EXAM Flashcards
(46 cards)
What is Chromatography?
A technique used to separate a mixture into its individual components based on differences in their composition (e.g., size, structure). It involves a stationary support and a mobile phase
What is the difference between Planar and Column Chromatography?
Planar: Stationary phase (SP) on a flat plate or paper, mobile phase (MP) moves by capillary action or gravity (e.g., TLC, paper chromatography)
Column: SP held in a narrow tube, MP forced through under pressure or by gravity (e.g., GC, LC, IC)
Name the primary separation mechanisms in column chromatography.
Mainly adsorption and partition. Others include size exclusion (gel filtration), ion exchange, ion pair, and affinity
What is a Chromatogram?
A plot showing the detector response over time. Peak positions can identify components, and peak areas can quantify them
What are common measures of migration/retention in column chromatography?
Retention time (tr), Void time (tm or t0), Capacity Factor (k’).
What is Void time (tm or t0)?
The time taken for the mobile phase to reach the detector (representing the volume outside the stationary phase pores).
How is Capacity Factor (k’) calculated and what does it indicate?
k’ = (tr – tm) / tm. It is a measure of chromatographic retention that corrects for the void time. An optimum range is 1 < k’ < 10
How is Selectivity (α) calculated and what does it indicate?
α = k’b / k’a or (tr(b) - tm) / (tr(a) - tm) for two components. It indicates the quality of separation between two components. α > 1 suggests separation, higher α means greater separation.
What is the principle of Adsorption Chromatography?
Separation based on the adsorption affinities of sample components for the surface of a solid stationary phase
What is Resolution (Rs)?
Rs measures the degree of peak overlap, taking into account peak width. It’s calculated as Rs = 2[tr(b) – tr(a)] / (Wa + Wb)
What Resolution (Rs) value indicates baseline resolution (complete separation)?
An Rs value of ≥ 1.5
Differentiate between Normal Phase and Reverse Phase Chromatography.
Normal Phase: Uses a polar stationary phase and a relatively non-polar mobile phase.
Reverse Phase: Uses a non-polar stationary phase (e.g., hydrocarbon) and a relatively polar mobile phase (e.g., water, methanol). Reverse phase is the most common type in HPLC
How is the Partition Coefficient (Kav) calculated in Gel Filtration Chromatography?
Kav = (Ve - Vo) / Vt
where Ve is elution volume, Vo is void volume, and Vt is total column volume.
What is the principle of Partition Chromatography?
Separation based on the equilibration (partitioning) of solutes between a liquid stationary phase (a thin film on a solid support) and a mobile phase
What is Gel Filtration Chromatography (Size Exclusion Chromatography)?
A technique that separates molecules based on their size (hydrodynamic radius) as they pass through a porous stationary phase
What is Ion Exchange Chromatography (IEC)?
An LC technique that separates molecules based on their charge, using a stationary phase with covalently attached ionic functional groups that reversibly interact with charged solutes
How does size affect elution in Gel Filtration Chromatography?
Larger molecules that cannot enter the pores elute first (at the void volume, Vo) [Previous conversation]. Smaller molecules that enter the pores elute later, with elution volume (Ve) increasing as size decreases
What are the two main types of Ion Exchange Chromatography?
Cation Exchange: Uses a negatively charged stationary phase and binds positively charged molecules.
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Anion Exchange: Uses a positively charged stationary phase and binds negatively charged molecules.
How is a protein’s charge related to its Isoelectric Point (pI) and buffer pH?
At pH < pI, the protein has a net positive charge. At pH > pI, it has a net negative charge. At pH = pI, it has a net zero charge.
How are molecules typically eluted from an Ion Exchange column?
By changing the pH of the mobile phase or by increasing the salt concentration, which competes with the bound molecules for binding sites on the stationary phase.
What is Affinity Chromatography?
A separation of molecules based on an interaction between the target molecule and a specific ligand immobilised on the stationary phase.
How are molecules typically eluted from an HIC column?
By decreasing the salt concentration of the mobile phase (high salt promotes binding to the hydrophobic stationary phase, low salt promotes elution)
How is Immobilised Metal Affinity Chromatography (IMAC) used?
IMAC uses immobilised metal ions (e.g., Ni2+) on the stationary phase to bind proteins that have specific metal-chelating residues, most commonly a poly-Histidine tag (His-tag), for purification
What is Hydrophobic Interaction Chromatography (HIC)?
A technique that separates molecules based on their surface hydrophobicity, using a stationary phase with hydrophobic ligands.
