Instrumental methods of analysis. Photometry.

1. 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. PCMAare dividedon: • Opticalmethods 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. • Thermalmethods are based on measurement of heat effectsof substances. • Mass spectrometric methods are based on studying of the ionized fragments ("splinters") of substances. • Kineticmethods 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

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 absorbanceof 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)

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 • competitivereactions (for ligand) • competitivereactions (complexing agent) • polymerization and dissociationreactions • 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 (100-200 to 380-400 nanometers) • Visible spectrum (380-400 to 780-800 nanometers)

33. Block diagram for a double-beam in-timescanning spectrophotometer with photo of atypical 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 thenСmin = 10-5mol/L

39. Accuracy of photometric definitiondepends from: • Specific features of photometric reaction or photometric compounds • Characteristics of the used device (usually makes 1 - 2 % relative)

40. Methods of quantitative analysis: 1. A method of calibrationchart !!!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”absorbanceis 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