9. Acylation reactions

Peak No.

(4) α-Amino-n-

(7) β-

(12) γ-Aminobutyric acid

(22) 3-Methyl-L-

(25) 1-Methyl-L-

(27) α-Aminoadipic

Table 4.

30

histidine

histidine

acid

butyric acid

Aminoisobutyric acid

Amino acid Abbrev. MW Formula + x

Gas Chromatography - Derivatization, Sample Preparation, Application

TBDMS

α-ABu 103.10 C16H37NO2Si2 331 274 34.36

β-ABu 103.10 C16H37NO2Si2 331 274 36.11

γ-ABu 103.10 C16H37NO2Si2 331 274 39.79

3MeHys 169.20 C19H39N3O2Si2 397 340 55.15

1MeHys 169.20 C19H39N3O2Si2 397 302 57.03

161.20 C24H53NO4Si3 503 446 58.06

(1) α-Alanine α-Ala 89.09 C15H35NO2Si2 317 260 31.69 (2) Glycine Gly 75.07 C14H33NO2Si2 303 246 32.63 (3) Sarcosine Sar 89.09 C15H35NO2Si2 317 260 33.85

(5) β-Alanine β-Ala 89.09 C15H35NO2Si2 317 260 35.58 (6) Urea 60.06 C13H32N2OSi2 288 231 36.01

(8) Valine Val 117.15 C17H39NO2Si2 345 186 36.15 (9) Leucine Leu 131.17 C18H41NO2Si2 359 200 37.71 (10) Norleucine 131.17 C18H41NO2Si2 359 200 38.8 (11) Isoleucine iLeu 131.17 C18H41NO2Si2 359 200 38.8

(13) Proline Pro 115.13 C17H37NO2Si2 343 184 39.87 (14) 2-Phenylglycine PhGly 151.17 C20H37NO2Si2 379 220 46.16 (15) 5-Oxoproline oPro 129.13 C17H35NO3Si2 357 300 46.18 (16) Methionine Met 149.20 C17H39NO2SSi2 377 320 46.68 (17) Serine Ser 105.09 C21H49NO3Si3 447 390 47.52 (18) Threonine Thr 119.12 C22H51NO3Si3 461 404 48.43 (19) Phenylalanine Phe 165.19 C21H39NO2Si2 393 336 50.35 (20) Aspartic acid Asp 133.10 C22H49NO4Si3 475 418 52.47 (21) Hydroxyproline HyPro 131.13 C23H51NO3Si3 473 314 53.23

(23) Glutamic acid Glu 147.13 C23H51NO4Si3 489 432 55.53 (24) Ornithine Orn 132.20 C23H54N2O2Si3 474 286 55.64

(26) Lysine Lys 146.19 C24H56N2O2Si3 488 300 58.02

(28) Histidine Hys 155.16 C24H51N3O2Si3 497 440 62.29 (29) Tyrosine Tyr 181.19 C27H53NO3Si3 523 302 63.29 (30) Arginine Arg 174.20 C24H56N4O2Si3 516 144 64.26 (31) Tryptophan Trp 204.22 C29H54N2O2Si3 546 244 67.98 (32) Cystine Cys 240.30 C28H64N2O4S2Si4 668 348 72.65 (33) Homocystine hCys 268.30 C32H72N2O4S2Si4 724 362 76.59

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

MW + x TBDMS Charact. ion

Ret. time

> 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 separation of chiral compounds, etc.

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:

$$\stackrel{\text{0}}{\underset{\text{Y:H}+\text{ R}}{\overset{\text{0}}{\text{C}}-\text{X}}} \stackrel{\text{0}}{\underset{\text{R}}{\overset{\text{0}}{\text{C}}}} \stackrel{\text{0}}{\underset{\text{R}}{\overset{\text{0}}{\text{C}}}} \stackrel{\text{0}}{\text{R}} \tag{19}$$

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:

$$\text{Y} \cdot \text{H} + \text{R} - \text{COOH} \rightarrow \text{R} - \text{COY} + \text{H}\_2\text{O} \tag{20}$$

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 (BOP), diethyl cyanophosphonate, O-benzotriazol-1-yl-N,N,N<sup>0</sup> ,N<sup>0</sup> -bis (tetramethylene)uronium hexafluorophosphate, 2,2<sup>0</sup> -dipyridyl disulfide + 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 chloride with an amine, for example, takes place as follows:


ð21Þ

ð22Þ

ð23Þ

ð24Þ

Since the reactivity of amides is lower than that of amines, the second hydrogen in the amine is more difficult to replace. Also, steric hindrance may negatively influence the reaction. The generation of a strong acid such as HCl is a disadvantage in the reaction with acyl halides, and usually the acid should be removed either by adding basic compounds such as Na2CO3 or MgCO3 or using pyridine as the reaction medium. The high reactivity of acyl halides is used for the acylation of compounds with less reactive hydrogens. Certain carbonyl cyanides react similarly to acyl chlorides.

