Photometric methods of analysisPhotometric methods of analysis

Assistant of the pharmaceutical chemistry department

Burmas Nataliya Ivanivnae-mail: Natashenka-Burmas@rambler.ru

PlanPlan

1. Main characteristics of photometric methods of analysis.

2. Molecular–absorption method: an essence, theoretical bases, usage in the pharmaceutical analysis.

3. Colorimetry: an essence, theoretical bases, usage in the pharmaceutical analysis.

4. Spectrophotometry : an essence, theoretical bases, usage in the pharmaceutical analysis.

5. The extraction-photometric analysis (EPMA): an essence, theoretical bases, usage in the pharmaceutical analysis.

6. Photometric titration: an essence, theoretical bases, usage in the pharmaceutical analysis.

1. Main characteristics of photometric methods of analysis

Molecular–absorption methodMolecular–absorption method is based on measurement of is based on measurement of absorption by molecules (or ions) substances of electromagnetic absorption by molecules (or ions) substances of electromagnetic radiation of an optical range.radiation of an optical range.

ColorimetryColorimetry is in which visible light was absorbed by a sample. The is in which visible light was absorbed by a sample. The concentration of analyte was determined visually by comparing the concentration of analyte was determined visually by comparing the sample’s color to that of a set of standards using Nessler tubes (as 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 described at the beginning of this chapter), or by using an instrument called a colorimeter.called a colorimeter.

PhotocolorimetryPhotocolorimetry is in which polychromatic light was absorbed by a is in which polychromatic light was absorbed by a samplesample

SpectrophotometrySpectrophotometry is in which monochromatic light was absorbed by is in which monochromatic light was absorbed by a samplea sample

UV - Spectrum (100-200 to 380-400 nanometers)UV - Spectrum (100-200 to 380-400 nanometers) Visible spectrum (380-400 to 780-800 nanometers)Visible spectrum (380-400 to 780-800 nanometers)

Block diagram for a double-beam in-timescanning spectrophotometer with photo of a

typical instrument.

Choice of optimum conditions of Choice of optimum conditions of spectrophotometry:spectrophotometry:

Choice absorption filters Choice absorption filters (in photometry)(in photometry) Choice of absorbanceChoice of absorbance

ААoptimaloptimal= 0.435= 0.435(less error) (less error)

А = 0.6 – 0.7А = 0.6 – 0.7 !!!! Not probably to measure absorbance!!!! Not probably to measure absorbance

2 < А < 0.032 < А < 0.03 Choice of thickness of a layerChoice of thickness of a layer - - not more 5 сmnot more 5 сm

А = А = l l C C The way of transformation of a defined component in The way of transformation of a defined component in

the photometricthe photometric compound compound

Choice of optimal wavelenghtChoice of optimal wavelenght ( (mmахах))

Sensitivity of photometric definitionSensitivity of photometric definition

А = А = l Cl C

CCminmin = А= Аminmin / / l l АА = 0.01 = 0.01 ll = 1 = 1 ccмм = 1000= 1000

thenthen ССminmin = 10 = 10-5-5 molmol//LL

The accuracy of photometric definitionThe accuracy of photometric definition depends from:depends from:

Specific features of photometric reaction or Specific features of photometric reaction or photometric compoundsphotometric compounds

Characteristics of the used device (usually makes Characteristics of the used device (usually makes 1 - 2 %1 - 2 % relative) relative)

Methods of quantitative analysis:Methods of quantitative analysis:

1. A method of calibration1. A method of calibration chartchart

!!!!!! The method can be applied, if:The method can be applied, if: The structure of standard and investigated The structure of standard and investigated

solutions are similarsolutions are similar The interval of concentration on calibration The interval of concentration on calibration

chart should cover of defined concentrationchart should cover of defined concentration

2. Comparison method (a method on one standard)2. Comparison method (a method on one standard)

!! !! The method can be used if:The method can be used if: DependenceDependence''s structure - property is strictly rectilinear and passes s structure - property is strictly rectilinear and passes

through the beginning of co-ordinates.through the beginning of co-ordinates. The concentration of standard and investigated solutions values of The concentration of standard and investigated solutions values of

analytical signals as much as possible similar and minimum analytical signals as much as possible similar and minimum different.different.

