INSTRUMENTAL METHODS OF ANALYSIS (CHM 303)-2UNITS

1
INSTRUMENTAL METHODS OF ANALYSIS (CHM 303)-2UNITS

• LECTURERS
• DR. (MRS.)T. F. AKINHANMI
• DR ADEDIJI

2
COURSE OUTLINE

• General Principles of Spectrometer
• Ultraviolet-visible absorption spectroscopy
  theory, quantitative application of UV
  measurement.
• IR spectrophotometrybasic theory, solid, liquid
  and gas samples, Group frequencies and
  quantitative uses.
• Molecular fluorescence spectroscopy
• Atomic spectroscopy, absorption and emission,
  flame atomization

3
GENERAL PRINCIPLE OF SPECTROMETERS

• SPECTROSCOPY/SPECTROMETRY
• deals with interactions of radiation and matter.
• is often used in physical and analytical
  chemistry for the identification of substances
  through the spectrum emitted from or absorbed by
  them.
• The absorption and emission of these radiations
  is associated with changes in the energy states
  of the interacting chemical species.

4

• absorption of e.m.r. by an atom, molecule or ion
  causes a transition from lower to higher energy
  level (only certain transitions are allowed).
• The energy of the radiation absorbed gives the
  energy difference between the two levels.
• each species has characteristic energy states,
  hence, spectroscopy can be used to identify
  interacting species.

5

• Spectroscopic Analytical Method Measurement and
  interpretation of electromagnetic radiation
  (e.m.r) absorbed, emitted or scattered by
  molecular or atomic species of interest.
• The instrument that performs such measurements is
  a spectrometer

6
A Schematic Diagram of a Spectrometer
7
Classification of spectrometric methods

• Based on region of electromagnetic radiation used
  e.g.
• ?-ray , X-ray, Ultra violet (UV), Visible, (V),
  Infra-red (IR), Microwave and radiofrequency

8
Classification of spectrometric methods..2

• Nature of excitation measured
• The type of spectroscopy depends on the physical
  quantity measured, i.e. energy absorbed or
  emitted.
• Examples
• Electromagnetic spectroscopy
• Electron spectroscopy
• Mass spectrometry
• Dielectric spectroscopy
• etc

9
Classification of spectrometric methods..3

• Nature of interaction
• Most spectroscopic methods are differentiated as
  either atomic or molecular based on whether or
  not they apply to atoms or molecules.
• Examples
• Atomic Absorption Spectroscopy (AAS), Electron
  Spin Resonance (ESR), Electron Paramagnetic
  Resonance (EPR), Electronic Ultra Violet-Visible
  (UV-VIS), Fluorescence, Infra-red (IR),
  Microwave, Nuclear Magnetic Resonance (NMR),
  Photo electron Spectroscopy (PES), Raman
  Spectroscopy.

10
Advantages of spectroscopic methods.

• Development of modern atomic theory
• A useful tool in the elucidation of molecular
  structure
• Ability to determine quantitative and qualitative
  analysis of inorganic and organic compounds.

11
Interaction of electromagnetic radiation with
matter

• Radiation from a suitable source (provides emr
  covering a range of frequencies) is passed
  through a sample
• Amount of radiation absorbed is related to the
  concentration of the analyte
• E.g. copper solution absorbs complementary colour
  yellow and transmits blue light when emr is
  passed through it.
• The amount of yellow light absorbed is measured
  and related to the concentration of copper
  solution.

12
Interaction of electromagnetic radiation with
matter..2

• When polychromatic light (different wavelength)in
  the Visible region is passed through an object,
  some may be absorbed, and some transmitted.
• transmitted wavelength is seen as colour
• Human eye is sensitive to the colour of white
  light (ROYGBIV)
• Interactions observed depend on energy of
  radiation and mode of detection

13
Electromagnetic Spectrum

• This is a broad range of radiations that extend
  from cosmic rays with wavelength as short as
  10-9nm to radio waves longer than 1000km.
• In between these two radiations and moving from
  short to long wavelengths are ?-rays, X-rays, UV
  rays( vacuum and near), visible (V), IR rays(near
  and far) and microwaves.
• The visible region is only a minute portion of
  the emr spectrum.

