Introduction to chromatography

Question 1

What is Chromatography

Answer 1

Method of seperating components withing a mixture that are ditrbuted between a mobile phase and stationary phase, based on their interaction and affinity with the stationary phase, and the resulting retention of those analytes.

Question 2

Why do we need chromatography and in particular why do we want to separate analytes?

Answer 2

• This provides analysis that is selective
• Can analyse one particular component: API, or exipient
• Gives, Qualitative, Quantative and preparative analysis.

Question 3

What is qualitative, quantative and preparative analysis

Answer 3

1. Qualitative Analysis - what is in that mixture
2. Quantitative analysis - How much of component being alalysed is in that mixture
3. Preparative analysis - helps us remove impurities if we know what they are

Question 4

What might the stationary, mobile phase be made of?

Answer 4

liquid, solid or gas

Question 5

What will the mobile phase usually be? and why?

Answer 5

• Liquid or gas - the mobile phase needs to be able to move

Question 6

what will the stationary phase usually be? and why?

Answer 6

• Adsorbent solid, or a liquid surrounding chemically attached to a solid backbone
• The stationary phase does not move

Question 7

What must be insured if SP and MP are both liquids?

Answer 7

• They must not be miscible

Question 8

How does seperation of the analytes in the mixture occur?

Answer 8

Selective interaction of the analyte with the stationary phase and less often the mobile phase

Question 9

What is the difference between adsorption and absorption?

Answer 9

• When the analyte is introduced into the system it will move from the mobiel phase to the stationary phase.
• This is called sorption
• Adosrption is when the analyte is not absorbed into the stationary phase
• Absorption - analyte is absorbed into the stationary phase

Question 10

Desorption is when the analyte comes back out the __ __ into the __ __

Answer 10

• Desorption is when the analyte comes back out the stationary phase into the mobile phase

Question 11

Describing the TLC plate in the image below

TLC plate

Answer 11

liquid - mobile phase
Paper - stationary phase
black spots - analyte
Seperation achieved as analytes travels different distances representing different components

Question 12

What does retention mean in chromatography?

Answer 12

It reflects how long a compound is retained by the stationary phase before it elutes (exits) and reaches the detector.

Question 13

What must happen to achieve retention?

Answer 13

• The analyte must spend some time in or on the stationary phase

Question 14

What equation can be used to measure retention?

Answer 14

kD = Csp/Smp
* KD = distribution constant
* Csp - concentration of analyte in stationary phase
* Cmp = concentration of analyte in mobile phase

Question 15

The rate of migration of an analyte is__ __ to its distribution constant.

Answer 15

The rate of migration of an analyte is inversely proportional to its distribution coefficient

Question 16

What are the two sorption mechanisms we need to know?

Answer 16

• Adsorption
• Partition - liquids adhered to solid backbones

Question 17

Adsorption chromatography

Answer 17

• Stationary phase is always solid but the mobile phase can be liquid (LC) or gas (GC)
• In GC, the mobile phase is non-interactive
• in LC, this mobile phase will also compete with the analyte for adsorption sites on stationary phase
• This can affect the retention of the analyte…

Question 18

What are the most common Polar and non-polar stationary phases?

Answer 18

Polar - Silica Gel, alumina, paper
Non-polar - Charcoal

Question 19

When will analytes have the most and the least retention?

Adsorption in liquid chromatography

Answer 19

• In NPC polar analytes will be retained the longest (elute slower)
• In RPC polar analytes will be retained the least (elute quicker)

Question 20

What is normal phase and reverse phase chromatography?

Answer 20

• If SP is more polar than MP then this is normal phase chromatography (NPC)
• If SP is less polar than MP then this is reverse phase chromatography (RPC)

Question 21

How does seperation occur in gas chromatography?

Adsorption in gas chromatography

Answer 21

• Seperation in adsorption chromatography is due to polarity differences between phases. The order of polarity of organic molecules can be generalised as in photo

Question 22

When might the eluton order be harder to predict?

Answer 22

molecules containing two or more functional groups.

Question 23

How is seperation achieved in partition chromatograpy?

Answer 23

• Seperation is achieved by differences in parition of analytes i.e how they distribute between those phases

Question 24

What are the phases made of in partition chromatography?

Answer 24

• Both phases are liquid.

Question 25

which phase is the bonded phase in partition chromatography? and what does bonded phase mean?

Answer 25

• typically Stationary Phase
• Means it is chemically bonded onto a sillica support

Question 26

What is gradient elution and how is it done?

Answer 26

In LC, gradient elution is done by changing the gradient of the mobile phase
In GC, the temperature alteration can result in gradient elusion