MOLECULAR ABSORPTION    SPECTROSCOPY :        THEORY,  INSTRUMENTATION &      APPLICATION       CHAPTER 2
COMPONENTS OF  INSTRUMENTS FOROPTICAL SPECTROSCOPY
General Design of Optical          InstrumentsAbsorptionEmission
Five Basic Optical Instrument Components1) Source – A stable source of radiant energy at the desired   wavelength (or  ra...
I. Sources of Radiation• Generate a beam of radiation that is stable and has  sufficient power.  A. Continuum Sources – em...
B. Line Sources – Emit a limited number lines orbands of radiation at specific wavelengths.- Used in atomic absorption spe...
II. Wavelength Selectors• Wavelength selectors output a limited,  narrow, continuous group of wavelengths  called a band.•...
A. Filters- Two types of filters:  1) Interference Filters  2) Absorption FiltersB. Monochromators- Wavelength selector th...
III. Radiation Transducer (Detectors)• Early      detectors     in      spectroscopic  instruments were the human eye,  ph...
A. Photon Detectors- Commonly useful in ultraviolet, visible, and nearinfrared instruments.- Several types of photon detec...
B. Thermal Detectors- Used for infrared spectroscopybecause photons in the IR region lackenergy to cause photoemission ofe...
IV. Sample Container• Sample containers, usually called cells or cuvettes must have  windows that are transparent in the s...
 Spectrometer  - is an instrument that provides information  about the intensity of radiation as a function  of wavelengt...
SUMMARY Types of source, sample holder and detector             for various EM region     REGION       SOURCE             ...
ULTRAVIOLET-VISIBLE  SPECTROSCOPY
In this lecture, you will learn:• Absorption process in UV/VIS region in  terms of its electronic transitions• Molecular s...
INSTRUMENTATION   Important components in a UV-Vis spectrophotometer                                                      ...
Instrumentation• UV-Visible instrument  1. Single beam  2. Double beam
Single beam instrument
• Single beam instrument -   One radiation source -   Filter/monochromator ( selector) -   Cells -   Detector -   Readout...
Single beam instrument• Disadvantages:  – Two separate readings has to be made on the    light. This result in some error ...
Double beam instrumentDouble-beam instrument with beams separated in space
• Double-beam instrument Advantages: 1. Compensate for all but most short-term    fluctuations in the radiant output of th...
ORGANIC               INORGANIC COMPOUNDS                SPECIES MOLECULAR SPECIES THATABSORB UV/VISIBLE RADIATION        ...
Definitions:• Organic compounds  – Chemical compound whose molecule contain carbon  – E.g. C6H6, C3H4• Inorganic species  ...
PERIODIC TABLE OFELEMENTS
ULTRAVIOLET-VISIBLE           SPECTROSCOPY• In UV/VIS spectroscopy, the transitions  which result in the absorption of EM ...
Molecular absorption• In molecules, not only have electronic  level but also consists of vibrational and  rotational sub-l...
Types of transitions• 3 types of electronic transitions  - ,  and n electrons  - d and f electrons  - charge transfer el...
What is σ,                       and n electrons?                                                    single covalent bond...
Sigma () electron Electrons involved in single bonds such as  those between carbon and hydrogen in alkanes. These bonds...
Pi () electron• Electrons involved in double and triple  bonds (unsaturated).• These bonds involve a pi () bond.• For ex...
• Examples of organic molecules containing   bonds.                       H      CH2CH3                             CH3  ...
n electron• Electrons that are not involved in  bonding between atoms are called n  electrons.• Organic compounds containi...
• Examples of organic molecules with non-  bonding electrons.                           ..     : NH2                 O:   ...
ABSORPTION BY ORGANIC COMPOUNDS• UV/Vis absorption by organic compounds  requires that the energy absorbed  corresponds to...
Electronic energy levels diagram           *                                       Antibonding                           ...
Electronic transitions               *   In alkanes               *   In alkenes, carbonyl compounds, alkynes, azo  ...
Electronic transitions       * transitions• The energy required to induce a          *  transition is large (see the a...
n        * transitions- Saturated compounds containing atoms with unshared  electron pairs (non-bonding electrons).- Comp...
