6. General comments regarding the main types of chemical reactions used in derivatization

Derivatizations as chemical reactions can be classified as follows: (1) reactions with formation of alkyl or aryl derivatives, (2) silylation reactions, (3) reactions with formation of acyl derivatives, (4) reactions of addition to carbon-hetero multiple bonds, (5) reactions with formation of cyclic compounds, and (6) other reactions specific to a certain analysis. The selection of the derivatization reaction is typically done based on the desired property to be brought to the analyte and its possible reactivity. For this reason, the reagent is selected to have moieties that add the desired property to the analyte and also to have the capability to react with the specific functional group of the analyte. The matrix of the sample also has a role in


acids, specific cryptands such as crown ethers can be used to solvate the alkali metal portion of an organic acid salts, allowing the anion to be freer and

desired product.

reaction as follows:

follows:

21

methylsulfinyl-methanide anion.

Derivatization Methods in GC and GC/MS DOI: http://dx.doi.org/10.5772/intechopen.81954

that can be used in this reaction is Hg(CN)2.

ation using diazomethane is assumed to take place as follows:

increasing the rate of nucleophilic substitution. One other approach for enhancing the alkylation efficiency is the use of phase transfer alkylation. This approach is based on the formation of a compound easily extractable in an organic phase and on the displacement of the equilibrium in the direction of the formation of the

One different way of enhancing the alkylation efficiency is the use of different alkylating reagents besides short-chain alkyl bromides or iodides. One example of a halide that is particularly reactive is pentafluorobenzyl bromide. This reagent can be used for the derivatization of a variety of compounds containing active hydrogens. Another reactive halide is 2-bromoacetophenone (phenacyl bromide). This reagent is used mainly for the alkylation of compounds containing more acidic hydrogens such as carboxylic acids. Another example of methylation using a special reagent R-X is applied on carbohydrates [15]. This methylation uses methylsulfinylmethanide anion. The reagent is prepared from dry DMSO and NaH or KH in a

–

Na<sup>þ</sup> þ H2 (8)

ð9Þ

ð10Þ

ð11Þ

ð Þ CH3 <sup>2</sup>SO þ NaH ! CH3 � SOCH2

Alkylfluoromethyl-sulfonates are even more reactive than sulfates, and the reaction may take place with the active hydrogen even from alcohols or amines as

A polyol or a monosaccharide dissolved in DMSO is easily methylated with

Other alkylating reagents are known (different X in R-X), also reacting in a nucleophilic substitution. For example, dimethyl sulfate can be used for alkylations.

Even tertiary amines, such as pyridine, also react with this type of reagent forming quaternary ammonium salts. The alkylation with alkylfluorosulfonates can be catalyzed as other alkylation reactions for increasing the reaction rate. A catalyst

Diazomethane is another common alkylating (methylating) reagent. The alkyl-

Diazomethane is a gaseous unstable substance, which cannot be stored for long periods of time. It is usually prepared in small quantities and used immediately with or without an intermediate step of dissolution in ether. The preparation can be done from different N-nitroso-N-alkyl compounds in a reaction with a base. A common preparation uses N-nitroso-N-alkyl-p-toluenesulfonamide (Diazald). Methylation with diazomethane may require addition of a Lewis acid catalyst such as BF3. The

Table 1.

Derivatization preferences for compounds containing active hydrogens.

the choice of a specific derivatization procedure. Initial matrix of the sample is not always suitable for derivatization, and in some cases preliminary sample preparation is necessary to change this matrix. The change can be as simple as drying the initial sample but can also be rather complex [14]. Table 1 gives a simplified view of preferences for the choice of a derivatization reagent for compounds containing active hydrogens [14].

Besides functionalities with active hydrogens, other functionalities can also be derivatized. Compounds containing carbonyls can be derivatized, for example, using condensation reactions. Some analytes may contain multiple functional groups such as the amino acids. Specific derivatization reactions can be selected for such cases.

