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Derivatization in GC & HPLC


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DERIVATIZATION OF HPLC AND GC Ms.Smita P.Shelke , Assistant Professor Gokhale Education Society’s Sir Dr M.S. Gosavi College Of Pharmaceutical Education & Research Prin.T.A.Kulkarni Vidyanagar , Nashik-422005 India (Maharashtra). Ph No. 0253 2232799 .


DERIVATIZATION :- Derivatization is the process of chemically modifying a compound to produce a new compound which has properties that are suitable for analysis using a GC and HPLC The chemical structure of the compound remains the same and just modifies the specific functional group of reacting compounds to derivative of deviating chemical and physical properties in order to make them detectable and analyzable Derivatization is needed in GC, HPLC , UV- Visible Spectroscopy etc.


WHAT DERIVATIZATION ACCOMPLISH ? Increase volatility eliminates the presence of polar OH, NH & SH groups Derivatization targets O, S, N & P functional groups . Increases detectability. Increases stability. To reduce adsorption of polar samples on active surfaces of column walls and solid support.


TYPES OF DERIVATIZATION : - Silylation. Alkylation. Acylation. Chiral derivatization.


1. SILYLATION :- Most prevalent method, readily volatizes the sample. Mechanism: This process produces silyl derivatives which are more volatile, more thermally stable. Replaces active hydrogen with Tri methyl Silyl Groups. Silylation occurs the nucleophilic attack(SN2). The better the leaving group, the better the silylation. Solvents: (functional groups) alcohol > Phenol > Carboxyl > Amine > Amide > Hydroxyl Advantages : - Ability to silylate a wide variety of compounds. Large number of silylating reagent available. Easily prepared. Disadvantages :- Silylation reagent are moisture sensitive. Must use aprotic organic solvents.


2. ALKYLATION : - Alkylation reduces molecular polarity by replacing active hydrogen with an alkyl group . These reagent are used to modify compounds with acidic hydrogen . Such as carboxylic acidic and phenols . These reagents makes esters, ethers, alkyl amines and alkyl amides. The principle reaction employed for preparation of these derivatives is nucleophilic displacement. Advantages :- Wide range of alkylation reagents available. Reaction condition may vary from strongly acidic to strongly basic. Some reaction can be done in aqueous solutions. Alkylation derivatives are generally stable. Disadvantage :- Limited to amines and acidic hydroxyls. Reaction conditions are frequently severe. Reagent are often toxic.

3. ACYLATION : - :

3. ACYLATION : - Acylation reduces the polarity of amino, hydroxyl, and thiol groups. In comparison to silylation reagent, the acylating reagents target highly polar, multifunctional compounds, such as carbohydrates and amino acids. Advantages : - Addition of halogenated carbons increased detectability by ECD. Derivatives are hydrolytically stable. Increases sensitivity by adding molecular weight. Acylation can be used as a first step to activate carboxylic acids prior to esterification Disadvantages :- Acylation derivatives can be difficult to prepare. Reaction product (acid by–product) often need to be removed before analysis. Acylation reagent are moisture sensitive. Reagents are hazardous and odorous.

4. Chiral derivatization : -:

4. Chiral derivatization : - These reagents target one specific functional group and produce individual diasteriomers of each of the enantiomers. There are two ways to separating enantiomers by chromatography: Separation on an optically active stationary phase. Preparation of diastereomeric derivatives that can be separated on a non stationary phase. REAGENTS : - TPC (N-trifluroacetyl-L-prolyl chloride) Used for optically active amines, most notable amphetamines. MCF [(-)methylchloroformate] Used for optically active alcohols. - If an optically pure reagent is used to prepare diasteriomeric derivatives, then only two derivatives are formed. The enantiomeric ratio is reflected in the relative peak size.


SPICIAL TYPES OF HPLC DERIVATIZATION : - For UV-Vis spectrophotometric detection. For flourimetric detection. For chiral analysis. According to when and where the derivatization is done Pre-column derivatization. Post-column derivatization.


PRE-COLUMN DERIVATIZATION :- Performed before the analytical separation is attained. Sample is derivatiszed manually or automatically and injected into the HPLC column. Separation of components occurs after derivatization Advantages :- Fewer equipment and reaction chemical restriction. Can be performed manually or automatically. No time restrictions on the kinetics of derivatization of reactions . Disadvantages :- Introduction of contaminants. Loss of analyte through adsorption. Sample degradation and incomplete reaction. Poorer precision due to increased complexity.


POST-COLUMN DERIVATIZATION :- Performed after analytical separation of compound but prior to detection. Addition pump is used for addition of derivatizing agent to the elute sample from column. Advantages :- Minimal artifact formation. Complete reaction is not essential as long as it is responsible and the chromatography of analyte remains unaffected. Disadvantages :- Band broadening. Added complexity for method development and routine use.


REFERANCES :- Mrs. Laurence Coppex , “ Derivatives for HPLC Analysis ” , Faculty of Chemistry and Pharmacy University of Genf. November 1999 - February 2000 Francis Orata , “ Derivatization Reactions and Reagents for Gas Chromatography Analysis”, Masinde Muliro University of Science and Technology, Kenya. Tom Kupiec , “ Quality-Control Analytical Methods: High-Performance Liquid Chromatography”, Analytical Research Laboratories Oklahoma City, Oklahoma

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