Miniaturized Planar Chromatography Using Office ... - ACS Publications

Feb 15, 2010 - cal tools are required to extract the full analytical power from this ... printer to spot analytes on UTLC plates and compared its perf...
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Miniaturized Planar Chromatography Using Office Peripherals Gertrud E. Morlock,*,† Claudia Oellig,† Louis W. Bezuidenhout,‡ Michael J. Brett,‡,§ and Wolfgang Schwack† Institute of Food Chemistry, University of Hohenheim, Garbenstrasse 28, 70599 Stuttgart, Germany, Department of ECE, University of Alberta, Edmonton, Canada, and NRC National Institute for Nanotechnology, Edmonton, Canada High-performance thin-layer chromatography is a separation technique commonly used to identify and quantify components in chemical mixtures. Sophisticated analytical tools are required to extract the full analytical power from this technique and especially for miniaturized planar chromatography its utility has not been harnessed. A new approach uses an elegant, simplified system assembled from ordinary consumer printers and scanners to perform separations on monolithic and nanostructured ultrathinlayer phases. This system is shown to outperform existing planar chromatographic tools for analysis on miniaturized plates. Analysis can be completed in a manner of minutes, running numerous samples in parallel at a reduced cost, with very low sample and reagent volumes, all using a familiar computer interface with common office peripherals. High-performance thin-layer chromatography (HPTLC) is a widely used technique for qualitative and quantitative analysis of liquid samples in diverse fields such as environmental, food, and pharmaceutical sciences.1 There is considerable overlap in the analyses that can be performed with HPTLC and high-performance liquid chromatography (HPLC), and to some degree with gas chromatography (GC).2 Compared to HPTLC, HPLC provides greater resolving power and more sensitive detection schemes, whereas HPTLC provides faster analysis and higher flexibility at a reduced cost of both chemicals and equipment. During HPTLC, unseparated analytes are held at the sorbent origin and remain accessible once the separation is complete. During HPLC or GC, such analytes would be captured at the column head without reaching the detector and thus could not be further analyzed. This ability to see everything on the plate, combined with always having fresh sorbent (no “memory” due to matrix effects on the sorbent), side-by-side sample analysis under extreme identical conditions, and the capability for multidetection especially with bioassays since the separated analytes are held fixed in the plate and organic * Corresponding author. Phone: +49-711-459-24094. Fax: +49-711-459-24096. E-mail: [email protected]. † University of Hohenheim. ‡ University of Alberta. § NRC National Institute for Nanotechnology. (1) Sherma, J. Anal. Chem. 2008, 80, 4253–4267. (2) Morlock, G.; Schwack, W. Trends Anal. Chem., 2010, submitted for publication.

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solvents are evaporated, represent some of the benefits of HPTLC over HPLC and GC.3 Ultrathin-layer chromatography (UTLC) is faster and further reduces the cost of HPTLC, but analyte spotting and quality imaging of developed plates has proven difficult due to the much thinner sorbent layer and the availability of equipment optimized only for use with the larger HPTLC plates.4,5 Subsequent reduction in resolution along with increased handling complexity has contributed to low rates of adoption in the community despite the benefits. We introduce a new approach to planar chromatography, which significantly reduces the complexity, expense, and analysis time of traditional planar chromatography laboratory setups by replacing existing equipment with simple office computer peripherals. Inexpensive thermal inkjet printers replace commercial applicators and flatbed scanners replace charged coupled device (CCD) based image capture devices. In keeping with the concept of faster, less expensive analyses, UTLC plates replace HPTLC plates as an integral part of this system. To prove this interdisciplinary concept, we modified a thermal inkjet printer to spot analytes on UTLC plates and compared its performance to a piezoelectric and an aerosol applicator. Thermal inkjet printers vaporize ink by activating heating elements in microscopic micromachined chambers contained in the print head; the vaporized ink is ejected and deposited on paper, and the chamber is refilled from the cartridge by capillary action.6 Droplet chamber volumes are typically in the picoliter range; personal use thermal inkjet printers activate multiple chambers simultaneously when printing, producing droplet volumes on the order of hundreds of picoliters. The ink can be replaced with other aqueous solutions, and consequently such printers have been adapted for printing solutions in biological and chemical applications,7 including monoclonal antibodies onto nylon membranes,8 DNA probes onto hybridization membranes,9 and derivatization reagents onto selected areas of HPTLC plates,10 but have never been used to directly apply analytes and to use it quantitatively. (3) Morlock, G.; Schwack, W. LC-GC Eur. 2008, 21, 366–371. (4) Hauck, H. E.; Bund, O.; Fischer, W.; Schulz, M. J. Planar Chromatogr. 2001, 14, 234–236. (5) Hauck, H. E.; Schulz, M. Chromatographia 2003, 57, S/313–S/315. (6) Le, H. P. J. Imaging Sci. Technol. 1998, 42, 49–62. (7) Calvert, P. Chem. Mater. 2001, 13, 3299–3305. (8) Nilsson, S.; Lager, C.; Laurell, T.; Birnbaum, S. Anal. Chem. 1995, 67, 3051–3056. (9) Goldman, T.; Gonzalez, J. S. J. Biochem. Biophys. Methods 2000, 42, 105– 110. (10) Morlock, G.; Stiefel, C.; Schwack, W. J. Liq. Chromatogr. Relat. Technol. 2007, 30, 2171–2184. 10.1021/ac902945t  2010 American Chemical Society Published on Web 02/15/2010

