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Lab-Training.com

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By

Dr. Saurabh Arora

Founder : Lab-Training.com

Director : Auriga Research Ltd.

E-mail : saurabharora@arbropharma.com

Dr. Deepak Bhanot

Vice President : Training & Development

E-mail : deepak.bhanot@aurigaresearch.com

Auriga Research Ltd.

Division of

Arbro Pharmaceuticals Ltd.

Analytical Division,

4/9 Kirti Nagar Industrial Area, New Delhi - 110015 (INDIA)

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Author’s Profile

Dr Saurabh Arora is a trained pharmacist with Master’s and Doctorate degrees in

pharmaceutics from reputed Indian institutes NPIER and Jamia Hamdard University

respectively.

Managing Director

Auriga Research Ltd.

He has setup 2 contract laboratories and a clinical research company along with managing and

growing the existing business. His organization has grown multiple folds and he hasbeen

fortunate to spearhead the growth initiatives backed by a team of over 250 employees..

Specialties: Formulation Development, Analytical Development, Chromatography, Mass

spectroscopy, GMP. GLP, GCP, Laboratory designing, Residue analysis, Project management,

International business and all that goes into growing and managing a business

Founder

Lab-Training.Com

Lab-Training.Com is developing and offering a series of free and paid E-Learning courses on

various analytical and laboratory techniques. He is responsible for the course concepts, course

content creation and review and course execution.

Founder

Food Safety Helpline

Food Safety Helpline has been established to help Food Business Operators implement the Food

Safety and Standards Act

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Dr Deepak Bhanot is a seasoned professional having nearly 30 years expertise beginning from

sales and product support of analytical instruments. After completing his graduation and post

graduation from Delhi University and IIT Delhi he went on to Loughborough University of

Technology, UK for doctorate research in analytical chemistry. His mission is to develop

training programs on analytical techniques and share his experiences with broad spectrum of

users ranging from professionals engaged in analytical development and research as well as

young enthusiasts fresh from academics who wish to embark upon a career in analytical industry.

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Lab-Training.com

� Knowledge grows when shared with others. Our belief in this has contributed immensely

towards growth of our web based portal for sharing our expertise and skills.

� Knowledge does not discriminate between national boundaries color of skin, religion,

caste, gender and creed.

Our world class infrastructure, manpower skills and over 25 years of experience is now

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Our e-learning courses, articles and certificate programmes have been appreciated by industries,

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demands for courses and articles on techniques of analytical interest and improvement of

laboratory activities. We are bound to upgrade our content keeping the needs of our clients and

followers in mind. It will be our endeavor to provide leadership in this key area of development.

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Table of Contents

Introduction to Atomic Absorption Spectroscopy course ............................................................................. 8

Scope of Spectroscopic Analysis ................................................................................................................ 10

Advantages of spectroscopic techniques ............................................................................................. 10

Application areas of spectroscopic analysis ........................................................................................ 11

Types of spectroscopic analysis .......................................................................................................... 11

Evolution of Atomic Absorption Spectroscopy .......................................................................................... 13

Introduction to AAS component parts ........................................................................................................ 16

Burner system........................................................................................................................................ 17

Monochromator .................................................................................................................................... 17

Detector .................................................................................................................................................. 17

Double Beam Schematic ....................................................................................................................... 18

Types of Light Sources in AAS .................................................................................................................. 19

Hollow Cathode Lamps ........................................................................................................................... 19

Multi element Hollow Cathode Lamps ................................................................................................... 20

Limitations of Hollow Cathode Lamps .................................................................................................... 20

Electrodeless Discharge Lamps ............................................................................................................... 21

Flame Atomic Absorption Spectroscopy .................................................................................................... 22

Nebuliser ................................................................................................................................................ 22

Spray Chamber ..................................................................................................................................... 23

Burner Head .......................................................................................................................................... 24

Graphite Furnace Atomic Absorption Spectroscopy .................................................................................. 25

Limitations of flame AAS ......................................................................................................................... 25

Benefits of graphite furnace analysis ...................................................................................................... 25

Graphite furnace components ................................................................................................................ 26

Limitations of graphite furnace analysis ................................................................................................. 27

Dispersion and Resolution of Light in Atomic Absorption Spectroscopy .................................................. 28

Interferences in Atomic Absorption Spectroscopy ..................................................................................... 31

Non-spectral interferences ................................................................................................................... 31

Spectral Interferences ........................................................................................................................... 32

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Background correction in Atomic Absorption Spectroscopy ..................................................................... 34

Background Correction ........................................................................................................................... 34

Zeeman Background Correction ............................................................................................................. 36

10 Interview questions in Atomic Absorption Spectroscopy ..................................................................... 38

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Introduction to Atomic Absorption Spectroscopy

course

It is possible to fly without motors, but not without knowledge and skill”

– Wilbur Wright

The overwhelming response to the free e-book HPLC and GC courses has encouraged us to

move ahead with the free AAS course. We understand that everyone has busy work schedules

and today's hectic life style leaves you little or no time to refer to voluminous books to learn any

new technique. However, for sustained growth learning has to be adopted as a lifelong habit.

