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


Table 2.

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

#### Figure 9.

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

Gas Chromatography - Derivatization, Sample Preparation, Application

Figure 8.

26

Some reagents used for trimethylsilylation.

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.

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

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-, imidazolyl, F3C-(CO)-N(CH3)-, etc. For example, a common reagent containing tert-butyldimethylsilyl group is N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA), which has the following structure:

The use of different groups than TMS may serve different purposes. For example, a fluorinated or brominated group may enhance significantly the detection sensitivity when using ECD or NCI-MS. Also, the stability toward hydrolysis of compounds silylated with different groups than TMS may be higher, and such silylation can be advantageous. This is, for example, the case of tert-butyldimethylsilyl group that is typically more stable to hydrolysis than trimethylsilyl.

As an example, silylation of amino acids with MTBSTFA is commonly used [22, 23], and it is preferred to the silylation generating TMS derivatives. The chromatogram of a set of amino acid standards with the concentration of 0.05 μmol/mL derivatized with MTBSTFA and separated on a DB-5MS chromatographic column (from Agilent) followed by MS analysis is shown in Figure 11. Details regarding the analyzed amino acids are given in Table 4.

In most situations, silylation generates only the desired derivatives. However, there are cases when the expected silylated compound is not formed, and either the silylation is not complete, or some compounds such as aldehydes, ketones, or esters with no obvious active hydrogen generate silylated compounds. Incomplete


silylation is usually the result of inappropriate reaction conditions. However, when compounds with multiple functionalities are silylated, it is possible to generate a variety of derivatized compounds, regardless of the intention to obtain fully

Chromatogram of a set of amino acid standards with the concentration of 0.05 μmol/mL derivatized with

In some cases, artifacts are formed due to the modification of the analyte under the influence of the reagents during derivatization. For example, when the silylation is done in basic or acidic conditions, the analytes that are sensitive to acidic or basic media may suffer unexpected transformations. The most frequent artifacts with compounds not containing obvious active hydrogens occur with aldehydes. Some aldehydes are able to undergo two types of chemical reactions with formation of OH groups, namely, enolization and acetal formation in the presence of water. The OH groups formed in this manner react with different silylating reagents and give the corresponding silylated products. Although the enolization or the acetal formation is negligible for the initial aldehyde, the reactions may be significantly displaced toward the formation of the silylated compounds of the enol or of the acetal. Artifacts can also be generated when the reaction is allowed to continue for an

silylated or partly silylated compounds.

MTBSTFA separated on a DB-5MS chromatographic column.

Examples of silyl groups different from TMS used in silylation reagents.

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

Figure 11.

29

Figure 10.

Table 3. Details regarding the analyzed phenols with the chromatogram shown in Figure 9. Derivatization Methods in GC and GC/MS DOI: http://dx.doi.org/10.5772/intechopen.81954

Figure 10.

The use of different groups than TMS may serve different purposes. For example, a fluorinated or brominated group may enhance significantly the detection sensitivity when using ECD or NCI-MS. Also, the stability toward hydrolysis of compounds silylated with different groups than TMS may be higher, and such silylation can be advantageous. This is, for example, the case of tert-butyldimethylsilyl group that is

As an example, silylation of amino acids with MTBSTFA is commonly used [22, 23], and it is preferred to the silylation generating TMS derivatives. The chromatogram of a set of amino acid standards with the concentration of 0.05 μmol/mL derivatized with MTBSTFA and separated on a DB-5MS chromatographic column (from Agilent) followed by MS analysis is shown in Figure 11. Details regarding the

In most situations, silylation generates only the desired derivatives. However, there are cases when the expected silylated compound is not formed, and either the silylation is not complete, or some compounds such as aldehydes, ketones, or esters

m/z Abrrev. No. Compound Ret.

Dimethylphenol

time

(19) 4-Methylcatechol 16.27 268 4-MeCa

(20) Hydroquinone 16.73 254 Hy

(21) 3-Methylcatechol 16.71 268 3-MeCa

(22) 5-Methylresorcinol 18.19 268 5-MeCa

m/z Abrrev.

diMePh

MeOPh

MeOPh

12.32 194 3,4-

20.18 282 2,5-

diMeRe

with no obvious active hydrogen generate silylated compounds. Incomplete

(2) o-Cresol 8.57 180 o-Cr (15) 3-Methoxyphenol 13.17 196 3-

(3) m-Cresol 8.76 180 m-Cr (16) 4-Methoxyphenol 13.47 196 4-

(4) p-Cresol 9.08 180 p-Cr (17) Catechol 13.88 254 Ca (5) 2-Ethylphenol 10.28 194 2-EtPh (18) Resorcinol 16.05 254 Re

diMePh

diMePh

diMePh

MeOPh

diMePh

dimePh

Details regarding the analyzed phenols with the chromatogram shown in Figure 9.

(10) 4-Ethylphenol 11.59 194 4-EtPh (23) 2-Methylresorcinol 18.66 268 2-MeRe (11) 4-Chlorophenol 11.71 185 4-ClPh (24) 4-Ethylresorcinol 19.90 282 4-EtRe

(25) 2,5-

Dimethylresorcinol

typically more stable to hydrolysis than trimethylsilyl.