We also studied the performance of a flatbed scanner for image capture compared to two integrated digital documentation systems. To make the comparisons, we applied a mixture of food dyes to UTLC plates using one of the three applicators, separated the dyes, digitized the results using either the scanner or the image capture devices, and analyzed the results using a customized commercial software package. We also evaluated the system using two different types of UTLC plates, monolithic silica gel plates4,5 (M-UTLC), and nanostructured silica plates11 (NS-UTLC). EXPERIMENTAL SECTION Reagents and Chemicals. We obtained the dyes used for our analysis as follows: curcumin (E100, ≈97% pure) from Merck, Darmstadt, Germany; Sunset Yellow FCF (E110, ≈90% pure) from Ringe and Kuhlmann, Hamburg, Germany; Ponceau 4R (E124, 80% pure, also known as Cochineal red A) from Ringe and Kuhlmann, Hamburg, Germany; Acid blue (E131, 50-51% pure, also known as Patent blue V) from Ringe and Kuhlmann, Hamburg, Germany; Azorubine (E122, purity grade not labeled) from Schuhmann and Son, Karlsruhe, Germany. Other reagents used were chromatography solvents (technical grade, distilled prior to use) and acetic acid (99-100% pure) from BASF, Ludwigshafen, Germany; glycerol (p.a. grade) from VWR, Darmstadt, Germany; 1,2-propanediol (per synthesis) from Merck, ultrapure water (>18 MΩ cm) supplied by a Synergy System from Millipore, Schwalbach, Germany. Chromatographic Plates. Two different types of ultrathinlayer chromatography plates were used for separations. Glassbacked UTLC plates (6 cm × 3.6 cm) coated with a 10 µm thick monolithic silica gel sorbent layer (M-UTLC) were obtained from Merck. Glass-backed plates (5 cm × 2 cm and 10 cm × 2 cm) were coated by depositing SiO2 hexagonal spiral nanostructures at a deposition angle of 84° as per Bezuidenhout et al.11 Preparation of Dye Solutions. For application by piezoelectric ejection and by spraying, dye standards were prepared by dissolving and diluting dyes in a methanol-ammonium acetate buffer of pH 6.8 (1 + 1, v/v) to obtain the final concentrations listed in Table S-2 in the Supporting Information. The buffer was prepared by dissolving 1.54 g of ammonium acetate in ultrapure water filled up to 500 mL, adjusting to pH 6.8 with acetic acid, and adding methanol to a final volume of 1000 mL. For application by inkjet printing, the dyes were dissolved in 15 g of glycerol, 15 mL of 1,2-propanediol, and 70 mL of heated ultrapure water. This alternate preparation was necessary to obtain the appropriate viscosity for inkjet printing.10 Thermal Inkjet Printing. The Pixma iP 3000 Bubble Jet printer from Canon, Krefeld, Germany, has four separate color cartridges that can be individually filled. We filled empty singleuse cartridges with 7 mL of dye solutions using a custom-made vacuum apparatus (Figure S-1B in the Supporting Information). Dye bands and spots were applied to UTLC plates by printing patterns drawn in vector illustration software (CorelDraw, Corel, Unterschleissheim, Germany). A pattern could be drawn and subsequently sent to the printer for reproduction, making it possible to print solutions in exact, miniaturized patterns at any position on the plate. Some modification and adaptations of the driver software (Canon Pixma iP 3000 Printer Driver v1.80 aDE (11) Bezuidenhout, L. W.; Brett, M. J. J. Chromatogr., A 2008, 1183, 179–185.

