Intro to chromatography

Question 1

How is chromatography used in clinical labs?

Answer 1

Used with different detectors such as MS, ECD, UV/vis, fluorescence

Question 2

Application of chromatography

Answer 2

• Drugs of abuse, therapeutic drug monitoring
• Steroid analysis/profiling
• AA, Organic acid, newborn screening
• Vitamin analysis

Question 3

Basics of chromatogrpahy.

Answer 3

Seperation of dissolved analytes depending on their relative attraction to various solid or liquid phases.

Phases: stationary or mobile

Question 4

Name seperation mechanims

Answer 4

• Adsorption
• Partition
• Ion-exchange
• Steric exclusion
• Affinity

Question 5

How can the analyte equilibrium betweent the two phases be defined?

Answer 5

Using the partition coefficient, k.

Analyte in stationary phase/analyte in mobile phase

Question 6

What is gas chromatography?

Answer 6

Uses greases, gums and resins as its stationary phase. With a mobile phase of helium and nitrogen

Seperation based on relative affinities to the coloumn material and gas

Question 7

What is size exclusion chromatography?

Answer 7

Stationary phase e.g. polyacrylamide gels, sephadex

Mobile phase is a buffer and seperation is based on shape and size

Question 8

What is thin layer chromatography?

Answer 8

Solid phase is silica or alumina

Mobile phase is solvent mixture

Question 9

What is ion exchange chromatography?

Answer 9

Stationary phase either gels, charged resins
Mobile phase is buffer and salts
Anion exchanger attract anions whilst cation exchanger attract cations

Question 10

What is high pressure liquid chromatography?

Answer 10

Stationary phase: silica base with or without functional groups
Mobile phase: usually a buffer and a solvent

Separation is based on polarity, by dipole dipole interactions with stationary phase hydroxyl groups

Also UPLC - ultra pressure LC

Question 11

Seperation theory; explain using reverse phase chromatography

Answer 11

Stationary phase is non polar and mobile phase is polar.

Non-polar analytes will like the stationary phase and elute last

Polar like the mobile phase best and will elute first (depending severity of polarity)

Question 12

Principles of RP seperation

Answer 12

• Greater the conc of organic substrate on the packed bed the stronger the retention
• The longest alkyl bonded phase gives the greatest retention
• Non-polar groups on sample increase retention
• Polar functional groups on sample reduce retention

Question 13

What is the plate model?

Answer 13

• Assumes that the column contains a large number of seperate layers of theoretical plates
• In each plate there is equilibration of the analyte between the mobile and stationary phase
• The analyte moves through the column by transfer between mobile and stationary phase at each plate

Therefore the more plates the better seperation

• Can be examined using height equivalent to a theoretical plate (HETP)
• HETP= Length of column/number of theoretical plates

Question 14

What does a short HETP suggest?

Answer 14

More plates are contained within a given length of column and the more efficient the column

Question 15

What is the Van Deemter equation?

Answer 15

Describes the different properties of the column influencing HETP
- HETP A+B/u+Cu
A=eddy diffusion
B= longitudinal diffusion
C= mass transfer
u= average velocity of the mobile phase

Question 16

What is eddy diffusion?

Answer 16

Analytes take different paths through the stationary phase at random, causin peak broadening as the different paths are different lengths

Question 17

How to decrease eddy diffusion?

Answer 17

The more regular the particle size and packing of the column, the less eddy diffusion occurs.

Question 18

What is longitudinal diffusion?

Answer 18

Conc of analyte is less at the edges of the band than the centre, so analyte diffuse from the centre of to the edges, causing band broadening

Question 19

How to decrease longitudinal diffusion?

Answer 19

• Increase the mobile phase velocity decreases the longitudinal diffusion as analyte spends less time on column
• However, increasing phase velocity increases back pressure of the system -UPLC more appopriate

Question 20

What is mass transfer?

Answer 20

• Analyte takes time to equilibriate between the two phases due to increased affinity to stationary phase
• If the mobile phase velocity is high and the anlyte has a strong affinity for the mobile phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary phase.

This causes band broadening

Question 21

How to minimise mass transfer?

Answer 21

• Fused core columns are aimed at reducing mass transfer effect
• Analytes can´t travel as far into the particles as it would in fully porous particle
• Also faster to re-equilibriate after changes in mobile phase as this does not travel into the particles as far either

Question 22

How will a polar and non polar act in a thin layer chromatography?

Answer 22

Polar compounds will interact greatly to the stationary phase whilst non-polar compounds will not form the necessary dipole dipole interactions in silica

Question 23

How is thin layer chromatography visualised?

Answer 23

UV light or using a staining agent