1. Instrumental methods of analysis. Photometry.
Lecture 12
Instrumental methods of analysis. Photometry.
Associate prof . L.V. Vronska
Associate prof . M.M. Mykhalkiv

2. Outline
Classification, advantages and lacks of physical-chemical methods of the analysis.
Optical methods of the analysis. Classification.
The fundamental law of absorption.
Electronic spectrum.
Photometric method of analysis: an essence, theoretical bases, usage in the pharmaceutical analysis.
Multiwave spectrophotometry .
Differential spectrophotometry.
The extraction-photometric analysis.
Photometric titration.

3. 1. Classification, advantages and lacks of physical-chemical methods (PCMA) of the analysis.
Physical and physical-chemical methods of the analysis are based on dependence application between measured physical properties of substances and qualitative (quantitative) composition

4. PCMA are divided on:
Optical methods are based on measurement of optical properties of substances.
Chromatographic methods are based on usage of ability of different substances to selective sorption.
Electrochemical methods are based on measurement of electrochemical properties of substances.
Radiometric methods are based on measurement of radioactive properties of substances.
Thermal methods are based on measurement of heat effects of substances.
Mass spectrometric methods are based on studying of the ionized fragments ("splinters") of substances.
Kinetic methods are based on measurement of dependence of speed of reaction from concentration of substance

5. Advantage of PCMA
High sensitivity - a low limit of detection (10-9 g) and definition
High selectivity
Rapid analysis methods
Automation and computerization is possibility
Analysis is possibility on distance
Possibility of the analysis without destruction of the sample
Possibility of the local analysis

6. Lacks of PCMA
Definition error is near ± 5 % (on occasion to 20 %), whereas - 0,01-0,005 % for gravimetry and 0,1-0,05 % for titrimetry
Reproducibility of results in separate methods is worse, than in classical methods of the analysis
It is necessary of usage of standards and standard solutions, graduation of equipment and plotting of calibration charts
Complexity of used equipment, its high cost, high cost of standard substances

7. 2. Optical methods of the analysis. Classification.
А) On investigated objects
The nuclear spectral analysis
The molecular spectral analysis

8. B) On the nature of interaction of electromagnetic radiation with substance
1. Absorption analysis
Atomic-absorption analysis
Molecular-absorption analysis
Turbidimetric analysis
2. The emissive spectral analysis
flame photometry
fluorescence analysis
The spectral analysis with usage of effect of combinational dispersion of light

9. 3. Other methods
nephelometric method
refractometric analysis
polarimetric analysis
interferometric analysis

10. C) On electromagnetic spectral range which use in analysis:
Spectroscopy (spectrophotometry) in UV and visible spectrum
IR - Spectroscopy
X-ray spectroscopy
Microwave spectroscopy

12. D) By the nature of energy jump
Electronic spectrum
Vibrational spectrum
Rotational spectrum

13. The characteristic of energy of quantum
Spectrum (method)
The characteristic of energy of quantum
Process
Radio-frequency (NMR, EPR)
Microwave
The optical UV
The visible
Infra-red (IR)
X-ray
Gamma radiation (nuclear-physical)
meters
meters nm nm
cm-1 m m
Change of electron spin and nuclear spin
Change of rotational conditions
Change of valence electron conditions
Change of vibrational conditions
Change of a condition of internal electrons
Nuclear reactions

14. 3. The fundamental law of absorption.
Reflection of light
sample
Dispersion of light
radiation source
Light absorption
luminescence

15. First law of light absorption
Each thin layer of constant thickness of a homogeneous environment absorbs an identical part of incident radiation
or:
The part of the light which is absorbed by a homogeneous environment, is directly proportional to a thickness of an absorbing layer:

16. Second law of light absorption
The part of the absorbed radiation is proportional to number of absorbing particles in volume of a solution, that is concentration

17. Bouguer-Lambert-Beer law
Reduction of intensity of light which has passed through a layer of light-absorbing substance is proportional concentration of this substance and a thickness of a layer

18. Quantitative characteristics of absorption
1. Transmittance - the ratio of the radiant power passing through a sample to that from the radiation’s source (T).
(I0)
(I) or

19. Diagram of Beer–Lambert absorption of a beam of light as it travels through a cuvette of width ℓ.

20. Optical density А (Absorbance)
An alternative method for expressing the attenuation of electromagnetic radiation is absorbance, A, which is defined as or

22. Bouguer-Lambert-Beer law
So:
The absorbance of a solution is proportional to concentration of light-absorbing substance and a thickness of a layer Or
The relationship between a sample’s absorbance and the concentration of the absorbing species
where: A – optical density (absorbance), ε – the molar absorptivity, C – concentration (molarity)

23. Additivity of optical densities
Beer’s law can be extended to samples containing several absorbing components provided that there are no interactions between the components. Individual absorbances, Ai, are additive. For a two-component mixture of X and Y, the total absorbance, Atot, is So
A = l(1С1 + 2С2 + …kСk)