In an effort to make your learning task easy we have embarked upon the e-book which comprises

of 10 Chapters. Each Chapter comprising of about 300 to 400 words will provide functional

aspects of AAS and also present useful practical tips. Reading a Chapter and understanding it

will not take more than about 10 min and you'll get ample time to assimilate the contents before

you move to the next Chapter.

Atomic Absorption Spectrometer

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AAS has emerged as a major analytical technique in diverse fields such as environmental

monitoring, mining and geology, oceanographic studies, studies on agricultural crops and soils,

pharmaceuticals, foods and beverages, petroleum and petrochemicals, forensic investigations and

hydro geological investigations.

The free program is designed to give an insight into the technique and once your interest is

captivated you can opt for our more elaborate online certificate program which will be

announced in due course. It will provide you an opportunity to interact with various learners and

experts across the globe.

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Scope of Spectroscopic Analysis

"If you want to increase your success rate, double your failure rate"

-- Thomas Watson, Sr. founder of IBM

Spectroscopic analysis is based on an atom or compound's interaction with electromagnetic

radiation of specific wavelength. Spectroscopy provides information on chemical identity of a

compound, quantity present and structure based on the technique selected and the wavelength of

electromagnetic spectrum. Commonly used spectroscopic techniques in any laboratory are UV –

VIS spectroscopy, FT – IR spectroscopy, Atomic Absorption spectroscopy and ICP/ ICP – MS

spectroscopy.

This topic will introduce you briefly to the different spectroscopic analysis techniques commonly

used in laboratories.

Advantages of spectroscopic techniques

• Rapid analysis – information is available in a matter of seconds as compared to minutes

or even hours in other conventional techniques

• Nondestructive – most spectroscopic methods are non-destructive in nature and there is

100%recovery of sample after analysis

• Micro analysis – generally the methods can be adapted to micro volume analysis when

quantity of sample is limited.

• High sensitivity –inherent sensitivity of spectroscopic techniques coupled with advances

in detection technology provide unparalleled sensitivity. Advancements in hyphenated

analytical techniques such as GC – IR, TGA – IR, GC-MS and LC – MS have lowered

detection and identification to levels which were not imagined earlier.

• Real-time monitoring – manufacturing processes can be monitored real-time using certain

spectroscopic techniques like FT – IR and corrective action can be initiated without the

need of sample withdrawal and off-line analysis.

• Spectroscopic detection has been adapted to a number of techniques such as HPLC

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Application areas of spectroscopic analysis

Application scope of spectroscopic analysis is virtually unlimited. Knowledge gained in

such analysis can contribute to

• Understanding constitution of matter from atoms to complex molecules

• Studies on diverse materials existing in nature from deep sea studies to space missions

• Investigations of crime samples

• Analysis and development of whole range of man-made materials of human consumption

• Studies on environmental samples

• Mineralogy

Types of spectroscopic analysis

UV – VIS Spectroscopy

Radiation in the UV and visible region of electromagnetic radiation interacts with organic

molecules or atoms selectively to give information on presence of absorbing entities. Absorption

radiation results in shifts of electrons within the electron levels of atoms and molecules.

FT –IR Spectroscopy

Radiation in the IR region results in changes in bonding in terms of vibration frequencies,

rotation and vibration energies depending on the wavelength within the IR region. Such

Scope of Spectroscopy

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information provides the basis for both qualitative and quantitative levels of IR absorbing

groups.

Atomic absorption Spectroscopy

Atomic absorption spectroscopy is based on absorption by ground state atoms of an element

present in the sample which is atomized in the flame or graphite furnace. Depending on

absorption of selected wavelength of the element the concentration is estimated. The technique

provides valuable information on concentration of required elements present in the sample.

Concentrations are possible in ppm or ppb levels depending on source of sample excitation.

ICP/ICP – MS

ICP uses a plasma source of excitation of sample. The temperature of the plasma is 2 to 3 orders

of magnitude above the flame AAS methods. The technique affords sensitivity upto ppb or even

sub ppb levels. ICP – MS technique further extends separation of ionized species based on

charge to mass ratio by a quadrupole mass selector. This facilitates analysis of a number of

elements that trace levels simultaneously.

