Chromatography and HPLC Flashcards
What are different detectors for Chromatography and HPLC?
- MS
- ECD
- UV/vis spectrophotometry
- Fluorescence
Batch analysis – not suitable for high throughput/urgent analyses
What are the uses of Chromatography in a clinical lab?
- Drugs of abuse, therapeutic drug monitoring
- Steroid analysis/profiling
- Amino acid, organic acids, newborn screening
- Vitamin analysis
What is Chromatography?
- Separation of dissolved analytes depending on their relative attraction to various solid or liquid phases
- One phase is fixed “stationary phase”
- One phase moves “mobile phase”
- Like attracts like
What are some separation mechanisms?
- Adsorption: Uses electrostatic, hydrogen bonding or dispersive interactions between a molecule and solid support
- Partition: Based on relative solubility
- Ion-exchange: Sign and magnitude of ionic charges
- Steric exclusion: Based on size
- Affinity: Based on bio-specific interactions e.g. antibody-antigen
What is Partition Co-efficient?
- An analyte is in equilibrium between the two phases. Mobile phase is in equilibrium with Stationary phase
- The equilibrium constant, k, is termed the partition coefficient;
- Can be calculated by:
(Analyte(moles) in the stationary phase) / (Analyte(moles) in the mobile phase)
What are the stationary phases and mobile phases in Gas Chromatography?
- Stationary phases: Greases, gums and resins
- Mobile phases are gases such as helium and nitrogen
Separation based on the relative affinities to the column material and gas
What are stationary and mobile phases in Size Exclusion Chromatography?
- Stationary phase e.g. polyacrylamide gels, sephadex
- Mobile phase is a buffer
Separation based on the size and shape of molecule
What are stationary and mobile phases in Thin Layer Chromatography?
- Solid phase is silica or alumina
- Mobile phase is a solvent mixture
What are stationary and mobile phases in Ion Exchange Chromatography?
- Stationary phase can be gels, charged resins
- Mobile phases include buffers and salts
- Cation and anion
What is Ion exchange theory?
Strong vs Weak Ion exchange
- To elute from strong ion exchanger you need to replace the analyte with large amounts of salt with the same charge
- To elute from a weak ion exchanger you can alter the pH, then the analytes are no longer bound. The charge of the ion exchanger alters rather than the analyte
What are stationary and mobile phases in High Pressue Liquid Chromatography?
- Stationary phases are many! Usually silica based with or without functional groups
- Mobile phases are usually a buffer and a solvent
Also UPLC – Ultra pressure (performance) liquid chromatography
What are features of the solvent in HPLC?
- Often have one aqueous and one organic
- Can have single mixed solvenet
- Can use isocratic or gradient elution
- Can have additive in depending on the application: buffer, salts and ion pain reagent (chromatography on a hydrophobic pair exchange. Make a bridge between analyte and stationary phase)
- For MS there Adduct promotion and acids – prevent analyte colonisation
What a features of the pump in HPLC?
If more than one pump is used, each with a different solvent, mixing of solvents can be achieved
What features of the HPLC columns?
- Usually stainless steel (high pressure <300 bar, ultra pressure <800 bar)
- Have a packing material inside
- Held in place by a frit which is a filter to stop particles coming out
What are guard columns?
- Guard columns protect the more expensive analytical column from particulates
- May have packing material inside or be just a filter
How are chromatography columns labelled?
Labelled with
- Type of column
- Length of column
- Diameter of column
- Particle size
Can use the length and diameter to calculate the volume of the column
What is inside the columns?
- Most often silica particle based.
- Older columns were irregular size particles but now very regular
- HPLC particles are usually 2.5 – 15 micrometres
- UPLC particles are smaller than this. Alternative types are available
What are functional groups that can be added to particles?
- C18
- C8
- C4
- Phenyl
- Amide
- CN
- PFP
- HILIC
What are the benefits of pores in Chromatography?
- The particles are very porous, like the pores of a sponge – 99% of chromatographic surface is inside the pores
- Mobile phase mustbe allowed into the pore in order for chromatographic retention of the analyte to take place.
How does reversed phase chromatography get carried out?
Mobile phase is polar and stationary phase is non-polar
- Most non-polar species like the stationary phase best and comes out last
- Somewhat polar species like the stationary phase somewhat and comes slows down a little
- Most polar species like the mobile phase best and comes out first
What are principles of reversed phase separation?
- The greater the concentration of organic substrate on the packed bed, the stronger the retention: 29%C > 20%C > 12%C
- The longest alkyl bonded phase gives the greatest retention: C18 > C8 > C4
- Non polar groups on sample increase retention: toluene elutes after benzene
- Polar functional groups on sample reduce retention: phenol elutes before benzene
What is back pressure?
- Back pressure is the pressure generated as the mobile phase moves through the column
- The smaller the particle, the greater the back pressure
- Other factors include Flow rate, Column dimensions and Solvent composition
What are the detectors used in chromatography?
Can be:
- UV-visible spectrometer
- Fluorescence detector
- Mass spectrometer
What is the plate model of Chromatography separation?
- This assumes that the column contains a large number of separate layers or theoretical plates.
- In each of these plates, there is equilibration of the analyte between the mobile and stationary phases.
- The analyte moves through the column by transfer between mobile and stationary phase at each plate
