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 synthesized Reagents ; 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 imidazoles Pentafluoro 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. textbook of chemical analysis

Vogels

Practical

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