Engineered Anisotropic Microstructures for Ultrathin-Layer

May 27, 2010 - S. R. Jim,† M. T. Taschuk,† G. E. Morlock,‡ L. W. Bezuidenhout,† W. ... proposed by Morlock et al.13 In this work, bands of tes...
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Anal. Chem. 2010, 82, 5349–5356

Engineered Anisotropic Microstructures for Ultrathin-Layer Chromatography S. R. Jim,† M. T. Taschuk,† G. E. Morlock,‡ L. W. Bezuidenhout,† W. Schwack,‡ and M. J. Brett*,†,§ Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Canada, Institute of Food Chemistry, University of Hohenheim, Stuttgart, Germany, and NRC National Institute for Nanotechnology, Edmonton, Canada The strong dependence of separation behavior on ultrathin-layer chromatography (UTLC) stationary phase microstructure motivates continued UTLC plate design optimization efforts. We fabricated 4.6-5.3 µm thick normal phase silica UTLC stationary phases with several types of in-plane macropore anisotropies using the glancing angle deposition (GLAD) approach to engineering nanostructured thin films. The separation behaviors of two new media, isotropic vertical posts and anisotropic bladelike films, were compared to that of anisotropic chevron media. Channel-like structures within the anisotropic media introduced preferential mobile phase flow directions that could be exploited to give separation tracks diagonal to the development direction. Extraction of chromatograms from these angled tracks required the development of a new analytical approach that involved a commercial flatbed film scanner and custom numerical image analysis software. GLAD stationary phase performance was quantified using the Dimethyl Yellow dye separated from a lipophilic dye mixture over migration distances less than ∼10 mm. The limits of detection were 10 ( 4 ng for the vertical posts and 11 ( 3 ng for the bladelike media. We obtained theoretical plate heights that varied with film microstructure between 12 and 28 µm. Unoptimized separation performance was comparable to that of other planar chromatography media. Macropore anisotropies engineered by GLAD may expand the capabilities of future UTLC stationary phases. Rapid analysis of many sample mixtures in parallel makes ultrathin-layer chromatography (UTLC) appealing in food chemistry, pharmaceutical analysis, environmental testing, and a variety of other rapid screening applications.1-3 As all analytes remain on the plate following separation, planar chromatographic methods such as UTLC also permit the use of multiple detection and * Corresponding author. Phone: 1-780-492-4438. Fax: 1-780-492-2863. E-mail: [email protected]. † University of Alberta. ‡ University of Hohenheim. § NRC National Institute for Nanotechnology. (1) Sherma, J. Anal. Chem. 2008, 80, 4253–4267. (2) Morlock, G. E.; Schwack, W. J. Planar Chromatogr.sMod. TLC 2007, 20, 399–406. (3) Poole, C. F. J. Chromatogr. A 2003, 1000, 963–984. 10.1021/ac101004b  2010 American Chemical Society Published on Web 05/27/2010

