Chiral Chromatography

Question 1

Importance of chirality in analytical toxicology?

Answer 1

> 50% of pharmaceuticals are chiral compounds and many of those are marketed as racemates consisting of an equimolar mixture of two enantiomers

Question 2

Properties of enantiomers?

Answer 2

Enantiomers of the same chiral molecule have similar physicochemical properties but may differ in their biological properties

Question 3

Metabolism of enantiomers?

Answer 3

Distribution, metabolism and excretion usually favour one enantiomer over the other, due to different biological activity chiral molecules can differ in toxicity

Question 4

Development of three point interaction model?

Answer 4

Development of chiral stationary material to separate enantiomers, understand metabolite and toxicity pathway of molecule, in the chiral stationary phase there are 3 active sites and hence 3 points of functionality - to facilitate chiral chromatography needs 3 types of interaction to get 3 different enantiomers

Question 5

Enantiomers being separated by three point interaction model?

Answer 5

a chiral molecule with an asymmetric carbon atom can present three groups that can match exactly three sites of the selector, if the enantiomer doesn’t fit nicely into the stationary material it won’t interact as well and so will be eluted first, however if it fits nicely into the stationary material it will interactions better and will be eluted later, however the other enantiomer (its mirror image) can present only two groups able to interact with only two sites of the selector - hence binding constant in enantiomer 1 will be higher than that of its mirror image (this is what allows enantiomers to be separated since they will interact differently)

Question 6

Example of a chiral stationary phase?

Answer 6

Macrolytic antibiotics eg vancomycin V (this is the chiral selector this can be incorporated into silica, silica is the base but incorporates this to facilitate chiral separation)

Question 7

HPLC mode of macrolytic antibiotic stationary phase?

Answer 7

Both normal phase and reversed phase HPLC

Question 8

Interactions used in macrolytic antibiotic stationary phase?

Answer 8

Hydrogen bonding, inclusion complexes, ionic, dipole and pi-pi interactions

Question 9

Size exclusion in macrolytic antibiotic stationary phase?

Answer 9

Have cavities only molecules of particular size and shape will be able to fit interacting with certain functionality, ionic and non polar interaction, need to fit to be retained in column will have longer retention time

Question 10

Cavity stationary phases?

Answer 10

Cyclodextrins (glucose units can choose larger quantity if want bigger cavity, so structure changes depending on the size of the molecule and interactions wanted), and crown ethers (functionality can be added, can interact with 2 or 3 functionalities, modify structure to get what we want)

Question 11

HPLC mode of the cavity stationary phases?

Answer 11

Reversed phase HPLC

Question 12

Interactions of the cavity stationary phases?

Answer 12

Size exclusion (whether molecule will fit or not) and hydrogen bonding

Question 13

Protein phases as stationary phase?

Answer 13

Eg albumins and ovomucoids bonded to silica (enzymes large biomolecules) eg cellobiohydrolase CBH

Question 14

What does CBH show selectivity to?

Answer 14

Shows particular selectivity to primary and secondary amines, good material to use to separate enantiomers of amphetamine

Question 15

Interactions of protein phases?

Answer 15

Polar and hydrophobic

Question 16

Separation of protein phases?

Answer 16

Wide variety of compounds

Question 17

Amphetamine as enantiomers?

Answer 17

Amphetamine exists as a pair of enantiomers which differ in pharmacological activity and metabolism,
S(+)-amphetamine higher stimulant activity, metabolises faster in humans, R(-)-amphetamine lower stimulant activity, dominant in urine

Question 18

Stimulant activity of enantiomers?

Answer 18

S(+)-amphetamine has twice as high stimulant activity than R(-)-amphetamine

Question 19

How is amphetamine most commonly synthesised?

Answer 19

Amphetamine is the most commonly synthesised as a racemic amphetamine, both S(+) and S(+)/R(-) amphetamine are prescription medicines

Question 20

How is amphetamine excreted?

Answer 20

Amphetamine can be exerted as a result of metabolism of methamphetamine and certain prescription drugs eg R(-) amphetamine is excreted as a result of administration of selegiline, S(+) amphetamine metabolises faster than R(-) enantiomer (excrete with R since S gets metabolised quicker and binds better to sites) If use chiral chromatography can prove illegal used, if use normal would just get one peak cannot tell the enantiomers so can’t prove amphetamine use

Question 21

Enantiomers of MDMA?

Answer 21

S(+)-MDMA more amphetamine like stimulant, metabolises faster in humans, R(-)-MDMA more hallucinogenic, dominant in urine

Question 22

Facts about MDMA?

Answer 22

No medical use, produced as racemate, metabolism favours S(+) enantiomer, preferential metabolism of s(+)-MDMA leads to enrichment of MDMA with R(-) enantiomer and formation of S(+) MDA (twice as much S(+) MDA is excreted in urine as compared to R(-) MDA

Question 23

Facts about MDA?

Answer 23

No medical use, produced as racemate, metabolism favours S(+) enantiomers, preferential metabolism of S(+)-MDA leads to enrichment of MDA with R(-) enantiomer

Question 24

How to know if MDMA or MDA have been ingested?

Answer 24

MDA metabolite of MDMA enriched with R must be consumed, if with S it has been metabolised from MDMA, when consuming racemate if see MDMA in urine with enantiomer it has bee abused and enriched with R