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Evolution of Atomic Absorption Spectroscopy

Trace metal studies on composition of materials has been the oldest branch of analytical

chemistry. Traditional gravimetric techniques still constitute the backbone of most undergraduate

educational laboratories but instrumental methods are fast replacing them due to advantages of

speed and precision. Atomic Absorption Spectroscopy to this day has maintained a top-notch in

most of the academic and industrial laboratories due to its affordability and range of applications.

Bunsen and Kirchoff studied the sodium spectrum and came to the conclusion that every element

has its own unique spectrum in the vapour phase implying that a metal in atomic state can absorb

radiation at same wavelength at which it emits it. This is the founding principle of atomic

absorption spectroscopy. In 1859 Kirchoff showed that the Fraunhofer lines in the sun’s

spectrum were atomic lines due to presence of various elements in the sun's atmosphere.

Spectrochemical analysis had its origins with the work of Bunsen and Kirchoff but found little

application until 1930’s. Modern Atomic Absorption Spectroscopy began in 1955 by a team of

Australian scientists led by Alan Walsh at CSIRO (Commonwealth Science and Industry

Research Organization) division of chemical physics, Australia. Walsh suggested the use of

hollow cathode lamps to provide the appropriate wavelength and use of a flame to generate

neutral atoms that would absorb the incident radiation in proportion to the concentration present

in the traversed path.

Early Day Atomic Absorption Spectrometer

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Early day instruments did not have much limitations but the technique had its own

inherent limitations such as

• Flame is not an ideal atomizer because of partial atomization, loss of sensitivity due to

background interference and only a fraction of sample reaching the flame due to

nebulization and passes quickly through the light path

• Sample has to be in liquid form and therefore solids will require pretreatment and

digestions

• Only one element can be analyzed at a time

Modern developments and advances in electronics and automation did not eliminate such

limitations but made it possible to increase laboratory throughput through features such as:

• Introduction of nitrous oxide – acetylene flame by Willis in 1965. It extended the number

of elements which could be determined due to higher flame temperatures.

• Introduction of techniques such as mercury hydride analysers afforded greater accuracy

and precision for analysis of metals like Hg,Pb,Sn,As,etc.

• High energy sources such as electrodeless discharge lamps for analysis of volatile

elements

Modern Day Atomic Absorption Spectrometer

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• Multi element lamps for faster analysis of number of elements in a sample

• Electrically heated graphite furnace analyzers for greater precision and handling of small

sample amounts for lower detection limits

• Introduction of background correction techniques

• Multi – lamp holders to expedite warm up prior to analysis

Several manufacturers utilize the advanced features and provide their advantages in

competitive environment. Some of the reputed manufacturers are :

• Perkin Elmer

• Agilent Technologies

• Analytik Jena

• Shimadzu

• Aurora Biomed

• Hitachi

• GBC Scientific Equipment

• BuckScientific

• Thermo Scientific

• Teledyne Leeman Labs

• Skyray Instrument

• PG Instruments

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Introduction to AAS component parts

"A day spent without learning something is a day wasted"

-- Anonymous

Atomic absorption occurs when a ground state atom absorbs light of a specific wavelength. The

amount of light absorbed is governed by Beer Lambert's law and will increase as the number of

atoms of the element in the light path increases. The component parts of Atomic Absorption

Spectrometer are similar to a UV -Vis spectrophotometer as both operate on same principle with

a basic difference that the sample cell of UV-Vis spectrophotometer is replaced by an

atomization source (flame or graphite furnace)

AAS Schematic Diagram

Light sources – Hollow Cathode Lamps

The light source commonly used is a hollow cathode lamp. A different element hollow cathode

lamp is required for each element determination. Cathode is made of same metal that is to be

estimated in the sample.

Single element lamps are used commonly though multi-element lamps are also available.

Lamps are made of glass with quartz windows and filled with an inert gas such as argon.

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Light sources – Electrodeless Discharge Lamps

Used for volatile elements such as As,Sb, Sn,Cd,Pb, etc

EDL’s have greater lamp life and high energy throughput

Burner system

The burner assembly comprises of nebulizer to reduce the liquid sample to a fine aerosol, a spray

chamber and a burner head which is used to generate a flame to produce atoms of the same

elements that are present in the sample.

Monochromator

A monochromator disperses the incident light beam and permits the selected wavelength to reach

the detector.

Detector

Detector commonly used is a photomultiplier tube which produces a signal proportional to the

amount of light received by it.

In this section you shall be introduced to two working configurations, namely single beam and

double beam instruments

Single Beam Schematic Diagram

Light from the source is modulated electronically or chopped mechanically by rotating chopper.

