Atomic Absorption Spectrometer (AAS)

PerkinElmer Aanalyst 100

January 5th -6th, 2015

Instrument Center
Science and Technology Faculty
Prince of Songkla University
Pattani Campus
 Contents

¾ Schedule

¾ Trainee List

¾ Part 1 Introduction and Application of AAS

¾ Part 2 Using and Maintenance of AAS

(Perkins Elmer Aanalyst 100)

 Schedule of
“Using Atomic Absorption Spectrometer (AAS) Perkins Elmer Aanalyst 100” Training
January 5th -6th, 2015
At Instrument Center, 3th floor, Building 25
Faculty of Science and Technology, Prince of Songkla University
*******************************

January 5th, 2015

08.15 - 08.30 am Registration
08.30 - 08.45 am Inauguration
08.45 – 10.15 am Introduction of AAS
Assoc.Prof.Dr. Boonsong Krisornpornsan
10.15 – 10.30 am Coffee Break
10.30 – 12.00 am Application of AAS
Assoc.Prof.Dr. Boonsong Krisornpornsan
12.00 – 01.00 pm Launch
01.00 – 02.30 pm Using and Maintenance of AAS (Perkins Elmer Aanalyst 100)
Mr.Pisipong Meunprasertdee
02.30 – 02.45 pm Coffee Break
02.45 – 03.00 pm Workshop 2 groups
03.00 – 04.30 pm Workshop #1 Preparation of standard and sample for using AAS
(Perkins Elmer Aanalyst 100)

January 6th, 2015

08.45 – 10.15 am Workshop # 2 Using AAS (Perkins Elmer Aanalyst 100)
10.15 – 10.30 am Coffee break
10.30 – 12.00 am Workshop # 2 Using AAS (Perkins Elmer Aanalyst 100)
12.00 am – 01.00 pm Launch
01.00 – 03.30 pm Workshop # 3 Maintenance Perkins Elmer Aanalyst 100
03.30 – 03.45 pm Coffee break
03.45 – 04.35 pm Test AAS principle and using
***********************************
 Training of
“Using Atomic Absorption Spectrometer (AAS) Perkins Elmer Aanalyst 100” Training
January 5th -6th, 2015
At Instrument Center, 3th floor, Building 25
Faculty of Science and Technology, Prince of Songkla University

*******************************

No Name - Surname Year/Degree/Major Code Signature

1 Dr. Areefen Rassamesard Lecturer, Physics 1

2 Miss Nussaba Boonyasak 4th year, Bachelor's degree, Food Science and 2
Nutrition
3 Miss Pawwida Wongna 4th year, Bachelor's degree, Food Science and 1
Nutrition
4 Mr. Bashir Algaily 2nd year, Master's degree, Applied Physics 2

5 Mr. Atthapon Saman 5th year, Bachelor's degree, Agricultural 1

Technology
6 Ms. Trinh Ngoc Thao Ngan 2st year, Master's degree, Food Science and 2
Nutrition
7 Dr.Sunaree Bordeepong Lecturer, Physics 1

8 Ms.Haleemoh Weasamaae 4th year, Bachelor's degree, Fishery Technology 2

9 Mr. Roslan Chemae 4th year, Bachelor's degree, Fishery Technology 1

10 Mr.Viroj Pinprom Graduate, Bachelor's degree, Chemistry and 2

Biology
11 Mrs.Saijai Maneerat Graduate, Master's degree, Soil Management 1
 Part 1
Introduction and Application of AAS
 05/01/58

Atomic Absorption Spectrophotometry

(AAS)

By

Assist. Prof. Dr. Boonsong Krisornpornsan

ผศ. ดร. บุญสง ไกรศรพรสรร

1

History of optics and light studies

His experiment
- lens,
l mirrors
i etc.
t

His study
- Ray of light

Ibn Alhazen
965-1039
(Father of optics) 2

1
 05/01/58

Historical background of spectroscopy

The first to turn

hi ttelescope
his l tto
the heavens.

- Moon, sun spots

etc.

