Introduction to Spectroscopy Atomic Absorption Spectroscopy _AAS

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Introduction to Spectroscopy Atomic Absorption Spectroscopy _AAS Powered By Docstoc
					Atomic Absorption Spectroscopy
           (AAS)
           Topics to be covered


¨Importance of elemental analysis .

¨ Introduction to spectroscopy.


¨ Atomic Absorption Spectroscopy (AAS
¨ Atomic Emission Spectroscopy (AES).

¨ Inductively Coupled Plasma Spectroscopy (ICP).
  Importance of elemental analysis
  Monitoring levels of certain elements in samples ( eg.
  pharmaceutical products and standards) to detect
  the concentrations of these elements.
             homodyalysis
             protonialdyalysis
             ringer lactate
  Monitoring the levels of the toxic elements in
  samples ( eg. cosmetics, food supplements, entire
  plant or part of it) to ensure it’s safety.

With these information, we can take steps to approve
  or withdraw products from the markets.
n Widely used in clinical chemistry and environmental
  laboratories.
      Elements (Heavy Metals)

The term heavy metal refers to any metallic
chemical element that has a relatively high density
and is toxic or poisonous at low concentrations.
They have a specific gravity that is at least 5 times
the specific gravity of water.
 Example: Arsenic 5.7,Cadmium 8.65 , Iron 7.9,
Lead 11.34 Mercury 13.546
Trace elements :
Heavy metals that are nutritionally essential for
a healthy life.
Examples are ( Iron, Manganese, Copper and
Zinc).
    Commonly encountered toxic
         heavy metals

¨ Arsenic                ¨ Cadmium



                        ¨ Iron
¨ Lead



¨ Mercury                ¨ Aluminum
                  Types of samples for analysis
     
                                ¨Food 
    ¨ Pharmaceutical            supplements
        product
                                             



      ¨ Standards               ¨Entire plant 
                                or part of it

                                   
       ¨ Cosmetics              ¨ Mixture of 
                                known & unknown
                                herb
            
                  Spectroscopy

n   Spectrometric methods are a large group of
    analytical methods
n   Spectroscopy is the science that deals with the
    interactions of radiation with matter (atomic and
    molecular).

n   The most widely used spectrometric methods
    are based on electromagnetic radiation (light,
    gamma rays, X-rays, UV, microwave, and radio-
    frequency).
    Electromagnetic Radiation:


¨   consists of discrete packets of energy, which
    we call photons
¨    A photon consists of an oscillating electric
    field component, E, and an oscillating
      magnetic field component, M.
¨ The characteristics of these fields are:

   Orthogonal ( perpendicular ) to each other
   Orthogonal to the direction of propagation
      of the photon




 They flip direction as the photon travels
 All photons (in a given, non-absorbing
  medium) travel at the same
  velocity, v.
     What is Frequency (n)?
      The number of flips, or oscillations, that occur in one
    second.
     What is A Wavelength (l)?
n        The physical distance in the direction of
    propagation over which the electric and magnetic fields
    of a photon make one complete oscillation.
                   Unit: Angstrom, nm, µm

n   Velocity Of Light = 2.99792 x 108 m/s

The electromagnetic nature of all photons is the same,
       but photons can have different frequencies
  The relationship between the light velocity, wavelength,
                    and frequency is :

                          v=nl
                    E = hn = hv / l 
The energy, E, of one photon depends on its frequency of
                        oscillation :


 where h is Planck's constant (6.62618x10-34 J·s)
   The relationship between the speed of light c ,
           wavelength, and frequency is :

