Chromatography Notes

Chromatography

• Derived from the Greek words chroma ("color") and graphein ("to write").

• Developed by Mikhail Tswett in 1906 for separating plant pigments.

• It is used for separation, identification, and determination of chemical components in mixtures.

• Process involves differential migration in a system with two phases: stationary and mobile.

Two Phases of Chromatography

• Stationary Phase (SP): Fixed phase; solid or liquid coated on a support. Interacts with solutes based on properties such as polarity or charge.

• Mobile Phase (MP): Liquid or gas that moves through the SP, carrying the solute. Elution strength is a critical property.

Elution

• Process of extracting material by washing with a solvent. Crucial for moving separated components off the stationary phase.

• Elute: action.

• Eluent: MP + solvent.

• Eluate: product of separation

Applications

• Resolution of mixtures, determination of homogeneity, comparison of substances. Used extensively in analytical chemistry.

• Purification and concentration of substances. Essential in pharmaceutical and biochemical research.

• Identification and quantification of substances in complex mixtures. Applicable in environmental monitoring and food analysis.

• Molecular structure identification. Can be coupled with techniques like mass spectrometry.

Classification of Chromatographic Methods

• Adsorption, partition, ion exchange, size exclusion, affinity, electrophoresis. Each method exploits different physicochemical properties.

Adsorption

• SP: Solid adsorbent. Analyte interacts directly with the solid surface.

• MP: Liquid or gas adsorbate.

• Examples: Thin Layer Chromatography, Column Chromatography

Partition

• SP: Liquid on inert solid. Separation based on solubility differences.

• MP: Liquid or gas.

• Examples: Paper Chromatography, High Pressure Liquid Chromatography (HPLC).

• Types: Liquid-Liquid (Normal & Reverse Phase).

Liquid-Liquid Partition

• Normal Phase: Polar SP, Non-polar MP. Suited for separating non-polar compounds.

• Reverse Phase: Non-polar SP, Polar MP. Ideal for polar compounds; most common HPLC mode.

Ion Exchange

• SP: Ion exchange resin (polymeric matrix). Resin contains fixed ions that exchange with mobile ions.

• MP: Liquid.

• Application: Inorganic ions separation. Also used in water purification and amino acid analysis.

Size Exclusion

• Separation based on molecular size. Also known as gel permeation or gel filtration chromatography.

• SP: Porous polymer. Molecules are separated based on their ability to enter pores.

• MP: Liquid or Gas.

• Types: Gel Filtration (hydrophilic SP), Gel Permeation (hydrophobic SP).

Affinity

• Ligand covalently bonded to solid support. Highly selective; exploits specific biological interactions.

• Matrix: Solid (e.g., agarose). Common in protein purification.

Electrophoresis

• Migration of charged molecules in an electric field. Rate of migration depends on charge, size, and shape.

• Application: Separation of biological substances (proteins, nucleic acids). Commonly used in DNA sequencing and protein analysis.

Chromatographic Techniques

• Column Chromatography (CC), Paper Chromatography (PC), Thin Layer Chromatography (TLC), Gas Chromatography (GC), High Performance Liquid Chromatography (HPLC).

Column Chromatography

• SP held in a narrow tube, MP forced through. Can be used for preparative and analytical purposes.

Paper Chromatography

• SP: Filter paper. Simple and inexpensive.

• MP: Various solvents. Choice of solvent affects separation.

• Determination: $R_f$ values. Used for compound identification.

• Methods: Descending, Ascending, Radial Chromatography.

Thin Layer Chromatography

• SP: Thin layer on a plate. Rapid and versatile.

• MP: Liquid.

• SP Examples: Silica Gel, Alumina, Polymide, Cellulose. Choice depends on the nature of the compounds.

• Detection: Charring, Iodine Vapor, UV Radiation. Methods to visualize separated compounds.

Gas Chromatography

• Sample vaporized and carried by inert gas (MP) through a column with liquid SP. Suitable for volatile compounds.

• Components: Carrier gas, sample injector port, column, detector, recorder.

Column

• Types: Packed, Capillary. Capillary columns offer higher resolution.

• Parts: Tubing, Solid Support, Stationary Phase.

High Performance Liquid Chromatography

• Methods: Affinity, adsorption, partition, ion exchange, and molecular exclusion. Most advanced chromatographic method and most commonly used analytical method in drug testing.

• SP: ODS Silica gel. Provides a non-polar surface for reverse-phase chromatography.

• Components: MP, solvent reservoir, pumps, injection valve, column, detector, integrator.

Ultra Performance Liquid Chromatography (UPLC)

• High efficiency, high resolution, speed of analysis, robustness. Utilizes smaller particles for increased resolution.

• Coupling with mass spectrometry (MS) brings further advantages in terms of selectivity, sensitivity, and high throughput for the analysis of complex samples.

Chromatographic Process and Evaluation

• Theoretical plates: Equilibrium between MP and SP. Measure of column efficiency.

• Types: Complete (TLC, PC), Continuous (HPLC, GC). Refers to how the separation is achieved.

Qualitative Evaluation of Chromatogram

• Retardation Factor ($R_f$) - Complete. Used in TLC and paper chromatography.

• Retention Time ($t_R$) - Continuous. Used in HPLC and GC.

• Retardation Volume ($V_R$) - Continuous. Volume of mobile phase required to elute a solute.

Retardation Factor ($R_f$)

• $R_f = \frac{distance \ travelled \ by \ solute}{distance \ travelled \ by \ solvent}$

• Complete chromatography.

Retention Time ($t_R$)

• Time from start to elution maximum.

• Continuous chromatography.

Retardation Volume ($V_R$)

• Volume of MP to elute compound.

• $V<em>R = t</em>R (min) \times flow \ rate (mL/min)$

• Continuous chromatography.