characterization modes, including mass spectrometry.1,4 UTLC stationary phases are typically comprised of thinner monolithic silica gels with finer pore sizes than thin-layer chromatography (TLC) or high-performance TLC (HPTLC) media.5,6 UTLC media therefore permit faster separations over shorter distances with better limits of detection. The strong dependence of separation behavior on stationary phase microstructure motivates continued UTLC plate design optimization efforts. Recent advances include isotropic electrospun nanofibrous media7 and nanostructured glancing angle deposited macroporous thin films.8 Glancing angle deposition (GLAD) is a single-step vacuum deposition process for fabricating thin films with porous columnar microstructures.9-12 GLAD films are suitable for sensing, optical, microfluidic, and other applications.11,12 The method precisely controls film porosity and morphology and can produce structural anisotropies in the plane as well as in the normal direction of a thin film. GLAD’s ability to engineer structural anisotropies over large areas distinguishes it from other approaches to fabricating UTLC stationary phases. A preliminary investigation of GLAD UTLC explored nanostructured SiO2 thin films with varied porosity and thickness (1-7 µm).8 Isotropic stationary phases were comprised of hexagonal helical structures. Anisotropic stationary phases possessed 5 µm thick chevron (“zig-zag”) architectures with channel-like features. Analyte migration along the anisotropic channel-like features was more rapid than across them. As GLAD UTLC plates may be developed over shorter distances and in less time than commercial UTLC media, they are amenable to the “Office Chromatography” concept recently proposed by Morlock et al.13 In this work, bands of test dye were applied to miniaturized GLAD UTLC plates using an ordinary (4) Salo, P. K.; Vilmunen, S.; Salomies, H.; Ketola, R. A.; Kostiainen, R. Anal. Chem. 2007, 79, 2101–2108. (5) Hauck, H. E.; Bund, O.; Fischer, W.; Schulz, M. J. Planar Chromatogr.sMod. TLC 2001, 14, 234–236. (6) Hauck, H. E.; Schulz, M. J. Chromatogr. Sci. 2002, 40, 550–552. (7) Clark, J. E.; Olesik, S. V. Anal. Chem. 2009, 81, 4121–4129. (8) Bezuidenhout, L. W.; Brett, M. J. J. Chromatogr. A 2008, 1183, 179–185. (9) Robbie, K.; Brett, M. J. J. Vac. Sci. Technol. A 1997, 15, 1460–1465. (10) Robbie, K.; Brett, M. Method of depositing shadow sculpted thin films. U. S. Patent 5,866,204, 1999. (11) Taschuk, M. T.; Hawkeye, M. M.; Brett, M. J. Glancing Angle Deposition. In Handbook of Deposition Technologies for Films and Coatings: Science, Applications and Technology, 3rd ed.; Martin, P., Ed.; William Andrew (Elsevier): Oxford, United Kingdom, 2010; pp 621-678. (12) Hawkeye, M. M.; Brett, M. J. J. Vac. Sci. Technol. A 2007, 25, 1317–1335. (13) Morlock, G. E.; Oellig, C.; Bezuidenhout, L. W.; Brett, M. J.; Schwack, W. Anal. Chem. 2010, 82, 2940–2946.

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Figure 1. Glancing angle deposition (GLAD) approach to fabricating columnar thin films. Modulation of the deposition angle (R) and azimuthal angle (φ) sculpts the columns into many useful morphologies.

inkjet printer and imaged with a desktop flatbed scanner following development. Such consumer electronics are cost-effective and perform well enough to be viable alternatives to the complex spotting and imaging systems typically used in TLC and HPTLC for the miniaturized plates used here. In this paper, we report the fabrication and performance of two new ∼5 µm thick GLAD UTLC SiO2 stationary phases. The effects of macropore anisotropies and the methods developed to characterize the unique separation behaviors on these media are investigated. Highly porous channel-like structures within the anisotropic media influenced analyte migration distance and direction. Developments performed diagonally to these structures resulted in angled separation tracks that required creation of a customized chromatogram extraction technique. The mobile phase migration anisotropy, separation performance, and limits of detection of the GLAD UTLC plates were assessed using normal phase separations of a colored lipophilic dye mixture. Theoretical plate heights for the Dimethyl Yellow dye component separated from the dye mixture varied with film microstructure between 12 and 28 µm. The limits of detection for the GLAD UTLC plates were about 10 ng per zone. These results are expected to improve with further optimization and encourage future efforts to engineer GLAD separation media. EXPERIMENTAL SECTION Glancing Angle Deposition (GLAD). Normal phase silica ultrathin-layer chromatography plates were nanoengineered through a physical vapor deposition technique known as glancing angle deposition (GLAD). This single-step approach to fabricating porous thin films has been described elsewhere.9-12 GLAD uses computer-controlled substrate motion to realize several columnar thin film morphologies (Figure 1). Deposition angle (R) manipulation modifies the film density and porosity;9,14,15 azimuthal angle (φ) modulation sculpts the columns into different geometries. Electron beam evaporation of SiO2 (99.99% pure, Cerac Inc., Milwaukee, Wisconsin, USA) onto 1.0 in. square glass substrates (Schott B270, S. I. Howard Glass, Worchester, Massachusetts, USA) was performed within a custom ultra high vacuum chamber (AXXIS, Kurt J. Lesker Co., Clairton, Penn(14) Tait, R. N; Smy, T.; Brett, M. J. Thin Solid Films 1993, 226, 196–201. (15) Krause, K. M.; Taschuk, M. T.; Harris, K. D.; Rider, D. A.; Wakefield, N. G.; Sit, J. C.; Buriak, J. M.; Thommes, M.; Brett, M. J. Langmuir 2009, 26, 4368–4376.

