**5. Chemical derivatization analysis**

Gas chromatography of volatile or nonpolar compounds may be done without derivatizing the sample; indeed, derivatives of compounds such as hydrocarbons or halogenated hydrocarbons cannot easily be prepared. It is possible to analyze polar compounds such as carboxylic acids and amines, without prior derivatization, on polar GC phases such as those based on polyethylene glycol. However, derivatization is useful in many instances where it may [31]:


However, there are some drawbacks in using derivatization process before GC analysis:


For these reasons, GC with derivatization is less frequently employed in quality control applications, where the purity of a single substance or the components in a formulation are being determined.

**67**

*Sample Preparation Techniques for Gas Chromatography DOI: http://dx.doi.org/10.5772/intechopen.84259*

1.Sample container and reaction vessel.

2.Heating and evaporation apparatuses.

3.Sample and reagent handling systems.

with aluminum-lined caps are used.

Anhydrous magnesium sulfate may be used too.

to leave a pluge of ca. 3 cm of the adsorbent [31].

as a proper starting point for the development of a method.

Derivatization reactions are usually simple chemical reactions which are likely to occur in nearly quantitative yield such as acylation, alkylation, and silylation. In silylation reactions, some derivatives like trimethylsilyl (TMS) and tertiarybutyldimethylsilyl (TBDMS) can be prepared from a wide range of functional groups including hydroxyl, carboxylic, amine, amide, thiol, phosphate, hydroxide, and sulfonic. In acylation processes, acetate formation of the analytes is prepared by some derivatizing agents such as acetic anhydride, trifluoroacetic anhydride (TFAA), pentafluoropropionic anhydride (PFPA), and heptafluorobutyl anhydride (HFBA). Alkylation reactions may be used to derivatize carboxylic acids, amines, sulfonic acids, phosphonic acids, phosphates, barbiturates, uracils, purines, penicillins, thiols, and inorganic anions [31].

Derivatization reactions require relatively simple apparatuses:

4.Removal systems for the exiting of the derivatizing reagents.

The certain standard procedures in derivatization are the following:

1.When volumes of reagent are small, reactions are carried out in 0.3 or 1 ml capacity Reacti-Vials or V-Vials. When volumes of solvents or reagents are greater, such as in aqueous phase reactions, then 3.5-ml screw-top sample tubes

2.The reagents or solvent are evaporated under a stream of nitrogen gas with the sample maintained at 60–80°C in a heating block. Obviously, less volatile reagents require heating at higher temperatures for their efficient removal. If the sample is volatile, evaporation at a low temperature for a longer time may be required, or it may be better to inject it without removing the reagents.

3.Drying is carried out by passing the sample through ca. 3 cm of anhydrous sodium sulfate contained in a Pasteur pipette plugged with cotton wool.

4.Dissolution of the derivatized sample prior to analysis is done by treating the sample with 2 ml of solvent for capillary column GC using the splitless injection mode (the volume may be adjusted if a split injection is used) or 100 μl for packed column GC. Since in most circumstances the derivatized compound should be clearly observed in relation to any interfering peaks from reagent residues, in injection 1 μl of product solution, 200 μg of material can be chosen

5.Removal of excess reagents is carried out by passage through a short column of Sephadex LH20. The sample is passed through a short column prepared by introducing Sephadex LH20 suspended in EtOAc/hexane (1:1, V/V) into a Pasteur pipette plugged with cotton wool and allowing the solvent to drain out

However, in some cases, the derivatization leads to sharper peaks and therefore to better separation and higher sensitivity. But the derivatization procedure requires *Sample Preparation Techniques for Gas Chromatography DOI: http://dx.doi.org/10.5772/intechopen.84259*

*Gas Chromatography - Derivatization, Sample Preparation, Application*

commercially available [30].

**5. Chemical derivatization analysis**

tion is useful in many instances where it may [31]:

present in mixtures of unknown compounds.

1.The cleanup of pesticide residues and chlorinated hydrocarbons.

5.The cleanup of cations, anions, metals, and inorganic compounds.

3.The separation of aromatic compounds from an aliphatic-aromatic mixture.

