Chromatography techniques and and its pharmaceutical application

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Pharmaceutical Analysis II

Chromatography
By: D.A
April, 2021

2. Introduction to chromatography
History
 Russian botanist Tswett M. (1872–1919)
◦ Used a column packed with a stationary phase of
calcium carbonate to separate colored pigments
from plant extracts.
◦ The sample was placed at the top of the column
and carried through the stationary phase using a
mobile phase of petroleum ether.
 Chroma-color, graphy-writing

3. Cont…
 Chromatography
◦ Process by which complex mixtures can be resolved and separated
 Mixture could be organic or inorganic .
 The name (from the Latin khromatos) implies color
◦ No direct connection except that the first compound separated by this process
were pigments.
◦ Is a separation process that is achieved by distributing the components of a
mixture between two phases, a stationary phase and a mobile phase
◦ Those components held preferentially in the stationary phase are retained
longer in the system than those that are distributed selectively in the mobile
phase.
◦ As a consequence, solutes are eluted from the system in the order of their
increasing distribution coefficients with respect to the stationary phase

4. Essential features of chromatographic separation
 Mixtures to be separated is introduced in to,Two phases
 Where each phase competes with each others
 Aiming at containing different components (analytes)
◦ As a result
 Each components will be distributed in the two competitive phase.
 According to certain distribution coefficients.
 These coefficients depends on physical properties of a molecules such as,
 Solubility
 Adsorption
 Volatility
 Molecular size and I
 onic charge in ionizable molecule.

5. Cont…
 Existence of two substance with exactly similar properties is
highly improbable
◦ Different substance will exhibit different properties.
◦ Such different causes chromatographic separation.
 Mobile phase
◦ is a moving phase that continuously flows through the
stationary phase and carries the analyte.
 Stationary phase
◦ The fixed or non-moving phase, immobile phase.

6. Cont…
 Separates components in mixture:
 Based on
 Polarity
 boiling point
 ionic group on ionizable molecule
 size
 There are different types of chromatography
 Based on the nature of stationary and mobile phase but more
particularly the nature of stationary phase

7. Partition Chromatography
 Liquid SP and Liquid/gas MP
 Molecules will partition into the
stationary phase based upon
affinity/polrity for stationary phase &
eventually partition into mobile phase
again
 Thin layer of the liquid SP is coated
onto inside of GC column or on small
particles on LC column

8. Adsorption Chromatography
 Solid SP and
Liquid/gas MP
 Very similar to partition
chromatography
 Adsorption just on
surface, partition into thin
layer
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9. Ion Exchange Chromatography
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 A solid polymeric stationary
phase containing replaceable ions
 Ionic liquid mobile phase
 Separation of either cations or
anions
 Separation based on relative
strength of ionic bond
 Solute ions of the opposite charge in
the mobile liquid phase are attached
to the resin by electrostatic force.
 Anion exchange has cations ((e.g., –
N(CH3)3+ on surface
 Cation exchange has anions (eg.
SO3-) on the surface

10. Molecular Exclusion Chromatography
 An inert gel which
acts as a molecular
sieve and a liquid
mobile phase is used.
 Separation based on
size
 Small molecules get
trapped in pores & take
longer to get out
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11. Affinity Chromatography
 Very selective
 Specific binding site is
used to concentrate
analyte on column
 Used a lot in biological
applications
 Eg. Antibody SP might
be used to separate
protein mix.
11

12. Chromatographic techniques
 Planar Cr.
 SP-plane support
◦ Paper chromatography
 Liquid SP-soaked in cellulose
paper
◦ Thin layer chromatography (TLC)
 adsorbent (Al2O3 or SiO2,
usually) coating a sheet of
plastic or glass
 Separated cpds-appear as
spot
12
 Column chromatography
 SP-in column
◦ Gas chromatography(GC)
◦ High performance liquid Cr.
(HPLC)
 Separated cpds-appear as a
peak

13. Cont…
 CC, TLC, & HPLC,
◦ May be of partition, adsorption, ion exchange, exclusive or affinity
 Paper chromatography
◦ Is primarily a partition process
 GC may be
◦ A partition process an in GLC
◦ An adsorption process as in GSC
 The term liquid chromatography
 Some times used to denote techniques in which the mobile phase is a
liquid,
 PC, CC, TLC, HPLC.

14. Application of chromatography
 It has wide application
◦ Evaluation of drugs and pharmaceutical products
◦ Drug extraction, isolation and purification
◦ Identification of active principal in plant research
 Application can be classified in three groups
◦ Preparative application
◦ Qualitative application
◦ Quantitative application
 Qualitative application
 The presence or absence of certain constituents
◦ Techniques of choice are,
 HPLC
 GC
 TLC

15. Cont…
 Gives valuable information about complexity.
◦ Number of spots in PC and TLC
◦ Number of peaks in GC and HPLC
 Used to check purity and identity
 Used to establish finger print for,
 Extracts, tincture, volatile oil or pharmaceutical preparation.
 So the purity and identity of a sample can be judged.
 Quantitative application
◦ Quantitative chromatography determines,
 Percentage of any components in a sample
◦ Either using
 Absolute amount
 Relative to another components
 All chromatographic techniques can applied.

16. Theoretical Concepts
 In a typical chromatographic system
◦ Different compounds move through the system
 With different rates of movements
 In the form of band or zones
 Different rates of movements (differential migration) takes
into consideration.
◦ The equilibrium distributions of a compounds between
 Stationary phase and
 Mobile phase
 The speed with which each compounds move through a system
◦ Determines by the number of molecules of that compound in mobile
phase at any instant .
 Sample molecules in the stationary phase do not move through the system.

