Chromatography P2 : Applications

What are the 2 planar chromo types

Paper and TLC

What are the specific stationary/mobile phases for paper and what they are used for.

• Stationary Phase: A layer of water bound to the cellulose fibers of the paper.

• Mobile Phase: The solvent in which the paper is dipped.

• Application: Used to assess the purity of a sample

What are the specific stationary/mobile phases for TLC and what they are used for.

• tHIN LAYER - DEF

• Stationary Phase: A sheet of glass, metal, or plastic coated with a thin layer of adsorbent (usually silica or alumina).

• Procedure: Samples are "spotted" at the base and dried. The sheet is placed in a tank with a shallow layer of solvent (mobile phase) which moves up.

• Detection: Colorless analytes are visualized using UV light or specific dyes

What is general process stages for column?

: 1. Equilibration, 2. Sample Application, 3. Elution, 4. Collection, 5. Regeneration .

Simple Column Chromatography Types

Gel filtration (size), Ion exchange (charge) Affinity (shape)

Gel Filtration - principle/S.P/MECHANISN/APPLICAION

• Principle: Separates molecules based on size (mass).

• Stationary Phase: Porous beads/particles.

• Mechanism (Crucial for Exam):

  • Large Molecules: Cannot enter the pores. They occupy the "void volume" (space between beads) and are washed through first.

  • Small Molecules: Penetrate the pores and have a longer distance to travel, so they elute last.

• Applications: Fractionation (separating by size) and Desalting (removing salts from samples)

Ion exchange - - principle/MECHANISm/APPLICAION

• Principle: Separates based on net charge. Relies on reversible interaction with an oppositely charged stationary phase²⁹.

• The "pI" Rule (Isoelectric Point):

  • pI: The pH at which a molecule has no net charge³⁰.

    pH < pI: Molecule becomes Positive (Cation) Binds to Cation Exchanger (which has negative beads)

    pH > pI: Molecule becomes Negative (Anion) Binds to Anion Exchanger (which has positive beads)

Applications: Protein purification, DNA/RNA separation, Water analysis (heavy metals), Drug purification

Draw coloumn in ion exchange

Affinity - Principle/S.P/Mechanism/Application

• Principle: Uses specific binding ("Lock and Key") or shape differences.

  Stationary Phase: An insoluble matrix with a specific ligand attached (e.g., antibody, enzyme substrate).

• Mechanism: The target analyte binds to the ligand while impurities wash away. The target is then eluted .

• Applications:

• Enzymes: Purified using bound substrates/inhibitors.

  Antibodies/Antigens: Immobilize one to purify the other.

• Hormones/Receptors: Purified from cell homogenates.

What are 2 instrumental chromo types

HPLC and Gas chromo GC

Description and Materials in hplc

• Description: Uses high pressure to force mobile phase through the column for high resolution and speed^40404040.

  Instrument Components (Order is key): Solvent Reservoir Pump Injector Port Column Detector Recorder

  Column Materials: Stainless Steel (durable, high pressure), PEEK (biocompatible), or Glass ⁴².

Draw HPLC

What are the S.P modes in HPLC

• CHOOSE RIGHT PHASE FOR THE JOB

• Normal Phase: Polar stationary phase + Non-polar mobile phase.

  Reverse Phase (Most Common): Non-polar stationary phase (e.g., C18 alkyl chains) + Polar mobile phase. Separates based on hydrophobicity.

• Chiral: Uses a chiral stationary phase to separate enantiomers

What are the detectors in hplc

• UV/Vis: Measures absorbance (most common)⁴⁷.

  Electrochemical: Measures current from oxidation-reduction⁴⁸.

  Fluorescence: Sensitive, but limited to fluorescent compounds⁴⁹.

  Mass Spectrometer (MS): Separates ions by mass-to-charge ratio ($m/z$)⁵⁰.

What is principle and components of GC

Principle: Partitions molecules between a liquid/polymer stationary phase and a gas mobile phase⁵¹.

Requirement: Samples must be volatile (vaporizable)⁵².

Instrument Components: Carrier Gas (Mobile Phase) Injector (Heated) Oven (Containing Column) Detector Waste

Draw GC

wHAT ARE THE COLOUMN TYPES IN GC

• WCOT: Wall Coated Open Tubular (liquid on wall).

• SCOT: Support Coated Open Tubular (liquid on support material on wall).

PLOT - Porous layer

Wat are the detectors in gc

• FID (Flame Ionisation): Burns sample in hydrogen flame. Very sensitive but destroys the sample .

• ECD (Electron Capture): Radioactive source. Specific for electron-capturing compounds like pesticides

Applications of GC

Applications: Environmental (Greenhouse gases, pesticides), Forensic (Arson accelerants, explosives), Clinical (Blood alcohol)