Transcript 12 Instrumental methods of analysis. Photometry

Lecture 12
Instrumental methods of analysis.
Photometry.
Associate prof . L.V. Vronska
Associate prof . M.M. Mykhalkiv
Outline
1. Classification, advantages and lacks of physicalchemical methods of the analysis.
2. Optical methods of the analysis. Classification.
3. The fundamental law of absorption.
4. Electronic spectrum.
5. Photometric method of analysis: an essence,
theoretical bases, usage in the pharmaceutical analysis.
6. Multiwave spectrophotometry .
7. Differential spectrophotometry.
8. The extraction-photometric analysis.
9. Photometric titration.
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
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
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




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
2. Optical methods of the analysis.
Classification.
А) On investigated objects
 The nuclear spectral analysis
 The molecular spectral analysis
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
3. Other methods
 nephelometric method
 refractometric analysis
 polarimetric analysis
 interferometric analysis
C) On electromagnetic spectral range which use
in analysis:
 Spectroscopy (spectrophotometry) in UV and
visible spectrum
 IR - Spectroscopy
 X-ray spectroscopy
 Microwave spectroscopy
D) By the nature of energy jump
 Electronic spectrum
 Vibrational spectrum
 Rotational spectrum
Spectrum (method)
The characteristic of
energy of
quantum
Process
101-10-1 meters
Change of electron spin
and nuclear spin
Microwave
10-1-10-3 meters
Change of rotational
conditions
The optical
UV
The visible
200-400 nm
400-750 nm
Radio-frequency
(NMR, EPR)
Infra-red (IR)
X-ray
Gamma radiation
(nuclear-physical)
Change of valence
electron conditions
10-13000 cm-1
Change of vibrational
conditions
10-8-10-10 m
Change of a condition
of internal electrons
10-10-10-13 m
Nuclear reactions
3. The fundamental law of absorption.
Reflection of light
radiation source
sample
Dispersion of light
Light absorption
luminescence
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:
I
I
 k1l
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
I
I
 k 2C
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
I  Io  e
 klC
Quantitative characteristics of
absorption
1. Transmittance - the ratio of the radiant
power passing through a sample to that from
the radiation’s source (T).
I
T
Io
or
(I0)
(I)
Diagram of Beer–Lambert absorption of a beam of light as it travels
through a cuvette of width ℓ.
Optical density А (Absorbance)
An alternative method for expressing the
attenuation of electromagnetic radiation is
absorbance, A, which is defined as
or
Io
I
 lg T   lg
 lg
A
Io
I
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
A  lC
where: A – optical density (absorbance), ε – the molar absorptivity,
C – concentration (molarity)
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 twocomponent mixture of X and Y, the total
absorbance, Atot, is
So
A  A1  A2  A3  ...  Ak
A = l(1С1 + 2С2 + …kСk)
permeate
 (the molar absorptivity)
Iron (ІІІ) rhodanate
103
Complex Ti with H2O2
103
Complex Ti with
chromotrope acid
Complex Cu with ammonia
105
5 102
Complex Cu with dithizon
5 104
Complex Al with aluminon
1,7 104
Complex Al with 2stilbazole
3,5 104
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
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
4. Electronic spectrum
 absorbance spectrum - a
graph of a sample’s
absorbance
of
electromagnetic radiation
versus wavelength (or
frequency or wavenumber).
 emission spectrum
is a graph of
emission intensity
versus wavelength
(or frequency or
wavenumber).
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)
Block diagram for a double-beam in-time
scanning spectrophotometer with photo of a
typical instrument.
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=lC
 Way of transformation of a defined component in
photometric compound
Choice of optimal wavelenght (mах)
Sensitivity of photometric definition
А=lC
Cmin = Аmin /  l
 А = 0.01
 l = 1 cм
  = 1000
then Сmin = 10-5 mol/L
Accuracy of photometric definition
depends from:
 Specific features of photometric reaction or
photometric compounds
 Characteristics of the used device (usually makes
1 - 2 % relative)
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
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
Cs tan dard As tan dard

