Sample Prep 2

Question 1

What is the least time consuming sample prep technique?

Answer 1

Protein precipitation

Question 2

What does protein precipitation involve?

Answer 2

Denaturation if proteins present in biomatrix by external stress such as a strong acid/base, heat or an organic solvent such as acetonitrile or methanol

Question 3

How much organic solvent do you add for protein precipitation?

Answer 3

3 parts organic solvent to one part biomatrix followed by cyclomixing and centrifugation

Question 4

What does centrifugation do to the sample?

Answer 4

Leads to formation of protein pellets and supernatant is separated for bio analytical quantification

Question 5

Denaturation causes an active change in the protein structure…

Answer 5

The drug bound to the protein becomes freely soluble in the Denaturation solvent ready for quantification

Question 6

What are the steps for protein precipitation?

Answer 6

1. Add 1.5ml acetonitrile
2. Vortex
3. Centrifuge
4. Transfer supernatant and dry
5. Reconstitute
6. Vortex and centrifuge
7. Add to autosampler vial and analyse using HP,C

Question 7

What is a protein precipitation plate?

Answer 7

A 96 well plate allowing for manual and robotic automation

They contain tubes with membrane/depth filter and are attachable to vacuum filtration

Question 8

What are the disadvantages of liquid-liquid extraction?

Answer 8

1. Time consuming
2. Large sample volume req
3. May require evaporation step prior to analysis to remove excess solvent
4. Large volumes of toxic organic solvents used
5. Need ultra pure solvents
6. Form emulsions?

Question 9

What is the process of LLE?

Answer 9

1. Add clean immiscible aq solvent phase
2. Shake/stir to allow molecules to partition
3. Phases settle and separate with gravity

Question 10

What is solid phase extraction?

Answer 10

A process by which components that are dissolved/suspended in a liquid mixture are separated from other components based on their physical and chemical properties

It is a column liquid-solid chromatography

Question 11

How does SPE work?

Answer 11

1. Sample is introduced into the column containing a bed and packing material
2. Solvent flows through the bed
3. Appropriate combo of stationary phases, sample components may pass directly through the column bed or be selectively retained

Question 12

What are the driving forces for SPE?

Answer 12

Gravity, pressure and vacuum

Sample must be liquid

Question 13

What are the steps of SPE?

Answer 13

1. Sample pre-treatment

• aq/non aq solvents may require dilution to reduce sample viscosity to ensure a free flowing sample
• pH adjustments

2. SPE column solvation

• a solvent is passed through the column to wet the sorbent and ensure interaction
• aq samples wetted by an organic solvent
• non aq wetted with matrix solvent

3. SPE column equilibration

• column is treated with a solvent that is as matrix like as possible to maximise retention of the analyse by the sorbent
• 0.5-2 ml/100mg of sorbent

4. Sample loading

• 1ml/min for a 1ml cartridge
• 7ml/min for a 6 ml cartridge

5. Interference elation

• a solvent which is miscible with the matrix, but in which the analytes are poorly soluble and interfering molecules are highly soluble

6. Analyte elution

• elution solvent in which analytes are soluble
• elution solvent should be compatible with the analysis technique