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sylvania, USA). The base pressure of the vacuum chamber was less than 6 × 10-7 Torr. The exact composition and surface chemistry of the evaporated GLAD films is difficult to assess, but hydrophilicity measurements (water contact angles) are consistent with thermally grown SiO2.16 All substrates were cleaned with a mild aqueous detergent solution (Citranox, Alconox, White Plains, New York, USA) prior to the deposition. All GLAD films were deposited at 1.0-1.3 nm s-1 with a deposition angle of R ) 84°. GLAD UTLC Plates. Vertical post films were designed to be isotropic in the plane of the substrate (Figure 2a and b). Continuous rapid substrate rotation (20 nm of film growth per rotation) caused the oblique evaporant flux (R ) 84°) to effectively arrive from all azimuthal directions rather than from a localized source. These films had 4.5 µm nominal thickness and columnar structures normal to the substrate surface. Anisotropic chevron (“zig-zag”) films were composed of layers of slanted columnar segments (Figure 2c and d). The columns in each layer were produced with constant φ (no substrate rotation) and inclined toward the apparent source position. Rapid ∆φ ) 180° rotations (over ∼10 nm of film growth) were conducted periodically during fabrication. After depositing a ∼500 nm thick layer of columns inclined in the φ ) 0° direction (a “zig” segment), a second ∼500 nm thick layer of columns inclined in the φ ) 180° direction (a “zag” segment) was deposited to complete one period of the chevron. Subsequent repetitions of this sequence produced chevron films with 5 µm nominal thickness and a 1 µm nominal period. In the limit of a small period, chevron films degenerate into serial bideposition (SBD) films (Figure 2e and f). Although deposited with a similar sequence of stepwise ∆φ ) 180° rotations (over ∼2 nm of film growth), the short “zig” and “zag” layers in the SBD film (each ∼12 nm thick) coalesced to form dense vertical bladelike structures. These films have a 4.5 µm nominal thickness and a 24 nm nominal period. Ultrathin-Layer Chromatography. UTLC experiments used separations of Test Dye Mixture III (CAMAG, Muttenz, Switzerland). The undiluted mixture contained 3 mg of Dimethyl Yellow, 0.5 mg of Oracet Red G, 2 mg of Sudan Blue II, 1.5 mg of Ariabel Red, 2 mg of Oracet Violet 2R, and 4 mg of Indophenol per milliliter of toluene (listed according to descending hRF values). Next, 25 nL spots of test dye mixture with varied concentrations (toluene dilutions) were applied 2.5 mm from the bottom of the GLAD UTLC plates by a modified robotic analyte dispensing system. This Automatic TLC Sampler 4 (ATS 4, controlled by the WinCats 1.4.2 Planar Chromatography Manager, CAMAG) dispensing system was modified by setting the firmware for a 100-µL syringe despite equipping the robotic arm with a 25-µL syringe. The spots were applied in “spray-on” mode at 300 nL s-1 dosage speed. The spotted plates were developed face-down for ∼90 s in a horizontal separation chamber (Desaga H-Chamber 50 mm × 50 mm, Sarstedt, Nu ¨ mbrecht, Germany; Figure 3). Porous glass frits (Desaga 50 mm frits, Sarstedt) supplied the toluene:n-hexane (4:3, v/v, both chromatography grade, VWR, Darmstadt, Germany) mobile phase loaded into the reservoir (0.7 mL) to the stationary phase layer. Also, 0.5-µL disposable glass capillaries (CAMAG) were placed beneath frits (16) Frieser, J. Electrochem. Soc. 1974, 121, 669–672.

Figure 2. SEM micrographs of macroporous GLAD thin film separation media. Micrographs of (a and b) isotropic vertical posts as well as anisotropic (c and d) chevron and (e and f) serial bideposition (SBD) media when viewed from an oblique angle and from above, respectively. Red arrow indicates the along-channel vector in anisotropic films. Top-down images of the (b) vertical post and (f) SBD films were imaged normal to the substrate surface. Pores within the top layer of the chevron film are apparent only when the film is viewed along the angled segments in this layer (∼53° to the substrate normal) (d). The inset in part d shows the film when viewed normal to the substrate.

