chromatographic methods of analysis
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Chromatographic
Methods of AnalysiPrepared by: Juangco, Cris-An
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Objectives
To enumerate different chromatographic methods used in testdrug substances and products.
To recognize theories involve in each type of chromatographicmethods.
To determine the qualitative and quantitative analysis performe
these methods. To apply these methods in analysis of pharmaceutical drugs.
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Introduction
Chromatography comes fromGreek words,
To write
color
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Introduction
Credited to Mikhail Tsvuses chromatography iseparated plant pigmenchlorophylls and xanthopassed them through apacked with calcium ca
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Thin-Layer Chromatography
Invariably termed in other literatures as:
open-column chromatography
drop chromatography
strip chromatography
spread-layer chromatography
surface chromatography
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Thin-Layer Chromatography: Theory
A uniform layer of dry
powdered (adsorbents)supported by a gla
aluminum foil or pla
TLC PlatesStationary P
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Thin-Layer Chromatography: Theory
The layer may be impregn
suitable materials suc
TLC PlatesStationary P
Buffering Ma
Silver Nitr
Ion-Exchange M
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Thin-Layer Chromatography: Theory
Mobile Phase: a solvent moving across the surface of
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Thin-Layer Chromatography: Theory
Chromatographic Chamber: usually glass for a clear ob
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Versatility of TLC
Simple Equipment
Short DevelopmentTime
Wide Choice ofStationary Phase
Constituents QuickRecovery
Distinct Separation
Easy Visualization
High detection l
Variable LThickne
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Experimental Techniques in TLC
Preparation of Thin Layer
Pouring of Layer
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Experimental Techniques in TLC
Preparation of Thin Layer
Pouring of Layer
Dipping
Spraying
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Experimental Techniques in TLC
Preparation of Thin Layer
Pouring of Layer
Dipping
Spraying
Spreading
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Experimental Techniques in TLC
Preparation of Thin Layer
Pre-coated Layer
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Choice of Adsorbents
Solubility of Substance
Nature of Compound
Reactivity of Compound
Chemical Activity of Compou
Binders
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Experimental Techniques in TLC
Solvent System
Choice of Solvent
Depends upon the (1) nature of the constituents to be separat(2) nature of the process involved
Solvents are arranged according to their eluotropic power for an ease of the solvents from the given adsorbents.
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Experimental Techniques in TLC
Purification of Adsorbent
Procedure
The iron-free layers may be achieved by providing the pre-coated plates a preliminary development with a mixture of methanol and chydrochloric acid* (9 : 1). By this process the entire iron gets migra
solvent front to the upper boundary of the TLC plate. Consequently, thare again dried and activated at 110C.
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Experimental Techniques in TLC
Spotting of the components
1. Sample is normally applied as a solution in a non-polar solvent as far a
2. The solvent employed for dissolving the sample must be easily volatile
3. The area of application should be smallest as far as possible so as to asharper resolution.
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Experimental Techniques in TLC
Spotting of the components
4. To maintain the size of the spot small repeated applications is made bthe solvent to evaporate after each application
5.Use of spotting templates,
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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 cleast 1 to 2 hours so that the vapors of the solvent(s) would pre-satu
adequately. This is done to obtain distinct separation of constituents, from and prevent evaporation of the solvent on TLC-plat
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Experimental Techniques in TLC
Development of Thin Layer
2. Protection Against Oxidation
Temperature:18-23CLight:Diffused daylight both natural and artificial
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Experimental Techniques in TLC
Development of Thin Layer
3. Visualization
Used of fluorescent indicators such as Examples: Barium diphenylamine2,7-dichlorofluorescein ; Fluorescein (0.2% w/v in Ethanol) ; Morin (0.1% ; Sodium fluorescinate (0.4% w/v in water) ; Rhodamine B ; Zinc Silicate ;silicate ; Methylumbelliferone (or 7-hydroxy-4-methyl coumarin).
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Detection of Components
ColoredSubstances
ColorlessSubstances
SpecificDetectingReagents
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Evaluation of TLC
Qualitative Evaluation
Rf Values
The Rf value represents the differences in rate of movement of the compcaused by their various partition coefficients i.e., their different solubilityand stationary phases.
=
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Evaluation of TLC
Quantitative Evaluation
Direct Methods
performed directly on the adsorbent layer.