for Windows XP) were required to produce high quality reproducible bands and spots. Warning flags for empty and missing cartridges were removed. Relevant driver settings (given as parameter (value)) were paper selection (normal paper); print quality (high); image type (none); change of color quality (printing color + 50, other cartridges - 50); intensity (+ 50); media type (printable media). We verified that the driver addressed only the cartridge containing the desired solution to be printed. Separate tests showed that 95% of printed ink came from the target cartridge using this combination of driver settings.10 The glass-backed UTLC plates were loaded onto the printer’s integrated CD-tray to directly print the dye solutions. Bands and spots were printed 4 mm from the bottom edge of the UTLC plate. Bands (as drawn in software) were 3 mm wide, 0.2-0.6 mm tall, and spaced 1 mm apart, yielding 13 tracks per M-UTLC plate (6 cm wide) and 23 tracks per NS-UTLC plate (10 cm wide). Spots (as drawn in the software) were 0.45-0.87 mm in diameter, spaced 2 mm centerto-center, yielding 26 tracks per M-UTLC plate and 46 tracks per NS-UTLC plate. We calculated the volume of solution dispensed by printing a 8 cm × 8 cm square and gravimetrically measuring the change in the ink-tank volume and using this volume per printed area ratio to determine the band and spot application volumes. The dye mixture solution density was 1.04 g/cm3. The dye volume printed ranged between 3.1 and 9.6 nL/band (dependent on band height), which equated to a postevaporation mass of 15-46 and 80-249 ng/band, depending on the dye concentration. Per spot, the printed volume ranged between 0.8 and 3.6 nL (dependent on the diameter), corresponding to a mass of 3-15 and 21-79 ng/spot, depending on dye concentration. Piezoelectric Dispensing. The PipeJet P9 Nanodispenser (BioFluidix, Freiburg, Germany) settings were chosen based on the information given in refs 12-14: 200 µm tube diameter; 18 mm tube length; 25-40 µm stroke length; 200-300 µm/s downstroke velocity; 50 Hz frequency. Because of software limitations, only spots could be applied with the Nanodispenser. Spots were applied 4 mm from the bottom edge of the UTLC plate, spaced 3 mm from the left edge and 3 mm apart center-to-center, yielding 19 tracks per M-UTLC plate (6 cm wide) and 16 tracks per NS-UTLC plate (5 cm wide). The dye mixture solution density was 0.84 g/cm3. The dispensed volume was again determined by gravimetric measurement of print volumes. Dispensed volumes were 8.2 or 10.6 nL/shot for the M-UTLC plates, giving postevaporation masses of 2-12 ng/shot, depending on dye concentration and method settings; dispensed volumes were 6.8 nL/shot for the NS-UTLC plates, corresponding to a mass of 1.4-1.7 ng/shot. Lower volumes were used for the NS-UTLC plates to compensate for the thinner, more porous sorbent layer. A range of 2-40 shots were applied per spot for the functional correlation, repeatability, and limits of quantitation (LOQ) studies. Pressurized Spray. We used the following settings to apply dye solutions to the plates with the Automatic TLC Sampler 4 (12) BioFluidix. PipeJet Nanodispensers P4.5/P9, Non-contact liquid handling for OEM applications, www.biofluidix.com, 2009. (13) Koltay, P.; Steger, R.; Bohl, B.; Zengerle, R. Sens. Actuators, A 2004, 116, 483–491. (14) Streule, W.; Lindemann, T.; Birkle, G.; Zengerle, R.; Koltay, P. JALA 2004, 9, 300–306.

