Separation Methods [Dr Green]

Question 1

List 3 methods of separation

Answer 1

Electrophoresis - capillary, isoelectric focusing Chromatography - gas, liquid, thin layer Membrane separation - e.g. dialysis

Question 2

What is chromatography?

Answer 2

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

Question 3

What is the stationary phase in chromatography?

Answer 3

Fixed in place either in a column or on a planar surface

Question 4

What is the mobile phase in chromatography?

Answer 4

Moves over or through the stationary phase, carrying with it the analyte mixture

Question 5

List 4 examples of chromatography?

Answer 5

Thin layer chromatography Column chromatography High pressure liquid chromatography Gas chromatography

Question 6

Define: Elution

Answer 6

Elution is the process of washing sample components through the stationary phase by continuous flow of the mobile phase (eluent) = column

Question 7

When does a peak appear on a chromatogram?

Answer 7

A peak appears when a separated component reaches the detector at the end of the column

Question 8

How are components identified on a chromatogram?

Answer 8

They are identified by their unique retention time (tR) under a certain set of separation conditions

Question 9

List 2 factors which could affect retention time (tR)

Answer 9

Velocity (flow rate) of mobile phase Chromatographic retention

Question 10

What is the equation to calculate the partition coefficient (Kc)?

Answer 10

Kc = [As]/[Am]

Question 11

What is the equation for the capacity factor/retention factor?

Answer 11

Question 12

Define the terms of the equation for the capacity/retention factor

Answer 12

k = capacity/retention factor

Kc = parition coefficient

V = volume of stationary/mobile phase

n = number of moles in stationary/mobile phase

Question 13

What is the equation to calculate retention time?

Answer 13

tR = t0 (1+k)

t0 = time taken for mobile phase to pass through column

tR1 = time taken for analyte 1 to go through column

Question 14

List 3 factors that cause band broadening (Rate Theory of Chromatography)

Answer 14

Longitudinal diffusion Resistance to mass transfer Eddy diffusion

Question 15

What causes longitudinal diffusion?

Answer 15

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

Question 16

What causes resistance to mass transfer?

Answer 16

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

Question 17

What causes eddy diffusion?

Answer 17

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

Question 18

How does low flow rate affect column efficiency?

Answer 18

Broadens peaks due to longitudinal diffusion

Question 19

How does large particle size affect column efficiency?

Answer 19

Increases eddy diffusion and mass transfer with particle size = peak broadening and decreases column efficiency

Question 20

How does an irregularly shaped stationary phase affect column efficiency?

Answer 20

Increase peak broadening due to eddy diffusion

Question 21

How does fast flow rate affect column efficiency?

Answer 21

Peak broadening due to resistance to mass transfer - diffusion in stationary phase

Question 22

Describe the 2 phases of HPLC

Answer 22

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

Question 23

List the 2 main types of HPLC detector

Answer 23

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

Question 24

List 3 situations where HPLC-MS is used

Answer 24

Drug development Pharmacokinetics Proteomics