The structure of standard and investigated solutions are as much The structure of standard and investigated solutions are as much as possible similar.as possible similar.

st

stхх A

СAС

х

dards

х

dards

A

A

С

C tantan

3. Method of molar or specific (concentration on 3. Method of molar or specific (concentration on % w/w) absorptivity% w/w) absorptivity

!! !! The method can be used if:The method can be used if: Strict linearity of dependence structure is an Strict linearity of dependence structure is an

analytical signal is observed.analytical signal is observed. The analytical device maintains requirements of The analytical device maintains requirements of

metrological checking.metrological checking.

х

хA

CE

AC х

х

4. Method of additives4. Method of additives

!!! The method can be applied, if:!!! The method can be applied, if: It is necessary to consider stirring It is necessary to consider stirring

influence of extraneous components of influence of extraneous components of sample on analytical signal of defined sample on analytical signal of defined substancesubstance

stх

х

stх

х

СС

С

A

A

хstх

хstх AA

AСС

Usage of UV – spectroscopy and Usage of UV – spectroscopy and spectrophotometry in visible spectrum:spectrophotometry in visible spectrum:

Identification and establishment of identity of Identification and establishment of identity of drugsdrugs

Quantitative definition of substance contain Quantitative definition of substance contain Cleanliness check Cleanliness check The express control of the forged drugs The express control of the forged drugs Research of the structure of new substancesResearch of the structure of new substances

2. Molecular–absorption method: an essence, theoretical bases, 2. Molecular–absorption method: an essence, theoretical bases, usage in the pharmaceutical analysis.usage in the pharmaceutical analysis.

Molecular–absorption methodMolecular–absorption method is based on measurement is based on measurement of absorption by molecules (or ions) substances of of absorption by molecules (or ions) substances of electromagnetic radiation of an optical range.electromagnetic radiation of an optical range.In analytical chemistry, atomic absorption In analytical chemistry, atomic absorption spectroscopy is a technique for determining the spectroscopy is a technique for determining the concentration of a particular metal element in a concentration of a particular metal element in a sample. The technique can be used to analyze the sample. The technique can be used to analyze the concentration of over 70 different metals in a solution. concentration of over 70 different metals in a solution. Although atomic absorption spectroscopy dates to the Although atomic absorption spectroscopy dates to the nineteenth century, the modern form was largely nineteenth century, the modern form was largely developed during the 1950s by a team of Australian developed during the 1950s by a team of Australian chemists. They were led by Alan Walsh and worked at chemists. They were led by Alan Walsh and worked at the CSIRO (Commonwealth Science and Industry the CSIRO (Commonwealth Science and Industry Research Organisation) Division of Chemical Physics Research Organisation) Division of Chemical Physics in Melbourne, Australia.in Melbourne, Australia.

PrinciplesPrinciplesThe technique makes use of absorption spectrometry to The technique makes use of absorption spectrometry to assess the concentration of an analyte in a sample. It assess the concentration of an analyte in a sample. It relies therefore heavily on Beer-Lambert law. In short, relies therefore heavily on Beer-Lambert law. In short, the electrons of the atoms in the atomizer can be the electrons of the atoms in the atomizer can be promoted to higher orbitals for a short amount of time by promoted to higher orbitals for a short amount of time by absorbing a set quantity of energy (i.e. light of a given absorbing a set quantity of energy (i.e. light of a given wavelength). This amount of energy (or wavelength) is wavelength). This amount of energy (or wavelength) is specific to a particular electron transition in a particular specific to a particular electron transition in a particular element, and in general, each wavelength corresponds to element, and in general, each wavelength corresponds to only one element. This gives the technique its elemental only one element. This gives the technique its elemental selectivity. As the quantity of energy (the power) put into selectivity. As the quantity of energy (the power) put into the flame is known, and the quantity remaining at the the flame is known, and the quantity remaining at the other side (at the detector) can be measured, it is possible, other side (at the detector) can be measured, it is possible, from Beer-Lambert law, to calculate how many of these from Beer-Lambert law, to calculate how many of these transitions took place, and thus get a signal that is transitions took place, and thus get a signal that is proportional to the concentration of the element being proportional to the concentration of the element being measured.measured.

