Intro to chromatography Flashcards
How is chromatography used in clinical labs?
Used with different detectors such as MS, ECD, UV/vis, fluorescence
Application of chromatography
- Drugs of abuse, therapeutic drug monitoring
- Steroid analysis/profiling
- AA, Organic acid, newborn screening
- Vitamin analysis
Basics of chromatogrpahy.
Seperation of dissolved analytes depending on their relative attraction to various solid or liquid phases.
Phases: stationary or mobile
Name seperation mechanims
- Adsorption
- Partition
- Ion-exchange
- Steric exclusion
- Affinity
How can the analyte equilibrium betweent the two phases be defined?
Using the partition coefficient, k.
Analyte in stationary phase/analyte in mobile phase
What is gas chromatography?
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
What is size exclusion chromatography?
Stationary phase e.g. polyacrylamide gels, sephadex
Mobile phase is a buffer and seperation is based on shape and size
What is thin layer chromatography?
Solid phase is silica or alumina
Mobile phase is solvent mixture
What is ion exchange chromatography?
Stationary phase either gels, charged resins
Mobile phase is buffer and salts
Anion exchanger attract anions whilst cation exchanger attract cations
What is high pressure liquid chromatography?
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
Seperation theory; explain using reverse phase chromatography
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)
Principles of RP seperation
- 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
What is the plate model?
- 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
What does a short HETP suggest?
More plates are contained within a given length of column and the more efficient the column
What is the Van Deemter equation?
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
What is eddy diffusion?
Analytes take different paths through the stationary phase at random, causin peak broadening as the different paths are different lengths
How to decrease eddy diffusion?
The more regular the particle size and packing of the column, the less eddy diffusion occurs.
What is longitudinal diffusion?
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
How to decrease longitudinal diffusion?
- 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
What is mass transfer?
- 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
How to minimise mass transfer?
- 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
How will a polar and non polar act in a thin layer chromatography?
Polar compounds will interact greatly to the stationary phase whilst non-polar compounds will not form the necessary dipole dipole interactions in silica
How is thin layer chromatography visualised?
UV light or using a staining agent