Galileo Galilei
(1564-1642) 3

A quantitative study of light

The first people

t study
to t d lilight
ht
scientifically.

- Prism
- Spectrum

Sir Isaac Newton

(1643-1727) 4

2
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Original studies of light

Newton’s contribution to spectroscopy

3
 05/01/58

Classification of electromagnetic radiation

Advancements in the study of light

-Fraunhofer lines in the

sun’s
sun s spectrum
- Developed a spactroscope
- Dratings

Joseph von Fraunhofer

(1787-1826)
German optician 8

4
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Color of light

-heat the element

Gustav Robert Kirchhoff Robert Wilhelm Eberhard

(1824-1887) Bunsen
German physicist (1811-1899)
German chemist
9

Emission spectra complement absorption spectra

10

5
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Hydrogen line spectrum

11

Scanning spectrophotometer (side view)

12

6
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Scanning spectrophotometer (top view)

13

Computer output from scanning spectrophotometer

14

7
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Hydrogen spectrum – The Balmer series

Wavelength color
( )
(nm)
656.2 red
486.1 blue
434.0 blue-violet
410 1
410.1 violet

Johann Jakob Balmer

(1825-1898)
15

Quantum properties of light

ΔE = nhν

ΔE – the change
in Energy

n= 1, 2, 3, …

h – (Planck’s
constant)

h = 6.626×10-34 Js

ν - frequency

Max Karl Ernst Ludwig Planck

(1858-1947)
German physicist
16

8
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Quantum model for the Hydrogen atom

Neils Henrik David Bohr

(1885-1962)
Danish physicist 17

Bohr’s Model

N l
Nucleus

Electron

Orbit

Energy Levels
18

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Excitation

+ energy
Excited electron

+E
Mo M*

19

Decay and Emission

Back to ground state

- E light

+E -E
Mo M* Mo
20

10
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Origins of atomic spectra

li ht
light
excited states

λ3

λ2 λ1

∆E = hƲ

21

Atomic Spectra

Every chemical element

has own unit spectral
fi
fingerprint.
i t

Emission spectrum of
hydrogen

Absorption spectrum of
hydrogen 22

11
 05/01/58

Emission spectroscopy

Electrons jjump
p
from higher levels
to lower ones.

Energy is released or
emitted in the form of
light
g of specific
p energy
gy
(or color).

Electrons must first be excited up to higher levels. This is

done either with heat (eg. Flame tests) or light (atomic
emission spectra).
23

Absorption spectroscopy

Electrons jump
from lower levels to
higher ones.
Energy is absorbed in
the form of light of
specific
p energy
gy ((or
color).

This is the method used in UV-Vis spectroscopy and

atomic absorption spectroscopy (AAS) and many others.

24

12
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A spectrum

Emission lines Absorption

p lines
C ti
Continuum

A spectrum = the amount of light given off by

an object at a range of wavelengths.
25

The colors come from the different energies,

frequencies
q and wavelengths
g of light.
g

They are properties of light and all interconnected.

26

13
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Calculations for energy levels

Z2
E = −2.178 × 10 −18 J ( )
n2

When Z = atomic number

of element
n = quantum
t number
b
& the units are Joules

27

Calculations the Balmer & Lyman series

Rydberg equation

1 1
ν = R( − )
n12 n22

When Ʋ = frequency
n = quantum number
R=R Rydberg
db constant
t t
= 3.29 x 10 Hz
15

1 Hz = 1 s-1

28

14
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Frequency (ν) V.S Wavelength (λ)

λν=c
C = speed of light
= 2.9979 X 108 m/s

29

Violet light Red light

has the has the
shortest longest
wavelength wavelength
of visible of visible
light and has
light, light This
light.
the highest also means
energy. it has the
lowest
frequency
1.2

and lowest
1.0
bsorbance

0.8
energy.
0.6
Ab

04
0.4

0.2

0.0
400 440 480 520 560 600 640 680 720 760

Wavelengths (nm)

The maximum absorption occurs 30

15
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How are color and light absorption are related?

