Pharmaceutical Analysis

Question 1

What are the 3 classic separation methods?

Answer 1

1. Electrophoresis
2. Chromatography
3. Membrane Separation

Question 2

What is chromatography?

Answer 2

When 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

Question 3

What is the stationary phase?

Answer 3

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

Question 4

What is the mobile phase?

Answer 4

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

Question 5

What are the 4 different examples of chromatography?

Answer 5

1. Thin Layer Chromatography
2. Column Chromatography
3. High Pressure Liquid Chromatography
4. Gas Chromatography

Question 6

How is chromatography used to separate?

Answer 6

1. Mobile or stationary phase
2. Both have two different compounds, two different sets of functional groups
3. One will interact with it in a different way from the other with the stationary phase

Question 7

What is elution? And what does it mean when it runs quicker down through the column?

Answer 7

1. The process of washing samples components through the stationary phase by continuous flow of the mobile phase
2. Less interaction with solvent
3. When component A or B gets to the detector, it makes a reading so we can see a graph

Question 8

What is a chromatogram?

Answer 8

1. A peak that appears when the saturated component reaches the detector at the end of a column
2. Components are then identified by unique retention time under a certain set of separation conditions
3. Factors such as tR (retention time) include:
   - velocity (flow rate) of mobile phase
   - chromatographic retention

Question 9

What does retention time normally depend on?

Answer 9

1. Amount of time the component spends in the stationary or mobile phase
2. More time in mobile means it passes through faster so the partition co-efficient is smaller
3. More time in stationary phase then partition co-efficient is larger

Question 10

What is the aim of resolution and where is it found?

Answer 10

1. Found in the chromatograph

2. It’s aim is how well it can separate two peaks

Question 11

What is baseline resolution?

Answer 11

When the detector goes to zero between the two peaks which is good resolution as you can clearly distinguish between the peaks after

Question 12

What is column efficiency?

Answer 12

The plate height that are relative to the retention time and affect the band broadening

Question 13

What is longitudinal diffusion?

Answer 13

1. When it runs down the column and doesn’t stay in one tight band, it longitudinally diffuses to separate into a broad band
2. The faster we run the column, the less our longitudinal diffusion, so it remains a tight band.

Question 14

What is resistance to mass transfer?

Answer 14

1. When the band broadens due to the resistance to diffusion of the molecule in the mobile and stationary phase
2. DependIrs on diffusion co-efficient of compound in each phase, diameter and shape of stationary phase
3. It’s parabolic flow profile: pressure driven flow

Question 15

What is Eddy diffusion?

Answer 15

1. When the mobile phase moves through the column which is packed with stationary phase
2. The more the column is packed- the lower eddy diffusion

Question 16

How do the parameters following increase or decrease column efficiency?

1. Low flow rate
2. Large particle size of stationary
3. Irregularly shaped stationary phase
4. Very fast flow rate

Answer 16

1. Low flow rate- broadens peak due to longitudinal diffusion
2. Large particle size of stationary phase- Increases eddy diffusion and mass transfer scales with particle size, this leads to peak broadening and decreases column efficiency
3. Irregularly shaped stationary phase- Eddy diffusion decreases with increasing particle size and packing uniformity- irregular shaped will increase peak broadening- impacts on diffusion due to mass transfer
4. Very fast flow rate- peak broadening due to resistance to mass transfer- diffusion in stationary phase

Question 17

What does having a more efficient column mean?

Answer 17

Narrower peaks and better resolution

Question 18

What does HPLC (high performance liquid chromatography) consist of?

Answer 18

Normal Phase:

1. Polar stationary phase, non polar mobile phase
2. Molecules elute in order of increasing polarity

Reverse Phase

1. Non-polar stationary phase, polar mobile phase
2. Molecules elute in order of decreasing polarity (most polar comes out first and least polar comes out last
3. About 80% HPLC separations

Question 19

Describe the normal phase in HPLC?

Answer 19

Consists of:
Stationary phase: Silica Gel with polar groups that are absorbed and retained

Mobile phase: hexane, dicloromethane, isopropanol, methanol (more polar mobile phase will elute compound more quickly)

Question 20

What is elution?

Answer 20

The process of extracting one material from another by washing with a solvent

Question 21

Describe the Reverse phase in HPLC?

Answer 21

Consists of:
Stationary phase: Octadecylsilane coated (ODS) Silica gel lipid groups adsorbed and retained

Mobile phase: Water, methanol, acetonitrile, tetrahydrofuran (the more lipophilic the mobile phase the faster elution of organic compounds)

Increasing in strength means increase in polarity

Question 22

What do you have to consider when it comes to elution of neutral components?

Answer 22

Balance between polarity and lipophilicity of compound: H-bonding capacity
Example: Rapid elution of prednisoline and betamethasone
- Much longer elution of esters

Question 23

What can you change for elution of ionisable compounds?

Answer 23

1. Altering pH of the mobile phase can control solvent strength
2. Works for weak electrolytes where solubility can be altered at working pH values in region of pKa

Question 24

What are the two main types of HPLC detectors?

Answer 24

1. One is responsive to physical and chemical properties of sample components- known as UV and fluorescence detectors
2. Responsive to changes in properties of the mobile phase- known as refractive index detector
   - non-specific and not as sensitive
   - only used when other detectors like sugars cannot be used

Question 25

What are the four types of HPLC detectors?

Answer 25

1. UV absorption detector (most popular) - fixed wavelength, variable wavelength with photo diode array - mobile phase must not absorb UV 2. Fluorescence detector- selective and sensitive 3. Refractive index detector - Universal - based on RI difference between solvent and solute - Moderately sensitive - very temperature sensitive 4. Mass Spectrometer - structural information

Question 26

What is HPLC-MS?

Answer 26

1. An industry standard technique for the identification of chemicals in mixtures 2. Very high sensitivity and specificity