Introducing 

Prezi AI.

Your new presentation assistant.

Refine, enhance, and tailor your content, source relevant images, and edit visuals quicker than ever before.

Loading content…
Transcript

Separation by HPLC

A closer look

What to know before starting

Chromatography

the separation of different components in a mixture

Choose LC Method

  • HPLC
  • GPC
  • IC
  • GC-MS
  • LC-MS
  • estimate best separation conditions
  • optimize method for selectivity, peak shape, run time, etc.

Method Development

  • 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

  • separate
  • quantify
  • identify

The New Comers

Major Parts

new things to consider

Column Selection

  • samples of biological origin
  • mixtures of enantiomers
  • preparative separations
  • hydrophobicity
  • steric selectivity
  • bonding material
  • ion exchange
  • sampler
  • pump
  • detector

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

Define Goals

- non-polar stationary phase (hydrocarbons)

- aqueous, moderately polar mobile phase (H2O + acetonitrile)

- Most polar compound elute first

- based on hydrophobic interactions

what are we looking for?

  • purpose of the method
  • compound of interest
  • degradation products
  • by-products
  • contaminants

Sample Type

regular or special?

  • regular samples are typical mixtures of small molecules
  • examples of special samples:

- isomers

- enantiomers

- biological samples

- macromolecules

Enantiomers

  • 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
Learn more about creating dynamic, engaging presentations with Prezi