Derivatisation in gas chromatography

Derivatization in gas chromatography

Presented By: Vidya Dhonde

M.Pharm sem-II

Guided By: Dr. Pratima Tatke

Contents:

Introduction Need of derivatization Types of derivatization Application Conclusion Reference

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 of Derivatization:

To permit analysis of compounds not directly responsible to analysis due to,

inadequate volatility or stability.

Improve chromatographic behaviour or detectability.

Many compounds do not produce a useable chromatograph (i.e. multiple peaks,

or one big blob), or the sample of interest goes undetected. As a result it may

be necessary to derivatize the compound before GC analysis is done.

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.

Types of Derivatization:

Alkylation:

Commonly used to add fluorinated groups (ECD)

Silylation :

readily volitizes the sample. Most prevalent method

Acylation:

Used as the first step to further derivatizations or as a method of protection of certain active hydrogens

GC Chiral Derivatization:

General Reaction

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

Alkylation:

Alkylation reduces molecular polarity by replacing active hydrogens with an alkyl group.

These reagents are used to modify compounds with acidic hydrogens, such as carboxylic acids and phenols.

These reagents make esters, ethers, alkyl amines and alkyl amides. Reagents containing fluorinated benzoyl groups can be used for ECD.

The principal reaction employed for preparation of these derivatives is nucleophilic displacement.

Alkylation is used to modify compounds with acidic hydrogens, such as carboxylic acids and phenols.

Alkylation can be used alone to form esters, ethers and amides- or they can be used in conjunction with acylation or silylation.

It is generally used to convert organic acids into esters. As the acidity of the active hydrogen decreases, the strength of the alkylating reagent must be increased.

The harsher the reaction conditions or reagents, the more limited the selectivity and applicability of this method.

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) for fats & oils.

Alkylation

Advantages

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.

Disadvantages

Limited to amines and acidic

hydroxyls.

Conditions frequently severe.

Reagents often toxic.

Optimization for particular

compounds usually necessary.

Silylation: silylation produces silyl derivatives which are more volatile, less stable, and more

thermally stable.

Silylation occurs through nucleophilic attack (SN2). The better the leaving group, the better the siliylation.

Silylation reagents will react with water and alcohols first. Care must be taken to ensure that both sample and solvents are dry.

Solvents should be as pure as possible. This will eliminate excessive peaks.

Pyridine is the most commonly used solvent. Although pyridine may produce peak tailing, it is an acid scavanger and will drive the reaction forward

In many cases, the need for a solvent is eliminated with silylating reagents.

Ease of reactivity of functional groups towards silylation. Many reagents require heating (not

in excess of 60 degrees C for about 10-15 minutes, to prevent breakdown). Hindered

products may require long term heating.

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 siliylation.

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

intermediate step of reaction mechanism.

Ease of reactivity of functional grps:Alcohol > Phenol > Carboxyl > Amine > Amide /hydroxyl

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

Silylation:

Advantages:

Wide range of applications

Variety of reagents available

Easily prepared

Excellent thermal stability

Excellent chromatographic

characteristics

Disadvantages:

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

Acylation: Acylation reduces the polarity of amino, hydroxyl, and thiol groups and adds

halogenated functionalities for ECD. In comparison to silylating reagents, the acylating reagents target highly polar, multifunctional compounds, such as carbohydrates and amino acids.

Acyl derivatives are formed with acyl anhydrides, acyl halides, and activated acyl amide reagents.

The anhydrides and acyl halides form acid by-products which must be removed before GC analysis.

Activated amide reagents, such as MBTFA, have the advantage of not yielding acid by-products.

Fluorinated acyl groups, going from trifluoracetyl to heptafluorobutyryl , can be used to increase retention times.

Acylation converts these compounds with active hydrogens into esters, thioesters, and amides. They are formed with acyl anhydride, acyl halide, and activated acyl amide reagents.

The anhydrides and acyl halide reagents form acid by-products, which must be removed before GC analysis. Acylations are normally carried out in pyridine, tetrahydrofuran or another solvent capable of accepting the acid by-product

Acylation:

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

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).

Acylation:

Advantages:

Hydrolytically stable.

Perfluro deriv. ↑ volatility.

↑sensitivity by added molecular weight.

↑detectability by ECD by added halogen

atoms.

Reacts with alcohols, thiols and amines

Can be used to activate -COOH for

esterification.

Disadvantages:

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.

APPLICATION: Pharmaceutical :Qualitative and quantitative analysis ,Volatile

compounds ,Complex mixture, Purity

Qualitative analysis: Retention time Relative retention time Retention volume specific retention volume Retention index Retention time plots

Quantitative analysis :

Analysis based on peak height Analysis based on peak area Area measurement by triangulation technique Area measurement by with at half the height

method Area measured by planimetry Area measurement using a boll and disc integrator Area measurement by cut and weigh method

OTHER APPLICATIONS: Food / flavour /fragrance

QC, solvent testing and

fingerprinting of fragrance

Petrochemicals

Natural gas analysis, refineries

mapping of oil reserves

Environmental

Detection of pollution ,water

discharge

References: Derivatization Reactions and Reagents for Gas Chromatography Analysis, Francis Orata,

Masinde Muliro, University of Science and Technology, Kenya pg. 83-99.

K. Szyrwińska1, A. Kołodziejczak1, Derivatization and Gas Chromatography– Low-Resolution Mass Spectrometry of Bisphenol pg. 63-73. Vol-12, 2007.

Dr. Mahajan SS. Instrumental methods of analysis, Fluorescence and Phosphorescence, Popular prakashan pvt ltd, 182-185.

Laurence M. Harwood, Christopher J. Moody Experimental organic chemistry: Principles and Practice (Illustrated ed). WileyBlackwell. pp. 180–185.

Jump up Christian B. Anfinsen, John Tileston Edsall, Frederic Middlebrook Richards Advances in Protein Chemistry;

Thank you!!!!!!