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Chapter

Derivatization Methods in GC
and GC/MS
Serban C. Moldoveanu and Victor David

Abstract

The first part of this chapter presents the main objectives for performing
derivatization of a sample to be analyzed by gas chromatography (GC) or gas
chromatography/mass spectrometry (GC/MS). The derivatization is typically
done to change the analyte properties for a better separation and also for enhancing
the method sensitivity. In GC/MS, derivatization may improve the capability of
compound identification. Examples illustrating such improvements are included.
The second part describes several types of derivatization that are more frequently
used in analytical practice. These include alkylation (e.g., methylation), formation
of aryl derivatives, silylation (e.g., formation of trimethylsilyl derivatives), acyla-
tion (e.g., reactions with acyl chlorides or with chloroformates), and several other
types of derivatizations. The chapter also presents typical derivatizations for
analytes with specific functional groups and discusses artifact formation in certain
derivatization reactions.

Keywords: gas chromatography, mass spectrometry, derivatization, alkylation,

aryl derivatives, silylation, acylation

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 fre-
quently 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

1
Gas Chromatography

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.
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 concen-
tration process. Also, the derivatization process may be combined with simulta-
neous extraction and concentration of the sample or may be followed by a second
preparation step before the chromatographic analysis. More frequently, the deriva-
tization 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 prop-
erty 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 prepa-
ration 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 effi-
ciency 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 decomposi-
tion products. Only when such criteria are satisfied can a specific chosen deriva-
tization be applied successfully.
The application of derivatization in chromatography is the subject of many stud-
ies. 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.
2
Derivatization Methods in GC and GC/MS
DOI: http://dx.doi.org/10.5772/intechopen.81954

2. Derivatization for improving separation in gas chromatography

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
GC separation are the following:
(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
simplified form as follows:

Y‐H þ R  X ! Y‐R þ HX (1)

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

Figure 1.
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).

3
Gas Chromatography

regarding polarity and implicitly in their boiling point, allowing separations

that are difficult to achieve otherwise. Also, derivatization may generate
more significant differences between the analytes and the matrix
components.
(c) Derivatization that replaces active hydrogens in the analyte to improve the
behavior of the analyte in the chromatographic separation. The
chromatographic column (e.g., a capillary column coated with a bonded
stationary phase) may display additional capability to interact with polar
molecules, besides the intended interactions due to its bonded phase. This
may come, for example, from the silica wall of the column. Secondary
interactions taking place with only a portion of the molecules of the analyte
generate peak tailing. This is exemplified in Figure 2 which shows a
hypothetical case of two different types of interaction between the column
and a specific molecular species.

(d) Derivatization for the improvement of stability of a compound. This

stability may refer to thermal stability, a property which overlaps to a
certain extent to what was described at point (a). However, even some
volatile compounds may be further thermally stabilized by derivatization.
Also, chemical stability can be enhanced by protecting specific groups in the
analyte using derivatization. For example, thiols can be protected using
derivatization against oxidation by the traces of oxygen in the heated
injection port of the GC.

The choice of the appropriate derivatization is not always a simple task. The
replacement of a hydrogen atom with a group of atoms may increase the molecular
weight of the derivatized analyte. In such cases, it must be verified that the increase
in the molecular weight by derivatization brings no or only a small increase in the
boiling point of the analyte. Most of the time, low molecular weight substituents
such as CH3 or Si(CH3)3 are preferable for GC analysis to the active hydrogens for
achieving the previously described goals. Large substituents may increase the boil-
ing point too much and make the compound not acceptable for GC analysis.
Besides replacement of active hydrogens, other derivatization reactions can be
utilized. For example, condensation reactions may decrease the boiling point and
improve the thermal stability of an analyte. However, the generation of new active
hydrogens must be avoided in condensation reactions or must be followed by a
second derivatization.

Figure 2.
Peak tailing due to multiple retention mechanisms.

4
Derivatization Methods in GC and GC/MS
DOI: http://dx.doi.org/10.5772/intechopen.81954

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. How-
ever, 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 sub-
stituents 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 enan-
tiomers can be done only using chromatographic columns having chiral stationary
phases. The derivatization of enantiomers with non-chiral reagents generates mole-
cules that remain enantiomers. This type of derivatization may improve the chro-
matographic 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 sepa-
ration 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.