## 7. Reactions with formation of alkyl or aryl derivatives

The formation of alkyl or aryl derivatives is applied to replace the active hydrogens from an analyte with an alkyl (R) or aryl (Ar) group. The replacement can be done in functionalities such as OH, COOH, SH, NH, or CONH. For example, the derivatization with short-chain alkyl bromides or iodides has numerous analytical applications for compounds such as steroids, amino acids, catecholamines, sulfonamides, phenols, barbiturates, organic acids, and mono- and oligosaccharides. A large number of reagents R-X are known, and in a simplified approach, it can be considered that R is carrying a specific property and X a specific reactivity, although the reactivity of a reagent is influenced by both R and X components of the molecule. The type of moiety R and that of reactive group X are guiding the selection process of selecting a reagent for a specific derivatization.

In most alkylation reactions, the analyte acts as a nucleophile (Y:, Y:H, Y:-) reacting in a substitution (SN) with the alkylating reagent R-X, which contains a leaving group X and an alkyl group R:

$$\text{Y:}\\\text{H} + \text{R}-\text{X} \rightarrow \text{Y}-\text{R} + \text{X:}\\\text{H} \tag{7}$$

Various reagents and conditions were utilized in the derivatizations for analytical purposes. As reagents R-X for alkylations, one of the most commonly used are the alkyl halides, especially alkyl iodides and alkyl bromides. Because some of the derivatizations can be slow and inefficient depending on the analyte and on the reagent, the reaction rate becomes an important parameter for the analytical applicability. The reaction with an alkyl halide for the preparation of methyl or ethyl substituents, for example, is frequently performed either with a specific methylation reagent, in the presence of a catalyst, or in some instances using a particular solvent. The enhancement of the alkylation efficiency can be achieved using several other procedures. For example, for the analytical alkylation of carboxylic

Derivatization Methods in GC and GC/MS DOI: http://dx.doi.org/10.5772/intechopen.81954

acids, specific cryptands such as crown ethers can be used to solvate the alkali metal portion of an organic acid salts, allowing the anion to be freer and increasing the rate of nucleophilic substitution. One other approach for enhancing the alkylation efficiency is the use of phase transfer alkylation. This approach is based on the formation of a compound easily extractable in an organic phase and on the displacement of the equilibrium in the direction of the formation of the desired product.

One different way of enhancing the alkylation efficiency is the use of different alkylating reagents besides short-chain alkyl bromides or iodides. One example of a halide that is particularly reactive is pentafluorobenzyl bromide. This reagent can be used for the derivatization of a variety of compounds containing active hydrogens. Another reactive halide is 2-bromoacetophenone (phenacyl bromide). This reagent is used mainly for the alkylation of compounds containing more acidic hydrogens such as carboxylic acids. Another example of methylation using a special reagent R-X is applied on carbohydrates [15]. This methylation uses methylsulfinylmethanide anion. The reagent is prepared from dry DMSO and NaH or KH in a reaction as follows:

$$\text{H} \text{ (CH}\_3\text{)}\_2\text{SO} + \text{NaH} \rightarrow \text{CH}\_3-\text{SOCH}\_2\text{"Na}^+ + \text{H}\_2\tag{8}$$

A polyol or a monosaccharide dissolved in DMSO is easily methylated with methylsulfinyl-methanide anion.

Other alkylating reagents are known (different X in R-X), also reacting in a nucleophilic substitution. For example, dimethyl sulfate can be used for alkylations. Alkylfluoromethyl-sulfonates are even more reactive than sulfates, and the reaction may take place with the active hydrogen even from alcohols or amines as follows:

$$\mathbf{k}^\* \text{--on} \begin{array}{c} \text{\"\text\textquotedblleft} \mathbf{c} \text{\"\text\textquotedblright} \mathbf{c} \text{\"\text\textquotedblright} \mathbf{c} \text{\"\text\textquotedblright} \mathbf{c} \text{\"\text\textquotedblleft} \mathbf{c} \text{\"\text\textquotedblright} \mathbf{c} \text{\"\text\textquotedblleft} \mathbf{c} \text{\"\text\textquotedblright} \mathbf{c} \text{\"\text\textquotedblleft} \mathbf{c} \text{\"\text\textquotedblright} \end{array} \tag{9}$$

ð10Þ

Even tertiary amines, such as pyridine, also react with this type of reagent forming quaternary ammonium salts. The alkylation with alkylfluorosulfonates can be catalyzed as other alkylation reactions for increasing the reaction rate. A catalyst that can be used in this reaction is Hg(CN)2.

