Chromatography

Question 1

Define chromatography.

Answer 1

A technique for the separation of a mixture by passing it through a medium in which the components move at different rates

Question 2

What is the solid phase in paper chromatography?

Answer 2

Cellulose

Question 3

What is the mobile phase in paper chromatography?

Answer 3

Any solvent (usually H2O / EtOH mix)

Question 4

What is the solid phase in thin layer chromatography?

Answer 4

Silica

Question 5

What is the mobile phase in thin layer chromatography?

Answer 5

Organic solvent

Question 6

What is Rf in thin layer chromatography?

Answer 6

The distance travelled by a compound divided by the distance travelled by the eluant

Question 7

What are normal phase methods of chromatography?

Answer 7

Techniques which utilise a polar stationary phase with a less polar mobile are referred to as normal phase methods.

Question 8

What are the limitations of normal phase chromatography?

Answer 8

• The polar stationary phase can degrade with repeated use
• The polar stationary phase can become contaminated with polar impurities leading to a change in performance on repeated use
• Requires the use of flammable and/or toxic organic solvents

Question 9

What is the difference in reversed phase chromatography compared to normal phase?

Answer 9

The stationary phase in inert and not polar

Question 10

Define the retention time

Answer 10

The time from the injection of the mixture onto the column until that component reaches the detector

Question 11

Define the adjusted retention time

Answer 11

The additional time for a solute to travel the length of the column.

Question 12

How can the peaks be well resolved?

Answer 12

Particles need to be small to create a large surface area and therefore less spaces between them. However this is expensive as higher pressure is required to push the solvent through the column

Question 13

If the column is isocratic, what does this mean?

Answer 13

It means that the column is eluted with a single eluent. (Constant composition)

Question 14

With isocratic systems, tailing often occurs. How can this be resolved?

Answer 14

Use a polarity gradient. With HPLC, this means starting with an aqueous eluent and slowly increasing the organic component until the final stage of the column is in organic solvent.

Question 15

What are the advantages and disadvantages of HPLC?

Answer 15

Adv

• Allows assessment of conc in mixtures
• High throughput/low cost

Disa

• Provides little data on nature of any analytes observed
• With UV detection any UV- inactive components will not be observed.

Question 16

What are the advantages and disadvantages of LCMS?

Answer 16

Adv

• Allows assessment of conc in mixtures
• Provides come info on the composition of peaks
• May observed UV-inactive components

Dis

• Only quantitative in relation to standards
• Structural info is limited in same was as MS

Question 17

What does the Beer Lambert Law show in chromatography?

Answer 17

The equation shows that because ‘Molar absorptivity/extinction coefficient’ and transmitted light intensity will be set by the detector, A (absorbance) is equal to c (concentration). So we can therefore measure the concentration of an unknown by comparing the absorbance of a known.