ð2Þ

5
Gas Chromatography

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-
isomers which can be separated on non-chiral stationary phases.
A discussion on the separation of enantiomers on chiral phases without deriva-
tization 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 separa-
tion 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 α-
chloropropionic, 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:

ð3Þ

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 methamphet-
amines 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]:

ð4Þ

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

6
Derivatization Methods in GC and GC/MS
DOI: http://dx.doi.org/10.5772/intechopen.81954

4. Derivatization for improving gas chromatographic detection with

other detectors than MS

Gas chromatography (not coupled with mass spectrometry, GC/MS being sepa-
rately 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-
mal conductivity detector (TCD), flame ionization detector (FID), nitrogen-
phosphorus detector (NPD), electron capture detector (ECD), flame photometric
detector (FPD), photoionization detector (PID), electrolytic conductivity (Hall), sul-
fur 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 derivatiza-
tion for improving detection does not deteriorate the separation. Preferably, both the
detection and the chromatographic separation are improved by the same derivatiza-
tion. 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.
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
halogenated compounds, derivatization can be used to enhance detection.
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
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 1012 mg of sample. A derivatization resulting in lowering the
ionization potential of the analyte may be beneficial for PID detection. However,
derivatization for enhancing PID response is not frequently used.
Some detectors such as electron capture detectors (ECD) may benefit very much
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 1013 mg of analyte in the detector compared to the best
sensitivity of FID that can be 108 to 1011 mg of analyte. The efficiency of the
process seems to be related to the ease of attaching an electron on the molecule. In
ECD this process can be written as follows:

A þ e– ! A– (5)

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

7
Gas Chromatography

in the order I > Br > Cl > F. Multiple substitutions seem to have a cumulative effect.
Besides halogens, nitro groups seem to have an effect similar to chlorine groups. For
aromatic compounds, the substituents affect the sensitivity of the ECD according to
their electron withdrawing capability. Strong electron withdrawing groups such as
NO2 increase the sensitivity of the detection, while electron donating groups reduce it.
A variety of substitution groups containing electronegative elements (halogens)
or nitro groups can be attached to an analyte. The procedure to attach these groups
is in most cases the typical substitution of an active hydrogen in the analyte Y-H
with a group R from a reagent R-X that has the appropriate active X group. Some
groups used for enhancing ECD (as well as NCI-MS) sensitivity following an alkyl-
ation or aryl derivatization reaction are shown in Figure 4, and several substitution
groups introduced by acylation, chloroformylation, or sulfonation used for the same
purpose are shown in Figure 5. Besides alkylation or aryl derivatization, other
derivatization techniques used to replace an active hydrogen are applied to intro-
duce into a molecule as a substituent containing halogens or nitro groups enhancing

Figure 4.
Substitution groups used in alkylation and aryl derivatization for enhancing ECD (and NCI-MS) detectability
(the masses are considered only for the most abundant isotope.).

Figure 5.
Substitution groups used in acylation chloroformation and sulfonation for enhancing ECD (and NCI-MS)
detectability.

8
Derivatization Methods in GC and GC/MS
DOI: http://dx.doi.org/10.5772/intechopen.81954

Figure 6.
Substitution groups used in silylation for enhancing ECD (and NCI-MS) detectability.

significantly the detectability of the derivatized analytes by ECD (as well as NCI-
MS). Silylation, for example, can be used for this purpose when silyl groups used for
derivatization contain halogens. Several silyl groups containing halogens that can be
attached to an analyte by silylation with special reagents are given in Figure 6 [4].

5. Derivatization for improving GC/MS qualitative and quantitative

analysis

The most powerful tool used for compound identification purposes is very likely
mass spectrometry (spectroscopy). This technique is capable to provide informa-
tion from very low amounts of material such as that eluting from a chromatographic
column and can be easily coupled with a gas chromatograph. Most analyses
performed with MS detection (GC/MS or GC/MS/MS) are using EI+ ionization
mode with electron impact at 70 eV. The electrons interact with the molecule A to
eject an additional electron leaving a positively charged species (with an odd num-
ber of electrons) of the type A▪+. The ions also receive energy during electron
impact and the excess of energy induces fragmentation. For most molecules, this
process can be written as follows:

A þ e– ! A▪þ þ 2e– and A▪þ ! Bi þ þ Ci ▪ (6)

The fragments Bi+ are commonly but not always with an even number of elec-
trons. The formation of molecular ions takes place with a range of internal energies,
and more than one fragmentation path is possible for a given molecule. Also, the
fragments can suffer further fragmentations. In general, the most abundant frag-
ment ion results from the fragmentations that form the most stable products (ion
and neutral radical). The abundance of a fragment ion is affected by its stability. For
this reason, the intensity of the response of a mass spectrometric detector can be
very different for different molecular species, and the prediction of this intensity is
difficult. As a result, the improvements in the sensitivity in EI + type mass
spectrometry (in GC/MS using EI+ ionization) are not usually sought (but not
impossible) through derivatization.
Derivatization for enhancing sensitivity is, however, frequently applied in NCI-
MS. In this technique, the electrons interact with the molecules of the CI gas which
is lowering their energy but without forming ions. The ionization of analyte mole-
cules takes place by interaction with the low-energy electrons or with already
formed negative ions by electron capture, dissociative electron capture, ion pair
formation, or ion molecule reaction. The ionization process with the formation of

9
Gas Chromatography

negative ions is efficient only for molecules with positive electron affinities. For this
reason, the sensitivity in NCI-MS is highly dependent on the electron affinity of the
analyte, similarly to the sensitivity in ECD. For enhancing the electron affinity, the
derivatization with reagents containing, for example, fluorinated moieties (indi-
cated in Figures 4–6) is practiced. The sensitivity of the analytical methods where
such derivatization is applicable can have very good sensitivity. For example,
derivatization with heptafluorobutyric anhydride of aromatic amines that are pre-
sent at low trace level in cigarette smoke leads to limit of detection (LOD) values as
low as 0.05 ng/cig. for compounds such as 4-aminobiphenyl [10, 11].
The fragmentation pattern generated by EI+ ionization mode that generates a
specific mass spectrum of a molecule is very likely the most utilized technique for
the identification of the molecular species. For this identification, large libraries of
mass spectra are available, and computer algorithms are used for automatic
searches. The identification of compounds using mass spectroscopy is not a simple
process even with the capabilities offered by the electronic searches in the mass
spectral libraries. This is particularly true for analysis of complex mixtures or when
the analyzed compound is present in traces. Some compounds do not have a very
characteristic mass spectrum, or during the chromatographic process, the separa-
tion is not achieved, and it is difficult to make an identification due to the spectra
overlapping. Also, numerous compounds may have a mass spectrum that matches
more than one compound (with a good quality fit). In such cases, a derivatization
with the purpose of obtaining a compound that forms more informative fragments
in the mass spectrum can be very useful.
The fragments from derivatized compounds can be used for the identification of
unknown compounds using library searches and even when the mass spectrum is
not available in the libraries. As an example, the derivatization by silylation allowed
the identification of a new pentacyclic triterpenoid present in several bioactive
botanicals [12]. An unidentified compound with MW = 456.7 was detected by LC/
MS/MS in a rosemary extract. The structure of the compound was elucidated after
silylation of the plant material based on the comparison of mass spectrum of the
unidentified compound with that of silylated betulinic acid. The new compound
was identified as (3β)-3-hydroxy-lupa-18,20(29)-dien-28-oic acid (or betul-18-en-
oic acid). The mass spectra of the two acids are shown in Figure 7.
The two mass units difference between different fragments from the mass
spectra of the two compounds allowed the identification of the new compound
structure. Neither free betulinic acid nor betul-18-en-oic acid are volatile, such
that the use of GC/MS for identification was possible only after derivatization.
Another special procedure that may be utilized for compound identification
based on mass spectra is the use of two parallel derivatizations, one of them
being done with an isotope-labeled reagent. Common labeling isotopes are
2
H (deuterium, d), 13C, 15N, etc. One such isotopic labeling can be done, for exam-
ple, using silylation with d18-N,O-bis(trimethylsilyl)-trifluoroacetamide (d18-
BSTFA). Derivatization of an aliquot of sample with regular BSTFA and another
with d18-BSTFA provides a pairing chromatogram with peaks at retention times
that have only small differences from the first but with spectra differing by a
number of units. The comparison of the spectra for corresponding peaks (based on
retention time) of a given compound allows the calculation of the number of silyl
groups attached to that compound. In addition, the fragmentation in the spectra can
be better interpreted allowing easier compound identification.
Derivatization in GC/MS analysis may have multiple other utilizations and ben-
efits. For example, quantitative analysis frequently utilizes isotopically labeled
internal standards. In an analysis with multiple analytes, addition of an isotopically
labeled internal standard for each analyte may become a complex process. When a