Diazomethane is another common alkylating (methylating) reagent. The alkylation using diazomethane is assumed to take place as follows:

$$\text{Y:}\\\text{H} + \text{H}\_2\text{C} = \text{N}^\circ = \overset{\text{i}}{\text{N}^\circ} \longrightarrow \text{CH}\_3-\text{N}^\* \equiv \overset{\text{i}}{\text{N}^\*} \longrightarrow \text{Y:} \overset{\text{i}}{\longrightarrow} \text{Y}-\text{CH}\_3 + \text{N}\_2 \tag{11}$$

Diazomethane is a gaseous unstable substance, which cannot be stored for long periods of time. It is usually prepared in small quantities and used immediately with or without an intermediate step of dissolution in ether. The preparation can be done from different N-nitroso-N-alkyl compounds in a reaction with a base. A common preparation uses N-nitroso-N-alkyl-p-toluenesulfonamide (Diazald). Methylation with diazomethane may require addition of a Lewis acid catalyst such as BF3. The

the choice of a specific derivatization procedure. Initial matrix of the sample is not always suitable for derivatization, and in some cases preliminary sample preparation is necessary to change this matrix. The change can be as simple as drying the initial sample but can also be rather complex [14]. Table 1 gives a simplified view of preferences for the choice of a derivatization reagent for compounds containing

Compound Amine Amide Alcohol Phenol Acid First derivatization preference Acylation Acylation Silylation Silylation Alkylation Second derivatization preference Alkylation Alkylation Acylation Acylation Silylation

Besides functionalities with active hydrogens, other functionalities can also be derivatized. Compounds containing carbonyls can be derivatized, for example, using condensation reactions. Some analytes may contain multiple functional groups such as the amino acids. Specific derivatization reactions can be selected for such cases.

The formation of alkyl or aryl derivatives is applied to replace the active hydrogens from an analyte with an alkyl (R) or aryl (Ar) group. The replacement can be done in functionalities such as OH, COOH, SH, NH, or CONH. For example, the derivatization with short-chain alkyl bromides or iodides has numerous analytical applications for compounds such as steroids, amino acids, catecholamines, sulfonamides, phenols, barbiturates, organic acids, and mono- and oligosaccharides. A large number of reagents R-X are known, and in a simplified approach, it can be considered that R is carrying a specific property and X a specific reactivity, although the reactivity of a reagent is influenced by both R and X components of the molecule. The type of moiety R and that of reactive group X are guiding the selection

In most alkylation reactions, the analyte acts as a nucleophile (Y:, Y:H, Y:-) reacting in a substitution (SN) with the alkylating reagent R-X, which contains a

Various reagents and conditions were utilized in the derivatizations for analytical purposes. As reagents R-X for alkylations, one of the most commonly used are the alkyl halides, especially alkyl iodides and alkyl bromides. Because some of the derivatizations can be slow and inefficient depending on the analyte and on the reagent, the reaction rate becomes an important parameter for the analytical applicability. The reaction with an alkyl halide for the preparation of methyl or ethyl substituents, for example, is frequently performed either with a specific methylation reagent, in the presence of a catalyst, or in some instances using a particular solvent. The enhancement of the alkylation efficiency can be achieved using several

other procedures. For example, for the analytical alkylation of carboxylic

Y:H þ R � X ! Y � R þ X:H (7)

7. Reactions with formation of alkyl or aryl derivatives

Derivatization preferences for compounds containing active hydrogens.

Gas Chromatography - Derivatization, Sample Preparation, Application

process of selecting a reagent for a specific derivatization.

leaving group X and an alkyl group R:

20

active hydrogens [14].

