Photometric methods of analysis
Assistant of the
pharmaceutical chemistry department
Burmas Nataliya Ivanivna
e-mail: Natashenka-Burmas@rambler.ru
Plan
1.
2.
3.
4.
5.
6.
Main characteristics of photometric methods of
analysis.
Molecular–absorption method: an essence,
theoretical bases, usage in the pharmaceutical
analysis.
Colorimetry: an essence, theoretical bases, usage
in the pharmaceutical analysis.
Spectrophotometry : an essence, theoretical bases,
usage in the pharmaceutical analysis.
The extraction-photometric analysis (EPMA): an
essence, theoretical bases, usage in the
pharmaceutical analysis.
Photometric titration: an essence, theoretical
bases, usage in the pharmaceutical analysis.
1. Main characteristics of photometric methods of
analysis






Molecular–absorption method is based on measurement of
absorption by molecules (or ions) substances of electromagnetic
radiation of an optical range.
Colorimetry is 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 is in which polychromatic light was absorbed by a
sample
Spectrophotometry is 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

The way of transformation of a defined component in
the 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
The 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:

The 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's structure - property is strictly rectilinear and passes
through the beginning of co-ordinates.

The concentration of standard and investigated solutions values of
analytical signals as much as possible similar and minimum
different.

The 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 is an
analytical signal is observed.

The analytical device maintains requirements of
metrological checking.
Aх
Cх 

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 the structure of new substances
2. Molecular–absorption method: 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.
In analytical chemistry, atomic absorption
spectroscopy is a technique for determining the
concentration of a particular metal element in a
sample. The technique can be used to analyze the
concentration of over 70 different metals in a solution.
Although atomic absorption spectroscopy dates to the
nineteenth century, the modern form was largely
developed during the 1950s by a team of Australian
chemists. They were led by Alan Walsh and worked at
the CSIRO (Commonwealth Science and Industry
Research Organisation) Division of Chemical Physics
in Melbourne, Australia.
Principles
The technique makes use of absorption spectrometry to
assess the concentration of an analyte in a sample. It
relies therefore heavily on Beer-Lambert law. In short, the
electrons of the atoms in the atomizer can be promoted to
higher orbitals for a short amount of time by absorbing a
set quantity of energy (i.e. light of a given wavelength).
This amount of energy (or wavelength) is specific to a
particular electron transition in a particular element, and
in general, each wavelength corresponds to only one
element. This gives the technique its elemental selectivity.
As the quantity of energy (the power) put into the flame
is known, and the quantity remaining at the other side (at
the detector) can be measured, it is possible, from BeerLambert law, to calculate how many of these transitions
took place, and thus get a signal that is proportional to the
concentration of the element being measured.
Analysis of liquids
A liquid sample is normally turned into an atomic gas in
three steps:
1.
Desolvation (Drying) – the liquid solvent is
evaporated, and the dry sample remains
2.
Vaporization – the solid sample vaporises to a gas
3.
Atomization – the compounds making up the
sample are broken into free atoms.
3. Colorimetry: an essence, theoretical bases,
usage in the pharmaceutical analysis.
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.
Instruments
Colorimetric equipment is similar to that used in
spectrophotometry. Some related equipment is also
mentioned for completeness. A tristimulus colorimeter
measures the tristimulus values of a color. A
spectroradiometer measures the absolute spectral radiance
(intensity) or irradiance of a light source. A
spectrophotometer measures the spectral reflectance,
transmittance, or relative irradiance of a color sample.
A spectrocolorimeter is a spectrophotometer that can
calculate tristimulus values. A densitometer measures the
degree of light passing through or reflected by a subject. A
color temperature meter measures the color 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,
usage in the pharmaceutical analysis.
In chemistry, spectrophotometry is the
quantifiable study of electromagnetic spectra.
It is more specific than the general term
electromagnetic spectroscopy in that
spectrophotometry deals with visible light,
near-ultraviolet, and near-infrared. Also, the
term does not cover time-resolved
spectroscopic techniques. Spectrophotometry
involves the use of a spectrophotometer.
A spectrophotometer is a photometer (a device for
measuring light intensity) that can measure intensity
as a function of the color (or more specifically the
wavelength) of light. Important features of
spectrophotometers are spectral bandwidth and linear
range of absorption measurement. Perhaps the most
common application of spectrophotometers is the
measurement of light absorption, but they can be
designed to measure diffuse or specular reflectance.
Strictly, even the emission half of a luminescence
instrument is a type of spectrophotometer. The use of
spectrophotometers is not limited to studies in physics.
They are also commonly used in other scientific fields
such as chemistry, biochemistry, and molecular
biology. They are widely used in many industries
including printing and forensic examination.
In short, the sequence of events in a
spectrophotometer is as follows:
1. The light source shines into a
monochromator.
2. A particular output wavelength is
selected and beamed at the sample.
3. The sample absorbs light.
4. 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)





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 a majority of metal ions (complexing
agent)
define a majority of a substance, which is
insoluble in water (Trimethoprimum in
composition of Biseptolum)
define impurities in drugs (salicylic acid in
Aspirinum)
define a biological-active substance in drugs
(from medicinal herbs) (heart glycosides,
alkaloids, flavanoids, components of essence)
5. 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!