Lecture 5: Sample Preparation and Extration 2 Flashcards
What are the 4 main sample preparation techniques we need to know?
Liquid-liquid extraction (LLE)
Solid-phase extraction (SPE)
QuEChERS
Spin columns
What is LLE?
Separation based on selective solubility of solute or analyte between immiscible liquid phases where solute passes to the liquid or preferred solubility (polarity – like goes with like).
Procedure for LLE
- Add extraction solvent.
- Stopper.
- Gently shake, invert, and open tap.
- Close tap.
- Place back in stand.
- Remove stopper.
- Open tap to release solvent.
What will you see in terms of layers in LLE?
If organic solvent has lower density than water, organic phase is top layer (E.g. diethyl ether, hexane, ethylacetate)
If organic solvent has greater density than water, organic phase is bottom layer (E.g. chloroform, dichloromethane)
What are the practical implications of LLE?
Extraction solvents can be selected according to their immiscibility with the sample solvent and to specifically extract solutes of a particular hydrophobicity.
Method selectivity offers possible high extraction recoveries and high chance of detecting solute (sensitivity).
Ionic (dissociated) solutes typically will remain in aqueous layer unless neutralised.
describe the equilibrate step in SPE
(to ensure stationary phase is in a state suitable for effective binding of the analytes – small volume of same conditioning solvent so a buffer or water is passed through column)
Describe the condition step in SPE
(to prepare stationary/solid phase to interact properly with analytes – pass a (polar) solvent through the column, for example, methanol)
What are the steps of SPE?
condition
equilibrate
load
wash
elute
describe the load step in SPE
(analyte and interferences) (introduce liquid sample (low flow rate) to stationary phase where analyte of interest bind while other components are washed away)
describe the wash step in SPE
(to remove unwanted matrix components so only target analytes remain – typically washed with solvent)
describe the elute step in SPE
(to release analytes – elution solvent passed through column which has stronger affinity for the analytes than the stationary phase causing the analytes to desorb and collect)
what does QuEChERS stand for?
Quick, easy, cheap, effective, rugged, safe
How does QuEChERS work?
- Involved analyte partitioning, ion pair, solvent dehydration and dispersive SPE (dSPE).
- Designed to be carried out with minimal lab equipment (no more than a vortex mixer, pipette and centrifuge).
- Homogenised samples (solid/liquid) are extracted with CAN solvent and cleaned using dSPE.
- Solid samples can be blended or pulverised prior to extraction.
describe the procedure for QuEChERS
- Homogenised/liquid sample
- ACN (acetonitrile) added (shake/vortex)
- Partitioning (add drying agent, buffer, and ion pair, then shake/centrifuge)
- dSPE clean up (shake/centrifuge)
- Analysis
What are the experimental conditions affecting SPE?
- Initial hydration of sample and pH (<80% water)
- Volume of ACN
- Mass of drying agent (magnesium sulfate)
- Mass of salt (counter ion)
- Initial extraction buffer (pH range)
- Temperature of extract (reduce temperature to encourage precipitation of material)
- Shaking time
- Centrifugation time and speed
- dSPE material (waxy/pigmented sample)
- concentration/solvent exchange (evaporate to dryness?)
ADV of SPE
- Quick, low cost, rugged
- Low solvent use
- Shown efficacy for more than just foodstuffs
DISADV of SPE
- Requires centrifuge
- Designed to remove interferences and not targeted analyte isolation
- Typically minor analyte concentration without evaporation and reconstitution
- Still relatively niche for sample types
- Recovery can be limited and repeatability affected in avoiding particulate interference from supernatant collection
Describe Spin columns - purpose, etc.
Ultrafiltration columns using centrifugation to increase efficiency of separation.
- Forces sample constituents through membrane in shorter time
- For immunoprecipitation applications and removal of protein prior to LC-MS
Procedure for spin columns
Sample application
centrifuge
remove material in the collection tube
invert spin filter
centrifuge
sample collection
Procedure for spin columns (large molecules)
Sample application - protein A in buffer containing 100nM NaCl
centrifuge
remove material in the collection tube
Application of low/no salt buffer
centrifuge
invert spin filter
centrifuge
sample collection of protein A in low/no salt buffer
