**2.2 Solid-phase microextraction (SPME)**

Miniaturization of analytical processes into microchip platforms designed for micro total analytical systems is a new and rapidly developing field. Solid-phase microextraction (SPME) is a modern technique that consists in direct extraction of the analytes with the use of a small-diameter fused silica fiber coated with an adequate polymeric stationary phase [10]. On the other hand, in two designs of SPME, a thin layer of sorbent is coated on the outer surface of fibers (fiber design), and the inner surface of a capillary tube (in-tube design) is covered. The fiber design can be used as an interface in both GC and HPLC, but in-tube design has been just applied as an easier approaching interface with HPLC. In fiber design, a thin film of liquid polymer or mixture of a solid sorbent with a liquid polymer has covered on the surface of a fused silica core fiber.

The properties of extraction process, by SPME, are as follows [2]:


SPME can be conducted in three modes [2]:


The SPME procedure is performed through two separate steps:


As discussed before, SPME has been introduced as a solventless extraction method, in which a fused silica fiber has been coated with a *thin* film of sorbent, to separate volatile analytes of interest from a matrix sample. Usually, the fiber is placed into a syringe needle which is protected for easy penetration into the sample and GC vial septa. Analyte extraction and analysis depend on the fiber type and its thermal desorption into a GC inlet. There are two approaches to SPME sampling of volatile organics:


**51**

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

immersion process.

Choosing between direct immersion and headspace SPME is relatively straightforward. Direct immersion SPME is warranted for liquid and solution samples which are used in solid-phase and liquid-liquid extraction methods. Headspace SPME is considered for extraction of volatile specious, with normal boiling point less than 200°C, from solid and liquid samples. For higher boiling point analytes, direct immersion SPME is probably necessary. Headspace is more preferred for especially complex or dirty samples due to fouling the fiber coating in a direct

SPME fibers have different coatings, and there is no single coating for extraction and separation of all volatile organics from a sample. Therefore, different types of coatings with different polarities are applied on SPME fibers. Currently, three types of fiber coatings are commercially available: (1) nonpolar, (2) semipolar, and (3) polar coatings. There are several SPME fiber coatings commercially available. These range in polarity from polydimethylsiloxane (PDMS), which is nonpolar, to "*carbowax-divinylbenzene*" (CW-DVB), which is highly polar. The nonpolar fibers are more commonly used for headspace SPME as the majority of volatile analytes tend to be nonpolar or slightly polar. The advantage of using different fiber polarities is that using a matched polarity fiber, as polar coated for a polar analyte, makes extraction selectivity be enhanced. On the other hand, there is less of a chance of extracting interfering compounds along with the analyte of interest, and an organic matrix is not a problem.

Fiber coating thickness is a second parameter that should be considered to select a fiber for both direct immersion and headspace SPME. The PDMS coating is commercially in hand in three thicknesses: 100, 30, and 7 μm. The 100-μm-thick fiber is generally applied for highly volatile compounds or when a larger organic matrix

Once the fiber is chosen, extraction conditions must be optimized. There are many variables such as (1) extraction time, (2) sample volume, (3) agitation,

1.As extraction time is increased, a plateau in peak area is reached. So, this

represents the time required for the system to reach the equilibrium and is the optimized extraction time. Most headspace SPME methods are completed in less than 5 min, while direct immersion SPME may require more than 30 min.

2.The sensitivity of a SPME method is proportional to the number of moles of analyte recovered from the sample. As the sample volume increases, analyte recovery increases too. But in very dilute samples, larger sample volume results

3.In many extraction methods, the agitation method affects both the extraction time and efficiency. In direct immersion SPME, agitation is often accomplished with a magnet and a stirrer. So, the stirring rate should be optimized and constant during the extraction process. Also, the fiber should be off-centered in the vial so that liquid is moving quickly around it. Agitation can also be achieved by physical movement of the fiber or by movement of the sample vial and/or sonication.

4.Extraction temperature can also be an important factor, especially in headspace SPME analyses. Despite of GC headspace analysis, increasing the

5.By modifying the sample matrix, the extraction recovery can be improved. There are two ways to modify the sample matrix: (a) adjusting the sample pH

temperature in SPME makes the extraction sensitivity decrease.

volume is used. The 7-μm-thick fiber is used for less volatile compounds.

Also, direct immersion SPME is highly matrix dependent.

in slower kinetics and higher analyte recovery.

(4) temperature, and (5) sample matrix.

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

*Gas Chromatography - Derivatization, Sample Preparation, Application*

is easily automated [11].

samples (i.e., single cells).

sample headspace for SPME sampling [13].

SPME can be conducted in three modes [2]:

desorption processes, respectively.

(disadvantage).

