MAAC: Chromatography 1

Question 1

Why is chromatography needed?

What types of analysis can be carried out?

Answer 1

To seperate analytes in a mixture and provide analysis that is selective

It can be used for quantatative, qualitative and preparative analysis.

Qualatative - To identify an analyte or confirm presence or abscence of a analyte

Quantatative - Measure quantatiy or dose

Preparative - Manufactured compound needs to be isolated or purified

Question 2

Why is chromatography needed in the pharmaceutical industry?

Answer 2

To monitor:

• Drug desgin and manufacture activity
  
  • To ensure drug made is what you think you have made
• Quality assurance
  
  • Ensure correct dose, no impuritites
• Clinical Trials
  
  • To investigate drug behaiour and metabolism

Question 3

In the pharmaceutical industry a combination of qualatative and quantatative analysis is extensively used. In terms of analysis of quality issues what are theses methods used for?

Answer 3

• Quality of actives (purify, identify. content)
• Quality of Excipents (Purify, identify, content)
• Cleanliness of macufacturing plant. Including between batches of different compounds/ processed (crosscontaminaition)
• Stablility testing. Including predication of drug degredation pathways

Question 4

What types of chromatography is there?

Answer 4

• TLC (thin layer C) or later HPTLC (high performance TLC)
• LC (liquid C) or later HLC
• GC (Gas C) or GLC (Gas Liquid C)
• CZE ( Capillary zone electophoresis)

Question 5

What 2 components make up a chromatography system? What phase can each of these be?

Answer 5

• Mobile phase (either liquid or gas)
• Stationary phase (Either absorbent solid or liquid attached to solid)

IF BOTH PHASES ARE LIQUID THEY MUST NOT BE MISSCIBLE

Question 6

Breifly describe how the analytes/ components in the mixture seperate

Answer 6

Seperation occurs by selective interaction of the analyte with the SP and MP

If the station phase is a porous solid, for example, the mobile phase flows through this and each component will have different interactions between the mobile phase and the station phase, which enables them to be separated. It’s normally differences in physicochemical properties of the components or analytes in the sample, which results in these different interactions and separation. So they have selective interactions with the station phase and the mobile phase. which enables them to be separated. It’s normally differences in physicochemical properties of the components or analytes in the sample, which results in these different interactions and separation. So they have selective interactions with the station phase and the mobile phase.

Question 7

What are the terms used to describe the interaction of the analytes with the stationary phase?

Answer 7

The interaction between the analyte and the stationary phase is described as a sorption process. This can then be subdivided into absorption and adsorption. Where absorption is when the analyte enters the volume of the stationary phase. And the more important process is adsorption. So this occurs on the surface of the stationary phase.

When the analyte is released from the stationary phase, as these interactions are reversible, it is known as desorption. The release process can be from or through a surface.

MP to SP = Sorption

SP to MP = Desorption

It’s these differences in rates of sorption and desorption of each of the individual components in the sample that results in the separation.

Question 8

Describe what a TLC chromatogram looks like and how it works

Answer 8

The sample is spotted at the bottom of a plate (SP) and the bottom of the plate placed in a solvent (MP). The mobile phase moves up the SP, allowing interaction of analytes with the SP and causing seperation into bands/ spots

The resulting chromatogtam is called TLC plate

Question 9

Describe the chromatogram of a GC or LC.

How does it work?

Answer 9

The retention time - time taken for individual analytes to move through the solid phase and reach a detector is measured. The seperate analytes are detected and plotted against the retention time.

The chromatogram is automatically produced by the instuemtn sodtware

Question 10

The selective retention of an analyte results in seperation. However to acheive retention, the analyte must spend sometime in or on the SP.

What equation is used?

Answer 10

Kd = Csp/ Cmp

The Kd is the distribution constant and is equal to the C of analyte distibuted in the SP phase and C in the MP phase

Question 11

What is the relationship between Kd and rate of migration

Answer 11

They are inversly proportional

So if high conc in stationary phase, the rate of migration through SP will be slower or greater retention in this phase

Question 12

Retention results from several different mechanism of sorption mechanisms. What are examples?

Answer 12

• Adsoption
• Partition
• Ion exchange
• Size exclusoin
• Affinity

Question 13

In adsporption chromatography, what phases are the SP and MP in LC and GC?

Answer 13

SP is always solid.

GC - MP is gas

LC - MP is Liquid

Question 14

What are examples of SP and how does their polairty vary?

Answer 14

• SP can be silica, aluminium, paper (cellulose), charchole
• All differ in polairy
• Charcol is non-polar

Question 15

How does the MP in GC and LC differ?

Answer 15

The MP in GC is non-interactive with the SP. It does not play a direct role in seperation so can be referred to as a carrier gas

THe MP is LC is interactive. It can compete with the analyte for adsorption sites on the SP

Question 16

In LC adsoprtion what do the following terms mean?

• Normal phase Chromatography
• Reverse Phase chromatography

Answer 16

1. NPC: This is where the SP picked is more polar than the MP. This means polar analytes will interact more with the SP and be retained for longer
2. RPC: This is where the SP picked is less polar than the MP and polar analytes will interact more with the mobile phase and will be retained less. Most common

Question 17

In GC these terms do not apply. Why?

How is retentioned determined instead?

Answer 17

The MP does non-interactive therefore is called a carrier gas

Instead the retention is based off the differences in polarity between the analytes and the SP phase. If the anaylte has a similar polarity to the SP phase it will interact more and be retined longer

Question 18

What is the order of polarity for the following?

• Alkenes
• Ether
• Carboxylic Acid
• Amine
• Ketone
• Aldehyde
• Alcohol
• Thiols

Answer 18

1. Alkane
2. Alkene
3. Ether
4. Ketone
5. Aldehyde
6. Thiol
7. Amine
8. Alcohol
9. Carbocylix

Question 19

In partion Chromatography what phase is the SP?

How does seperation work?

Answer 19

• SP is bonded phase - Liquid chemically bonded to silica support
• Different SP can be used with varrying polarity so again Normal and reverse bonded phase Chromatography can be carried out
• Seperation is due to differences in solubility of anayltes in the SP
• Polar, ionic and ionsaible molecules can easuly be analysed

Question 20

What is Gradient ELution partition chromatography useful for?

Describe how it is carried out in both GC and LC

Answer 20

Useful to seperate analytes wiht very different properties without having to use different methods

GC -

LC -

Question 21

What is ion suppression?

Answer 21

The mobile phase normally has a speicific pH required for analysis

Thus the ionisation of analytes such as WA/WB can be supressed and their retjetion manipulated such that seperations can be acheived

HA — A- + H+

BOH — B+ + OH-

HA and BOH will partition into non-polar SP where as A- and B+ will partion into polar SP

The less ionisation the more retainion on a non-polar SP