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(ATS 4) controlled via WinCats 1.4.2 Planar Chromatography Manager from CAMAG, Muttenz, Switzerland: 3 mm band length; 4 mm track distance for bands, 2.5 mm track distance for spots; and 40 nL/s dosage velocity. Bands and spots were applied 6 mm from the bottom edge on M-UTLC plates and 3 mm from the bottom edge on NS-UTLC plates, spaced 8 and 4 mm from the left edge, respectively. Dispensed volumes were determined directly from the calibrated ATS 4 device settings. The dye mixture solution density was 0.84 g/cm3. For the precision (repeatability) study, typical application volumes were 0.3-0.6 µL, depending on the application mode (bands or spots), and whether M-UTLC or NS-UTLC plates were used; postevaporation masses ranged between 21 and 42 ng per band/spot. Application volumes for the functional correlation measurements were between 0.1 and 1.6 µL, corresponding to 7-120 ng/band for the M-UTLC plates and 12-57 ng/band for the NS-UTLC plates. The LOQ measurements had application volumes of 0.1-1.0 µL, resulting in deposited masses between 7 and 70 ng per band/spot. The mass deposited using the ATS 4 was controlled by adjusting the application volume, whereas multiple shots and larger printed band heights were used to control the deposited mass for the PipeJet and Inkjet applicators, respectively. Chromatography. After application, the start zone was dried in a warm air stream for 0.5 min. Both M-UTLC and NS-UTLC plates were developed at the same time in a 10 cm × 5 cm twinthrough chamber (CAMAG) using an ethyl acetate-methanol (3 + 1, v/v) mobile phase. The mobile phase migrated 25 mm in 10 min on the M-UTLC plates and 10 mm in under a minute on the NS-UTLC plates, at which point the development was halted by removal from the chamber. The plates were then dried under a warm air stream for 0.5 min. Documentation. Plates were documented for archiving and software analysis by digitizing them via either digital photography or scanning with a flatbed scanner. The Scanjet G2410 (HewlettPackard, Bo¨blingen, Germany) uses a built-in fluorescent tube and a linear charge-coupled device (CCD) array to scan and capture the plate image with 1200 dpi resolution and 16-bit color depth per channel. Plates were placed face down on top of the glass and scanned, with some dye colors (e.g., E124) requiring a sheet of white paper backing the UTLC plate for optimum capture to compensate for the translucent plates. The scanner was operated in high definition mode, varying the contrast and grayscale to obtain an optimum signal-to-noise ratio. With the DigiStore 2 Documentation System (CAMAG), plates were illuminated in reflectance mode with UV (254 nm) and visible/white light. The plate image was captured by a Baumer Optronic DXA252 12-bit per channel color depth CCD, with a 100 µm spatial resolution (100 000 pixels in a 5 cm × 2 cm frame and 216 000 pixels in a 6 cm × 3.6 cm frame). With the use of the TLC Visualizer Documentation and Evaluation System (CAMAG), plates were illuminated in reflectance mode with UV (254 nm) and visible/white light and also captured by a Baumer Optronic DXA252 CCD. For both systems, the following capture settings were used: 3 ms exposure time in the visible range; 200 ms exposure time in the UV range; and a gain of 1. To gain optimal images, we varied the spot amplification, 2942

