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Journal of Chromatography A, 1364 (2014) 261–270

Contents lists available at ScienceDirect

Journal of Chromatography A

j o ur na l ho me page: www.elsev ier .com/ locate /chroma

ilica-based nanofibers for electrospun ultra-thin layerhromatography

oni E. Newsome, Susan V. Olesik ∗

epartment of Chemistry and Biochemistry, The Ohio State University, 100 West 18th Avenue, Columbus, OH 43210, United States

r t i c l e i n f o

rticle history:eceived 29 January 2014eceived in revised form 19 August 2014ccepted 21 August 2014vailable online 28 August 2014

eywords:lectrospinningilica nanofiberlanar chromatographyltra-thin layer chromatographyligned nanofiber

a b s t r a c t

Nanofibrous silica-based stationary phases for electrospun ultra-thin layer chromatography (E-UTLC) aredescribed. Nanofibers were produced by electrospinning a solution of silica nanoparticles dispersed inpolyvinylpyrrolidone solutions to create composite silica/polymer nanofibers. Stationary phases werecreated from as-spun nanofibers, or the nanofibers were heated either to crosslink the polyvinylpyrroli-done or to calcine and selectively remove the polymer. As-spun, crosslinked, and calcined nanofiberswith similar mat thicknesses (23–25 �m) were evaluated as stationary phases for E-UTLC separationsof laser dyes and amino acids and compared to commercial silica TLC plates. As-spun nanofiber platesoffered fast mobile phase velocities, but like other polymer-based nanofibers, separations were only com-patible with techniques using nonsolvents of the polymer. Crosslinked nanofibers were not as limitedin terms of chemical stability, but separations produced tailed spot shapes. No limitations in terms ofmobile phases, analyte solvents, and visualization techniques were observed for calcined nanofibers.

mino acid Highly efficient separations of amino acids were performed in 15 mm on calcined nanofiber plates, withobserved plate heights as low as 8.6 �m, and plate numbers as large as 1400. Additional alignment of thenanofibers provided shorter analysis times but also larger spot widths. The extension of stationary phasesto silica-based nanofibers vastly expands the range of mobile phases, analyte solvents, and visualizationtechniques which can be used for E-UTLC separations.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Originating in the 1950s, thin layer chromatography (TLC) orlanar chromatography is a widely used technique in synthetichemistry as well as the food science, pharmaceutical, and environ-ental industries [1]. The parallel nature of planar chromatography

nd simple equipment required allow high sample throughputt low cost. Typical stationary phases for TLC are comprised oforbent particles attached to a solid support, such as silica gel,unctionalized silica, cellulose, magnesium oxide, or aluminumxide attached to an aluminum or glass substrate [2–5]. High-erformance thin layer chromatography (HPTLC) and ultra-thin

ayer chromatography (UTLC) have also been developed in addi-ion to conventional TLC [3,6]. HPTLC utilizes sorbent particlesith smaller dimensions and size distributions as well as overall

late dimensions. It offers shorter analysis times, lower detection

imits, and reduced sample and mobile phase volumes. Trend-ng toward miniaturization, UTLC was introduced in 2001 using

∗ Corresponding author. Tel.: +1 614 292 0733; fax: +1 614 688 5402.E-mail address: olesik.1@osu.edu (S.V. Olesik).

ttp://dx.doi.org/10.1016/j.chroma.2014.08.065021-9673/© 2014 Elsevier B.V. All rights reserved.

monolithic silica sorbent layers fixed to a glass substrate withoutbinder [7]. As opposed to conventional TLC which utilizes sor-bent layers with thicknesses of 100–400 �m, UTLC utilizes sorbentlayers with thicknesses of only 5–25 �m [3]. UTLC layers furtherimprove sensitivity and decrease analysis time and separation dis-tance compared to HPTLC, but the miniaturized media are morechallenging to handle [3]. Monolithic silica gel UTLC plates (10 �mthick) were available from Merck (EMD/Millipore in North Amer-ica), however their manufacture was discontinued [8]. To the best ofour knowledge, UTLC plates are no longer commercially available.However, UTLC continues to develop further. Stationary phasesfrom electrospun nanofibers [9–14], carbon-nanotube-templatedforests [15–17], and nanostructured thin films [3,18–26] are underdevelopment.

The use of electrospun nanofiber stationary phases in UTLC wasrecently introduced by our group and is referred to as E-UTLC [9].Electrospinning is a simple and cost-effective process which relieson repulsive electrostatic forces to produce fibers from a polymer

solution. A high electric potential is created between a polymersolution and a conductive collector. At a critical voltage, the surfacetension of the polymer solution is overcome and a jet of polymer isejected from the polymer droplet toward the grounded collector.

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62 T.E. Newsome, S.V. Olesik / J. C

sing the electrospinning approach, nanofibers with diameter inhe range of 50–900 nm can be produced [27]. In E-UTLC, electro-pinning is used to produce mat of randomly-placed nanofibersbout 25 �m thick and the nanofibrous mat is used as an ultra-thintationary phase for UTLC separations. Advantages of this methodnclude the absence of a binder material between the nanofibertationary phase and the solid support, as well as easily tunableat thicknesses and nanofiber diameters [9,10]. Additionally, faster

eparations and enhanced efficiencies have been reported in a shorteparation distance. Because of the vast range of polymers thatan be electrospun, this method is readily capable of producinganofibers with a variety of functionalities [27].

So far, only electrospun polymer and carbon nanofibers haveeen utilized as stationary phases in E-UTLC. Electrospun station-ry phases comprised of polymers include polyacrylonitrile (PAN)9,11,13], polyvinylalcohol (PVA) [12], and cellulose acetate [14],hile those comprised of carbon nanofibers have been electro-

pun from an epoxide polymer followed by pyrolysis to glassyarbon [10]. In addition, varieties of PAN nanofiber stationaryhases have been reported. Compared to using randomly-placedanofibers, the use of highly-aligned PAN nanofibers was studied

n aligned electrospun ultra-thin layer chromatography (AE-UTLC)nd demonstrated increased speed of analysis as well as increasedfficiency [11]. Additionally, a photoluminescent indicator has beendded to PAN nanofibers to aid in the visualization of UV-activeompounds [13]. However, separations using these polymer-basedtationary phases are restricted to mobile phases, analyte solvents,nd visualization techniques that are nonsolvents of the polymer14]. As such, the range of analytes which can be separated and

ethods used for separation and visualization are limited withhese polymer-based devices. On the other hand, carbon nanofiber-UTLC devices do not have these solvent limitations. However,ue to the color of carbon, conventional staining techniques areifficult if not impossible, limiting carbon nanofiber E-UTLC to UVetection only once analytes have been desorbed from the station-ry phase. Extension of E-UTLC to silica-based nanofibers would bef particular interest not only because silica remains the most com-only used stationary phase in TLC but also because silica-based

tationary phases would not be limited to particular solvents orisualization techniques as are the current electrospun polymernd carbon nanofiber stationary phases, respectively [2,28].