10
Derivatization Methods in GC and GC/MS
DOI: http://dx.doi.org/10.5772/intechopen.81954

Figure 7.
Mass spectrum of silylated betulinic acid and that of silylated betul-18-en-oic acid.

derivatization is involved in the analysis, this can be done with a non-labeled

reagent for the analytes in the sample, while the internal standards are obtained by
derivatization of standards with the same reagent but isotopically labeled. Such
technique has been proven to be very successful, for example, in the analysis of
multiple amino acids (but using an LC/MS/MS procedure [13]).

6. General comments regarding the main types of chemical reactions

used in derivatization

Derivatizations as chemical reactions can be classified as follows: (1) reactions

with formation of alkyl or aryl derivatives, (2) silylation reactions, (3) reactions
with formation of acyl derivatives, (4) reactions of addition to carbon-hetero mul-
tiple bonds, (5) reactions with formation of cyclic compounds, and (6) other reac-
tions specific to a certain analysis. The selection of the derivatization reaction is
typically done based on the desired property to be brought to the analyte and its
possible reactivity. For this reason, the reagent is selected to have moieties that add
the desired property to the analyte and also to have the capability to react with the
specific functional group of the analyte. The matrix of the sample also has a role in

11
Gas Chromatography

Properties

Compound Amine Amide Alcohol Phenol Acid

First derivatization preference Acylation Acylation Silylation Silylation Alkylation

Second derivatization preference Alkylation Alkylation Acylation Acylation Silylation

Table 1.
Derivatization preferences for compounds containing active hydrogens.

the choice of a specific derivatization procedure. Initial matrix of the sample is not
always suitable for derivatization, and in some cases preliminary sample prepara-
tion is necessary to change this matrix. The change can be as simple as drying the
initial sample but can also be rather complex [14]. Table 1 gives a simplified view of
preferences for the choice of a derivatization reagent for compounds containing
active hydrogens [14].
Besides functionalities with active hydrogens, other functionalities can also be
derivatized. Compounds containing carbonyls can be derivatized, for example, using
condensation reactions. Some analytes may contain multiple functional groups such
as the amino acids. Specific derivatization reactions can be selected for such cases.

7. Reactions with formation of alkyl or aryl derivatives

The formation of alkyl or aryl derivatives is applied to replace the active hydro-
gens from an analyte with an alkyl (R) or aryl (Ar) group. The replacement can be
done in functionalities such as OH, COOH, SH, NH, or CONH. For example, the
derivatization with short-chain alkyl bromides or iodides has numerous analytical
applications for compounds such as steroids, amino acids, catecholamines, sulfon-
amides, phenols, barbiturates, organic acids, and mono- and oligosaccharides. A
large number of reagents R-X are known, and in a simplified approach, it can be
considered that R is carrying a specific property and X a specific reactivity, although
the reactivity of a reagent is influenced by both R and X components of the mole-
cule. The type of moiety R and that of reactive group X are guiding the selection
process of selecting a reagent for a specific derivatization.
In most alkylation reactions, the analyte acts as a nucleophile (Y:, Y:H, Y:-)
reacting in a substitution (SN) with the alkylating reagent R-X, which contains a
leaving group X and an alkyl group R:

Y:H þ R  X ! Y  R þ X:H (7)

Various reagents and conditions were utilized in the derivatizations for analyti-
cal purposes. As reagents R-X for alkylations, one of the most commonly used are
the alkyl halides, especially alkyl iodides and alkyl bromides. Because some of the
derivatizations can be slow and inefficient depending on the analyte and on the
reagent, the reaction rate becomes an important parameter for the analytical appli-
cability. The reaction with an alkyl halide for the preparation of methyl or ethyl
substituents, for example, is frequently performed either with a specific methyla-
tion reagent, in the presence of a catalyst, or in some instances using a particular
solvent. The enhancement of the alkylation efficiency can be achieved using several
other procedures. For example, for the analytical alkylation of carboxylic