Some examples of absorption due to       n   * transitions       Compound   max (nm)   maxH2O                 167      ...
n      * transitions- Unsaturated compounds containing atoms  with unshared electron pairs (nonbonding  electrons)- These...
      * transitions- Compounds with unsaturated functional  groups to provide the  orbitals.- These result in some of t...
Examples n       * and    *             H         O      H      C     C             H         H       * at 180 nm   ...
MOLECULAR SPECIES THAT ABSORB      UV/VISIBLE RADIATION (A) Absorption by organic compounds2 types of electrons are respon...
Absorption by organic compounds• The shared electrons in single bonds, C-C or  C-H ( electrons) are so firmly held.  Ther...
Absorption by organic compounds      CHROMOPHORES Unsaturated organic functionalgroups that absorb in the UV/VIS          ...
Typical organic functional groups  that serve as chromophores Chromophores      Chemical structure   Type of transition Ac...
Absorption by organic compounds            AUXOCHROME• Groups such as –OH, -NH2 & halogens  that attached to the double bo...
Effect of Multichromophores         on Absorption• More chromophores in the same molecule  cause bathochromic effect ( shi...
Absorption by organic compounds• Factors that influenced the : i) Solvent effects (shift to shorter : blue    shift) ii)...
Absorption spectra for typical organic            compounds
Important terminologies• Hypsochromic shift (blue shift)  - Absorption maximum shifted to shorter • Bathochromic shift (r...
Terminology for Absorption Shifts          Nature of Shift            Descriptive TermTo Longer Wavelength        Bathochr...
(B) Absorption by inorganic species• Involving d and f electrons absorption• 3d & 4d electrons  - 1st and 2nd transition m...
Absorption spectra of some transition-metal ions and rare                       earth ions Most transition metal ions are ...
Absorption by inorganic species• 4f & 5f electrons  - Ions of lanthanide and actinide elements  - Their spectra consists o...
(C) Charge transfer absorption Absorption involved transfer of electron from  the donor to an orbital that is largely  as...
Absorption spectra of aqueous charge transfer                 complexes
Quantitative Analysis• The fundamental law on which absorption  methods are based on Beer’s Law (Beer-  Lambert Law).
Measuring Absorbance• You must always attempt to work at the  wavelength of maximum absorbance  (max).• This is the point...
Quantitative Analysis• Calibration curve method• Standard addition method
• Calibration curve method - A general method for determining the concentration of a substance in an unknown sample by com...
Standard Calibration Curve          AbsorbanceHow to measure the concentration of unknown?   • Practically, you have measu...
How to produce standard calibration curve                                   Absorbance• Prepare a series of  standard solu...
• The slope of the line, m:          m = y2 – y1              x2 – x1• The intercept, b:          b = y – mx• Thus, the eq...
Concentration, x    y = mx + b                     5                    10                    15                    20    ...
Standard addition method- used to overcome matrix effect- involves adding one or more incrementsof a standard solution to ...
Standard Addition PlotAbsorbance
How to produce standard              addition curve?1. Add same quantity of unknown sample to a series of flasks.2. Add va...
Standard Addition                  MethodsSingle-point standard      Multiple standard   addition method         addition ...
Standard addition- if Beer’s Law is obeyed,A = bVstdCstd       +    bVxCx        Vt                 Vt = kVstdCstd     +...
Standard Addition- Plot a graph: A vs Vstd   A = mVstd + bwhere the slope m and intercept b are:m = kCstd    ;     b = kVxCx
• Cx can be obtained from the ratio of these  two quantities: m and b     b = kVxCx     m kCstd     Cx = bCstd          mVx
Example:• 10 ml aliquots of raw-water sample were  pipetted into 50.0 ml volumetric flasks. Then,  0.00, 5.00, 10.00, 15.0...
The results obtained:      Vol. of std added   Absorbance             (ml)             (A)             0              0.21...
Absorbance vs Vol. of std added                                    1.2                                     1              ...
• The unknown concentration of the analyte  in the solution is then calculated:    Csample = -(Vstd)0Cstd                V...
