**4. Nanofiber based SPE method development**

What is the starting point in electrospun nanofiber based SPE method development? The questions can be classified into four as;


Of all these, the most important aspect is to come up with a feasible electrospun nanofiber based SPE device as that serves as a platform for a optimal SPE method development.

To date, fabricated SPE devices that employ electrospun nanofibers as a sorbent bed have been based on polystyrene or nylon polymers. This has resulted in a classification of electrospun nanofiber based SPE devices into two as: polystyrene type (polymer fibers of a relatively low mechanical strength); and, nylon type (polymer fibers of a relatively high mechanical strength) [59].

#### **4.1. Polystyrene type SPE devices**

**Figure 5.** SEM image of electrospun nanofiber composite membrane containing molecular imprinted nanoparticles

**Figure 6.** Schematic representation of molecularly imprinted nanofibers with binding sites specific for 2,4-D template

Chronakis and co-workers reported a simplified approach that allowed the generation of template defined sites directly during electrospinning [58]. The electrospun nanofibers were prepared from a solution mixture of PET and polyallylamine in the presence of a template molecule, 2,4-dichlorophenoxyacetic acid (2,4-D). Polyallylamine was used to provide functional groups that interacted with the template during the electrospinning process, and PET was used as the supporting matrix to ensure easy fiber formation and to minimize the conformational change of the polymers when the nanofibers were subjected to different

[45].

12 Advances in Nanofibers

molecules [58].

In 2007, Kang and co-workers were the first to report the use of electrospun polymer nanofibers for packed sorbent SPE [60]. They manually packed 1 mg of polystyrene nanofibers into a 200 µl micro pipette tip to form a micro column as shown in Fig.7. (a) and (b) as published in their 2009 article. Solvents were pushed through the electrospun nanofiber based SPE device manually by the pressure of air forced by a gas tight plastic syringe (2 mL) (Fig.7 (c)). The device demonstrated a leap forward regarding the use electrospun nanofibers for miniaturized SPE devices. Although the packing process involved the use of simple homemade tools, it seems the packing operation is not that simple as reproducibility relies very much on operator experience. Nevertheless, the study created a platform for further research as it clearly demonstrated that nanofibers allowed the miniaturization of SPE devices without compro‐ mising the extraction efficiency. The packing procedure took advantage of the ease of rolling polystyrene nanofibers into nanofiber clews. Several reports followed where either polystyr‐ ene or polystyrene copolymer nanofibers were employed as the sorbent bed packed in micro column format [61-66].

**Figure 7.** Micro column packed with polystyrene nanofibers and designed setup: (a) Schematic representation of packed fiber solid phase extraction device: (1) Pipette tip, (2) nanofibers packed, (3) eppendorf certrifuge tube, (4) de‐ sorption solution; and (b) Photograph of the micro-column; and (c) Photograph of a manual device [63].

All reports seem to have focused mainly on demonstrating the feasibility of using electrospun nanofibers as a SPE sorbent material since the experiments were biased towards obtaining maximum recovery.

For a better understanding of the use of electrospun nanofibers as a sorbent bed, it would be necessary to determine the mechanical strength, packing density and retention characteristics of the sorbent bed. In an effort to determine the mechanical strength of electrospun polystyrene fibers as a function of the effect of the force applied in the manual packing process, in our research group we viewed the top side of the micro column sorbent bed under scanning electron microscopy (SEM) (see Fig.8. (b-c)). The SEM morphology confirmed that the force applied did not induce any breakup or significant flattening of the electrospun polystyrene fibers.

in all existing formats and configurations for both SPE and SPME [4]. Furthermore, the use of electrospun fibers introduces an aspect of simplicity and miniaturization to the fabrication of