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acids, specific cryptands such as crown ethers can be used to solvate the alkali
metal portion of an organic acid salts, allowing the anion to be freer and
increasing the rate of nucleophilic substitution. One other approach for enhancing
the alkylation efficiency is the use of phase transfer alkylation. This approach is
based on the formation of a compound easily extractable in an organic phase and
on the displacement of the equilibrium in the direction of the formation of the
desired product.
One different way of enhancing the alkylation efficiency is the use of different
alkylating reagents besides short-chain alkyl bromides or iodides. One example of a
halide that is particularly reactive is pentafluorobenzyl bromide. This reagent can
be used for the derivatization of a variety of compounds containing active hydro-
gens. Another reactive halide is 2-bromoacetophenone (phenacyl bromide). This
reagent is used mainly for the alkylation of compounds containing more acidic
hydrogens such as carboxylic acids. Another example of methylation using a special
reagent R-X is applied on carbohydrates [15]. This methylation uses methylsulfi-
nylmethanide anion. The reagent is prepared from dry DMSO and NaH or KH in a
reaction as follows:

ðCH3 Þ2 SO þ NaH ! CH3  SOCH2 – Naþ þ H2 (8)

A polyol or a monosaccharide dissolved in DMSO is easily methylated with

methylsulfinyl-methanide anion.
Other alkylating reagents are known (different X in R-X), also reacting in a
nucleophilic substitution. For example, dimethyl sulfate can be used for alkylations.
Alkylfluoromethyl-sulfonates are even more reactive than sulfates, and the
reaction may take place with the active hydrogen even from alcohols or amines as
follows:

ð9Þ

ð10Þ

Even tertiary amines, such as pyridine, also react with this type of reagent
forming quaternary ammonium salts. The alkylation with alkylfluorosulfonates can
be catalyzed as other alkylation reactions for increasing the reaction rate. A catalyst
that can be used in this reaction is Hg(CN)2.
Diazomethane is another common alkylating (methylating) reagent. The alkyl-
ation using diazomethane is assumed to take place as follows:

ð11Þ

Diazomethane is a gaseous unstable substance, which cannot be stored for long

periods of time. It is usually prepared in small quantities and used immediately with
or without an intermediate step of dissolution in ether. The preparation can be done
from different N-nitroso-N-alkyl compounds in a reaction with a base. A common
preparation uses N-nitroso-N-alkyl-p-toluenesulfonamide (Diazald). Methylation
with diazomethane may require addition of a Lewis acid catalyst such as BF3. The

13
Gas Chromatography

methylation of partly acetylated sugars and amino sugars using diazomethane and
BF3 in ether leads to the methylation of the free OH groups without the migration or
substitution of the existent acyl groups.
A common alkylation of acidic analytes such as carboxylic acids, phenols, and
thiols is performed using another type of alkylating reagent, namely, N,N-
dimethylformamide dialkyl acetals. N,N-Dimethylformamide dimethyl acetal
(Methyl-8®) is commonly used for methylations. For a compound containing a
COOH group, the reaction with this reagent takes place as follows:

ð12Þ

The compounds with acidic hydrogens can also be alkylated (methylated) using
trimethyl orthoacetate, alkyl-p-tolyltriazenes (R─NH─N═N─C6H4─CH3), and
O-alkyl isoureas are also used for the formation of analytes containing acidic
hydrogens, imino esters, etc.
Alcohols can also act as alkylating reagents in particular when the analyte con-
tains a more acidic hydrogen. Catalyst such as HCl, BF3, CF3 COOH or a cation
exchange resin in H+ form is also frequently added to facilitate the reaction. The
addition of HCl can be made as a water solution or as gaseous HCl that does not
bring additional water to the reaction medium. The formation of alkyl or aryl
derivatives of acids is a particularly important reaction known as esterification.
Derivatization by esterification has been used with acids as the analyte and the
alcohol as the reagent and also with the alcohol as the analyte and the acid the
reagent. The esterification can be viewed either as the acid alkylation or as the
acylation of the alcohol (see also the esterification mechanism). This reaction is
typically catalyzed by strong acids and can be written as follows:

ð13Þ

The mechanism of ester formation can be summarized by the following series of

reactions:

ð14Þ

The esterification efficiency can be improved by removing the water formed in

this reaction. This can be done using a chemical reagent or distillation when the
compounds of interest boil above 100°C. Among the materials able to eliminate
water are desiccants such as anhydrous MgSO4, molecular sieves, or substances that
react with water such as CaC2, (CH3)2C(OCH3)2 (2,2-dimethoxypropane), and
even an appropriately chosen acid anhydride that reacts faster with water than with
the reacting alcohol. The derivatization also may be performed in the presence of
SOCl2 (thionyl chloride), which reacts with the water assisting in its removal, and
when present in excess, may react with the alcohols forming alkyl chlorides or with
the acids forming acyl chlorides. Chloride is a better leaving group in a nucleophilic

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alkylation reaction, and the efficiency of alkylation increases. Acids also can be
esterified using a mixture of an alcohol and an acyl halide.
One procedure for the formation of esters with less active organic acids applies
the addition of dicyclohexylcarbodiimide (DCCI) in the derivatization process, to
facilitate esterification. The reaction can be performed by adding to the acids that
need to be analyzed the appropriate alcohol and DCCI usually in a solvent such
as pyridine. Dicyclohexylurea, which is formed in the reaction, is not soluble in
pyridine and can be separated. Besides DCCI, other carbodiimides can be used in
the reaction of acids and alcohols. Among these are carbonyldiimidazole (CDI),
6-chloro-1-p-chlorobenzensulfonyloxybenzotriazole (CCBBT), 1-(3-dimethylami-
nopropyl)-3-ethylcarbodiimide (EDAC), etc. Also, 2-chloro-1-methylpyridinium
iodide, 2,4,6-triisopropylbenzenesulfonyl chloride, trialkyloxonium fluoroborate,
etc. can be used to facilitate esterification.
Transesterification is another technique applicable for obtaining certain alkyl
derivatives of acids (or acyl derivatives of alcohols). The reaction can be written as
follows:

ð15Þ

Transesterification can be catalyzed by acids (or Lewis acids) such as HCl, BF3,
and H2SO4 or by bases such as CH3OK, CH3ONa, or C4H9ONa. The basic catalysts
are commonly used for the methanolysis of triglycerides, followed by the analysis of
the fatty acid methyl esters using GC or GC/MS [16].
A special alkylation can be achieved online during the heating in the injection
port of a gas chromatograph using tertraalkylammonium hydroxides or alkylary-
lammonium hydroxides. Tetramethylammonium hydroxide (TMAH) is the most
common reagents of this type. The reaction takes place as follows (Δ indicates
heating):

ð16Þ

Numerous other reactive compounds may be used for replacing active hydro-
gens in specific compounds. For example, epoxides, aziridines, and episulfides react
easily with compounds with active hydrogens. Formation of a second group
containing an active hydrogen may preclude the use of such reagents for analytical
purposes.
Besides the desired derivatives, certain unexpected compounds that can be
considered artifacts for the particular analysis can also be formed in alkylation
reactions. The artifacts may be formed from unexpected interactions of the reagent
with the analyte or may be a result of undesired effects of the catalysts or medium
used for derivatization. In some cases, the control of the alkylation process may be
difficult. Longer or shorter reaction times or intervals between derivatization and
analysis may lead to errors, even when an internal standard is used for quantitation.
One common case of artifact formation occurs during the reaction with com-
pounds containing O-acyl or N-acyl groups, such as previously acylated

15
Gas Chromatography

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
positions.
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
artifacts by unexpected alkylations.
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
aryl halide is substituted with electron attracting groups such as NO2.

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 com-
pounds 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:

ð17Þ

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

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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 some-
times 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 sub-
strates 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 com-
pounds. 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 hydro-
gens 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:

ð18Þ

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 by-

17
Gas Chromatography

Figure 8.
Some reagents used for trimethylsilylation.

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

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Compound Functional group Decreasing reactivity

(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

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

(12) Secondary amine NH

(13) Indole NH

Table 2.
Several functional groups that can be silylated (listed in the approximate order of decreasing ease of silylation).