31

Just as light can be split into its various colors, so

these colors can be recombined to form white light.

Mixing all these colored lights would give us white,

but we can also use combinations of fewer colors to
get white.

32

16
 05/01/58

We can see that red, blue and green light add together
to make white light. But where green overlaps
magenta, white light is also formed. Same with red and
cyan. And with yellow and blue.

We call these colors ‘complementary’

33

Complementary colors are on the opposite sides of the

color wheel.

They help us understand the colors we see when light

is absorbed. 34

17
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If we take blue light away from white light (absorb

blue), what color remains?

35

The absorption spectra of individual atoms

have very distinct lines showing which
energies of light have been absorbed as the
electrons jump to higher levels.

36

18
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The absorption spectra of compounds

tends to have broad overlapping regions
for all the possible electron jumps.

37

This is the type of absorption spectra which is

obtained using a using a UV-Vis spectrophotometer.

The position of the peak shows the wavelength of the

light most strongly absorbed and what % is absorbed.
38

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This substance is absorbing the red/orange light

(~600 nm) and transmitting the rest which our eye
mixes and interprets as blue/green.

39

White light shining on a white object.

All light is reflected.

40

20
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White light shining on a red object.

Red light is reflected- other colors absorbed

(particularly green and similar colors).
41

This is a more realistic view of this. The reflected

colors combine and are mixed in the brain to give
the overall red color.

42

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White light shining on a blue object.

43

44

22
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White light shining on a black object.

All light is absorbed. Black is the absence of

light.

White light shining on

a grey object. A
small amount of each
color is reflected.
45

Atomic Spectroscopy

Emission
Mo + heat M* Mo + light

Absorption
Mo + light M*

46

23
 05/01/58

Atomic Spectroscopy (AS)

1. Atomic Absorption Spectroscopy (AAS)

2. Flame Atomic Emission Spectroscopy (FAES)

3. Inductively Coupled Plasma Atomic Emission

Spectroscopy (ICP)

47

Basic principle

AAS is an analytical technique that measures

the concentrations of elements. It makes use
of the absorption of light by these elements in
order to measure their concentration.

-จํานวนหรือปริมาณของ unexcited atoms

ที่จะ absorb แสง ซึง่ emit มาจาก excited
atoms ของ element ชนิดเดียวกัน
48

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 05/01/58

- AAS quantifies the absorption of ground state

atoms in the gaseous state.
- The atoms absorb ultraviolet or visible light and
make transitions to higher electronic energy
levels. The analyte concentration is determined
from the amount of absorption.
- Concentration measurements are usually
determined from a working curve after calibrating
the instrument with standards of known
concentration.
concentration
- Atomic absorption is a very common technique
for detecting metals and metalloids in a samples.

49

detectable

50

25
 05/01/58

AAS

AAS have 4 p
principal
p components
p

1. A light source (Continuum and line source)

(usually a Hallow Cathode Lamp (HCL))

2. An atom cell (atomizer)

3. A monochromator

4. A detector, and read out device

51

Schematic Diagram of an AAS

Light source Atomizer Monochromator Detector and

readout device

52

26
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53

Quartz window HCL

HCL contains a
tungsten anode and a
Pyrex body hollow cylindrical
cathode made of the
element to be
determined. These are
sealed in a glass tube
filled with an inert gas
(Ne or Ar).

54

27
 05/01/58

How HCL works?

Applying a potential difference between the anode and the
cathode leads to the ionzation of some gas atoms (Ne/Ar).
These gaseous ions bombard the cathode and eject metal atoms
from the cathode in a process called sputtering. Some sputtered
atoms are in excited states and emit radiation characteristic of the
metal as they fall back to the ground state.

The shape of the cathode which is hollow cylindrical concentrates

the emitted radiation into a beam which passes through a quartz
window all the way to the vaporized sample.

Since atoms of different elements absorb characteristic

wavelengths of light. Analyzing a sample to see if contains a
55
particular element means using light that element.