The disadvantage of generating a strong inorganic acid in the acylation with acyl halides also can be avoided by having, instead of the acyl halide, an anhydride. The

The acid resulting together with the acylated compound is not a strong acid such as HCl. The anhydrides of trifluoroacetic acid (TFA), pentafluoropropionic anhydride (PFPA), and heptafluorobutyric (HFBA) acids are commonly used for derivatization of alcohols, phenols, amines, etc., with the purpose of enhancing detectability (by ECD or NCI-MS) and also for improving the chromatographic behavior (higher volatility, better thermal stability, better separation). The volatility of fluorinated compounds allows the GC applications. The reactivity of the perfluorinated anhydrides increases in the order HFBA < PFPA < TFA. However, the differences are not significant. Once formed, the heptafluorobutyrates are more stable to hydrolysis than the trifluoroacetates. An inert solvent such as CH2Cl2, ether, ethyl acetate, acetone, tetrahydrofuran or in CH3CN, etc. can be used as a medium for the reaction with perfluoroanhydrides. For the neutraliza-

tion of the acids formed during derivatization, the basic compounds such as

In order to avoid the formation of water or of a strong acid in the acylation reaction, certain amides such as N-methyl-bis(trifluoroacetamide), bis(trifluoroacetamide), or 2,2,2-trifluoro-N-methyl-N-(2,2,2-trifluoroacetyl)acetamide (MBTFA) can be used as reagents. Acylation of amines takes place at room temperature. Solvents such as CH3CN, pyridine, DMSO, or THF can be used as a reaction medium:

One other procedure successfully applied to obtain acyl derivatives is the use of acyl imidazoles as reagents. This class of compounds reacts with analytes containing alcohol, primary and secondary amino groups, or thiols. The reaction generates as a

triethylamine, pyridine, or even solid NaHCO3 can be utilized.

by-product imidazole:

33

reaction of Y:H with an anhydride takes place as follows:

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

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

Group Group structure Mass of

Gas Chromatography - Derivatization, Sample Preparation, Application

2,2-Dimethylpropionyl-(pivaloyl)

Pentafluoropropionyl

(Pentafluorophenyl)-

(Pentafluorophenoxy)-

acetyl

acetyl

Table 5.

32

the group

Formyl 29 Formic acid Steroids

Propionyl 57 Propionic anhydride Alcohols

Butyryl 71 Butyric anhydride Alcohols

Heptafluorobutyryl 197 Heptafluorobutyric

Trichloroacetyl 145 Trichloroacetic

Pentafluorobenzoyl 195 Pentafluorobenzoyl

Some common groups present in acylating reagents used in derivatizations for GC analysis [14].

Acetyl 43 Acetyl chloride,

Trifluoroacetyl 97 N-Methyl-bis

Example of reagents

acetic anhydride

85 Pivaloyl chloride, pivalic anhydride

147 Pentafluoropropionic anhydride (PFPA)

anhydride

chloride, pentafluorobenzoylimidazole

209 (Pentafluorophenyl) acetyl chloride

225 (Pentafluorophenoxy) acetyl chloride

anhydride (HFBA), heptafluorobuty rylimidazole

(trifluoroacetamide), bis(trifluoroacetamide), trifluoroacetic acid (TFA)

Common analytes

Alcohols

Alcohols

Amino acids

Alcohols, amines, amino acids

Alcohols, amines, amino acids

Alcohols

Alcohols, amides

Alcohols, amides

Alcohols

$$\begin{array}{ccccc} \text{Y} & \stackrel{\text{H}}{\longrightarrow} & \text{O} \\ \text{Y} & \stackrel{\text{H}}{\longrightarrow} & \text{R} \end{array} \begin{array}{ccccc} \text{O} & \stackrel{\text{H}}{\longrightarrow} & \text{O} \\ \text{H} & \stackrel{\text{H}}{\longrightarrow} & \text{O} \\ & & \stackrel{\text{H}}{\longrightarrow} & \text{O} \end{array} \tag{21}$$

Since the reactivity of amides is lower than that of amines, the second hydrogen in the amine is more difficult to replace. Also, steric hindrance may negatively influence the reaction. The generation of a strong acid such as HCl is a disadvantage in the reaction with acyl halides, and usually the acid should be removed either by adding basic compounds such as Na2CO3 or MgCO3 or using pyridine as the reaction medium. The high reactivity of acyl halides is used for the acylation of compounds with less reactive hydrogens. Certain carbonyl cyanides react similarly to acyl chlorides.