Herein, we report the first example to extend E-UTLC toilica-based nanofiber stationary phases. Using an electrospinningrotocol optimized for minimum nanofiber diameter and homoge-ous morphology [29], silica-based nanofibers were produced bylectrospinning a solution of silica nanoparticles dispersed in aolyvinylpyrrolidone solution to create composite silica/polymeranofibers. They were further processed by heat treatment eithero crosslink the polyvinylpyrrolidone or to calcine and selec-ively remove the polymer. The as-spun, crosslinked, and calcinedanofibers were applied to the UTLC separations of laser dyes andmino acids. The performance of the silica-based E-UTLC platesas compared to commercially available silica HPTLC plates. The

bbreviation, E-UTLC, will be used throughout to denote UTLCith randomly-placed fibers while AE-UTLC will denote UTLC with

ligned fibers.

. Materials and methods

.1. Materials

Silica nanoparticles (SiO2 NPs), AngstromSphere monodis-ersed silica powder, 250 nm with a particle size standard deviationf <10%, were purchased from Fiber Optic Center Inc. (New Bedford,A). Polyvinylpyrrolidone (PVP), average Mw 1,300,000, K 85–95,

togr. A 1364 (2014) 261–270

was purchased from Acros Organics through Fisher Scientific (Pitts-burgh, PA). Reagent alcohol (90% ethanol, 5% methanol, and 5%2-propanol; HPLC grade, 99.8% purity) was purchased from FisherScientific. Methanol was purchased from Macron Chemicals (St.Louis, MO).

Laser dyes were purchased from Exciton Inc. (Dayton, OH);the dyes included rhodamine 590 chloride, rhodamine 101 chlo-ride, rhodamine 610 chloride, rhodamine 610 perchlorate, andpyrromethene 597. The amino acids include arginine, asparticacid, threonine, and phenylalanine; arginine and aspartic acidwere purchased from Sigma–Aldrich (St. Louis, MO), threoninewas purchased from Calbiochem through EMD Millipore (Billerica,MA), and phenylalanine was purchased from Eastman ChemicalCompany (Kingsport, TN). Ethyl acetate (EtOAc) and methanolwere purchased from Macron Chemicals (St. Louis, MO). Hep-tane, glacial acetic acid (HOAc), 1-propanol, 2-propanol, acetone,methyl ethyl ketone, nitric acid, sulfuric acid, and phosphoricacid were purchased from Fisher Scientific. Ninhydrin, n-butanol(n-BuOH), hydrocortisone, androsterone, and cholesterol were pur-chased from Sigma–Aldrich. Ethanol was purchased from DeconLabs, Inc. (King of Prussia, PA). Ferric chloride (FeCl3) was purchasedfrom Mallinckrodt Chemicals. Commercially available Merck silicagel HPTLC plates were purchased from EMD Chemicals (Gibbstown,NJ) with diameters of 4.0–8.0 �m, 60 A medium pore size, and layerthickness of 150–200 �m on glass plates.

2.2. Instrumentation

The apparatus used for electrospinning has been describedelsewhere [31]. As-spun composite SiO2/PVP nanofibers werecrosslinked using a Lindberg/Blue tube furnace (Model TF55030A,Waltham, MA) and were calcined using a Lindberg/Blue M tubefurnace (Model STF55346C-1, Waltham, MA). The scanning elec-tron microscopes (SEM) included a Hitachi S-4300 SEM (HitachiHigh Technologies America, Inc., Pleasonton, CA) and a Quanta 200Series SEM (FEI Company, Hillsboro, OR). Each sample was sputtercoated with gold to create a conductive surface for SEM imaging.Digital images were taken with a Canon A650IS 12.1 MP digitalcamera.

2.3. Electrospinning SiO2/PVP nanofibers

SiO2/PVP electrospinning solutions were prepared as describedelsewhere [29]. PVP solutions were prepared in reagent alcohol(10.0 wt%) and SiO2 NP dispersions were prepared by dispersingdry SiO2 NPs in reagent alcohol (20.0 wt%). Electrospinning solu-tions were prepared by mixing the SiO2 dispersion and PVP solutionat a 2:3 weight ratio (SiO2 dispersion: PVP solution).

The apparatus and optimized method used to produce electro-spun SiO2/PVP nanofibers for all experiments except for alignedelectrospun nanofibers have been described in detail [29]. Matthickness was determined via SEM image analysis of the cross-section of the nanofibrous mat using a vertical sample stage; allSEM images of the electrospun nanofibers were analyzed usingImageJ software (available from National Institutes of Health, NIH,at http://rsbweb.nih.gov/ij/).

To create aligned SiO2/PVP nanofibers, the collection plate wasattached to a high speed spinning drum [11]. The speed of thedrum was determined using a NIST-certified, Monarch Instrument

PLT200 pocket laser tachometer (Cole-Parmer, Vernon Hills, IL). Atip to collector distance of 10 cm was used. A 105 cm × 3.5 cm pieceof 0.003 in. thick sanded stainless steel shim stock was wrappedaround the drum to serve as the nanofiber collector.

hromatogr. A 1364 (2014) 261–270 263

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Table 1Average nanofiber diameter and mat thickness of the randomly-placed as-spun,crosslinked, and calcined SiO2/PVP nanofiber stationary phases and the aligned-calcined SiO2/PVP nanofiber stationary phase.

SiO2/PVP nanofiber Nanofiber diameter(nm) [29]

Mat thickness(�m)

As-spun 380 ± 100 24 ± 3Crosslinked 380 ± 110 24 ± 2

nanofibers were electrospun for 30 min to achieve a stationaryphase with a similar mat thickness (24 ± 2 �m). This stationaryphase is shown in Fig. 1(A–C). Initial experiments were performedutilizing the as-spun nanofiber stationary phases spun for 30 min

T.E. Newsome, S.V. Olesik / J. C

.4. Thermal crosslinking and calcining of the composite SiO2/PVPanofibers

SiO2/PVP nanofibers were thermally crosslinked to producensoluble nanofibers (crosslinked nanofibers) using an optimized

ethod [29]. As-spun SiO2/PVP nanofibers collected for 1.0 h wererosslinked by heating to 200 ◦C at 1.0 ◦C/min under constant airow in a tube furnace. SiO2/PVP nanofibers were calcined to selec-ively remove the polymer matrix (calcined nanofibers) [29]. Thes-spun SiO2/PVP nanofibers collected for 2.5 h were calcined byeating to 465 ◦C at 0.5 ◦C/min under constant air flow.

