the fragments and
their relative abundance reveal
information about the structure
of the molecule.
Electron Impact Ionization
A high-energy electron can dislodge an
electron from a bond, creating a
radical cation (a positive ion with an
unpaired e-).
 Separation of Ions
• Only the cations are deflected
by the magnetic field.
• Amount of deflection depends on m/z.
• The detector signal is proportional
to the number of ions hitting it.
• By varying the magnetic field, ions
of all masses are collected and
counted.
 The Mass Spectrum
Masses are graphed or tabulated
according to their relative
abundance.
 The GC-MS
A mixture of compounds is
separated by gas
chromatography, then identified
by mass spectrometry.
 High Resolution MS
• Masses measured to 1 part in 20,000.
• A molecule with mass of 44
could be C3H8, C2H4O, CO2, or
CN2H4.
• If a more exact mass is 44.029, pick
the
correct structure from the table:
C3H8 C2H4O CO2 CN2H4
44.06260 44.02620 43.98983
44.03740
 Molecules
with
Heteroatom
s
• Isotopes: present in their usual
abundance.
• Hydrocarbons contain 1.1% C-13, so
there will be a small M+1 peak.
• If Br is present, M+2 is equal to M+.
• If Cl is present, M+2 is one-third of M+.
• If iodine is present, peak at 127, large
gap.
• If N is present, M+ will be an odd
number.
• If S is present, M+2 will be 4% of M+.
Isotopic
Abundance

81
Br
Mass
Spectrum
with Sulfur
Mass
Spectrum
with
Chlorine
Mass
Spectrum
with
Bromine
 Mass
Spectra of
Alkanes
More stable carbocations will be
more abundant.
 Mass
Spectra of
Alkenes
Resonance-stabilized cations favored.
Chapter 12
 Mass
Spectra of
Alcohols
• Alcohols usually lose a water molecule.
• M+ may not be visible.
 Introduction to Chromatography
Definition
Chromatography is a separation technique based on the different interactions of
compounds with two phases, a mobile phase and a stationary phase, as the
compounds travel through a supporting medium.

Components:

mobile phase: a solvent that flows through the supporting medium

stationary phase: a layer or coating on the supporting medium that interacts

with the analytes

supporting medium: a solid surface on which the stationary phase is bound or

coated
□ Chromatography permit the scientist to separate
closely related components of complex mixtures.

□ In all chromatographic separations the sample is

transported in a mobile phase, which may be a gas, a
liquid, or a supercritical fluid. This mobile phase is
then forced through
an immiscible stationary phase, which is fixed in
place in a column or on a solid surface. The two
phases are chosen so that the components of the
sample distribute themselves between the mobile
and stationary phase to varying degrees.

□ The sample is introduced at the head of a column,

whereupon the components of the sample
distribute
themselves between the two phases. Introduction of
additional mobile phase (the eluent) forces the
solvent containing a part of the sample down the
column.
The analytes interacting
most strongly with the
stationary phase will take
longer to pass through the
system than those with
weaker interactions.

These interactions are

usually chemical in nature,
but in some cases physical
interactions can also be
used.
Types of Chromatography

1.) The primary division of chromatographic techniques is based on the type of

mobile phase used in the system:

Type of Chromatography Type of Mobile

Phase Gas chromatography (GC) gas
Liquid chromatograph (LC) liquid

2.) Further divisions can be made based on the type of stationary phase used in the
system:

Gas Chromatography
Name of GC Method Type of Stationary Phase
Gas-solid chromatography solid, underivatized
support Gas-liquid chromatography liquid-coated
support
Bonded-phase gas chromatographychemically-derivatized support
Types of Chromatography

Liquid Chromatography
Name of LC Method Type of Stationary Phase
Adsorption chromatography solid, underivatized
support
Partition chromatography liquid-coated or derivatized
support Ion-exchange chromatography support
containing fixed charges Size exclusion chromatography
porous support
Affinity chromatography support with immobilized ligand
3.) Chromatographic techniques may also be classified based on the type of support
material used in the system:

Packed bed (column)

chromatography Open tubular
(capillary) chromatography Open
bed (planar) chromatography

In column forced under pressure.

chromatography, the
stationary phase is held in
a narrow tube through
which the mobile phase is
In planar chromatography, the
stationary phase is supported on a
flat plate or in the interstices of a
paper; here, the mobile phase
moves through the stationary
phase by capillary action or under
the influence of gravity.
Theory of Chromatography

1.) Typical response obtained by chromatography (i.e., a chromatogram):

□If a detector that responds to solute concentration is

placed at the end of the column and its signal is
plotted as function of time (or of volume of the added
mobile phase), a series of peaks is obtained. Such a
plot, called a chromatogram.