14
Electromagnetic Spectrum2

• They have similar nature and travel with the
  speed of light but have different frequencies
  wavelength and produces different effect on
  matter.

15
Electromagnetic spectrum. The small visible range
range (shaded) is shown enlarged at the riht.

• Electromagnetic spectrum. The small visible range
  (shaded) is shown enlarged at the right

16
Properties of Electromagnetic Radiation (Emr)

• E.m.r
• A form of energy transmitted through space at
  very high velocities.
• is described as a wave with wavelength,
  frequency, velocity and amplitude.
• treated as discrete packets of energy or
  particles called photons or quanta.

17
Wave theory of electromagnetic radiation

• Emr is considered as waves consisting of electric
  and magnetic fields which vary periodically and
  in a direction perpendicular to the direction of
  propagation.

18
Definition of terms

• Frequency is the number of cycles per second
  (s-1)
• Wavenumber is the number of waves per cm (cm-1)
• Wavelength is the linear distance between
  successive maxima or minima of a wave or distance
  between the peaks in cm.
• Amplitude Maximum displacement from the zero or
  rest position i.e. length of the electrical field
  vector at the wave maximum.

19
Definition of terms2

• Relationship between wave parameters velocity
  (c) Frequency(?) x wavelength(?)
• c is the velocity of light 3 x 1010cm/s or 3 x
  108m/s)
• Wavelength 1/wavenumber
• Wavenumber 1/? Frequency/velocity

20
Definition of terms3

• Period Time in seconds that it takes for
  successive maxima or minima to pass a point in
  space
• Refractive Index(?) Measures the extent of
  interaction between emr and the medium through
  which it is passing .

21
Excercise

• Problem 1 Calculate the wavenumber of a beam of
  IR radiation with a wavelength of 3µm.
• 1/ ? wavenumber 1/3 µm 1/3x10-6m-1 or
  1/3x10-4cm-1 3,333 cm-1 or 3.333 x 103cm-1
• Problem II The frequency of a radiation is
  3x1012s-1. Calculate the wavelength of the
  radiation. c 3x108m/s
• Answer 10-4m

22
Particulate or Quantum nature of emr

• Emr
• Considered to be made up of photons or stream of
  energy packets (quanta) travelling through space
  at a constant velocity c, when in a vacuum.
• The energy of a photon is related to the
  frequency or wavelength by the expression
• E hv hc/? (h Planks constant 6.63x
  10-34Js)
• frequency (v) and velocity (c) are directly
  proportional to photon energy while wavelength
  (?) is inversely proportional to energy
• A photon has zero mass and energy hv.

23
ABSORPTION OF RADIATION 

• Energy can be absorbed through three processes.
• They all involve raising the molecule to a higher
  internal energy level.
• Increase in energy is equal to absorbed energy.
• E hv
• Particles exist in the lowest energy state
  (ground state) at room temperature.

24
Absorption radiation processes

• Rotational Transition This gives information
  about the rotational energy level of molecules.
• Molecules rotate about various axes, when they
  absorb e.m.r. and are raised to a higher
  rotational energy level.
• Occurs in the far IR and microwave regions.
• Rotational transition produces only discrete
  absorption lines in the spectrum with wavelength
  of each line corresponding to a particular
  transition.

25
Absorption radiation processes2

• Vibrational transition Atoms or groups within
  the molecule vibrate relative to each other.
• Energy of vibration occurs at definite quantized
  levels.
• The molecule may absorb a discrete amount of
  energy and be raised to a higher vibrational
  energy level in a vibrational transition.
• can occur in addition to rotational transition.

26
Absorption radiation processes3

• Numerous discreet transitions are obtained giving
  rise to a spectrum of peaks or envelops of
  unresolved fine structure.
• The wavelength at which these peaks occur can be
  related to the vibrational modes within the
  molecule.
• Gives information about the molecular structure
  of a compound.
• Occurs in the near IR region.