.5. Thin layer chromatography

UTLC plates were prepared from as-spun nanofibers by cuttinghe original 6.5 cm × 11.0 cm mat collected on shim stock into three3 cm × 6 cm plates. UTLC plates were prepared from crosslinkedanofibers by cutting the crosslinked 2.3 cm × 11.0 cm mat into

∼2 cm × 6 cm plate. UTLC plates were prepared from the cal-ined nanofibers and aligned-calcined nanofibers by cutting theats into ∼1.5 cm × 2.5 cm plates. Analytes were spotted onto the

ottom of UTLC plates, 2 mm above the solvent immersion linehich was 3 mm from the bottom of the plate, using fused silica

apillary tubing (i.d. 100 �m id, Polymicro Technologies, Phoenix,Z); the volume spotted was 50 nL, unless otherwise noted. 5 �Molutions of laser dyes in EtOAc were used on as-spun nanofiberlates, and aqueous solutions of amino acids were used on therosslinked nanofiber plates (15 mM) and on the calcined nanofiberlates and commercial silica plates (30 mM). A rectangular devel-pment chamber (volume = 80 mL) was used with 1 mL of mobilehase for UTLC stationary phases and 2 mL for commercial station-ry phases. Prior to development, the chamber was equilibrated for5 min. The optimization of the chamber size and the spotting cap-

llary size is described in the Optimization of E-UTLC section (Fig. S1)n the Supplemental Information (SI).

After development, visualization of analytes was recorded using Canon A650IS 12.1 MP digital camera. For laser dye separations,nalytes were visualized using a Spectroline digital documenta-ion system (Spectroline, Westbury, NY) which consisted of theamera mounted on a CC-81 cabinet using a GL-1301 univer-al camera adapter with a 58 mm adapter ring and an ENF-280 C65 nm/254 nm UV lamp (8 W). During image documentation, laseryes were visualized using UV light (� = 254 nm). For amino acideparations, stationary phases were sprayed evenly with freshlyrepared ninhydrin solution (0.3 g of ninhydrin in 100 mL n-BuOHnd 3 mL HOAc) using a TLC reagent nebulizer (Kimble-Chaseineland, NJ) and heated for 10 min at 110 ◦C [30]. The sameanon camera was used for documentation using ambient lighting.hromatograms were obtained from digital images using ImageJensitometric analyses and were analyzed using PeakFit (version, SPSS Inc.). All reported results were based on at least three mea-urements.

.6. Post-separation visualization method compatibility

For post-separation visualization method compatibility experi-ents, 50 nL of the aqueous amino acid solutions (described above)

nd solutions of hydrocortisone, androsterone, and/or cholesterol5 mM dissolved in methanol) were spotted onto TLC plates using50 �m capillary tubes and allowed to dry for at least an hour. Elec-rospun PAN E-UTLC plates were fabricated following a methodescribed elsewhere [9]. After visualization, plates were imaged

nder ambient lighting using the Canon digital camera mentionedbove. Visualization methods investigated include the use of phos-horic acid [31], ferric chloride/sulfuric acid [31], sulfuric acid [32],nd nitric acid vapor [2]. Caution must be used with acid-charring

Calcined 300 ± 90 25 ± 2Aligned-calcined 300 ± 120 23 ± 3

reagents, and spraying these reagents must be carried out inside ofa fume hood [6].

3. Results and discussion

3.1. Preparation of electrospun SiO2/PVP nanofiber stationaryphases

In HPTLC and TLC separations, band dispersion decreases withdecreasing particle diameters and with increasing particle uni-formity [6]. Therefore, various electrospinning parameters wereinvestigated with the goal of minimizing nanofiber diameter andmaximizing the amount of SiO2 within the nanofibers, while atthe same time keeping a homogenous dispersion of nanoparti-cles within the composite nanofibers [29]. For as-spun SiO2/PVPnanofibers, an average nanofiber diameter of 380 nm was achieved.There was no change in average nanofiber diameter after thermalcrosslinking of the nanofibers, and calcined nanofibers decreasedin average nanofiber diameter to 300 nm (Table 1).

3.1.1. As-spun SiO2/PVP nanofiber stationary phasesTo prepare E-UTLC stationary phases with composite SiO2/PVP

nanofibers, the effect of electrospinning time on mat thicknesswas studied. Fig. S2A illustrates that the total mat thickness of as-spun SiO2/PVP nanofibers increased linearly with electrospinningtime up to a limiting time which in this case is around 30–35 min.Thus far E-UTLC stationary phases consisting of mats with thick-ness of 15–25 �m have worked well [9–14]. Consequently as-spun

Fig. 1. (A) Image of the as-spun nanofiber stationary phase, and SEM images revea-ling (B) nanofiber morphology and diameter (380 nm) viewed from the top-downand (C) stationary phase thickness (24 �m) viewed from the mat edge.

264 T.E. Newsome, S.V. Olesik / J. Chromatogr. A 1364 (2014) 261–270

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n the separation of laser dyes to test the viability of this phasehickness for ULTC separations. No tailing was observed in thesexperiments suggesting that a 30 min collection time for a 24 �mat thickness was satisfactory.

.1.2. Crosslinked SiO2/PVP nanofiber stationary phasesDue to differences in mass loss and mat shrinkage during ther-

al treatment, collection times were separately optimized forrosslinked nanofiber and calcined nanofiber stationary phases. Inrder to obtain stationary phases with a consistent thickness foromparison, the effect of total electrospinning time on mat thick-ess after thermal crosslinking and calcining was also studied. Asemonstrated in Fig. S2B, the mat thickness of the crosslinkedanofiber mats also increased with increasing electrospinning timeimilar to as-spun nanofibers. However longer electrospinningimes were required for crosslinked nanofibers compared to as-pun nanofibers to achieve a similar mat thickness. This differenceas not attributed mass loss of the polymer as only about 0.5–1.0%

f PVP was lost during thermal treatment according to thermalravimetric analysis (TGA) but rather it was attributed to com-ression of the mat during thermal crosslinking [29]. To obtainrosslinked nanofiber stationary phases with a thickness consis-ent with the as-spun nanofibers, SiO2/PVP nanofibers which wereo be thermally crosslinked were electrospun for 1.0 h and sub-equently heated to 200 ◦C for thermal crosslinking; this yieldedrosslinked nanofiber stationary phases with a thickness 24 ± 1 �mFig. 2A–C). Note that the nanofiber diameter did not change fromhe temperature increase during crosslinking.