SELF-EXERCISEThe chromium in an aqueous sample was determined by pipetting10.0 ml of the unknown into each of 50.0 mL volu...
Visible SpectroscopyThe portion of the EM spectrum from 400-800 isobservable to humans- we (and some other mammals)have th...
Visible SpectroscopyWhen white (continuum of λ)light passes through, or isreflected by a surface, those λsthat are absorbe...
Visible SpectroscopyOrganic compounds that are “colored” are typically those withextensively conjugated systems (typically...
Visible Spectroscopy                               lycopene, max = 474 nm                                        O       ...
CHM260 - UV-VIS
CHM260 - UV-VIS
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CHM260 - UV-VIS

  1. 1. MOLECULAR ABSORPTION SPECTROSCOPY : THEORY, INSTRUMENTATION & APPLICATION CHAPTER 2
  2. 2. COMPONENTS OF INSTRUMENTS FOROPTICAL SPECTROSCOPY
  3. 3. General Design of Optical InstrumentsAbsorptionEmission
  4. 4. Five Basic Optical Instrument Components1) Source – A stable source of radiant energy at the desired wavelength (or  range).2) Sample Container – A transparent container used to hold the sample (cells, cuvettes, etc).3) Wavelength Selector – A device that isolates a restricted region of the EM spectrum used for measurement (monochromators, prisms & filters).4) Detector/Photoelectric Transducer – Converts the radiant energy into a useable signal (usually electrical).5) Signal Processor & Readout – Amplifies or attenuates the transduced signal and sends it to a readout device as a meter, digital readout, chart recorder, computer, etc.
  5. 5. I. Sources of Radiation• Generate a beam of radiation that is stable and has sufficient power. A. Continuum Sources – emit radiation over a broad wavelength range and the intensity of the radiation changes slowly as a function of wavelength. This type of source is commonly used in UV, visible and IR instruments. - Deuterium lamp is the most common UV source. - Tungsten lamp is the most common visible source. - Glowing inert solids are common sources for IR instruments.
  6. 6. B. Line Sources – Emit a limited number lines orbands of radiation at specific wavelengths.- Used in atomic absorption spectroscopy- Usually provide radiation in the UV and visibleregion of the EM spectrum.- Types of line source: 1) Hollow cathode lamps 2) Electrodeless discharge lamps 3) Lasers-Light – amplification by stimulatedemission of radiation
  7. 7. II. Wavelength Selectors• Wavelength selectors output a limited, narrow, continuous group of wavelengths called a band.• Two types of wavelength selectors: 1) Filters 2) Monochromators
  8. 8. A. Filters- Two types of filters: 1) Interference Filters 2) Absorption FiltersB. Monochromators- Wavelength selector that can continuouslyscan a broad range of wavelengths- Used in most scanning spectrometersincluding UV, visible, and IR instruments.
  9. 9. III. Radiation Transducer (Detectors)• Early detectors in spectroscopic instruments were the human eye, photographic plates or films. Modern instruments contain devices that convert the radiation to an electrical signal.• Two general types of radiation transducers: a. Photon detectors b. Thermal detectors
  10. 10. A. Photon Detectors- Commonly useful in ultraviolet, visible, and nearinfrared instruments.- Several types of photon detectors are available: 1. Vacuum phototubes 2. Photomultiplier tubes 3. Photovoltaic cells 4. Silicon photodiodes 5. Diode array transducers 6. Photoconductivity transducers
  11. 11. B. Thermal Detectors- Used for infrared spectroscopybecause photons in the IR region lackenergy to cause photoemission ofelectrons.- Three types of thermal detectors: 1. Thermocouples 2. Bolometers 3. Pyroelectric transducers
  12. 12. IV. Sample Container• Sample containers, usually called cells or cuvettes must have windows that are transparent in the spectral region of interest.• There are few types of cuvettes: - quartz or fused silica - silicate glass - crystalline sodium chlorideQuartz or fused silica- required for UV and may be used in visible regionSilicate glass- Cheaper compared to quartz. Used in UV.Crystalline sodium chloride- Used in IR.
