chromatographic methods of analysis

8/10/2019 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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The layer may be impregn

suitable materials suc

TLC PlatesStationary P

Buffering Ma

Silver Nitr

Ion-Exchange M


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Mobile Phase: a solvent moving across the surface of


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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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Pouring of Layer

Dipping

Spraying


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Pouring of Layer

Dipping

Spraying

Spreading


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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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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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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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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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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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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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2. Protection Against Oxidation

Temperature:18-23CLight:Diffused daylight both natural and artificial


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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


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


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


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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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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Pump

To convey mobile phase at high prcontrolled flow rate


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Sample Injection

To convey mobile phase at high pr

controlled flow rate


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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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Detector

Monitors the mobile phase coming out of thecolumn.


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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

chromatographic methods of analysis

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