27
Absorption radiation processes4

• Electronic transition Electrons of a molecule
  may be raised to a higher electronic energy which
  corresponds to electronic transitions.
• Occurs in the UV and V regions.
• A spectrum of broad bands of absorbed wavelength
  is obtained.

28
(No Transcript)
29
SPECTROMETRY INSTRUMENTATION

• Basic components of a spectrometer
• Source of radiation
• Spectral selection device (Monochromator)
• Cell
• Detector
• Read out device

30
Source of radiation

• A source of radiation must meet the following
  requirements
• have high intensity
• stable with little or no short and long term
  drifts.
• should be relatively cheap.
• easily available
• cover a wide spectrum

31
Type of Spec. Type of Spec. Example of Radiation Source Notes
Absorption Colorimeter Xenon lamp etc
Photometer Tungsten filament lamp The tungsten filament is an excellent source of visible and near-IR light. It operates at atemp of 2900K and produces useful radiation in the range of 320-2500nm which covers the entire Vis., parts of Ir and UV region. Do not have enough output in the mid-IR region(4000-200cm-1) to be used for IR spec. The deuterium arc lamp is employed for UV spec. Here, the electric discharge causes D2 to dissociate and emit UV light over the range of 160-375nm.
Spectrophotometer UV-Vis Deuterium arc lamp, Tungsten filament lamp The tungsten filament is an excellent source of visible and near-IR light. It operates at atemp of 2900K and produces useful radiation in the range of 320-2500nm which covers the entire Vis., parts of Ir and UV region. Do not have enough output in the mid-IR region(4000-200cm-1) to be used for IR spec. The deuterium arc lamp is employed for UV spec. Here, the electric discharge causes D2 to dissociate and emit UV light over the range of 160-375nm.
Infrared Electric heating of an inert solid e.g silicon carbide called Globar(heated to 150K) or Nernst glower(Zr and Yt oxides) The tungsten filament is an excellent source of visible and near-IR light. It operates at atemp of 2900K and produces useful radiation in the range of 320-2500nm which covers the entire Vis., parts of Ir and UV region. Do not have enough output in the mid-IR region(4000-200cm-1) to be used for IR spec. The deuterium arc lamp is employed for UV spec. Here, the electric discharge causes D2 to dissociate and emit UV light over the range of 160-375nm.
Atomic absorption spectral methods Flame and non flame AAS Hollow cathode lamp
Atomic emission spectral methods Arc spec. Sample in arc
Atomic emission spectral methods Spark spec. Sample in Spark
Atomic emission spectral methods Flame emission spec. Sample in flame
Atomic emission spectral methods Atomic fluorescence spec. Discharge lamp
32
Spectral Selection Device.

• Selects the small portion of wavelength which is
  required for absorption by the analyte from a
  wide range of spectrum.
• Examples are optical filters, prisms and
  diffraction gratings.
• Prisms and diffraction gratings are called
  monochromators and they are used to disperse
  light into its component wavelength.
• A light of one wavelength is said to be
  monochromatic.

33
Spectral Selection Device2

• Optical filters
• Functions by absorbing large portions of the
  spectrum while transmitting relatively limited
  wavelength regions.
• Used when the spectral purity of the radiation is
  not important in colorimeters, photometers,
  emission spec. etc.
• A colorimeter usually comes with a number of
  filters and the appropriate one is selected.
• The filter is normally the colour complement of
  the solution of the analysis.
• Sensitivity of the measurement depends on the
  filter.
• Advantages include (1) simplicity (2) low cost
  (3) high light transmittance.

34
Spectral Selection Device3

• Monochromator All monochromators contain
• an entrance slit which provides a narrow image
  of radiation source
• a collimating lens or mirror to produce a
  parallel beam of radiation
• a prism or grating as a dispersing element
• a focusing element which projects a series of
  rectangular images of the entrance slit upon a
  plane surface
• exit slit which isolates the desired spectral
  band.
• the width of the exit slit determines what range
  of wavelengths is passed on to the sample. The
  narrower the slit width, the narrower is the
  bandwidth(range of wavelength) emerging from the
  monochromator.