  13. 13.  Spectrometer - is an instrument that provides information about the intensity of radiation as a function of wavelength or frequency. Spectrophotometer - is a spectrometer equipped with one or more exit slits and photoelectric transducers that pemits the determination of the ratio of the radiant power of two beams as a function of wavelength as in absorption spectroscopy.
  14. 14. SUMMARY Types of source, sample holder and detector for various EM region REGION SOURCE SAMPLE DETECTOR HOLDERUltraviolet Deuterium lamp Quartz/fused silica Phototube, PM tube, diode arrayVisible Tungsten lamp Glass/quartz Phototube, PM tube, diode arrayInfrared Nernst glower Salt crystal e.g. Thermocouples, (rare earth oxides crystal sodium bolometers or silicon carbide chloride glowers)
  15. 15. ULTRAVIOLET-VISIBLE SPECTROSCOPY
  16. 16. In this lecture, you will learn:• Absorption process in UV/VIS region in terms of its electronic transitions• Molecular species that absorb UV/VIS radiation• Important terminologies in UV/VIS spectroscopy
  17. 17. INSTRUMENTATION Important components in a UV-Vis spectrophotometer 5 1 2 3 4 Signal Source Sample  selector Detector processor lamp holder & readoutUV region:-Deuterium lamp; Quartz/fused silica Prism/monochromator Phototube,H2 discharge tube PM tube, diode arrayVisible region:- Tungsten lamp Glass/quartz Prism/monochromator Phototube, PM tube, diode array
  18. 18. Instrumentation• UV-Visible instrument 1. Single beam 2. Double beam
  19. 19. Single beam instrument
  20. 20. • Single beam instrument - One radiation source - Filter/monochromator ( selector) - Cells - Detector - Readout device
  21. 21. Single beam instrument• Disadvantages: – Two separate readings has to be made on the light. This result in some error because the fluctuations in the intensity of the light do occur in the line voltage, the power source and in the light bulb btw measurements. – Changing of wavelength is accompanied by a change in light intensity. Thus spectral scanning is not possible.
  22. 22. Double beam instrumentDouble-beam instrument with beams separated in space
  23. 23. • Double-beam instrument Advantages: 1. Compensate for all but most short-term fluctuations in the radiant output of the source as well as for drift in the transducer and amplifier. 2. Compensate for wide variations in source intensity with . 3. Continuous recording of transmittance or absorbance spectra.
  24. 24. ORGANIC INORGANIC COMPOUNDS SPECIES MOLECULAR SPECIES THATABSORB UV/VISIBLE RADIATION CHARGE TRANSFER
  25. 25. Definitions:• Organic compounds – Chemical compound whose molecule contain carbon – E.g. C6H6, C3H4• Inorganic species – Chemical compound that does not contain carbon. – E.g. transition metal, lanthanide and actinide elements. – Cr, Co, Ni, etc• Charge transfer – A complex where one species is an electron donor and the other is an electron acceptor. – E.g. iron (III) thiocyanate complex
  26. 26. PERIODIC TABLE OFELEMENTS
  27. 27. ULTRAVIOLET-VISIBLE SPECTROSCOPY• In UV/VIS spectroscopy, the transitions which result in the absorption of EM radiation in this region are transitions between electronic energy levels.
  28. 28. Molecular absorption• In molecules, not only have electronic level but also consists of vibrational and rotational sub-levels.• This result in band spectra.
  29. 29. Types of transitions• 3 types of electronic transitions - ,  and n electrons - d and f electrons - charge transfer electrons
  30. 30. What is σ,  and n electrons? single covalent bonds (σ)H + O + H H O H or H O H lone pairs(n) O C O or O C O double bonds () N N or N N triple bond ()
  31. 31. Sigma () electron Electrons involved in single bonds such as those between carbon and hydrogen in alkanes. These bonds are called sigma () bonds. The amount of energy required to excite electrons in  bond is more than UV photons of wavelength. For this reason, alkanes and other saturated compounds (compounds with only single bonds) do not absorb UV radiation and therefore frequently very useful as transparent solvents for the study of other molecules. For example, hexane, C6H14.