4.The cleanup of steroids, esters, ketones, glycerides, alkaloids, and carbohydrates.

As discussed in previous sections, the sufficient amount of a sorbent, which is loaded with the sample extract, has packed the SPE cartridge. Then, the analyte of interest is eluted through the column by an efficient eluting solvent, and the other contaminants are remained on the cartridge. The packing compound may be an inorganic material like either Florisil or one of many stationary phases which are

Gas chromatography of volatile or nonpolar compounds may be done without derivatizing the sample; indeed, derivatives of compounds such as hydrocarbons or halogenated hydrocarbons cannot easily be prepared. It is possible to analyze polar compounds such as carboxylic acids and amines, without prior derivatization, on polar GC phases such as those based on polyethylene glycol. However, derivatiza-

2.Stabilize compounds which are unstable at the temperatures required for GC.

4.Yield information with regard to the number and type of functionalities

5.Improve the behavior of compounds toward selective detectors such as electron capture or nitrogen-selective detectors and mass spectrometry.

However, there are some drawbacks in using derivatization process before GC

1.The derivatizing agent may be difficult to remove and interfere in the analysis, and this is particularly disadvantageous when the purity of a compound is

2.The derivatization conditions may cause unintended chemical changes in a

For these reasons, GC with derivatization is less frequently employed in quality control applications, where the purity of a single substance or the components in a

3.The derivatization step increases the time required for analysis.

1.Increase the volatility and decrease the polarity of polar compounds.

3.Improve the separation of groups of compounds on GC column.

2.The separation of nitrogen compounds from hydrocarbons.

**66**

analysis:

being assessed by GC.

formulation are being determined.

compound, for example, dehydration.

Derivatization reactions are usually simple chemical reactions which are likely to occur in nearly quantitative yield such as acylation, alkylation, and silylation. In silylation reactions, some derivatives like trimethylsilyl (TMS) and tertiarybutyldimethylsilyl (TBDMS) can be prepared from a wide range of functional groups including hydroxyl, carboxylic, amine, amide, thiol, phosphate, hydroxide, and sulfonic. In acylation processes, acetate formation of the analytes is prepared by some derivatizing agents such as acetic anhydride, trifluoroacetic anhydride (TFAA), pentafluoropropionic anhydride (PFPA), and heptafluorobutyl anhydride (HFBA). Alkylation reactions may be used to derivatize carboxylic acids, amines, sulfonic acids, phosphonic acids, phosphates, barbiturates, uracils, purines, penicillins, thiols, and inorganic anions [31].

Derivatization reactions require relatively simple apparatuses:


The certain standard procedures in derivatization are the following:


However, in some cases, the derivatization leads to sharper peaks and therefore to better separation and higher sensitivity. But the derivatization procedure requires more time and effort. Assadi et al. studied the determination of chlorophenols in water samples using simultaneous dispersive liquid-liquid microextraction and derivatization followed by gas chromatography-electron-capture detection [32]. In this research, dispersive liquid-liquid microextraction (DLLME) and derivatization coupled to gas chromatography-electron-capture detector (GC-ECD) was simultaneously applied for quantitative investigation of chlorophenols (CPs) in water sample. In this method, 500 μl of acetone, as disperser solvent, containing 10.0 μl of chlorobenzene, as extracting solvent, and 50 μl of anhydride acetic acid, as derivatizing reagent, was quickly injected into 5.00 ml of water sample containing CPs (analytes) and K2CO3 (0.5%, w/v) by a syringe. So, during a few seconds of time, the analytes were both derivatized and extracted simultaneously. Then, the mixture was centrifuged, and 0.50 μl of precipitated phase containing concentrated analytes was analyzed by GC-ECD instrument [32].

