Derivatization of GCSuraj C.Suraj C.AACPAACP

PPT. Package 2

Overview

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Overview

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Introduction

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Introduction• 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.NOTE: A modified analyte in this case will be the

product, which is known as the derivative. NOTE: The derivative may have “similar or closely

related” structure, but not the same as the original non-modified chemical compound. 6

Why ?

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Why ? To permit analysis of compounds not directly

amenable to analysis due to, for example, inadequate volatility or stability

Improve chromatographic behaviour or detectability

NOTE: Derivatization is a useful tool allowing the use of GC and GC/MS to be done on samples that would otherwise not be possible in various areas of chemistry such as medical, forensic, and environmental

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Outcome/Accomplish

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Why ? Impart Volatility

Detect Volatility

Reduction in column absorption

Improve detectability

Accentuate differences among the

compounds

Analysis of non-volatile products

Stabilization of compounds for GC

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Points to beNOTED

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Points to be NOTED• Volatility

• Volatile or eluted out :

Without thermal decomposition

Or molecular rearrangement

• Functional groups with active Hydrogen

• Derivatization either ↑ or ↓ volatility12

Points to be NOTED• Generally derivatization is aimed at improving on

the following aspects in GC:

i. Suitability

ii. Efficiency

iii.Detectability

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General Reaction

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General Reaction• The most commonly used derivatization

procedures involve the “substitution of active

hydrogens” on the compound to be derivatized

with a variety of functional groups.

• These functional groups impart the desired

characteristics to the compound, while

eliminating the adverse effects. 15

General ReactionR1—AH + R2—D → R1 —AD + R2—H

Where,

atom “A” = Oxygen, Sulfur, Nitrogen or similar

atoms

atom “D” = Functional group on the derivatization

reagent16

Derivatization

Reagents

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Derivatization Reagents• Definition

• Criteria for selection:

Produce more than 95% derivatives

No structural or molecular alterations

No sample loss

Non – interacting derivatives

Stable derivatives with time 18

Methods

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Types

Alkylation

Silylation

Acylation

Chiral Derivatization

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Alkylation

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AlkylationINTRODUCTION:

•Represents the replacement of active hydrogen by

an aliphatic or aliphatic-aromatic (e.g., benzyl) group

in process referred to as “ESTERIFICATION”.

RCOOH + PhCH2X → RCOOCH2Ph + HX

Where, X = Halogen groupR’ = Alkyl substitution

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AlkylationNEED:

Conversion “organic acids into esters”, especially methyl esters that produce of better chromatograms than the free acids.To prepare ethers, thioethers and thioesters, N-alkylamines, amides and sulphonamides.Alkyl esters formed offer “excellent stability” and can be isolated and stored for extended periods if necessary.NOTE: Use of inorganic acids (HCl/SCl) for fats & oils.

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REAGENTS

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AlkylationADV:

Wide range of reagents avail.Reaction condition can vary from strongly acidic to strongly basic.Some reactions can be done in aqueous systems.Derivatives are generally stable.

DISADV:Limited to amines and acidic hydroxyls.Conditions frequently severe.Reagents often toxic.Optimization for particular compounds usually necessary.

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Acylation

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AcylationINTRODUCTION:

•An acyl group is introduced to an organic compound.

•In the case of a carboxylic acid, the reaction

involves the introduction of the acyl group and the

loss of the hydroxyl group.

CH3OCOCOCH3 + HOR → CH3OCOR´ + HOCOCH3

Where, R = alkyl grp

R’ = another alkyl substitution27

Acylation

NEED:

•Compounds that contain active hydrogens (e.g., -

OH, -SH and -NH) can be converted into esters,

thioesters and amides, respectively, through

acylation.