Figure 3. Horizontal UTLC separation chamber configured for miniaturized GLAD UTLC plate formats.

to position them ∼1 mm higher and into direct contact with the UTLC plate. Additional mobile phase (0.7 mL) was added to the conditioning trough beneath the GLAD UTLC plate. The developed plates were dried for ∼5 s with a 1200 W warm air hair drier (that produced a temperature of ∼60 °C) immediately upon removal from the chamber. Digitization of Developed GLAD UTLC Plates. Highresolution JPEG images (1200 dpi, RGB mode, 8-bits per channel) of the developed GLAD UTLC plates were captured in transmission mode by a flatbed film scanner (4800 × 9600 dpi, CanoScan 5600F, Canon, Mississauga, Ontario, Canada). Identical scanner settings were used to digitize all developed plates including those used to assess the limits of detection (see the Supporting Information). No additional image enhancements were applied before chromatogram extraction. Chromatogram Extraction. Individual separation tracks were isolated from the scanned images of the developed UTLC plates using Photoshop (Photoshop CS3 Extended, version 10.0.1, Adobe Systems, San Jose, USA). In cases where the separation track was diagonal to the development direction, a diagonal track region

Figure 4. Schematic representation of chromatogram extraction from a diagonal separation track. (a) As the separation track was not parallel to the development direction (blue arrow), it had to be isolated from the scan of the developed UTLC plate using an angled mask (black). This track (dotted black line) also deviated from the alongchannel direction (red arrow) by an angle ∆θ. The horizontal black line marks the migration front position. (b) Row-averaging the pixel darkness values across the width of the track for every position along the development direction enabled chromatogram extraction.

was defined within the image (Figure 4a). Pixels outside of this region were masked and excluded from subsequent analysis. Track widths were constant along their lengths and were visually selected to include the full width of the largest spot (typically the Dimethyl Yellow spot). Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

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Chromatograms were extracted from the images using a custom script written for the MATLAB mathematical analysis software platform (MATLAB R2009a, version 7.8.0.347, MathWorks Inc., Natick, Massachusetts, USA). This script calculated the lightness (L) associated with each pixel according to the conventions used to convert an image from RGB (red, green, blue) mode to HSL (hue, saturation, lightness) mode:17 1 L ) [max(R, G, B) + min(R, G, B)] 2

(1)

where R, G, and B are the normalized red, green, and blue channel values. Darkness (D) was then defined as D)1-L

(2)

Pixel darkness values were averaged across the rows contained within the separation track for every position along the development direction. Output chromatograms plotted the pixel darkness (row-averaged perpendicular to the plate edges) along the development direction (Figure 4b). Measurement of the Limits of Detection. Decreasing concentrations of dye mixture were separated to assess the limits of detection for the GLAD UTLC plates. Signal-to-noise ratios (SNRs) for the separated Dimethyl Yellow dye component were calculated numerically using a variant of the “total peak area” method18 for the darkness signals described above. Regions adjacent to the corresponding chromatogram peak were identified by eye; linear fits to these regions were used to evaluate background levels and single pixel row noise (σ). Chromatogram peak area (A) was numerically integrated. After scaling the noise to the full peak width (w) using w1/2, the signal-to-noise ratio becomes SNR )

A σ√w

(3)

The reported limits of detection (LOD) use 99% confidence intervals (SNR ) 3). RESULTS AND DISCUSSION Instrument Configuration for UTLC. Although inkjet printing is the preferred mode of sample application on miniaturized plate formats,13 the ATS 4 was used because the limited solvent resistance of commercially available inkjet printers precluded nonaqueous samples. Since a standard ATS 4 instrument cannot handle sample volumes less than 100 nL, modifications were necessary to achieve the small volumes desired for the GLAD UTLC plates. The 100-µL syringe in the ATS 4 was replaced with a 25-µL syringe, but the system firmware remained set for a 100µL syringe. The narrower inner diameter of the smaller syringe reduced the instrumental volume limit by a factor of 4 and permitted the 25 nL sample spots used in these experiments. Under normal operating conditions, ATS 4 spot volume precision inclusive chromatography and evaluation is better than 1.8% RSD. However, we expect that with our modifications to the instrument, (17) Foley, J. D.; Van Dam, A. Fundamentals of Interactive Computer Graphics; Addison-Wesley: Reading, MA, 1984; pp 617-619. (18) Baedecker, P. A. Anal. Chem. 1971, 43, 405–410.

