1. General comments

Two specific trends can be noticed in modern chemical analysis. One is the continuous demand for more sensitive and accurate analytical methods. The other is the desire for simpler methods that require as little as possible human intervention. One of the various procedures to make the analytical methods more sensitive and accurate is the use of specific chemical changes (e.g., derivatization) applied on the analytes or even on the whole sample. However, these changes frequently involve more human intervention than the direct use of advanced instrumentation. For this reason, the methods involving chemical changes such as derivatizations are not necessarily the first choice when selecting an analytical method. Nevertheless, in many cases, the benefits of derivatization are more important than the disadvantage of requiring human intervention, and for this reason, derivatization is still frequently used in the analytical practice. Also, modern GC, GC/MS (or GC/MS/MS) instrumentation may offer autosampling with the capability of adding reagents to the sample, as well as stirring, heating, and injecting the sample at specific time intervals in the GC system. This type of instrumentation may reduce significantly the human handling involved in derivatization.

Various chemical changes can be performed on an analyte in order to make it suitable for a specific method of analysis. The most common is derivatization, but other chemical changes can be utilized, for example, pyrolytic decomposition and, in the case of polymers, polymer fragmentation using reagents. The choice depends on the nature of the analyte, the sample matrix, the intended changes in the analyte properties, and the analytical method to be used.

2. Derivatization for improving separation in gas chromatography

GC separation are the following:

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

simplified form as follows:

Figure 1.

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For GC analysis, the effect of derivatization can be beneficial in a variety of circumstances. Some of the most common uses of derivatization for improving the

(a) Derivatization that replaces active (polar) hydrogen atoms in the analyte to decrease its boiling point. The active hydrogens in a chemical compound typically enhance the capability to form hydrogen bonds and increase the compound polarity. For this reason, many compounds containing active (polar) hydrogens are not volatile, the volatility being necessary for using GC or GC/MS as a core analytical method. Derivatization can be used to replace active hydrogens from an analyte Y-H (or Y:H) in functional groups such as OH, COOH, SH, NH, and CONH. These reactions can be written in a

In reaction (1), the reagent R-X contains an "active" group X and a group R that carries a desired property (e.g., lack of polarity for GC). Group R in the reagent can be a low molecular mass fragment such as CH3 or C2H5, a shortchain fluorinated alkyl in alkylation reactions, Si(CH3)3 or other silyl groups in silylations, COCH3 or short-chain fluorinated acyl groups in acylations, etc. An example of a chromatogram resulting from the GC/MS analysis of a silylated tobacco sample is given in Figure 1. Tobacco contains many hydroxy acids such as malic, trihydroxybutanoic, citric, quinic, glucuronic, and chlorogenic. Also, it contains monosaccharides (e.g., glucose, fructose), disaccharides (e.g., sucrose), and even trisaccharides. None of these compounds are volatile, having numerous active hydrogens. The replacement of these hydrogens with Si(CH3)3 by silylation renders these compounds vola-

tile, and they can be analyzed by GC/MS as seen in Figure 1.

(b) Derivatization for enhancing the separation. Specific moieties added to an analyte may be necessary for enhancing the separation. This is frequently practiced for general GC separations and is also very useful for the

GC/MS chromatogram of a silylated tobacco sample, with separation on a DB-5 MS column from Agilent (Agilent Technologies Inc., Wilmington, DE, USA) (Note: an internal standard I.S. was added to the sample).

separation of chiral molecules (see Section 4). The derivatized analytes may have significantly different properties from each other, for example,

<sup>Y</sup>‐<sup>H</sup> <sup>þ</sup> <sup>R</sup> � <sup>X</sup> ! <sup>Y</sup>‐<sup>R</sup> <sup>þ</sup> HX (1)

The addition of a reagent on a sample may produce a chemical reaction only with the analytes without affecting the matrix. However, it is also possible that some matrix components are derivatized unintentionally. Usually, it is preferable to have only the analytes derivatized since in this way a better separation from the matrix is expected. Some derivatizations are used in the sample cleanup or concentration process. Also, the derivatization process may be combined with simultaneous extraction and concentration of the sample or may be followed by a second preparation step before the chromatographic analysis. More frequently, the derivatization is done to change the analyte properties for the core analytical procedure (GC, GC/MS, etc.).

Derivatization can be applied before the core chromatographic process or after it. Precolumn derivatization takes place before the separation and postcolumn derivatization after it. In GC precolumn derivatization is much more common and most derivatizations are performed "offline." There are however derivatizations that can be done "online," for example, in the injection port of the GC such as some methylations using tetramethyl ammonium hydroxide (TMAH). Postcolumn derivatizations are performed only for enhancing the detectability of the analytes. Typically, they must be done "online" and should be completed in the specific time frame needed by the analyte to reach the detector.

