Separation Methods [Dr Green] Flashcards
List 3 methods of separation
Electrophoresis - capillary, isoelectric focusing Chromatography - gas, liquid, thin layer Membrane separation - e.g. dialysis
What is chromatography?
Components of a mixture are separated based on differences in the rate at which they are carried through a stationary phase by a gaseous or liquid mobile phase Components will flow at the rate of the mobile phase unless they partition into stationary phase
What is the stationary phase in chromatography?
Fixed in place either in a column or on a planar surface
What is the mobile phase in chromatography?
Moves over or through the stationary phase, carrying with it the analyte mixture
List 4 examples of chromatography?
Thin layer chromatography Column chromatography High pressure liquid chromatography Gas chromatography
Define: Elution
Elution is the process of washing sample components through the stationary phase by continuous flow of the mobile phase (eluent) = column
When does a peak appear on a chromatogram?
A peak appears when a separated component reaches the detector at the end of the column
How are components identified on a chromatogram?
They are identified by their unique retention time (tR) under a certain set of separation conditions
List 2 factors which could affect retention time (tR)
Velocity (flow rate) of mobile phase Chromatographic retention
What is the equation to calculate the partition coefficient (Kc)?
Kc = [As]/[Am]
What is the equation for the capacity factor/retention factor?
Define the terms of the equation for the capacity/retention factor
k = capacity/retention factor
Kc = parition coefficient
V = volume of stationary/mobile phase
n = number of moles in stationary/mobile phase
What is the equation to calculate retention time?
tR = t0 (1+k)
t0 = time taken for mobile phase to pass through column
tR1 = time taken for analyte 1 to go through column
List 3 factors that cause band broadening (Rate Theory of Chromatography)
Longitudinal diffusion Resistance to mass transfer Eddy diffusion
What causes longitudinal diffusion?
The concentration of the analyte is less at the edges of the band that at the centre and so analyte diffuses out from the centre to the edges = band broadening
What causes resistance to mass transfer?
Band broadening due to resistance to diffusion of the molecule in the mobile and stationary phase ( amount of time taken to equilibrate between the 2 phases) Depends on diffusion coefficient of compound in each phase, diameter and shape of stationary phase
What causes eddy diffusion?
The mobile phase moves through the column which is packed with stationary phase so solute molecules will pass through the stationary phase at random = broadening of solute band, because different paths are of different lengths
How does low flow rate affect column efficiency?
Broadens peaks due to longitudinal diffusion
How does large particle size affect column efficiency?
Increases eddy diffusion and mass transfer with particle size = peak broadening and decreases column efficiency
How does an irregularly shaped stationary phase affect column efficiency?
Increase peak broadening due to eddy diffusion
How does fast flow rate affect column efficiency?
Peak broadening due to resistance to mass transfer - diffusion in stationary phase
Describe the 2 phases of HPLC
Normal phase: Polar stationary phase (silica), non-polar mobile phase (hexane) Molecules elute in order of increasing polarity Reverse phase: Non-polar stationary phase (ODS coated silica gel), polar mobile phase (water, methanol) Molecules elute in order of decreasing polarity
List the 2 main types of HPLC detector
Responsive to physical and chemical changes of sample components e.g. UV, fluorescent Responsive to changes in properties of the mobile phase e.g. refractive index detector
List 3 situations where HPLC-MS is used
Drug development Pharmacokinetics Proteomics