This helps isolate and remove sample cell emissions from light emitted by the source. The

specific wavelength isolated by monochromator is led to the detector and the electrical signal

generated is proportional to the elemental concentration in the sample.

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Double Beam Schematic

Double Beam AAS Schematic Diagram

Light beam from source is split into two teams by the chopper. One beam passes directly

through the flame and the other beam passes round the flame. Detector response represents the

ratio of sample and reference beams. Fluctuations in light intensity are eliminated electronically

to get greater reliability of results.

The sensitivity is lower than single beam instruments but the popular acceptance of double beam

configuration is due to advantage of elimination of background changes in the atomizer.

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Types of Light Sources in AAS

"A man is but the product of his thought, what he thinks, he becomes"

-- M.K. Gandhi

Light sources are generally of two types. You'd be familiar with ‘continuum light sources’ such

as Sun or a light bulb which emit electromagnetic radiation in the wavelength range from about

250 to 700 nm in the visible region which we see as normal white light. The white light

comprises of several different wavelengths which constitute the colours of the rainbow. The

other type of light sources are ‘line sources’ which emit light of a specific wavelength and it is

such light sources which are used in Atomic Absorption Spectroscopy. Now you shall be

introduced to such light sources

Hollow Cathode Lamps

A hollow cathode lamp gives a high intensity, narrow line wavelength of element to be

determined

Hollow Cathode Lamp Schematic

Hollow Cathode Lamp

The hollow cathode lamp consists of a glass cylinder filled with an inert gas usually Argon or

Neon at low pressure. The cathode is made from metal which is to be determined.. The emission

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line of the lamp corresponds with the absorption wavelength of the analyte. The end window of

the lamp is usually made of Quartz or Pyrex that transmits the spectral lines of the element to be

determined.

Following stages are involved in light emission from Hollow cathode lamp:

• Sputtering – filled gas is ionized when potential difference is applied between the anode

and the cathode. Positively charged inert gas ions strike the negatively charged cathode

and dislodge metal atoms.

• Excitation – sputtered metal atoms are excited to impact with the ionized gas

• Emission – light of wavelength specific to the element comprising the cathode is emitted

when the atom decays from the excited state to the normal state

Hollow cathode lamps have a shelf life as well as usage lifetime defined in milliampere hours.

Increasing current increases lamp intensity but excessive current reduces lamp life and also

results in self absorption broadening ,i.e, atoms in the hollow cathode lamp begin to absorb light

emitted from the hollow cathode lamp itself. This leads to lower absorbance and reduction in the

linear range of calibration curve.

Multi element Hollow Cathode Lamps

The cathode of mult ielement lamps is made from alloying compatible elements without

overlapping line spectra. Examples of such lengths are Ca-Mg,Cu-Fe-Ni, Cu-Fe-Mn-Zn, etc. All

elements of multi element hollow cathode lamps can be determined sequentially without need for

change of lamps in between. Multi element lamps provide advantages of cost, speed of analysis

but the sensitivity is lower in comparison to individual element determination by single element

lamp

Limitations of Hollow Cathode Lamps

• Hollow cathode lamps have a shelf life

• With the exception of multi element lamps the lamp needs to be changed for

determination of different elements

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• Sputtering deposits metal atoms on sides and end windows which affects lamp life and

more so for volatile elements

• Some cathode materials liberate hydrogen on heating which contributes to continuum

background emission

Electrodeless Discharge Lamps

For most elements hollow cathode lamp is a satisfactory light source. In case of volatile elements

reduced lamp life and low intensity can be overcome by use of high energy throughput

electrodeless discharge lamps. Electrodeless discharge lamps are commonly available for Sb, As,

Bi, Cd, Cs, Pb, Hg, K, Rb, Sn, Te, etc.

Electrodeless Discharge Lamp Schematic

Electrodeless Discharge Lamp

An EDL consists of a quartz bulb filled with an inert gas containing the element or a salt of the

element for which the lamp is to be used. The bulb is placed inside a ceramic cylinder on which

antenna for a RF generator is coiled. When an RF field is applied to the bulb, the inert gas is

ionised and the coupled energy excites the vaporized atoms inside the bulb and causes emission

of characteristic light. EDL’s offer advantage of lower detection limits. The useful life of an

EDL is considerably longer than that of a hollow cathode lamp of same element.

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Flame Atomic Absorption Spectroscopy

"Formal education will make you a living, self education will make you a fortune"

-- Jin Rohn

Sample atomisation produces ground state atoms that are necessary for atomic absorption to take

place. This involves application of thermal energy to break the bonds that hold the atoms

together.