Gas Chromatography - Derivatization, Sample Preparation, Application

time

(1) Phenol 6.88 166 Ph (14) 3,4-

analyzed amino acids are given in Table 4.

(6) 2,5-Dimethylphenol 10.70 194 2,5-

(7) 3,5-Dimethylphenol 11.07 194 3,5-

(8) 2,4-Dimethylphenol 11.20 194 2,4

(9) 2-Methoxyphenol 11.28 196 2-

(12) 2,6-Dimethylphenol 11.79 194 2,6-

(13) 2,3-Dimethylphenol 12.02 194 2,3-

Table 3.

28

No. Compound Ret.

Examples of silyl groups different from TMS used in silylation reagents.

#### Figure 11.

Chromatogram of a set of amino acid standards with the concentration of 0.05 μmol/mL derivatized with MTBSTFA separated on a DB-5MS chromatographic column.

silylation is usually the result of inappropriate reaction conditions. However, when compounds with multiple functionalities are silylated, it is possible to generate a variety of derivatized compounds, regardless of the intention to obtain fully silylated or partly silylated compounds.

In some cases, artifacts are formed due to the modification of the analyte under the influence of the reagents during derivatization. For example, when the silylation is done in basic or acidic conditions, the analytes that are sensitive to acidic or basic media may suffer unexpected transformations. The most frequent artifacts with compounds not containing obvious active hydrogens occur with aldehydes. Some aldehydes are able to undergo two types of chemical reactions with formation of OH groups, namely, enolization and acetal formation in the presence of water. The OH groups formed in this manner react with different silylating reagents and give the corresponding silylated products. Although the enolization or the acetal formation is negligible for the initial aldehyde, the reactions may be significantly displaced toward the formation of the silylated compounds of the enol or of the acetal. Artifacts can also be generated when the reaction is allowed to continue for an

extended period of time. Other uncommon reactions with a specific silylation reagent and analyte may occur. An example of an uncommon reaction is the ring

The formation of acyl derivatives is applied for replacing the active hydrogens from an analyte in functionalities such as OH, SH, NH [11, 24], CONH, etc. The acylation is also used for reducing polarity and improving the behavior of the analytes in the chromatographic column. Acylation may confer a better volatility of the analytes, although not as marked as for silylation or methylation. Only the derivatization with acetyl groups or with fluorinated acyl groups (not heavier than heptafluorobutyryl) improves volatility, while other heavier acyl groups are not suitable for this purpose. Acetylation, for example, can be used for compounds such

as monosaccharides and amino acids to allow GC analysis. The detectability

improvement on the other hand is a very common purpose for acylation. Acylation with fluorinated compounds is frequently used for enhancing detectability in GC with ECD or NCI-MS detection. Other uses of acylation include the enhancement of

Most acylation reactions are nucleophilic substitutions where the analyte is a nucleophile (Y:, Y:H, Y:-) reacting with the acylating reagent RCO-X that contains a leaving group X and an acyl group RCO: as shown in the following reaction:

Some common acyl groups present in acylation reagents are indicated in Table 5. As shown in Table 5, the acyl groups in the reagent can be attached to various "X" groups. One such group is OH and among the acylating reagents are some free acids. When nucleophile is an alcohol, the reaction is known as esterification and has been discussed in Section 7. The acylation with acids can be applied besides alcohols to certain thiols, phenols, amines, etc. and can be written as follows:

The reaction can be displaced toward the formation of the acyl derivatives by eliminating the water using compounds such as anhydrous MgSO4, molecular sieve, or substances that react with water such as CaC2, or (CH3)2C(OCH3)2. Dicyclohexylcarbodiimide (DCCI) also is used for modifying the yield of the desired product. The reaction with reagents containing a carboxylic acid reactive group also can be done in the presence of 2,4,6-trichlorobenzoyl chloride or with various sulfonyl chlorides such as 2,4,6-triisopropyl-benzenesulfonyl chloride or 2,4,6-trimethylbenzenesulfonyl chloride. The reaction of amines with acids can be displaced toward the formation of the amides using a peptide coupling reagent such as benzotriazol-1-yl-oxy-tris(dimethyl-amino)-phosphonium hexafluorophosphate

triphenylphosphine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDAC), etc. Common acylating reagents are acyl halides such as chlorides or bromides, which are reactive compounds suitable for acylation. The reaction of an acyl chlo-

(BOP), diethyl cyanophosphonate, O-benzotriazol-1-yl-N,N,N<sup>0</sup>

(tetramethylene)uronium hexafluorophosphate, 2,2<sup>0</sup>

ride with an amine, for example, takes place as follows:

31

Y : H þ R � COOH ! R � COY þ H2O (20)

,N<sup>0</sup> -bis


ð19Þ

opening of flavanones.

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

9. Acylation reactions

separation of chiral compounds, etc.


Table 4.

Details regarding the analyzed amino acids with the chromatogram shown in Figure 11.

extended period of time. Other uncommon reactions with a specific silylation reagent and analyte may occur. An example of an uncommon reaction is the ring opening of flavanones.