  32. 32. Pi () electron• Electrons involved in double and triple bonds (unsaturated).• These bonds involve a pi () bond.• For exampel: alkenes, alkynes,conjugated olefins and aromatic compounds.• Electrons in  bonds are excited relatively easily; these compounds commonly absorb in the UV or visible region.
  33. 33. • Examples of organic molecules containing  bonds. H CH2CH3 CH3 C C H H C H propyne C C C C H C H H H H H C Cethylbenzene benzene C C H H H 1,3-butadiene
  34. 34. n electron• Electrons that are not involved in bonding between atoms are called n electrons.• Organic compounds containing nitrogen, oxygen, sulfur or halogens frequently contain electrons that re nonbonding.• Compounds that contain n electrons absorb UV/VIS radiation.
  35. 35. • Examples of organic molecules with non- bonding electrons. .. : NH2 O: C R H3C H C C .. : Br .. Haminobenzene Carbonyl compound If R = H  aldehyde 2-bromopropene If R = CnHn  ketone
  36. 36. ABSORPTION BY ORGANIC COMPOUNDS• UV/Vis absorption by organic compounds requires that the energy absorbed corresponds to a jump from occupied orbital to an unoccupied orbital of greater energy.• Generally, the most probable transition is from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO).
  37. 37. Electronic energy levels diagram * Antibonding Unoccupied levels * Antibonding   * n  * n  *   *Energy n Nonbonding  Bonding Occupied levels  Bonding
  38. 38. Electronic transitions   * In alkanes   * In alkenes, carbonyl compounds, alkynes, azo compoundsIncreasingenergy n  * In oxygen, nitrogen, sulfur and halogen compounds n  * In carbonyl compounds
  39. 39. Electronic transitions  * transitions• The energy required to induce a  * transition is large (see the arrow in energy level diagram).• Never observed in the ordinarily accessible ultraviolet region.• This type of absorption corresponds to breaking of C-C, C-O, C-H, C-X, ….bonds
  40. 40. n * transitions- Saturated compounds containing atoms with unshared electron pairs (non-bonding electrons).- Compounds containing O, S, N and halogens can absorb via this type of transition.- Absorption are typically in the  range, 150 - 250 nm region and are not very intense.-  range: 100 – 3000 cm-1mol-1- Absorption maxima tend to shift to shorter  in polar solvents. e.g. H2O, CH3CH2OH
  41. 41. Some examples of absorption due to n * transitions Compound max (nm) maxH2O 167 1480CH3OH 184 150CH3Cl 173 200CH3I 258 365(CH3)2O 184 2520CH3NH2 215 600
  42. 42. n * transitions- Unsaturated compounds containing atoms with unshared electron pairs (nonbonding electrons)- These result in some of the most intense absorption in  range, 200 – 700 nm- Unsaturated functional group - to provide the  orbitals-  range: 10 – 100 Lcm-1mol-1
  43. 43.  * transitions- Compounds with unsaturated functional groups to provide the  orbitals.- These result in some of the most intense absorption in  range, 200 – 700 nm-  range: 1000 – 10,000 Lcm-1mol-1
  44. 44. Examples n * and  * H O H C C H H   * at 180 nm n  * at 290 nm
  45. 45. MOLECULAR SPECIES THAT ABSORB UV/VISIBLE RADIATION (A) Absorption by organic compounds2 types of electrons are responsible:i. Shared electrons that participate directly in bond formation ( and  bonding electrons)ii. Unshared outer electrons (nonbonding or n electrons)
  46. 46. Absorption by organic compounds• The shared electrons in single bonds, C-C or C-H ( electrons) are so firmly held. Therefore, not easily excited to higher E levels. Absorption (  *) occurs only in the vacuum UV region (  180 nm).• Electrons in double & triple bonds (electrons) are more loosely held. Therefore, more easily excited by radiation. Absorptions (   *) for species with unsaturated bonds occur in the UV/VIS region (   180 nm)
  47. 47. Absorption by organic compounds CHROMOPHORES Unsaturated organic functionalgroups that absorb in the UV/VIS region.
