5.2 Column chromatography

Column Chromatography

• It is a separation technique where a stationary phase is packed in a column.
• A mobile phase moves through, carrying the sample
• It separates mixture components based on their interaction with the stationary phase

Basic Components

• Includes a stationary and mobile phase

Stationary phase

• A solid or liquid fixed inside the column
• Sample components interact to varying degrees with it
• Common materials include:
  • Silica gel (SiO2) in normal-phase chromatography
  • Alumina (Al2O3) for separating polar compounds
  • Resins or polymers in ion-exchange and size-exclusion chromatography

Mobile phase

• Is a liquid or gas that flows through the column, carrying sample components
• Facilitates movement by dissolving or suspending the sample
• Solvents or buffer systems are chosen based on sample properties and chromatography type
• It can be polar (e.g., water, methanol) or non-polar (e.g., hexane, chloroform)

Principles of Separation

• Based on differential interaction of sample components with the stationary phase.
• Components that interact strongly with the stationary phase move slower
• Components that interact weakly move faster with the mobile phase

Separation Factors and Types

• influenced via:
• Absorption (Affinity) Strength: Stronger interactions result in slower movement
• Solubility: Higher solubility in the mobile phase leads to faster movement
• Polarity

Normal-phase chromatography

• Has a polar stationary phase (e.g. silica)
• and a non-polar mobile phase
• Polar compounds are retained more

Reverse-phase chromatography

• Has a non-polar stationary phase (e.g. C18)
• and a polar mobile phase
• Non-polar compounds are retained more

Size & Shape

• In size-exclusion chromatography, larger molecules elute first
• Smaller molecules get trapped in porous beads

Process of Column Chromatography

Packing the Column

• The stationary phase is loaded into the column and equilibrated with the mobile phase

Sample Application

• A small volume of the concentrated sample is applied to the top of the column

Elution

• Mobile phase (eluent) is passed through the column
• Moves sample components downward and causes separation at different speeds.

Fraction Collection & Analysis

• Separated components exit the column in distinct fractions
• Fractions can be collected and analysed using techniques like UV spectrophotometry, mass spectrometry, or HPLC detection

Types of Column Chromatography

• Absorption Chromatography
  • Based on surface interactions (e.g., silica gel binding)
• Partition Chromatography
  • Separation based on solubility differences
• Ion-Exchange Chromatography
  • Uses charged stationary phase to separate ionic compounds
• Size-Exclusion Chromatography (SEC)
  • Separates based on molecular size, with smaller molecules eluting later.
• Affinity Chromatography
  • Uses specific binding interactions (e.g., antibody-antigen, enzyme-substrate)

Applications of Column Chromatography

Protein Purification

• Used in biochemistry and molecular biology
• Isolates proteins based on size, charge, or affinity

Pharmaceutical Analysis

• Used to separate and purify drug compounds

Environmental Testing

• Detects contaminants in water and soil

Food industry

• Analyses food additives, flavours, and toxins

Metabolomics & Clinical Diagnostics

• Identifies biomolecules in blood or urine samples

Advantages

• Separates complex mixtures effectively
• Scalable for both small and large sample volumes
• Compatible with a variety of detection methods

Disadvantages

• Can be time consuming
• Requires careful solvent and stationary phase selection
• Some separations may require multiple purification steps

Size-Exclusion Chromatography (Gel-Filtration Chromatography)

• Separates molecules based on size
• Useful for sorting proteins with different molecular weights

Mechanism

• Stationary phase: cross-linked gel particles with pores of specific sizes
• Smaller molecules enter the pores, delaying movement
• Larger molecules are too big to enter the pores and elute first
• Separation depends on cross-linking on the gel

Gel Materials

• Gel particles are made from one of two types of polymers:
  • Sephadex or Sepharose: Carbohydrate-based polymers (dextran or agarose)
  • Bio-Gel: Polyacrylamide-based polymer
• Level of cross-linking determines pore size and molecular size range that can be separated

Exclusion Limit

• Is the size of a molecule that is too large to enter the pores
• Molecules at or above the exclusion limit elute immediately
• Smaller molecules elute later as they move through the pores

Process of Separation

Column Preparation

• The stationary phase (gel beads) is packed into a column

Sample Application

• A mixture of molecules is applied to the top of the column

Elution with Mobile Phase

• The mobile phase (buffer or solvent) flows through the column
• Large molecules travel around the gel beads and elute first
• Smaller molecules enter the pores and take longer to pass through the column

Collection & Analysis

• Fractions are collected and analyzed to determine molecular size distribution

Advantages

• Separates molecules purely based on size, independent of charge or affinity
• Gentle on proteins preserving their structure and function
• Can estimate molecular weight by comparing unknown samples to standards
• Compatible with other techniques such as spectroscopy and mass spectrometry

Disadvantages

• Limited resolution - Works best for molecules with significant size differences
• Requires proper column calibration with molecular weight standards
• Dilution of sample - Fractions collected are often in a larger volume than the original sample

Gel-Filtration (Figure 5.5)

• Larger molecules are excluded from the gel and move more quickly through the column
• Small molecules have access to the interior of the gel beads, so they take a longer time to elute
• The protein concentration is usually measured by UV absorption as the samples elute from the column

Affinity Chromatography

• Is a technique based on specific binding interactions between molecules
• The stationary phase has a polymer covalently linked to a ligand that specifically binds the target protein
• Proteins with an affinity for the ligand bind to the column
• The bound protein can be eluted by introducing free ligands or altering pH/ionic strength

Key Components

Stationary Phase

• Polymer matrix with a covalently attached ligand that specifically interacts with target the protein.
• Unlike size-exclusion chromatography, the polymer is not cross-linked and does not separate by size