.1.3. Calcined SiO2/PVP nanofiber stationary phasesSelective removal of PVP from the SiO2/PVP nanofibers by cal-

ination has been studied by our group [29]. The final temperaturend ramp rate in the heating program were selected in order toaximize the amount of PVP removed from the nanofibers whileaintaining various characteristics in the resulting surface whichere optimal for use in UTLC. Desired characteristics includedanofiber mat size in which the final calcined nanofiber mat areaas large enough for UTLC separations (at least 1 cm × 2 cm) and

he highest degree of strength and robustness to withstand hand-ing of the stationary phase during TLC analysis. The calciningonditions which met these criteria were a final temperature of65 ◦C and a ramp rate of 0.5 ◦C/min. To maximize the amount of

Fig. 3. (A) Image of the calcined nanofiber stationary phase, and SEM images revea-ling (B) nanofiber morphology and diameter (380 nm) viewed from the top-downand (C) stationary phase thickness (24 �m) viewed from the mat edge.

PVP removed from the nanofibers, the highest possible tempera-ture was desired. Only about 2/3 of the polymer was removed fromnanofibers calcined at 450 ◦C, and while nearly the entire amountof polymer was removed from nanofibers calcined at 475 ◦C, theresulting mats were so brittle that nanofibers broke down into silicananoparticles upon minimum handling or with any kind of contactwith solvent. Therefore, calcined nanofiber stationary phases wereprocessed at 465 ◦C to leave a small amount of polymer (7.7%) in thenanofiber scaffold providing the stationary phase with sufficientrobustness to withstand handling and contact with mobile phaseduring UTLC. Additionally, compared to higher ramp rates, usinga 0.5 ◦C/min ramp rate kept the calcined nanofiber mat relativelyflat and free from fracture which provided a mat with sufficientarea for UTLC separations. To obtain calcined nanofiber stationaryphases with a thickness consistent with as-spun and crosslinkednanofiber stationary phases for comparison, SiO2/PVP nanofiberswhich were to be calcined were electrospun for 2.5 h for a matthickness of 25 ± 3 �m (Fig. 3A–C).

3.1.4. Aligned-calcined SiO2/PVP nanofiber stationary phasesPrevious work illustrated that aligned electrospun nanofibers in

AE-UTLC provided 2–2.5 times shorter analysis time with improvedreproducibility (up to 2 times higher) and efficiency (up to 100times greater) compared to using non-aligned nanofibers in E-UTLC. Accordingly, alignment of the SiO2/PVP nanofibers wasinvestigated using a rotating drum method [11] for the fabricationof aligned-calcined nanofiber stationary phases. As the compositeSiO2/PVP nanofibers have never been aligned, the electrospinningparameters for aligning the pure polymer nanofibers were usedas a starting point for studying the alignment of the compositenanofibers. To achieve the highest degree of alignment in the com-posite nanofibers, the effect of drum rotational speed on nanofiberalignment was studied. A collection time of 30 min was used toinvestigate drum speeds of 1000, 1250, and 1500 rpm. No signifi-cant variation in nanofiber diameter was observed, however therewas a difference in the quality of alignment of the nanofibers (Fig.S3). The degree of alignment was quantified by determining thepercentage of nanofibers which deviated from the direction of

alignment by 10◦, 20◦, and 30◦ [11]. These results are presentedin Fig. S4. Each rotational speed examined provided a high degreeof alignment as ∼80–85% of nanofibers were aligned within 30◦.However, the highest rotational speed of 1500 rpm consistently

T.E. Newsome, S.V. Olesik / J. Chroma

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rovided a higher degree of alignment compared to 1000 and250 rpm, with 62% of fibers aligned within 10◦ and 84% of fibersligned within 20◦. Thus, aligned SiO2/PVP nanofibers were pro-uced at 1500 rpm and were subsequently calcined to produceligned-calcined nanofiber stationary phases. To obtain stationaryhases with a thickness consistent to the other SiO2/PVP nanofibertationary phases, SiO2/PVP nanofibers that were to be aligned andalcined were electrospun using a total collection time of 2.5 h at

10 cm tip to collector distance. This provided an aligned-calcinedtationary phase with a thickness of 23 ± 3 �m and an averageanofiber diameter of 300 nm similar to the non-aligned calcinedanofibers (Table 1, Fig. 4).

.2. Separation of laser dyes on As-spun nanofibrous E-UTLCtationary phases

Initially, a set of five laser dyes, rhodamine 590 chloride,hodamine 101 chloride, rhodamine 610 perchlorate, rhodamine10 chloride, and pyrromethene 597, was separated on as-spunanofiber UTLC plates to investigate the efficacy of using thentreated composite SiO2/PVP nanofibers as a stationary phase. Toptimize the mobile phase for this separation, neat solvents that areonsolvents of PVP were investigated as mobile phases, includingthyl acetate, methyl ethyl ketone, tert-butyl methyl ether, toluene,exane, and heptane. Of these, EtOAc provided the highest selectiv-

ty. The retardation factor (Rf) of each analyte was calculated [2]; thealues are listed in Table 2 for separations using an EtOAc/heptaneobile phase at two different ratios (10:90 and 20:80, v/v).A mixture of 10:90 EtOAc/heptane (v/v) mobile phase provided

he best separation in terms of selectivity over a 35 mm separationistance in 5–6 min, and the two rhodamine 610 dyes co-eluteds expected since these analytes only differ by their counterion.omparing the separation of laser dyes using the as-spun E-UTLC

able 2etardation factors of laser dyes using the as-spun nanofiber stationary phase. Elec-rospun mats consisted of randomly-placed as-spun nanofibers (not aligned).

