Medicine - Chromatographic Techniques

Question 1

Uses of chromatography

Answer 1

Analyse
Identify
Purify
Quantify

Question 2

Common features of chromatography

Answer 2

All have a stationary phase (solid or liquid supported by a solid)
And a mobile phase (liquid or gas)
The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates dependent on their attraction to the mobile phase and the stationary phase

Question 3

What is meant by development when referring to chromatographic techniques?

Answer 3

Elution

Question 4

What is the solute referred to as?

Answer 4

The euate

Question 5

What is the mobile phase referred to as?

Answer 5

The elluant

Question 6

Describe the process of column chromatography in 5 steps

Answer 6

1 - load the stationary phase material into the column
2 - Equilibrate column stationary phase with the mobile phase
3 - Load sample in as small a volume as possible
4 - Add more mobile phase to the column
5 - Collect sample fractions from the column

Question 7

What are the two possibilities of substance the column can be loaded with in terms of mobile and stationary phases? (In column chromatography)

Answer 7

The column is either loaded and filled with mobile phase, which is then flushed through the column,
Or
More usually, loaded with a slurry of stationary phase and mobile phase together (care taken to avoid bubbles)

Question 8

When is sand used in column chromatography?

Answer 8

Sand is used if cotton wool is used in place of a sinter disc

Question 9

Describe how molecules of analyte are separated in column chromatography in terms of stationary and mobile phases

Answer 9

Molecule with high affinity for the stationary phase will take longer to travel through the column.
Thus, molecules of different polarities separate out

Question 10

Stuff to monitor in normal phase chromatography and how to monitor it

Answer 10

Monitor fractions:

• colour
• weight
• TLC
• derivatives
• spectroscopy

Question 11

What one factor determines the type of chromatography it is?

Answer 11

The type of solid/stationary phase determines the type of chromatography

Question 12

Most common stationary phase in normal phase chromatography? Give particle sizes

Answer 12

Silica gel (Si-OH) is mostly commonly used

• 40-63um particle size
• high surface area (~800m2/g)

Alumina is less commonly used
50-200um particle size

Question 13

Describe how use of column chromatography as a purification tool would happen in 4 steps

Answer 13

1) Collect fractions
2) Determine component of interest by methods such as colour, TLC, UV, fluorescence
3) Pool fractions
4) Column can then be washed with eluting solvent and re-used or discarded

Question 14

Use of chromatography in laboratories and in industry?

Answer 14

Used to separate an purify compounds on scale from mg to kg

Question 15

What is the stationary phase in TLC?

Answer 15

Usually silica, but alumina and cellulose are used too

Question 16

What does TLC stand for?

Answer 16

Thin layer chromatography

Question 17

What is the driving force in TLC?

Answer 17

Mobile phase moves through stationary phase by capillary action

Question 18

What is the elutropic series?

Answer 18

A scale of compounds starting at high polarity (water, acetic acid) down to low polarity (cyclohexane, n-hexane)

Question 19

What is flash column chromatography used (a lot of the time) for?

Answer 19

Gas separation

Question 20

In flash column chromatography, what affects solvent flow? Discuss

Answer 20

Particle size affects solvent flow
70-230 silicia gel for gravity columns
Smaller particles (higher mesh values) need a pump to flow the mobile phase

Question 21

Describe the instrumentation in flash column chromatography

Answer 21

Solvent reservoir 
Pump (causes fast elution, can use gas instead) 
Column 
Detector (fraction collector) 
Recorder 

All of this is in a closed system

Question 22

Elution expected in order of organic classes for flash column chromatography?

Answer 22

Increasing polarity (more slow)

Saturated hydrocarbons 
Unsaturated hydrocarbons 
Ethers
Esters 
Halides 
Ketones
Aldehydes 
Amines 
Alcohols 
Acids and bases

Question 23

Name 7 uses of TLC

Answer 23

• Quality control and purity evaluation
• synthetic chemistry for identifying compounds
• To select a solvent system for column chromatography
• To follow the progress of a reaction
• To follow purification process
• Basic identity to check on pharmaceutical raw materials
• Detect impurities in pharmaceutical raw materials and formulations

Question 24

What interactions go on in TLC and normal phase chromatography?

Answer 24

Weak polar interactions i.e.
Dipole-dipole
Hydrogen bonds

Question 25

Name the specific stationary phase used sometimes in TLC to detect things with no colour

Answer 25

F254 = fluorescent reagent (shine UV light and it looks green)

Question 26

Name some ways you would detect colourless components when doing TLC

Answer 26

• UV light
• chemical treatment (dip, spray) e.g. Ninhydrin
• chemical vapour e.g. Iodine

Question 27

TLC colour for UV light (indirect)

Answer 27

Dark spot on yellow/green background

Question 28

TLC colour for iodine vapour

Answer 28

Brown

Spraying with starch makes it permanent

Question 29

In TLC, what does potassium permanganate detect?

Answer 29

Sugars and sugar-like molecules

Question 30

What colour spot does Ninhydrin cause in TLC? What is it used to detect?

Answer 30

Purple/pink spot with amines

Used for gentamycin

Question 31

What colour does alkaline tetrazolium cause in TLC when it detects something? What does it detect?

Answer 31

Blue spots with corticosteroids