HCL emission process

1. Ionization 2. Sputtering

+ + Mo
- Neo Ne+ - Ne+

3. Excitation 4. Emission
+ M*
M + M*
- Mo
Ne+
- Mo light

56

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For example with sodium,

A lamp containing sodium emits light from
excited sodium atoms that produce the light mix
of wavelengths to be absorbed by any sodium
atoms from the sample.

A beam of the electromagnetic radiation

emitted from excited sodium atoms is passed
through the vaporized sample. Some of the
radiation is absorbed by the sodium atoms in
the sample. The greater the number of atoms
there is in the vapor, the more radiation is
absorbed.
57

Considerations

1. Type of HCL

2. Position of HCL

3. Current adjust of HCL

58

29
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Atomizer

Elements to be analyzed needs to be in

atomic sate.

Atomization is separation of particles into

individual molecules and breaking molecules
into atoms. This is done by exposing the
analyze to high temperatures in a flame or
graphite furnace
furnace.

59

The role of the atom cell is to primarily

dissolved a liquid sample and then the solid
particles are vaporized into their free gaseous
ground state form. In this form atoms will be
available to absorb radiation emitted from the
light source and thus generate a measurable
signal proportional to concentration.

There are two types of atomization: Flame and

Graphite furnace atomization.

60

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61

Flame

Flame AA can only analyze solutions, Where it

uses a slot type burner to increase the path
l
length,
th and
d th
therefore
f tto iincrease th
the ttotal
t l
absorbance

Sample solutions are usually introduced into a

nebulizer the sample is dispersed into tiny
droplets which can be readily broken down in
droplets,
the flame.

62

31
 05/01/58

Flame Atomizers

p p techniques
1. Used in all atomic spectroscopic q

2. Converts analyze into free atoms in the form

of vapor phase free atoms

3. Heat is required

4. Routes for sample introduction

63

Various flame atomization techniques 64

32
 05/01/58

Types of flames used in Atomic spectroscopy

Fuel and Oxidant Temperature, oC

Gas/air 1700-1900
Gas/O2 2700-2800
H2/air 2000-2100
H2/O2 2550-2700
Acetylene/air 2100-2400
2100 2400
Acetylene/O2 3050-3150
Acetylene/N2O 2600-2800

65

Processes that take place in flame

Solution of
analyze
Nebulization
Spray
Desolvation
Solid/gas
aerosol
Volatilization Excited hƲ molecular
molecules
Gaseous
molecules
Dissociation Excited atoms hƲ atomic
Atoms
Ionization Excited ions hƲ ionic
Atomic ions

66

33
 05/01/58

Effect of flame temperature on excited state population

E* , g * = 3
Excited state

Absorption
n
Emission
∆E
Eo , g o = 2
ground state
Energy difference
Atoms in excited state
Temperature (K)
N* = g*e-∆E/kT
Atoms in ground state No g o Boltzmann
constant
Statistical factor (1.381x10-23)
67

The lowest excited state of a sodium atom lies

3.371X10-19 J/atom above the ground state.
The degeneracy of the excited state is 2, while
that of the ground state is 1.
1 Let
Let’s
s calculate the
fraction of sodium atoms in the excited state in
an acetylene-air flame at 600 K.
N* = g*e-∆E/kT
No g o
N* = 2e-3.371x10-19/(1.381x10-23*2600)
No 1
= 1.67x10-4

That is, less than 0.02 % of the atoms are in the excited state. 68

34
 05/01/58

Thus 99.996
99 996 % of Na atoms are in the ground state
state.

Atomic emission uses excited atoms.

Atomic absorption uses ground state atoms.

69

How would the fraction of atoms in the

excited state change if the temperature were
2 610 K instead?

