Introduction to

Spectroscopy (AAS)

MED2105
1
2 Today’s lecture

Definition of Spectroscopy
Basic principles of Spectroscopy

Uses of Atomic Absorption Spectroscopy

Instrumentation of Atomic Absorption
Spectroscopy

Standard addition method

External calibration method
3 Introduction

Light
Electromagnetic radiation
Wavelength: the distance in a wave
where the shape of the wave repeats and
is usually determined by the distance
between corresponding points of the
same phase
4 Introduction

The Electromagnetic Spectrum

Full range of electromagnetic radiation
wavelengths
Visible light has wavelengths ranging from 400 nm
(_________) to 780 nm (_______)
___________is a combination of all the colors of
light. If we pass it through a clear glass prism, it
splits up to form the different visible colors of light
5 Introduction

The Electromagnetic Spectrum

Visible spectrum Infrared
Ultraviolet Microwaves
X-Rays Radio waves
Gamma Rays
6 Introduction

Why each of them have different properties?

∵ differences in λ and thus energy

E is the energy of the light (unit: kcal/mol)

h is the Planck’s constant
c is the speed of light (what is the value?)
λ is the wavelength in nanometers
Energy of a photon is _______________to its
wavelength. The shorter the wavelength, the
__________the energy of the photon.
7 Introduction

Frequency = number of times per second

that a crest passes a given point
What is the unit?
∵ λv = c where v is frequency
The equation may be rewritten as:
E=h∙ν
Longer waves have ________frequencies,
shorter waves have ________frequencies.
As the frequency increases, the energy of
the light ________.
8 Spectroscopy

∵ Atoms and molecules interact with light

Spectroscopy
The study of interaction between matter
(i.e., molecules, atoms, nuclei) and
electromagnetic radiation (i.e., photons)
Provide detailed information on their
structure, composition and interactions
Summary: absorption of a photon's energy
by ________, and the emission of photons by
_________ as a molecule relaxes
9 Spectroscopy

Remember all electrons

have a series of energy
levels they can
occupy?
The lowest energy level
is the “___________."
In order for an electron
to move from one level
to a higher level it must
absorb energy equal to
the difference in the
levels.
10 Spectroscopy

An electron can absorb a

photon of light that strikes it
only if that photon has the
exact energy to change the
electron to a higher allowed
energy level.

An electron already at a
higher level can emit a
photon of light having exact
energy to change that
electron to one of its lower
allowed levels.
11 Spectroscopy

Light interacts with matter in two ways:

a)Absorption
The most common type
Occurs when the incident
electromagnetic radiation is
completely absorbed by atoms and
molecules in sample
b) Emission
Matter itself emits electromagnetic
radiation that is triggered by an
external source of energy such as
flames or even electromagnetic
radiation of higher energy
12 Spectroscopy

Absorption spectrum
Study of light absorbed by molecules
When white light is passed through a sample,
under the right conditions, the electrons of the
sample absorb some wavelengths of light.
The light coming out of the sample will be missing
those wavelengths corresponding to the energy
levels of the electrons in the sample.
Spectrum with black lines
13 Spectroscopy

Emission spectrum
The electrons of the sample are promoted to
very high energy levels by some methods
(examples?)
As these electrons return to lower levels, they
emit light.
14 Spectroscopy

a) Qualitative Spectroscopy
The spectrum of a chemical species is unique
to that species
Identify chemical species by measuring a
spectrum and comparing it with spectra for
known chemical species to find a match.
b) Quantitative Spectroscopy
At any given temperature, the same number
of photons will always be absorbed or emitted
by the same number of atoms or molecules in
a given period of time (which law?)
Provide a direct measure of the number of
atoms or molecules present in a sample
15 Spectroscopy
 Atomic absorption
16
spectrometry (AAS)
17 Atomic absorption
spectrometry (AAS)
Different areas of chemistry
Forensic science
Environmental science
Food technology
Pharmaceuticals
Agriculture
Pathology
Industry
Mining
18 Atomic absorption
spectrometry (AAS)
Absorption in Forensic science
Determination of trace elements
Elemental profiles of biological samples
Trace elements in artificial fibers
Determination of poisoning
Hair analysis for heavy metals poisons
Determinations of ammunition
manufacturers
Discrimination of objects or elements
19 Atomic absorption
spectrometry (AAS)
Hallow cathode Monochromator
lamp Detector
Atomizer
Sample
20 Atomic absorption
spectrometry (AAS)
Radiation source: hollow cathode lamp
Contain the element to be determined
Sealed in a glass tube filled with an inert
gas, e.g., neon or argon
21 Atomic absorption
spectrometry (AAS)
Atoms of the metal to be tested are present
within the lamp, and when the lamp is on,
these atoms are supplied with energy, which
causes them to elevate to the excited states.
Upon returning to the ground state, exactly
the same wavelengths of light that are useful
in the analysis are emitted.