The complete atomisation assembly comprises of:

• Nebuliser

• Spray chamber

• Burner Head

Each of the components of the atomisation assembly are discussed in detail below:

Nebuliser

AAS Nebulizer Schematic

AAS Nebulizer

Nebuliser converts the liquid sample into a fine spray or aerosol. In order to provide efficient

nebulisation for different sample solutions (aqueous or organic, acids or bases, etc) the nebuliser

should be adjustable and corrosion resistant. Stainless steel is commonly used but for corrosive

solutions other corrosion resistant materials such as inert plastic, Pt/Ir or Pt/Rh alloy are also

used. High sensitivity in combination with inert ceramic bead can be used to enhance

nebulisation efficiency for lowest detection limits.

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Spray Chamber

Aerosol from the nebuliser is led to the mixing or spray chamber. In this chamber the aerosol is

mixed with fuel and oxidant gases and carried to the burner head. Only a fraction of the sample

introduced by the nebuliser is used for analysis. An impact device prevents larger droplets from

reaching the burner as these would delay sample vaporisation and atomisation through short

transit through the flame. Only fine sized droplets are carried to the burner head

An impact device such as a flow spoiler or an impact bead is aligned at the exit of the aerosol

stream of the nebulizer. A flow spoiler is more efficient at removing large droplets whereas the

impact bead removes fewer large droplets and exhibits better sensitivity since more sample is led

to the burner. However, the increased number of large droplets may have undesirable effects and

increase interference.

Glass and ceramic impact beads can cause memory and contamination problems compared to the

chemically inert flow spoiler and for this reason flow spoiler is preferred for routine work and for

greater sensitivity impact bead is preferable. The excess sample is removed from the pre-mix

chamber through a drain. The drain uses a liquid trap to prevent combustion gases from escaping

through the drain line. The inside of the spray chamber is coated with wettable plastic material to

provide free drainage of excess sample and prevent burner chamber memory. A freely draining

burner chamber rapidly reaches equilibrium typically in less than two seconds for response to

sample changes.

Spray Chamber

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Burner Head

Burner heads are constructed of titanium to provide extreme resistance to heat and corrosion. A

10 cm single slot burner is recommended for air- acetylene flames. Its long length provides best

sensitivity. A special 5 cm burner head is recommended for nitrous oxide –acetylene flame

applications. The flame can be rotated to provide reduced sensitivity.

Single slot 5 cm air-acetylene burner head is available when reduced sensitivity is required. It

can be rotated to provide further sensitivity redaction and it has a wide slot to prevent clogging

A 3- slot burner head is designed for analysis of samples having high concentration of dissolved

solids.

Majority of elements can be an analysed using air – acetylene flames which have high

temperature range of 2150° C – 2300° C. Nitrous oxide – acetylene flames attain temperatures of

2600°C- 2800° C and can be used for analysing refractory elements which form stable oxides at

lower temperatures.

Burner Head

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Graphite Furnace Atomic Absorption Spectroscopy

"The only source of knowledge is experience"-- Albert Einstein

Flame atomic absorption spectroscopy is a well established and precise method for elemental

analysis giving concentration results in mg/L (ppm) levels. However, better sensitivity is

achievable using electro- thermal atomisation with a graphite furnace.

Limitations of flame AAS

• Burner – nebuliser is a rather inefficient sampling device. Majority of the sample gets

drained and the small fraction reaching the flame has a short residence in the light path

• High sample consumption of the order of 3-5ml/min

• Matrix interferences limit applications particularly in analysis of biological and

geological samples

• Analysis limited to ppm concentration ranges

Benefits of graphite furnace analysis

• Entire sample is atomised and the atoms are retained in the atomisation graphite tube for

extended user controlled time periods

• Microlitre quantities of sample are sufficient and the quantity can be increased to 50 –

100 µl to enhance sensitivity

• Temperature programming steps help remove the solvent and major matrix interferences

• Detection limits typically 100 - 1000 times better than flame techniques are achievable

thereby giving routinely analysis in µg/l(ppb levels)

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Graphite furnace components

Graphite tube – serves as a sample cell as well as a heating element

Electrical contact cylinders – provide electrical c

heating of tube and sample

Water cooling housing – serves to cool the assembly

Inert gas – protects heated tube from atmospheric oxidation. External gas stream surrounds the

outside of the tube and internal gas f

during atomisation to increase sample residence time and improve signal output.