Laser dye Retardation factor, Rf (RSD, %)

EtOAc:Heptane(10:90)

EtOAc:Heptane(20:80)

Rhodamine 590 chloride 0.0 (N/A) 0.0 (N/A)Rhodamine 101 chloride 0.09 (6.0) 0.14 (12)Rhodamine 610 perchlorate 0.79 (3.7) 0.92 (1.6)Rhodamine 610 chloride 0.79 (3.0) 0.92 (1.2)Pyrromethene 597 0.99 (0.7) 0.99 (1.4)

togr. A 1364 (2014) 261–270 265

and as-spun AE-UTLC with PAN nanofiber stationary phases, therewas a higher degree of selectivity using optimum mobile phaseconditions as noted by the wide range in Rf observed for as-spunnanofibers (0.0–0.99) compared to those observed for both E-UTLC(0.42–0.69) and AE-UTLC PAN (0.30–0.50) with PAN nanofibers[9,11]. The observed wide range in Rf was likely due to the additionof the silica to the polymer nanofiber composite, as this high selec-tivity was more comparable to that observed using commercialsilica TLC plates where Rf can range from 0.08 to 0.81 under opti-mum mobile phase conditions [9]. This highlights the importance ofincorporating silica into E-UTLC stationary phases, as the presenceof silanol groups enables a wide range of interactions with ana-lytes, such as proton donor or acceptor interactions, dipole–dipoleinteractions, induced dipole interactions, and interactions based ondispersion forces [28]. However, the untreated as-spun nanofiberstationary phases were only compatible with a limited number ofsolvents which are nonsolvents of PVP; these various solvents com-mon to TLC are listed in Table S1 as compatible or incompatiblewith this stationary phase. Although this separation establishedthe suitability of as-spun nanofibrous E-UTLC and AE-UTLC sep-arations, it also highlighted the limitations of keeping the polymerin the as-spun nanofiber matrix without crosslinking or removal bycalcination.

3.3. Separation of amino acids on calcined nanofibrous E-UTLCstationary phases

Separations using the as-spun stationary phases were only com-patible with mobile phases, analyte solvents, and visualizationtechniques which were nonsolvents of non-crosslinked PVP. Assuch, the as-spun silica-based nanofibers were either calcined toremove the polymer from the nanofiber or crosslinked to createnanofibers which were compatible with a wider variety of reagentscommon to TLC. Silica-based stationary phases are well suited forthe separation of a wide variety of analytes including amino acids.Separations of amino acids on silica have been achieved using awide variety of mobile phase compositions, many of which includewater [30,31,32]. The separation of arginine (Arg), aspartic acid(Asp), threonine (Thr), and phenylalanine (Phe) was studied on cal-cined nanofiber E-UTLC stationary phases. To optimize the mobilephase, the retention behavior of each amino acid was studied undera wide range of mobile phase compositions [33]. Neat solventsrepresenting different selectivity groups were evaluated, includ-ing n-butanol, 1-propanol, 2-propanol, methanol, ethanol, acetone,methyl ethyl ketone, and ethyl acetate. None of these had sufficientsolvent strength to achieve satisfactory analyte migration for sep-aration to occur. Subsequently, mixtures of several solvents withwater were evaluated, including methanol/water, ethanol/water,1-propanol/water, 2-propanol/water, and n-butanol/water/aceticacid. Due to the restricted miscibility of n-butanol (∼7–8%) inwater [34], acetic acid was added to create a fully miscible mobilephase. Different proportions of water or water/acetic acid tothe neat solvents were examined, and separations utilizing n-butanol/water/acetic acid provided the highest selectivity in theshortest separation distance. The effect of changing the ratio ofn-BuOH to H2O/HOAc in mobile phase composition was studied(Fig. 5A), keeping the ratio of H2O to HOAc even. Mobile phasescomprised of 70:15:15 and 60:20:20 n-BuOH/H2O/HOAc (v/v/v)provided the best resolution. Thus, mobile phases with 60% and 70%n-BuOH with different ratios of H2O to HOAc were studied to fur-ther optimize mobile phase composition. Among compositions thatoffered the best resolution, 60:30:12 n-BuOH/H2O/HOAc (v/v/v)

was determined the optimum mobile phase for calcined nanofibers,as this particular composition had the most reproducible Rf values(1–6% RSD) and smallest relative final spot widths (wb, peak widthat base) between 1.3 and 1.6 mm. Separations were performed

266 T.E. Newsome, S.V. Olesik / J. Chroma

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A

0.00

0.20

0.40

0.60

0.80

1.00

35 40 45 50 55 60 65 70 75 80 85 90 95 100

Rf

% BuOH (BuOH/H2O/HAc)

C

0.00

0.20

0.40

0.60

0.80

1.00

35 40 45 50 55 60 65 70 75 80 85 90 95 100

Rf

% BuOH (BuOH/H2O/HAc)

B

Fcs

ips

3E

nsTcsl

ig. 5. Retardation factors of Arg (�), Asp (�), Thr (�), and Phe (�) using the (A) cal-ined nanofiber and (B) crosslinked nanofiber E-UTLC plates and the (C) commercialilica HPTLC plate. The volume ratio of H2O to HOAc was 1:1.

n a short distance of 15 mm in 9–10 min. This mobile phase com-osition was used for all other studies with calcined nanofibertationary phases.

.4. Separation of amino acids on crosslinked, nanofibrous-UTLC stationary phases

Rather than selective removal of the polymer from theanofibers via calcination, the use of crosslinked nanofiber E-UTLCtationary phases was investigated for the separation of Arg, Asp,

hr and Phe. As-spun SiO2/PVP E-UTLC nanofibers were thermallyrosslinked to obtain silica-based composite nanofibers that weretable in common reagents used as TLC mobile phases and ana-yte solvents [29]. Like the calcined nanofiber stationary phases,

togr. A 1364 (2014) 261–270

the retention behavior of the amino acids was studied under awide range of mobile phase compositions using neat solvents rep-resenting different selectivity groups. Similarly, none of these neatsolvents had sufficient solvent strength for separation to occur.Comparing mixtures of these solvents with water or water/aceticacid, mixtures of n-BuOH/H2O/HOAc provided the best selectiv-ity. The mobile phase composition was optimized by studying theeffect of changing the ratio of n-BuOH to H2O/HOAc in the mobilephase while keeping the ratio of H2O to HOAc even (Fig. 5B). Using a15 mm separation distance, 90:5:5 n-BuOH/H2O/HOAc (v/v/v) wasdetermined to be the optimum mobile phase for the crosslinkednanofiber plates as this composition provided the best selectiv-ity and resolution. The mobile phase traveled much quicker onthe crosslinked nanofiber E-UTLC plates compared to the cal-cined nanofiber E-UTLC plates, taking only 3–4 min to travel a15 mm separation distance. Interestingly, even with the polymerstill present in the nanofiber, the crosslinked nanofiber platesshowed the same retention order as the calcined nanofiber platessuggesting that the silica functionalities were largely responsiblefor analyte selectivity. However, all analytes were less stronglyretained on the crosslinked nanofiber plates, as it only took theaddition of 10% of H2O/HOAc to n-BuOH to observe ample migra-tion of each analyte compared to the 30–40% of H2O/HOAc neededfor calcined nanofiber plates (Fig. 5B and A, respectively). Theweaker retention on the crosslinked nanofiber E-UTLC stationaryphase compared to the calcined nanofiber E-UTLC stationary phasecan be attributed to the large difference in density between thetwo materials. The calcined E-UTLC nanofibrous phase is approxi-mately 25% more dense than the crosslinked nanofiber stationaryphase [29]. Therefore, the volume of stationary phase availablefor analyte adsorption is greater for the calcined nanofiber platethan the crosslinked nanofiber plate even though the two sta-tionary phases have the same mat thickness. As a consequenceof the lower volume of stationary phase, the retention on thecrosslinked nanofiber plates was weaker than on the calcinednanofiber plates.