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the resolution, and the automatic background correction to increase sensitivity. Evaluation. All plate images were evaluated regardless of the method of capture with VideoScan Digital Image Evaluation software (v1.02, CAMAG), with the following evaluation parameters: Savitsky Golay filter width of mostly 9 or 11; lowest slope as baseline correction mode; and color filters applied for evaluation. A densitogram was obtained by converting each color channel into a grayscale image and mapping each separation lane onto a two-dimensional curve for each channel as follows: the pixels for each line across the width of the lane were averaged, and each of these averaged values was then plotted along the length of the channel to obtain an equivalent chromatogram. The channels could be combined to obtain the separation densitogram or analyzed individually. The results were then quantified by measuring either the height of the peaks or the area under the peaks on the densitograms for each dye band/spot. RESULTS AND DISCUSSION Sample Application by an Inkjet Printer. The first component of the apparatus is the inkjet printer. Applying analyte solutions to UTLC plates is problematic due to the low volumes and small spots needed for optimal resolution as a result of the thin sorbent layer and short development distances. The lownanoliter volumes required approach the limits of current automated HPTLC spotting technology because the relatively thicker layers of HPTLC do not require such low volumes for sufficiently small starting zones. Hence, we modified a Canon inkjet printer (Pixma IP 3000, Canon) by inserting print cartridges filled with sample and standard solutions to print directly onto UTLC plates placed into the printer’s CD tray (Figure S-1 in the Supporting Information). To ensure homogeneous flow through the inkjet nozzles, all solutions had to be adjusted for optimal viscosity and surface tension. The driver software also had to be adjusted to ignore cartridge level and presence indicators. To evaluate reliable sample application by the inkjet printer, different mixtures of water-soluble food dyes (E100, E110, E122, E124, and E131, Table S-1 in the Supporting Information) were spotted on UTLC plates using either the inkjet printer, a piezoelectric ejector with low volume ejections (PipeJet Nanodispenser P9, BioFluidix), or an aerosol applicator manufactured specifically for HPTLC use (ATS 4, Automatic TLC Sampler 4, CAMAG), and all are shown in Figure 1. The application was performed both as bands and spots, with the exception of the piezoelectric ejector limited to spots only. We estimated the range of volumes applied with each applicator to be 0.8-9.6 nL for the inkjet printer, 6.8-10.6 nL for the piezoelectric dispenser, and 0.1-1.6 µL for the aerosol applicator. For comparison, we established calibration plots for each applicator by applying increasing quantities of analyte in six steps, starting at the limit of quantification (LOQ) and evaluating the subsequently separated dye bands and spots (Table S-2 in the Supporting Information). The linearity of such calibration plots can be used as an indicator of the dynamic homogeneity of samples deposited by the applicator15 and to establish the most (15) International Conference on Harmonisation (ICH). Harmonised Tripartite Guideline on Validation of Analytical Procedures: Text and Methodology Q2(R1), 2005, www.ich.org (search for Q2(R1)).

Figure 1. Application techniques on ultrathin layers (5-10 µm) and sharpness of the zones applied in the low nanoliter range by (A) PipeJet Nanodispenser P9, (B) Automatic TLC Sampler 4 (ATS 4), and (C) inkjet printer Pixma iP 3000 (bubble-jet). Table 1. (A) Summary of the Three Application Methods Used to Apply Analyte to the M-UTLC Platesa and (B) Summary of the Two Types of UTLC Plates Analyzed, Using the Inkjet Printer As an Applicatorb A application linearity R2 (maximum % RSD)

precision % RSD, n g 8 (g S/N)

applicator

deposition volume

spots

bands

spots

bands

inkjet printer piezoelectric dispenser pneumatic spray

0.8-9.6 nL 6.8-10.6 nL 0.1-1.6 µL

g0.9913 (3.2%) g0.9940 (2.3%) g0.9917 (6.4%)

g0.9988 (1.3%) n/a g0.9804 (5.1%)

3.1-6.0% (6) 1.3-3.4% (9) 2.7-7.3% (9)

2.9-3.8% (10) n/a 4.5-8.7% (21)

B UTLC plate

stationary phase

application linearity R2 (maximum % RSD)

M-UTLC NS-UTLC

monolithic silica gel, 10 µm thick nanostructured silica, 5 µm thick

g0.9988 (1.3%) g0.9997 (1.3%)

precision % RSD, n g 8 (g S/N)

LOQ range (amount/band)

2.9-3.8% (10) 2.7-3.3% (16)

26-69 ng 21-84 ng

a The minimum R2 for the dye spots in a given separation is shown. b Bandwise application, peak area evaluation. LOQs differed with the dyes; the range of LOQ for the dye mixture are given.