35
Types of monochromator
Two types of monochromators (a) Bunsen
prism monochromator, and (b) Czerney-Tumer
grating monochromator.
36
Spectral Selection Device3

• Prisms
• Radiation is admitted through an entrance slit,
  collimated by a lens and then strikes the surface
  of the prism at an angle.
• Refraction occurs at both faces of the prism.
• The dispersed radiation is then focused on a
  slightly curved surface containing the exit slit.
  
• The desired wavelength can be caused to pass
  through this slit by rotation of the prism.

37
Spectral Selection Device4

• Prisms (Contd.)
• The spectral purity of the radiation energy is
  determined mainly by the dispersion character of
  the prisms.
• The angular dispersion of prism is a function of
  temp.
• When light passes through a prism the light bends
  and the different colors that make up white light
  become separated.
• Each color has a particular wavelength and each
  wavelength bends at a different angle.

38
Spectral Selection Device5

• Diffraction gratings
• consist of a series of closely spaced parallel
  grooves coated with aluminum to make it
  reflective.
• the aluminum top is protected with a thin layer
  of silica (SiO2 ) to prevent the metal surface
  from tarnishing due to oxidation which would
  reduce its reflectivity.
• gratings give exceptionally high resolutions of
  spectral lines.
• Dispersion power better than prism.

39
Sample Containers

• Sample Containers
• usually called cells or cuvette.
• The sample container should not absorb the
  radiation.
• The most common cuvettes for measuring Vis and UV
  spectra are 1.00cm pathlength .
• Glass cells are suitable for Visible light
  measurement but not for UV spec. Quartz is
  transparent through the normally accessible UV-V
  region.
• For IR spec. liquid samples use cells commonly
  constructed of NaCl, KBr which transmit IR
  radiation. Quartz is used in near IR
• For solid sample, they are ground into fine
  powder, mixed with nujol. The mull obtained is
  pressed between two IR windows (KBr) or the
  sample could be mixed directly in KBr

40
Sample Containers2

• reference sample a suitable reference sample is
  required for qualitative spec.
• Must be a solvent or a reagent blank containing
  all reagents (except the analyte) in a cell
  identical to the sample cell.
• For atomic spec., no special sample containers
  are used since the samples have to be atomized.
• For non-flame AES, sample is placed on the
  electrode.
• For FAES and FAAS sample is aspirated.
• For non-flame AAS, the sample is pipetted unto
  heated surface.

41
Detector

• Detector
• Used to turn a level of illumination or photons
  into an electrical signal so that the samples
  transmittance or reflectance can be measured.
• Type determined by region of measurement
• Requirements of a detector
• must respond over a broad wavelength range
• sensitive to low levels of radiant power
• Rapid response to the radiation
• produce an electrical signal that can be readily
  amplified
• have a relatively low noise level.
• Types of Detector
• Photon detector e.g. phototube and
  photomultiplier
• Thermal detector e.g. thermocouple and bolometer

42
Detector2

• Phototube
• Made of a photocathode in the form of a half
  cylinder with an anode rod at its axis and it is
  enclosed in a glass envelope or silica.
• When a photon enters the window of the tube and
  strikes the cathode , electron is emitted
• The electron is attracted to the anode resulting
  in a flow of current which can be amplified and
  measured.
• The current flow is directly proportional to the
  incident radiation
• Used majorly in single beam spectrometer.
• Has the advantages of
• (1) offering good sensitivity (2) enabling
  modest slit width (3) long life (4) good
  stability (5) requires simple electronics.