A large amount of tailing of the analyte spots was observedon the crosslinked nanofiber plates when using the same ana-lyte volumes and concentrations which were used on the calcinednanofiber plates. Sample overloading is a common source of tailingwhen using commercially available TLC plates. Therefore, reducingthe amount of sample loaded onto the crosslinked nanofiber plateswas investigated by decreasing the concentration and volume ofanalyte applied. After decreasing both the analyte concentrationand then the applied volume by half, tailing was still observed.Analyte spots were no longer visible after further decreasing theconcentration and/or volume as this was probably under the detec-tion limit of ninhydrin visualization using these detection methods[35]. This could suggest that the loading capacity for the crosslinkedplate was more than four times lower than that for the calcinednanofiber plate. Consequently, crosslinked nanofiber plates with anincreased volume of stationary phase were examined by increasingthe mat thickness above 25 �m. Stationary phases were preparedfrom mats collected at longer electrospinning times of 1.5, 2.0, and2.5 h which resulted in mat thicknesses of 30 ± 3 �m, 36 ± 3 �m,and 41 ± 2 �m, respectively. Stationary phases collected at longertimes were not examined as the nanofibers in these mats containedmorphological deformities (beaded nanofibers) which would likelyhinder chromatographic performance. Tailing was still observed inthe thicker crosslinked stationary phases even when the analyteconcentration and applied volume were decreased. Therefore thepoor chromatographic performance observed with the crosslinked

nanofiber E-UTLC stationary phases was attributed to the hetero-geneity of functionalities present in the stationary phase (from themixture of both PVP and silica functionalities) and the deactivationof silica sorption sites by the polymer [33].

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T.E. Newsome, S.V. Olesik / J. C

Performing amino acid separations using the minimum amountf analyte required for ninhydrin visualization and a separationistance of 15 mm, tailed developed spot widths ranged from 2.1o 2.7 mm on the crosslinked nanofiber plates. These values were

uch larger than what was achieved with the calcined nanofiberlates. Interestingly, even with weaker retention and larger rela-ive spot widths, the selectivity of the four amino acids was highnough that a separation to a resolution of 1.2–1.7 was achieved inhis short separation distance. However, compared to the calcinedanofiber stationary phases, the larger relative spot widths on therosslinked nanofiber stationary phases resulted in decreased chro-atographic efficiency as lower plate numbers (46–430) and larger

late heights (30–77 �m) were observed.

.5. Effect of alignment on calcined, nanofibrous AE-UTLCtationary phases

.5.1. Mobile phase velocityEven though the efficiencies observed on the crosslinked

anofiber E-UTLC stationary phases were notably lower than thosebserved on the calcined nanofiber E-UTLC stationary phases,eparations on the crosslinked nanofiber E-UTLC plates were sig-ificantly faster. The mobile phase traveled much quicker usingrosslinked nanofiber E-UTLC plates, taking only 3–4 min to travel5 mm compared to 9–10 min for the calcined nanofiber E-UTLClates. Therefore, differences in mobile phase velocities of the var-

ous silica-based nanofiber plates were further characterized andompared to a commercially available silica HPTLC plate. Mobilehase transport through nanofibrous E-UTLC stationary phases haseen studied using the Lucas–Washburn equation which describeshe capillary flow transportation of mobile phase through porous

edia [9–13]. The Lucas–Washburn equation has been applied tohe mobile phase migration in traditional TLC stationary phases and-UTLC stationary phases. For chromatographic phases the veloc-ty constant, �, replaces the term �R/2� in the Lucas–Washburnquation (see equations in SI). Therefore plots of migration distanceersus t0.5 were assembled for the silica-based E-UTLC plates andhe commercial silica plate in order to determine � for each of thelates examined (Fig. S5). The mobile phase used for these studiesas hexane to allow comparison with other E-UTLC plates under

he same conditions.These plots show the linear dependence predicted by the

ucas–Washburn equation which verified the applicability of thisquation to the capillary flow of mobile phase through electrospunilica-based stationary phases and demonstrated the homogene-ty of the silica-based nanofiber E-UTLC stationary phases. Thisependence was similar to that observed in other polymer- andarbon-based E-UTLC stationary phases [9–13]. The � values for thes-spun, calcined, and crosslinked nanofiber E-UTLC plates wereetermined to be 0.026, 0.012, and 0.029 cm2/s, respectively, and.016 cm2/s for the commercial HPTLC plate (r2 > 0.991). The � valuebserved for the HPTLC plate agrees well with previously reportedork [9,10,13]. The increased � for the as-spun and crosslinkedanofiber plates relative to the HPTLC plate directly corresponds tohe increased mobile phase velocities observed, and they are simi-ar to the reported values for other polymer and carbon nanofiber-UTLC stationary phases [9,10,13]. A similar analysis for aligned-alcined nanofiber plates provided a � value of 0.019 cm2/s, whichs ∼60% greater than the non-aligned calcined nanofiber plates.

.5.2. Separation of amino acids on calcined nanofibrousE-UTLC stationary phases

Separations of the amino acid mixture on the aligned-calcinedanofiber plates using 60:30:12 n-BuOH/H2O/HOAc required about

min to reach a 15 mm separation distance which was notablyaster than the calcined nanofiber E-UTLC plates and HPTLC plates.

togr. A 1364 (2014) 261–270 267

However, applied spots were observed to be slightly distorted dueto wicking of the analytes during sample application. This distor-tion from wicking took on an elliptical shape in the direction ofnanofiber alignment. As this is also the direction of development inAE-UTLC, developed spots were also elliptically shaped. Nonethe-less, these wb values were still smaller than the tailed spot widthsobserved on crosslinked nanofiber plates for Asp, Thr, and Phe.However, even though the difference was not statistically signifi-cant using a t-test, developed spot widths were shown to be overallslightly larger on calcined nanofiber AE-UTLC plates compared tocalcined nanofiber E-UTLC plates, with the wb ranging from 1.4 to2.0 mm. As a consequence, plate heights as low as those observedon the calcined nanofiber E-UTLC plates were not achieved on thealigned-calcined nanofiber plates, as the resulting plate numberswere in the range of 260–480 and plate heights were in the rangeof 19–23 �m.

