chromatography intro

Question 1

why is separation science so important?

Answer 1

to see Does a product contain the correct amount of the desired compound / acceptable levels of impurities

Question 2

to measure the amount of a certain product what must happen first?

Answer 2

to measure the levels of these components they must first be separated!

Question 3

what is a limitation of separation science?

Answer 3

Related impurities with same FG or λmax

Question 4

are ionised or un-ionised drugs separable in polar solvents?

Answer 4

ionised

Question 5

how can separation in mixtures be achieved?

Answer 5

exploitation of physiochemical properties

Question 6

what does chromatography separate mixtures based on

Answer 6

their interaction with stationary and mobile phases

Question 7

what is TLC?

Answer 7

thin layer chromatography-allows us to quickly separate and visualise components of a mixture with simple equipment

Question 8

what is TLC made from?

Answer 8

usually made from a thin strip of aluminium coated with a solid stationary phase such as silica

Question 9

how does the mobile phase move in TLC?

Answer 9

by capillary action

Question 10

how are results viewed in TLC?

Answer 10

Separated components are viewed under UV or with a chemical stain.

Question 11

what does the mobile phase usually consist of?

Answer 11

typically consists of a mixture of non-polar(hydrophobic) and slightly polar solvents such as dichloromethane, petrol ether, ethyl acetate and methanol.

Question 12

what does the stationary phase usually consist of?

Answer 12

Silica is commonly used as the stationary phase. The silanol groups make the stationary phase polar(hydrophilic).

Question 13

do polar compounds travel far?

Answer 13

Silica is commonly used as the stationary phase. The silanol groups make the stationary phase polar(hydrophilic).

Question 14

how do you optimise TLC separation?

Answer 14

different amounts of mobile and stationary phase

Question 15

what is Ninhydrin?

Answer 15

is used to show amino acids, and develops pink or brown spots when heated.

Question 16

what do Phosphomolybdic acid and potassium permanganate do?

Answer 16

are strong oxidising agent which result in yellow / brown spots. They react with alcohols, amines, sulphides and other groups which are sensitive to oxidation.

Question 17

what does Bromocresol green do?

Answer 17

stains compounds which are acidic such as carboxylic acids (pKa <5).

Question 18

what does Iodine vapour do?

Answer 18

has a high affinity for unsaturated and aromatic compounds. Brown spots develop on the plate after a short time

Question 19

what does 2,4-Dinitrophenylhydrazine do?

Answer 19

(Brady’s Reagent) generates orange spots with aldehydes.

Question 20

is TLC a quantatitive technique?

Answer 20

no

Question 21

how does HPLC address the major limitations of TLC?

Answer 21

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).

Question 22

how does HPLC work?

Answer 22

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

Question 23

what are the two ways of UV detection?

Answer 23

Variable wavelength detector

Diode array detector

Question 24

how does RPLC occur?

Answer 24

By alkylating the silica, CHn chains are bonded to the silica particles creating a non-polar environment.

Question 25

By modifying the composition of the mobile phase what can we do?

Answer 25

increase/decrease retention time

Question 26

what effect do ionised molecules have on the stationary phase?

Answer 26

they interact weakly