Question

1.   What is the order of elution of: ethyl acetate (88.11 MM), acetic acid (60.05 g/mol), and dimethylamine (45.08 g/mol), off the following columns? The pKas of these are: 25, 4.75, 10.7, respectively, and you are using a neutral running buffer.

a)   Reverse phase/hydrophobic    __________ then __________then __________

b)   Anion exchange       __________ then __________then __________

c)   Gel exclusion           __________ then __________then __________

Answer 1

From the pKa values acetic acid is more polar and ethyl acetate is least polar.

a) In hydrophobic column polar compounds eluted first and non-polar compounds eluted last.

Reverse phase/hydrophobic acetic acid then dimethyl amine then ethyl acetate

b) Anion exchaneg column is polar column so it has more attraction towards polar compounds and they eluted last and non-polar compounds eluted first.

Anion exchange ethylacetate then dimethyl amine then acetic acid

c)Elution using Gel exclusion depends on size of the molecule, small molecules spend more time in column and they eluted last and larger molecules eluted first.

Gel exclusion ethyl acetate then acetic acid then dimethyl amine.