                      C=nl
  When light passes through other media, the velocity
                     of light ¯
Since the energy of a photon is fixed, the frequency of
              a photon does not change.
 Thus for a given frequency of light, the wavelength
         must ¯ as the velocity decreases.
                       n = c / v
   The decrease in velocity is quantitated by the
  refractive index, n, which is the ratio of c to the
       velocity of light in another medium, v:
Electromagnetic Spectrum:
1- Absorption of Radiation

n   When radiation passes through a layer of
    solid, liquid, or gas, certain frequencies may
    be absorbed, a process in which
    electromagnetic energy is transferred to the
    sample.
n   Absorption promotes these particles
    from their ground state to more higher-
    energy excited state.
n   Tow types of absorption spectra:
    – Atomic absorption spectrum.
    – Molecular absorption spectrum.
Beer’s Law
n   Many compounds absorb radiation. The
    diagram below shows a beam of
    monochromatic radiation of radiant power P0 
    directed at a sample solution.

n   Absorption takes place and the beam of
    radiation leaving the sample has radiant
    power P.
n   The amount of radiation absorbed may be
    measured in a number of ways:

    – Transmittance, T = P / P0
      % Transmittance, %T = 100 T

    – Absorbance,
       n A = log10P0 / P
        A = log10 1 / T
        A = log10 100 / %T
        A = 2 - log10 %T 
n   The last equation, A = 2 - log10 %T , is
    worth remembering because it allows
    you to easily calculate absorbance from
    percentage transmittance data.
n   The relationship between absorbance
    and transmittance is illustrated in the
    following diagram:
n   The equation representing the Beer’s law:
                      A=εbc
n   Where
    – A is absorbance (no units, A = log10 P0 / P ).

    – ε is the molar absorbtivity (is a measure of
      the amount of light absorbed per unit -1
                                         -1
      concentration) with units of L mol cm .
           the                the sample that
    – b is pathpath length of cuvette in which is,
      the       length of the                  the
      sample is contained. We will express this
      measurement in centimeters.


    – c is the concentration of the-1.
      solution, expressed in mol L
                                    compound in
n   Beer’s law tells us that absorbance depends
    on the total quantity of the absorbing
    compound in the light path through the
    cuvette. If we plot absorbance against
    concentration, we get a straight line passing
    through the origin (0,0).
The working curves are used to

• Determine the concentration of an
  unknown sample.

• To calibrate the linearity of an
  analytical instrument.
 What are the Processes by which a molecule
 can absorb radiation?

1- Rotational transition :
    The molecule rotate about various axes, the
  energy of rotation being at definite energy
  levels, so the molecule may absorb radiation
and be raised to a higher rotational energy level .
2- Vibrational transition:
The atoms or group of atoms within a
molecule vibrate relative to
each other. The molecule may then absorb a
discrete amount of energy
 and be raised to a higher vibrational energy
level..
3- Electronic transition:
The electrons of molecule may be raised to a
higher electron Energy.




  The three types of internal energy are
                quantized
Rotational transitions: low energy E [long λ (microwave or
far-infrared)]
Vibrational transitions: takes place at high energy E [ near,
far infrared region]
Electronic transitions: takes place at higher energy E
[visible and U.V region]
   Type of Radiation    Frequency Range (Hz)     Wavelength Range      Type of Transition
gamma-rays             1020-1024               <1 pm                nuclear
X-rays                 1017-1020               1 nm-1 pm            inner electron
Ultraviolet            1015-1017               400 nm-1 nm          outer electron

Visible                4-7.5x1014              750 nm-400 nm        outer electron

                                                                    outer electron molecular
near-infrared          1x1014-4x1014           2.5 µm-750 nm
                                                                        vibrations
Infrared               1013-1014               25 µm-2.5 µm         molecular vibrations
                                                                    molecular rotations,
Microwaves             3x1011-1013             1 mm-25 µm
                                                                        electron spin flips*
radio waves            <3x1011                 >1 mm                nuclear spin flips*
                   Cont. Introduction to
                     Spectroscopy
 TRANSITION         SPECTRUM     TECHNIQUE             MAIN USE



   Electronic         UV-vis   UV-vis spectroscopy     Quantitative
    Transitions                                           Analysis
                               Atomic Absorption
                                   Spectroscopy        Quantitative
                                                          Analysis
  Vibrational          IR       IR Spectroscopy      Functional groups
    Transitions                                         Structural
                                                         Identification



