Module 1

Question 1

What is Chromatography?

Answer 1

A physical method of separation in which the components to
be separated are distributed between two phases, one of which
is stationary (stationary phase) while the other (the mobile
phase) moves in a definite direction

Question 2

The stationary phase may be

Answer 2

solid, liquid, liquid supported on a solid

Question 3

The stationary phase may be packed in

Answer 3

column,
spread as a layer, or distributed as a film

Question 4

the mobile phase may be

Answer 4

gaseous or liquid

Question 5

Dead time (tM)

Answer 5

time for mobile phase to pass through the
column

Question 6

elution process

Answer 6

The elution process involves introducing a sample into a column where a mobile phase washes the solutes down. Solutes distribute between the mobile phase and the stationary phase, achieving equilibrium. Separation occurs based on retention, where solutes with stronger interactions with the stationary phase elute later, while weaker interactions result in earlier elution

Question 7

Adsorption separation mechanism

Answer 7

Solid SP + Liquid or gaseous MP, Solute molecules get adsorbed on the surface of the SP

Question 8

Partition mechanism of separation

Answer 8

SP = liquid bonded/adsorbed to a solid surface, MP = Liquid, gas, or supercritical fluid, Partitioning of a solute between MP and SP stationary on a solid support according to distribution coeff K

Question 9

Ion exchange chromatography separation mechanism

Answer 9

SP = solid resin to which anions such as SO3- or cations such as N(CH3)3+ are covalently attached; MP = liquid, Solute ions of opposite charge are attracted to the SP

Question 10

Size exclusion chromatography (gel permeation; gel filtration)

Answer 10

SP = porous gel; MP = liquid or gas, Gel pores are small; large molecules excluded; small ones
pass through

Question 11

Affinity chromatography separation method

Answer 11

SP = Group-specific liquid covalently bonded to solid; MP = liquid
Solute has affinity for bonded stationary phase, ex: Antibody-antigen interactions

Question 12

Adjusted retention time

Answer 12

tR’ = tR – tM

Question 13

Retention factor

Answer 13

small k, k= (tR-tM)/tM, + retained on the column= the larger retention factor

Question 14

selectivity

Answer 14

alpha= tR’ (B)/tR’(A)= k2/k1, greater α means better separation, also equals to large K2/K1

Question 15

The distribution coefficient

Answer 15

big K, distribution of analyte between sp and mp, K=Cs/Cm, Cs is conc in sp

Question 16

Retention factor and distribution coefficient

Answer 16

k=K*(Vs/Vm)

Question 17

To improve resolution

Answer 17

increase ΔtR
Design a separation that produces narrower peaks (smaller w)