Chromatography 2

Question 1

List the Structural factors which govern the rate of elution of compounds from HPLC columns for neutral compounds

Answer 1

The balance between polarity and lipophilicity is the major factor

On normal phase columns more polar compounds will be retained longer

On reverse phase columns more lipophilic compounds will be retained longer

Altering pH of the mobile phase does not influence this

Question 2

State a good indicator of how polar a compound is likely to be?

Answer 2

The number of OH groups cf other non-polar groups such as Me, are a good indicator to how polar a compound is likely to be. Also the type of OH (1o, 2o or 3o) indicate how well that OH can contribute to hydrogen bonding e.g. a tertiary OH group will be more sterically hindered than a primary OH and therefore will form weaker Hydrogen bonds.

Question 3

What would you predict the order of polarity of the following compound to be ?
T O N M see Structure on slide 3

Answer 3

O N T M

Question 4

On a ODS column with a methanol water (70/30) mobile phase what order would you expect them to elute?
T O N M ( see slide for structures)

Answer 4

O N T M

Question 5

Facts about Ionizable compounds

Answer 5

An additional factor that can be used to control the solvent strength of a mobile phase is pH (usually used in reverse phase where the mobile phase is often aqueous

Question 6

The majority of drugs are ionisable, and the degree of ionisation is dependant on what?

Answer 6

PH
Thus you can use pH to alter retention times of ionizable compounds and make them travel faster or slower through the column.

Question 7

If you were running aspirin through a reverse-phase column with an aqueous mobile phase, what would you expect to happen to the elution time if you increased the pH (made it more basic)?

Answer 7

Become ionised and therefore more polar. Will stay longer in mobile phase and elute faster.

Question 8

If you had a basic drug that was eluting too fast from a reverse-phase column. What could you do to increase its retention time?

Answer 8

Make the mobile phase more acidic

Question 9

Facts about Liquid Chromatography-Mass Spectroscopy (LCMS)

Answer 9

Hyphenated technique – Can separate and the use of HPLC/MS has the advantage of aiding in the structural identification of the separated analytes.

Question 10

What is the mobile phase in Gas Chromatography?

Answer 10

is a gas, e.g. helium,

Question 11

What is the stationary phase in Gas chromatography?

Answer 11

The stationary phase can be either liquid adsorbed onto a solid (most common – called GLC), or organic species bonded to a solid surface (older less used technique).

Question 12

Separation in Gas Chromatography is mainly based on….

Answer 12

mainly based on the volatility of the substances in the mixture.

Question 13

Compounds with reasonable volatility and heat stability can be separated by gas chromatography. Good for small organic molecules. True/ False?

Answer 13

True

Question 14

What is the most widely used gas chromatography?

Answer 14

GAS-LIQUID CHROMATOGRAPHY (GLC)

Question 15

What is the mobile phase used in GLC?

Answer 15

Gas

Question 16

The stationary phase in GLC is ……

Answer 16

Liquid

the stationary phase is a liquid that is retained on the surface of an inert solid by adsorption or chemical bonding

Question 17

Separation of the components in GLC is dependant on two factors namely,

Answer 17

1. The volatility of the component at column temperature

2. The polarity (solubility) of the component in the stationary phase

Question 18

State why samples with highest volatility and lowest solubility in the stationary phase, come out first in GLC?

Answer 18

The exact position of the equilibrium depends on the characteristics of the stationary phase, the sample, and the column temperature. As the vapour is carried away the solute comes back out of the stationary phase to re-establish the equlibrium. This is why samples with highest volatility and lowest solubility in the stationary phase, come out first.

Question 19

Which of the following would be expected to elute first from GC and why?
Methanol
Ethanol
Propanol
Butanol
Pentanol,

Answer 19

Methanol because it is the smallest molecule and it has the lowest boiling point. Therefore it will come out of the column first because it is the most volatile