Lecture 15 Chromatography Flashcards
What is chromatography?
A physical separation technique
What are the two phases of chromatography?
Mobile and stationary phases
In chromatography, define the mobile phase.
Phase that carries the sample through a stationary phase; usually a gas or liquid
In chromatography, define the stationary phase.
A fixed phase through/over which the mobile phase (sample) passes; usually solid
How would a particle in mobile phase move if it had a higher affinity for the stationary phase?
The particles would migrate slower than those with a lower affinity (migrates faster).
Define Ion Exchange Chromatography
Separates analytes based on their charge using a charged stationary phase; performed in vertical columns.
Which protein (pos or neg charged) has a HIGH affinity for the column?
Negatively charged
Define partition chromatography
Based on the differential distribution of solutes between two immiscible liquids; liquid or gas stationary phase.
Absorbed, coated, or chemically bonded to the surface or particles of a column.
Define adsorption chromatography
Analytes separated by adsorption and desorption of solutes at the surface of a solid particle
Define adsorption
A solid molecule holding a gas or liquid molecule on its surface
Define desorption
The release of the molecule.
What physical forces are used within adsorption chromatography?
Hydrogen bonding and dispersive interactions
Define affinity chromatography
Analytes are separated via liquid chromatography using biological interaction. An affinity ligand (binding agent) is placed into the column and the pH and ionic strength are adjusted to achieve optimal binding.
Define size-exclusion chromatography
Separates molecules based on size. Small molecules enter pores of medium and are removed from flow.
Low resolution, usually saved for final purification.
Define planar chromatography
Compounds travel differing distances based on how strongly they partition between the stationary and mobile phase (Rf; aides in ID of analytes).
Define column chromatography
Stationary phase coated onto particles which are placed in or coated onto a tube or column.
Can be gas chromatography or liquid chromatography.
Define column-gas chromatography (GC)
Carrier gas (He, H, or N) moves solutes to detector where they are separated via retention time and quantitated by comparing peak area/height to a known standard.
In column-gas chromatography, what is a packed column?
Filled with support materials that are used uncoated
In column-gas chromatography, what is a capillary column?
Non-volatile liquid is coated or chemically bonded with the support particles or directly onto the wall.
In column-gas chromatography, what is the resolution?
How well the analytes are separated by chromatography.
In column-gas chromatography, how are the peaks characterized?
Characterized based on retention time and width.
How does column-liquid chromatography differ from column-gas chromatography?
(1) Requires a solvent reservoir and pump
2) Columns much smaller diameter (material: fused silica
