chromatography phases Flashcards
resin
The stationary phase suport material (e.g. silica)
ligand
the molecule bonded to the resin to effect a separation
mobile phase
the liquid passing around or through the stationary phase (or gas for gas liquid chromatography)
analyte
a molecule injected into the mobile phase for separation
how to separate small molecules
thin layer chromatography
gas liquid chromatography
high pressure liquid chromatography
how to separate larger molecules
ion exchange chromatography
affinity chromatography
gel permeation chromatography
hydrophobic interaction chromatography
what are normal and reversed phase chromatography examples of
partition chromatography
normal and reversed phase chromatography
liquid/liquid separations on a solid matrix. Silica is usually the solid matrix to which is bonded in normal phase a monolayer of polar material (e.g. alkyl amine) and in reversed phase a monolayer of organic material
liquid surrounds the silica beads. Analytes applied to the column depending on their Kd (dissociation constant) will prefer to partition into either the monolayer of liquid on the surface of the bead or partition into the mobile phase. When a new mobile phase is introduced into the column a new partition can take place.
why do we have high pressure in reverse and normal phase chromatography
the small particle size requires high pressure to drive the mobile phase through the stationary phase in the column. ultra high pressure liquid chromatography needs higher pressure due to smaller beads than high pressure liquid chromatography. The smaller the beads the greater number of theoretical plates(resolving power)
Parts of HPLC equipment
The column separates the material applied
- autosamplers allow repeated injections
- column heaters help reproducibility
- pumps regulate flow rate and allow gradients to be produced
- detectors register analytes as they emerge from the resin. There are many different detectors e.g. UV/ visible, mass spectrometers
- data management- computers control the elements of the system and record the results
normal phase chromatography stationary phase, mobile phase, order of elution and applications
stationary phase-alkyl amine bonded to silica
mobile phase-organic e.g. hexane
order of elution- least polar first and most polar last
applications-compounds with low water solubility e.g. phospholipids, steroids or fat soluble vitamins
reverse phase chromatography stationary phase, mobile phase, order of elution and applications
stationary phase- C5,C8,C18 bonded to silica
mobile phase starting conditions- high polarity solvent e.g. 10% methanol in water - organic analytes will partition onto the resin
mobile phase finishing conditions- low polarity solvent e.g. 100% methanol introduced gradually as a gradient. Organic analytes will partition onto the mobile phase depending on the Kd.
order of elution-most polar first and least polar last
applications- wide range
Hydrophilic interaction chromatography
- A type of normal phase chromatography
- The resin has a polar surface typically silica retaining a monolayer of water and the starting mobile phase is organic e.g. >80% methanol
- polar molecules in the sample will partition into the water bonded to the surface of the silica. Other interactions can include ion exchange effects. Analytes which have a polar nature will be retained and non polar will elute
- material retained on the column are eluted with a decreasing gradient of organic solvent
AKTA
protein purification system
- made mostly of plastic which is biologically compliant-metal ions in normal HPLC bind to cystine in proteins so using plastic is better.
- column is packed with biologicaly benign stationary phase
- lower pressure than HPLC due larger stationary phase
can HPLC be used for protein purification
yes but has to be converted
e.g.PEEK tubing must be added