43
Detector3

• Photomultiplier (PMT)
• More sensitive than a phototube for UV-V regions
  and used in most double beam scanning
  spectrophotometer.
• made up of photocathode which has an additional
  electrode (called dynode) held at about 70-100V
  positive with respect to the cathode, which
  attracts the photoelectrons.
• The dynode is coated with antimony, cesium or
  beryllium-copper alloys.
• It emits several secondary electrons, usually
  four or five for each photoelectron that strikes
  it.
• These emitted electrons are attracted to a 2o
  dynode with a further current increase and so on
  for more dynodes where even more electrons are
  knocked off and accelerated towards a third
  dynode.

44
Detector4

• Photomultiplier (PMT) (Contd.)
• process is repeated several times with the result
  that more than 106 electrons are finally
  collected for each photon (photoelectron)
  striking the first surface.
• Electrical amplification of PMT final output
• Spectrometers using phototubes and
  photomultiplier tubes are called
  SPECTROPHOTOMETERS and the measurement is known
  as SPECTROPHOTOMETRY

45
Detector5

• Problems of PMT
• Photomultiplier tubes are easily damaged by
  exposure to strong radiation and can only be used
  for measurement of low radiation energy.
• This can lead to either saturation of the
  detector or permanent damage of either the
  photocathode or dynode surface.
• can be avoided by using a spectrometer having a
  solenoid or mechanically operated shutter which
  interrupts the light beam.

46
Detector6

• Thermocouple
• Infrared radiation is detected by measuring the
  temperature rise of a blackened material placed
  in the path of beam.
• Transduces heat to electrical signals
• Thermocouple - a junction between two different
  electrical conductors
• Thermopile - a group of thermocouples

47
Detector7

• Bolometer
• Has a conducting element whose electrical
  resistance varies with temperature
• Made from thin strips of metal e.g. Ni or Pt,
• or semiconductors consisting of oxides of Ni or
  Co (called thermistors)

48
Readout devices

• Analogue meter Value displayed is proportional
  to the magnitude of signal from the detector and
  it is proportional to the concentration of the
  analyte.
• Digital meter Shows actual value
• Chart recorder keeps permanent records of the
  signals and also gives record of background
  noise.
• Computer readout
• Camera e.g. in AES.

49
Design of Spectrometer

• Two main designs
• Single beam
• Double beam
• Single beam designs are very simple and have very
  efficient intensity with high resolution
• Double beam design is well suited for continuous
  recording of absorption spectra

50
Schematic diagram of a single beam
spectrophotometer
51
Schematic diagram of a double beam scanning
spectrophotometer
52
Design of Spectrometer2

• Double beam design
• Takes continuous measurement of the light
  emerging from the sample and the reference cells.
  
• Incident beam is passed alternately through the
  sample and reference cuvette by the rotating
  beam.
• PMT develops alternating current
• output of the detector is proportional to the
  ratio of intensities of the two beams (sample and
  reference)
• The ratio is proportional to the absorbance

53
Design of Spectrometer3

• Advantages of double beam instrument
• compensates for changes in lamp intensity
• instrumental variations affect sample and
  reference similarly.
• reduction in manual manipulations
• However, the double beam system is more complex
  resulting in a loss of light efficiency.

54
ULTRAVIOLET-VISIBLE SPECTROPHOTOMETRY

• Electronic Spectra and Molecular structure
• Principle Absorption of emr in the UV-V regions
  of the spectrum resulting in changes in the
  electronic structure of ions and molecules.
• The electronic transitions are due to the
  absorption of radiation by specific types of
  groups, bonds and functional groups within the
  molecule.
• The wavelength of absorption and intensity are
  dependent on the type of bonds.

55
Electronic Spectra and Molecular structure

• Origin of Absorption Spectra
• The absorption of radiation is due to molecules
  containing electrons which can be raised to
  higher energy levels by the absorption of
  radiation (electronic spectroscopy).
• Electrons in a molecule can be classified into
  four types
• Closed - shell electrons (not involved in
  bonding)
• Electrons with a single bond (s bond).
• These need radiations of high energy and short
  wavelength e.g.
• (- CH2-CH2-) found in saturated hydrocarbons.

