3. Derivatization for chiral separation in gas chromatography

The compounds with structures that are mirror images to each other are indicated as enantiomers, and their molecules are not superimposable, having the property called chirality. Chirality is commonly caused by the existence in the molecule of at least one tetrahedral carbon atom substituted with groups that are different. However, chiral molecules may be generated with a phosphorus or a sulfur chiral atom. Not only chiral centers (such as an asymmetric carbon) generate enantiomers, but a chiral axis or a chiral plane can lead to enantiomers. The chirality in an enantiomer is specified using the symbols R and S based on specific rules. For the assignment of a symbol R or S to a chiral carbon, the substituents are arranged in a sequence a > b > c > d. For the four atoms directly attached to the asymmetric carbon, a higher atomic number outranks the lower, and a higher atomic mass outranks the lower mass. For the same atoms directly attached to the asymmetric carbon, the priorities are assigned at the first point of difference. After the sequence is established, the molecule is oriented in space with the group "d" of the lowest priority behind the asymmetric carbon. When viewed along the C─d bond (from C) and the three substituents a, b, and c are oriented clockwise, the compound contains an R asymmetric carbon, and it contains an S asymmetric carbon for counterclockwise arrangement.

More than one asymmetric carbon can be present in a molecule, as in the case of carbohydrates. The stereoisomers generated by more than one asymmetric carbon can be mirror image one to the other (enantiomers) or may have different steric arrangements being diastereoisomers. These types of molecules are schematically shown in Figure 3.

The (S,S)- and the (R,R)-compounds from Figure 3 are enantiomers, while the (S,R)-compound is a diastereoisomer to both (S,S)- and to (R,R)-compounds (it is an enantiomer to the (R,S)-compound). The gas chromatographic separation of enantiomers can be done only using chromatographic columns having chiral stationary phases. The derivatization of enantiomers with non-chiral reagents generates molecules that remain enantiomers. This type of derivatization may improve the chromatographic separation from other molecules, but the derivatized compounds of remaining enantiomers cannot be separated except on chiral stationary phases. Sometimes, better separation can be obtained even between the enantiomers (on chiral chromatographic columns) after derivatization. One such example is the separation of (R)- and (S)-nornicotine derivatized with isobutyl chloroformate on a chiral Rt-BDEXsm column with separation improved compared to that of underivatized enantiomers [6]. The derivatization reaction is indicated below:

Figure 3. Compounds with two chiral centers.

Diastereoisomers can be separated on chromatographic columns with non-chiral stationary phases which offer a much wider possibility to select the column. For this reason, an alternative procedure toward the separation of enantiomers is using derivatization with chiral reagents. This type of derivatization generates diastereoisomers which can be separated on non-chiral stationary phases.

4. Derivatization for improving gas chromatographic detection with

mal conductivity detector (TCD), flame ionization detector (FID), nitrogenphosphorus detector (NPD), electron capture detector (ECD), flame photometric detector (FPD), photoionization detector (PID), electrolytic conductivity (Hall), sulfur chemiluminescence, nitrogen chemiluminescence, aroyl luminescence detector (ALD), atomic emission detector (AED), helium ionization detector (HID), vacuum ultraviolet (VUV) absorbance, infrared Doppler (IRD) absorption, FID with catalytic conversion of all analytes in CH4 (e.g., Polyarc system [9]), etc. The derivatization with the purpose of improving detectability in GC is determined by the type of detector utilized. Most derivatizations are performed precolumn, even if they are applied only with the purpose of improving detection. However, it is important that the derivatization for improving detection does not deteriorate the separation. Preferably, both the detection and the chromatographic separation are improved by the same derivatization. Some specific postcolumn reactions applied to the analytes are part of certain types of detectors such as chemiluminescence detectors, atomic emission detectors (AED), and FID with catalytic conversion into CH4. Some of these chemical changes in

the analytes are not necessarily classified as derivatization reactions.

derivatization for enhancing PID response is not frequently used.

ECD this process can be written as follows:

15

halogenated compounds, derivatization can be used to enhance detection.

common. An adverse result occurs for the NPD detectors when silylation is performed on the sample. Besides a possible reduction in the NPD response on silylated compounds containing nitrogen, a drastic decrease in the lifetime of the detector may occur, probably due to the excess of silylating reagent that commonly is injected with a derivatized sample and affects the alkali active element of the NPD. The response of the photoionization detector (PID) depends on the ionization potential of the analyte, and compounds with higher ionization potential are not sensitive in PID, while those with lower ionization potential may have excellent sensitivity, as low as 10�<sup>12</sup> mg of sample. A derivatization resulting in lowering the ionization potential of the analyte may be beneficial for PID detection. However,