8.SPME is compatible with chromatographic analytical systems, and the process

9.In conventional SPE, the analyte can be extracted exhaustively (>90%) into the solid phase from a sample medium, while small amount of sample (1–2%) has been introduced into the analytical equipment. But in SPME, although the analyte extraction is non-exhaustive and its small portion has been extracted into the solid phase (about 2–20%), all sample can be injected into the analytical instrument. So, besides high concentration ability and selectivity, SPME possesses another advantage in the ability of using trace analyses [12].

10. SPME facilitates unique investigations, such as extraction from very small

11. In SPME, changes in the sample matrix may affect quantitative results

12. SPME can be used to extract semivolatile organics from environmental

waters and biological matrices as long as the sample is relatively clean. Since extraction of semivolatile organics by SPME from dirty matrices is difficult, one strategy for doing it is to heat the sample to drive the compound into the

1.Direct extraction, in which the coated fiber is immersed in the aqueous sample.

2.Headspace configuration, for sampling air or the volatiles from the headspace above an aqueous sample. However, headspace techniques are more applicable

3.Membrane protection configuration, in which the coated fiber is protected with a membrane, for analyzing the analytes in too much dirty samples.

a.At first, the solid sorbent is immersed into the sample medium for a specific period of time. This step is used for both of fiber and in-tube designs.

b.Then, the solid sorbent, either fiber or in-tube design, is interfaced with GC and HPLC (or capillary electrophoresis) instruments for thermal and solvent

As discussed before, SPME has been introduced as a solventless extraction method,

1.Direct sampling. In this approach, the fiber is placed directly into the sample

2.Headspace sampling. In this approach, the fiber is placed in the headspace of

in which a fused silica fiber has been coated with a *thin* film of sorbent, to separate volatile analytes of interest from a matrix sample. Usually, the fiber is placed into a syringe needle which is protected for easy penetration into the sample and GC vial septa. Analyte extraction and analysis depend on the fiber type and its thermal desorption into a GC inlet. There are two approaches to SPME sampling of volatile organics:

to volatile organics than to the semivolatile organic compounds.

The SPME procedure is performed through two separate steps:

**50**

matrix.

the sample.

Choosing between direct immersion and headspace SPME is relatively straightforward. Direct immersion SPME is warranted for liquid and solution samples which are used in solid-phase and liquid-liquid extraction methods. Headspace SPME is considered for extraction of volatile specious, with normal boiling point less than 200°C, from solid and liquid samples. For higher boiling point analytes, direct immersion SPME is probably necessary. Headspace is more preferred for especially complex or dirty samples due to fouling the fiber coating in a direct immersion process.

SPME fibers have different coatings, and there is no single coating for extraction and separation of all volatile organics from a sample. Therefore, different types of coatings with different polarities are applied on SPME fibers. Currently, three types of fiber coatings are commercially available: (1) nonpolar, (2) semipolar, and (3) polar coatings. There are several SPME fiber coatings commercially available. These range in polarity from polydimethylsiloxane (PDMS), which is nonpolar, to "*carbowax-divinylbenzene*" (CW-DVB), which is highly polar. The nonpolar fibers are more commonly used for headspace SPME as the majority of volatile analytes tend to be nonpolar or slightly polar. The advantage of using different fiber polarities is that using a matched polarity fiber, as polar coated for a polar analyte, makes extraction selectivity be enhanced. On the other hand, there is less of a chance of extracting interfering compounds along with the analyte of interest, and an organic matrix is not a problem.

Fiber coating thickness is a second parameter that should be considered to select a fiber for both direct immersion and headspace SPME. The PDMS coating is commercially in hand in three thicknesses: 100, 30, and 7 μm. The 100-μm-thick fiber is generally applied for highly volatile compounds or when a larger organic matrix volume is used. The 7-μm-thick fiber is used for less volatile compounds.

Once the fiber is chosen, extraction conditions must be optimized. There are many variables such as (1) extraction time, (2) sample volume, (3) agitation, (4) temperature, and (5) sample matrix.


or its salt content and (b) dissolving the solid sample in a proper solvent like water or a strongly aqueous solution. In similarity with classical liquid-liquid extractions, modifying the pH can change the extraction behavior.

Also, the SPME-GC injection system must be optimized. When the SPME interfacing GC is used, the GC injection system is typically done under splitless conditions. Since there is not any solvent and accommodation of the sample solvent, there is no need of specific small internal diameter glass liners, which are often used [2]. The main consideration is to transfer the analytes in the shortest possible time out of the fiber coating and in focusing the analytes into the sharpest bands possible. For semivolatile compounds, inlet optimization is very simple, and classical splitless inlet conditions can be used. A typical condition would be a temperature of about 250°C; a sufficient head pressure can maintain optimum GC column flow and an initial column temperature at least 100°C below the normal boiling point of the analyte. For volatile analytes, the optimization of the inlet is more difficult. So, keeping the initial column temperature at enough low level to refocus these analytes is often not possible, without cryogenics. The inlet must therefore be optimized to provide the fastest possible desorption and transfer to the GC column, while the GC column is maintained as cool as possible to achieve any focusing that is possible.