**Figure 8.** (a) Photograph of micro column SPE device, SEM images of (b) sorbent bed (c) magnification of top edge (d)

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To date, methods of SPE disk fabrication involve a complicated multi step process in which microparticles are tightly held together within an inert fiber matrix, such as polytetrafluoro‐ ethylene [67]. This has limited the range of disk sorbent chemistries that are available. Two possible ways of simplifying the disk sorbent fabrication process would be; (i) to incorporate nanoparticles into nanofibers and pack the fibers in disk format or (ii) to fabricate nanofibers

In 2010, our group reported an alternative polystyrene fiber based SPE device fabrication procedure [68]. The experimental approach consisted of copolymerization with subsequent electrospinning of the resultant polymer. 10 mg of electrospun polystyrene fibers were packed in a disk format (5 mm × 1 mm) (Fig.9.). The study clearly demonstrated how the use of electrospun fibers can simplify SPE disk fabrication as the sorbent material was packed using simple homemade tools. It is expected that in the near future, a wide range of chemistries will

of the material with the chemistry of interest and pack them in disk format.

SPE devices.

zoomed in top surface.

One of the main drawbacks of the micro column packing procedure is consistency as it relies very much on the experience of the researcher. The main challenge is the rolling up of a uniform size of fiber clews. Without precise control of the size of fiber clews as well as the force applied, it would be difficult to maintain a uniform packing density and sorbent bed height, thus inconsistency of flow characteristics. Since electrospun nanofibers are applicable as a HPLC stationary phase, adopting this packing method for HPLC columns would be a challenge. This is in light of the fact that, without precise control of the packing procedure, band broadening could result due to multiple flow paths as a function of non uniform fiber orientation. Despite all these challenges, the development of the micro column packing procedure was invaluable to electrospun nanofiber based SPE sorbent research as it was used as a platform for evaluating the feasibility of using electrospun nanofibers as a sorbent bed.

Two important aspects in the development of SPE technology are simplicity of sorbent fabrication and miniaturization of devices. It is a fact that electrospun fibers could be packed

mising the extraction efficiency. The packing procedure took advantage of the ease of rolling polystyrene nanofibers into nanofiber clews. Several reports followed where either polystyr‐ ene or polystyrene copolymer nanofibers were employed as the sorbent bed packed in micro

**Figure 7.** Micro column packed with polystyrene nanofibers and designed setup: (a) Schematic representation of packed fiber solid phase extraction device: (1) Pipette tip, (2) nanofibers packed, (3) eppendorf certrifuge tube, (4) de‐

All reports seem to have focused mainly on demonstrating the feasibility of using electrospun nanofibers as a SPE sorbent material since the experiments were biased towards obtaining

For a better understanding of the use of electrospun nanofibers as a sorbent bed, it would be necessary to determine the mechanical strength, packing density and retention characteristics of the sorbent bed. In an effort to determine the mechanical strength of electrospun polystyrene fibers as a function of the effect of the force applied in the manual packing process, in our research group we viewed the top side of the micro column sorbent bed under scanning electron microscopy (SEM) (see Fig.8. (b-c)). The SEM morphology confirmed that the force applied did not induce any breakup or significant flattening of the electrospun polystyrene

One of the main drawbacks of the micro column packing procedure is consistency as it relies very much on the experience of the researcher. The main challenge is the rolling up of a uniform size of fiber clews. Without precise control of the size of fiber clews as well as the force applied, it would be difficult to maintain a uniform packing density and sorbent bed height, thus inconsistency of flow characteristics. Since electrospun nanofibers are applicable as a HPLC stationary phase, adopting this packing method for HPLC columns would be a challenge. This is in light of the fact that, without precise control of the packing procedure, band broadening could result due to multiple flow paths as a function of non uniform fiber orientation. Despite all these challenges, the development of the micro column packing procedure was invaluable to electrospun nanofiber based SPE sorbent research as it was used as a platform for evaluating

Two important aspects in the development of SPE technology are simplicity of sorbent fabrication and miniaturization of devices. It is a fact that electrospun fibers could be packed

the feasibility of using electrospun nanofibers as a sorbent bed.

sorption solution; and (b) Photograph of the micro-column; and (c) Photograph of a manual device [63].

column format [61-66].

