Separation Methods [Dr Green] Flashcards

1
Q

List 3 methods of separation

A

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

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2
Q

What is chromatography?

A

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

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3
Q

What is the stationary phase in chromatography?

A

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

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4
Q

What is the mobile phase in chromatography?

A

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

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5
Q

List 4 examples of chromatography?

A

Thin layer chromatography Column chromatography High pressure liquid chromatography Gas chromatography

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6
Q

Define: Elution

A

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

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7
Q

When does a peak appear on a chromatogram?

A

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

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8
Q

How are components identified on a chromatogram?

A

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

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9
Q

List 2 factors which could affect retention time (tR)

A

Velocity (flow rate) of mobile phase Chromatographic retention

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10
Q

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

A

Kc = [As]/[Am]

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11
Q

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

A
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12
Q

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

A

k = capacity/retention factor

Kc = parition coefficient

V = volume of stationary/mobile phase

n = number of moles in stationary/mobile phase

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13
Q

What is the equation to calculate retention time?

A

tR = t0 (1+k)

t0 = time taken for mobile phase to pass through column

tR1 = time taken for analyte 1 to go through column

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14
Q

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

A

Longitudinal diffusion Resistance to mass transfer Eddy diffusion

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15
Q

What causes longitudinal diffusion?

A

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

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16
Q

What causes resistance to mass transfer?

A

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

17
Q

What causes eddy diffusion?

A

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

18
Q

How does low flow rate affect column efficiency?

A

Broadens peaks due to longitudinal diffusion

19
Q

How does large particle size affect column efficiency?

A

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

20
Q

How does an irregularly shaped stationary phase affect column efficiency?

A

Increase peak broadening due to eddy diffusion

21
Q

How does fast flow rate affect column efficiency?

A

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

22
Q

Describe the 2 phases of HPLC

A

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

23
Q

List the 2 main types of HPLC detector

A

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

24
Q

List 3 situations where HPLC-MS is used

A

Drug development Pharmacokinetics Proteomics