Spin Orientation      Radio          NMR                 Structure
                                                         Identification
¨ Which molecules or atoms can absorb radiation?
¨ Molecules:
n     For absorption to occur there must be change in the dipole
      moment (polarity) of the molecule.
       i.e polar covalent bond in which a pair of electrons is
      shared unequally.
         eg: of a molecule that can not exhibit a dipole
      moment.
             N º N          Can not exhibit a dipole and will not absorb in the I.R region.
        eg. of a molecule that can exhibit a dipole moment.
            O= C =O    Unsymmetrical diatomic molecule, does have a permanent dipole     
          and so                will absorb light.

         OÞCÜO Vibration mode ® symmetry and no dipole moment
         OÜ CÜ O By induced dipole ® dipole moment and the molecule can absorb I.R radiation.
¨ Atoms:
Incase of atoms only electronic transition occurs.


                    P


                    O



                    N



                   M



                    L



                   K
    Atomic absorption spectra              Molecular absorption spectra

1- The outer most electrons               The outer most electrons occupy s,
    occupy one of the atomic orbitals     p or n electronic energy in the
   and have its energy levels [K, L,       ground state.
     M,   N, ......
     s, s,p s,p,d s,p,d,f ]
    2- Upon excitation electrons are      Upon excitation electrons
    promoted to any permissible higher p* or s* energy.
                               raised to
     atomic energy levels                 levels
    3-Since there are no bonds there      Since there are bonds, there are
    are no vibrational or rotational      vibrational and rotational energy
    energy levels in either the ground or levels in both the ground and
    excited state.                         excited states
    4- The analytical wavelength is the   The analytical wavelength is the
    resonance wavelength of the            lmax.
     analyte
    5- The spectra are line form.          The spectra are in the form
                                           of bands due to the presence
                                           of very close, superimposed
                                           and unresolved vibrational and
                                           rotational energy levels in the
Atomic Absorption Spectroscopy (AAS)

AAS was employed in the 1950’s
Used for qualitative and quantitative detection.
 It’s used for the determination of the presence and
concentrations of metals in liquid samples.
Metals that can be detected include Fe, Cu, Al, Pb,
Ca, Zn, Cd and many more.
Concentrations range is in the low mg/L (ppm)
range.
Elements that are highlighted in
   pink are detectable by AAS
    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 suitable                             2. The element
for a certain element                             in the sample
                                                                         6. The detector
                                                 will be atomized
                                                                       measures the change
                                                      by heat
                                                                           in intensity


                                                                      7. A computer data
                                                                     system converts the
                                                                    change in intensity into
                                                                        an absorbance
The disadvantage of both the HCL and laser is that they
have narrow-band light sources and so only one element is
                 measurable at a time.
 2. Laser:
They are intense enough to excite atoms to higher
energy levels. This allows AA and atomic fluorescence
measurements in a single instrument.
Note:
Simple dedicated AA instruments often replace the
monochromator with a bandpass interference filter.
A. Flame Atomic Absorption Spectroscopy:


 n   The technique requires a liquid sample to be
     aspirated, aerosolized, and mixed with
     combustible gases, such as acetylene and air
     or acetylene and nitrous oxide.
 n   The mixture is ignited in a flame whose
     temperature ranges from 2100 to 2800 ºC.
          1. Nebulizer:
                                                                                       4. The mixture flows
                                                                                       immediately into the
                                                                                           burner head.

                                  5. It burns as a smooth,
                                    laminar flame evenly
                                 distributed along a narrow
                                             slot.


    1. mixes acetylene (the fuel)
              and oxidant
        (air or nitrous oxide).                                                  3. The result is a heterogeneous
                                                                               mixture of gases (fuel + oxidant) and
                                                                               suspended aerosol (finely dispersed
                                                                                             sample).