  48. 48. Typical organic functional groups that serve as chromophores Chromophores Chemical structure Type of transition Acetylenic -CC-   * Amide -CONH2   *, n  * Carbonyl >C=O   *, n  * Carboxylic acid -COOH   *, n  * Ester -COOR   *, n  * Nitro -NO2   *, n  * Olefin >C=C<   *
  49. 49. Absorption by organic compounds AUXOCHROME• Groups such as –OH, -NH2 & halogens that attached to the double bonded atoms cause the normal chromophoric absorption to occur at longer  (red shift).
  50. 50. Effect of Multichromophores on Absorption• More chromophores in the same molecule cause bathochromic effect ( shift to longer ) and hyperchromic effect (increase in intensity).• In conjugated chromophores, * electrons are delocalized over larger number of atoms. This cause a decrease in the energy of  * transitions and an increase in  due to an increase in probability for transition.
  51. 51. Absorption by organic compounds• Factors that influenced the : i) Solvent effects (shift to shorter : blue shift) ii) Structural details of the molecules
  52. 52. Absorption spectra for typical organic compounds
  53. 53. Important terminologies• Hypsochromic shift (blue shift) - Absorption maximum shifted to shorter • Bathochromic shift (red shift) - Absorption maximum shifted to longer 
  54. 54. Terminology for Absorption Shifts Nature of Shift Descriptive TermTo Longer Wavelength BathochromicTo Shorter Wavelength HypsochromicTo Greater Absorbance HyperchromicTo Lower Absorbance Hypochromic
  55. 55. (B) Absorption by inorganic species• Involving d and f electrons absorption• 3d & 4d electrons - 1st and 2nd transition metal series e.g. Cr, Co, Ni & Cu - Absorb broad bands of VIS radiation - Absorption involved transitions between filled and unfilled d-orbitals with energies that depend on the ligands, such as Cl-, H2O, NH3 or CN- which are bonded to the metal ions.
  56. 56. Absorption spectra of some transition-metal ions and rare earth ions Most transition metal ions are colored (absorb in UV-VIS) due to d  d electronic transitions
  57. 57. Absorption by inorganic species• 4f & 5f electrons - Ions of lanthanide and actinide elements - Their spectra consists of narrow, well- defined characteristic absorption peaks.
  58. 58. (C) Charge transfer absorption Absorption involved transfer of electron from the donor to an orbital that is largely associated with the acceptor. an electron occupying in a  or  orbital (electron donor) in the ligand is transferred to an unfilled orbital of the metal (electron acceptor) and vice-versa. e.g. red colour of the iron (III) thiocyanate complex
  59. 59. Absorption spectra of aqueous charge transfer complexes
  60. 60. Quantitative Analysis• The fundamental law on which absorption methods are based on Beer’s Law (Beer- Lambert Law).
  61. 61. Measuring Absorbance• You must always attempt to work at the wavelength of maximum absorbance (max).• This is the point of maximum response, so better sensitivity and lower detection limits.• You will also have reduced error in your measurement.
  62. 62. Quantitative Analysis• Calibration curve method• Standard addition method
  63. 63. • Calibration curve method - A general method for determining the concentration of a substance in an unknown sample by comparing the unknown to a set of standard sample of known concentration.
  64. 64. Standard Calibration Curve AbsorbanceHow to measure the concentration of unknown? • Practically, you have measure the absorbance of your unknown. Once you know the absorbance value, you can just read the corresponding concentration from the graph.
  65. 65. How to produce standard calibration curve Absorbance• Prepare a series of standard solution with known concentration.• Measure the absorbance of Calibration standard the standard solutions.• Plot the graph Abs vs concentration of std.• Find the “best’ straight line. Stock solution 100 ppm
  66. 66. • The slope of the line, m: m = y2 – y1 x2 – x1• The intercept, b: b = y – mx• Thus, the equation for the least-square line is: y = mx + b
  67. 67. Concentration, x y = mx + b 5 10 15 20 25• From the least-square line equation, you can calculatethe new y values by substituting the x value.• Then plot the graph.