N* = 2e-3.371x10-19/(1.381x10-23*2610)
No 1
= 1.74x10-4

The fraction of atoms in the excited state is

still less than 0.02 %, but that fraction has
changed by 100*(1.74-1.67)/1.67 = 4 %
70

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Ratio of excited- to ground-state atoms for lines with

varying excitation energies at increasing temperature

element Line g*/go E (J) Ratio of excited- to ground-state atoms

(nm)
2000 K 4000 K 6000 K 8000 K
Cs 852.1 2 2.34x10-19 4.19x10-4 2.90x10-2 1.19x10-1 2.41x10-1

Na 589.1 2 3.38x10-19 9.65x10-8 4.39x10-3 3.38x10-2 9.37x10-2

Ca 422.7 3 4.66x10-19 1.40x10-7 6.47x10-4 1.08x10-2 4.41x10-2

Zn 213.9 3 9.13x10-18 1.30x10-14 1.98x10-7 4.90x10-5 7.70x10-4

71

Atomization devices

Atomization:
1. A process of forming free atoms by heat
2 Atomizers
2. At i are ddevices
i th
thatt carry outt atomization:
t i ti

2.1 Continuous (constant temperature with time)

-Flame
-Plasma

2.2 Non-continuous (temperature varies with time)

-Electrothermal
-Spark discharge
72

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Sample introduction systems

In continuous atomizers sample is constantly

introduced in form of droplets, dry aerosol, vapor.

Nebulizer: A device for converting the solution into

fine spray or droplets

Continuous sample introduction is used with

continuous nebulizers in which a steady state
atomic population is produced. Sample is
introduced in fixed or discrete amounts.

73

Discontinuous samplers are used with continuous

atomizers

1. Discrete samples are introduced into atomizers in many

ways:

Electrothermal atomizers
A syringe is used .
A transient signal is produced as temperature changes
with time and sample is consumed.

2. Indirect insertion (probe)

2
Sample is introduced into a probe (carbon rod) and
mechanically moved into the atomization region vapor
cloud is transient because sample introduced is limited.

74

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3. Flow injection
The analyte is introduced into the carrier stream into a
nebulizer as mist

4. Hydride generation
The volatile sample is stripped from the analyte solution
and carried out by a gas into the atomizer. This strip is
followed by chemically converting the analyte to hydride
vapor form.

5. With Arc spark

Solids are employed.

6. Laser microbe technique

A beam of laser is directed onto a small solid sample, gets
vaporized, atomized by relative heating, Either sample is
probed by encoding system or vapor produced is swept into
a second absorption or fluorescence. 75

Nebulization gas is always compressed, usually

acts as the oxidant, it is oxygen in flame and argon
in plasma

Nebulization chambers p produce smaller droplets

p
and remove or drain larger droplets called aerosol
modifiers.

Aspiration rate is proportional to compressed gas

pressure. The pressure drops through capillary, here
¼ capillary
ill di
diameters
t are recommended.d d Thi
This iis
inversely proportional to viscosity of the solution.

Peristaltic and/or syringe pumps could be used.

76

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Oxidant and fuel are usually brought into

nebulization chamber through a separate port.
They mix and pass the burner head called
premixed burner system.

Add organic solvents to reduce the size of the

drop.

77

The atomic absorption spectrometer

Sample Introduction System

Nubulizer

Capillary

Solution
78

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The fine mist of droplets is mixed with fuel

(acetylene), and oxidant (air or nitrous oxide)
and burned.

The flame temperature is important because it

influences the distribution of atoms. It can be
manipulated by oxidant and fuel ratio
ratio.

79

Monochromators

This is a very important part in an AAS. It is used to

separate out all of the thousands of lines. Without a
good monochromator, detection limits are severely
compromised.

A monochomator is used to select the specific

wavelength of light which is absorbed by the sample,
and to exclude other wavelengths
wavelengths. The selection of
the specific light allows the determination of the
selected element in the presence of others.

80

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Detector and read out device

The light selected by the monochromator is directed

onto a detector that is typically a photomultiplier
tube, whose function is to convert the light signal into
an electrical signal proportional to the light intensity.

The processing of electrical signal is fulfilled by a

signal amplifier. The signal could be displayed for
readout or further fed into a data station for printout
readout,
by the requested format.

81

Calibration curve

A calibration curve is used to determine the

unknown concentration of an element in a solution.
The instrument is calibrated using several solutions
of known concentrations. The absorbance of each
known solution is measured and then a calibration
curve of concentration vs absorbance is plotted.