The disadvantage of generating a strong inorganic acid in the acylation with acyl halides also can be avoided by having, instead of the acyl halide, an anhydride. The reaction of Y:H with an anhydride takes place as follows:

ð22Þ

The acid resulting together with the acylated compound is not a strong acid such as HCl. The anhydrides of trifluoroacetic acid (TFA), pentafluoropropionic anhydride (PFPA), and heptafluorobutyric (HFBA) acids are commonly used for derivatization of alcohols, phenols, amines, etc., with the purpose of enhancing detectability (by ECD or NCI-MS) and also for improving the chromatographic behavior (higher volatility, better thermal stability, better separation). The volatility of fluorinated compounds allows the GC applications. The reactivity of the perfluorinated anhydrides increases in the order HFBA < PFPA < TFA. However, the differences are not significant. Once formed, the heptafluorobutyrates are more stable to hydrolysis than the trifluoroacetates. An inert solvent such as CH2Cl2, ether, ethyl acetate, acetone, tetrahydrofuran or in CH3CN, etc. can be used as a medium for the reaction with perfluoroanhydrides. For the neutralization of the acids formed during derivatization, the basic compounds such as triethylamine, pyridine, or even solid NaHCO3 can be utilized.

In order to avoid the formation of water or of a strong acid in the acylation reaction, certain amides such as N-methyl-bis(trifluoroacetamide), bis(trifluoroacetamide), or 2,2,2-trifluoro-N-methyl-N-(2,2,2-trifluoroacetyl)acetamide (MBTFA) can be used as reagents. Acylation of amines takes place at room temperature. Solvents such as CH3CN, pyridine, DMSO, or THF can be used as a reaction medium:

$$\overset{\text{R}}{\overset{\text{R}}{\text{N}}}\_{\text{R}} + \overset{\text{F}\_3\text{C}}{\overset{\text{R}}{\overset{\text{C}}{\text{N}}}}\_{\text{O}} \overset{\text{C}}{\overset{\text{C}}{\overset{\text{C}}{\text{N}}}}\_{\text{O}} \overset{\text{C}}{\overset{\text{C}\text{F}\_3}{\overset{\text{C}}{\text{N}}}}\_{\text{R}} \overset{\text{R}}{\overset{\text{R}}{\overset{\text{C}}{\text{N}}}}\_{\text{R}} \overset{\text{R}}{\overset{\text{C}}{\overset{\text{C}}{\text{N}}}}\_{\text{F}\_1} \overset{\text{C}}{\overset{\text{C}}{\overset{\text{C}}{\text{N}}}}\_{\text{F}\_2\text{C}} \overset{\text{O}}{\overset{\text{C}}{\overset{\text{C}}{\text{N}}}}\_{\text{N}} \overset{\text{O}}{\overset{\text{C}}{\overset{\text{C}}{\text{N}}}}\_{\text{N}} \overset{\text{O}}{\overset{\text{C}}{\overset{\text{C}}{\overset{\text{C}}{\text{N}}}}}\_{\text{H}} \overset{\text{O}}{\overset{\text{C}}{\overset{\text{C}}{\overset{\text{C}}{\text{N}}}}}\_{\text{H}} \overset{\text{O}}{\overset{\text{C}}{\overset{\text{C}}{\overset{\text{C}}{\overset{\text{C}}}}}}\_{\text{H}} \overset{\text{O}}{\overset{\text{C}}{\overset{\text{C}}{\overset{\text{C}}{\overset{\text{C}}}}}}\_{\text{H}} \overset{\text{O}}{\overset{\text{C}}{\overset{\text{C}}{\overset{\text{C}}{\overset{\text{C}}}}}}\_{\text{H}} \overset{\text{O}}{\overset{\text{C}}{\overset$$

One other procedure successfully applied to obtain acyl derivatives is the use of acyl imidazoles as reagents. This class of compounds reacts with analytes containing alcohol, primary and secondary amino groups, or thiols. The reaction generates as a by-product imidazole:

ð24Þ

Succinimidyl esters also can be used for acylation purposes. Amines and the amino group in amino acids also can be acylated using urethane-protected α-amino acid-N-carboxyanhydrides or oxycarbonyl-amino acid-N-carboxyanhydrides. Alkylketenes and their dimers may be used for acylation.

aldehydes and ketals with ketones, and although most of such compounds are not stable enough to be suitable for derivatization, cyclic acetals and ketals may be stable and used for analytical purposes. A common reaction of carbonyl compounds is with amines. The initial addition reaction usually continues with water elimination forming a substituted imine or a Schiff base. Similar to the reaction of amines is the reaction with substituted hydroxylamines or hydrazines. A typical reaction of derivatization of carbonyl compounds is that using dinitrophenylhydrazine (DNPH). The derivatized compound can be analyzed either by LC [27] or by

The groups R<sup>a</sup> and Rb can be H or alkyl or various other substituents.

in the presence of catalytic amounts of acetic acid. The resulting substituted

Another reagent that can be used for ketone derivatization is N-aminopiperidine

β-Diketones may react differently with hydrazines generating pyrazole deriva-

Several other classes of compounds similar to hydrazines react with the carbonyl

compounds. Among these are hydrazones (NH2─N═CR2), hydrazides (NH2NH-COR), and semicarbazide (NH2NH-CONH2). Hydroxylamines also react with carbonyl compounds forming oximes. Hydroxylamine itself, hydroxylamine hydrochloride (STOX® reagent), or derivatives such as H2N-OSO3H in a solvent

When the reaction is performed with hydroxylamine, the generated oxime contains an active hydrogen. This can be further derivatized, for example, by

For derivatization purposes other reagents can be used, such as substituted hydroxylamines like methoxyamine hydrochloride NH2OCH3•HCl (MOX® reagent) and O-(pentafluorobenzyl)-hydroxylamine hydrochloride (FLOROX® reagent). The reaction of a ketone or aldehyde with FLOROX is shown below:

ð26Þ

ð27Þ

ð28Þ

ð29Þ

GC/MS [28]. The reaction takes place as follows:

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

hydrazone can be used in GC analysis:

like pyridine can be used in this reaction:

silylation in a reaction with a common silylation reagent.

tives as shown below:

35

A special type of acylation is that using chloroformates. Carbonic acid, <sup>O</sup>═C(OH)2, can form amides, esters, halides, etc., due to the presence of two OH groups bonded to the CO group. Carbonic acid ester halides, also called chloroformates or chloroformate esters, with the formula R─O─C(═O)─X, where R is an alkyl or aryl group and X is F, Cl, Br, or I, can react with various compounds containing active hydrogens, such as acids [25], amines, alcohols, thiols, and amino acids. Amino acids, for example, in the presence of an alcohol in water form carbamate esters (urethanes) reacting as follows [26]:

ð25Þ

The formation in reaction (25) of the alcohol Ra –OH may lead to traces of a resulting compound with both substituted radicals being Ra . For this reason it is typically recommended to perform the reaction in the presence of an alcohol having the same radical as the chloroformate reagent (Ra = R<sup>b</sup> ). Chloroformates containing in the alkyl or aryl group halogen substituents are particularly reactive. Even tertiary amines can react with specific chloroformates, such as pentafluorobenzoyl chloroformate or with trichloroethyl chloroformate, by displacing an alkyl group connected to the nitrogen atom and forming the carbamate ester.

Similar in many respects to that of acyl derivatives R–CO–X are the reactions of sulfonyl derivatives R–SO2–X. Sulfonyl halides are in general less reactive than halides of carboxylic acids. The reaction of a sulfonyl derivative may take place with alcohols, phenols, amines, etc. The reactivity toward the sulfonyl sulfur is RNH2 > CH3COOR > H2O > ROH.

High reactivity toward active hydrogens in alcohols, amines, etc. can also be achieved using reagents with other functionalities. These functionalities include isocyanates, isothiocyanates, carbonyl azides, etc. These reactions can be seen as a replacement of an active hydrogen with a CO-R group or CS-R group as it occurs in other acylations.