No specific derivatization is usually recommended to improve sensitivity when using nonselective detectors such as TCD and FID. However, in some cases when the detector is not sensitive to a specific analyte, such as formaldehyde or heavily

In case of NPD detector, derivatization with nitrogenous compounds can be done, which should give a higher sensitivity. However, this type of derivatization is not very

Some detectors such as electron capture detectors (ECD) may benefit very much

With some exceptions, ECD response can be correlated with the electron affinity of the analyte [4]. In general, the halogen substituents increase the sensitivity in ECD

<sup>A</sup> <sup>þ</sup> <sup>e</sup>– ! <sup>A</sup>– (5)

from certain derivatization types. ECD (as well as negative chemical ionization mass spectrometry or NCI-MS) can be extremely sensitive, but they are selective to compounds that are able to form more stable negative ions. ECD, for example, can have sensitivity as low as 10�<sup>13</sup> mg of analyte in the detector compared to the best sensitivity of FID that can be 10�<sup>8</sup> to 10�<sup>11</sup> mg of analyte. The efficiency of the process seems to be related to the ease of attaching an electron on the molecule. In

Gas chromatography (not coupled with mass spectrometry, GC/MS being separately presented) used as an analytical technique can involve various detectors. The variety of such detectors is rather large, and several types include the following: ther-

other detectors than MS

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

A discussion on the separation of enantiomers on chiral phases without derivatization is beyond the purpose of this chapter. Numerous publications are dedicated to this subject, including papers published in general chromatography journals or in dedicated journals (e.g., Chirality), books (see, e.g., [7]), and information on the web.

The separation after derivatization with a pure enantiomer reagent is based on formation of diastereoisomers that can be separated on regular stationary phases. Depending on the nature of the analyte and of the derivatization, different separation techniques can be applied. A variety of common columns are used for such GC separations. The choice of the column is again dependent on the analyte and the derivatization procedure. For example, α-substituted organic acids such as αchloropropionic, α-bromocaproic, etc. can be derivatized with a specific enantiomer of an amino acid ester (e.g., ethyl 2-aminopropanoate) in the presence of a peptide coupling reagent (benzotriazol-1-yl-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate or BOP) in a reaction of the type:

The derivatized acids that are now diastereoisomers (R,S) and (S,S) can be separated on a common capillary column (e.g., a DB-1701 column from Agilent). Another example of derivatization with a chiral reagent is that of methamphetamines with (R)-menthyl chloroformate. This derivatization allows the separation of over-the-counter (R)-methamphetamine from the illicit (S)-methamphetamine. The reaction of the (R)-enantiomer is indicated below [8]:

The separation of the (R,R) and (S,R) derivatives was possible on a non-chiral column for a GC/MS analysis.

Diastereoisomers can be separated on chromatographic columns with non-chiral stationary phases which offer a much wider possibility to select the column. For this reason, an alternative procedure toward the separation of enantiomers is using derivatization with chiral reagents. This type of derivatization generates diastereo-

A discussion on the separation of enantiomers on chiral phases without derivatization is beyond the purpose of this chapter. Numerous publications are dedicated to this subject, including papers published in general chromatography journals or in dedicated journals (e.g., Chirality), books (see, e.g., [7]), and information on

The separation after derivatization with a pure enantiomer reagent is based on formation of diastereoisomers that can be separated on regular stationary phases. Depending on the nature of the analyte and of the derivatization, different separation techniques can be applied. A variety of common columns are used for such GC separations. The choice of the column is again dependent on the analyte and the derivatization procedure. For example, α-substituted organic acids such as αchloropropionic, α-bromocaproic, etc. can be derivatized with a specific enantiomer of an amino acid ester (e.g., ethyl 2-aminopropanoate) in the presence of a peptide coupling reagent (benzotriazol-1-yl-oxy-tris(dimethylamino)-phosphonium

The derivatized acids that are now diastereoisomers (R,S) and (S,S) can be separated on a common capillary column (e.g., a DB-1701 column from Agilent). Another example of derivatization with a chiral reagent is that of methamphetamines with (R)-menthyl chloroformate. This derivatization allows the separation of over-the-counter (R)-methamphetamine from the illicit (S)-methamphetamine.

The separation of the (R,R) and (S,R) derivatives was possible on a non-chiral

ð3Þ

ð4Þ

isomers which can be separated on non-chiral stationary phases.

Gas Chromatography - Derivatization, Sample Preparation, Application

hexafluorophosphate or BOP) in a reaction of the type:

The reaction of the (R)-enantiomer is indicated below [8]:

column for a GC/MS analysis.

14

the web.
