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BP701T

INSTRUMENTAL METHODS OF ANALYSIS

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Unit – III

Introduction to chromatography

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BP701T INSTRUMENTAL METHODS OF ANALYSIS 45 Hours

Scope:

This subject deals with the application of instrumental methods in qualitative and quantitative analysis of drugs. This subject is designed to impart a fundamental knowledge on the principles and instrumentation of spectroscopic and chromatographic techniques. This also emphasizes on theoretical and practical knowledge on modern analytical instruments that are used for drug testing.

 

Objectives:

Upon completion of the course the student shall be able to

  • Understand the interaction of matter with electromagnetic radiations and its applications in drug analysis
  • Understand the chromatographic separation and analysis of drugs.
  • Perform quantitative & qualitative analysis of drugs using various analytical instruments.

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THIN LAYER CHROMATOGRAPHY

  • Principle
  • Materials used to perform TLC
  • Experimental technique
  • Application
  • Advantages
  • Disadvantages

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Principle

  • The sample is dissolved in in a volatile solvent
  • Sample is applied with the help of capillary on to the base line drawn on the solid adsorbent.
  • The plate is dipped in to the solvent working as mobile phase.
  • As the mobile phase rises up the TLC plate by capillary action, the components dissolve in the solvent and move up the TLC plate.
  • In adsorption separation will occur on the basis of differences in adsorption (preferential adsorption). Weekly adsorbed component will separate first and strongly later on.
  • In partition , if the component is soluble in the mobile phase it will move with the solvent and vise versa .
  • The movement of the solute is measured relative to that of solvent is expressed as the retardation factor.

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  • This technique manipulates POLARITY
    • More polar substances bind strongly to the adsorbent and elute SLOWER
    • Less polar substances bind weakly to the adsorbent and elute FASTER
  • The strength of interactions between the adsorbent and eluting components vary approximately in this order:

Salt formation > coordination > H-bonding >

dipole-dipole > van der Waals

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Polarity decreases

More Polar

Less Polar

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OH

OH

OH

OH

OH

OH

OH

Mobile

phase

analytes

Silica Gel

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analytes

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More Polar

Less Polar

Adsorbs stronger and separate very slowly

Adsorbs weakly and separate very fast

Sample to be applied on this area

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Adsorption of Solutes

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The adsorption strength of compounds increases with increasing polarity of functional groups, as shown below:

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-CH=CH2, -X, -OR, -CHO, -CO2R, -NR2, -NH2, -OH, -CONR2, -CO2H.

(weakly adsorbed) (strongly adsorbed)

(nonpolar) (more polar)

Elution Strength of Mobile Phase (e)

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Elution strength is generally considered to be equivalent to polarity. A solvents elution strength depends on Intermolecular Forces between the solvent and the analytes and between the solvent and the stationary phase.

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A more polar (or more strongly eluting solvent) will move all of the analytes to a greater extent, than a less polar, weakly elution solvent.

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For example, the elution strength of hexane is very low; e = 0.01.

the elution strength of ethyl acetate is higher; e = 0.45

the elution strength of ethanol is even higher; e = 0.68

Thin Layer Chromatography - Calculation of Rf’s

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The Rf Value

  • A given compound will always travel a fixed distance relative to the distance the solvent travels
  • This ratio is called the Rf value and is calculated in the following manner:

. distance traveled by substance . �distance traveled by solvent front

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THIN LAYER CHROMATOGRAPHY

Calculation of Rf’s

The Rf is defined as the distance the center of the spot moved divided by the distance the solvent front moved (both measured from the origin)

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Materials used in TLC�

  • Glass Plate
  • Adsorbents
  • Oven for activation of plate
  • Developing chamber
  • Mobile Phase
  • A device for applying the adsorbent layer
  • Storage facility for the prepared plate

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Materials used in TLC

Glass Plate

Hooper

A device for applying the adsorbent layer

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Developing chamber

Mobile phase

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Stationery phase�

Stationery phase

Description

Application

Silica gel G

Silica gel with average particle size 15µm containing ca 13% calcium sulfate binding agent

Used in wide range pharmacopoeial test

Silica gel G254

Silica gel G with fluorescence added

Same application with Silica gel G where visualization is to be carried out under UV light.

