Chromatographic
Methods of Analysis
Prepared by: Juangco, Cris-Anne III A.
Objectives
To enumerate different chromatographic methods used in testing
drug substances and products.
To recognize theories involve in each type of chromatographic
methods.
To determine the qualitative and quantitative analysis performed by
these methods.
To apply these methods in analysis of pharmaceutical drugs.
Introduction
Chromatography comes from the
Greek words,
color
To write
Introduction
Credited to Mikhail Tsvet when he first
uses chromatography in 1906 when he
separated plant pigments such as
chlorophylls and xanthophylls. He
passed them through a column
packed with calcium carbonate.
Type of Chromatographic Methods
Thin-Layer Chromatography
Gas Chromatography
High Performance Liquid Chromatography
Size Exclusion Chromatography
Thin-Layer Chromatography
Invariably termed in other literatures as:
open-column chromatography
drop chromatography
strip chromatography
spread-layer chromatography
surface chromatography
Thin-Layer Chromatography: Theory
TLC Plates
Stationary Phase
A uniform layer of dry, finely
powdered (adsorbents) material
supported by a glass,
aluminum foil or plastics.
Thin-Layer Chromatography: Theory
TLC Plates
Stationary Phase
The layer may be impregnated with
suitable materials such as:
Buffering Materials
Silver Nitrate
Ion-Exchange Materials
Thin-Layer Chromatography: Theory
Mobile Phase: a solvent moving across the surface of the plate.
Thin-Layer Chromatography: Theory
Chromatographic Chamber: usually glass for a clear observation.
Versatility of TLC
Simple Equipment
Distinct Separation
Short Development
Time
Wide Choice of
Stationary Phase
Constituents Quick
Recovery
Easy Visualization
High detection limit
Variable Layer
Thickness
Experimental Techniques in TLC
Preparation of Thin Layer
Pouring of Layer
Experimental Techniques in TLC
Preparation of Thin Layer
Pouring of Layer
Dipping
Spraying
Experimental Techniques in TLC
Preparation of Thin Layer
Pouring of Layer
Dipping
Spraying
Spreading
Experimental Techniques in TLC
Preparation of Thin Layer
Pre-coated Layer
Choice of Adsorbents
Solubility of Substance
Nature of Compound
Reactivity of Compound
Chemical Activity of Compound with
Binders
Experimental Techniques in TLC
Solvent System
Choice of Solvent
Depends upon the (1) nature of the constituents to be separated and
(2) nature of the process involved
Solvents are arranged according to their eluotropic power for an ease eluotropication
of the solvents from the given adsorbents.
Experimental Techniques in TLC
Activation of Adsorbent
Procedure
First by air-drying the TLC plates for a duration of 30 minutes and then in a hot-air
oven maintained at 110 C for another 30 minutes and subsequently cooling them in a
dessicator. This drying process helps a great extent in rendering the adsorbent layer
active. In order to achieve very active layers, silica gel and alumina coated plates may
be heated up to 150 C for a duration of 4 hours and cooling them in a dessicator.
Experimental Techniques in TLC
Purification of Adsorbent
Procedure
The iron-free layers may be achieved by providing the pre-coated and air-dried
plates a preliminary development with a mixture of methanol and concentrated
hydrochloric acid* (9 : 1). By this process the entire iron gets migrated with the
solvent front to the upper boundary of the TLC plate. Consequently, the purified plates
are again dried and activated at 110C.
Experimental Techniques in TLC
Spotting of the components
1. Sample is normally applied as a solution in a non-polar solvent as far as possible,
2. The solvent employed for dissolving the sample must be easily volatile-in-nature
3. The area of application should be smallest as far as possible so as to achieve a
sharper resolution.