### 10. Other derivatization reactions

A variety of other derivatization reactions are reported in the literature (see, e.g., [1]) and used for GC and GC/MS analyses. Among these are the addition to hetero multiple bonds in functional groups such as C═O, C═S, C═N, or C☰N. Many such reactions are additions to multiple bonds. Such reactions are, for example, the additions to the C═O groups in aldehydes and ketones. Reagents containing active hydrogens in groups such as NH2, OH, H2N-NH-, etc. can react, for example, with aldehydes and ketones. Alcohols, for example, form hemiacetals or acetals with

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

Succinimidyl esters also can be used for acylation purposes. Amines and the amino group in amino acids also can be acylated using urethane-protected α-amino acid-N-carboxyanhydrides or oxycarbonyl-amino acid-N-carboxyanhydrides.

chloroformates or chloroformate esters, with the formula R─O─C(═O)─X, where R is an alkyl or aryl group and X is F, Cl, Br, or I, can react with various compounds containing active hydrogens, such as acids [25], amines, alcohols, thiols, and amino acids. Amino acids, for example, in the presence of an alcohol in water form

ð25Þ

–OH may lead to traces

. For this reason

).

A special type of acylation is that using chloroformates. Carbonic acid, <sup>O</sup>═C(OH)2, can form amides, esters, halides, etc., due to the presence of two OH groups bonded to the CO group. Carbonic acid ester halides, also called

Alkylketenes and their dimers may be used for acylation.

Gas Chromatography - Derivatization, Sample Preparation, Application

carbamate esters (urethanes) reacting as follows [26]:

The formation in reaction (25) of the alcohol Ra

carbamate ester.

other acylations.

34

RNH2 > CH3COOR > H2O > ROH.

10. Other derivatization reactions

of a resulting compound with both substituted radicals being Ra

it is typically recommended to perform the reaction in the presence of an alcohol having the same radical as the chloroformate reagent (Ra = R<sup>b</sup>

Chloroformates containing in the alkyl or aryl group halogen substituents are particularly reactive. Even tertiary amines can react with specific chloroformates, such as pentafluorobenzoyl chloroformate or with trichloroethyl chloroformate, by displacing an alkyl group connected to the nitrogen atom and forming the

sulfonyl derivatives R–SO2–X. Sulfonyl halides are in general less reactive than halides of carboxylic acids. The reaction of a sulfonyl derivative may take place with

High reactivity toward active hydrogens in alcohols, amines, etc. can also be achieved using reagents with other functionalities. These functionalities include isocyanates, isothiocyanates, carbonyl azides, etc. These reactions can be seen as a replacement of an active hydrogen with a CO-R group or CS-R group as it occurs in

A variety of other derivatization reactions are reported in the literature (see, e.g., [1]) and used for GC and GC/MS analyses. Among these are the addition to hetero multiple bonds in functional groups such as C═O, C═S, C═N, or C☰N. Many such reactions are additions to multiple bonds. Such reactions are, for example, the additions to the C═O groups in aldehydes and ketones. Reagents containing active hydrogens in groups such as NH2, OH, H2N-NH-, etc. can react, for example, with aldehydes and ketones. Alcohols, for example, form hemiacetals or acetals with

alcohols, phenols, amines, etc. The reactivity toward the sulfonyl sulfur is

Similar in many respects to that of acyl derivatives R–CO–X are the reactions of

aldehydes and ketals with ketones, and although most of such compounds are not stable enough to be suitable for derivatization, cyclic acetals and ketals may be stable and used for analytical purposes. A common reaction of carbonyl compounds is with amines. The initial addition reaction usually continues with water elimination forming a substituted imine or a Schiff base. Similar to the reaction of amines is the reaction with substituted hydroxylamines or hydrazines. A typical reaction of derivatization of carbonyl compounds is that using dinitrophenylhydrazine (DNPH). The derivatized compound can be analyzed either by LC [27] or by GC/MS [28]. The reaction takes place as follows:

ð26Þ

The groups R<sup>a</sup> and Rb can be H or alkyl or various other substituents.

Another reagent that can be used for ketone derivatization is N-aminopiperidine in the presence of catalytic amounts of acetic acid. The resulting substituted hydrazone can be used in GC analysis:

$$\widehat{\sum}\_{\mathsf{R}} \mathsf{w} \leftarrow \mathsf{w} \xrightarrow{\mathsf{R}} \mathsf{x} \xrightarrow{\mathsf{R}} \overline{\mathsf{x} \quad \mathsf{c} \mathsf{w}} \quad \mathsf{c} \leftarrow \mathsf{w} \quad \mathsf{+} \mathsf{c} \xrightarrow{\mathsf{a}} \mathsf{x} \quad \mathsf{\*}$$