Properties

Table 1.

methylation of partly acetylated sugars and amino sugars using diazomethane and BF3 in ether leads to the methylation of the free OH groups without the migration or substitution of the existent acyl groups.

alkylation reaction, and the efficiency of alkylation increases. Acids also can be

One procedure for the formation of esters with less active organic acids applies the addition of dicyclohexylcarbodiimide (DCCI) in the derivatization process, to facilitate esterification. The reaction can be performed by adding to the acids that need to be analyzed the appropriate alcohol and DCCI usually in a solvent such as pyridine. Dicyclohexylurea, which is formed in the reaction, is not soluble in pyridine and can be separated. Besides DCCI, other carbodiimides can be used in the reaction of acids and alcohols. Among these are carbonyldiimidazole (CDI), 6-chloro-1-p-chlorobenzensulfonyloxybenzotriazole (CCBBT), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDAC), etc. Also, 2-chloro-1-methylpyridinium iodide, 2,4,6-triisopropylbenzenesulfonyl chloride, trialkyloxonium fluoroborate,

Transesterification is another technique applicable for obtaining certain alkyl derivatives of acids (or acyl derivatives of alcohols). The reaction can be written as

Transesterification can be catalyzed by acids (or Lewis acids) such as HCl, BF3, and H2SO4 or by bases such as CH3OK, CH3ONa, or C4H9ONa. The basic catalysts are commonly used for the methanolysis of triglycerides, followed by the analysis of

A special alkylation can be achieved online during the heating in the injection port of a gas chromatograph using tertraalkylammonium hydroxides or alkylarylammonium hydroxides. Tetramethylammonium hydroxide (TMAH) is the most common reagents of this type. The reaction takes place as follows (Δ indicates

Numerous other reactive compounds may be used for replacing active hydrogens in specific compounds. For example, epoxides, aziridines, and episulfides react

Besides the desired derivatives, certain unexpected compounds that can be considered artifacts for the particular analysis can also be formed in alkylation reactions. The artifacts may be formed from unexpected interactions of the reagent with the analyte or may be a result of undesired effects of the catalysts or medium used for derivatization. In some cases, the control of the alkylation process may be difficult. Longer or shorter reaction times or intervals between derivatization and analysis may lead to errors, even when an internal standard is used for quantitation. One common case of artifact formation occurs during the reaction with com-

easily with compounds with active hydrogens. Formation of a second group containing an active hydrogen may preclude the use of such reagents for analytical

pounds containing O-acyl or N-acyl groups, such as previously acylated

ð15Þ

ð16Þ

esterified using a mixture of an alcohol and an acyl halide.

etc. can be used to facilitate esterification.

Derivatization Methods in GC and GC/MS DOI: http://dx.doi.org/10.5772/intechopen.81954

the fatty acid methyl esters using GC or GC/MS [16].

follows:

heating):

purposes.

23

A common alkylation of acidic analytes such as carboxylic acids, phenols, and thiols is performed using another type of alkylating reagent, namely, N,Ndimethylformamide dialkyl acetals. N,N-Dimethylformamide dimethyl acetal (Methyl-8®) is commonly used for methylations. For a compound containing a COOH group, the reaction with this reagent takes place as follows:

$$\text{Y}-\text{COOH} + \text{(CH}\_3\text{N}-\text{CH}\_3\overset{\text{OCH}\_3}{\underset{\text{OCH}\_3}{\rightleftharpoons}} \text{Y}-\text{COOCH}\_3 + \text{(CH}\_3\text{N}-\text{CH}=\text{O} + \text{CH}\_3\text{OH}\tag{12}$$

The compounds with acidic hydrogens can also be alkylated (methylated) using trimethyl orthoacetate, alkyl-p-tolyltriazenes (R─NH─N═N─C6H4─CH3), and O-alkyl isoureas are also used for the formation of analytes containing acidic hydrogens, imino esters, etc.

Alcohols can also act as alkylating reagents in particular when the analyte contains a more acidic hydrogen. Catalyst such as HCl, BF3, CF3 COOH or a cation exchange resin in H+ form is also frequently added to facilitate the reaction. The addition of HCl can be made as a water solution or as gaseous HCl that does not bring additional water to the reaction medium. The formation of alkyl or aryl derivatives of acids is a particularly important reaction known as esterification. Derivatization by esterification has been used with acids as the analyte and the alcohol as the reagent and also with the alcohol as the analyte and the acid the reagent. The esterification can be viewed either as the acid alkylation or as the acylation of the alcohol (see also the esterification mechanism). This reaction is typically catalyzed by strong acids and can be written as follows:

$$\text{R} \text{---} \text{COOH} + \text{R}^{\text{e}} \text{---} \text{OH} \xrightarrow[\text{+} \text{H}^{+}]{} \text{R} \text{---} \text{COOR}^{\text{a}} + \text{H}\_{2}\text{O} \tag{13}$$