### **6. Superheated water extraction**

When the temperature of liquid water is increased under pressure, between 100 and 374°C, its polarity is reduced significantly, and so, it can be applied as an extracting solvent for a wide variety of analytes. Its most interested application has been to determine PAHs, PCBs, and pesticides from environmental samples. Although it gives comparable results to Soxhlet extraction, the organic solvent consumptions have been significantly decreased, and quicker extractions were achieved. Unlike supercritical fluid extraction (SFE), unless the pressure is decreased and steam is applied, n-alkanes cannot be extracted. Other superheated water applications include the separation of required oils from plant substances where it preferably extracts the more important natural oxygenated compounds than steam distillation. The aqueous extract can be enriched via different methods such as solvent extraction, SPE, SPME, and extraction disk. On the other hand, the extraction can be coupled to LC or GC instruments, as online methods. In many cases the superheated water extraction is cleaner, faster, and cheaper than the conventional extraction methods [33].

The pressures, which are needed to keep a condensed state of water, are moderate in 15 bar at 200°C and 85 bar at 300°C. At any pressure, if the pressure falls below the boiling point of liquid water, superheated steam is produced. This superheated state possesses a significantly lower dielectric constant than that of the liquid state and also has gas-like diffusion velocity and viscosity properties. Consequently, superheated water behaves completely different from an extraction liquid solvent.

Superheated water has been widely used as an analytical extraction solvent. The changes in the polarity of water with increasing temperature have been also exploited in superheated water chromatographic methods [34].

Ozel et al. studied the analysis of volatile components from *Ziziphora taurica* subsp. *taurica* by steam distillation, superheated water extraction, and direct thermal desorption with GC·GC-TOFMS [35]. In this research, volatile compounds from the leaves of *Ziziphora taurica* subsp. *taurica* have been separated by steam distillation, superheated water extraction, and direct thermal desorption methods. The volatile constituents were analyzed by a perfect two-dimensional gas chromatography-time-of-flight mass spectrometry instrument. Some other researchers reported that superheated water is a powerful alternative extractor for separation of essential oils, because of its ability in working at low temperatures and obtaining higher speed extractions. Therefore, this makes the decomposition of volatile and heat-sensitive analytes be avoided. Extra advantages of the use of SWE are its simplicity, low cost, and friendly environment [36].

**69**

**Figure 4.**

*Schematic diagrams of two modules in SDME.*

*Sample Preparation Techniques for Gas Chromatography DOI: http://dx.doi.org/10.5772/intechopen.84259*

Single-drop microextraction (SDME) has witnessed incessant growth in the range of applications of sample preparation for trace organic and inorganic analysis. In SDME, a Teflon rod (or needle of a syringe) with a spherical recess at its one end is loaded with 8 μl of organic solvent (n-octane) containing the internal standard (n-dodecane) and immersed in aqueous sample taken in a 1 ml vial for a known period of time while being stirred. Thereafter, the rod is exited from the solution, and with a GC syringe, 1 μl of extract is injected into the GC column for analysis [37]. The stirrer rate of donor aqueous phase affects the solvent extraction speed and homogeneity of the obtained extract. SDME is comparable to SPME in terms of speed, precision, and sensitivity. But it is much cheaper than SPME and provides narrower peaks because in SDME, the solvent evaporation is faster than the analyte desorption from the fiber in SPME. However, in SDME, just little portion of extract is used to inject the GC column. By using a GC syringe instead of Teflon rod, the inconvenience of its filling can be eliminated. So, 1 μl of extract can be retracted back into the syringe after extraction process and injected directly into the GC column. Thus, the GC microsyringe can be used without any modification, and all other devices are general laboratory equipment. The GC microsyringe with a bend tip can hold the organic drop in place at controlled stirring rate. So, a number of instrumental analysis methods can be coupled to single-drop microex-

There are two modules in SDME: direct immersion single-drop microextraction (DI-SDME) and headspace SDME. Their schematic diagrams are shown in **Figure 4**. The direct immersion SDME is just applied for liquid samples containing nonpolar or relatively polar analytes. To stabilize solvent drop during the extraction process, any insoluble and special materials must be removed from the sample medium, and a proper organic solvent with the least solubility in water, high boiling point, and high affinity to extract the analyte of interest should be chosen. Also at a moderated stirring rate, the drop must not be dislodged. However, DI-SDME is more favorable to match with GC method because of using water-immiscible solvent in the

**7. Single-drop microextraction**

traction procedures.