Alumina (Al2O3)

surface area of about 200 m/g and a pore size of about 60 Å

Basic and neutral compounds, Terpenes, Alkaloids, Steroids, Aliphatic compounds, and Aromatic compounds

Cellulose

Cellulose powder of less than 30µm particle size.

Identification of tetracycline's

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MOBILE PHASE

  • TLC Solvents or Solvent Systems.
  • A single solvent or mixture of two solvents can work as mobile phase in TLC .
  • e.g. petroleum ether, carbon tetrachloride, chloroform, ethyl acetate, hexane can used as mobile phase.
  • The ability of mobile phase to move up is depend on the polarity itself
  • Volatile organic solvents is preferably used as mobile phase.

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MOBILE PHASE

SOLVENT

POLARITY INDEX

Hexane

0

Butanol

3.9

Chloroform

4.1

Methanol

5.1

Ethanol

5.1

Acetonitrile

5.8

Air

9.0

Water

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TLC plate preparation

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How to perform TLC?

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TLC Plate Preparation

Methods used to apply adsorbent

Spreading

Spraying

Dipping plate in slurry

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TLC Plate Preparation

  • TLC plates are usually commercially available, with standard particle size ranges to improve reproducibility.
  • They are prepared by mixing the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulphate (gypsum) and water. This mixture is spread as thick slurry on an uncreative carrier sheet, usually glass, thick aluminum foil, or plastic.
  • The thickness of the adsorbent layer is typically around 0.1 – 0.25 mm for analytical purposes
  • Around 0.5 – 2.0 mm for preparative TLC.

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Materials used in TLC

Glass Plate

Spreading the slurry by Hooper

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Developing chamber

Mobile phase

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Activation of plate

Glass Plate

  • Plate is kept for drying in oven at 100OC.
  • This step is called as activation of plate.
  • By doing this surface area of the adsorbent increases.

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Drawing a Line and circle to apply the sample

Glass Plate

Circle to apply sample

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Developing chamber

Mobile phase

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Materials used in TLC

Glass Plate

Circle to apply sample

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Developing chamber

Mobile phase

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Methods of Plate Development

  • Ascending development
  • Descending Development
  • Two dimensional Development

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Application of sample and development of Chromatogram by Ascending way

High

Polar

Less

Polar

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Development of Chromatogram by Descending way

High

Polar

Less

Polar

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Development of chromatogram by descending way

Mobile Phase

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Two dimensional chromatography

Solvent front

A

C

B

Solvent

A

B

C

AB

is

immersed in mobile phase

AC

is

immersed in mobile phase

X

X

Sample

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Physical Methods:

Ultraviolet Light—some organic compounds illuminate or fluoresce under short-wave UV light:

TLC Visualization Methods

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Chemical methods

Iodine Vapor—forms brown/ yellow complexes with organic compounds

Fluorescent Indicators—compounds fluoresce when placed under UV light

Silver Nitrate Spray (for Alkyl Halides)—dark spots form upon exposure to light

Sulfuric Acid Spray + Heat—permanent charred spots are produced

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Iodine Vapor—forms brown/ yellow complexes with organic compounds

Fluorescent Indicators—compounds fluoresce when placed under UV light

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Silver Nitrate Spray (for Alkyl Halides)—dark spots form upon exposure to light

Sulfuric Acid Spray + Heat—permanent charred spots are produced

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Applications of TLC

  • Qualitative analysis: -
  • If the separated components are colored then identification is very easy. All the visualizing agents used in paper chromatography (Detecting agents or indicators) can be used in TLC.
  • From Rf value qualitative analysis can be performed.

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Quantitative analysis

The size of the spot increases with the amount. Square root of the spot area is find out from that amount of solute can be found out.

Potentiodensitometry of the plate is carried out.

Flurometry or emission Spectroscopy is also used.

Separated spot is removed by knife edge. Its dissolved in proper solvent and its amount is finding out by volumetric analysis.

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Other Applications

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TLC

TLC can is applicable in the field of medicinal preparations, pharmaceutical preparations, natural product extract and related compounds.

Assaying the radiochemical purity of radiopharmaceuticals.

Determination of the pigments in plants.

In forensic science Laboratory detection of pesticides and insectides in food, poison etc.

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