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the precision of our 25 nL spots will be somewhat worse. Sample spots were sprayed at twice the typical dosage speed in order to prevent drying of the 25 nL sample volumes at the end of the stainless steel needle. Spotwise application was started and ended with one spot placed outside the plate to ensure homogeneously applied spots on the plate. The plate was developed in an adjusted horizontal separation chamber (Figure 3). Two glass spacers and an elevated frit position allowed proper UTLC plate alignment and mobile phase transport. Separation of this lipophilic dye mixture is usually performed with toluene on TLC or HPTLC layers. For the less active nanostructured layers however, the mobile phase had to be adjusted to be more nonpolar. A mixture of toluene and n-hexane in the ratio of 4:3 (v/v) led to a comparable separation. The face-down development and the addition of mobile phase to the conditioning trough beneath the GLAD UTLC plate provided the vapor phase saturation necessary to obtain a flat migration front. The flatbed film scanner was equipped with a lamp built into the lid of this scanner and permitted transmission mode measurements of separation patterns on the translucent plates. High-quality scans were possible since the transmitted light underwent minimal scattering within the ultrathin layers. Macropore Anisotropies on UTLC Thin Film Media. The engineered GLAD thin film UTLC media were characterized with a scanning electron microscope (SEM) and exhibited macroporous structures (>50 nm pore size) dictated by their deposition protocols (Figure 2). This isotropic vertical post film was 4.6 µm thick (4.5 µm nominal) (Figure 2a and b). It was composed of ∼200 nm diameter columns with ∼200 nm spacing, randomly distributed over the substrate surface. The surface area enhancement of this film is estimated to be ∼1000 m2 m-2 (film surface area to footprint area).15 The anisotropic chevron film was 5.4 µm thick (5.0 µm nominal) with a ∼1 µm spatial period (Figure 2c and d) and was designed to be similar to those described by Bezuidenhout and Brett.8 Porous channel-like structures ran in a direction normal to the plane of the chevrons. Perpendicular to this “along-channel” direction, the film was less porous. Similar channel-like structures were also observed within the 4.6 µm thick (4.5 µm nominal) anisotropic SBD film (Figure 2e and f). The columnar bladelike features in this film were approximately 200 nm thick, twice the thickness of the chevron film. The spacing between these bladelike structures in the SBD films was also wider than that between the features in the chevron film (∼300 versus ∼100 nm). UTLC on Isotropic and Anisotropic Thin Film Media. Dye mixture separations were used to assess thin film media performance. The Dimethyl Yellow, Sudan Blue II, and Ariabel Red dyes were best resolved. Separation distances varied with structure and development direction (Figure 5). Separation distances were approximately 7, 5, and 12 mm for the isotropic vertical post film (Figure 5a), anisotropic chevron film (along-channel direction, Figure 5c), and anisotropic SBD film (along-channel direction, Figure 5e), respectively. This trend is consistent with the previous finding that migration occurs most rapidly through GLAD UTLC media that possess the largest macropores.8 Decreased porosity perpendicular to the channel-like structures reduced transverse broadening and may allow more separation tracks on an aniso-

Figure 5. Scanned images of developed GLAD UTLC plates. Undiluted dye mixtures were separated on (a) vertical post, (b and c) chevron, and (d and e) SBD films. Dyes migrated more rapidly in the along-channel direction (c and e) than in the across-channel direction (b and d). Migration anisotropies (see text) of ∼4.5 and ∼6.5 were measured for the chevron and SBD films, respectively. Plates developed in the upward direction. Red arrow indicates the alongchannel vector in anisotropic films. The images were enhanced for presentation only.

tropic plate developed in the along-channel direction than on an isotropic plate of the same size. Reduced porosity in the acrosschannel direction limited mobile phase migration and separation quality achieved in the across-channel direction of the chevron (Figure 5b) and SBD films (Figure 5d). To quantify these effects, we define migration anisotropy (MA) in terms of the along- and across-channel migration distances obtained for the same (∼90 s) development time:

MAi )

Zi,along Zi,across

(4)

