chromatography intro Flashcards
why is separation science so important?
to see Does a product contain the correct amount of the desired compound / acceptable levels of impurities
to measure the amount of a certain product what must happen first?
to measure the levels of these components they must first be separated!
what is a limitation of separation science?
Related impurities with same FG or λmax
are ionised or un-ionised drugs separable in polar solvents?
ionised
how can separation in mixtures be achieved?
exploitation of physiochemical properties
what does chromatography separate mixtures based on
their interaction with stationary and mobile phases
what is TLC?
thin layer chromatography-allows us to quickly separate and visualise components of a mixture with simple equipment
what is TLC made from?
usually made from a thin strip of aluminium coated with a solid stationary phase such as silica
how does the mobile phase move in TLC?
by capillary action
how are results viewed in TLC?
Separated components are viewed under UV or with a chemical stain.
what does the mobile phase usually consist of?
typically consists of a mixture of non-polar(hydrophobic) and slightly polar solvents such as dichloromethane, petrol ether, ethyl acetate and methanol.
what does the stationary phase usually consist of?
Silica is commonly used as the stationary phase. The silanol groups make the stationary phase polar(hydrophilic).
do polar compounds travel far?
Silica is commonly used as the stationary phase. The silanol groups make the stationary phase polar(hydrophilic).
how do you optimise TLC separation?
different amounts of mobile and stationary phase
what is Ninhydrin?
is used to show amino acids, and develops pink or brown spots when heated.
what do Phosphomolybdic acid and potassium permanganate do?
are strong oxidising agent which result in yellow / brown spots. They react with alcohols, amines, sulphides and other groups which are sensitive to oxidation.
what does Bromocresol green do?
stains compounds which are acidic such as carboxylic acids (pKa <5).
what does Iodine vapour do?
has a high affinity for unsaturated and aromatic compounds. Brown spots develop on the plate after a short time
what does 2,4-Dinitrophenylhydrazine do?
(Brady’s Reagent) generates orange spots with aldehydes.
is TLC a quantatitive technique?
no
how does HPLC address the major limitations of TLC?
The stationary phase is now packed into a stainless -steel tube, and the particle size, pore size and size distribution carefully regulated.
Increased speed – solvent is pumped (up to 350 bar)
•Increased surface area
•Resolution (separating ability) is greatly improved.
•Compounds leaving the column (eluting) can be measured quantitatively (e.g. UV detector).
how does HPLC work?
injected in
passed through detector
if interact with mobile phase- move quickly and hit detector first
if interact with stationary phase more will be slower and hit detector last
what are the two ways of UV detection?
Variable wavelength detector
Diode array detector
how does RPLC occur?
By alkylating the silica, CHn chains are bonded to the silica particles creating a non-polar environment.
By modifying the composition of the mobile phase what can we do?
increase/decrease retention time
what effect do ionised molecules have on the stationary phase?
they interact weakly
