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,

Chroma
color
Graphein
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
Easy Visualization
Time
Wide Choice of
High detection limit
Stationary Phase
Constituents Quick Variable Layer
Recovery 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 110°C.
 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: 18-23°C
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

Specific
Colored Colorless
Detecting
Substances Substances
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.

  𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑜𝑓 𝑐𝑒𝑛𝑡𝑒𝑟 𝑜𝑓 𝑠𝑝𝑜𝑡 𝑓𝑟𝑜𝑚 𝑡h𝑒 𝑠𝑡𝑎𝑟𝑡𝑖𝑛𝑔 𝑝𝑜𝑖𝑛𝑡

𝑅𝑓 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑚𝑡h𝑒 𝑠𝑡𝑎𝑟𝑡𝑖𝑛𝑔 𝑝𝑜𝑖𝑛𝑡
Evaluation of TLC

Quantitative Evaluation

Direct Methods
performed directly on the adsorbent layer.

Measure-
ment of Densito- Spectro-
Spot Areas metry photometry
 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 non-
volatile 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 2 3
Cross-linking
Gel Features Hydrophillic Voids in gels
nature
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 Silica Gel
Crosslinked
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 mobile-
phase,
• 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.