The mechanism of ester formation can be summarized by the following series of reactions:

$$\begin{array}{c} \text{O} \\ \stackrel{\text{H}}{\underset{\text{R}}{\rightleftharpoons}} \text{O} \xrightarrow{+\text{H}^{+}} \begin{array}{c} \text{O} \\ \stackrel{\text{H}}{\underset{\text{R}}{\rightleftharpoons}} \text{O} \end{array} \xrightarrow{\text{H}^{+}} \begin{array}{c} \text{O} \\ \stackrel{\text{H}}{\underset{\text{R}}{\rightleftharpoons}} \text{O} \end{array} \xrightarrow{\text{H}^{+}} \begin{array}{c} \text{O}^{-} \\ \stackrel{\text{H}^{+}}{\underset{\text{R}}{\rightleftharpoons}} \text{O} \end{array} \xrightarrow{\text{H}^{+}} \begin{array}{c} \text{O}^{-} \\ \stackrel{\text{H}^{+}}{\underset{\text{R}}{\rightleftharpoons}} \text{O} \end{array} \xrightarrow{\text{H}^{+}} \begin{array}{c} \text{O}} \text{O} \\ \stackrel{\text{H}^{+}}{\underset{\text{R}}{\rightleftharpoons}} \text{O} \end{array} \xrightarrow{\text{H}^{+}} \begin{array}{c} \text{O}} \text{O} \\ \stackrel{\text{H}^{+}}{\underset{\text{R}}{\rightleftharpoons}} \text{O} \end{array} \tag{14}$$

The esterification efficiency can be improved by removing the water formed in this reaction. This can be done using a chemical reagent or distillation when the compounds of interest boil above 100°C. Among the materials able to eliminate water are desiccants such as anhydrous MgSO4, molecular sieves, or substances that react with water such as CaC2, (CH3)2C(OCH3)2 (2,2-dimethoxypropane), and even an appropriately chosen acid anhydride that reacts faster with water than with the reacting alcohol. The derivatization also may be performed in the presence of SOCl2 (thionyl chloride), which reacts with the water assisting in its removal, and when present in excess, may react with the alcohols forming alkyl chlorides or with the acids forming acyl chlorides. Chloride is a better leaving group in a nucleophilic

#### Derivatization Methods in GC and GC/MS DOI: http://dx.doi.org/10.5772/intechopen.81954

methylation of partly acetylated sugars and amino sugars using diazomethane and BF3 in ether leads to the methylation of the free OH groups without the migration or

A common alkylation of acidic analytes such as carboxylic acids, phenols, and

The compounds with acidic hydrogens can also be alkylated (methylated) using

Alcohols can also act as alkylating reagents in particular when the analyte contains a more acidic hydrogen. Catalyst such as HCl, BF3, CF3 COOH or a cation exchange resin in H+ form is also frequently added to facilitate the reaction. The addition of HCl can be made as a water solution or as gaseous HCl that does not bring additional water to the reaction medium. The formation of alkyl or aryl derivatives of acids is a particularly important reaction known as esterification. Derivatization by esterification has been used with acids as the analyte and the alcohol as the reagent and also with the alcohol as the analyte and the acid the reagent. The esterification can be viewed either as the acid alkylation or as the acylation of the alcohol (see also the esterification mechanism). This reaction is

The mechanism of ester formation can be summarized by the following series of

The esterification efficiency can be improved by removing the water formed in this reaction. This can be done using a chemical reagent or distillation when the compounds of interest boil above 100°C. Among the materials able to eliminate water are desiccants such as anhydrous MgSO4, molecular sieves, or substances that react with water such as CaC2, (CH3)2C(OCH3)2 (2,2-dimethoxypropane), and even an appropriately chosen acid anhydride that reacts faster with water than with the reacting alcohol. The derivatization also may be performed in the presence of SOCl2 (thionyl chloride), which reacts with the water assisting in its removal, and when present in excess, may react with the alcohols forming alkyl chlorides or with the acids forming acyl chlorides. Chloride is a better leaving group in a nucleophilic

trimethyl orthoacetate, alkyl-p-tolyltriazenes (R─NH─N═N─C6H4─CH3), and O-alkyl isoureas are also used for the formation of analytes containing acidic

