8. Silylation reactions

Silylation is the chemical reaction of replacing a reactive hydrogen atom in OH, COOH, SH, NH, CONH, POH, SOH, or enolisable carbonyl with a silyl group, most frequently with trimethylsilyl (TMS). A large number of analytical methods involve silylation applied to alcohols including carbohydrates [17], phenols [18], amines, sterols [19], etc. The purpose of silylation in chromatography is mainly to reduce the polarity of the analyte, increase its stability, and improve the GC behavior. The differences in the mass spectra of the silylated compounds as compared to the initial analyte may also be an advantage for detectability. However, the mass spectra of many silylated compounds may not be available in common mass spectral libraries. Also, the silylated compounds plus the commonly present excess of silylating reagent may deteriorate some types of stationary phases such as that of Carbowax (polyethylene glycol)-type columns, and for this reason, their separation cannot be done on such columns.

Silylation can be performed on specific analytes or directly on complex samples such as a plant material (see, e.g., [12]). The silylating agent and the solvent can play the double role of extractant and silylating reagent. Many publications describe the use of silylation reactions for analytical purposes (e.g., [1, 5, 20]). The reaction of an analyte Y:H with the formation of a TMS derivative can be written as follows:

$$\begin{array}{cccc} \text{CH}\_3 & \text{CH}\_3\\ \text{Ar-H} + \text{H}\_3\text{C} - \underset{\text{light}}{\text{Si}} - \text{X} & \underset{\text{H}\_3\text{C}}{\longrightarrow} - \underset{\text{H}\_3}{\text{Si}} - \text{Y} + \text{H}\mathbf{X} \\\\ \text{CH}\_3 & \underset{\text{CH}\_3}{\text{CH}\_3} \end{array} \tag{17}$$

The molecular weight for TMS is 73.047 calculated considering in the elemental composition of only the masses of the most abundant isotope. Numerous reagents have been synthesized to be used in silylations. Various aprotic solvents can be used as medium for silylation. The analysis can be focused on one analyte or on a mixture of analytes. The main factors contributing to the increase of the efficiency and the rate of the silylation reaction are the silyl donor ability of the reagent and the ease of

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

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

Gas Chromatography - Derivatization, Sample Preparation, Application

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

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

Silylation is the chemical reaction of replacing a reactive hydrogen atom in OH, COOH, SH, NH, CONH, POH, SOH, or enolisable carbonyl with a silyl group, most frequently with trimethylsilyl (TMS). A large number of analytical methods involve silylation applied to alcohols including carbohydrates [17], phenols [18], amines, sterols [19], etc. The purpose of silylation in chromatography is mainly to reduce the polarity of the analyte, increase its stability, and improve the GC behavior. The differences in the mass spectra of the silylated compounds as compared to the initial analyte may also be an advantage for detectability. However, the mass spectra of many silylated compounds may not be available in common mass spectral libraries. Also, the silylated compounds plus the commonly present excess of silylating reagent may deteriorate some types of stationary phases such as that of Carbowax (polyethylene glycol)-type columns, and for this reason, their separation cannot be done on such columns.

Silylation can be performed on specific analytes or directly on complex samples such as a plant material (see, e.g., [12]). The silylating agent and the solvent can play the double role of extractant and silylating reagent. Many publications describe the use of silylation reactions for analytical purposes (e.g., [1, 5, 20]). The reaction of an analyte Y:H with the formation of a TMS derivative can be written as follows:

The molecular weight for TMS is 73.047 calculated considering in the elemental composition of only the masses of the most abundant isotope. Numerous reagents have been synthesized to be used in silylations. Various aprotic solvents can be used as medium for silylation. The analysis can be focused on one analyte or on a mixture of analytes. The main factors contributing to the increase of the efficiency and the rate of the silylation reaction are the silyl donor ability of the reagent and the ease of

ð17Þ

aryl halide is substituted with electron attracting groups such as NO2.

positions.

artifacts by unexpected alkylations.

8. Silylation reactions

24

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

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

Silylation reagents can be used pure or in mixtures of two or even three reagents. The reagent mixtures may provide a more efficient silylation for specific compounds. For example, silylation of 3,4-dimethoxyphenylethylamine with BSA leads 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 Table 2.

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 Table 3.

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 follows:

$$\overset{\text{O}}{\text{C}\_{3}\text{C}-\overset{\text{Si(CH}\_{3})\_{3}}{\text{C}=\text{N}---\text{Si(CH}\_{3})\_{3}}} + 2\overset{\text{H}}{\text{H}\_{2}\text{O}} \xrightarrow[\overset{\text{F}}{\text{C}\_{3}\text{C}-\overset{\text{O}}{\text{C}}-\overset{\text{NH}\_{2}}{\text{NH}\_{2}}} + 2\overset{\text{O}}{(\text{H}\_{3}\text{C})\_{3}\text{Si}} \xrightarrow{\text{O}} \text{OH} \tag{18}$$

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 with a water content higher than about 10%.

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

Figure 8.

Some reagents used for trimethylsilylation.

by-product is NH3, for BSTFA the by-product is N-TMS-trifluoroacetamide, and for TMSI the by-product is imidazole. When the silylation reagent generates an acid as a by-product of the reaction, this may interfere with the silylation. For this reason, silylation can be promoted by any acid acceptor used as solvent or present in the solvent. Among such solvents are pyridine, triethylamine, and to a lower extent DMF. They can be used as both solvents and acid acceptors. Mixtures of solvents are commonly used for both enhancing solubility and promoting silylation. For example, formamide in the presence of pyridine may react with an acidic by-product generating CO and an ammonium salt. The addition of basic compounds to the silylation reaction may also influence the

efficiency of the silylation. Also, some compounds may act as catalysts for

Chromatogram of a set of phenol standards in DMF with the concentrations between 2.0 and 2.5 μg/mL derivatized with BSTFA, separated on a BPX-5 chromatographic column followed by MS analysis.

imidazolyl, F3C-(CO)-N(CH3)-, etc. For example, a common reagent

trifluoroacetamide (MTBSTFA), which has the following structure:

Although the TMS derivatives are by far the most commonly used in the derivatization for analytical purposes, other substituents in the silyl group can be used as reagents. Several such groups are indicated in Figure 10. The groups can be present in a variety of reagents connected to leaving groups "X-" such as Cl-,

Compound Functional group Decreasing reactivity

Several functional groups that can be silylated (listed in the approximate order of decreasing ease of silylation).

(1) Primary alcohol OH (2) Secondary alcohol OH (3) Tertiary alcohol OH (4) Phenol OH (5) Thiophenol SH (6) Aliphatic acid COOH (7) Aromatic acid COOH (8) Primary amine NH2 (9) Thiol SH (10) Amide CONH2

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

(11) N-TMS amide CONH-Si(CH3)3

(12) Secondary amine NH (13) Indole NH

containing tert-butyldimethylsilyl group is N-methyl-N-(tert-butyldimethylsilyl)-

silylation.

27

Figure 9.

Table 2.
