Chromatography Techniques Overview

Basic Principles of Chromatography (FDSC 624)

• Chromatography is a separation technique based on differential distribution
• Different methods exist, categorized by the type of differential distribution.

Types of Differential Distribution

• Differential Distribution by Different Chemical States of the Same Matter:

• Distillation

• Sublimation

• Crystallization

• Zone melting

• Precipitation

• Differential Distribution by Different Chemical Phases:

• Absorption (partition)

• Adsorption

• Liquid-liquid extraction (LLE)

• Solid-phase extraction (SPE)

• Supercritical fluid extraction (SFE)

• Solid-phase microextraction (SPME)

• Differential Distribution by Different Chemical Environments or Locations:

• Separation based on different migration rates under a field

• Electric field: electrophoresis, and field-flow fractionation

• Gravitational field: centrifugation and filtration

• Thermal field: Thermal diffusion

• Membrane (semi-permeable): dialysis, osmosis, and ultra-filtration

Chromatographic Techniques

• Classical Column Chromatography

• Uses a packed column for separation

• Separation occurs based on differences in affinity to stationary/mobile phase

• Thin Layer Chromatography (TLC)/Planar Chromatography

• Uses a planar stationary phase

• Separation occurs based on differences in affinity to stationary/mobile phase

• High Performance Liquid Chromatography (HPLC)

• Uses a packed column for separation

• Separation occurs based on differences in affinity to stationary/mobile phase and size

• Gas Chromatography (GC)

• Uses a packed column for separation

• Separation occurs based on differences in boiling point

• Supercritical Fluid Chromatography (SFC)

• Uses a supercritical fluid as the mobile phase

• Separation occurs based on differences in interaction with the stationary/mobile phase

Chromatographic Definitions

• Migration time: time it takes for the non-retained species to travel through the column
• Retention time:
• Time for analyte A
• Time for analyte B
• Adjusted retention time: time corrected to exclude the migration time of the nonretained species
• Peak Width (WA, WB): measure of band spreading
• Column Length: length of the column packing material
• Flow rate: volume of mobile phase passing through the column per unit time.
• Volume of stationary phase: Volume of the stationary phase in the column
• Concentration of analyte: concentration of analyte in mobile and stationary phase

Chromatographic Relationships

• Linear mobile-phase velocity: speed of the mobile phase
• Volume of mobile phase: the amount of mobile phase used.
• Retention factor: measure of the analyte's affinity to the stationary phase
• Distribution constant: measure of the analyte's distribution between the two phases.
• Selectivity factor: measure of the selectivity of the separation.
• Resolution: measure of the separation between two peaks.
• Number of Plates: measure of column efficiency.
• Plate height: measure of the height per theoretical plate in the column.

Rate Theory

• Plate height (H): describes the efficiency of a column; minimize
• Eddy diffusion (A): term showing how mobile phase spreads out
• Molecular diffusion (B): term showing how the analyte molecules move through the column
• Mass transfer (C): term showing how the movement of analyte between the mobile and stationary phases
• Velocity of mobile phase (u): measures how fast the mobile phase moves.

Eddy Diffusion

• Packing factor (λ)
• Diameter of packed particles (d_p)

Molecular Diffusion

• Obstruction factor (Ψ)
• Diffusion coefficient (D_M)

Mass Transfer

• Retention factor (k)
• Diffusion coefficient (D_s)
• Average film thickness (d_f)

Chromatography: Peak Resolution

• Poor resolution (a)
• More separation (b)
• Less band spread (c)

Quantitative Analysis

• Peak areas
• Peak heights
• Calibration and standards
• Internal standard method

Liquid Chromatography (HPLC)

• Uses a pressurized polar solvent to carry a sample through a tubular column filled with non-polar packing.
• Allows separation of organic species.
• Qualitative and quantitative analysis supported by various detectors
• Different modes of separation categorized:
• Adsorption Chromatography
• Partition Chromatography
• Ion-exchange Chromatography
• Size-exclusion Chromatography

Adsorption Chromatography

• Stationary phase is a solid
• Separations occur through adsorption/desorption steps.
• Common stationary phases: silica and alumina.

Partition Chromatography

• Separation based on solute partitioning between two liquid phases
• Separation based on relative solubility.
• Normal phase: polar stationary phase, non-polar solvent.
• Reverse phase: non-polar stationary phase, polar solvent.

Ion-exchange Chromatography

• Stationary phase has a positively or negatively charged surface.
• Separation based on charged solute molecules in mobile phase binding opposingly charged surfaces on stationary phase
• Commonly using weak exchange resins.

Size-exclusion Chromatography

• Separation based on molecular size.
• Using gels with controlled pore size.
• Larger molecules elute first (shorter distance to travel).

Basic HPLC Theory

• All organic molecules have a characteristic retention time.
• Different modes of operation: normal phase, reverse phase, ion-exchange and gel-permeation
• Chromatographs have several common components:
• Solvent/pump (mobile phase)
• Injection port/valve
• Columns (stationary phase)
• Detectors (up to four)
• Output (analog or digital)

HPLC System Design

• Components: pump, filter, injector, column, detector, data recorder, waste solvent reservoir.

HPLC Pump

• Reciprocating, cam-driven, single-piston pumps. Uses a simple pulse dampener to create a consistent solvent flow.

Injections Valve

• High-pressure, fluoropolymer material to accommodate different sample loops, syringes and automated sampler ports.

Tubing and Fittings

• Conventional stainless steel; high pressure and solvent-resistance; for GPC/SEC work, and NPC.
• Fluoropolymer (PEEK); reduced ionic contamination for bio-assays & RPC.

Columns & Fittings

• Packed columns with 3-10 micron silica-based stationary phases to provide maximum data quality and minimum time using metal or polymer high-pressure fittings and seals.

Column

• Normal or reverse phases
• Gel permeation, ion-exchange

Normal Phase Columns

• Uses hydrophilic stationary phase and non-polar solvent.
• Useful for low polarity and petrochemical samples, tests for molecular weight determinations.

Reverse Phase Columns

• Uses hydrophobic stationary phase and polar solvent.
• Useful for high polarity and hydrophilic samples. Most popular form of HPLC.

Solvent Selection

• Normal phase: hexane, methylene chloride, THF, DMF, DMSO
• Reverse phase: 90% water, methanol & acetonitrile; some add small amounts of aprotic solvents (THF).

HPLC Detectors

• UV/Vis (fixed & variable wavelengths)
• Refractive Index
• Fluorescence
• Evaporative Light Scattering
• Electro-Chemical

LC Detectors: Differential Refractive Index

• Looks at the difference between a static reference cell and a sample flow cell
• Specialized for sugars and carbohydrates, polymer (molecular weight) determinations.

Filter Fluorescence

• Use excitation and emission filters to allow analysis of drugs, natural products, vitamins, and specialized chemicals at a PPT level
• Used for poly-aromatic analyses and environmental work

HPLC: Important Features

• Solvent pump rate (micro-LC, analytical, semi-prep)
• System pressure rating (normal phase, reversed phase)

Chromatographic Controls

• Higher flow rate = closer peaks
• More polar solvent = faster separation
• Higher pressure = closer peaks
• Lower flow, polarity, pressure = better resolution
• Controlled flow = peaks with minimum resolution loss

Test your knowledge on various chromatography techniques including reversed phase, normal phase, ion-exchange, and gel-permeation chromatography. This quiz covers principles, materials, and solvent interactions essential for successful chromatographic separation. Perfect for students and professionals in chemistry and biochemistry fields.