where Zi,along and Zi,across are the migration distances for the ith component in the dye mixture separated in the along- and across-channel directions on separate plates with the same anisotropic stationary phase. As the migration distance in a particular direction depends on the size of the macropores in that direction, media with macropore anisotropies exhibit MA > 1. Therefore, the isotropic vertical post film has a MA of 1, and a perfectly anisotropic medium would have an infinite MA. Dimethyl Yellow migration anisotropies were ∼4.5 (migration distance in Figure 5c versus that in 5b) and ∼6.5 (migration distance in Figure 5e versus that in 5d) for the chevron and SBD films, respectively. UTLC on Anisotropic Media with Diagonally-Oriented Channel-Like Structures. Diluted dye mixture (50%; 1.5 mg of Dimethyl Yellow, 0.25 mg of Oracet Red G, 1 mg of Sudan Blue II, 0.75 mg of Ariabel Red, 1 mg of Oracet Violet 2R, and 2 mg of Indophenol per milliliter of toluene) was separated on anisotropic UTLC plates with their channel-like structures oriented at a 45° angle to the plate edges and development direction (Figure 6) (see the Supporting Information). We estimate an orientation error of approximately ±2° associated with the loading of the substrates into the film deposition chamber. Following UTLC development, the dye separation tracks were found to incline toward the alongchannel direction but did not become fully aligned with it, in agreement with our previous report.8 The separation tracks in the chevron and SBD films deviated from the along-channel direction by angles of ∆θ ) 10° ± 2° and ∆θ ) 5° ± 2°, respectively (as

Figure 6. Scanned images of diagonally developed GLAD UTLC plates. Dye separation tracks (dotted black line) on (a) chevron and (b) SBD media deviated from the along-channel direction (red arrow) by angles of ∆θ ) 10° ( 2° and ∆θ ) 5° ( 2° when the channel-like structures were oriented at 45° to the development direction (blue arrow). The horizontal black line marks the migration front position. Diluted dye mixtures (50%) were separated. The images were enhanced for presentation only.

defined in Figure 4). We believe that these deviations are correlated with the migration anisotropy values. A higher migration anisotropy value and a smaller separation track deviation angle both indicate that the SBD media exhibits stronger macropore anisotropy than the chevron media. This finding is consistent with the scanning electron micrographs collected for the SBD and chevron films (Figure 2). A geometric relationship between the deviation angle and the migration anisotropy may exist. If the separation track vector corresponding to the 45° channel features is decomposed into projections in the along- and across-channel directions, then

tan ∆θ )

Zi,across ) MAi-1 Zi,along

(5)

From the Dimethyl Yellow migration anisotropies reported above, eq 5 predicts separation track deviation angles for the chevron and SBD films of ∆θ ) 12.5° and ∆θ ) 8.7°, respectively. While the predicted deviation angles are different from the measured values, this simple treatment captures most of the trend observed on media with channel-like features oriented at 45° to the development direction. In its current form, it cannot describe media with their channel-like features oriented in different directions. For instance, if the along-channel and development directions are parallel, the track deviation should be ∆θ ) 0°; if they are perpendicular, the track deviation should be ∆θ ) 90°. Further work is required to resolve the numerical disagreement between the theoretical and measured track deviations and describe arbitrarily oriented channel-like structures. Mobile Phase Migration Front Identification. The morphology of macroporous columnar GLAD thin films is known to change following the introduction and evaporation of liquids.19-21 As the liquid evaporates, capillary forces cause the columns to bend into each other and “clump” together. Surface forces between adjacent columns cause them to remain clumped together long after the film has dried. Although the resultant clumps can only be viewed with SEM, the change in the GLAD thin film optical properties due to clumping can be seen by the unaided eye (Figure 7). The migration front was equivalent to the boundary of the clumped region on the plate and remains visible several months after the separation had been performed. The clumping phenomenon therefore offers an automatic means of permanently (19) Fan, J.-G.; Dyer, D.; Zhang, G.; Zhao, Y.-P. Nano Lett. 2004, 4, 2133–2138. (20) Fan, J.-G.; Zhao, Y.-P. Appl. Phys. Lett. 2007, 90, 013102. (21) Fan, J.-G.; Fu, J.-X.; Collins, A.; Zhao, Y.-P. Nanotechnology 2008, 19, 045713.

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Figure 7. Photograph of the migration front automatically preserved on a GLAD UTLC plate developed and dried 5 months earlier. Clumping of columnar grains within the wetted region (bottom) causes the optical properties to change from those of the unperturbed film (top). The interface between these regions is visible to the naked eye when lit from behind by ambient light. The migration front curvature on the anisotropic SBD film is due to the diagonal orientation of the channel-like features and the resultant preferential mobile phase flow to the right. The blue arrow marks the development direction and the horizontal black line marks the migration front position.