A wide variety of derivatization reagents and procedures are described in the literature, with the reagents carrying specific moieties that provide a desired property to the analytes, as well as with specific reactive groups that permit the reaction with the analyte. Multiple step derivatizations as well as derivatizations followed by a second one are known.

Derivatization is not always the first step in sample preparation. Sample preparation typically includes other operations, besides derivatization. Some of these steps are more complex such as sample cleanup or concentration and others more simple such as pH adjustments, addition of proton acceptors or donors, change of the medium (from one solvent to another), and addition of catalysts to enhance the derivatization, and these may be necessary for a successful derivatization.

Although derivatization is performed in order to make possible or to improve the results of a chemical analysis, there are also some disadvantages of using derivatization. Besides the potential need of more manpower for the analysis, the addition of more operations applied on the sample (including the analytes) can be a source of additional errors. In particular the involvement of a chemical reaction that may not be perfectly controlled can bring significant errors in the analytical results. To minimize the potential errors when using derivatization, specific aspects of the derivatization must be considered in its choice, such as the efficiency of the chemical reaction used in the derivatization, the stability of the derivatized analytes, the availability of reagents and necessary equipment, and the time necessary for performing the analysis. For a given analyte or group of analytes, the reaction with the derivatization reagent must be complete or at least close to complete, must take place in a length of time that is not prohibitive, and must have very little loss of the analyte with formation of artifacts or decomposition products. Only when such criteria are satisfied can a specific chosen derivatization be applied successfully.

The application of derivatization in chromatography is the subject of many studies. Numerous derivatizations have been reported in journals (e.g., J. Chromatogr. A and B, J. Chromatogr. Sci., J. Sep. Sci., Chromatographia, etc.), in various books [1–5], in application notes of instrument manufacturers, as well as on the web.

other chemical changes can be utilized, for example, pyrolytic decomposition and, in the case of polymers, polymer fragmentation using reagents. The choice depends on the nature of the analyte, the sample matrix, the intended changes in the analyte

The addition of a reagent on a sample may produce a chemical reaction only with the analytes without affecting the matrix. However, it is also possible that some matrix components are derivatized unintentionally. Usually, it is preferable to have only the analytes derivatized since in this way a better separation from the matrix is expected. Some derivatizations are used in the sample cleanup or concentration process. Also, the derivatization process may be combined with simultaneous extraction and concentration of the sample or may be followed by a second preparation step before the chromatographic analysis. More frequently, the derivatization is done to change the analyte properties for the core analytical procedure

Derivatization can be applied before the core chromatographic process or after it. Precolumn derivatization takes place before the separation and postcolumn derivatization after it. In GC precolumn derivatization is much more common and most derivatizations are performed "offline." There are however derivatizations that can be done "online," for example, in the injection port of the GC such as some methylations using tetramethyl ammonium hydroxide (TMAH). Postcolumn derivatizations are performed only for enhancing the detectability of the analytes. Typically, they must be done "online" and should be completed in the specific time

A wide variety of derivatization reagents and procedures are described in the literature, with the reagents carrying specific moieties that provide a desired property to the analytes, as well as with specific reactive groups that permit the reaction with the analyte. Multiple step derivatizations as well as derivatizations followed by

Derivatization is not always the first step in sample preparation. Sample preparation typically includes other operations, besides derivatization. Some of these steps are more complex such as sample cleanup or concentration and others more simple such as pH adjustments, addition of proton acceptors or donors, change of the medium (from one solvent to another), and addition of catalysts to enhance the

Although derivatization is performed in order to make possible or to improve

The application of derivatization in chromatography is the subject of many studies. Numerous derivatizations have been reported in journals (e.g., J. Chromatogr. A and B, J. Chromatogr. Sci., J. Sep. Sci., Chromatographia, etc.), in various books [1–5],

in application notes of instrument manufacturers, as well as on the web.

derivatization, and these may be necessary for a successful derivatization.

the results of a chemical analysis, there are also some disadvantages of using derivatization. Besides the potential need of more manpower for the analysis, the addition of more operations applied on the sample (including the analytes) can be a source of additional errors. In particular the involvement of a chemical reaction that may not be perfectly controlled can bring significant errors in the analytical results. To minimize the potential errors when using derivatization, specific aspects of the derivatization must be considered in its choice, such as the efficiency of the chemical reaction used in the derivatization, the stability of the derivatized analytes, the availability of reagents and necessary equipment, and the time necessary for performing the analysis. For a given analyte or group of analytes, the reaction with the derivatization reagent must be complete or at least close to complete, must take place in a length of time that is not prohibitive, and must have very little loss of the analyte with formation of artifacts or decomposition products. Only when such criteria are satisfied can a specific chosen deriva-

properties, and the analytical method to be used.

Gas Chromatography - Derivatization, Sample Preparation, Application

frame needed by the analyte to reach the detector.

(GC, GC/MS, etc.).

a second one are known.

tization be applied successfully.

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