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2 - Thin layer chromatography (TLC)

2 - Thin layer chromatography (TLC)

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17.2 Thin layer chromatography (TLC)



TLC is an easy technique for separation and identification of unknown compounds and requires simple apparatus. They readily provide qualitative information

and it is also possible to obtain quantitative data with careful attention. A variety of

small molecules such as amino acids, sugars, organic acids, and lipids are separated

by thin layer chromatography (TLC).

Chemicals required:

silica gel for TLC

butanol

acetic acid

ninhydrin as spraying agent

Preparation of solvent system: Solvent system is prepared using butanol, acetic acid,

and distilled water in the ratio of 50:10:40 mL. Pour about 100 mL of the solvent

system into the glass tank to a depth of 5 cm. Allow it to stand for 1 h with a lid over

the top of the tank.

Preparation of sample:

Sample 1: 50 mg of tryptophan was dissolved in 1 mL of water.

Sample 2: 50 mg of methionine was dissolved in 1 mL of water.

Sample 3: 50 mg of proline was dissolved in 1 mL of water.

Preparation of spraying reagent (w/v): 2% ninhydrin in acetone was prepared and

used as a spraying agent.

Preparation of silica gel and silica gel plates:

1. About 40 g of silica gel is dissolved in 100 mL of distilled water and is mixed

thoroughly to become slurry.

2. Glass plates are cleaned with ethanol and coat the glass plates with the above

silica gel slurry uniformly with the help of a spreader.

3. The plates are dried at room temperature at overnight and then heat the plates in

an oven for 1 h.

Procedure:

1. Leave 2 cm from one end of the glass plate and apply the sample containing

amino acid with the help of micropipette as small spots.

2. Allow the sample spots to dry and place the glass plates inside the solvent chamber.

3. The solvent rises by capillary action.

4. The chamber was covered with a lid and grease was applied to prevent the entry

of air into the chamber until the solvent reaches top of the glass plate.

5. Remove the glass plates from the tank, dry and proceed for the identification of

the separated compounds.

6. Spray with ninhydrin reagent for identification of amino acids.

Calculation:

The RF value of each sample was calculated as

RF =



Distance traveled by solute

Distance traveled by solvent



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140



CHAPTER 17  Analytical techniques



17.3  GAS CHROMATOGRAPHY (GC)

17.3.1 INTRODUCTION

Gas chromatography involves separation and analyses of different constituents of

mixtures by a mobile gas phase passing over a stationary adsorbent. The technique

is similar to column chromatography, except that the mobile phase is replaced by a

moving gas that is called the carrier gas.

Gas chromatography is a powerful tool for the analyses of organic materials. It is

very handy for low levels of pesticides and other contaminants of the environment.

Gas chromatography can be of two types: gas–liquid chromatography (GLC) and

gas–solid chromatography.



17.3.2 PRINCIPLE

In GLC the separation is brought about by partitioning the sample between a mobile

gas phase and a thin nonvolatile liquid layer coated on some inert solid particles,

while gas–solid chromatography is based upon selective adsorption of constituents

of the sample on a solid of large surface area used as the stationary phase.

When a mixture of volatile material transported by a carrier gas is led through a

column containing an adsorbent solid phase or more commonly an absorbing liquid

phase coated over a solid material, each volatile component is partitioned between

the carrier gas and the solid or the liquid.

Depending upon the retention time in the column, the volatile components emerge

from the column at different times and are finally detected by a suitable detector. If

the carrier gas used the rate of flow and the temperature of the column is kept constant, the retention time (ie, the time taken by each component of mixture to traverse

through the column) for each constituent of the mixture will always be the same.

It usually shows a linear relationship with the boiling point of the compound and

is a characteristic for the constituents concerned under the given set of conditions for

a given column. Thus, it is possible to identify the compound from its characteristic

retention time on a particular column and under a given set of conditions. Quantitative estimation can be carried out from the extent of peak area recorded by the detector recorder system.

Apparatus used for gas chromatography is a simple tube of about 4 mm in diameter and about 120 cm to many meters in length. It is made of stainless steel or glass

and is usually bent or coiled so that it could be accommodated in a small space. The

tube or the column is packed with particles of some suitable adsorbent or in the case

of GLC the fixed phase is a nonvolatile liquid coated over some solid support (particles of diatomaceous earth, crushed fire bricks, etc.).

As mobile phase, the gases used may be argon, helium, nitrogen, or hydrogen –

hydrogen is usually not preferred because of fire hazards. There has been a recent

trend to use capillary-gas chromatography in which instead of the column a very

thin capillary, about 0.25 mm in diameter and usually 50 m in length made of glass,

stainless steel, or some organic polymer, is employed. The inside of the capillary



17.3 Gas chromatography (GC)



wall is coated with the stationary liquid phase. These capillary columns are superior

to packed columns in terms of separation efficiency. They can separate up to several

components from a single sample.

To maintain a constant rate of flow of the carrier gas, there is a flow meter and

an adjustment device that regulates the flow of the carrier gas into the column. The

sample is introduced through a self-sealing silicon rubber partition into a chamber

that is heated to bring about evaporation of the sample. The temperature of the chamber must not be so high as to decompose the sample.