archiving the migration front position on developed GLAD UTLC plates without manual marking. Initial work also suggests that a very slight but characteristic shift in the darkness baseline intensity may denote the migration front in the extracted chromatogram. Investigation and modification of the clumping phenomenon through post-deposition processing remain active areas of research in our group and others. Effects of Film Morphology on Separation Behavior. Chromatograms were extracted from scans of the developed UTLC plates by considering the analyte and mobile phase migration along the development direction. (In diagonal developments, this direction was different from the separation track direction, as shown in Figure 4.) The resultant chromatograms also reflect the strong dependence of separation behavior on film microstructure (Figure 8). Along-channel separations on the chevron and SBD media are comparable to those on the vertical post media. While the Dimethyl Yellow peak (hRF ∼ 60) appeared well-resolved, the peaks corresponding to the Sudan Blue II (hRF ∼ 25) and Ariabel Red (hRF ∼ 15) dye components were only partially resolved. The other dye components were too faint to produce identifiable peaks. The low resolution of all components is attributed to the relatively large initial spot size. Appropriate RGB color channel mixing may isolate individual dye components within the scanned image and improve this result. Rapid spot migration in the along-channel direction on the SBD media compensates for increased spot elongation and results in separation performance similar to that of the vertical post media. Increased peak broadening observed in the along-channel direction on the chevron media (∼40% compared to the vertical post and SBD media) is believed related to the increased structure tortuosity relative to the vertical post and SBD media. Nonuniform 5354

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Figure 8. Chromatograms from dye separations on isotropic and anisotropic GLAD UTLC media. Vertical post (a, solid green), alongchannel SBD (b, solid red), diagonal channel SBD (c, dotted red), along-channel chevron (d, solid blue), and diagonal channel chevron (e, dotted blue) media. Peaks corresponding to the start zone (start), Ariabel Red (AR), Sudan Blue II (SB), and Dimethyl Yellow (DY) are identified. Double peaks observed at the starting point in some of the curves are associated with ring-shaped spots. Darkness curves offset for clarity.

mobile phase flow around the angled segments in the chevron film may enhance eddy diffusion of the analytes. Chromatograms corresponding to the diagonal developments conducted on the chevron and SBD media exhibit some interesting trends. Despite increased peak broadening, the chromatograms show increased peak separation and suggest reduced analyte retention on the plate. Although the separation track vector is different from the along-channel direction, spot elongation still appears to have occurred preferentially in the highly porous alongchannel direction. This effect is most obvious in the diagonally

Table 1. GLAD UTLC Stationary Phase Figures of Merit Based upon the Dimethyl Yellow Component Separated from Undiluted and Diluted (50%) Test Dye Mixtures migration distance (mm) stationary phase structure (and development direction) vertical posts chevron (along-channel) chevron (diagonal channel) SBD (along-channel) SBD (diagonal channel)

undiluted

diluted

undiluted

diluted

undiluted

diluted

10 ± 4

6.3 ± 0.1 3.8

540 ± 50 170

9.6 ± 0.6 5.27 ± 0.05

270 ± 25 150 ± 5 167 ± 6 260 ± 25 330 ± 50

12 ± 1 22

11 ± 3

5.64 ± 0.07 3.50 ± 0.07 3.02 ± 0.06 7.2 ± 0.1 5.33 ± 0.05

21 ± 2 23.5 ± 0.5 18 ± 1 28 ± 2 17 ± 2

()

Hi )

plate height H (µm)

LOD (ng)

developed chevron plate (Figure 6a) as it has the greatest separation track deviation from the along-channel direction. Changes in the observed spot broadening geometry combined with the pixel row averaging technique developed for diagonal separations may account for some of these chromatogram features. Quantitative Measures of GLAD UTLC Plate Performance. Darkness signals associated with the Dimethyl Yellow dye component in the dye mixture were used to calculate the limits of detection and estimated separation efficiency of the thin film separation media (Table 1). The limits of detection were found to be 10 ±4 ng and 11 ±3 ng for the vertical post and SBD media, respectively (Figure 9). These unoptimized values are an order of magnitude higher than those achieved on other UTLC media.6 Separation efficiency is described in terms of the theoretical plate number (Ni) and theoretical plate height (Hi):22,23 Ni ) 16

plate number N

Zi wi

2

Zi wi2 ) Ni 16Zi

(6a)

(6b)