The sample solution is fed into the instrument

instrument, and
the absorbance of the element in this solution is
measured. The unknown concentration of the
element is then calculated from the calibration curve.
82

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Absorbance V.S Concentration

83

Interferences

The concentration of the analyte element is

considered to be proportional to the ground state
atom
t population
l ti iin th
the fl
flame, any ffactor
t ththatt affects
ff t
the ground state atom population can be classified
as an interference.

Factors that may affect the ability of the instrument

to read this parameter can also be classified as an
interference.

84

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The different interferences that are encountered in

atomic absorption spectroscopy are:

1. Absorption of source radiation: Element other than the one of interest

may absorb the wavelength being used.

2. Ionization interference: the formation of ions rather than atoms causes

lower absorption of radiation. This problem is overcome by adding
ionization suppressors.

3. Self absorption: the atoms of the same kind that are absorbing
radiation will absorb more at the center of the line than at the wings, and
thus resulting in the change of shape of the line as well as its intensity.

4. Background absorption of source radiation: This is caused by the

presence of a particle from incomplete atomization. This problem is
overcome by increasing the flame temperature.

5. Transport interference: rate of aspiration, nebulization, or transport of

the sample (e.g viscosity, surface tension, vapor pressure, and density)85

Atomic emission spectroscopy

Atomic emission spectroscopy is also an analytical technique

that is used to measure the concentrations of elements in
samples.
p

It uses quantitative measurement of the emission from

excited atoms to determine analyte concentration.

The analyte atoms are promoted to a higher energy level by

the sufficient energy that is provided by the high temperature
of the atomization sources.

The excited atoms decay back to lower levels by emitting

light . Emission are passed through monochromators or filters
prior to detection by photomultiplier tube.
86

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The instrumentation of atomic emission

spectroscopy is the same as that of atomic
absorption but without the presence of a radiation
source.

In atomic emission the sample is atomized and

the analyte atoms are excited to higher energy
levels all in the atomizer.

87

Schematic diagram of an atomic emission spectrometer

88

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The source of energy in atomic emission could

be a flame like the one used in atomic absorption,
or an ICP.

1. The flame (1700-3150 oC) is most useful for

elements with relatively low excitation energies like
sodium, potassium and calcium.

2. The flame (6000-8000 oC) has a very high

temperature and is useful for elements of high
excitation energies.

89

Comparison between atomic absorption and

emission spectroscopy

absorption emission
1. Measure trace metal 1. Measure trace metal
concentrations in complex matrices. concentrations in complex matrices.
2. Atomic absorption depends upon 2. Atomic emission depends upon
the number of ground state atoms. the number of excited state atoms.
3. It measures the radiation 3. It measures the radiation emitted
absorbed by the ground state by the excited state atoms.
atoms.
4. Presence of a light source (HCL). 4. Absence of the light source.
5. The temperature in the atomizer 5. The temperature in the atomizer
is adjusted to atomize the analyte is big enough to atomize the analyte
atoms in the ground state only. atoms and excite them to a higher
energy level.
90

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AAS applications

They are many applications for atomic absorption:

1 Clinical analysis: Analyzing metals in biological fluids such as blood
1.
and urine.

2. Environmental analysis: Monitoring our environment: e.g finding out

the levels of various elements in rivers, seawater, drinking water,
air, soil and petrol.

3. Pharmaceuticals

4. Industry

5. Mining

6. Food, Cosmetics, Hair, Plant, Animal etc.

91

The use of AAS

1. The type and current of HCL

2 The wavelength for analysis
2.
3. The flame
4. Chemical treatment
5. Interferences

92

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Detection limit (DL)

3X

noise
-Standardization by IUPAC
-DL = Conc. Producing signal that is 3 times
noise level
-DL
DL = 3x
3 stdtd d
dev off bl
blank
k
-Defines signal to noise ratio
-Provides information on:
1. Total instrument performance
2. Lower limit of concentration measurement 93

47