Сх
Aх
Aх  С st
Сх 
Ast
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
Cх 
Aх

Aх
Cх 
E
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
Aх
Сх

Aх  st С х  С st
Aх
С х  Сst 
Aх  st  Aх
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
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:
A  A1  A2  ...  An
Each “partial” absorbance is equal
Ai   i  Ci  l
1. Analysis of two componential mix, when
light absorption curves both substance
bridge along all spectrum, but on it is
partite maximums of absorption
A  A1  A2
1
1
1
A 2  A1  A2
2
2
If we consider Beer’s Law
A  1  C1  l   2  C2  l
1
1
1
A  1  C1  l   2  C2  l
2
2
2
Molar absorptivity of first component
A1  1  c1  l
1
11 
1
A1
1
c1  l
Molar absorptivity of another component
A2    2   c2  l
1
 2 
1
1
A2
1
c2  l
The obtained results is substituted into
system of the equations and solve it. Its
decision can be presented next formulas :
C1 
C2 
A1  22  A2   21
11  22  12  21
;
A2  11  A1   12
 11  22   12  21
.
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
 1 /  2 and _  2 /  1
1
must be maximal
2
2
2
Modern spectrophotometer for UV and visible
spectrum
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
 In this case concentration of first
substance is calculated on
measured absorbance А at
wavelength 1:
C1  A1 /(11  l ).
Concentration of second substance in mix is
calculated through concentration С1
A2   12  C1  l   22  C 2  l ;
C2 
A2   1 2  C1  l
 22  l
3. Analysis of two componential mix, when
it is separate maximums of light
absorption for each substance in
spectral range
C1 
A1
 11  l
at wavelength 2
at wavelength 1
C2 
A 2
 2 2  l
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.
Normal spectrophotometry
Differential spectrophotometry
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)
Main characteristic of absorption in
differential spectrophotometry
Ratio of light intensities
Ix
I comparison
is named conditional transmittance
Т х _ relative 
Іх
І compar
Relative (conditional)
transmittance
Т х  Іо
Тх
Т х _ relative 


І comparison Tcompar  І о Т compor
Іх
Т х _ relative 
Тх
Т compar
 lg T  A
Ах _ relative  Ах  Аcompar ,
Ах _ relative    С х  l  Аcompar
Ах _ relative    С х  l  Аcompar   (C x  C0 )l
 Measured experimentally absobance
is
difference of absorbances of investigated
solution and comparison solution.
Quantitative analysis:
 A method of calibration chart
 Calculated method (modified comparison
method)
Ax _ relat
Аст _ relat
Сх 

С х  Сcompar
С s tan d  Сcompar
Ах _ relat
Аs tan_ relat
,
(С s tan  Сcompar )  Сconpar
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.
8. The extraction-photometric
analysis (EPMA)
 it is hybrid method of analysis, in which
combine extraction (as method excretion,
separation
and
concentrating)
and
spectrophotometry
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)
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)

Photometric
formation
reaction
of
ionic
associate
[SbCl6]- + R+ = R+[SbCl6]extraction in toluene
or benzene
R – acidic or basic dye
 max= 660 nm (= 5104)
 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)
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)
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)
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.
 Condition of titrimetric reaction usage
in
spectrophotometry is linear
relation between absorbance and
concentration
А=lC
Reactions which is used in spectrophptometric
titration:
 Acid-base
 Complexing
 Redox
Calculation of titrant volume:
 On titration curve
 On system of the equations:
Ae. p.  a1  b1 Ve. p.
Ve. p.
a2  a1

b1  b2
Ae. p.  a2  b2 Ve. p.
 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
   AB   B   A
Photometric titration curve of solution
Fe2+ by standard solution of K2Cr2O7
A
e.p.
V (K2Cr2O7)
Photometric titration curve of solution
KMnO4 by standard solution of Fe2+
A
e.p.
V (Fe2+)
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
 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
Acorrected
V0  Vtitrant.
 Аmeasured 
V0
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
Thanks for your attention!