Analysis of liquidsAnalysis of liquids

A liquid sample is normally turned into an atomic gas in A liquid sample is normally turned into an atomic gas in three steps:three steps:

1.1. Desolvation (Drying) – the liquid solvent is Desolvation (Drying) – the liquid solvent is evaporated, and the dry sample remainsevaporated, and the dry sample remains

2.2. Vaporization – the solid sample vaporises to a gasVaporization – the solid sample vaporises to a gas

3.3. Atomization – the compounds making up the Atomization – the compounds making up the sample are broken into free atoms.sample are broken into free atoms.

3. Colorimetry: an essence, theoretical bases, 3. Colorimetry: an essence, theoretical bases, usage in the pharmaceutical analysis.usage in the pharmaceutical analysis.

ColorimetryColorimetry in which visible light was absorbed in which visible light was absorbed by a sample. The concentration of analyte was by a sample. The concentration of analyte was determined visually by comparing the sample’s determined visually by comparing the sample’s color to that of a set of standards using Nessler color to that of a set of standards using Nessler tubes (as described at the beginning of this tubes (as described at the beginning of this chapter), or by using an instrument called a chapter), or by using an instrument called a colorimeter.colorimeter.

InstrumentsInstruments

Colorimetric equipment is similar to that used in Colorimetric equipment is similar to that used in spectrophotometry. spectrophotometry. Some related equipment is also Some related equipment is also mentioned for completeness.mentioned for completeness. A tristimulus colorimeter A tristimulus colorimeter measures the tristimulus values of a color. A measures the tristimulus values of a color. A spectroradiometer measures the absolute spectral radiance spectroradiometer measures the absolute spectral radiance (intensity) or irradiance of a light source. A (intensity) or irradiance of a light source. A spectrophotometer measures the spectral reflectance, spectrophotometer measures the spectral reflectance, transmittance, or relative irradiance of a color sample. transmittance, or relative irradiance of a color sample.

A A spectrocolorimeterspectrocolorimeter is a spectrophotometer that is a spectrophotometer that can can calculatecalculate tristimulus values. A densitometer measures tristimulus values. A densitometer measures the degree of light passing through or reflected by a the degree of light passing through or reflected by a subject. A subject. A color temperature metercolor temperature meter measures the color measures the color temperature of an incident illuminant.temperature of an incident illuminant.

Two spectral reflectance curves. The object in question reflects light with shorter wavelengths while absorbing those in others, lending it a blue appearance.

4. Spectrophotometry : an essence, theoretical bases, 4. Spectrophotometry : an essence, theoretical bases, usage in the pharmaceutical analysis.usage in the pharmaceutical analysis.

In chemistry, spectrophotometry is the In chemistry, spectrophotometry is the quantifiable study of electromagnetic spectra. quantifiable study of electromagnetic spectra. It is more specific than the general term It is more specific than the general term electromagnetic spectroscopy in that electromagnetic spectroscopy in that spectrophotometry deals with visible light, spectrophotometry deals with visible light, near-ultraviolet, and near-infrared. Also, the near-ultraviolet, and near-infrared. Also, the term does not cover time-resolved term does not cover time-resolved spectroscopic techniques. Spectrophotometry spectroscopic techniques. Spectrophotometry involves the use of a spectrophotometer. involves the use of a spectrophotometer.