56
Electronic Spectra and Molecular structure2

• Paired non bonding outer electrons (n) on atoms
  such as Cl, O or N (lone pairs).
• This can be excited by UV-V radiation.
• electrons in double or triple bonds(p orbitals).
• Readily excited and account for majority of
  electronic spectra in the UV-V region.
• absorption of radiation results in electronic
  transition to an anti bonding orbital.

57
Possible electronic transitions of p, s, and n
electrons are
58
An Electronic Spectrum
59
An Electronic Spectrum(Skoog et al., 2004)
60
ABSORPTION BY CHROMOPHORES

• The absorbing groups in a molecule are known as
  CHROMOPHORES (any functional group that absorbs
  emr whether or not a colour is thereby produced).
  
• A molecule containing a chromophore is called a
  CHROMOGEN.
• Chromophores are responsible for absorptions in
  the UV-V regions e.g. C C, C O, - N N-,R-NO2
  e.t.c.
• E.g. ketones exhibit n-p and p-p transitions.

61
Absorption Xteristics of some typical chromophores

• Chromophore Example Transition ?max
  
• C C ethylene p p 190
• C 0 acetone p p 270
• n - p 285
  
• -N N - azo p p 285-400
• Benzene p p 200

62
ABSORPTION BY CHROMOPHORES2

• Enhancement of absorption of radiation
• The positions (wavelength, ? ) and intensities of
  the absorption bands are affected by
• substituents close to the chromophores
• conjugation with other chromophores
• Solvent

63
Enhancement of absorption Effects of
Substituents

• An auxochrome is defined as a group of atoms
  that could enhance the colour imparting
  properties of a chromophore without being itself
  a chromophore.
• possesses electron-donating properties
• Examples are -OH, OR, -NH2, -NR2, etc.
• Effects of auxochrome
• Bathochromic shift (red shift) - displacement of
  the absorption maximum to a longer wavelength

64
Enhancement of absorption Effects of
Substituents2

• Effects of auxochrome (Contd.)
• Hypsochromic shift(blue shift) a shift to a
  shorter wavelength .
• Hyperchromism increased intensity of absorption
  .
• Hypochromism reduction of absorption intensity.

65
Calculation of ? max

• 
  
  ?(nm)
• Base value for heteronuclear diene C O
  234
• homonuclear diene C C
  254
• Increments
• 
  
  
• Double bond extending 30
• Alkyl sub or ring residue 5
• Exocyclic double bond 5
• Polar gp OAC 0
• OAlk
  6
• SAlk
  
  30
• Cl,Br
  
  5
  
• N (Alk)
  
  60
• solvent
  0
• Calculated ? max
•  

66
Enhancement of absorption Effects of Conjugation

• Conjugation multiple bonds are separated by
  just one single bond each. E.g.
• C C C C -
  C C
• Ethylene (175nm)
  1,3-butadiene(217nm)
• The double bond in ethylene is localized while it
  is not in 1, 3 butadiene.
• Overlap of the p orbitals and reduction in the
  energy gap between adjacent orbitals give rise to
  bathochromic effect in 1, 3 butadiene.

67
(No Transcript)
68
Enhancement of absorption Effects of
Conjugation2

• Absorption by aromatic compounds Aromatic
  compounds also exhibit conjugation.
• As substituted groups are added to the benzene
  ring, bathochromic shift and increase in
  intensity results e.g OH,-OR,-NH2.
• Polynuclear compounds have increased conjugation
  and absorb at longer ? (e.g.indicator dyes ).
• They absorb in the visible region. Loss of proton
  or electron will change electron distribution,
  hence colour change.
• Ph- H2O ? Hph OH-

69
Enhancement of absorption Effects of Solvents

• Solvent effect
• spectrum of the species is affected by nature of
  solvent in which it is dissolved
• Peaks resulting from n -p transitions are
  shifted to shorter wavelengths (blue shift) with
  increasing solvent polarity.