Quartz windows – at each end of the tube help to seal the tube and allow light to pass through

Power supply programmer – controls current supplied to tube as covered by user program

serves as a sample cell as well as a heating element

provide electrical connection to the tube.Current flow provides

serves to cool the assembly

protects heated tube from atmospheric oxidation. External gas stream surrounds the

outside of the tube and internal gas flow purges the tube. Flow is reduced or completely stopped

during atomisation to increase sample residence time and improve signal output.

at each end of the tube help to seal the tube and allow light to pass through

controls current supplied to tube as covered by user program

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onnection to the tube.Current flow provides

protects heated tube from atmospheric oxidation. External gas stream surrounds the

low purges the tube. Flow is reduced or completely stopped

during atomisation to increase sample residence time and improve signal output.

at each end of the tube help to seal the tube and allow light to pass through

controls current supplied to tube as covered by user program

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Transverse heating provides uniform heating of graphite tube across its length. In end to end

heating there can be temperature gradient along the tube length. L’vov platforms delay the

vaporization and atomisation of the sample until furnace atmosphere has reached equilibrium

conditions.

Stabilised temperature platform furnace (STPF) was pioneered in 1970’s by Perkin Elmer. It is a

combination of graphite tube quality, design and operational parameters to improve atomisation

and detection. Tube lifetime improvement is provided by using high-quality graphite for the

tubes., Platforms maximise power heating to virtually eliminate interferences and internal gas

stop increases sensitivity.

Limitations of graphite furnace analysis

• Longer analysis time in comparison to flame analysis

• Lesser number of elements analysed by furnace technique – around 40 as compared to

about 70 in flame technique

• Higher cost of graphite furnace assembly but it is also available as a switching option

with flame operation in most commercial instruments

• Higher and more complex background levels require expensive background correction

options

Graphite Furnace Components

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Dispersion and Resolution of Light in Atomic

Absorption Spectroscopy

"Learn something new. Try something different. Convince yourself that you have no

limits"

-- Brian Tracy

Monochromator

A monochromator is a device that isolates and transmits a band of wavelength from a wider

range of wavelengths available at the inlet slit. The dispersion of light can be obtained by means

of a prism or diffraction grating. The Czerny- Turner monochromator using a pair of concave

mirrors and a plane grating is most widely used in atomic absorption spectroscopy.

Czerny – Turner Monochromator

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Broadband beam reaches the entrance slit positioned at the focal length of the collimating mirror

and the parallel beam is diffracted by the plane grating and after reflection from the second

mirror is focused on the exit slit. As each color (wavelength) arrives at a separate point in the

exit plane, a series of images are focused on the exit slit. As the exit slit has a finite width, parts

of nearby images overlap. The light leaving the exit slit contains the image of entrance slit along

with images of nearby colors Rotation of dispersion grating causes the band of colors to move

relative to fixed exit slit so that the desired entrance slit image can be centered on the exit slit.

Thus the range of colors leaving the exit slit is a function of slit width. Slit size is variable,

though usually not continuously.

Mirrors

Mirrors used in the monochromator must be highly reflecting in the wavelength range of interest.

This can be achieved by polishing the front surface with aluminium, silver or gold. The metal

layer is covered with a protective coating that prevents the metal from tarnishing.

Grating

The dispersion of light takes place on the grating. Parallel beam striking the grating leaves the

grating at slightly different wavelengths. The angle of dispersion at the grating is controlled by

the density of lines on the grating, i.e. number of lines/mm. High dispersion is achieved by

increasing the line density. In order to isolate desired line from nearby lines narrower exit slit is

used. The use of a wider slit width allows more light thereby enhancing sensitivity but at the cost

of resolution.

Blaze angle governs the efficiency of the grating. The slope of the triangular groove in a ruled

grating is adjusted to enhance the brightness of a particular diffraction order. The further

removed a given wavelength of light is from the wavelength for which the grating is blazed the

greater will be the extent of light loss at that wavelength.

The wavelength range normally used in atomic absorption spectroscopy is from 185nm to about

900 nm. With a grating blazed somewhere in the middle of this range significant energy fall–of

occurs at the wavelength extremities due to the energy inefficiencies of the diffraction process.

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Dual blazed gratings with a blaze in both UV and visible regions offer better energy efficiency

over the entire wavelength range.

Monochromator Parameters

Slit Width

Slit width is the width in millimetres of the entrance and exit slist of the monochromator. Narrow

slit width gives better resolution. In standard monochromator design both entrance and exit slits

have equal width. Wider the slit widths more wavelengths passes through the monochromator.

Research grade instruments have user controlled slit widths.

Monochromator Focal Length

Greater the focal length of collimating mirrors the larger their resolution. The resolving power of

a monochromator is governed by both focal length and slit width.