reliable quantification mode (peak height or area after bandwise or spotwise application). When the dyes were applied as bands, the inkjet printer outperformed the ATS 4 by a larger coefficient of determination and smaller relative standard deviation, whereas with spotwise application the PipeJet performed slightly better than the inkjet printer and ATS 4 (Table 1A). The PipeJet is limited, however, to operating only as a spotwise applicator and has a higher volume limit than the inkjet printer making it difficult to apply sufficiently small analyte spots. We measured the consistency of each applicator, its precision, by analyzing the same dyestuff mixture applied nine times on the same plate (Table S-3 in the Supporting Information). The inkjet printer was more consistent than the aerosol applicator when depositing bands, although the ATS 4 had a higher signal-to-noise ratio (Table 1A). With the deposition of spots on M-UTLC plates, the PipeJet was once again better than the other two applicators. Results for the NS-UTLC plates were comparable. To conclude from the summarized results (Table 1A), such an inkjet printer can easily be modified to precisely deposit small amounts of liquid with no contact between the printer head and plate. The inkjet printer performed as good as or better than the two commercial systems, at a substantially reduced capital and operative cost, and with the ability to print lower volumes than

the other two applicators. Comparable piezoelectric-driven printers could be used for liquids that are not compatible with the heat from thermal inkjet printing. Miniaturized Plate Image Capture Using a Flatbed Scanner. The next component of the apparatus is the flatbed scanner. Unlike opaque HPTLC plates, UTLC plates are semiopaque, making evaluation by densitometric absorption almost impossible due to partial transmission of light through the plate. The small spot size and reduced migration distances on UTLC plates also require significantly higher resolution than HPTLC for quantitative measurements. We captured the same plates with a flatbed scanner (Scanjet G2410, Hewlett-Packard) and two digital documentation systems (TLC Visualizer and Digistore2, CAMAG) to compare the performance of the scanner in appropriate context. The scanner uses a linear CCD array to scan the image, while the two documentation systems capture an image of the plate via a CCD camera.16 Generally, commercial software for TLC analysis (VideoScan Digital Image Evaluation, CAMAG) was used to evaluate and quantify the results. Plate images were optimized for a high signal-to-noise (S/N) ratio first by adjusting the physical scanner/camera settings and then by postcapture software opera(16) Morlock, G. CAMAG Bibliogr. Serv. CBS 2005, 94, 9-10, www.camag.com (after registration).

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Table 2. Influence of Digital Documentation System and Evaluation Method (Peak Area vs Peak Height) on Signal-to-Noise Ratio (S/N), Determined by Evaluation of Two Separated Dye Spots Using Each Systema S/N (area) documentation system and specific settings DigiStore 2 TLC Visualizer automatic background correction image enhancement (64 integrated images) spot amplification higher resolution (1200 dpi) flatbed scanner high-definition mode (high) white paper backdrop just for E124

E124

S/N (height)

E131

E124

E131

5

5

10

11

13

12

17

20

20

13

39

28

110

46

79

25

a Dyes were applied on UTLC plates using the inkjet printer and then separated prior to documentation.

tions including background correction, image enhancement, and spot amplification. The S/N ratio is a good indicator of the quality of the captured plate image and also of the subsequent quantitative analysis. The flatbed scanner outperformed both commercial TLC documentation systems (Table 2), in particular for area measurements, where the E131 signal was more than 3 and 9 times stronger than obtained by the TLC Visualizer and the Digistore2, respectively. Hence, coupled with TLC-specific evaluation software, the flatbed scanner makes an easy to use digital documentation system capturing miniaturized UTLC plates at a fraction of the cost. Performance of Miniaturized Plates. Separations on TLC or HPTLC layers might also be performed; however, the focus was laid on a new approach that can cope with miniaturized planar formats like UTLC because the achievement of reliable results on miniaturized plates is not possible with currently available, oversized HPTLC instrumentation. The UTLC plate is an integral component in the proposed system. Four completely different approaches for manufacturing UTLC plates have been reported.2,4,11,17 The two available plate types used for this study add a degree of flexibility to the system without affecting its simplicity in use. We tested their performance when integrated in the system using the inkjet printer for bandwise sample application followed by peak area evaluation using the flatbed scanner for documentation, which provided the best results for UTLC analysis. The coefficient of determination (R2) from calibration curves was g0.9988 for the M-UTLC plates and g0.9997 for the NS-UTLC plates, both with % RSD e 1.3% (Table 1B, details in Table S-2 in the Supporting Information). Repeatabilities were high for both plates, with relative standard deviations of less than 3.3% and 3.8% (Table 1B, details in Table S-3 in the Supporting Information). Limits of quantitation (LOQ, at S/N of 10) were comparable for the two plate types. For spotwise application, the LOQ were between 9 and 26 ng/zone for M-UTLC plates and between 10 and 33 ng/zone for NS-UTLC plates, depending on the dye (17) Clark, J. E.; Olesik, S. V. Anal. Chem. 2009, 81, 4121–4129.

