Pharmaceutical Analysis

Question 1

What is electrophoresis? (4)

Answer 1

Movement of charged particles in an applied electric field (from - to +)
Can separate on size and charge of molecules
HIgher the charge - the greater the movement
Smaller molecules will move further

Question 2

Explain dialysis (membrane separation)

Answer 2

Separation through membrane dependent on size

Small molecules are removed down a concentration gradient

Question 3

Name 4 methods of separation

Between a stationary and mobile phase

Answer 3

Ion exchange chromatography
Gas chromatography
Liquid chromatography
Thin layer chromatography (TLC)

Question 4

Explain chromatography

Answer 4

Components of a mixture are separated based on differences in the rate (affinity) at which they are carried through a stationary phase by a gaseous or liquid mobile phase

Question 5

What is the stationary phase?

Answer 5

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

Question 6

What is the mobile phase?

Answer 6

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

Question 7

Define: Elution time

Answer 7

The time between the start of the separation and the time at which the solute elutes (removes)

Question 8

Define: Chromatogram

Answer 8

A visible record (e.g. graph) which shows the results of separating the components of a mixture by chromatography
PICTURE

Question 9

Define: Retention

Answer 9

The action of absorbing or continuing to hold a substance

Question 10

Define: Resolution

Answer 10

A quantitative measure of how well 2 elution peaks can be differentiated in a chromatographic separation (how well the peaks are separated)

The difference in retention times between 2 peaks divided by the combined widths of the elution peaks

Question 11

Name the 2 types of HPLC

Answer 11

Normal phase

Reverse phase

Question 12

Describe the normal phase of HPLC

Answer 12

Stationary phase: polar - silica
Mobile phase: non-polar - hexane, DCM, methanol
Molecules elute in order of increasing polarity

Question 13

Describe the reverse phase of HPLC

Answer 13

Stationary phase: Non-polar - silica gel, ODS
Mobile phase: Polar - water, methanol
Molecules elute in order of decreasing polarity

Question 14

Define: Elution

Answer 14

The process of washing sample components through the stationary phase by continuous flow of the mobile phase

Question 15

Define: Eluent

Answer 15

Solvent running through the column

Question 16

Define: Eluate

Answer 16

Analyte being separated

Question 17

List 2 factors which can affect retention time (tR)

Answer 17

Flow rate of mobile phase

Composition of mobile and stationary phase

Question 18

How are individual compounds identified in a chromatogram?

Answer 18

By their retention time (tR)

Question 19

What is Kc?

Answer 19

Kc = the partition coefficient
An analyte is in equilibrium between the two phases
The molar concentration of analyte in the stationary phase divided by the molar concentration of the analyte in the mobile phase
EQUATION

Question 20

What is the retention factor (k’) often used to describe?

Answer 20

The rate of an analyte on a column

Also known as capacity factor

Question 21

What is the formula of the retention factor (k’)?

Answer 21

EQUATION

Question 22

What is the formula for resolution?

Answer 22

EQUATION

Question 23

Define the terms of the resolution formula

Answer 23

t2 = resolution time of 2nd peak
t1 = retention time of 1st peak
W2 = width at base of 2nd peak
W1 = width at base of 1st peak

Question 24

List 3 factors that induce and affect band broadening (Rate Theory of Chromatography)

Answer 24

Path of the molecule through the column
Flow rate
Packing of the stationary phase

Question 25

List the 3 main factors of peak broadening (irregular misshapen peaks)

Answer 25

Longitudinal diffusion
Resistance to mass transfer
Eddy diffusion

Question 26

What is longitudinal diffusion?

Answer 26

Analyte concentration is higher at the centre of the band and so it diffuses out to the edges
Slow flow rate increase longitudinal diffusion
Results in broader peaks
PICTURE

Question 27

How does resistance to mass transfer come about?

Answer 27

Analyte needs time to partition between mobile and stationary phase
Band broadening due to resistance to diffusion of the molecule in the mobile and stationary phase
High flow rate causes increased broadening
PICTURE

Question 28

How does eddy diffusion come about?

Answer 28

Broadening of peaks due to different analytes taking different paths through the stationary phase
Decreases with increasing particle and packing uniformity
Independent of flow rate

Question 29

Name the 2 types of HPLC detector

Answer 29

Responsive to physical and chemical properties of sample components (e.g. UV, fluorescence detectors)
Responsive to changes in properties of the mobile phase (e.g. refractive index detector)