                                                              2. A negative pressure is formed
                                                              at the end of the small diameter,
                                                                   plastic nebulizer tube®
                                                                         (aspiration).
6. Liquid sample not flowing                                      Note:
into the flame collects in the                                    When do we use NO2 ?
            waste.
            2. Ignition:

The process of lighting the AAS flame involves:
    turning on first the fuel then the oxidant and then
   lighting the flame with the instrument's auto
   ignition system.

The flame breaks down the analyte's matrix ® create
   the elemental form of the analyte atom.
During combustion, atoms of the element of
     Interest in the sample are reduced to free,

unexcited ground state atoms, which absorb
  light at characteristic wavelengths,
     as shown in the figure.
    Optimization is accomplished by :


•   Aspirating a solution containing the element

•   Adjusting the fuel/oxidant mix until the
    maximum light absorbance is achieved.

•   Careful control of the fuel/air mixture is
    important because each element's response
    depends on that mix in the burning flame.
          3. Lamp (Hollow Cathode Lamb):
Consists of a cathode and an anode. The cathode is
   made of the element of interest




1.   A large voltage across the anode and cathode will
     cause the inert gas to ionize.
2.   The inert gas ions will then be accelerated into the
     cathode, sputtering off atoms from the cathode.
3.    Both the inert gas and the sputtered cathode
     atoms will in turn be excited by collisions with each
     other.
4- When these excited atoms decay to lower
   energy levels they emit a few spectral
   lines characteristic of the element of
   interest.
5- The light is emitted directionally through
   the lamp's window, a window made of a
   glass transparent in the UV and visible
   wavelengths.
6- The light can then be detected and a
   spectrum can be determined.
:              4. Monochromator
    The light passes from the HCL through the element in
     the sample to the monochromator.
       It’s function is:
       It isolates the specific light of the element of
     interest from the other background lights and
     transfers it to the photomultiplier tube (detector).
          5. Photomultiplier Tube (PMT)

:
    Before an analyte is aspirated, a measured signal is
     
    generated by the PMT as light from the HCL passes
    through the flame. When analyte atoms are present in
    the flame--while the sample is aspirated--some of that
    light is absorbed by those atoms. This causes a
    decrease in PMT signal that is proportional to the
    amount of analyte
.




                                               PMT
 The PMT detects the amount of reduction of
the light intensity due to absorption by the
analyte, and this can be directly related to
the amount of the element in the sample.

     The PMT converts the light signal into an
electrical
     signal and a computer system translates
it into
     absorbance.
    Photomultiplier Tube

Light                    Photocathode
                  e-
    Dynode
                         Dynode
   Dynode



               Current

Convert light energy to electrical energy
     Gases used in the FAAS:

Different flames can be achieved using different
 mixtures of gases, depending on the desired
 temperature and burning velocity.

Some elements can only be converted to atoms at high
 temperatures.

Some metals form oxides that do not re-dissociate into
  atoms. To inhibit their formation, conditions of the
  flame may be modified to achieve a reducing, non-
  oxidizing flame.
    Table of the characteristics of various
                   flames.


                            Max. flame speed (cm/s)   Max. temp. (oC)

      Air-Coal gas                    55                   1840

       Air-propane                    82                   1925

      Air-hydrogen                   320                   2050

Air-50% oxygen-acetylene             160                   2300

Oxygen-nitrogen-acetylene            640                   2815

    Oxygen-acetylene                 1130                  3060

    Oxygen-cyanogen                  140                   4640

 Nitrous oxide-acetylene             180                   2955

  Nitric oxide-acetylene              90                   3095

Nitrogen dioxyde-hydrogen            150                   2660

 Nitrous oxide-hydrogen              390                   2650
      B. Graphite Furnace Atom Absorption
      Spectroscopy:

   
      (GFAAS) Graphite Furnace Atomic Absorption
      Spectroscopy
       (ETAAS) Electro thermal Atomic Absorption
      Spectrometry
        