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analyzed (Table 3). When the dye mixture was applied as bands, the LOQ were approximately 2-3 times higher but still comparable between plates. The difference in LOQ between the two application modes could be attributed to the evaluation method, as in the bandwise mode only the homogeneous middle part of the band was evaluated, comprising approximately 60% of the aliquot, whereas the entire zone was evaluated in the spotwise mode. With the use of office peripherals, the ultrathin layer architecture can now be expressed quantitatively (Table 1B), which is helpful for further layer structure improvements. The S/N ratio, and hence the LOQ, could be further improved for both plate types by applying electronic color filters to the captured plates (Figure 2) and by optimizing the software for each color. For example, the S/N ratio for E124 at 37 ng/band could be improved 4-fold to 79 in this manner, yielding an LOQ of about 5 ng/band, compared to the limit of 21 ng/band obtained without color filtering. Accelerated Chromatography Using Nanostructured Plates. There were some notable differences between the separations on the M-UTLC and NS-UTLC plates. The migration distance was much shorter on the NS-UTLC plates compared to the M-UTLC plates; the NS-UTLC plate development took far less than 1 min to cover a 3 mm migration distance (Figure 3B), whereas on the M-UTLC plate development took 10 min over a 25 mm migration distance. This was most likely due to the differences in the plate morphologies: the NS-UTLC plate sorbent layer was a 5 µm thick silica film with a distinct channeled topology (Figure 3A, left) with an approximate specific surface area of 150 m2/g;11 the M-UTLC plate sorbent layer was a 10 µm thick monolithic silica gel with a tight porous structure (Figure 3A, right) and a higher specific surface area (350 m2/g).4,5 As a result of the shorter migration distance, the colored zones laid closer together on the NS-UTLC plates, reducing the selectivity (a measure of the resolution) and separation number (a measure of the number of compounds that can be resolved) relative to the M-UTLC plates by factors of 1.4 and 2, respectively (Table S-4 in the Supporting Information). Conversely, the NS-UTLC plates consistently had a higher repeatability (Table S-3 in the Supporting Information). Although the performance of the nanostructured UTLC plates was on par with the monolithic UTLC plates, there is the potential for improved performance from the nanostructured plates through optimization of the nanostructure architecture for targeted applications. CONCLUSIONS We have demonstrated how an inexpensive inkjet printer could be modified to apply analyte solutions to ultrathin-layer chromatography plates, how the developed plates could be documented using a simple to use desktop scanner, and how this setup outperforms expensive, complex commercial systems when used with miniaturized UTLC plates. The versatility in plate design afforded by NS-UTLC adds a strong asset to the separation scientists’ tool chest. In future work, the engineering of more efficient NS-UTLC plates could be realized by tailoring composition, morphology, structure, and pore size to specific applications based on the nature of the compounds being separated. For example, GLAD films were recently optimized for structure, periodicity, and porosity for application as photonic crystals,18 sensors,19 and optical filters.20

Table 3. LOQ of Dyes on M-UTLC and NS-UTLC Platesa M-UTLC plates

NS-UTLC plates

dye

hRF- value

S/N

amount (ng/spot)

S/N

amount (ng/band)

hRF- value

S/N

amount (ng/spot)

S/N

amount (ng/band)

E122 E124 E131

57 22 30

10 10 11

26 11 9

10 10 10

69 30 26

63 30 45

14 14 10

33 11 10

13 9 10

84 21 34

a

Dyes were applied using the inkjet printer, separated, and captured using the flatbed scanner system.

Figure 2. Digital evaluation of E124 (red dye), E131 (blue dye), and E100 (yellow dye) in only a few seconds by transformation of the plate image into analog curves; track 3 (A) without and (B-D) with employment of different electronic filters to separate colors postchromatographically (B, yellow filter f red/blue dyes; C, red filter f blue dyes; D, blue filter f yellow/red dyes).