Solid samples have to be dissolved in some solvent, whereas gaseous samples

require special sample introduction valves. The detectors, placed at the exit of separation chamber, detect and measure the small amounts of separated components

present in the stream of the carrier gas leaving the column. Normally, three types

of detectors are employed in gas chromatography: thermal conductivity detectors,

flame ionization detectors, and electron capture detectors.



17.3.3 DETECTORS

17.3.3.1  Thermal conductivity detector

Thermal conductivity detectors are the most widely used detectors in gas chromatography. These detectors use heated metal filament (or thermisters that are made

of some semiconductor of fused metal oxides) to sense small changes in thermal

conductivity of the carrier gas. Thermal conductivity of the carrier gas only gives an

essentially constant signal. The presence of vapors of the different components of the

mixture in carrier gas brings about changes in the thermal conductivity proportional

to their amount in the stream. This brings about changes in the resistance of the filament which is measured. The recorder that records these changes is equipped with an

automatic device that traces the magnitude of these changes on a graph sheet along

with the retention time.



17.3.3.2  Flame ionization detector

Flame ionization detectors are based on the measurement of electrical conductivity

of gases. At normal temperatures and pressure, gases act as a bad conductor or insulators but when ionized they act as a good conductor of electrical current.

Gases and vapors as they emerge from the separation column are mixed with hydrogen and burned in air to produce a flame which ionizes the component molecules

in the carrier gas. The burning jet is the negative electrode, while the anode is usually

a small loop of wire extending into the tip of the flame across which a small voltage

is applied.

The ions produced are collected at the electrodes and a current is generated which

is proportional to the number of the component molecules ionized. Only the carrier

gas burning with hydrogen produces an essentially constant signal but when the components of the mixture being analyzed emerge ionization occurs and a higher current

is observed. The detector is equipped with an automatic recording device that records

these fluctuations and transmits them to a graph sheet along with the retention time.



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CHAPTER 17  Analytical techniques



17.3.3.3  Electron capture detector

Electron capture detector is based on the phenomenon of electron capture of compounds

having an affinity for free electrons. A 3-ray source is used to obtain slow electrons by

ionization of the carrier gas (nitrogen is preferred) passing through the detector.

These electrons as they flow toward the anode under a fixed potential give rise to

a steady current. When the component molecules of the mixture being analyzed come

out of the separation column and pass through the detector these electrons are trapped.

The net result is replacement of the electrons by negatively charged ions of much

greater mass and corresponding reduction in the flow of electric current proportional

to the concentration of electron capturing component in the carrier gas. These changes are detected by using a suitable circuit and recorded with the help of an automatic

device that traces a graph along with the retention time.



17.4  HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

(HPLC)

Determination of quality parameters under stress conditions using HPLC (Fig. 17.2)

Principle: Separation of mixture of compound into individual components through

equilibrium distribution between stationery and mobile phases.



FIGURE 17.2  High-Performance Liquid Chromatography.



17.4 High-performance liquid chromatography (HPLC)



It is a separation technique that involves

• injection of a small volume of liquid sample

• into a tube (column) packed with tiny particles (3–5 mm in diameter called

stationery phase)

• where individual components of the sample are moved down the packed tube

(column) with a liquid (mobile phase) forced through the column by high

pressure delivered by a pump.

These components are separated from one another by the column packing that

involves various chemical and or physical interactions between their molecules and

the packing particles. These components are detected at the exit of this tube (column)

by a flow through device (detector) that measures their amount. An output from this

detector is called a “liquid chromatogram.”



17.4.1  ROLE OF FIVE MAJOR HPLC COMPONENTS

Pump: The role is to force a liquid (called the mobile phase) through the liquid chromatography at a specific flow rate expressed in milliliters per minute. The normal

flow rates are 1–2 mL/min. Pumps can reach pressures in the range of 6000–9000

psi (400–600 bars).

Based on the requirement of one or two pumps, pumps are of two types:

1. Isocratic pump: It delivers constant mobile phase composition (solvent is

premixed) and remains constant with time.

2. Gradient pump: It delivers variable mobile phase composition (binary gradient

delivers two solvents). Mobile phase solvent composition increases with time.

Autosampler (injector): The injector serves to introduce the liquid sample into

the flow stream of the mobile phase (sample volume ranges between 5 and 20 mL)

automatically. The injector must also be able to withstand the high pressures of the

liquid system.

Column: Column is considered the “heart of the chromatograph.” Column stationery phase separates the sample components of interest by interaction between the

sample components and the column packing material. The small particles inside the

column cause high back pressure at normal flow rates. Analytical column internal

diameter ranges from 1.0 to 4.6 mm and lengths from 15 to 250 mm. Columns are

packed using high pressure to ensure that they are stable. There are two major separation modes that are used to separate most compounds.

1. In reverse phase chromatography, the column packing material is nonpolar (C18,

C8, C3, phenyl, etc.) and the mobile phase is water (buffer) or water miscible

organic solvents (eg, methanol, acetonitrile). The organic solvent increases the

solvent strength and elutes compounds that are very strongly retained. The packing

material in C18 column is octa dodecyl silane which has highly stable bonds.

2. In normal phase chromatography, the column packing is polar and the mobile

phase is nonpolar (eg, hexane, isooctane, ethyl acetate).



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