3.6. Post-separation visualization method compatibility

All three types of silica-based nanofibers stationary phases wereassessed in terms of chemical stability to test their compatibilitywith various post-separation visualization techniques which arecommon to TLC. Electrospun PAN UTLC plates [9] and commercialsilica TLC plates were also assessed for comparison. Visualizationreagents included ninhydrin to visualize amino acids and nitric acidvapor, ferric chloride/sulfuric acid, sulfuric acid, and phosphoricacid to visualize various steroids, including hydrocortisone, andros-terone, and/or cholesterol. The results are summarized in Table 3and two examples are depicted in Fig. S6 showing the difference inplate compatibility with the visualization reagents nitric acid vaporand ferric chloride/sulfuric acid.

The as-spun nanofiber plates are incompatible with all of thevisualization reagents used. The PAN polymer nanofiber plateswere also incompatible with all of the visualization reagents usedexcept for ninhydrin, typically due to distortion of spot shapes ordissolution of the nanofiber mat. Conversely, calcined nanofiberplates were completely compatible with all of the visualizationreagents used and no issues of spot shape distortion or wrin-kling of the mat were encountered. These experiments served tohighlight the increased chemical stability and resulting increasedsolvent compatibility of the silica-based nanofiber E-UTLC plates.Consequently, visualization techniques which are compatible withE-UTLC separations has been vastly expanded.

3.7. Band broadening

In TLC, the plate height, H and plate number, N, values dependon the position of each analyte zone in the chromatogram. Thisis due to the decrease in mobile phase velocity as a function oftime and thus migration distance using traditional capillary-flowdriven TLC development techniques. Phenylalanine was chosen todemonstrate the relationships between plate number and migra-tion distance and between observed plate height and migrationdistance on the calcined nanofiber E-UTLC plate, as this was themost efficient of the silica-based stationary phases examined.As shown in Fig. 6A, N linearly increased as separation distanceincreased between 10 and 40 mm, and this relationship beganto deviate from linearity around 50 mm. Correspondingly, H ini-tially decreased as separation distance increased between 10 and30 mm, remained relatively the same at 40 mm, and then noticeablyincreased at 50 mm (Fig. 6B).

Since the flow of mobile phase in E-UTLC is capillary-driven

like traditional TLC and HPTLC, it is expected that an increasein H would be observed at a given distance, and for PAN andcarbon nanofiber E-UTLC this may occur at distances furtherthan 55–60 mm. Attributed to the slower mobile phase velocity

268 T.E. Newsome, S.V. Olesik / J. Chromatogr. A 1364 (2014) 261–270

Table 3Compatibility of as-spun, calcined, and crosslinked silica-based nanofiber plates and PAN nanofiber plates (C = compatible, I = incompatible) with different visualizationreagents used to derivatize amino acids (ninhydrin only) or steroids (cholesterol, hydrocortisone, and/or androsterone). Colors of developed analyte spots are noted if visible.Appearance on commercial silica TLC plates is also described for comparison.

Visualization reagentand analyte

Appearance oncommercial silica plate

PAN nanofibers SiO2/PVP nanofiber type

As-spun Calcined Crosslinked

Ninhydrin Purple, red, orange C Purplea I Nanofibers dissolved C Purple, red C Purple, redHNO3 vapor Peach, tan I Nanofibers dissolved I Nanofibers dissolved C Tan C Tanb

FeCl3/H2SO4 Brown, blue I Brown, bluea,c I Nanofibers dissolved C Brown, blue C Brown, bluea

10% H2SO4 Tan, pink, purple I Nanofibers dissolved I Nanofibers dissolved C Purple C PinkH3PO4 Tan, yellow I Nanofibers dissolved I Nanofibers dissolved C Tand C Tand

a Spot shape distorted.b Nanofiber mat wrinkled.

o3cdp

3c

pc

FiB

c Stationary phase detached from substrate and partially dissolved.d Dissolved if stationary phase oversprayed with reagent.

bserved on this E-UTLC stationary phase as discussed in Section.5, calcined nanofiber E-UTLC plates have a smaller Hmin valueompared to the AE-UTLC PAN plates and carbon nanofiber E-UTLCevices. Therefore, separations utilizing calcined nanofiber E-UTLClates are recommended at separation distances of 40 mm or less.

.8. Comparison of calcined, nanofibrous E-UTLC plates toommercial HPTLC plates

Separations on the calcined nanofiber E-UTLC plates were com-ared to those on commercial silica HPTLC plates. The sameonditions described in Section 3.3 were used (100 �m capillary

0

500

1000

1500

2000

2500

3000

3500

4000

0 10 20 30 40 50 60

N

Distance (mm)

A

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

0 10 20 30 40 50 60

H (

µm

)

Distance (mm )

B

ig. 6. Change in efficiency of phenylalanine with increasing migration distancen terms of (A) plate number, N, and (B) plate height, H, using a 60:30:12 n-uOH/H2O/HOAc (v/v/v) mobile phase and the calcined nanofiber UTLC plate.