14 Advances in Nanofibers

maximum recovery.

fibers.

in all existing formats and configurations for both SPE and SPME [4]. Furthermore, the use of electrospun fibers introduces an aspect of simplicity and miniaturization to the fabrication of SPE devices.

To date, methods of SPE disk fabrication involve a complicated multi step process in which microparticles are tightly held together within an inert fiber matrix, such as polytetrafluoro‐ ethylene [67]. This has limited the range of disk sorbent chemistries that are available. Two possible ways of simplifying the disk sorbent fabrication process would be; (i) to incorporate nanoparticles into nanofibers and pack the fibers in disk format or (ii) to fabricate nanofibers of the material with the chemistry of interest and pack them in disk format.

In 2010, our group reported an alternative polystyrene fiber based SPE device fabrication procedure [68]. The experimental approach consisted of copolymerization with subsequent electrospinning of the resultant polymer. 10 mg of electrospun polystyrene fibers were packed in a disk format (5 mm × 1 mm) (Fig.9.). The study clearly demonstrated how the use of electrospun fibers can simplify SPE disk fabrication as the sorbent material was packed using simple homemade tools. It is expected that in the near future, a wide range of chemistries will be introduced on disk SPE devices fabricated using a similar approach that will ultimately lead to a routine disk fabrication technology.

Although the procedure involved a manual compression stage, it was presumably simplified as it did not involve the rolling up of fiber clews. Unlike the micro column SPE device where some degree of flattening was observed, for this device, flattening was not observed as viewed under SEM (see Fig.9. b-c). This suggested that the force applied in the packing process was lower compared to that applied for the micro column SPE device. A possible explanation could be that the wire (0.5 mm diameter) employed in the micro column SPE device fabrication procedure by virtue of having a smaller contact surface compared to the glass rod (5 mm diameter) employed in the disk SPE device fabrication procedure resulted in increased pressure.

In 2011, Bagheri and co-workers reported an alternative fabrication procedure for a nylon type SPE device in which a polypyrrole-nylon 6 blend was employed as a sorbent bed [75]. Similar to the nylon 6 disk sorbent fabrication procedure, a nanofiber sheet (1× 1 cm) was cut out directly from the aluminium foil (see Fig.11.). However, the extraction procedure was not a flow through process as the sorbent bed was held by a wire and dipped into a sample solution. Although they referred to their device as micro SPE, it could be regarded as a SPME device as it was equilibrium based. Nevertheless, it is presented in this context as the fabrication procedure takes advantage of the mechanical strength of nylon nanofibers to illustrate the dependence of electrospun nanofiber evice fabrication on nanofiber mechanical stability.

**Figure 10.** The fiber-filter solid phase extraction (SPE) device: gas tight syringe (1); sample solution (2); filter (3); elec‐

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trospun mat using nylon 6 nanofibers (4); and, collecting tube (5) [70].

**Figure 11.** (a) Image of the polypyrrole-polyamide nanofiber sheet used for micro-solid phase extraction (µ-SPE). (b)

The only report of electrospun nanofiber based micro extraction in a packed syringe (MEPS) was by Bagheri and co-workers [76]. Their packing procedure involved the manual compres‐ sion of 8.1 mg of polyrrole/nylon 6 nanofiber blend into a 1 mL insulin injection syringe. The electrospun nanofiber sorbent bed was laid flat at the bottom of the syringe barrel between

Sample extraction: (A) magnetic bar; (B) nanofiber sheet; (C) thin holding wire; (D) vial; and, (E) vial cap [75].

**Figure 9.** (a) Photograph of disk (I) SPE device, images of (b) sorbent bed (c) zoomed in top surface [68].

Despite the observed slight flattening of the electrospun polystyrene fibers in the micro column packing format, percolation of the solvents was not hindered which suggested that the packing procedure was adequate for fundamental experiments. To date, it seems, the only way of fabricating SPE devices that use electrospun nanofibers of a relatively low mechanical strength (as represented by polystyrene) would be via a mechanical compression stage. A possible way of improving on consistency could be to automate the mechanical compression stage.