Pb * → Pb + hv
22 Atomic absorption
spectrometry (AAS)
Differences?
Atomisation of the sample
Create a supply of free analyte atoms in the
ground state and exposing this atom
population to light of the characteristic
wavelength for that element
1) Flame Atomization
Suck a solution of the sample into a flame
2) Graphite Furnace Atomization
Electrothermal atomisation is where a
drop of sample is placed into a graphite
tube that is then heated electrically
23 Atomic absorption
spectrometry (AAS)
Atomisation of the sample

If you have an aqueous solution of

CuCl2, after atomization, what will you
get?

_____________________________________
24 Atomic absorption
spectrometry (AAS)
Flame atomizer
simplicity, low cost
a flame that has enough energy to both
volatilize and atomize the sample
25 Atomic absorption
spectrometry (AAS)
Graphite Furnace Atomization
Heat the hollow graphite tube by passing a
controlled strong electric current in a
programmed series of steps
1) remove the solvent and major matrix
components
2) atomize the sample to generate the
ground state atoms
26 Atomic absorption
spectrometry (AAS)
A typical graphite furnace program
consists of three stages:

1)Drying
80 – 200 °C
Once the sample has been injected
into the graphite tube, it is dried to a
solid residue
The solvent is evaporated
27 Atomic absorption
spectrometry (AAS)
A typical graphite furnace program
consists of three stages:

2) Ashing or Charring
350 –1600 –C
Any organic material in the sample is
converted to CO2 and H2O, and
volatile inorganic materials are
vaporized
Analyte is remained in the sample
28 Atomic absorption
spectrometry (AAS)
A typical graphite furnace program
consists of three stages:
3) Atomization
2000 – 3000 °C
Furnace is rapidly heated to a high
temperature to vaporize the residues
from the ashing stage
Creates a cloud of free atoms in the
optical path
The absorbance is measured during
this stage
29 Atomic absorption
spectrometry (AAS) Differences

https://www.who.int/ipcs/assessment/public_health/lead_blood.pdf
30 Atomic absorption
spectrometry (AAS)

Monochromator
Select the specific wavelength of light
which is absorbed by the sample and to
exclude other wavelengths

Detector
Measure the intensity of the light beam
Record the reduction as absorption
Show on output device
31 Atomic absorption
spectrometry (AAS)

Interferences can be negative

(sample absorbs less than it should)
or positive (sample absorbs more
than it should)
Can be caused by contaminants
within the sample that absorb at
the same wavelength as the
analyte, and thus can cause
inaccurate measurements
32 Atomic absorption
spectrometry (AAS)
Interferences examples
Ionization: atoms are ionized at the
temperature of the flame/furnace,
which decreases the amount of free
atoms
Incomplete atomization
Oxide formation
Anion interference
Cross contamination and contamination
of the sample
33 Atomic absorption
spectrometry (AAS)
Sample preparation
Consider laboratory environment, the vessel
holding the sample, storage of the sample,
and pretreatment of the sample
Clean environment: clean rooms, and closed,
clean vessels for transportation of the sample
Conserved in terms of pH, constituents, and
any other properties that could alter the
contents
The material of the vessel walls can adsorb
some of the analyte leading to poor results
Acidifying the solution with nitric acid
34 Atomic absorption spectrometry
(AAS) Summary