A spectrophotometer is a photometer (a device for A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity measuring light intensity) that can measure intensity as a function of the color (or more specifically the as a function of the color (or more specifically the wavelength) of light. Important features of wavelength) of light. Important features of spectrophotometers are spectral bandwidth and linear spectrophotometers are spectral bandwidth and linear range of absorption measurement. Perhaps the most range of absorption measurement. Perhaps the most common application of spectrophotometers is the common application of spectrophotometers is the measurement of light absorption, but they can be measurement of light absorption, but they can be designed to measure diffuse or specular reflectance. designed to measure diffuse or specular reflectance. Strictly, even the emission half of a luminescence Strictly, even the emission half of a luminescence instrument is a type of spectrophotometer. The use of instrument is a type of spectrophotometer. The use of spectrophotometers is not limited to studies in physics. spectrophotometers is not limited to studies in physics. They are also commonly used in other scientific fields They are also commonly used in other scientific fields such as chemistry, biochemistry, and molecular such as chemistry, biochemistry, and molecular biology. They are widely used in many industries biology. They are widely used in many industries including printing and forensic examination.including printing and forensic examination.

In short, the sequence of events in a In short, the sequence of events in a spectrophotometer is as follows:spectrophotometer is as follows:

1.1. The light source shines into a The light source shines into a monochromator.monochromator.

2.2. A particular output wavelength is A particular output wavelength is selected and beamed at the sample.selected and beamed at the sample.

3.3. The sample absorbs light.The sample absorbs light.

44. . The extraction-photometric The extraction-photometric analysisanalysis (EPMA)(EPMA)

it isit is hybridhybrid method of analysismethod of analysis, , in in whichwhich combinecombine extractionextraction ( (asas methodmethod excretion, separation and excretion, separation and concentratingconcentrating) ) andand spectrophotometry.spectrophotometry.

EPMA is usedEPMA is used, , whenwhen::

analyze analyze complex mixcomplex mix define define substancesubstance, which is , which is slightly solubleslightly soluble in in

waterwater, but , but freely solublefreely soluble in select organic solventin select organic solvent define define substancesubstance, which is , which is very small quantity into very small quantity into

investigated objectinvestigated object define define impurities in presence main componentsimpurities in presence main components immediately definition investigated component is immediately definition investigated component is

impossible (impossible (light absorption curves both substance light absorption curves both substance bridge along all spectrumbridge along all spectrum))

define define colourless substancecolourless substance (use coloured extraction (use coloured extraction reagent)reagent)

ChoiceChoice of photometric reactionof photometric reaction inin EPMAEPMA::

Photometric reaction of formationPhotometric reaction of formation colouredcoloured metal complexesmetal complexes::

PbPb2+2+ + 2 + 2HH22DzDz = = PbPb((HDzHDz))22 + 2+ 2HH++

extractionextraction inin CHClCHCl33

oror CClCCl44

maxmax= 520 nm= 520 nm (= 7(= 7101044))

Extraction has Extraction has extraction efficiency equalextraction efficiency equal R=99,9%R=99,9% is necessary for usage of is necessary for usage of extraction-photometric method of analysis extraction-photometric method of analysis thatthat

It receive It receive by choiceby choice:: Solvent-extracting agentSolvent-extracting agent Extraction reagentExtraction reagent Photometric reactionPhotometric reaction Chemical factors of extraction (рН, ionic Chemical factors of extraction (рН, ionic

strength, solution composition)strength, solution composition)

Advantages ofAdvantages of Е ЕPPМА:МА: High sensitivityHigh sensitivity, because of:, because of:

- high molar - high molar absorptivity of extracted complexes of extracted complexes

- concentrating of solution by extraction method- concentrating of solution by extraction method High selectivityHigh selectivity (pre-award separation, (pre-award separation,

excretion of defined component from mix)excretion of defined component from mix) Rapid analysisRapid analysis (in comparison with classical (in comparison with classical

method of precipitation) method of precipitation) Relative simplicity of instrumentation Relative simplicity of instrumentation

((separatory funnel, spectrophotometer), spectrophotometer)