There are some examples for applying SPME as sample preparation step before GC detection of different analytes in a variety of samples. Some of them are pointed below.

Goncalves et al. studied solid-phase microextraction-gas chromatography- (tandem) mass spectrometry as a tool for pesticide residue analysis in water samples at high sensitivity and selectivity with confirmation capabilities. In this study, for SPME extraction a *"poly(dimethylsiloxane)-poly(divinylbenzene)"*-coated fiber was selected [14].

Yonamine et al. studied solid-phase microextraction-gas chromatography-mass spectrometry and headspace-gas chromatography of tetrahydrocannabinol (THC), amphetamine, methamphetamine, cocaine, and ethanol in saliva samples. In this study, at first saliva samples were submitted to an initial headspace procedure for ethanol determination by a GC-flame ionization detector. Then, two consecutive fiber solid-phase microextractions were carried out: THC was extracted by submersing a polymeric fiber, and amphetamine, methamphetamine, and cocaine were subsequently extracted after alkalinization [15].

### **2.3 Molecularly imprinted polymer (MIP) adsorbent in SPE and SPME**

Molecularly imprinted polymer (MIP) is an alternative kind of sorbent which can be applied for *solid-phase extractions* and solid-phase microextractions. MIP is a polymeric sorbent which is produced in the presence of a target analyte, as a molecular template. Once the template is washed and removed through the polymer, some selective recognition sites has been remained in the polymeric sorbent for selective extraction of the analyte target. By using MIP as the sorbent, the surface contact area between the sorbent and the sample is much greater than in the coated fiber or coated inner surface tubing SPME procedures described earlier [2]. MIP inherent advantages include reusability, simplicity, low cost, high affinity and selectivity for target molecule, and physical and chemical stability over a wide range of experimental conditions and solvents [16].

Some of those applications are discussed below.

Djozan et al. studied preparation and evaluation of solid-phase microextraction fibers based on monolithic molecularly imprinted polymers for selective extraction of diacetylmorphine and analogous compounds. The main purpose of this research

**53**

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

was to develop a technique for fabrication of a monolithic and robust solid-phase microextraction on the basis of MIP interfacing with GC and GC-MS analysis for selective extraction and structural analysis of diacetylmorphine, respectively. On the other hand, a very simple approach has been developed for the fabrication of SPME fiber from diacetylmorphine-imprinted polymers which were subsequently used for

extraction of diacetylmorphine and then analyzed with GC and GC-MS [16].

syringe can be applied several times for different samples [17].

tative extractions can be achieved than those of SPME procedures.

Some of SBSE applications with GC analysis are discussed below.

(PDMS) prepared by sol-gel method was used as extraction phase [19].

**2.4 Stir bar sorptive extraction (SBSE)**

acid anhydride as acetylation reagent [18].

the potential to prepare thermally stable coatings [19].

approaching equilibrium.

extraction (HSSE) [2].

**2.5 Soxhlet extraction**

Rehim studied new trend in sample preparation [17]: online microextraction in packed syringe for liquid and gas chromatography applications and determination of local anesthetics in human plasma samples using gas chromatography-mass spectrometry. In this study, local anesthetics in plasma samples were used as model substances, and the method was developed and validated for microextraction in packed syringe (MEPS) online with GC-MS. MEPS and SPE procedures have some differences. In MEPS method, the sorbent packing is placed directly into the syringe, not into a separate column, as it is done in SPE. So, a separate robot does not need for applying the sample into the separation phase, as it is done in SPE. Also, the packed

Stir bar sorptive extraction (SBSE) is used for the extraction of trace amounts of organics from aqueous food, environmental, and biological samples. A stir bar has been covered with a sorbent phase and placed into the sample solution to separate the analyte of interest. Although SBSE procedures are not exhaustive, more quanti-

The coated stir bar is usually used to stir the sample solution for a specialized period of time, depending on the sample volume and stirring speed, until

SBSE improves on the low concentration capability of in-sample solid-phase microextraction (IS-SPME). Also, SBSE can be applied to headspace sorptive

Nakamura et al. studied simultaneous determination of alkylphenols and bisphenol A in river water by stir bar sorptive extraction with in situ acetylation and thermal desorption-gas chromatography-mass spectrometry. In this study, SBSE was used for the sample enrichment of seven alkylphenols and bisphenol A in river water. Also, in situ derivatization in aqueous samples was performed with acetic

The extraction phase on the stir bar in SBSE is critical for the performance of both extraction and thermal desorption. The sol-gel coating technology possesses