ð12Þ

ð13Þ

ð14Þ

thiols is performed using another type of alkylating reagent, namely, N,Ndimethylformamide dialkyl acetals. N,N-Dimethylformamide dimethyl acetal (Methyl-8®) is commonly used for methylations. For a compound containing a

COOH group, the reaction with this reagent takes place as follows:

Gas Chromatography - Derivatization, Sample Preparation, Application

typically catalyzed by strong acids and can be written as follows:

substitution of the existent acyl groups.

hydrogens, imino esters, etc.

reactions:

22

alkylation reaction, and the efficiency of alkylation increases. Acids also can be esterified using a mixture of an alcohol and an acyl halide.

One procedure for the formation of esters with less active organic acids applies the addition of dicyclohexylcarbodiimide (DCCI) in the derivatization process, to facilitate esterification. The reaction can be performed by adding to the acids that need to be analyzed the appropriate alcohol and DCCI usually in a solvent such as pyridine. Dicyclohexylurea, which is formed in the reaction, is not soluble in pyridine and can be separated. Besides DCCI, other carbodiimides can be used in the reaction of acids and alcohols. Among these are carbonyldiimidazole (CDI), 6-chloro-1-p-chlorobenzensulfonyloxybenzotriazole (CCBBT), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDAC), etc. Also, 2-chloro-1-methylpyridinium iodide, 2,4,6-triisopropylbenzenesulfonyl chloride, trialkyloxonium fluoroborate, etc. can be used to facilitate esterification.

Transesterification is another technique applicable for obtaining certain alkyl derivatives of acids (or acyl derivatives of alcohols). The reaction can be written as follows:

$$\mathfrak{n}-\text{c}^{\mathsf{O}^{\bullet}}\_{\mathsf{O}^{\bullet}} + \mathfrak{n}-\text{o}\mathfrak{n} \longrightarrow \mathfrak{n}-\text{c}^{\mathsf{O}^{\bullet}}\_{\mathsf{O}^{\bullet}} + \mathfrak{n}-\text{o}\mathfrak{n} \tag{15}$$

Transesterification can be catalyzed by acids (or Lewis acids) such as HCl, BF3, and H2SO4 or by bases such as CH3OK, CH3ONa, or C4H9ONa. The basic catalysts are commonly used for the methanolysis of triglycerides, followed by the analysis of the fatty acid methyl esters using GC or GC/MS [16].

A special alkylation can be achieved online during the heating in the injection port of a gas chromatograph using tertraalkylammonium hydroxides or alkylarylammonium hydroxides. Tetramethylammonium hydroxide (TMAH) is the most common reagents of this type. The reaction takes place as follows (Δ indicates heating):

Numerous other reactive compounds may be used for replacing active hydrogens in specific compounds. For example, epoxides, aziridines, and episulfides react easily with compounds with active hydrogens. Formation of a second group containing an active hydrogen may preclude the use of such reagents for analytical purposes.

Besides the desired derivatives, certain unexpected compounds that can be considered artifacts for the particular analysis can also be formed in alkylation reactions. The artifacts may be formed from unexpected interactions of the reagent with the analyte or may be a result of undesired effects of the catalysts or medium used for derivatization. In some cases, the control of the alkylation process may be difficult. Longer or shorter reaction times or intervals between derivatization and analysis may lead to errors, even when an internal standard is used for quantitation.