β-Diketones may react differently with hydrazines generating pyrazole derivatives as shown below:

$$\text{"}\_{\text{"}\_{\text{"}\_{\text{"}\_{\text{"}\_{\text{"}\_{\text{"}\_{\text{"}\_{\text{"}\_{\text{"}\_{\text{"}\_{\text{"}\_{\text{"}\_{\text{"}\_{\text{"}\_{\text{"}\_{\text{"}\_{\text{?}}}}}}}}}}}}}}} \bigfrown{}\_{\text{"}\_{\text{"}\_{\text{"}\_{\text{"}\_{\text{"}\_{\text{"}\_{\text{?}}}}}}} \bigfrown{\bigfrown{\text{"}\_{\text{"}\_{\text{"}\_{\text{?}}}}}} \stackrel{\text{~}\_{\text{"}\_{\text{"}\_{\text{?}\_{\text{?}}}}} \bigfrown{\bigfrown{\text{"}\_{\text{?}}}}} \stackrel{\text{~}\_{\text{"}\_{\text{?}\_{\text{?}\_{\text{?}}}}} \bigfrown{\bigfrown{\text{"}}\_{\text{"}\_{\text{?}\_{\text{?}}}}} \tag{28}$$

Several other classes of compounds similar to hydrazines react with the carbonyl compounds. Among these are hydrazones (NH2─N═CR2), hydrazides (NH2NH-COR), and semicarbazide (NH2NH-CONH2). Hydroxylamines also react with carbonyl compounds forming oximes. Hydroxylamine itself, hydroxylamine hydrochloride (STOX® reagent), or derivatives such as H2N-OSO3H in a solvent like pyridine can be used in this reaction:

$$\overset{\text{R}^{\mathsf{g}}}{\overset{\text{R}^{\mathsf{g}}}{}} \text{c} = \text{o} + \overset{\text{H}\_{\mathsf{H}}}{} \text{e} - \text{o} \text{H} \cdot \overset{\text{HCl}}{\longrightarrow} \overset{\text{R}^{\mathsf{g}}}{} \overset{\text{R}^{\mathsf{g}}}{} \text{e} - \overset{\text{H}}{} \overset{\text{OH}}{} + \text{HCl} + \text{H}\_{2}\text{O} \tag{29}$$

When the reaction is performed with hydroxylamine, the generated oxime contains an active hydrogen. This can be further derivatized, for example, by silylation in a reaction with a common silylation reagent.

For derivatization purposes other reagents can be used, such as substituted hydroxylamines like methoxyamine hydrochloride NH2OCH3•HCl (MOX® reagent) and O-(pentafluorobenzyl)-hydroxylamine hydrochloride (FLOROX® reagent). The reaction of a ketone or aldehyde with FLOROX is shown below:

Another reaction with formation of new cycles is that of amino acids with

This reaction has been successfully used for the analysis of amino acids in proteins [29, 30]. p-Bromophenyl isothiocyanate has been used in a similar

A variety of aromatic cycles can be formed in reactions involving bifunctional compounds. Addition reactions to hetero multiple bonds in bifunctional molecules frequently lead to cyclic compounds. For example, formaldehyde can react with tryptophan or tryptamine generating a β-carboline derivative as

The new compound can be analyzed by GC, usually after further derivatization

A typical reaction leading to pyrazoles is the reaction of hydrazines with diketones such as 2,4-pentandione (acetylacetone). For example, the reaction between hydrazine or methylhydrazine and acetylacetone takes place as

The resulting compound can be analyzed using a GC separation.

Activated carbonyl groups such as those in hexafluoroacetone are known to react with difunctional compounds. The reaction may take place with an amino acid

Amino acids can react with an activated anhydride such as trifluoroacetic

The reaction takes place by heating the amino acids with an excess of TFAA.

The reaction mixture is then dissolved in ethyl acetate and analyzed by GC.

ð34Þ

ð35Þ

ð36Þ

ð37Þ

ð38Þ

phenyl isothiocyanate leading to a thiohydantoin derivative:

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

reaction.

follows:

follows:

as follows:

37

anhydride (TFAA):

by silylation of the carboxyl group.

The oximes existing in sin- and anti- forms can produce double peaks in the GC chromatographic separations. To avoid this effect, oximes can be converted into nitriles when treated with acetic anhydride in the presence of CH3COONa. This reaction was used in the derivatization of carbohydrates when a simultaneous acetylation takes place (Wohl degradation). The reaction can be written as follows:

$$\begin{array}{ccccc} \text{R} - \text{C} - \text{OH} + \text{NH}\_2\text{OH} & \begin{array}{c} \text{C} - \text{N} - \text{OH} \\ \text{R} \end{array} & \begin{array}{c} \text{C} - \text{N} - \text{OH} \\ \text{R} \end{array} & \begin{array}{c} \text{CH}\_2\text{COOCH}\_2 \\ \text{C} \end{array} \\ \end{array} & \begin{array}{c} \text{CH}\_2\text{COOCH}\_2 \\ \text{R} \end{array} & \begin{array}{c} \text{CH}\_2\text{COOCH}\_2 \\ \text{R} \end{array} & \begin{array}{c} \text{CH}\_2\text{COOCH}\_2 \\ \text{R} \end{array} \\ \end{array} \end{array} \quad (31)$$

The transformation of the oximes into nitriles generates one single compound from the two (syn- and anti-) isomers and can be used to simplify the chromatograms of sugars derivatized as oximes.