Experimental Techniques in TLC
Spotting of the components
4. To maintain the size of the spot small repeated applications is made by allowing
the solvent to evaporate after each application
5.Use of spotting templates,
Experimental Techniques in TLC
Development of Thin Layer
1. Equilibration of the Chamber
It may be achieved by allowing the solvent system to remain in the chamber for at
least 1 to 2 hours so that the vapors of the solvent(s) would pre-saturate the latter
adequately. This is done to obtain distinct separation of constituents, uniform solvent
from and prevent evaporation of the solvent on TLC-plates.
Experimental Techniques in TLC
Development of Thin Layer
2. Protection Against Oxidation
Temperature: 18-23C
Light: Diffused daylight both natural and artificial
Experimental Techniques in TLC
Development of Thin Layer
3. Visualization
Used of fluorescent indicators such as Examples: Barium diphenylamine sulphonate ;
2,7-dichlorofluorescein ; Fluorescein (0.2% w/v in Ethanol) ; Morin (0.1% w/v in Ethanol)
; Sodium fluorescinate (0.4% w/v in water) ; Rhodamine B ; Zinc Silicate ; Calcium
silicate ; Methylumbelliferone (or 7-hydroxy-4-methyl coumarin).
Detection of Components
Colored
Substances
Colorless
Substances
Specific
Detecting
Reagents
Evaluation of TLC
Qualitative Evaluation
Rf Values
The Rf value represents the differences in rate of movement of the components duly
caused by their various partition coefficients i.e., their different solubility in the mobile
and stationary phases.
=
Evaluation of TLC
Quantitative Evaluation
Direct Methods
performed directly on the adsorbent layer.
Measurement of
Spot Areas
Densitometry
Spectrophotometry
Evaluation of TLC
Quantitative Evaluation
Indirect Methods
the separated constituents are quantitatively removed from, the adsorbent and
subsequently estimated after elution
Colorimetry ; Fluorimetry ; Radiometry ; Flame-photometry ; UV-Spectrophotometry ;
Gravimetry ; Polarography ; Vaporphase Chromatography;
Application in Pharmaceutical Analysis
Presence of Impurities in Drug Substances
Related Substances Present in Official Drugs
Foreign Alkaloids Present in Alkaloidal Drugs
Foreign Steroids Present in Steroidal Drugs
Ninhydrin Positive Substances Present in Official Amino Acids
Gas Liquid Chromatography
Gas chromatography makes use, as the
stationary phase, a glass or metal column filled
either with a powdered adsorbent or a nonvolatile liquid coated on a non-adsorbent powder.
The mobile-phase consists of an inert-gas loaded
with the vaporized mixture of solutes flowing
through the stationary phase at a suitable
temperature. In the course of the passage of the
vapor of the sample through the column
Advantages of GLC
High frequency separation
High Degree of Detection
Rapid speed of analysis
Accurate and Precise Results
Ease of analysis
Instrumentation
Gas can be hydrogen, helium, nitrogen or air.
Carrier Gas Tank
Instrumentation
Pressure Regulator and Flowmeter
Instrumentation
Depends upon the sample to be injected.
Sample Injection System
Instrumentation
Made up of stainless steel, copper or glass that is
coiled with length varying from 120 cm to 150 cm
and ID of 4.0mm
Separation Column
Instrumentation
Maintain invariant-temperature
Thermal Compartment
Instrumentation
Can be TCD, FID, ECD, NP-FID, FPD and PID.
Detector
Instrumentation
Device that facilitates simultaneous
measurements.
Integrator
Evaluation of GLC
Quantitative Evaluation
Area Normalization
Features:
Very suitable for routine type of samples where the variations in composition are only
marginal i.e., in such cases where the response factors need to be checked
periodically only when necessary.
An obligatory condition of this method being that all the components of the sample
should elute and be recorded
Evaluation of GLC
Quantitative Evaluation
Internal Standard Method
Features:
It gives very accurate and precise results,
It completely eliminates possibility of error caused due to loss of some part of the
sample (other than the determined components) during the initial preparation stage,
It eliminates error due to incomplete elution of all the sample components, and
It eliminates error caused due to inaccurate measurement of sample size before
injection
Evaluation of GLC
Quantitative Evaluation
Comparison Method
The comparison method makes use of a purely synthetic blend containing the
component to be determined in the same order of concentration as expected in the
sample.