□Chromatogram is useful for both qualitative and

quantitative analysis. The positions of peaks on the
time axis may serve to identify the components of
the sample; the areas under the peaks provide a
quantitative measure of the amount of each
component.
 chromatogram - concentration versus elution time

Wh

Wb

Inject

Where:
tR = retention time (retained molecule)
tM = void time (the length of time it takes unretarded molecule to flow through the
column)
Wb = baseline width of the peak in time
units Wh = half-height width of the peak
in time units
Note: The separation of solutes in chromatography depends on two factors:

(a) a difference in the retention of solutes (i.e., a difference in their time

or volume of elution
(b) a sufficiently narrow width of the solute peaks (i.e, good efficiency for
the separation system)

Peak width & peak position

determine separation of peaks

A similar plot can be made in terms of elution volume instead of elution time. If
volumes are used, the volume of the mobile phase that it takes to elute a peak
off of the column is referred to as the retention volume (VR) and the amount of
mobile phase that it takes to elute a non-retained component is referred to as
the void volume (VM).
2.) Solute Retention:

The time it takes after sample injection for the analyte peak to reach
the detector is called the retention time and is given the symbol tR.

The time tM for the unretained species to reach the detector is called
the dead time.

The rate of migration of the unretained species is the same as the

average rate of motion of the mobile phase molecules.

The average linear rate of solute migration ν is

ν = L/tR

where, L is the length of the column packing.

The average linear rate of movement u of the molecules of the

mobile phase is u = L/tM

Where tM, the dead time.

A solute’s retention time or retention volume in chromatography is

directly related to the strength of the solute’s interaction with the
mobile and stationary phases.
□The effectiveness of a chromatographic column in separating
two solutes depends in part upon the relative rates at which the
two species are eluted.

□These rates are determined by the magnitude of the equilibrium

constants for the reactions by which the solutes distribute themselves
between the mobile and stationary phases.

□The distribution equilibria involved in chromatography involve the

transfer of an analyte between the mobile and stationary phases.
Amobile Astationary

□The equilibrium constant K for this reaction is called the distribution

constant, the partition ratio, or the partition coefficient,

K = cS/cM

where cs is the molar concentration of the solute in the stationary

phase and cM is its molar concentration in the mobile phase. K is
constant over a wide range of solute concentrations.
Capacity factor (k’): more universal measure of retention, determined from tR or VR.

k’ = (tR –tM)/tM

k’ = (VR –VM)/VM

□ capacity factor is useful for comparing results obtained on different

systems since it is independent on column length and flow-rate.

□ When the retention factor for a solute is much less than unity (less than 1),
elution occurs so rapidly that accurate determination of the retention times is
difficult and separation is poor. When the retention factor is larger than
perhaps 20 to 30, elution times become
inordinately long. Ideally, separations are performed under conditions in which
the retention factors for the solutes in a mixture lie in the range between 2
and 10 (separation is optimum).
The value of the capacity factor is useful in understanding the retention
mechanisms for a solute, since the fundamental definition of k’ is:
moles Astationary phase
k’ =
moles Amobile phase

k’ is directly related to the strength of the interaction between a solute with the stationary
and mobile phases.

Moles Astationary phase and moles Amobile phase represents the amount of solute present
in each phase at equilibrium.

Equilibrium is achieved or approached at the center of a chromatographic peak.

A simple example relating k’ to the interactions of a solute in a column is
illustrated for partition chromatography:

KD
A (mobile A (stationary phase)
phase)

where: KD = equilibrium constant for the distribution of A between

the mobile phase and stationary phase

Assuming local equilibrium at the center of the chromatographic peak:

[A]stationary phase Volumestationary phase

k’ =
[A]mobile phase Volumemobile phase

Volumestationary phase
k’ = KD
Volumemobile phase
As KD increases, interaction of the solute with the stationary phase becomes more
favorable and the solute’s retention (k’) increases
 Volumestationary phase
k’ = KD
Volumemobile phase

Separation between two solutes requires different KD’s

for their interactions with the mobile and stationary
phases.
3.) Efficiency:

Efficiency is related experimentally to a solute’s peak width.

