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High Pressure Thin-Layer Chromatography Principles and Practice

BY: Debasis Sahu | Category: Biology | Submitted: 2010-11-14 08:36:59
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Article Summary: "Thin Layer Chromatography (TLC) is a simple, rapid, versatile, sensitive, inexpensive analytical technique for the separation of substances. Whereas the mobile phase is a liquid, containing a single solvent or a mixture of solvents and the stationary phase is an active solid, known as sorbent. It is also known as planar chromato.."


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Introduction

Thin-Layer Chromatography (TLC) is a simple, rapid, versatile, sensitive, inexpensive analytical technique for the separation of substances. Whereas the mobile phase is a liquid, containing a single solvent or a mixture of solvents and the stationary phase is an active solid, known as sorbent. It is also known as planar chromatography. TLC can be used both an analytical and a preparative technique.

Advantages of TLC
TLC has many advantages over other chromatographic methods for separation of simple mixtures-
• Simple
• Rapid
• Inexpensive
• Multiple sample application
• Volume of solvent used per sample is less

Principle
Adsorption

Solute and solvent molecule compete for 'sites' on the adsorbent, to be adsorbed, the solute molecule must first displace a solvent molecule. Molecules with polar functional groups or those capable of hydrogen-bonding will have a strong affinity for the adsorbent surface and will be strongly retained. The adsorbent surface consists of discrete adsorption sites. In the case of silica, these are hydroxyl (-OH) groups, the grouping SI-OH is known as silanol group.

Partition
The ability of a solute to distribute itself between two phases according to the distribution or partition coefficient
K=Cs/Cm

Ion-exchange
If the process involves the exchange of negatively charged ions, it is known as anion-exchange. The complementary process is known as cation-exchange.

Size-exclusion
Retention of solutes on the basis of size or shape.

Factors Affecting Retention
Intermolecular forces

All such forces are electrostatic in origin and based on Columbus law of attraction between unlike and repulsion between like forces.
• Coulombic forces between ions
• Dipole-dipole interaction: Forces arising from interaction between molecules having
permanent dipole
• Dipoles are induced by neighbouring molecules
• Forces between neutral atoms or molecules
• Hydrogen bonding interactions

Steps Involved in TLC/HPTLC Analysis

The analytical procedure for trace organic substances in natural matrices involves
• Sampling and sample preservation
• Sample preparation and clean-up
• Selection of TLC/HPTLC plates
• Application of samples
• Selection of solvent system
• Separation of the compounds of interest
• Detection and quantification
• Data reduction, record keeping and quality control

TLC/HPTLC Plates
Coating Materials

• Silica gel
• Alumina
• Cellulose
• Polyamide-separation affected mainly due to hydrogen bonding between the solute and the amide group, used for separation of phenols, carboxylic acids, aromatic nitro compounds alkaloids, heterocyclic compounds.
• Sephadex- modified dextran gel used to separate hydrophilic solutes such as peptides and nucleic acid based on size by gel filtration
• Ion exchangers
• Mixed layers
• Impregnated layers
• Chiral TLC plates

Identification of Compounds
Position of separated spots on chromatograms are characterized by the Rf value of each substance, where
Rf = Distance traveled by solute
Distance traveled by solvent

Quantification
Requirements for Quantitation

• Sample preparation must be reproducible
• Accurate and precise application of samples
• Separation of specified component
• Standards and samples are always chromatographed on the same plate
• Identity of the component

Method of Quantification
Scraping and elution: Scraping the separated analyte, recovery of the compound by elution and analyzed by titration, electroanalytical, GC, UV-VIS.

Visual comparison: Samples and standards are chromatographed side by side on the same plate and compare the spot size and intensity.

Densitometry: It can be carried out directly on the plate in different modes viz., absorption, transmission and fluorescence. A calibration curve consisting areas of the standards versus amount analyte spotted is constructed and the amount of analyte in the sample is interpolated from the curve.

Applications
HPTLC finds lots of applications in the fields of
1. Pharmaceuticals (Drug monitoring in biological fluids, impurity profiles).
2. Forensic (Drug of abuse, poisons, adulterations)
3. Environment (pesticide residues in crops, food products).


In conclusion, HPTLC is the enhancement to the basic TLC method including automation of the different steps, to increase the resolution and to allow more accurate and precise quantitative measurements.

About Author / Additional Info:
Written by Debasis Sahu and Shikha Sharma

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