Measure-ment of
Spot Areas
Densito-metry
Spectro-photomet
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Evaluation of TLC
Quantitative Evaluation
Indirect Methods
the separated constituents are quantitatively removed from, the ads
subsequently estimated after elution
Colorimetry ; Fluorimetry ; Radiometry ; Flame-photometry ; UV-SpectrGravimetry ; Polarography ; Vaporphase Chromatography;
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Application in Pharmaceutical Analysi
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 Acid
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Gas Liquid Chromatography
Gas chromatography makes use, as thestationary phase, a glass or metal column filled
either with a powdered adsorbent or a non-volatile liquid coated on a non-adsorbent powder.The mobile-phase consists of an inert-gas loaded
with the vaporized mixture of solutes flowingthrough the stationary phase at a suitabletemperature. In the course of the passage of the
vapor of the sample through the column
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Advantages of GLC
High frequency separation
High Degree of Detection
Rapid speed of analysis
Accurate and Precise Results
Ease of analysis
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Instrumentation
Carrier Gas Tank
Gas can be hydrogen, helium, nitro
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Instrumentation
Pressure Regulator and Flo
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Instrumentation
Sample Injection Syst
Depends upon the sample to be
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Instrumentation
Separation Column
Made up of stainless steel, copper ocoiled with length varying from 120 c
and ID of 4.0mm
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Instrumentation
Thermal Compartme
Maintain invariant-tempera
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Instrumentation
Detector
Can be TCD, FID, ECD, NP-FID, FP
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Instrumentation
Integrator
Device that facilitates simulta
measurements.
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Evaluation of GLC
Quantitative Evaluation
Area Normalization
Features: Very suitable for routine type of samples where the variations in comp
marginal i.e., in such cases where the response factors need to be checperiodically only when necessary.
An obligatory condition of this method being that all the componentsshould elute and be recorded
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Evaluation of GLC
Quantitative EvaluationInternal Standard Method
Features: It gives very accurate and precise results, It completely eliminates possibility of error caused due to loss of some
sample (other than the determined components) during the initial prep It eliminates error due to incomplete elution of all the sample compone It eliminates error caused due to inaccurate measurement of sample siz
injection
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Evaluation of GLC
Quantitative Evaluation
Comparison Method
The comparison method makes use of a purely synthetic blend containin
component to be determined in the same order of concentration as expecsample.
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Application in Pharmaceutical Analysi
Assay of Drugs
Determination of specific organic impurities
Determination of Related Substances in official drugs
Determination of water in drugs
Determination of chloroform
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High Performance Liquid Chromatograph
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.
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High Performance Liquid Chromatography: Th
Bonded-Phase Supports
Bonded-Phase Supports: The bonded-phase supports usually overcome pthe nagging problems which is mostly encountered with adsorbed-liquid pthe molecules, comprising the stationary phase, i.e., the surfaces of the siliare covalently bonded to a silica-based support particle.
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HPLC: Instrumentation
Solvent Reservoir
1 dm3
glass bottle having a lid anddiameter tube to convey the mobile
the reservoir to the degass
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HPLC: Instrumentation
Degasser
subjecting the mobile phase unde
distillation, spurging with fine spraygas of low solubility such as Argon o
by heating and ultrasonic sti
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HPLC: Instrumentation
Pump
To convey mobile phase at high prcontrolled flow rate
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HPLC: Instrumentation
Sample Injection
To convey mobile phase at high pr
controlled flow rate
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HPLC: Instrumentation
HPLC column
Material : Stainless-steel (highly polis
External Diameter : 6.35 mm (or 0.Internal Diameter : 4-5 mm (usual : 4
Length : 10-3 cm (usual : 25 cm).
Packings can be styrene-divinyl copoPorous layer beads and porous-silica
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HPLC: Instrumentation
Detector
Monitors the mobile phase coming out of thecolumn.
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HPLC: Instrumentation
Detector
Monitors the mobile phase coming out of thecolumn.
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Applications in Pharmaceutical Analysis
Isolation of Natural Active Compounds
Control of microbiological processes
Assays of Drugs
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Size Exclusion Chromatography
a means of separation which is exclusively dependent on the exchangmolecules between the solvent of the mobile-phase and the same solve
pores of the column-packing material
Gel FeaturesCross-linking
natureHydrophillic Voids
1 2 3
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Size Exclusion Chromatography: Theory
The efficiency and ability of a gel to slow down the movement of various sdownwards in a packed column with the respective gel entirely depends omolecular size of the substance in relation to the pore sizes prevailing withmatrix
The liquid phase which is absorbed by the synthetic polymer granules (e.gmostly available in a wide range as solvent for solute molecules in contactand is affected by pH, ionic strength and its concentration.
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Materials
AgaroseAgarose,
CrosslinkedSilica Ge
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Size Exclusion Chromatography: Apparatus
The apparatus for size-exclusion chromessentially comprises of a chromatograp
generally made up of glass having a diame
ratio of between 1 : 10 and 1 : 20, packeappropriate separation material (e.g., diffe
Sephadex)
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Applications in Pharmaceutical Analysis
Determination of Relative Component Composition
Determination of Molecular Weight
Assays of Drugs for impurities
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Thank You!Prepared by: Juangco, Cris-An
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