DERIVATIZATION IN GAS CHROMATOGRAPHY

Presented by

Sowmya k balan

TOPICS TO BE DISCUSSED

• DEFINITION • CRITERIA FOR THE COMPOUNDS • REASONS FOR DERIVATIZATION• TYPES OF DERIVATIZATION• DERIVATIZATION REACTIONS

DEFINITION

• Derivatization is the process of chemically

modifying a compound to produce a new

compound which has properties that are

suitable for analysis using gas

chromatography.

CRITERIA / PROPERTIES OF SAMPLE

• VOLATILITY

• POLARITY

• THERMOSTABILITY

The ideal derivatization procedure will:

Accomplish the desired modification.

Proceed quantitatively, or at least reproducibly.

Produce products that are readily distinguishable and separable from the starting materials.

Proceed rapidly with simple and straightforward laboratory techniques, and will be both selective and applicable to a number of similar compounds.

Involve reagents and reactions that present no unusual hazards.

Compounds containing functional groups with active hydrogen (-

COOH, -OH, -NH and -SH) are usually derivatized for analysis by

gas chromatography.

REASONS FOR DERIVATIZATION

• Improve peak shape• Suitable volatility • Better sensitivity and detectability • Increased thermostability • Relocalization of interfering peaks

Types of derivatization

• Pre column derivatization ; For improve

separation by column

• Post column derivatization ; For improve

response by detector.

Precolumn derivatisation:

• Here the compound is converted into more volatile and thermostable derivatives.

• Improve the separation and less tailing will be seen.

In the following condition, pre derivatization is done:

• The compound is less volatile• The compounds are thermolabile

• To reduce tailing• To improve separation factor

Post column derivatization:

• Improve the response shown by detector

• The compound may be converted in such a way that their ionisation or affinity towards electrons is increased.

Derivatization reactions:

• Esterification• Acylation• Perfluoroacylation• Alkylation • Silylation• Condensation• Chiral derivatization

ESTERIFICATION:

• Use to prepare derivatives of carboxylic acids.

• Eg . Analgesics , Prostaglandins , amiono acids etc. • Derivatization – Fischer Esterification Method under

acidic conditions

R-COOH + R’- OH R-COOR’

Methyl ester frequently synthesizedReagents ; diazomethane ,diazoethane

ACYLATION:Acylation Reactions

Popular reaction for the production of volatile derivatives of highly polar and involatile organic materials.

Advantages.:

• Reduces the polarity of the substance

• Improve the peak shape and reduce peak tailing.

• Improves the stability of those compounds that are thermally labile.

• Disadvantage• Acylation derivatives difficult to prepare• Reagents are moisture sensitive and hazardous• Acid byproducts need to be removed before analysis

Applications

• Render extremely polar materials such as sugars amenable to separation by GC

• Used to derivatize amines, amides, alcohols, thiols, phenols, enols, and glycols etc…….

ACYLATING REAGENTS

Fluorinated anhydrides fluoro acyl imidazolesPentafluoro propanolol

PERFLUOROACYLATION:

• Increases the molecular weight of the sample

relative to the analogous hydrocarbon.

• Increases the retention time.

eg; Direct acylation of the trifluoroacetic anhydride in

trifluoroacetic acid followed by methylation with

diazomethane in methanol.

ALKYLATION

• Used to derivatize functional groups such as alcohols,

phenols, primary and secondary amines, imides etc in which

there is labile hydrogen

• Reduces molecular polarity by replacing active hydrogen with

alkyl group

• Principal reaction ; nucleophillic displacement

.eg. Williamson ether synthesis….. Alcohol/phenol +

alkyl/benzyl halide in presence of base.

 

• Alkylating reagents

• Dialkyl acetals ( DMF)

• Tetrabutyl ammonium hydroxide(TBH)

• Pentafluoro benzyl bromide

• Advantage

• Wide range of reagents available

• Reactions done in aqueous solutions

• Derivatives formed are stable

• Disadvantage• Limited to amines and acidic hydroxyls• Reaction conditions are severe• Reagents ate toxic

Silylation • Most prevalent method• Produces silyl derivatives which are more volatile

and thermally stable• Occurs through nucleophillic attack• Used for the analysis of the non volatile polar

compounds.• Order of functional group by silyl acceptor ability is

alcohols > Phenols > carboxylic acids > amines > amides

• Reagents• Hexamethyl disilane (HMDS)• Tri methyl clorosilane(TMCS• Advantage• Easily prepared• Large number of reagents available• Disadvantage• Reagents are moisture sensitive• Must use aprotic organic solvents( pyridine)

• Sample or solution of sample is heated with slight

excess of silylating agent. Reaction must be carried

out in the sealed vial with inert closure. Solvent used

must be free from active hydrogen. Popular solvent

like hexane, ether, benzene will dissolve the reagent

but are not conductive to rapid reactions

Condensation

• Usually done for aldehydes or ketones present in the

sample.

• Commonly used reagent is methoxylamine and is

used to protect keto groups in steroids by forming

methoximes .

GC CHIRAL DERIVATIZATION

• They target one specific functional group and produce individual diasteromers

• Enantiopure derivatization reagents• menthyl chloroformate(MCF)

• N trifluro acetyl L propyl chloride (TPC)

REFERENCES

Practical pharmaceutical chPractical pharmaceutical chPrinciples of instrumental analysis,5th edition,Skoog,Holler.

Vogel’s textbook of chemical analysis

Practical pharmaceutical chemistry,A.H.Beckett,J.B.Stenlake