  68. 68. Standard addition method- used to overcome matrix effect- involves adding one or more incrementsof a standard solution to sample aliquotsof the same size.- Each solution is diluted to a fixed volumebefore measuring its absorbance.
  69. 69. Standard Addition PlotAbsorbance
  70. 70. How to produce standard addition curve?1. Add same quantity of unknown sample to a series of flasks.2. Add varying amounts of standard (made in solvent) to each flasks, e.g. 0, 5, 10, 15 mL).3. Fill each flask to line, mix and measure.
  71. 71. Standard Addition MethodsSingle-point standard Multiple standard addition method addition method
  72. 72. Standard addition- if Beer’s Law is obeyed,A = bVstdCstd + bVxCx Vt Vt = kVstdCstd + kVxCxk is a constant equal to  b Vt
  73. 73. Standard Addition- Plot a graph: A vs Vstd A = mVstd + bwhere the slope m and intercept b are:m = kCstd ; b = kVxCx
  74. 74. • Cx can be obtained from the ratio of these two quantities: m and b b = kVxCx m kCstd Cx = bCstd mVx
  75. 75. Example:• 10 ml aliquots of raw-water sample were pipetted into 50.0 ml volumetric flasks. Then, 0.00, 5.00, 10.00, 15.00 and 20.00 ml respectively of a standard solution containing 10 ppm of Fe3+ were added to the flasks, followed by an excess of aqueous potassium thiocyanate in order to produce the red iron- thiocyanate complex. All the resultant solutions were diluted to volume and the absorbance of each solution was measured at the same.
  76. 76. The results obtained: Vol. of std added Absorbance (ml) (A) 0 0.215 5.00 0.424 10.00 0.625 15.00 0.836 20.00 1.040Calculate the concentration of Fe3+ (in ppm)in the raw-water sample
  77. 77. Absorbance vs Vol. of std added 1.2 1 0.8Absorbance b = 0.24 0.6 Slope, m = 0.0382 (Vstd)0 = -6.31 ml 0.4 0.2 0 -10 -5 0 5 10 15 20 25 Vol. of std Note: From the graph, extrapolated value represents the volume of reagent corresponding to zero instrument response.
  78. 78. • The unknown concentration of the analyte in the solution is then calculated: Csample = -(Vstd)0Cstd Vsample Cx = bCstd mVx
  79. 79. SELF-EXERCISEThe chromium in an aqueous sample was determined by pipetting10.0 ml of the unknown into each of 50.0 mL volumetric flasks.Various volumes of a standard containing 12.2 ppm Cr were addedto the flasks, following which the solutions were diluted to the mark. Volume of Volume of Absorbance unknown (mL) standard (mL) 10.0 0.0 0.201 10.0 10.0 0.292 10.0 20.0 0.378 10.0 30.0 0.467 10.0 40.0 0.554i) Plot a suitable graph to determine the concentration of Cr in theaqueous sample.
  80. 80. Visible SpectroscopyThe portion of the EM spectrum from 400-800 isobservable to humans- we (and some other mammals)have the adaptation of seeing color at the expense ofgreater detail. 400 500 600 700 800 , nm Violet 400-420 Indigo 420-440 Blue 440-490 Green 490-570 Yellow 570-585 Orange 585-620 Red 620-780
  81. 81. Visible SpectroscopyWhen white (continuum of λ)light passes through, or isreflected by a surface, those λsthat are absorbed areremoved from the transmittedor reflected light respectively.What is “seen” is thecomplimentary colors (thosethat are not absorbed).This is the origin of the “colorwheel”.
  82. 82. Visible SpectroscopyOrganic compounds that are “colored” are typically those withextensively conjugated systems (typically more than five).Consider b-carotene. b-carotene, max = 455 nm λmax is at 455 nm – in the far blue region of the spectrum . This is absorbed. The remaining light has the complementary color of orange.
  83. 83. Visible Spectroscopy lycopene, max = 474 nm O H N N H O indigoλmax for lycopene is at 474 nm – in the near blue region ofthe spectrum this is absorbed, the compliment is now red.λmax for indigo is at 602 nm – in the orange region of thespectrum. This is absorbed, the compliment is now indigo!
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