Alcohols, amines, and thiols also can react at other hetero multiple bonds leading to analytical applications. This addition may occur at the isocyanates (─N═C═O), ─C═O group in an amide, at a nitrile, at CS2, or at other groups. One example is the addition under special conditions of alcohols to dimethylformamide. The resulting acetals are very reactive and are used themselves as reagents, as shown previously for N,N-dimethylformamide dimethyl acetal (see reaction 12). Another example is the reaction of CS2 with alcohols in the presence of a base, leading to the formation of xanthates. Amines also react with CS2, and the formed isothiocyanate can be analyzed using GC analysis. The reaction takes place as follows:

ð32Þ

Formation of new cycles from noncyclic compounds or replacement of old cycles with new ones that are more stable or have a desired property is also exploited in sample processing using derivatization. Epoxides, for example, can be formed in the reaction of a compound with a carbon–carbon double bond and a peroxy acid. Among the peroxy acids more frequently used for the formation of epoxides are peracetic, performic, perbenzoic, trifluoroperacetic, and 3,5 dinitroperoxybenzoic acids. However, in this reaction a mixture of enantiomers is formed, as shown below for a cis olefin:

The separation of the epoxides may be easier to achieve than that of olefins, and this type of derivatization has been utilized, for example, for better separation of various cis and trans unsaturated fatty esters.

ð30Þ

ð31Þ

ð32Þ

ð33Þ

The oximes existing in sin- and anti- forms can produce double peaks in the GC chromatographic separations. To avoid this effect, oximes can be converted into nitriles when treated with acetic anhydride in the presence of CH3COONa. This reaction was used in the derivatization of carbohydrates when a simultaneous acetylation takes place (Wohl degradation). The reaction can be written as follows:

Gas Chromatography - Derivatization, Sample Preparation, Application

The transformation of the oximes into nitriles generates one single compound from the two (syn- and anti-) isomers and can be used to simplify the chromato-

Alcohols, amines, and thiols also can react at other hetero multiple bonds leading to analytical applications. This addition may occur at the isocyanates (─N═C═O), ─C═O group in an amide, at a nitrile, at CS2, or at other groups. One example is the addition under special conditions of alcohols to dimethylformamide. The resulting acetals are very reactive and are used themselves as reagents, as shown previously for N,N-dimethylformamide dimethyl acetal (see reaction 12). Another example is the reaction of CS2 with alcohols in the presence of a base, leading to the formation of xanthates. Amines also react with CS2, and the formed isothiocyanate can be

Formation of new cycles from noncyclic compounds or replacement of old cycles with new ones that are more stable or have a desired property is also exploited in sample processing using derivatization. Epoxides, for example, can be formed in the reaction of a compound with a carbon–carbon double bond and a peroxy acid. Among the peroxy acids more frequently used for the formation of epoxides are peracetic, performic, perbenzoic, trifluoroperacetic, and 3,5-

dinitroperoxybenzoic acids. However, in this reaction a mixture of enantiomers is

The separation of the epoxides may be easier to achieve than that of olefins, and this type of derivatization has been utilized, for example, for better separation of

analyzed using GC analysis. The reaction takes place as follows:

grams of sugars derivatized as oximes.

formed, as shown below for a cis olefin:

various cis and trans unsaturated fatty esters.

36

Another reaction with formation of new cycles is that of amino acids with phenyl isothiocyanate leading to a thiohydantoin derivative:

ð34Þ

This reaction has been successfully used for the analysis of amino acids in proteins [29, 30]. p-Bromophenyl isothiocyanate has been used in a similar reaction.