Figure 9.
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:

19
Gas Chromatography

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 chro-
matogram 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

No. Compound Ret. m/z Abrrev. No. Compound Ret. m/z Abrrev.
time time

(1) Phenol 6.88 166 Ph (14) 3,4- 12.32 194 3,4-

Dimethylphenol diMePh

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

MeOPh

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

MeOPh

(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

(6) 2,5-Dimethylphenol 10.70 194 2,5- (19) 4-Methylcatechol 16.27 268 4-MeCa
diMePh

(7) 3,5-Dimethylphenol 11.07 194 3,5- (20) Hydroquinone 16.73 254 Hy

diMePh

(8) 2,4-Dimethylphenol 11.20 194 2,4 (21) 3-Methylcatechol 16.71 268 3-MeCa
diMePh

(9) 2-Methoxyphenol 11.28 196 2- (22) 5-Methylresorcinol 18.19 268 5-MeCa

MeOPh

(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

(12) 2,6-Dimethylphenol 11.79 194 2,6- (25) 2,5- 20.18 282 2,5-
diMePh Dimethylresorcinol diMeRe

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

dimePh

Table 3.
Details regarding the analyzed phenols with the chromatogram shown in Figure 9.

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Figure 10.
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

21
Gas Chromatography

Peak Amino acid Abbrev. MW Formula + x MW + x Charact. Ret.

No. TBDMS TBDMS ion time

(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

(4) α-Amino-n- α-ABu 103.10 C16H37NO2Si2 331 274 34.36

butyric acid

(5) β-Alanine β-Ala 89.09 C15H35NO2Si2 317 260 35.58

(6) Urea 60.06 C13H32N2OSi2 288 231 36.01

(7) β- β-ABu 103.10 C16H37NO2Si2 331 274 36.11

Aminoisobutyric
acid

(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

(12) γ-Aminobutyric γ-ABu 103.10 C16H37NO2Si2 331 274 39.79

acid

(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

(22) 3-Methyl-L- 3MeHys 169.20 C19H39N3O2Si2 397 340 55.15

histidine

(23) Glutamic acid Glu 147.13 C23H51NO4Si3 489 432 55.53

(24) Ornithine Orn 132.20 C23H54N2O2Si3 474 286 55.64

(25) 1-Methyl-L- 1MeHys 169.20 C19H39N3O2Si2 397 302 57.03

histidine

(26) Lysine Lys 146.19 C24H56N2O2Si3 488 300 58.02

(27) α-Aminoadipic 161.20 C24H53NO4Si3 503 446 58.06

acid

(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

Table 4.
Details regarding the analyzed amino acids with the chromatogram shown in Figure 11.

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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.

9. Acylation reactions

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:

ð19Þ

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:

Y : H þ R  COOH ! R  COY þ H2 O (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. Dicyclohex-
ylcarbodiimide (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-trimethyl-
benzenesulfonyl 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,N0 ,N0 -bis
(tetramethylene)uronium hexafluorophosphate, 2,20 -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 chlo-
ride with an amine, for example, takes place as follows:

23
Gas Chromatography

Group Group structure Mass of Example of Common

the group reagents analytes

Formyl 29 Formic acid Steroids

Acetyl 43 Acetyl chloride, Alcohols

acetic anhydride

Trifluoroacetyl 97 N-Methyl-bis Alcohols

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

Propionyl 57 Propionic anhydride Alcohols

Butyryl 71 Butyric anhydride Alcohols

2,2-Dimethyl- 85 Pivaloyl chloride, Amino

propionyl-(pivaloyl) pivalic anhydride acids

Pentafluoro- 147 Pentafluoropropionic Alcohols,

propionyl anhydride (PFPA) amines,
amino
acids

Heptafluorobutyryl 197 Heptafluorobutyric Alcohols,

anhydride (HFBA), amines,
heptafluorobuty amino
rylimidazole acids

Trichloroacetyl 145 Trichloroacetic Alcohols

anhydride

Pentafluorobenzoyl 195 Pentafluorobenzoyl Alcohols,

chloride, amides
pentafluorobenzoyl-
imidazole

(Pentafluorophenyl)- 209 (Pentafluorophenyl)- Alcohols,

acetyl acetyl chloride amides

(Pentafluorophenoxy)- 225 (Pentafluorophenoxy)- Alcohols

acetyl acetyl chloride

Table 5.
Some common groups present in acylating reagents used in derivatizations for GC analysis [14].

