Module 12: Chromatography Flashcards
Principle of chromatography
Separation of compounds based upon interaction with two phases (stationary and mobile)
Peak area
Quantity detected
Used to measure concentration
Clinical uses of chromatography
Toxicology, amino acids, glycohemoglobin
Types of separation
Ion exchange (anion and cation) Steric exclusion Adsorption Partition Affinity
Anion exchange chromatography
Stationary phase is positive
Selects anions from the sample
Cation exchange chromatography
Stationary phase is negative
Selects cations from the sample
Steric exclusion chromatography
Gel filtration, gel permeation
Stationary phase contains small pores that allow separation based on size and shape
Adsorption chromatography
Solid phase has adsorptive sites (sticky) to capture compounds
Partition chromatography
Separation based on polarity
Often used in HPLC and GC (GLC) systems
Normal phase in partition chromatography
Stationary = polar Mobile = non-polar
Reverse phase in partition chromatography
More common than normal phase
Stationary = non-polar
Mobile = polar
Affinity chromatography
Uses specific binding proteins (e.g. antibodies) to capture analyte
3 chromatography methods
Paper/thin layer chromatography (TLC)
Gas liquid chromatography (GLC)
High performance liquid chromatography
Paper/thin layer chromatography (TLC) stationary and mobile phases
Stationary: paper, can be mounted on glass or plastic support
Mobile: liquid solvent
Paper/thin layer chromatography (TLC) clinical uses
Drug screens, amino acid screens
Racing factor/retention factor
Distance compound moves/total solvent from distance
Used to identify compounds
Gas liquid chromatography (GLC) stationary and mobile phases
Stationary: porous inert solid coated with liquid
Mobile: inert gas
Gas liquid chromatography (GLC) clinical uses
Alcohol analysis (sample compounds must be volatile) Can be coupled with a mass spectrometer for further identification of compounds
High performance liquid chromatography stationary and mobile phases
Stationary: solid, or liquid coated in solid
Mobile: liquid
High performance liquid chromatography clinical uses
Drugs and glycohemoglobins
Retention time
Time from sample injection to peak height in detector
Factors affecting retention time
Sample composition Column temperature (hotter=faster) Column packing (stationary phase material and amount) Flow rate Column length
Chromatography resolution
Relative separation of two different peaks
Factors affecting chromatography resolution
Column length
Column temperature
Stationary and mobile phase composition
Column efficiency
Overall performance based on column packing
Internal standard
Structurally similar reference compound of known concentration added to all samples to account for system variations
Higher column temperature speeds up or slows down chromatography retention time?
Speeds up
How is chromatography resolution affected by column length?
Longer is better
How is chromatography resolution affected by column temperature?
Lower is better