tubes in the 80 mL development chamber), except that an increasedvolume of mobile phase was needed for HPTLC plate developmentcompared to the E-UTLC plates because of the larger stationaryphase thickness (2 mL and 1 mL, respectively). Similar to the E-UTLCplates examined, the retention behavior of the amino acids wasstudied under a wide range of mobile phase compositions usingneat solvents representing different selectivity groups. 70:15:15n-BuOH/H2O/HOAc (v/v/v) was determined to be the optimummobile phase for the commercial plates as this composition pro-vided the best selectivity. This mobile phase took 7–8 min totravel 15 mm, the same distance used for calcined nanofiber andcrosslinked nanofiber stationary phases; however, a resolved sep-aration of the four analytes was not possible within this distancefor any of the mobile phase compositions examined due to thelarger relative developed spot widths and low selectivity betweenAsp and Thr. Therefore, separations were performed on the com-mercial plate over increasing distances (above 15 mm) using the70:15:15 n-BuOH/H2O/HOAc (v/v/v) mobile phase, and the short-est distance required for a resolved separation between all fouranalytes was 50 mm. Separations at these distances took over 1 hto perform (typically ∼70 min). Table S2 compares the retardationfactors and plate numbers observed for separations on calcinednanofiber E-UTLC plates to those on silica HPTLC plates using theoptimized TLC conditions described above. As anticipated, the com-mercial silica plates showed the same order of retention as thesilica-based calcined nanofiber plates: Arg, Asp, Thr, Phe. Otherthan the low selectivity between Asp and Thr, the commercialplates showed comparable retention strength of the amino acidsrelative to the calcined nanofiber plates as noted by the similarrange of Rf over the same mobile phase compositions (Fig. 5C andA, respectively). Nevertheless, plate numbers were consistentlyhigher for calcined nanofiber plates developed to 15 mm comparedto commercial silica plates developed to both 15 and 50 mm. Nwas 16–52 times larger at a similar separation distance, and ittook development to over 3 times that distance on the commercialsilica plate to achieve plate numbers of the same order of mag-nitude (N was still ∼2–6 times larger for the calcined nanofiberE-UTLC plates). To factor out the total separation distance, plateheights were also compared (Fig. 7). H values as small as 8.6 �mwere observed on calcined nanofiber E-UTLC plates which were4–14 times smaller compared to commercial plates. The significantincrease in N and decrease in H demonstrate that the efficiencyof the calcined nanofiber E-UTLC plates was superior to the effi-ciency of the commercial HPTLC plates. This was attributed to thedecreased developed spot widths obtained with the smaller sized

sorbent compared to those observed on the commercial silica plateswith significantly larger particles. The difference between the aver-age developed spot widths of the two plates is demonstrated in Fig.S7. The observed wb on calcined nanofiber E-UTLC plates ranged

T.E. Newsome, S.V. Olesik / J. Chroma

0

50

100

150

200

250

Arg Asp Th r Phe

Pla

te h

eig

ht,

H (

µm

)

F1

fsiwTmmatssbaabwtEctcc

F((p

ig. 7. Plate heights (H) on commercial HPTLC plates developed to 50 mm (�) and5 mm (�) and the calcined nanofiber E-UTLC plate developed to 15 mm (�).

rom 1.3 to 1.6 mm at a 15 mm development distance, which wasignificantly smaller than those on observed on commercial sil-ca plates which ranged from 1.6 to 3.6 mm and 2.3 to 4.3 mm

hen developed to distances of 15 mm and 50 mm, respectively.he decrease in observed wb is also illustrated in the sample chro-atograms shown in Fig. 8 for the separations of the amino acidixture using the commercial HPTLC plate developed to 50 mm

nd the calcined nanofiber E-UTLC plate developed to 15 mm. Fromhese chromatograms, it is clear that due to the size of wb a resolvedeparation of all four amino acids would be difficult if not impos-ible on the commercial silica plates developed to 15 mm. This cane better described by the separation number, SN, which is defineds the number of zones which can be separated on a plate over

given distance (Zf − Z0) [2]. SN was experimentally determinedy plotting spot widths at half height and extrapolating the spotidths at half height for Rf = 1 and Rf = 0 [21]. Using a separation dis-

ance of 15 mm, a SN of 8–9 was achieved with calcined nanofiber-UTLC plates and a SN of only 3 was achieved with the commer-ial HPTLC plates using the methods described above. Increasing

he separation distance to 50 mm, a SN of 9 was achieved withommercial HPTLC plates, while a SN of ∼20 was possible for thealcined nanofiber plates.

0 5 10 15

Distance (mm )

B

1

2 3

4

0 10 20 30 40 50

Distance (mm )

A

12

3

4

ig. 8. Chromatograms for the separation of (1) Arg, (2) Asp, (3) Thr, and (4) Phe onA) a commercial HPTLC plate using a 70:15:15 n-BuOH/H2O/HOAc mobile phase andB) a calcined nanofiber E-UTLC plate using a 60:30:12 n-BuOH/H2O/HOAc mobilehase.

togr. A 1364 (2014) 261–270 269

The plate heights achieved on calcined nanofiber E-UTLC plates(Fig. 7) are comparable to the range of H values which have beenreported using other E-UTLC devices. In the separation of a drugmixture on PAN nanofiber E-UTLC and AE-UTLC plates using UVvisualization, H values as low as 34 �m and 15 �m were observed,respectively [11]. While the H values observed here were also supe-rior to those observed on PVA nanofiber plates in the separationof amino acids using ninhydrin visualization (as low as 30 �mwas observed), other published E-UTLC separations have achievedlower H values in the separation of FITC-labeled amino acids usingcarbon nanofiber [10] and PVA nanofiber [12] E-UTLC plates (as lowas 0.1 �m and 2 �m, respectively). The plate heights observed hereemploying a capillary for manual analyte application were betterthan those reported for GLAD SiO2 UTLC plates (12–28 �m) [18].

4. Conclusions

This was the first time E-UTLC has been extended to station-ary phases composed of silica, the most commonly used surfacein TLC. Three silica-based nanofiber plates of similar mat thick-ness were evaluated: as-spun, crosslinked, and calcined nanofibers.Although the as-spun nanofiber E-UTLC plates offered fast mobilephase velocity, separations were limited to mobile phases, analytesolvents, and visualization techniques which were nonsolvents ofnon-crosslinked PVP. Crosslinked nanofibers were equally fast andwere not as limited in terms of chemical stability, but separationsof amino acids produced tailed spot shapes compared to calcinednanofibers owing to the heterogeneity of the stationary phase.Although slower mobile phase velocities were observed, no limita-tions in terms of mobile phases, analyte solvents, and visualizationtechniques were observed for calcined nanofibers. After optimizingcapillary tube size and chamber type, highly efficient separations ofamino acids were accomplished in 15 mm on calcined nanofiber E-UTLC plates. With plate heights as low as 8.7 �m, the plate heightsachieved were significantly lower on the calcined nanofiber E-UTLC plates compared to the silica HPTLC plates which requireda separation distance of 50 mm. Alignment of calcined nanofibersproduced separations which were about two times faster than thenon-aligned counterparts and notably faster than the HPTLC plates.By extending E-UTLC stationary phases to silica-based nanofibers,the range of mobile phases, analyte solvents, and visualizationtechniques which can be used for E-UTLC separations has beenwidely expanded and method development has been streamlinedto TLC techniques which are used with conventional silica station-ary phases.

Acknowledgments

The authors would like to thank the National Science Founda-tion (CHE-1012279) for funding this research. We would also liketo thank the undergraduate students who assisted in SEM imageanalyses, Chris Brue and Danielle Hopping.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.chroma.2014.08.065.

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