Usage of ЕPМА in analysis of Usage of ЕPМА in analysis of pharmaceutical drugs:pharmaceutical drugs:

define a majority of metal ions define a majority of metal ions (complexing (complexing agent)agent)

define a majority of a substance, which is define a majority of a substance, which is insoluble in waterinsoluble in water (Trimethoprimum in (Trimethoprimum in composition of Biseptolum)composition of Biseptolum)

define impurities in drugs define impurities in drugs (salicylic acid in (salicylic acid in Aspirinum)Aspirinum)

define a biological-active substance in drugs define a biological-active substance in drugs (from (from medicinal herbs)medicinal herbs) (heart glycosides, (heart glycosides, alkaloids, flavanoids, components of essence)alkaloids, flavanoids, components of essence)

5. Photometric titration5. Photometric titration

Photometric titration is titration method in Photometric titration is titration method in which end point of titration (e.p.t) is which end point of titration (e.p.t) is determined by photometry or determined by photometry or spectrophotometry method.spectrophotometry method.

Method isMethod is based on determination e.p.t based on determination e.p.t on on jump of solution absorbance jump of solution absorbance in equivalence in equivalence point.point.

Condition of titrimetric reaction usage Condition of titrimetric reaction usage in in spectrophotometry is linear spectrophotometry is linear relation between absorbance and relation between absorbance and concentrationconcentration

А = А = l C l C

Reactions which is used in spectrophptometric Reactions which is used in spectrophptometric titrationtitration::

Acid-baseAcid-base

ComplexingComplexing

RedoxRedox

Calculation of titrant volumeCalculation of titrant volume::

On titration curveOn titration curve

On system of the equationsOn system of the equations::

..11.. pepe VbaA

..22.. pepe VbaA 21

12.. bb

aaV pe

Curves of specrtofotometric titrationCurves of specrtofotometric titration can can be the be the different formdifferent form. Their character depends . Their character depends on what components of reaction absorb at the on what components of reaction absorb at the chosen wavelength.chosen wavelength.

А + В А + В АВ АВChange of solution absorbance is defined by Change of solution absorbance is defined by

value of molarvalue of molar absorptivityabsorptivity

ABAB

PhotometricPhotometric titrationtitration curvecurve ofof solution solution FeFe2+2+ by standard solution ofby standard solution of KK22CrCr22OO77

V (K2Cr2O7)

A

e.p.

PhotometricPhotometric titrationtitration curvecurve ofof solution solution KMnOKMnO44

by standard solution ofby standard solution of FeFe2+2+

V (Fe2+)

A

e.p.

The spectrophotometric titration can be applied The spectrophotometric titration can be applied whenwhen

TitrantTitrant or or defined substancedefined substance or or product of product of reactionreaction absorb lightabsorb light. .

If titrant or defined substance or product of If titrant or defined substance or product of reaction reaction don’t absorb lightdon’t absorb light so we so we use indicatorsuse indicators – substances, which don’t absorb light, but form – substances, which don’t absorb light, but form compound with defined substance (АInd), titrant compound with defined substance (АInd), titrant (ВInd) or a reaction product (АВInd) which (ВInd) or a reaction product (АВInd) which absorb lightabsorb light

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

0

.0

V

VVАA titrant

measuredcorrected

Advantages of specrophotometricAdvantages of specrophotometric titrationtitration::

The higher selectivity and possibility definition The higher selectivity and possibility definition of several components of sample;of several components of sample;

Possibility of Possibility of titration of the paintedtitration of the painted solutions; solutions; The higher sensitivityThe higher sensitivity in comparison with in comparison with

classical method of analysisclassical method of analysis Possibility of Possibility of usage of reactions which don’t usage of reactions which don’t

come to an end in e.p.come to an end in e.p. or reactions which have or reactions which have small equilibrium constantsmall equilibrium constant

The higher accuracyThe higher accuracy

Thanks for your attention!Thanks for your attention!