**7. Single-drop microextraction**

*Gas Chromatography - Derivatization, Sample Preparation, Application*

was analyzed by GC-ECD instrument [32].

**6. Superheated water extraction**

conventional extraction methods [33].

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

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

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

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

exploited in superheated water chromatographic methods [34].

simplicity, low cost, and friendly environment [36].

**68**

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 microextraction procedures.

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

**Figure 4.** *Schematic diagrams of two modules in SDME.*

drop. The searches are shown that N-octane and toluene possess the best extraction efficiency for nonpolar substances, while chloroform is more favorable to extract polar alkaloids, and then they can be analyzed by GC techniques [37]. One limitation of direct immersion SDME is the instability of the droplet at high stirring rates. Although high stirring rates enhance the extraction efficiency, to avoid the problem caused by elevated stirring speeds, a 1-μl microsyringe (instead of a more common 10-μl one) with some modification of its tip was used by Ahmadi et al. [38].

Ionic liquids have been established as alternative to organic solvents because of their high boiling point and viscosity which allow production of larger and more reproducible extraction drops. HPLC is a preferred method for analyzing ionic liquid extract, but their nonvolatility causes them unsuitable for GC analysis. To couple ionic liquid-based SDME to GC instrument, the extract is introduced via a removable interface which prevents entering of ionic liquid into the GC column, while the analytes can be entered quantitatively into the capillary column [37].

HS-SDME in which the organic droplet is held above the aqueous sample solution is most suitable for the consideration of volatile or semivolatile analytes [39]. The advantages of HS-SDME include the following: (1) Headspace SDME permits quick stirring of the sample solution with no concerning on the droplet stability. (2) The effects of nonvolatile matrix interferences are reduced, even if they are not eliminated. (3) In this mode, the analytes are distributed between three phases: the aqueous sample, headspace, and organic droplet. Since an elevated stirring rate of the sample solution enhances the mass transfer between the three phases. (4) In comparison with HS-SPME, HS-SDME shows to have the same precision and rate of analysis as HS-SPME. However, HS-SDME procedure possesses two special advantages over HS-SPME. At first, the approach of choosing solvents is wider. Second, the solvent cost (on the basis of several microliters) is negligible in comparison with the cost of commercially available fibers in SPME [39]. Alternatively, the use of SDME for headspace analysis seems relatively difficult, because of the requirement of the higher boiling point solvents. Although the most suitable solvents for gas chromatography should have relatively high vapor pressures or low boiling points, the limit of these solvents is obvious: they would evaporate too quickly in the headspace during extraction. Therefore, the select of suitable solvents should be the first decision in HS-SDME techniques.

## **8. Conclusions**

Many methods are available for the treatment of volatile substances prior to instrumental analysis. In this chapter, the major methods which are leading to GC analysis have been explained. It has been observed that yet the classical techniques such as purge and trap, static headspace extraction, and liquid-liquid extraction act as important roles in chemical analysis of all sample types. New methods, such as SPME and membrane extraction, possess some advantages like convenient in automation and field sampling and reduction of solvent consumption, as well. If the analyst may be confronted with every difficulty, there is an appropriate available method to solve and face it. As a consequence, the main and primary analytical problem is to select the best sample preparation technique.