Mobile Phase

• A buffer or solvent that washes away unbound molecules
• Later, an elution buffer containing free ligands releases the bound protein

Ligand

• A molecule that binds specifically to the target protein
• Can be highly specific (binding a single protein) or group-specific (binding a class of proteins)

Process

Column Preparation

• The polymer-bound ligand is packed into a column

Sample Application

• The mixture is loaded onto the column

Binding Step

• Target protein binds specifically to the immobilised ligand
• Unbound molecules pass through

Wash Step

• Unbound proteins are removed by washing with a buffer

Recovery

• Purified protein is collected in fractions

Advantages

• Highly selective - Targets specific proteins with minimal contamination
• Produces very pure proteins - Often eliminates the need for further purification steps
• Efficient - Can purify proteins in a single step
• Can be tailored - Ligands can be designed for individual proteins or groups of similar proteins

Disadvantages

• Expensive - Designing specific ligands can be costly
• Limited capacity - Only works for proteins that have a known ligand-binding property
• Elution conditions may affect protein structure - Harsh pH or salt changes can denature proteins

Applications

• Protein purification (e.g. isolating enzymes, antibodies, and receptors)
• Drug discovery (e.g. identifying drug-protein interactions)
• Biochemical research (e.g. studying ligand-receptor interactions)
• Purification of recombinant proteins (e.g. using His-tagged proteins with Ni2+ affinity chromatography

Principle of affinity chromatography

• In a mixture of proteins, only one binds to a substance, called the substrate
• The substrate is attached to the column matrix
• Once the other proteins have been washed out, the desired protein can be eluted by adding a solution of high salt or by adding free substrate

Ion-Exchange Chromatography

• Separates substances based on charge
• Uses an ion-exchange resin in the stationary phase
• This contains charged ligands that interact with oppositely charged molecules
• Is less specific than affinity chromatography - separation depends on the net charge of molecules rather than specific binding

Types of Ion Exchange Chromatography

Cation Exchange Chromatography

• Resin has a net negative charge and binds positively charged molecules (cations)
• Counterions (e.g. Na+, K+) initially occupy binding sites and are exchanged with cations

Anion Exchange Chromatography

• Resin has a net positive charge and binds negatively charged molecules (anions)
• Counterions (e.g. Cl-) are displaced by anions

Process of Ion-Exchange Chromatography

Column Equilibration

• Ion-exchange resin is equilibrated with a buffer of appropriate pH and ionic strength
• Resin is initially bound to counterions (e.g. Na+, K+, or Cl-)

Sample Application

• Mixture of proteins is loaded onto the column
• Proteins with a net charge opposite to the resin bind to it
• Proteins with the same charge as the resin or no net charge elute freely

Washing Step

• Unbound proteins are washed away with a buffer

Elution Step

• Bound proteins are eluted by either:
  • Changing the buffer pH or increasing salt concentration

Fraction Collection & Analysis

• Separated proteins are collected in different fractions

Advantages

• Highly effective for purifying charged proteins (e.g. separating acidic from basic proteins)
• Can be finely controlled by adjusting pH and salt concentration
• Can be used in protein analysis, purification, and water treatment applications

Limitations

• Requires careful buffer selection to optimize separation
• Limited selectivity compared to affinity chromatography - only separates based on charge, not specific interactions
• Elution conditions may affect protein stability - changes in pH or high salt concentrations can alter protein structure

Applications

• Purification of proteins, peptides, and nucleic acids based on charge differences
• Water purification is done when removing charged contaminants
• Analysis of amino acids and biomolecules in research and diagnostics

High-Performance Liquid Chromatography (HPLC)

• Sophisticated technique used for high-resolution separation of molecules based on polarity
• It uses high pressure to improve efficiency and speed

Types of HPLC

Reverse-Phase Liquid Chromatography (RP-LC)

• Is is the Most common
• Stationary phase is nonpolar (e.g. C18 silica coated with hydrophobic groups)
• Mobile phase is polar (e.g. water, methanol, acetonitrile)
• Nonpolar compounds interact more strongly with the stationary phase and elute later
• Polar compounds elute first, as they move more easily with mobile phase
• Widely used for separating hydrophobic compounds (e.g. lipids, drugs, peptides)

Normal-Phase Liquid Chromatography (NP-LC)

• Stationary phase is polar (e.g. silica gel)
• Mobile phase is nonpolar (e.g. hexane, toluene)
• Polar compounds interact with the stationary phase and elute later
• Nonpolar compounds elute first, moving freely with the mobile phase
• Used for separating polar compounds, such as sugars and some pharmaceuticals

Advantages

Higher efficiency & faster separations

• Can be completed in minutes instead of hours

Highly adjustable

• Flow rate and solvent composition can be controlled precisely

Greater resolution

• Allows separation of very similar compounds

Automated & reproducible

• Widely used in research, pharmaceuticals, and diagnostics

Limitations

Expensive equipment and maintenance

• Requires specialized pumps, columns, and detectors

High pressure required

• Can wear down the column over time

Solvent selection is crucial

• Improper solvents can affect separation quality

Fast Protein Liquid Chromatography (FPLC) vs. HPLC

• HPLC is widely used for small molecules, drugs, and hydrophobic proteins
• FPLC is a variant of HPLC designed for protein purification, using milder conditions to prevent protein denaturation
• FPLC uses polar components, making it safer for protein separation

Applications of HPLC

• Pharmaceutical analysis- Drug purity, dosage form testing
• Food industry- Detection of contaminants, vitamins, and food additives
• Environmental testing- Water and soil contaminant analysis
• Clinical & biomedical research- Separation of biomolecules (e.g. amino acids, peptides, hormones)