Dispersion

The dispersion of a monochromator is characterized as the width of band of wavelengths per unit

of slit width, i.e, nm of wavelengths per mm of slit width

Spectral Bandwidth

Spectral bandwidth is the width of the triangle at the points where the light has reached half the

maximum value defined as Full Width at Half Maximum (FWHM)

Stray Light

Stray light is light other than selected wavelength reaching the detector.

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Interferences in Atomic Absorption Spectroscopy

Interference is a phenomena that leads to changes in intensity of the analyte signal in

spectroscopy. Interferences in atomic absorption spectroscopy fall into two basic categories,

namely, non-spectral and spectral.

Non-spectral interferences affect the formation of analyte items and spectral interferences result

in higher light absorption due to presence of absorbing species other than the analyte element.

Interference in Atomic Absorption Spectroscopy

Non-spectral interferences

Matrix interference

When a sample is more viscous or has different surface tension than the standard it can result in

differences in sample uptake rate due to changes in nebulization efficiency. Such interferences

are minimized by matching as closely as possible the matrix composition of standard and sample

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Chemical interference

If a sample contains a species which forms a thermally stable compound with the analyte that is

not completely decomposed by the energy available in the flame then chemical interference

exists. Refractory elements such as Ti, W, Zr, Mo and Al may combine with oxygen to form

thermally stable oxides. Analysis of such elements can be carried out at higher flame

temperatures using nitrous oxide – acetylene flame instead of air-acetylene to provide higher

dissociation energy. Alternately an excess of another element or compound can be added e.g. Ca

in presence of phosphate produces stable calcium phosphate which reduces absorption due to Ca

ion. If an excess of lanthanum is added it forms a thermally stable compound with phosphate and

calcium absorption is not affected.

Ionization interference

Ionization interference is more common in hot flames. The dissociation process does not stop at

formation of ground state atoms. Excess energy of the flame can lead to excitation of ground

state atoms to ionic state by loss of electrons thereby resulting in depletion of ground state atoms.

In cooler flames such interference is encountered with easily ionized elements such as alkali

metals and alkaline earths. Ionisation interference is eliminated by adding an excess of an

element which is easily ionized thereby creating a large number of free electrons in the flame and

suppressing ionization of the analyte. Salts of such elements as K, Rb and Cs are commonly used

as ionization suppressants.

Spectral Interferences

Spectral interferences are caused by presence of another atomic absorption line or a molecular

absorbance band close to the spectral line of element of interest. Most common spectral

interferences are due to molecular emissions from oxides of other elements in the sample.

The main cause of background absorption is presence of undissociated molecules of matrix that

have broad band absorption spectra and tiny solid particles, unvaporized solvent droplets or

molecular species in the flame which may scatter light over a wide wavelength region. When this

type of non-specific adsorption overlaps the atomic absorption of the analyte, background

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absorption occurs. The problem is overcome by measuring and subtracting the background

absorption from the total measured absorption to determine the true atomic absorption.

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Background correction in Atomic Absorption

Spectroscopy

"Tell me and I'll forget : show me and I may remember; involve me and i'll understand"

-- Chinese Proverb

Before you move to background correction it is necessary to understand what is background

absorption. The main reason for background absorption is presence of undissociated molecules

of matrix that have broadband absorption spectra and tiny solid particles in the flame which may

scatter light over a wide wavelength region. When this type of non-specific adsorption overlaps

the atomic absorption wavelength of the analyte the ground state absorption is cut. The problem

is overcome by measuring and subtracting the background absorption from the total of measured

absorption to determine true atomic absorption component

Background Correction

A number of background correction approaches have been proposed but we shall limit our

discussion to two main approaches which have found widespread application in commercial

instruments

Continuuum Deuterium Source Background Correction

Continuum background correction measures and compensates for any background component

present in atomic absorption measurements

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D2 Lamp Background Correction Schematic

The broadband contimuum source emits light over a broad spectrum of wavelengths instead of

specific line.Spectral interferences which are caused by atoms with absorption lines very close to

the analyte absorption line or by fine structure in the molecular absorption profile can result in

either positive or negative errors in the measurement of the concentration of the analyte.

Fortunately spectral interferences are rare in flame atomic absorption which is a distinct

advantage over emission techniques

A common inexpensive technique for background correction in flame Atomic Absorption

Spectroscopy is deuterium background correction. The correction is effective over the

wavelength range of 180nm -420nm. Background level becomes significant at lower wavelength

range.

The cathode lamp and the deuterium lamp are sequentially pulsed with a chopper or

electronically with delay of about 2ms. When hollow cathode lamp is on and deuterium lamp off

total absorbance (AA + BG) is measured. When the HCL is off and the deuterium lamp on the

continuum energy recorded is (BG). The atomic signal is automatically calculated by subtracting

background from total absorbance.