#### **4.3. Nylon type SPE devices**

In 2009, Xu and co-workers were the first to report the fabrication of SPE devices that rely on relatively mechanically strong electrospun nanofibers as the sorbent bed [69]. Nylon 6 nanofiber sorbent beds were "packed" by cutting out circular portions (1.5 cm × 120-150 µm × 1.5 mg) of the nanofiber sheet (see Fig.10.).

There have been several other reports on the use of nylon 6 nanofibers packed in the same format [70-74]. Similar to the first report by Xu and co-workers [69], all these reports seem to have focussed on demonstrating the feasibility of using electrospun nylon 6 nanofibers as a SPE sorbent material.

be introduced on disk SPE devices fabricated using a similar approach that will ultimately lead

Although the procedure involved a manual compression stage, it was presumably simplified as it did not involve the rolling up of fiber clews. Unlike the micro column SPE device where some degree of flattening was observed, for this device, flattening was not observed as viewed under SEM (see Fig.9. b-c). This suggested that the force applied in the packing process was lower compared to that applied for the micro column SPE device. A possible explanation could be that the wire (0.5 mm diameter) employed in the micro column SPE device fabrication procedure by virtue of having a smaller contact surface compared to the glass rod (5 mm diameter) employed in the disk SPE device fabrication procedure resulted in increased

**Figure 9.** (a) Photograph of disk (I) SPE device, images of (b) sorbent bed (c) zoomed in top surface [68].

of improving on consistency could be to automate the mechanical compression stage.

Despite the observed slight flattening of the electrospun polystyrene fibers in the micro column packing format, percolation of the solvents was not hindered which suggested that the packing procedure was adequate for fundamental experiments. To date, it seems, the only way of fabricating SPE devices that use electrospun nanofibers of a relatively low mechanical strength (as represented by polystyrene) would be via a mechanical compression stage. A possible way

In 2009, Xu and co-workers were the first to report the fabrication of SPE devices that rely on relatively mechanically strong electrospun nanofibers as the sorbent bed [69]. Nylon 6 nanofiber sorbent beds were "packed" by cutting out circular portions (1.5 cm × 120-150 µm

There have been several other reports on the use of nylon 6 nanofibers packed in the same format [70-74]. Similar to the first report by Xu and co-workers [69], all these reports seem to have focussed on demonstrating the feasibility of using electrospun nylon 6 nanofibers as a

to a routine disk fabrication technology.

pressure.

16 Advances in Nanofibers

**4.3. Nylon type SPE devices**

SPE sorbent material.

× 1.5 mg) of the nanofiber sheet (see Fig.10.).

**Figure 10.** The fiber-filter solid phase extraction (SPE) device: gas tight syringe (1); sample solution (2); filter (3); elec‐ trospun mat using nylon 6 nanofibers (4); and, collecting tube (5) [70].

In 2011, Bagheri and co-workers reported an alternative fabrication procedure for a nylon type SPE device in which a polypyrrole-nylon 6 blend was employed as a sorbent bed [75]. Similar to the nylon 6 disk sorbent fabrication procedure, a nanofiber sheet (1× 1 cm) was cut out directly from the aluminium foil (see Fig.11.). However, the extraction procedure was not a flow through process as the sorbent bed was held by a wire and dipped into a sample solution. Although they referred to their device as micro SPE, it could be regarded as a SPME device as it was equilibrium based. Nevertheless, it is presented in this context as the fabrication procedure takes advantage of the mechanical strength of nylon nanofibers to illustrate the dependence of electrospun nanofiber evice fabrication on nanofiber mechanical stability.

**Figure 11.** (a) Image of the polypyrrole-polyamide nanofiber sheet used for micro-solid phase extraction (µ-SPE). (b) Sample extraction: (A) magnetic bar; (B) nanofiber sheet; (C) thin holding wire; (D) vial; and, (E) vial cap [75].