70
Solvents for UV

• Water 205
• CH3C?N 210
• C6H12 210
• Ether 210
• EtOH 210
• Hexane 210
• MeOH 210
• Dioxane 220

• THF 220
• CH2Cl2 235
• CHCl3 245
• CCl4 265
• benzene 280
• Acetone 300
• Various buffers for HPLC, (check before using)

71
Level of participation!!!

• What compounds show UV spectra?
• Think of any unsaturated compounds Conjugated
  double bonds are strong absorbers
• Transition metal complexes, inorganics
• Highlight factors that can enhance absorption by
  chromophores
• Auxochromes, conjugation and solvent

72
Absorption process

• When light is absorbed by a sample, the radiant
  power or intensity of the beam is decreased.
• The radiant intensity P refers to the energy per
  second per unit area of the beam.
• Radiant power Po strikes a sample of pathlength
  b.
• The intensity of the beam emerging from the other
  side of the sample is P.
• Some of the light may be absorbed by the sample
  hence, P lt Po.

73
Beer-Lambert Law

• The Beer-Lamberts Law states that absorbance (A)
  is proportional to the (i)concentration, c (ii)
  pathlength, b of the absorbing specie (analyte).
• A?c
• A?b
• A ecl
• Linear absorbance with increased
  concentration--directly proportional
• Makes UV useful for quantitative analysis and in
  HPLC detectors
• Must demonstrate linearity in validating response
  in an analytical procedure.

74
(No Transcript)
75
Transmittance and Absorbance

• Relevant expressions for solving quantitative
  problems
• T P/P0
• T P/P0 x100 T x100
• T T/100
• A log 1/T log 100/T
• A log100-log T
• A 2- log T
• T Antilog (2 A)
• Transmittance and absorbance range
• Transmittance range is between 0 and 1
• Transmittance (0-100)
• Absorbance range (0 - infinity)
• Absorbance gt 2 measured with difficulty in
  practice (a thinner cell or dilute sample used in
  such situations)

76
Beer-Lambert Law Limitations

• Real limitations to Beer-Lamberts Law
• Holds in dilute solutions (0.01M)
• Above a certain concentration the linearity
  curves down, loses direct proportionality--Due to
  molecular associations at higher concentrations.
  

77
Beer-Lambert Law Limitations2

• Instrumental Deviations
• Instability of light source
• Fluctuations in power supply voltage
• Non linear response of detector/ amplifier system
• Use of polychromatic light
• Stray light

78
Beer-Lambert Law Limitations3

• Chemical Deviations
• Shift in position of a chemical or physical
  equilibrium E.g.
• C6H5COOH H2O C6H5COO- H3O
• ?max 273nm ?max
  268nm
• e273 970 e268
  560

79
Beer-Lambert Law Limitations4

• Solution to limitations of Beers law in simple
  acid base equilibrium
• Solution can be buffered
• Measurement can be made at wavelength
  corresponding to isosbestic point (wavelength at
  which the molar absorptivity of the
  interconvertible materials is same).
• Absorbance at the isosbestic point a function of
  overall concentration and not equilibrium
  concentration

80
(No Transcript)
81
Applications of Beer-Lambert

• Single Compounds
• Concentration of unknown solution (Cu) can be
  determined by comparing the absorbance of the
  unknown solution (Au) with that of a standard
  solution (conc-Cs) and (absorbance-As) containing
  the same absorbing species
• A ebc
• Concentration of unknown solution (Cu) is
  calculated using Au Cu
• As Cu

82
Worked example

• Problem I
• 0.1 solution of Cu(II)SO4 gives an absorbance of
  0.55. Determine (i) absorbance (ii) Transmittance
  when the solution is doubled. Cell path 1.00cm
• Au Cu x As 2x0.1 x 0.55 0.011
• Cs
  0.1
• T antilog (2 - A) 100 1.0257
  98.9743

