Separation by HPLC
A closer look
What to know before starting
Chromatography
the separation of different components in a mixture
- estimate best separation conditions
- optimize method for selectivity, peak shape, run time, etc.
- number of compounds
- chemical structures
- molecular weights
- pKa values
- UV spectra
- concentration range
- sample solubility
Theoretical vs. Empirical
- chemical composition (separation of compounds of similar structure)
- data from literature
verses
- initial chromatographic separation
- disregard chemical structure info
The Traditional Way
the enlightened trial and error
HPLC
- To minimize the use of information on sample structure and carry out method development for most samples in the same general way
- Favored conditions for an initial separation are used as basis for the next experiment
- Repeat process until acceptable separation is achieved
high performance liquid chromatography
2 types:
- Normal Phase
- Reverse Phase
Function
Major Parts
Column Selection
- samples of biological origin
- mixtures of enantiomers
- preparative separations
- hydrophobicity
- steric selectivity
- bonding material
- ion exchange
Normal Phase
- polar stationary phase (cyano or amino functional groups)
- non-polar, non-aqueous mobile phase (hexane) + slightly polar mobile phase (IPA, chloroform)
- Least polar compound elute first
- poor reproducibility of retention time
Reverse Phase
- non-polar stationary phase (hydrocarbons)
- aqueous, moderately polar mobile phase (H2O + acetonitrile)
- Most polar compound elute first
- based on hydrophobic interactions
- purpose of the method
- compound of interest
- degradation products
- by-products
- contaminants
- regular samples are typical mixtures of small molecules
- examples of special samples:
- isomers
- enantiomers
- biological samples
- macromolecules
- compounds that are mirror images
- cannot be separated based on polarity or other chemical properties in reverse phase
- sample treatment - derivatization
- tedious
- possible racemization
- mobile phase - chiral derivatizing agent
- expensive
- not available for all chiral compounds
- solid phase - chiral column
Three Main Conditions that affect retetion
- mobile phase
- stationary phase
- temperature