Figure 3. (A) Micrographs (left) of a nanostructured ultrathin silica plate (deposition angle 84°, thickness 5 µm, isotropic structure) and (right) of a monolithic silica gel plate: cracks are a result of the cleaving process for micrograph preparation. (B) Chromatogram obtained on a nanostructured plate (plate size 10 cm × 2 cm, bandwidth 3 mm) for separation of E124 (red dye; down) and E131 (blue dye; up) in 40 s isochronic for 22 runs in parallel.

In addition to significantly reducing equipment costs, the system enables fast but precise automated sample application, ultrafast development on the ultrathin sorbent layers, and simple documentation and analysis using a desktop scanner coupled with powerful software tools. This interdisciplinary synergistic approach, combining the newest developments in chromatography with modern print and media technology, can ideally be realized via planar chromatography. This approach, while proven here, is still at its infancy, and there is great potential for further improvements and integration. Instead of developing the plate in a tank, the mobile phase could be printed as a band onto the bottom of the plate by changing the driver settings. An infrared light source could be integrated in the flatbed scanner to rapidly evaporate the few microliters of mobile phase, and an ultraviolet light source (254/366 nm) could be added to the white light source to enable imaging of UV-active or native fluorescent (18) Kennedy, S. R.; Brett, M. J.; Toader, O.; John, S. Nano Lett. 2002, 2, 59– 62. (19) Hawkeye, M. M.; Brett, M. J. J. Appl. Phys. 2006, 100, 044322. (20) Steele, J. J.; Gospodyn, J. P.; Sit, J. C.; Brett, M. J. IEEE Sens. J. 2006, 6, 24–27.

Figure 4. (A) Concept of dual printer/scanner unit combining print and image-capturing technologies with miniaturized planar chromatography; modified inkjet printer for application of analyte solutions as well as the few microliters of mobile phase to ultrathin-layer chromatography plates; use of a desktop scanner with different light bulbs for plate drying (IR light) and documentation of the developed plates by UV 254 and 366 and white light (visible); (B) workflow of the operations.

compounds as shown for TLC/HPTLC.21,22 The whole system could then be integrated into one dual printer/scanner unit (21) Mustoe, S.; McCrossen, S. J. Planar Chromatogr. 2001, 14, 252–255. (22) Halkina, T.; Sherma, J. Acta Chromatogr. 2006, 17, 250–260.

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(Figure 4) combining print and image-capturing technologies with chromatography. After loading the ink wells with appropriate solutions, the entire separation and analysis could be performed in a couple of minutes with a few mouse clicks, without ever stepping away from the computer, on a system that takes up the same space and power as a office peripheral. This shift of the TLC process onto miniaturized plates with a customized nanostructure, operated and analyzed with an inexpensive, simplified equipment setup that produces faster separations at a lower cost with reduced sample and reagent volumes is analogous to the shift of liquid chromatographic and capillary electrophoresis processes onto microfluidic lab-on-a-chip platforms, including the ability to manage dozens of samples in parallel at one go. The ease of analysis, flexibility, affordability, and analytical capacity of this approach could encourage the adoption of ultrathin-layer chromatography as a common analytical technique.

well as the Natural Sciences and Engineering Research Council of Canada, Micralyne Inc., the Alberta Ingenuity Fund, the Alberta Informatics Circle of Research Excellence, and the NRC National Institute for Nanotechnology for providing funding and support for this work. The authors also thank G. Braybrook and D.-A. E. Rollings for SEM imaging.

ACKNOWLEDGMENT The authors thank Merck, Darmstadt, Germany, and CAMAG, Muttenz, Switzerland, for plates and equipment, respectively, as

Received for review December 23, 2009. Accepted January 27, 2010.

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SUPPORTING INFORMATION AVAILABLE Modified thermal inkjet printer and cartridge filling station; table of the structure formulae of food dyes; table of the functional correlations and table of the repeatabilities of dyes on M-UTLC and NS-UTLC plates applied by printing and spraying; and table comparing the selectivity and separation number on M-UTLC and NS-UTLC plates. This material is available free of charge via the Internet at http://pubs.acs.org.

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