Derivatization in GC

SURAJ C. | M.P.A. | February 24, 2014

DERIVATIZATION IN GC A REVIEW

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SURAJ C. AACP PAGE 1

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.

NEED FOR DERIVATIZATION

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.

OUTCOME OF DERIVATIZATION

1. Impart Volatility

The main reason for derivatization is to impart volatility to otherwise nonvolatile

compounds.

The low volatility may result from the size of the molecule and the resultant large

dispersion forces holding the molecule together.

Smaller molecules may have a low volatility due to the strong intermolecular

attractions between polar groups.

In the latter case, masking the polar groups by derivatization can yield dramatic

increases in volatility.

2. Detect Volatility

Derivatization can also be used to decrease volatility to allow analysis of very low

molecular weight compounds, to minimize losses in manipulation and to help separate

sample peaks from solvent peak.

3. Reduction in column absorption

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Polar samples tend to adsorb on the active surfaces of the column walls and the solid

support.

Reduction of this adsorption can be accomplished by derivatization.

4. Improve detectability

In general, the halogenated substituents increase electron affinity in the following

order I > Br> Cl > F (Though they show little increase in volatility), thereby improving

volatility to a certain extent, as in case of use of ECD detectors for halogenated

compounds derived.

5. Accentuate differences among the compounds

Derivatization serves to accentuate the differences in the sample compounds to

facilitate the chromatographic separation.

6. Analysis of non-volatile products

In short, the primary goal is to convert the non-volatile compounds to volatile counter-

parts to improve detection & thereby analysis.

7. Stabilization of compounds for GC

Sometimes, some compounds become unstable due to high temperature or higher

volatile nature of the compound in the analysis.

Thereby, in such case stability hastens and thus leading to change in results.

Thus, derivatization may also be followed to eradicate instability problems and thereby

improved analysis in GC.

REQUIREMENTS OF GC

Volatility

Volatile or eluted out :

Without thermal decomposition

Or molecular rearrangement

Functional groups with active Hydrogen

Derivatization either ↑ or ↓ volatility

Generally derivatization is “aimed at improving” on the following aspects in GC:

I. Suitability

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TO make the analyte suitable enough to be detected well & measured

efficiently without any decomposition in the process of analysis.

II. Efficiency

To produce good peak resolution and symmetry for easy identification and

practicability in GC analysis & reduce Interactions.

III. Detectability

Achieved either by ↑ the bulk of the compound or by introducing onto the

analyte compound, atoms or functional groups that interact strongly with the

detector and hence improve signal identification.

(Ex: halogen add in ECD & TMS ether for identifying the well fragmented peaks).

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.

R1—AH + R2—D → R1 —AD + R2—H

Where,

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

atom “D” = Functional group on the derivatization reagent

DERIVATIZATION REAGENTS

Definition:

May be defined as “a substance that is used to chemically modify a compound

to produce a new compound which has properties that are suitable for analysis in GC

or LC.”

Criteria for selection:

Produce more than 95% derivatives

No structural or molecular alterations

No sample loss

Non – interacting derivatives

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Stable derivatives with time

METHODS OF DERIVATIZATION

*Alkylation *Silylation *Acylation *Chiral Derivatization

ALKYLATION

INTRODUCTION:

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 group

R’ = Alkyl substitution

NEED:

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.

COMMONLY USED:

*BF3(in Methanol) *Methyl 8® *Reagent MethElute™Reagent

*Diazomethane *Pentafluorobenzyl Bromide

1. Boron Trifluoride in Methanol

ADV: 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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Used primarily for esterification procedures with fatty acids;

however, phenolic hydroxyls may be derivatized.

2. Dimethylformamide Dialkylacetals

Used for carboxylic acid esterification. Analytical applications

have been expanded to include alcohols, phenols, steroid

carbonyls, amino acids, primary and secondary amines, and

thiols.

3. Trimethylanilinium Hydroxide (TMPAH):

MethElute™ Reagent – 0.2M TMPAH in Methanol.

Used for on-column methylation of amines, hydroxyls and

carboxyls.

4. Diazomethane:

Most versatile reagent for preparation of methyl esters; fast and quantitative with no

organic byproducts.

Diazomethane and its precursors are toxic and dangerous.

ACYLATION

INTRODUCTION:

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 substitution

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.

BF3•CH3OH

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BENEFITS 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).

COMMONLY USED:

ADV: Hydrolytically stable.

Perfluro deriv. ↑ volatility.

↑sensitivity by added mol.wt.

↑detectability by ECD by added

halogen atoms.

Reacts with alcohols, thiols and

amines

Can 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-sensitive

Reagents are hazardous and

odorous.

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1. Perfluoro Acid Anhydrides:

Produce perfluoroacyl derivatives of alcohols,

thiols and amines.

Derivatives are relatively stable to hydrolysis.

Derivatives are useful for ECD, FID and TCD

detection.

Usually used with basic solvent.

Produce characteristic MS fragmentation.

Are widely used for drug analysis.

Produce acid byproducts which must be

removed before GC analysis.