- an efficient system will produce narrow peaks

Efficiency is related theoretically to the various kinetic processes that are

involved in solute retention and transport in the column
- determine the width or standard deviation (σ) of peaks

Wh

Dependent on the amount of time that a solute spends in the column (k’ or tR)
 Methods for Describing Column Efficiency

Two related terms are widely used as quantitative measures of chromatographic

column efficiency:
(1) plate height H and
(2) plate count plates (number of theoretical plates) N.
The two are related by the
equation N = L/H
where L is the length (usually in centimeters) of the column packing.

The efficiency of chromatographic columns increases as the plate count

becomes greater and as the plate height becomes smaller.

A chromatographic column is made up of numerous discrete but contiguous

narrow layers called theoretical plates. At each plate, equilibration of the solute
between the mobile and stationary phase was assumed to take place.
Movement of the solute down the column was then treated as a stepwise
transfer of equilibrated mobile phase from one plate to the next.
Number of theoretical plates (N): compare efficiencies of a system for solutes that
have different retention times

The larger the value of N is for a column, the better the column will be able
to separate two compounds.

- the better the ability to resolve solutes that have small differences in
retention
- N is independent of solute retention
- N is dependent on the length of the column

N can be calculated from two time measurements tR and Wb; to obtain H,

the length of the column packing L is usually known. The plate count
is then given by
N = 16 (tR/Wb)2
Another method for approximating N, is to determine Wh, the width of
peak at half its maximum height. The plate count is then given by
N = 5.54(tR/ W)h2
The plate count N and the plate height H are widely used in the
literature and by instrument manufactures as measures of column
performance.
Plate height or height equivalent of a theoretical plate (H or HETP): compare efficiencies of
columns with different lengths:

H = L/N

where: L = column length

N = number of theoretical plates for the column

Note: H simply gives the length of the column that corresponds to one theoretical
plate.

H can be also used to relate various chromatographic parameters (e.g., flow rate,
particle size, etc.) to the kinetic processes that give rise to peak broadening:

Why Do Bands Spread?

a. Eddy diffusion
b. Mobile phase mass transfer
c. Stagnant mobile phase mass transfer
d. Stationary phase mass transfer
e. Longitudinal diffusion
a.) Eddy diffusion – a process that leads to peak (band) broadening due to the
presence of multiple flow paths through a packed column.

As solute molecules travel through the

column, some arrive at the end sooner
then others simply due to the different path
traveled around the support particles in the
column that result in different travel
distances.

Longer path arrives at end of column after (1).

b.) Mobile phase mass transfer – a process of peak broadening caused by
the presence of different flow profile within channels
or
between particles of the support in the column.

A solute in the center of the

channel moves more quickly than
solute at the edges, it will tend to
reach the end of the channel first
leading to band-broadening

The degree of band-broadening due to eddy diffusion and mobile

phase mass transfer depends mainly on:

1) the size of the packing material

2) the diffusion rate of the solute
c.) Stagnant mobile phase mass transfer – band-broadening due to differences in the
rate of diffusion of the solute molecules between the
mobile phase outside the pores of the
support (flowing mobile phase) to the
mobile phase within the pores of the support (stagnant mobile
phase).

Since a solute does not travel

down the column when it is in
the stagnant mobile phase, it
spends a longer time in the
column than solute that remains
in the flowing mobile phase.

The degree of band-broadening due to stagnant mobile phase

mass transfer depends on:

1) the size, shape and pore structure of the packing material

2) the diffusion and retention of the solute
3) the flow-rate of the solute through the column
d.) Stationary phase mass transfer – band-broadening due to the movement of
solute between the stagnant phase and the stationary phase.

Since different solute molecules

spend different lengths of time
in the stationary phase, they
also spend different amounts of
time on the column, giving rise
to
band-broadening.

The degree of band-broadening due to stationary phase mass

transfer depends on:

1) the retention and diffusion of the solute

2) the flow-rate of the solute through the column
3) the kinetics of interaction between the solute
and the stationary phase
e.) Longitudinal diffusion – band-broadening due to the diffusion of the solute along
the length of the column in the flowing mobile phase.