480 ± 140 340 ± 20

21 ± 6 16 ± 1

color channel mixing, and two-dimensional peak intensity fitting may further understanding of anisotropic GLAD UTLC media and improve separation performance. Although measurements of the angular deviation (between the separation track and the along-channel direction) are consistent with our simple analytic treatment (eq 5), additional experiments with increased measurement accuracy are required to refine this model. Studies of separation tracks on GLAD thin films with arbitrarily oriented channel-like structures should provide insight into the mechanism of capillary-driven flow through anisotropic media and facilitate design of these UTLC plates. To further exploit the advantages offered by the GLAD approach, additional engineering of nanostructured separation media is required. The strength of the macropore anisotropies could be designed to maximize analyte resolution or to permit more samples to be analyzed in parallel on smaller UTLC plates. Additional degrees of freedom may be realized through postdeposition modification of film architecture and surface chemistry. Encapsulation of GLAD films within microchannels may compliment the advantages offered by microfluidics.24 All of these capabilities make GLAD a promising platform for the study and design of new analytical separation techniques.

where Zi and wi are the migration distance and base width for the peak corresponding to the ith component in the dye mixture. The obviously low plate numbers calculated for the GLAD UTLC media (150-540, Table 1) are related to the very short migration distances in these experiments (3-10 mm, Table 1). While the plate heights (12-28 µm, Table 1) are significantly greater than those reported for the reversed phase electrospun polymer UTLC plates,7 they are comparable or better than those of normal phase TLC, HPTLC, and early monolithic silica gel UTLC plates.3,6,23 Further study is required to resolve the variations in separation performance, especially between the diluted and undiluted dye separations. The relatively large start zone (similar in size for diluted and undiluted dye mixture) may contribute to the unexpected decrease in separation performance for the diluted dye spots. Potential Opportunities. The initial unoptimized results presented here are promising and appear consistent with other normal phase silica UTLC media.6 Enhancements to the chromatogram extraction method such as track width optimization, (22) Sherma, J. Basic TLC Techniques, Materials, and Apparatus. In Handbook of Thin-Layer Chromatography, 3rd ed.; Sherma, J., Ed.; Fried, B.; Marcel Dekker: New York, 2003; pp 5. (23) Fried, B.; Sherma, J. Thin-Layer Chromatography, 4th ed.; CRC Press (Marcel Dekker): New York, 1999; pp 1-24.

Figure 9. Limits of detection for the Dimethyl Yellow dye. Spots in the (a) vertical post and (b) SBD films. Insets show the measurable peak associated with the 9.4 ng Dimethyl Yellow peak. The limits of detection for the dye on these films were 10 ( 4 ng and 11 ( 3 ng, respectively, with a 3σ criterion used. Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

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CONCLUSIONS Using glancing angle deposition (GLAD), we engineered 4.6-5.3 µm thick normal phase silica UTLC plates with varied macropore architectures: isotropic vertical posts as well as anisotropic chevron and blade-like media. Channel-like structures have been shown to affect separation behaviors on anisotropic media. We have defined the migration anisotropy ratio and separation track deviation angle to characterize the impact of the macropore anisotropy. GLAD stationary phase performance was quantified using dye separations and a customized chromatogram extraction technique. Theoretical plate numbers and plate heights measured for the separated Dimethyl Yellow dye component were within 150-540 and 12-28 µm, respectively. Unoptimized limits of detection of Dimethyl Yellow dye were approximately 10 ± 4 ng for the vertical posts and 11 ± 3 ng for the blade-like media. While these quantitative results indicate that anisotropic stationary phases may have a place within the analytical chemistry toolbox, further exploration and optimization are required to leverage the new dimensions of control offered by GLAD separation media. (24) Bezuidenhout, L. W.; Flaim, E.; Elias, A. L.; Brett, M. J. Proceedings of µTAS 2009: 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Jeju, Korea, Nov. 1-5, 2009.

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ACKNOWLEDGMENT The authors are grateful for the financial support provided by the Natural Sciences and Engineering Research Council (NSERC) of Canada, Micralyne Inc. (Edmonton, Canada), and Alberta InnovatessTechnology Futures. The equipment provided by CAMAG (Muttenz, Switzerland), the expert scanning electron microscopy work of G. Braybrook (University of Alberta Earth and Atmospheric Sciences SEM Lab), and the assistance of S. Fernando (Glancing Angle Deposition Laboratory, University of Alberta) in preparing the Supporting Information are also much appreciated. SUPPORTING INFORMATION AVAILABLE Video of a rapid dye separation performed on an anisotropic GLAD UTLC plate with diagonally oriented channel-like structures; scanner settings used to digitize all developed UTLC plates; and image enhancements used only to prepare the developed plate images for presentation. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review April 15, 2010. Accepted May 12, 2010. AC101004B