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
How does changes in number of plates affect the operation?
- The more theoretical plates, the better separation you get
- Another way to express this is to look at the height equivalent to a theoretical plate (HETP)
- HETP = Length of column / number of theoretical plates
- This is reported in millimeters, and the shorter the HETP, the more plates are ‘contained’ within a given length of column and the more efficient the column
What is the Rate theory of Chromatography?
- The HETP is dependent on different properties of the column. These are described in the Van Deemter equation
HETP = A + B/u + Cu
- A = Eddy diffusion
- B = Longitudinal diffusion
- C = Mass transfer
- u = average velocity of the mobile phase
What are factors that affect seperation?
- Eddy diffusion
- Longitudinal diffusion
- Mass Transfer
What is Eddy diffusion?
- Analyte molecules take different paths through the stationary phase at random.
- This causes peak broadening as the different paths are different lengths.
- The more regular the particle size and packing of the column, the less Eddy diffusion is observed
- Modern UPLC columns with very small particle sizes have much less Eddy diffusion than more traditional columns
What is Longitudinal Diffusion?
- The concentration of analyte is less at the edges of the band than at the centre, so analyte diffuses from the centre to the edges.
- This causes band broadening
- Increasing mobile phase velocity can decrease the effect of longitudinal diffusion as the analyte spends less time on the column.
- Higher mobile phase velocity increases the back pressure on the system, so a UPLC system may be needed to take advantage of higher velocities
What is Mass transfer?
- The analyte takes time to equilibrate between mobile and stationary phases
- If the velocity of the mobile phase is high and the analyte has a strong affinity for the mobile phase, the analyte in the mobile phase will move ahead of the analyte in the stationary phase
- This causes band broadening
- The higher the mobile phase velocity, the worse the band broadening
How can mass transfer effect be altered?
- Fused core columns are aimed at reducing mass transfer effect
- The analytes can’t travel as far into the particle as it would in a fully porous particle
- They are also faster to re-equilibrate after changes in mobile phase as this doesn’t travel into the particle as far either