5. The monochromator isolates the light of
      the element of interest from the
                                                4. The graphite tube is
                                              permanently flushed with         1. The source of light (lamp)
  background lights to the PMT. The PMT      argon while it is in operation    emits light with a wavelength
   tube measures the change intensity.                                          specific to the element of
                                                                                          interest




2. A controlled voltage is applied at
the ends of the graphite tube, which                       3. Samples are deposited in the graphite tube
      is heated rapidly to high                            ® heated to vaporize and atomize the analyte
    temperatures (up to 2600°C).                           ® atoms absorb ultraviolet or visible light and
                                                           make transitions to higher electronic energy
                                                                              levels.
vSmall sample
vFormation of stable Thermal oxide (Al, Mo, Ti)
vResidual elements
vBiological samples
vEffective atomization
vSample stay for Long time in graphite tube>>>hig
sensetivety
vSuitable for solid samples
vIonic interferase rather than chemical or physical
due to high temp.
    Mercury Cold Vapor

 -Free mercury atoms can exist at room temperature
can be analysed using atomic absorption without
employing flame and graphite furnace techniques at
high temperatures.
-Mercury is reduced in solution using stannous
chloride or sodium borohydride in a closed
system.
 -The reaction quantitatively releases mercury
(from the sample solution) and is carried by a
stream of air or argon through a quartz
sample sell placed in the light path of an AA
instrument for analysis.

 The detection limit for mercury by this cold
vapor technique is approximately 0.02 mg/L.
Hydride Generation Atomic Absorption
    Spectroscopy (HGAASC.):

  It’s used for elements that are forming volatile
hydrides (e.g. As, Se, pb, Sb, Te, Sn, Bi)
 (HAAS) is very useful in case of interferences, poor
reproducibility, and poor detection limits.
                                       4. The PMT detects the amount of
                                     reduction of the light intensity due to
                                     absorption by the analyte and can be              1. The HCL emits the light
                                      directly related to the amount of the                with a wavelength
                                              element in the sample                   characteristic to the element
                                                                                               of interest




  3. The monochromator isolates                                      2. The Light passes from the HCL
 analytical lines' photons passing                                     through the optical cell to the
      through the optical cell                                           monochromator then to the
and removes the scattered light of                                                detector
other wavelengths from the optical
                 cell
                            sample flow in the (HGAAS)

4. In the optical cell the flame is ignited automatically by                           3. The liquid mixture flows
              the air/C2 H 4 and the gaseous                                       into a gas/liquid separator where
metal hydride form decomposes into the elemental form                        the hydride and some gaseous hydrogen are
                   which can absorb the                                    purged (via a high purity inert gas) into the optical
 HCL's beam. The light passes to the Mon. and then to                                  cell via a gas transfer line
                           the PMT



                                                          Optical cell




                                                                               Volatile Hydride
                                                                                                         Gas liquid separator



                                                                                           Mixing
                                                                           peristaltic     reaction
                                                                             pump
                                                                                             coil
     1. The metal oxyanions reacts with                             2. The peristaltic pump and the flowing reagents along
     Sodium Borohydride and HCl and                                   with the tubing of specific lengths controls the time
     produces a volatile hydride: H2Te,                                       from reagent mixing and separation of
           H2Se, H3As, H3Sb, etc.                                                  the volatile hydride from the
                                                                        liquid and sending the sample to the optical cell.
         AAS Interferences

   
 Any factor that affects the ground state population
  
  of the analyte element.
  Factors that may affect the ability of the
  instrument to read this parameter.

A) Spectral interferences: due to radiation
  overlapping than of the light source.

B) Formation of compounds that do not dissociate in
  the flame.
 The most common example is the formation of
  calcium and strontium phosphates.
C) Ionization of the analyte reduces the
signal.
This is commonly happens to barium, calcium,
strontium, sodium and potassium.
D) Matrix interferences: due to differences
between surface tension and viscosity of test
solutions and standards
.
E) Broadening of a spectral line
              1. Doppler effect
              2. Lorentz effect
             3. Quenching effect
  4. Self absorption or self-reversal effect

				
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