17. Cont…
 There fore, compound that spends most of their time in the
stationary phase
 Move through the system rather slowly
 Compound whose molecules are found in the mobile phase
most of the time
 Move through the system more rapidly.
 Molecules of solvent or mobile phase move through the system
 At faster possible rate
 Except in gel chromatography
 Partition chromatography
 Some times called liquid to liquid chromatography (LLC)

18. Cont…
 Sample solution is introduced into two phase
 Namely liquid-liquid system
 One of the phase kept as a stationary phase
 On a suitable support
 Filter paper, cellulose powder etc
 Have the ability to absorb and fix the stationary phase
 The other moves past through the stationary liquid and support
material.
 The two phases should be immiscible
 Adsorption chromatography
 Some times liquid-solid chromatography (LSC)
 Stationary phase is solid
 Such as alumina or silica gel

19. Cont…
 The mobile phase is liquid
 Separation occurs when components get,
 Adsorbed on the solid phase with different strength
 In theory, adsorption chromatography,
 More complex than partition chromatography

20. Thin layer Chromatography (TLC)
 The term “thin-layer chromatography”, introduced by N.A
Izamailov and M.S Shraiber in 1938,
 means a chromatographic separation process in which the
stationary phase consists of a thin layer applied to a solid
substrate or “support”.
 In TLC, the sample solution is applied as a spot or band on
the origin of the layer spread on a support (plate).
 After evaporation of the sample solvent, the plate is placed
in a sealed chamber containing a solvent chosen as a
mobile phase.

21. Cont…
 Development occurs as the mobile phase moves through the
layer, and the components of the sample move at different rates to
create the separation.
 Environmental, pharmaceutical, biomedical and food samples are
among the sample types commonly analyzed by TLC

22. Cont…
.
 Advantages of TLC include
 Rapid analysis time because many samples can be analyzed
simultaneously,
 Low solvent usage,
 A high degree of accuracy and precision for instrumental TLC, and
 Sensitivity in the nanogram or pictogram range.
 Coupling of TLC with other analytical methods such as high
performance liquid chromatography (HPLC), mass
spectrometry (MS), and Fourier transform infrared spectrometry
(FTIR) provides enhanced opportunities for sample analysis.

23. Cont…
Data Analysis
 The position of a substance zone (spot) in a thin-layer chromatogram can be
described with the aid of the retardation factor Rf. This is defined as the quotient
obtained by dividing the distance between the substance zone and the starting
line by the distance between the solvent front and the starting line

24. Cont…
 This value is calculated as follows:
 The Rf value is characteristic for the compound under those particular
chromatographic conditions. The response of a compound to a particular detection
reagent (e.g. spray) is also characteristic of the substance.
Figure : (a) A TLC plate before elution (spot 1 contains one application, spot 2, two
applications and spot 3,three applications); (b) Running a TLC chromatogram; (c) Example
after elution.
82
.
0
8
.
6
6
.
5
)
( 


b
a
pink
Rf
41
.
0
8
.
6
8
.
2
)
( 


a
c
blue
Rf

25. Cont…
 Application of TLC analysis
 Application of TLC include analysis of the following types: starting raw
,material (plant extracts ,extracts of animal origin, fermentation mixtures);
intermediates( crude products, reaction mixtures, mother liquors, and
secondary products);
 Pharmaceutical raw material ( identification, purity testing , assay,
separation of closely related compounds, stability testing); formulated
products ( identification, purity testing, assay, separation of closely related
compounds, stability testing under storage and stress, content uniformity
test, dissolution test); and
 Analysis of drugs and their metabolites in biological media such as urine,
plasma, or gastric fluid(pharmacological , toxicological, pharmacokinetic,
metabolic, bioequivalence, forensic and compliance and pharmacodynamic
studies).

26. Qualitative Analysis
 TLC is often used by BP monographs as part of a number of identity tests
performed on pure drug substances.
 Table 1 lists a few compounds which have their identity checked by TLC and
a variety of location reagents and mobile phases to illustrate the fact that
there is much less uniformity about TLC methodology than there is in the
case of HPLC and GLC methodology.

27. Stationary phase Mobile phase Visualizatio
n reagents
Comments
Framycein sulphate
Methyl prednisolone
Aprotinin
Levamisole
Pentagastrin
Silica gel +
carbomer binder
Silica gel GF254
Silica gel
Silica gel with
fluorescent
indicator
Silica gel G
10%w/v
KH2PO4
Ether/toluene
/butanol-1-ol
saturated with
water
(85:10:50
Acetate buffer
Toluene/aceto
ne/13.5M
ammonia
(60:40:1)
Analyte is
examined by
TLC in three
different
mobile phases
Naphthalen
ediol /
H2SO4
UV light
254nm then
ethanolic
sulfuric acid
(20%),+
heat to
1200C
Ninhydrin
spray
UV light
254 nm
4-
dimethylam
ino-
benzaldhyd
e in
methanol/H
Cl
Rf and color of the
sample are
compared with a
pure standard. The
resolution of the
analyte from
streptomycin is
checked
Rf and color of the
sample are
compared with a
pure standard. Also
Rf of an oxidation
product is used as
an additional check
Rf and color of the
spot obtained is
matched to that of a
standard
Rf and the size of the
spot obtained is
matched to that of a
standard
The Rf of the analyte
in three different
mobile phases is
determined and the
color of its spot is
matched to that of
the standard