83
Applications of Beer-Lambert2

• Application to mixtures
• A mixture containing two or more absorbing
  species at a given wavelength has its total
  absorbance to be the sum of all absorbing species
  (provided each sample obeys Beers law)
• Atotal A1 A2 A3An
• e1b1c1 e2b2c2 e3b3c3. enbncn
• Subscripts refer to absorbing components

84
Applications of Beer-Lambert3

• Mixture with components absorbing at different
  wavelengths
• Measurement of absorbance is taken at different
  wavelengths (provided all species obey Beers law
  at the wavelengths of measurement).
• Consider a mixture of two compounds a and b
  absorbing strongly at ?1 and ?2respectively, the
  concentration of each compound is calculated by
  solving 2 simultaneous Beer- Lambert law
  equations
• A ?1 ea?1bca eb?1bcb
• A ?2 ea?2bca eb?2bcb

85
Characteristics of Spectrophotometer

• Spectrophotometer has the following important
  characteristics
• Wide applicability Numerous inorganic and
  organic species absorb in the UV-Vis range.
• non-absorbing species can be analysed after
  conversion to absorbing species by suitable
• chemical treatments e.g. complexing agents
• High sensitivity. Analysis for concentration in
  the range of 10-4 to
• 10-5M are common.
• Moderate to high selectivity. Under some certain
  conditions, it may be possible to locate a
  wavelength region in which the analyte is the
  only absorbing components in a sample.
• Good accuracy Here the relative error in
  concentration measurements lies in the range of 1
  to 3. This can be decreased to a few tenths of a
  percent.
• Ease and convenience. They are easily and rapidly
  performed with the modern instruments

86
Procedure for carrying out spectrophotometric
analysis

• Before spectrophotometric analysis can be
  undertaken, it is necessary to develop a
  procedure
• Selection of Wavelength
• Variables that influence absorbance
• The nature of the solvent
• The pH of the solution
• The temperature
• High electrolyte concentration
• Presence of interfering species

87
Procedure for carrying out spectrophotometric
analysis2

• Determination of the relationship between
  absorbance and concentration
• Calibration Curve--- It is a way of determining
  the relationship between absorbance and
  concentration (at different concentrations).
• Steps to be taken
• Prepare various concentrations of standard
  solution of analyte
• Measure the absorbance of each standard solution
• Plot a calibration graph i.e. absorbance against
  concentration
• Measure absorbance of unknown solution
• Extrapolate the concentration of unknown solution
  from graph with measured absorbance

88
Standard calibration curve

• Standard addition---It is a way of determining
  the relationship between absorbance and
  concentration.
• standards are prepared.
• Increasing volume of the standards (starting from
  0mL) are added to fixed amount of the sample
  solution and made up to a uniform mark.
• This method eradicates matrix effects due to
  difference in standards and sample

89
Sources of Error in Ultraviolet-Visible Analysis

• Choosing the wavelength and bandwidth
• Beers law applies for monochromatic radiation.
• The wavelength of the maximum absorption should
  be used.
• This renders the effect of using light that is
  not perfectly monochromatic to insignificance,
  since the molar absorptivity is fairly constant
  over a small range of wavelength
• The monochromator bandwidth should be as large as
  possible but small compared with the band being
  measured giving a smaller signal to noise ratio.

90
Instrumental errors

• Reliability for most spectrophotometers is
  between A0.4-0.9.
• Too low absorbance means Io coming through the
  sample is smaller to the intensity coming through
  the reference, giving large relative uncertainty
  in the measured difference between them.
• Too high absorbance means too little light
  reaches the detector, (low signal-to- noise ratio
  and reduced precision).
• Instrumental noise (dark current noise) occurs
  which is caused by factors such as thermal motion
  of electrons in electronic components, noise in
  the readout device, etc

91
Instrumental errors2

• placement and cleaning of the cuvette
  (fingerprints, dust etc)
• stray light (light with wavelengths outside the
  narrow bandwidth) expected from the
  monochromator.
• The source of the stray light is majorly
  radiation source or improperly closed
  instruments.
• Others include errors arising from sample
  preparation, uncertainities in the wavelength
  setting and source fluctuations.