The only report of electrospun nanofiber based micro extraction in a packed syringe (MEPS) was by Bagheri and co-workers [76]. Their packing procedure involved the manual compres‐ sion of 8.1 mg of polyrrole/nylon 6 nanofiber blend into a 1 mL insulin injection syringe. The electrospun nanofiber sorbent bed was laid flat at the bottom of the syringe barrel between two SPE frits. It is quite unfortunate that the authors did not publish the resultant SPE device otherwise it would have contributed towards a better understanding of the behaviour of the sorbent bed under compression. Similar to conventional MEPS [77] (see Fig.12.) procedure that proceeds via a draw eject cycle, a variable speed stirring motor was employed to drive solvents through the electrospun nanofiber MEPS device. Despite the fact that the authors did not publish their fabricated device, their contribution was invaluable as they were the first to demonstrate an electrospun nanofiber based MEPS device.

at the stage of determining the breakthrough volume. Simplification of the electrospun nanofiber based SPE process has been achieved in our group by employing a syringe pump

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**Figure 14.** Syringe pump driven semi automated systems (a) micro column SPE device (b) modified disk (I) and disk (II) SPE device (s) (c) syringe pump (d) polypropylene syringe (e) PVC tubing (f) polypropylene adaptor (g) glass adaptor.

It is expected that in future, further simplification of electrospun nanofiber based SPE opera‐ tion may be achieved employing robotic solid phase extraction systems. Despite the fact that robotic SPE systems like the Hamilton Micro Lab 2200 [20] and the Zymark Py Technology II® [21] achieved minimal success contrary to expectation, the knowledge gained could be useful

**Figure 15.** Most common robots for automated SPE (a)Zymark Rapid Trace System (b) Tomtec Quadra System (c) Gil‐

An optimal sorbent could provide a platform for fast analyte mass-transfer kinetics, which depends on the physicochemical properties of the sorbent (surface area, pore structure and

as a step towards a new direction in automated electrospun nanofiber based SPE.

for semi automation.

son SPE 215 System.

**5. Theoretical aspects**

**Figure 12.** Representation of a MEPS syringe from SGE Analytical Science [77]

In our group, we fabricated a miniaturised version of an electrospun nylon 6 nanofiber based disk SPE device (Fig.13.a). The device was fabricated by cutting out circular portions of 5 mm diameter from the nanofiber sheet (Figure.13.b) and stacking them up to an optimal sorbent mass of 4.6 mg (5× 350 µm disks).

**Figure 13.** (a) Photograph of disk (II) SPE device, (b) photograph of an electrospun nylon 6 nanofiber mat showing regions from which disks were cut and (c) SEM image of sorbent bed zoomed in top surface [59].

Operationally, solvents have been driven through the SPE sorbent bed manually by a micro‐ pipette, syringe or by a vacuum manifold. All these approaches seem to be tedious particularly at the stage of determining the breakthrough volume. Simplification of the electrospun nanofiber based SPE process has been achieved in our group by employing a syringe pump for semi automation.

**Figure 14.** Syringe pump driven semi automated systems (a) micro column SPE device (b) modified disk (I) and disk (II) SPE device (s) (c) syringe pump (d) polypropylene syringe (e) PVC tubing (f) polypropylene adaptor (g) glass adaptor.

It is expected that in future, further simplification of electrospun nanofiber based SPE opera‐ tion may be achieved employing robotic solid phase extraction systems. Despite the fact that robotic SPE systems like the Hamilton Micro Lab 2200 [20] and the Zymark Py Technology II® [21] achieved minimal success contrary to expectation, the knowledge gained could be useful as a step towards a new direction in automated electrospun nanofiber based SPE.

**Figure 15.** Most common robots for automated SPE (a)Zymark Rapid Trace System (b) Tomtec Quadra System (c) Gil‐ son SPE 215 System.