24.  (the molar absorptivity)
permeate
 (the molar absorptivity)
Iron (ІІІ) rhodanate
103
Complex Ti with H2O2
Complex Ti with chromotrope acid
105
Complex Cu with ammonia
5 102
Complex Cu with dithizon
5 104
Complex Al with aluminon
1,7 104
Complex Al with 2-stilbazole
3,5 104

25. Physical Limitations to Beer’s Law
NOT monochromaticity of light:
A = lС.
NOT parallelism of light.
Temperature.
NOT identical value of refraction of solutions.
NOT proportionality of a photocurrent and intensity of a light

26. Chemical Limitations to Beer’s Law
Dilution of solution (than more of reagent excess, it is less deviation from the law);
рН of medium: state of metal ion
stability of complex ions
competitive reactions (for ligand)
competitive reactions (complexing agent)
polymerization and dissociation reactions
ox-red reactions

27. 4. Electronic spectrum
absorbance spectrum - a graph of a sample’s absorbance of electromagnetic radiation versus wavelength (or frequency or wavenumber).

29. emission spectrum is a graph of emission intensity versus wavelength (or frequency or wavenumber).

31. 5. Photometric method of analysis: an essence, theoretical bases, usage in the pharmaceutical analysis
Molecular–absorption method is based on measurement of absorption by molecules (or ions) substances of electromagnetic radiation of an optical range:
Colorimetry in which visible light was absorbed by a sample. The concentration of analyte was determined visually by comparing the sample’s color to that of a set of standards using Nessler tubes (as described at the beginning of this chapter), or by using an instrument called a colorimeter.
Photocolorimetry - in which polychromatic light was absorbed by a sample
Spectrophotometry - in which monochromatic light was absorbed by a sample
UV - Spectrum ( to nanometers)
Visible spectrum ( to nanometers)

33. Block diagram for a double-beam in-time scanning spectrophotometer with photo of a typical instrument.

35. Choice of optimum conditions of spectrophotometry:
Choice absorption filters (in photometry)
Choice of absorbance
Аoptimal= 0.435
(less error)
А = 0.6 – 0.7
!!!! Not probably to measure absorbance
2 < А < 0.03
Choice of thickness of a layer not more 5 сm
А =  l C
Way of transformation of a defined component in photometric compound

36. Choice of optimal wavelenght (mах)

38. Sensitivity of photometric definition
А =  l C
Cmin = Аmin /  l
А = 0.01
l = 1 cм
 = 1000
then Сmin = 10-5 mol/L

39. Accuracy of photometric definition depends from:
Specific features of photometric reaction or photometric compounds
Characteristics of the used device (usually makes % relative)

40. Methods of quantitative analysis:
1. A method of calibration chart
!!! The method can be applied, if:
Structure of standard and investigated solutions are similar
The interval of concentration on calibration chart should cover of defined concentration

41. 2. Comparison method (a method on one standard)
!! The method can be used if:
Dependence structure - property is strictly rectilinear and passes through the beginning of co-ordinates
Concentration of standard and investigated solutions values of analytical signals as much as possible similar and minimum different
Structure of standard and investigated solutions are as much as possible similar

42. 3. Method of molar or specific (concentration on % w/w) absorptivity
!! The method can be used if:
Strict linearity of dependence structure - an analytical signal is observed
The analytical device maintains requirements of metrological checking

43. 4. Method of additives
!!! The method can be applied, if:
It is necessary to consider stirring influence of extraneous components of sample on analytical signal of defined substance

44. Usage of UV – spectroscopy and spectrophotometry in visible spectrum:
Identification and establishment of identity of drugs
Quantitative definition of substance contain
Cleanliness check
The express control of the forged drugs
Research of new substances structure

45. 6. Multiwave spectrophotometry
The absorbance of any system containing limited number of painted components which chemically one don’t react with another, is equal sum of absorbance of mix components at the same wavelength:

46. Each “partial” absorbance is equal

47. 1. Analysis of two componential mix, when light absorption curves both substance bridge along all spectrum, but on it is partite maximums of absorption

48. If we consider Beer’s Law

49. Molar absorptivity of first component

50. Molar absorptivity of another component

51. The obtained results is substituted into system of the equations and solve it. Its decision can be presented next formulas :

52. Optimal conditions for two-wave spectrophotometry
Percentage error С/С must be the least, value of absorbances must be in the range of 0,3-1, and ratio
must be maximal

53. Modern spectrophotometer for UV and visible spectrum

54. 2. Analysis of two componential mix, when light absorption curves both substance bridge, but on it is spectral range, where absorption one of substance may neglect

55. In this case concentration of first substance is calculated on measured absorbance А at wavelength 1:

56. Concentration of second substance in mix is calculated through concentration С1

57. 3. Analysis of two componential mix, when it is separate maximums of light absorption for each substance in spectral range

58. at wavelength 1
at wavelength 2

59. 7. Differential spectrophotometry
Differential spectrophotometry is used for:
Increases of precision of analyses at definition of considerable quantities of substance;
For elimination of extraneous influence of another components and an exception of reagent absorption.

