Chromatography and Solvent Polarity

Thin-Layer Chromatography (TLC)

• Planar chromatographic methods include thin-layer chromatography (TLC), paper chromatography (PC), and electrochromatography.
• Each method uses a thin layer of material, either self-supporting or coated on a glass, plastic, or metal surface.
• TLC is faster, has better resolution, and is more sensitive than paper chromatography.

TLC Principles

• Thin-layer chromatography is carried out on silica gel or alumina dispersed on an inert support (flat glass plates).
• The mobile phase is drawn over the surface by capillary action.
• TLC and liquid chromatography are similar in theory and stationary/mobile phases.
• TLC is faster and less expensive than HPLC.

TLC Technique Steps

• Preparation of the plate: A slurry of the active material is uniformly spread over the plate using a spreader. Air-dry overnight or oven-dry at 80-90°C for 30 minutes. Commercial plates can be conventional or high-performance.
• Sample application: This is a critical step. Manual application uses a capillary tube or syringe. Mechanical dispensers are commercially available.
• Chromatogram development: The plate is placed in a closed chamber (tank) lined with filter paper to avoid direct contact between the sample and the solvent system. The developing solvent travels up the plate, dissolving the sample and carrying it up.
• Locating the spots: Colored compounds are located visually. For colorless compounds, iodine or sulfuric acid are used. Ninhydrin is used for amino acids and 2,4-dinitrophenylhydrazine is used for aldehydes and ketones.

TLC Applications

• Identification of unknown compounds
• Analysis of reaction mixtures
• Monitoring chemical reactions
• Determination of the purity of a compound
• Quantitative determination of an unknown concentration (external standard method)
• Quantitative determination of an unknown concentration (internal standard method).

High-Performance Liquid Chromatography (HPLC)

• HPLC is a fast technique using liquid as the mobile phase.
• HPLC uses pressure for fast flow.
• HPLC is used for organic and inorganic analysis.

HPLC Advantages

• Speed
• High resolution
• High accuracy
• High sensitivity
• Automation is possible

HPLC Disadvantages

• Expensive instrumentation
• Requires skill
• Not universally applicable

HPLC Instrumentation

• Solvent mixing valve
• Pump
• Sample injection valve
• Column
• Detector
• Recorder

Mobile Phase Reservoir

• Often contains a filtration system for filtering dust and particulate matter.
• Equipped with a degasser to remove dissolved gases (usually oxygen and nitrogen).

HPLC Pump

• Generates a flow of the mobile phase through the HPLC injector, column, and detector
• Requirements include: generation of pressures up to 6000 lbs/in2
• Pulse-free output
• Flow rate ranging from 0.1 to 10 ml/min
• Made of corrosion-resistant materials

Types of HPLC Pumps

• Constant pressure pumps: simple, inexpensive, easy to operate, free from pulsations, resulting in smooth baselines. However, flow rate must be constantly monitored.
• Constant flow pumps: the ability to repeat elution volume and peak area (regardless of viscosity changes and column blockage), up to the pressure limit of the pump. This type is the most widely used in all common HPLC applications.

Mobile Phase

• Must solvate the analyte molecules and the solvent they are in.
• Be suitable for the analyte to transfer between the sample and solvent during separation.
• Be readily available
• Compatible with the instrument (pumps, seals, fittings, detector, etc)
• Compatible with the stationary phase
• Adequate purity
• Not too compressible (causes pump/flow problems)
• Filtered. Free of gases

Stationary Phases

• HPLC stationary phases are packed columns made from stainless steel.
• Columns have lengths of 3-50 cm and diameters of 3-10 mm.
• Packed with stationary phase: 1 to 20 µm diameter
• Particles: silica gel, alumina.
• Microbore/capillary columns (open tubular column)

Isocratic versus Gradient Elution

• Isocratic elution: constant mobile phase composition.
• Gradient elution : varying mobile phase composition. Using multiple pumps.
• Gradient elution is commonly used for complex mixtures

HPLC Detectors

• Bulk property detectors: Respond to some physical property of the mobile phase (e.g. refractive index, dielectric constant, density).
  • Advantages: universal application
  • Disadvantages: low sensitivity and limited range
• Solute property detectors: Respond to some physical or chemical property of solutes (e.g., UV absorbance, fluorescence).
  • Advantages: High sensitivity and wide range.
• Disadvantages: More selective; may require multiple detectors;

Standard Absorbance Detector

• Single beam UV-VIS instrument with a flow-through cell
• Uses Hg vapor lamps and 254 nm
• Can use any UV-VIS with a special flow cell.
• For non-destructive analysis.

Diode Array Detector (DAD)

• Versatile research-grade HPLC instrument
• Scans a range of wavelengths every few seconds.

Refractive Index Detector

• Universal HPLC detector; non-destructive.
• Responds to changes in refractive index.
• Temperature sensitive (needs regulated temperature).

Evaporative Light Scattering Detector (ELSD)

• Universal detector; destructive.
• Useful for large molecules.
• Analytes desolvated in the detector.

Other GC Detectors

• Mass Spec
• Electrochemical
• Conductivity
• Fluorescence

HPLC Recorder

• Recorder that plots the separation of individual components over time.

Common HPLC Techniques

• Partition chromatography
• Normal phase chromatography
• Reverse phase chromatography
• Adsorption chromatography
• Ion chromatography
• Size exclusion chromatography
• Ion-pair chromatography
• HILIC chromatography
• Affinity chromatography
• Chiral chromatography

Types of Chromatography

• Adsorption chromatography
• Partition chromatography

Partition Chromatography

• Normal phase chromatography: polar stationary phase, nonpolar mobile phase.
• Reverse phase chromatography: nonpolar stationary phase, polar mobile phase.

Partition Chromatography

• Mostly bonded-phase packings are prepared by reaction of an organochlorosilane with the -OH groups on the silica particles.
• In reversed phase chromatography, the organic group R is typically a straight-chain octyl- (C8) or octyldecyl- (C18) group.

GC Theory

• Separation based on the partition of solutes between a stationary phase and an inert mobile phase (gas).
• Vaporization of the sample before injection into the column.
• Components separate based on their interaction with the stationary phase.
• Elution in order of increasing boiling points (and polarities).

GC Column

• Various lengths and diameters; often coiled.
• Temperature control is crucial.
• Types: packed columns (inert support material); capillary columns (liquid film coated inside).

GC Stationary Phases

• Low volatility
• Thermal stability of column at operating temperature
• Chemical inertness
• Characteristc solvents for the separation.

GC Detectors

• General function: monitors the carrier gas as it emerges from the column
• Types: thermal conductivity detector (TCD), flame ionization detector (FID), electron capture detector (ECD).

Sample Injection

• Methods: On-column and split-splitless injectors, injection volume, injection techniques.

This quiz covers essential concepts related to polarity indices of solvents in chromatographic analysis. It examines the role of solvent mixtures, mobile and stationary phases, and their impact on solute behavior during chromatography. Test your understanding of these critical topics!