Limitations of Continuum D2 background correction :

• The D2 lamp has a finite lifetime and requires periodic replacement

• The D2 lamp and HCL light may not view the same portion of atom cloud in the flame

due to time lag. This could become significant at high background levels.

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• Proper alignment of both light sources is required for right background correction

• Correction is limited to the specific wavelength range of the two lamps

• Intensities of both lamps if not similar will result in errors

Zeeman Background Correction

Zeeman Background Correction Schematic

Zeeman Background Correction is used mainly in graphite furnace atomic absorption systems.

When an atom is placed in a magnetic field and its absorption of observed in polarised light, the

normal single line is split into three components – б-, π and б +displaced symmetrically about

the normal position

Magnetic Field Off

Magnetic Field on

Free atoms show Zeeman splitting in a magnetic field but molecules, liquid droplets or solid

particles show no Zeeman splitting and so advantage can be taken of polarised light. The π

component is linearly polarised parallel to the magnetic field while the б components are

circularly polarised perpendicular to the magnetic field. A polariser is positioned in the optical

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system to remove the π components of the transmitted radiation. This affords background

measurement at the exact analyte wavelength when magnetic field is applied. Since the

background is measured at the analyte wavelength and not averaged as in D2 system structural

molecular background and spectral interferences are easily corrected.

In AC modulated Zeeman system the combined atomic and background absorption is measured

while magnetic field is off. When the magnetic field is on, the detector measures only the

background absorption as the π component is not detected. The difference between the two is the

Zeeman corrected atomic absorption signal

The magnetic field may be applied to the graphite tube with either transversally( perpendicular)

or longitudinal ( parallel) to the optical axis. A major advantage of longitudinal Zeeman

background correction is that the polariser is not needed to eliminate the π component, thereby

providing higher light throughput by eliminating polarizer absorption

Advantages of Zeeman background correction :

• Corrects high levels of background

• Corrects at exact analytical absorption line

• Requires only a single standard light source. Alignment problems of multiple light

sources are not encountered

Limitations of Zeeman background correction :

• More expensive than continuum background correction

• Loss in sensitivity for some elements due to splitting of б and π components which may

overlap

So far you have been introduced to the basic concepts and principles of Atomic Absorption

Spectorscopy. The next Chapter is a set of questions which you may face at time of interview

when you apply for a suitable opening in a laboratory equipped with an Atomic Absorption

Spectrometer.

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10 Interview questions in Atomic Absorption

Spectroscopy

In this Chapter we have posed before you some typical questions which you could come across

during interview sessions when you apply for a job involving use of atomic absorption

spectrometers. We have deliberately not provided you with tailored made answers and the

objective is to motivate you to look for the answers. We are sure that if you review the earlier

Chapters on the topic you'll find all your answers. This exercise will encourage you to search for

solutions that you are faced with not only in atomic absorption spectroscopy but in other areas of

your activity as well.

Q1. Explain briefly the principle of operation of an Atomic Absorption Spectrometer?

Q2. What is the difference in operation principle of AAS and AES?

Q3. Why is nitrous oxide used as oxidant in some applications?

Q4. What are the advantages of using Electrodeless discharge lamps over Hollow cathode lamps

as light sources for analysing some elements?

Q5. What are the advantages of double beam over single beam operation?

Q6. What is the role of a flow spoiler or an impact bead in the spray chamber? Explain the

benefits of each over the other.

Q7. What is the role of a monochromator in the atomic absorption spectrometer?

Q8. What do you understand by deuterium background correction and what are its limitations?

Q9. Explain the benefits of graphite furnace analysis over flame analysis?

Q10. What are the limitations in analysis of analysis of volatile elements such as As, Sn, Pb,Sb

,etc What alternate sample treatment option is commonly used for such analysis?

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Conclusion

We believe that you enjoyed the free e- course on AAS. The course provided you an insight into

the components of AAS system and their individual contribution towards the overall accuracy

and precision of your results. Apart from a general introduction the course was designed keeping

in mind the requirements of the AAS user. Without going into mathematical treatment of the

subject an attempt has been made to convey the basics concepts and offer practical tips on

effective utilization of the AAS system.

In case the e- course has awakened your desire to go deeper into the subject you are welcome to

join the Certificate Course on AAS which shall be available round the year after its launch. For

more details on this advanced programme please await our announcement on the site.

Once again we take the opportunity to thank you for your interest. Please feel free to participate

actively by contributing articles in areas off your interest and offer your valuable comments and

suggestions.

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