Application in Pharmaceutical Analysis
Assay of Drugs
Determination of specific organic impurities
Determination of Related Substances in official drugs
Determination of water in drugs
Determination of chloroform
High Performance Liquid Chromatography
Invented due to the limitations of GC and LC.
Advantages
Capable of handling macromolecules,
Suitable for pharmaceutical compounds,
Efficient analysis of labile natural products,
Reliable handling of inorganic or other ionic species, and
Dependable analysis of biochemicals.
High Performance Liquid Chromatography: Theory
Bonded-Phase Supports
Bonded-Phase Supports: The bonded-phase supports usually overcome plethora of
the nagging problems which is mostly encountered with adsorbed-liquid phases. Here
the molecules, comprising the stationary phase, i.e., the surfaces of the silica particles,
are covalently bonded to a silica-based support particle.
HPLC: Instrumentation
1 dm3 glass bottle having a lid and a 1/8 inch
diameter tube to convey the mobile phase from
the reservoir to the degassers
Solvent Reservoir
HPLC: Instrumentation
subjecting the mobile phase under vacuum,
distillation, spurging with fine spray of an inert
gas of low solubility such as Argon or Helium or
by heating and ultrasonic stirring.
Degasser
HPLC: Instrumentation
To convey mobile phase at high pressure and
controlled flow rate
Pump
HPLC: Instrumentation
To convey mobile phase at high pressure and
controlled flow rate
Sample Injection
HPLC: Instrumentation
Packings can be styrene-divinyl copolymers,
Porous layer beads and porous-silica particles
Material : Stainless-steel (highly polished surface)
External Diameter : 6.35 mm (or 0.25 inch),
Internal Diameter : 4-5 mm (usual : 4.6 mm), and
Length : 10-3 cm (usual : 25 cm).
HPLC column
HPLC: Instrumentation
Monitors the mobile phase coming out of the
column.
Detector
HPLC: Instrumentation
Monitors the mobile phase coming out of the
column.
Detector
Applications in Pharmaceutical Analysis
Isolation of Natural Active Compounds
Control of microbiological processes
Assays of Drugs
Size Exclusion Chromatography
a means of separation which is exclusively dependent on the exchange of solute
molecules between the solvent of the mobile-phase and the same solvent within the
pores of the column-packing material
1
Gel Features
Cross-linking
nature
Hydrophillic
Voids in gels
Size Exclusion Chromatography: Theory
The efficiency and ability of a gel to slow down the movement of various substances
downwards in a packed column with the respective gel entirely depends on the
molecular size of the substance in relation to the pore sizes prevailing within the gel
matrix
The liquid phase which is absorbed by the synthetic polymer granules (e.g., Sephadex) is
mostly available in a wide range as solvent for solute molecules in contact with the gel
and is affected by pH, ionic strength and its concentration.
Materials
Agarose
Agarose,
Crosslinked
Silica Gel
Size Exclusion Chromatography: Apparatus
The apparatus for size-exclusion chromatography
essentially comprises of a chromatographic column
generally made up of glass having a diameter to height
ratio of between 1 : 10 and 1 : 20, packed with an
appropriate separation material (e.g., different grades of
Sephadex)
Size Exclusion Chromatography: Application of
sample
The sample is normally applied to the column by adopting one of the five following
methods, namely:
Directly to the drained-bed-surface with permitting the bed to dry,
Layered beneath the mobile-phase, provided the sample is denser than the mobilephase,
Using a flow adaptor,
Using a syringe through a septum, and
Using an injection valve.
Applications in Pharmaceutical Analysis
Determination of Relative Component Composition
Determination of Molecular Weight
Assays of Drugs for impurities
Thank You!
Prepared by: Juangco, Cris-Anne III A.