A variety of aromatic cycles can be formed in reactions involving bifunctional compounds. Addition reactions to hetero multiple bonds in bifunctional molecules frequently lead to cyclic compounds. For example, formaldehyde can react with tryptophan or tryptamine generating a β-carboline derivative as follows:

$$\left\langle \bigcap\_{\lambda \mathbf{u}} \bigwedge\_{\mathbf{u}\_1 \dots \mathbf{u}\_m}^{\mathbf{u}\_1 \dots \mathbf{u}\_m} \longrightarrow \bigvee\_{\mathbf{u} \preccurlyeq \mathbf{u}} \right\rangle^{\mathbf{u}} \tag{35}$$

The new compound can be analyzed by GC, usually after further derivatization by silylation of the carboxyl group.

A typical reaction leading to pyrazoles is the reaction of hydrazines with diketones such as 2,4-pentandione (acetylacetone). For example, the reaction between hydrazine or methylhydrazine and acetylacetone takes place as follows:

ð36Þ

The resulting compound can be analyzed using a GC separation.

Activated carbonyl groups such as those in hexafluoroacetone are known to react with difunctional compounds. The reaction may take place with an amino acid as follows:

$$\mathbf{c}^{\alpha\_{\rho}}\_{\text{c}} \overset{\text{\tiny\text{\textquotedblleft}}{\text{\textquotedblleft}}}{\text{\textquotedblleft}} \text{\textquotedblright} \mathbf{c} \overset{\text{\textquotedblleft}}{\text{\textquotedblleft}} \text{\textquotedblright} \mathbf{c} \overset{\text{\textquotedblleft}}{\text{\textquotedblleft}} \text{\textquotedblright} \mathbf{c} \overset{\text{\textquotedblleft}}{\text{\textquotedblleft}} \text{\textquotedblright} \mathbf{c} \overset{\text{\textquotedblleft}}{\text{\textquotedblleft}} \text{\textquotedblright} \mathbf{c} \overset{\text{\textquotedblleft}}{\text{\textquotedblleft}} \text{\textquotedblright} \mathbf{c} \overset{\text{\textquotedblleft}}{\text{\textquotedblleft}} \text{\textquotedblright} \mathbf{c} \overset{\text{\textquotedblleft}}{\text{\textquotedblleft}} \text{\textquotedblright} \mathbf{c} \overset{\text{\textquotedblleft}}{\text{\textquotedblleft}} \text{\textquotedbl} \mathbf{c} \overset{\text{\textquotedblleft}}{\text{\textquotedblleft}} \text{\textquotedbl} \mathbf{c} \overset{\text{\textquotedblleft}}{\text{\textquotedblleft}} \text{\textquotedbl} \mathbf{c} \overset{\text{\textquotedblleft}}{\text{\textquotedbl}} \mathbf{c} \overset{\text{\textquotedblleft}}{\text{\textquotedbl}} \mathbf{c} \overset{\text{\textquotedblleft}}{\text{\textquotedbl}} \mathbf{c} \overset{\text{\textquotedblleft}}{\text{\textquotedbl}} \mathbf{c} \overset{\text{\textquotedblleft}}{\text{\textquotedbl}} \mathbf{c} \overset{\text{\textquotedbl}}{\text{\textquotedbl}}}{\text{\textquotedbl}} \mathbf{c} \overset{\text{\textquotedbl}}{\text{\textquotedbl}}}{\text{\text{\textqu$$

Amino acids can react with an activated anhydride such as trifluoroacetic anhydride (TFAA):

$$\text{C}^{\text{O}}\_{\text{C}}\text{C}^{\text{OH}}\_{\text{H}} + \text{H}\_{\text{O}}\text{C}^{\text{O}} \overset{\text{O}^{\text{O}}}{\text{C}^{\text{O}}}\_{\text{O}} \overset{\text{O}^{\text{O}}}{\text{C}^{\text{O}}}\_{\text{O}} \overset{\text{O}^{\text{O}}}{\text{C}^{\text{O}}}\_{\text{O}} \overset{\text{O}^{\text{O}}}{\text{C}^{\text{O}}}\_{\text{O}} + \text{H}\_{\text{O}}\text{C}^{\text{O}} \overset{\text{O}^{\text{O}}}{\text{C}^{\text{O}}}\_{\text{H}}$$

The reaction takes place by heating the amino acids with an excess of TFAA. The reaction mixture is then dissolved in ethyl acetate and analyzed by GC.

Numerous other types of derivatization reactions were used for making the analytes suitable for GC and GC/MS analyses. These include formation of various cyclic types of compounds such as azines, siliconides, boronates, etc., that are thermally stable and do not have polar hydrogens such that GC or GC/MS analysis is possible. In addition to reagents that add specific moieties to the analytes, oxidation and reduction were sometimes used for the analyte modification (see, e.g., [4]).