2. Perfluoroacylimidazoles

Produce perfluoro derivatives of alcohols,

amines and thiols.

Quantitatively acylate indol alkylamines.

Derivatives are relatively stable to hydrolysis.

Derivatize both primary and secondary amines

Produce no acidic byproducts.

Reagents are very reactive with water.

Substance to be derivatized must be dry.

Cannot use in protonated solvents.

3. MBTFA N-Methyl-bis(Trifluoroacetamide):

Forms trifluoroacetyl derivatives of alcohols,

amines and thiols.

Reacts with both primary and secondary

amines.

Reactions with amines generally complete in

30 minutes at room temperature.

Reacts more slowly with alcohols than amines.

Byproduct is stable and volatile.

Excellent for mono-, di- and trisaccharides.

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SILYLATION

INTRODUCTION:

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.

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.

MECHANISM:

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 silylation.

This results to the production of a bimolecular transition state in the intermediate

step of reaction mechanism.

NOTE: Moisture sensitive, thereby should be tightly stored.

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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

COMMONLY USED:

o BSA

o BSTFA Frequently used with TMCS Catalyst

o MSTFA

o HMDS Usually used together for carbohydrates

o TMCS

o TMSI Good for volatile carboxylic acids

o TMSDEA

o MTBSTFA Frequently used with TBDMCS Catalyst

1. BSA:

Strong silyl donor.

– Similar to BSTFA and MSTF

Reacts with all active hydrogen compounds.

– Alcohols, phenols, carboxylic acids, amines, amides, thiols.

Usually requires anhydrous condition.

– TMCS 1% - 10% frequently used as catalyst.

ADV: Wide range of applications.

Variety of reagents available.

Easily prepared.

Excellent thermal stability.

Excellent chromatographic

characteristics.

DISADV: Moisture-sensitive.

TMS & TBD-MCS derivatives

are easily hydrolyzed.

No aqueous solutions.

Must use aprotic org. solvents.

Reacts with column materials.

Silicone residues build up in GC detectors.

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Sometimes reacts quantitatively under mild conditions.

Reaction products often interfere with volatile derivatives.

Silicon fouling of detectors is common.

2. BSTFA:

Strong silyl donor.

– Similar to BSA and MSTFA

Frequently used with TMCS 1 - 10%.

Alone and with TMCS, most commonly used derivatizing agent


– Alcohol, phenols, carboxylic acids, amines, amides, thiols

Usually requires anhydrous condition.

Often reacts quantitatively under mild conditions.

Reaction products more volatile than those from BSA.

Much less detector fouling than with BSA.

3. MSTFA:

Strong silyl donor.

– Similar to BSA and BSTFA – always monovalent

Frequently used with TMCS 1 - 10%.


– Alcohols, phenols, carboxylic acids, amines, amides, thiols

Most volatile reagent and reaction product.

– Used for derivatizing small volatile molecules

Better than BSA in avoiding detector fouling.

Usually requires anhydrous conditions.

4. TMSDEA:

Strongly basic silylating agent.

Very volatile reagent.

Excellent for derivatizing low molecular weight carboxylic acids.

Reaction can be driven to completion by removal of

diethylamine. (B.P. : 55°C)

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Good for preparation of TMS standards.

Relatively weak silyl donor.

5. TMCS & HMDS

Weak silyl donors.

Among the oldest (first) silylating reagents.

Usually used in combination with each other.

Excellent for derivatization of sugars and simple carbohydrates.

Usually combined with pyridine and other solvents.

TMCS can form derivatives of sodium salts of acids and phenols.

6. Ready-to-Use Tri-Sil® Reagents

REAGENT DESCRIPTION Formulations APPLICATION

Tri-Sil® 48999 A reagent-catalyst-solvent mixture for one-step derivatization.

HMDS: TMCS: Pyridine

(3:1:9)

Carbohydrates, phenol, sterol, org. acid, alcohol. (Not recommended for 3-keto steroids)

Tri-Sil® Concentrate 49005

A concentrated reagent - catalyst system.

HMDS: TMCS (3:1)

Same as above, but offers greater latitude in applications.

DERIVATIZATION SOLVENTS

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GC CHIRALD ERIVATIZATION

INRODUCTION

Chiral Chemistry:

Isomers: molecules that have the same molecular formula but a different

arrangement of atoms.

Chiral centers or asymmetric carbons: carbon atoms having four different

groups or atoms attached.

Diastereomers: Stereoisomers that are not mirror images of each other.

Diastereomers may have different chemical and physical properties and can

usually be separated by classical methods.

Enantiomers: Isomers that are mirror images of each other but cannot be superimposed.

Enantiomers have identical chemical and physical properties except for their

ability to rotate the plane of polarized light. Special techniques must be used for

separation and identification

Derivatization 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.

METHODS 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 acid

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SUMMARY

Choice of derivatization technique depends upon:

Available reagent

Sample

Derivatives must be suitable, detectable and efficient for GC analysis.

For acid analytes, the first choice for derivatization is esterification.

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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Derivatization in GC