Guan et al. studied determination of organophosphorus pesticides in cucumber and potato by stir bar sorptive extraction. In this study, organophosphorus pesticides (OPPs) in vegetables were determined by SBSE and capillary GC with thermionic specific detection (GC-TSD). Hydroxy-terminated polydimethylsioxane

Soxhlet extraction was accepted as a standard method for the extraction of semivolatile and nonvolatile organics by the US Environmental Protection Agency (EPA 3540C0) and also the extraction of fat in cacao products by the Association of Official Analytical Chemists (AOAC 963.15). Soxhlet extraction was *introduced by Franz Ritter von Soxhlet in 1879*. It had been the most extensive applied technique

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

*Gas Chromatography - Derivatization, Sample Preparation, Application*

or its salt content and (b) dissolving the solid sample in a proper solvent like water or a strongly aqueous solution. In similarity with classical liquid-liquid

extractions, modifying the pH can change the extraction behavior.

Also, the SPME-GC injection system must be optimized. When the SPME interfacing GC is used, the GC injection system is typically done under splitless conditions. Since there is not any solvent and accommodation of the sample solvent, there is no need of specific small internal diameter glass liners, which are often used [2]. The main consideration is to transfer the analytes in the shortest possible time out of the fiber coating and in focusing the analytes into the sharpest bands possible. For semivolatile compounds, inlet optimization is very simple, and classical splitless inlet conditions can be used. A typical condition would be a temperature of about 250°C; a sufficient head pressure can maintain optimum GC column flow and an initial column temperature at least 100°C below the normal boiling point of the analyte. For volatile analytes, the optimization of the inlet is more difficult. So, keeping the initial column temperature at enough low level to refocus these analytes is often not possible, without cryogenics. The inlet must therefore be optimized to provide the fastest possible desorption and transfer to the GC column, while the GC column is maintained as cool as possible to achieve any focusing that is possible. There are some examples for applying SPME as sample preparation step before GC detection of different analytes in a variety of samples. Some of them are pointed

Goncalves et al. studied solid-phase microextraction-gas chromatography- (tandem) mass spectrometry as a tool for pesticide residue analysis in water samples at high sensitivity and selectivity with confirmation capabilities. In this study, for SPME extraction a *"poly(dimethylsiloxane)-poly(divinylbenzene)"*-coated fiber was

Yonamine et al. studied solid-phase microextraction-gas chromatography-mass spectrometry and headspace-gas chromatography of tetrahydrocannabinol (THC), amphetamine, methamphetamine, cocaine, and ethanol in saliva samples. In this study, at first saliva samples were submitted to an initial headspace procedure for ethanol determination by a GC-flame ionization detector. Then, two consecutive fiber solid-phase microextractions were carried out: THC was extracted by submersing a polymeric fiber, and amphetamine, methamphetamine, and cocaine were

**2.3 Molecularly imprinted polymer (MIP) adsorbent in SPE and SPME**

Molecularly imprinted polymer (MIP) is an alternative kind of sorbent which can be applied for *solid-phase extractions* and solid-phase microextractions. MIP is a polymeric sorbent which is produced in the presence of a target analyte, as a molecular template. Once the template is washed and removed through the polymer, some selective recognition sites has been remained in the polymeric sorbent for selective extraction of the analyte target. By using MIP as the sorbent, the surface contact area between the sorbent and the sample is much greater than in the coated fiber or coated inner surface tubing SPME procedures described earlier [2]. MIP inherent advantages include reusability, simplicity, low cost, high affinity and selectivity for target molecule, and physical and chemical stability over a wide range

Djozan et al. studied preparation and evaluation of solid-phase microextraction fibers based on monolithic molecularly imprinted polymers for selective extraction of diacetylmorphine and analogous compounds. The main purpose of this research

**52**

below.

selected [14].

subsequently extracted after alkalinization [15].

of experimental conditions and solvents [16].

Some of those applications are discussed below.

was to develop a technique for fabrication of a monolithic and robust solid-phase microextraction on the basis of MIP interfacing with GC and GC-MS analysis for selective extraction and structural analysis of diacetylmorphine, respectively. On the other hand, a very simple approach has been developed for the fabrication of SPME fiber from diacetylmorphine-imprinted polymers which were subsequently used for extraction of diacetylmorphine and then analyzed with GC and GC-MS [16].

Rehim studied new trend in sample preparation [17]: online microextraction in packed syringe for liquid and gas chromatography applications and determination of local anesthetics in human plasma samples using gas chromatography-mass spectrometry. In this study, local anesthetics in plasma samples were used as model substances, and the method was developed and validated for microextraction in packed syringe (MEPS) online with GC-MS. MEPS and SPE procedures have some differences. In MEPS method, the sorbent packing is placed directly into the syringe, not into a separate column, as it is done in SPE. So, a separate robot does not need for applying the sample into the separation phase, as it is done in SPE. Also, the packed syringe can be applied several times for different samples [17].