•Highly polar and volatile derivatives

•Stability from the thermal decomposition 28

AcylationBenefits of Acylation:

•Improve analyte stability by protecting unstable groups.•Provides volatility on substances such as carbohydrates or amino acids, which have many polar groups that they are non-volatile and normally decompose on heating.•Assists in chromatographic separations which might not be possible with compounds that are not suitable for GC analysis.•Compounds are detectable at very low levels with an electron capture detector (ECD). 29

REAGENTS

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AcylationADV:

Hydrolytically stable.Perfluro deriv. ↑ volatility.↑sensitivity by added mol.wt.↑detectability by ECD by added halogen atoms.Reacts with alcohols, thiols and aminesCan be used to activate -COOH for esterification.

DISADV:Derivatives are frequently difficult to prepare.Reaction products often must be removed before analysis.Reaction must be done in non-aqueous system.Reagent are moisture-sensitiveReagents are hazardous and odorous. 31

Silylation

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SilylationINTRODUCTION:

•Introduction of a “silyl group” into a molecule, usually

in substitution for active hydrogen such as dimethylsilyl

[SiH(CH3)2], t-butyldimethylsilyl [Si(CH3)2C(CH3)3] and

chloro-methyl-dimethylsilyl [SiCH2Cl(CH3)2].

•Replacement of “active hydrogen” by a silyl group

reduces the polarity of the compound and reduces

hydrogen bonding. 33

SilylationINTRODUCTION………..Contd..:

•Many hydroxyl and amino compounds regarded

as non-volatile or unstable at 200 – 300 °C have

been successfully analyzed in GC after silylation.

•The silylated derivatives are more volatile and

more stable and thus yielding narrow and

symmetrical peaks (Kataoka, 2005).34

SilylationMECHANISM:

•Replacement of the active hydrogen (in -OH, -COOH, -NH, -NH2, and –SH groups) with a trimethylsilyl group. •Silylation then occurs through nucleophilic attack (SN2), where the better the leaving group, the better the siliylation. •This results to the production of a bimolecular transition state in the intermediate step of reaction mechanism. 35

SilylationMECHANISM ………. Contd….:

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SilylationMECHANISM ………. Contd….:

NOTE: Moisture sensitive, thereby should be tightly stored.NOTE: Solvents used should be as pure and as little as possible as it will eliminate excessive peaks and prevent a large solvent peak.NOTE: Ease of reactivity of functional grps:

Alcohol > Phenol > Carboxyl > Amine > Amide /hydroxyl

NOTE: For alcohols, the order will be as follows:Primary > Secondary > Tertiary 37

REAGENTS

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SilylationADV:

Wide range of applicationsVariety of reagents availableEasily preparedExcellent thermal stabilityExcellent chromatographic characteristics

DISADV:Moisture-sensitiveTMS & TBD-MCS derivatives are easily hydrolyzedNo aqueous solutions.Must use aprotic org. solventsReacts with column materialsSilicone residues build up in GC detectors

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Derivatization

Solvents

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Derivatization Solvents• Definition

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GC Chiral Derivatizatio

n

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SilylationINTRODUCTION

•Involves reaction of an enatiomeric molecule with an enantiomerically pure Chiral Derivatizing Agent (CDA) to form two “diastereomeric” derivatives that can be separated in this case using GC.•Any molecule having asymmetric carbon is called as “CHIRAL” molecule. NOTE: Chirality of analyte molecules requires special consideration in their analysis and separation techniques. 43

SilylationMETHODS OF SEPARATION

Separation on an optically active stationary phase.

Preparation of diastereomeric derivatives that can be

separated on a non chiral stationary phase.

REAGENTS

TPC :- N-trifluoroacetyl-L-prolyl chloride

ITPC :- (S)-(–)-N-(Trifluoroacetyl)-prolylchloride

MTPA :- (–)-α-Methoxy-rifluoromethyl-phenylacetic acid44

Summary

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Summary

INTRODUCTION

•Choice of derivatization technique depends upon:Available reagentSample•Derivatives must be suitable, detectable and efficient for GC analysis.•For acid analytes, the first choice for derivatization is esterification.

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Summary

INTRODUCTION

•Nearly all functional groups which present a problem in gas chromatographic separation can be derivatized by silylation reagents.•Chiral GC complex due to different reaction rates, but could be reduced by proper selection of reagents.

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