Chromatography Flashcards
What is the basic principle of chromatography?
The partitioning of analyses between a stationary phase and a mobile phase
What happens to compounds with partition coefficient favouring the mobile phase?
They migrate faster than compounds that favour the stationary phase
What is a eluent?
Mobile phase
What is elution?
The motion of solute and solvent through the stationary phase
What is effluent?
The mobile phase (and solute) collected at thread of the chromatography
What is resolution?
The degree of separation
What did the mobile phase move through the stationary phase via?
Capillary action
What is the distance travelled by the analyte determined by?
It’s relative affinity for the stationary versus the mobile phase
What is the stationary phase?
A plate (glass,plastic,aluminium) coated with adsorbent SiO2
What percentage of weight volume of solvent is the sample dissolved in?
1-2% w/v
What is the most common stationary phase?
Silica gel
Name two other polar stationary phases?
Cellulose and alumina
What do more polar compound absorb more onto?
The stationary phase and less time in the mobile phase
What is the Rf usually quoted as?
Rfx100 value
What is the intensity of a spot on a TLC plate related logarithmically to?
The concentration of the analyte producing it
As polarity increases what happens to the Rf value for each compound in the mixture?
The Rf value increases
How is the sample visualised after separation?
Use if UV light is a non-destructive method
Need to use silica gel plate impregenated with fluorescent material
What wavelength does the sample illuminate at?
254 nm
Name a general purpose developing reagent?
Iodine spray
What does iodine produce?
Brown spots with many organic compounds
What is potassium permanganate used for?
Detection of sugars and sugar- like molecules
What is Ninhydrin solution used for?
Detection of primary and tertiary amines.
Used in the identification of aminoglycoside antibiotics such as gentamycin.
Also used in conjunction with Dragendorff reagent
What % is ethanol/sulfuric acid used in producing fluorescent spots from corticosteroids?
20%
What are applications of TLC?
Limit tests- example such as impurities in hydrocortisone acetate
What are strengths of TLC?
Flexible: mobile and stationary phase, spray reagents.
Robust and cheap
Entire chromatogram can be seen
Used for many drugs impurities
Can be run in batches and can be automated
What are limitations of TLC?
Limited number of theoretical plates available for separation
Limited sensitivity
Unsuitable for volatile compounds
Requires a degree of operator skill
What is chromatography?
Is one of the best methods for separating and measuring the components of a mixture