60. Normal spectrophotometry
Differential spectrophotometry

61. Essence of differential spectrophotometry:
Absorbances of investigated and standard solution are measured on ratio to solvent investigated component with concentration С0 (it is low fidelity equal concentration of investigated solution) instead of ratio to pure solvent (its absorbance is equal practical zero)

62. Main characteristic of absorption in differential spectrophotometry
Ratio of light intensities
is named conditional transmittance

63. Relative (conditional) transmittance

64. Measured experimentally absobance is difference of absorbances of investigated solution and comparison solution.

65. Quantitative analysis:
A method of calibration chart
Calculated method (modified comparison method)

66. Advantage of differential spectrophotometry
Considerable range expansion of defined contents (high concentration);
Relative error is equal 0,05-2 %, that much more low, than in photometry.

67. 8. The extraction-photometric analysis (EPMA)
it is hybrid method of analysis, in which combine extraction (as method excretion, separation and concentrating) and spectrophotometry

68. EPMA is used, when: analyze complex mix
define substance, which is slightly soluble in water, but freely soluble in select organic solvent
define substance, which is very small quantity into investigated object
define impurities in presence main components
immediately definition investigated component is impossible (light absorption curves both substance bridge along all spectrum)
define colourless substance (use coloured extraction reagent)

69. Choice of photometric reaction in EPMA:
Photometric reaction of formation coloured metal complexes:
Pb2+ + 2H2Dz = Pb(HDz)2 + 2H+
extraction in CHCl3
or CCl4
max= 520 nm (= 7104)

70. [SbCl6]- + R+ = R+[SbCl6]-
Photometric reaction of ionic associate formation
[SbCl6]- + R+ = R+[SbCl6]-
extraction in toluene or benzene
R – acidic or basic dye
max= 660 nm (= 5104)

71. Extraction has extraction efficiency equal R=99,9% is necessary for usage of extraction-photometric method of analysis that
It receive by choice:
Solvent-extracting agent
Extraction reagent
Photometric reaction
Chemical factors of extraction (рН, ionic strength, solution composition)

72. Advantages of ЕPМА: High sensitivity, because of:
- high molar absorptivity of extracted complexes
- concentrating of solution by extraction method
High selectivity (pre-award separation, excretion of defined component from mix)
Rapid analysis (in comparison with classical method of precipitation)
Relative simplicity of instrumentation (separatory funnel, spectrophotometer)

73. Usage of ЕPМА in analysis of pharmaceutical drugs:
define majority of metal ions (complexing agent)
define majority of substance, which is insoluble in water (Trimethoprimum in composition of Biseptolum)
define impurities in drugs (salicylic acid in Aspirinum)
define biological-active substance in drugs (from medicinal herbs) (heart glycosides, alkaloids, flavanoids, components of essence)

74. 9. Photometric titration
Photometric titration is titration method in which end point of titration (e.p.t) is determined by photometry or spectrophotometry method.
Method is based on determination e.p.t on jump of solution absorbance in equivalence point.

76. Condition of titrimetric reaction usage in spectrophotometry is linear relation between absorbance and concentration
А =  l C

77. Reactions which is used in spectrophptometric titration:
Acid-base
Complexing
Redox

78. Calculation of titrant volume:
On titration curve
On system of the equations:

79. Curves of specrtofotometric titration can be the different form
Curves of specrtofotometric titration can be the different form. Their character depends on what components of reaction absorb at the chosen wavelength.
А В  АВ
Change of solution absorbance is defined by value of molar absorptivity

81. Photometric titration curve of solution Fe2+ by standard solution of K2Cr2O7
e.p.
V (K2Cr2O7)

82. Photometric titration curve of solution KMnO4 by standard solution of Fe2+
e.p.
V (Fe2+)

83. The spectrophotometric titration can be applied when
Titrant or defined substance or product of reaction absorb light.
If titrant or defined substance or product of reaction don’t absorb light so we use indicators – substances, which don’t absorb light, but form compound with defined substance (АInd), titrant (ВInd) or a reaction product (АВInd) which absorb light

84. As during titration occur solution dilution in cell (cuvette), than for taking into account of solution volume increase is necessary to plot of photometric titration curve on coordinate Аcorrected – С

85. Advantages of specrophotometric titration:
The higher selectivity and possibility definition of several components of sample;
Possibility of titration of the painted solutions;
The higher sensitivity in comparison with classical method of analysis
Possibility of usage of reactions which don’t come to an end in e.p. or reactions which have small equilibrium constant
The higher accuracy

86. Thanks for your attention!