The degree of band-broadening due

to longitudinal diffusion depends
on:

1) the diffusion of the solute

2) the flow-rate of the solute
through the column
Van Deemter equation: relates flow-rate or linear velocity to H:
H = A + B/μ + Cμ

where:

μ = linear velocity (flow-rate x Vm/L)

H = total plate height of the column
A = constant representing eddy diffusion &
mobile phase mass transfer
B = constant representing longitudinal diffusion
C = constant representing stagnant mobile
phase
& stationary phase mass transfer

One use of plate height (H) is to relate these kinetic process to band
broadening to a parameter of the chromatographic system (e.g., flow-rate).

This relationship is used to predict what the resulting effect would be of varying
this parameter on the overall efficiency of the chromatographic system.

Number of theoretical plates(N) (N) = 5.54 (tR/Wh)2 peak width (Wh)

H = L/N
Plot of van Deemter equation shows how H changes with the linear velocity
(flow-rate) of the mobile phase

μ optimum

Optimum linear velocity (μopt) - where H has a minimum value and the point
of maximum column efficiency.

μopt is easy to achieve for gas chromatography, but is usually too small
for liquid chromatography requiring flow-rates higher than optimal to
separate compounds
4.) Measures of Solute Separation:

separation factor (α) – parameter used to describe how well two solutes are
separated by a chromatographic system:

α = k’2/k’1 k’ = (tR –
tM)/tM where:
k’1 = the capacity factor of the first solute
k’2 = the capacity factor of the second solute,

A value of α 1.1 is usually indicative of a good separation

Does not consider the effect of column efficiency or peak widths, only retention.
 The separation factor α of a column for the two species A
and B is defined as
α = KB/KA

where KB is the distribution constant for species B

and KA is the distribution constant for species A.

When α is greater than unity, good separation will result.

α A relationship between the selectivity factor and retention

factors: α = k`B/k`A

Where k`B and k`A are the retention factors.

An expression for the determination of α from an

experimental chromatogram:
resolution (RS) – resolution between two peaks is a second measure of how
well two peaks are separated:
tr2 – tr1
RS =
(Wb2 + )/2
where: Wb1
tr1, Wb1 = retention time and baseline width
for the first eluting peak
tr2, Wb2 = retention time and baseline width for the
second eluting
peak

Rs is preferred over α since

both retention (tr) and column
efficiency (Wb) are considered
in defining peak separation.

Rs 1.5 represents baseline

resolution, or complete
separation
of two neighboring solutes □
ideal case.
Rs 1.0 considered
adequate for most
separations.
 APPLICATIONS OF CHROMATOGRAPHY

Chromatography has grown to be the premiere method for separating closely related
chemical species. In addition, it can be employed for qualitative identification and
quantitative determination of separated species.

Qualitative Analysis: A chromatogram provides only a single piece of qualitative

information about each species in a sample, namely, its retention time or its position on
the stationary phase after a certain elution period. It is a widely used tool for recognizing
the presence or absence of components of mixtures containing a limited number of
possible species whose identities are known.

Quantitative Analysis: Chromatography can provide useful quantitative information about

the separated species. Quantitative column chromatography is based upon a comparison
of either the height or the area of the analyte peak with that of one or more standards.

Analyses Based on Peak Height: The height of a chromatographic peak is obtained by

connecting the base lines on either side of the peak by a straight line and measuring he
perpendicular distance from this line to the peak. Accurate results are obtained with
peak heights only if variations in column conditions do not alter the peak widths.

Analyses Based on Peak Areas: Peak areas are a more satisfactory analytical variable than
peak heights. On the other hand, peak heights are more easily measured and, for narrow
peaks, more accurately determined. Most modern chromatographic instruments are
equipped with digital electronic integrators that permit precise estimation of peak areas.
 Calibration and Standards

The most straightforward method for quantitative

chromatographic analyses involves the preparation of a series
of standard solutions that approximate the composition of the
unknown. Chromatograms for the standards are then obtained
and peak heights or areas are plotted as a function of
concentration. A plot of the data should yield a straight line
passing through the origin.

The Internal Standard Method

The highest precision for quantitative chromatography is obtained

by use of internal standards because the uncertainties
introduced by sample injection are avoided. In this procedure,
a carefully measured quantity of an internal standard
substance is introduced into each standard and sample, and
the ratio of analyte to internal standard peak areas (or heights)
serves as the analytical parameter. For this method to be
successful, it is necessary that the internal standard peak be
well separated from the peaks of all other components of the
sample.