24
Derivatization Methods in GC and GC/MS
DOI: http://dx.doi.org/10.5772/intechopen.81954

ð21Þ

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 neutraliza-
tion 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(trifluoroa-
cetamide), 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. Sol-
vents such as CH3CN, pyridine, DMSO, or THF can be used as a reaction medium:

ð23Þ

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

25
Gas Chromatography

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.
A special type of acylation is that using chloroformates. Carbonic acid,
O═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 = Rb).
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

26
Derivatization Methods in GC and GC/MS
DOI: http://dx.doi.org/10.5772/intechopen.81954

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 elimina-
tion 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 Ra 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:

ð27Þ

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

tives as shown below:

ð28Þ

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:

ð29Þ

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:

27
Gas Chromatography

ð30Þ

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:

ð31Þ

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-
grams 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:

ð33Þ

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.

28
Derivatization Methods in GC and GC/MS
DOI: http://dx.doi.org/10.5772/intechopen.81954

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 bifunc-
tional 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:

ð35Þ

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 reac-
tion 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:

ð37Þ

Amino acids can react with an activated anhydride such as trifluoroacetic

anhydride (TFAA):

ð38Þ

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.

29
Gas Chromatography

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]).

11. Derivatization reactions involving solid-phase reagents or

derivatization on a solid support

Solid-phase reagents are polymeric materials with specific groups that are
reactive and can be transferred to the analyte molecule producing derivatization.
For an analyte of the form Y:H, the reaction with a solid-phase reagent can be
written as follows:

Y:H þ R‐Polymer ! R  Y þ H‐Polymer (39)

Solid-phase reagents must work analogously to the corresponding small-

molecule reagents containing the group R (a tag). Reagents that are insoluble in
certain solvents at high concentrations can often provide a high ratio of analyte/
substrate in a polymeric microenvironment that yields a high kinetic rate for the
heterogeneous reaction.
A variety of materials can be used as solid support, such as specifically bound
reagents on a silica support (used, e.g., for online derivatization in HPLC analysis),
ion exchange resins, as well as other supports [31]. One example of solid-phase
support that can produce derivatization is trifluoroacetyl nylon 6,6. This solid-
phase reagent can be obtained from poly(hexamethylene adipamide) (nylon 6,6)
and trifluoroacetyl anhydride. This solid-phase reagent can be used in amine
derivatization in a reaction as follows:

ð40Þ

This derivatization of the amine is done by mixing the solid-phase reagent with a
solution of amine solution in CH3CN. Following derivatization, the solid-phase
reagent is separated by centrifugation, and the solution is concentrated by evapo-
rating part of the solvent and analyzed by GC (an amine internal standard must be
used in this procedure). However, some such derivatizations require a long time of
interaction between the solid-phase reagent and the analytes and found only limited
applications.
(Another) alternative of derivatization of specific analytes is using the reaction
between the reagent and the analyte both adsorbed on a solid support. This type of
derivatization has been used, for example, in connection with a solid-phase
microextraction (SPME) technique [32]. In this technique a reagent is initially
adsorbed in the SPME fiber, followed by exposure to the analytes. The derivatized
analytes are further desorbed in the injection port of the GC and analyzed using a
detector such as MS. For example, formaldehyde from air can be analyzed using a

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

polydimethylsiloxane (PDMS) fiber containing o-(2,3,4,5,6-pentafluorobenzyl)-

hydroxylamine hydrochloride as a reagent. After exposing the fiber to the air
sample contaminated with formaldehyde, the derivatization agent reacts with
formaldehyde absorbed onto the coating forming an oxime. The oxime is thermally
desorbed in a GC injector port and analyzed by GC with ECD [33].

Author details

Serban C. Moldoveanu1* and Victor David2

1 R.J. Reynolds Tobacco Co., Winston-Salem, NC, USA

2 University of Bucharest, Bucharest, Romania

*Address all correspondence to: moldovs@rjrt.com

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License (http://creativecommons.org/licenses/
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

31
Gas Chromatography

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