One common case of artifact formation occurs during the reaction with compounds containing O-acyl or N-acyl groups, such as previously acylated

carbohydrates, glycolipids, or glycoproteins, in particular when the reaction is done with short-chain alkyl bromides or iodides. When the OH groups of different sugars or NH2 groups of amino sugars were already protected with acyl groups, it was noted that, depending on the catalyst and the chosen medium, these acyl groups can be replaced by alkyl groups, or they may migrate from one position (such as C1) to other positions.

silylation of different functional groups in the analyte. The solvent (or mixture of solvents) used as a medium and the compounds present or added in the silylation medium may also play a role for silylation efficiency. The reagent excess is sometimes important for displacing the equilibrium in the desired direction, and usually an excess up to ten times larger than stoichiometrically needed is used for silylation. Temperature also increases reaction rate, as expected, and heating of the sample with the reagents at temperatures around 70°C for 15 to 30 min is common. Some

The approximate order of the increasing silyl donor ability for the reagents shown in Figure 8 is HMDS < TMCS < MSA < TMSA < TMSDEA < TMSDMA < MSTFA < BSA < BSTFA < TMSI. This order may be different on particular sub-

The reagent mixtures may provide a more efficient silylation for specific compounds. For example, silylation of 3,4-dimethoxyphenylethylamine with BSA leads

In general, the silylation of OH and COOH groups takes place with better results than that of NH2, CONH, or NH groups. Excellent results are obtained, for example, for the analysis of phenols after silylation [19]. A chromatogram of a solution containing a mixture of phenols at concentrations between 2.0 and 2.5 μg/mL in DMF, derivatized with BSTFA, separated on a BPX-5 chromatographic column (SGE Anal. Sci.), followed by MS analysis in single-ion monitoring (SIM) mode is shown in Figure 9. Details regarding the analyzed phenols are given in

Besides organic active hydrogens, several inorganic compounds with active hydrogens can also react with silylating reagents. Among these are H2O, H2O2, and strong inorganic acids. Also, some salts of the acids may be silylated. The reaction of silylating reagents with water imposes that water should be at the low level in the matrix or the solution of the analytes. The reaction with water takes place as

In many solvents used as medium for derivatization, the trimethylsilanol formed in the reaction with water is separated as a distinct layer of solvent. The formation of two layers impedes a proper sampling of the derivatized material in the GC/MS instrument. In addition to that, the presence of an excess of water suppresses the derivatization of other compounds. The silylation is not recommended on samples

The silylation reaction is commonly performed in a solvent that does not have active hydrogens. The most commonly used solvents as a medium for silylation are dimethylformamide (DMF), pyridine, and acetonitrile. The main role of the solvent is to dissolve the analyte and the reagents. The by-product HX of silylation shown in reaction (17) can be an acid, a base, or a neutral compound. As examples, for TMCS the by-product is HCl, for HMDS the

with a water content higher than about 10%.

ð18Þ

to the substitution of only one active hydrogen in the NH2 group, while the silylation with BSA in the presence of 5% TMCS produces silylation of both hydrogens in the NH2 [21]. A common silylating mixture is BSTFA with 1% TMCS. One of the determining factors regarding the silylation efficiency is the nature of the molecule Y:H that is being silylated (the analyte) and plays a crucial role in the choice of the derivatization conditions. It was noticed experimentally that the decreasing ease of silylation follows approximately the order shown in

Silylation reagents can be used pure or in mixtures of two or even three reagents.

reagents used for trimethylsilylation are shown in Figure 8 [14].

Derivatization Methods in GC and GC/MS DOI: http://dx.doi.org/10.5772/intechopen.81954

Table 2.

Table 3.

follows:

25

strates where other reagents or reagent mixtures may be more reactive.

Oxidation is another common side reaction when using Ag2O as a catalyst. The oxidation effect of Ag2O can be seen on free sugars as well as when attempting to permethylate peptides. Sulfhydryl groups are particularly sensitive to oxidation with Ag2O as a catalyst. The use of methylsulfinyl carbanion as a methylating reagent may also produce undesired side reactions with certain esters generating methylsulfinylketones. Also, strong alkylating reagents may produce undesired artifacts by unexpected alkylations.

The derivatization with the purpose of obtaining aryl derivatives is similar in many respects to the alkylation reaction. The reaction takes place with compounds containing active hydrogens. Simple aryl halides are generally resistant to be attacked by nucleophiles and do not react similar to alkyl halides. This low reactivity can be significantly increased by changes in the structure of aryl halide or in the reaction conditions. The nucleophilic displacement can become very rapid when the aryl halide is substituted with electron attracting groups such as NO2.