Principles:
Atoms of different elements absorb
characteristic wavelengths of light
Use light from that particular element
that you are investigating
E.g., a lamp containing lead emits light
from excited lead atoms that produce
the right mix of wavelengths to be
absorbed by any lead atoms from the
sample
35 Atomic absorption spectrometry
(AAS) Summary

Principles:
Sample is atomized
Atomized: converted into ground state
free atoms in the vapour state
A beam of electromagnetic radiation
emitted from excited lead atoms is
passed through the vaporized sample.
Some of the radiation is absorbed by
the lead atoms in the unknown sample
36 Atomic absorption
spectrometry (AAS)

Principles:
The amount of light absorbed is
proportional to the number of lead
atoms in the unknown sample
Construct a calibration curve by running
several samples of known lead
concentrations under the same
conditions as the unknown sample

Analyze the concentrations of lead in all the

standard solutions and unknown sample(s)
37 Exercises

What
method
is this?
38 Exercises
 External calibration method
39
Standard addition method
40 Purposes of two methods

A quantitative analysis approach often used

in analytical chemistry
Find out the amount / concentration of a
particular substance (analyte) in a matrix
Important in fundamental research and in
many applied fields of study
Calibration curve: a graph showing the
analytical response as a function of the known
quantity of analyte
Use it to interpret response for __________
quantities
41 1) External calibration method

Multiple-point external standardization

Volumetric flask of a stock solution

Calculations are performed using the dilution

equation: C1V1 = C2V2
A series of solutions are prepared in which each
successive solution is more dilutethan the
previous
42 1) External calibration method

Volumetric flasks with increasing concentrations

of Cu²+ (external standards)

Each standard and sample, along with a blank,

is run on the gas chromatograph/AAS/HPLC-MS
Signal (peak area/absorbance…) is recorded
for each concentration of standard, sample
and blank
43 1) External calibration method

The general steps involved in using standard

analyte concentration and detector
response data to calculate an unknown
analyte concentration:

1) Create a scatter plot of the data

2) Create a linear regression line (trend line)
3) Obtain a regression equation and R-
squared value
4) Rearrange the regression equation to
calculate the unknown analyte
concentration
44 1) External calibration method
Example #1 : straight line (the method’s
sensitivity remains constant) (y = mx)

Example #2: A spectrophotometric method for

the quantitative analysis of Pb (II) ions in blood
has a normal calibration curve of

y= mx + b
45 1) External calibration method

Advantages:
Analyze a series of samples using a single
calibration curve. This is important advantage
when we have many samples to analyze
Simple to perform
Quick

Limitation:
Matrix effects
46 Example of external calibration
method Exercises
A student prepared standard lead
solutions for comparison and the
absorbance of each solution was
measured. A road-side soil sample
was also prepared. 10 mL of this
prepared soil sample was placed
in a 100 mL volumetric flask and
enough water was added to
make it up to the mark. The
absorbance of this diluted soil
sample was recorded. He plotted
the calibration curve. Find the
concentration of lead in the 10 mL
undiluted sample in mg/L.
47 Example of external calibration
method
48 2) Standard addition method

Highly recommended to compensate for

matrix interferences in the analysis
Complex matrix samples such as
biological fluids, soil samples….
Why we need to pay attention to matrix?
The matrix may contain other
components that interfere with the
analyte signal causing inaccuracy in
the determined concentration
Aliquots of standard (spiking) are added
to unknown samples
49 2) Standard addition method

1) Solutions of known concentration are

prepared (standards).
2) To each of those standards a known and
same amount of the sample is added before
completing volume with the solvent.
50 2) Standard addition method
3) Instrumental signal of the solutions (including
blank)are recorded and create a calibration curve.

4) Sample concentration by extrapolation of the linear

equation to y = 0.
51 2) Standard addition method
Example #1: Finding amount of riboflavin in soy milk
52 2) Standard addition method
You calculate this!

Example #2: You measure this!

A plot of the signal intensities of the

solutions vs. the added concentrations
yields a straight line.
53 2) Standard addition method

Example:

The concentration of the analyte in

unknown sample is determined from the
point at which the extrapolated line crosses
the concentration axis at zero signal.
54 2) Standard addition method