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Direct production of microstructured surfaces for planar chromatography using 3D printing Niall P. Macdonald, Sinead A. Currivan, Laura Tedone, and Brett Paull Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04546 • Publication Date (Web): 26 Jan 2017 Downloaded from http://pubs.acs.org on January 27, 2017

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Analytical Chemistry

Direct production of microstructured surfaces for planar chromatography using 3D printing. Niall P. Macdonald1,2, Sinead A. Currivan2, Laura Tedone2, and Brett Paull1,2* 1

ARC Centre of Excellence for Electromaterials Science, University of Tasmania, Sandy Bay, Hobart 7001, Tasmania,

Australia. 2

Australian Centre for Research on Separation Science, School of Physical Sciences, University of Tasmania, Sandy

Bay, Hobart 7001, Tasmania, Australia. *Corresponding author: [email protected] Tel: +61 3 6226 6680 Fax: +61 3 6226 2858 ABSTRACT: Through optimization of the printing process and orientation, a suitably developed surface area has been realized upon a 3D printed polymer substrate, to facilitate chromatographic separations in a planar configuration. Using an Objet Eden 260VS 3D printer, polymer thin layer chromatography platforms were directly fabricated without any additional surface functionalization, and successfully applied to the separation of various dye and protein mixtures. The print material was characterized using gas chromatography coupled to mass spectrometry, and spectroscopic techniques such as infrared and Raman. Preliminary studies included the separation of colored dyes, whereby the separation performance could be visualized optically. Subsequent separations were achieved using fluorescent dyes and fluorescently tagged proteins. The separation of proteins was affected by differences in isoelectric point (pI), and the ion exchange properties of the printed substrate. The simple chromatographic separations are the first achieved using an unmodified 3D printed stationary phase.

Additive manufacturing technology, commonly referred to as 3D printing, allows the direct fabrication of physical objects from digital 3D designs. A range of techniques are currently available, including fused deposition modelling (FDM), stereolithography (SLA), inkjet/polyjet (i3DP), two photon lithography, selective laser sintering, and layered hydrospinning. A recent detailed review of 3D printing technology has been published by Vaezi et al.1. The impact of 3D printing has been significant in the field of biomedical engineering, in particular bio-printing2,3, imaging4, diagnostics, and scaffolds for cells5. Further insight to the impact of 3D printing on biotechnology and chemical sciences can be found in the review by Gross et al.6. While not a new technology, first developed in 19867, there has been a surge of interest in the utility of 3D printing in chemical science research, since the early 2000’s. FDM was first demonstrated as a method for fabricating templates for microfluidic devices with poly(dimethylsiloxane)8, and in 2012 for fabricating chemical reaction-ware9. As the resolution and accessibility of SLA evolved, as has the complexity of devices that have been fabricated, with more recent reports demonstrating direct printing of 3D micromixers10, and devices with integrated valves11. A recent review on the use of 3D printing in the field of microfluidics has been published by Waheed et al.12. In the area of materials science 3D printing is also having considerable impact, with considerable interest in the printing of new multi-material objects and composites13. In the field of analytical chemistry, a number of reviews have been published recently, highlighting the impact of 3D printing6,14, however, application within the field of separation science, and specifically chromatography, is rather limited to-date. Two reports

on the use of 3D printing of actual chromatography columns (not the stationary phase) have been published15,16, together with a recent theoretical study showing the use of i3DP to produce porous media potentially suitable for preparative chromatographic separations17. However, to-date the actual separation of solutes on an unmodified 3D printed substrate has not yet been reported in the literature. Thin layer chromatography (TLC) has traditionally been performed with a sorbent consisting of inorganic (silica, alumina) or organic materials (cellulose, polyamide), which have been fixed against a supporting plate. In classical TLC, mobile phase movement is driven by capillary forces, while the separation is achieved via differential partitioning between the mobile and solid phases18. TLC remains a very common technique in industrial applications, for example in the separations of amino acids, and for in-process synthesis analysis, however, it has some limitations to overcome, such as the flow profile, which can result in lengthy separations with a low efficiency, (e.g. tailing)19. Additional forces, such as applied pressure 19,20 or the influence of an electric field can be applied to reduce separation times, and to improve separation efficiency19, however, this requires specialised equipment and technical expertise. Recent advances in the field include the use of novel materials, approaches, and miniaturisation, as outlined in a number of recent reviews21-23. In the interest of polymeric and customisable stationary phases, the inclusion of porous polymer monoliths are necessary, as they are plate localised, customisable, and cheap to fabricate24,25. The benefit of porous polymer layers has been also demonstrated, using post-polymerisation modification

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reactions26, where the porous polymer plates could be used in pressurised electro-chromatographic modes19,27, or in the 2D TLC separations of proteins and peptides25. 3D printing offers the ability to directly prepare the planar substrate for separations in a relatively short space of time, at relatively low cost, with no further modification necessary. The fabrication process can be easily customised, in order to produce plates of different thicknesses, lengths, and widths. In this work, we report for the first time, a polymeric substrate for planar separations fabricated using a 3D printing approach, wherein no additional surface modifications were required for application. The printed substrate was applied in the separation of model solutes, such as water soluble dyes, fluorescent dyes, and proteins (fluorescently tagged myoglobin (pI 6.8, 7.2) and lysozyme (pI 11.35)). The printed substrate was compared to TLC separations achieved on a traditional cellulose substrate. The simple separations reported are the very first demonstrated separations achieved using a directly printed unmodified substrate. Experimental Materials and methods. Veroclear-RGD810 print material and SUP707™ water-soluble support were purchased from Stratasys Ltd. (Minnesota, USA). NaOH, myoglobin (equine heart), lysozyme (chicken egg), DMSO, and fluorescamine were purchased from Sigma Aldrich (Sydney, Australia). Phenol red and bromothymol blue were purchased from BDH Chemicals (Queensland, Australia) and used as received. Deionised water was provided by a Merck Millipore purification system (Massachusetts, USA). Instrumentation. GC-MS analysis was carried out on a QP2010 (Shimadzu Corp., Kyoto, Japan), using a SLB-5ms 30 m × 0.25 mm × 0.25 µm capillary column (Supelco Inc., Pennsylvania, USA). Data was collected and processed by means of GC-MS solution software (Shimadzu Corp., Kyoto, Japan). The TLC plates were designed using SolidWorks 2014-2015 (Dassault Systèmes SE, France) and were printed using an Objet Eden 260VS professional 3D printer (Stratasys Ltd., Minnesota, USA), using 16 µm layers; resolution 600 x 600 x 1600 DPI (XYZ). FTIR was performed on a Bruker Vertex 70 (Bruker Optics, Victoria, Australia) using a Platinum ATR (pure diamond) cell, and a DLaTGS detector. The scan range was selected to be between 400 and 4, 000 cm1, using 32 scans for the background, and sample analysis. Raman spectroscopy was performed using a Renishaw inVia Raman microscope (Renishaw, Gloucestershire, UK), using a 50 x objective, a laser of 830 nm, and an extended scan type, with a grating of 1, 200 l/mm. The scan range was between 200 and 3, 600 cm-1/Raman shift. Apparent contact angle images were taken using a Canon 60D DSLR (Canon Inc, Tokyo, Japan) with a 70mm F2.8 EX DG Macro lens (Sigma Corp, Kawasaki, Japan). Droplets of 6 µL (DI water) were dispensed with an eVol XR digital analytical syringe (Trajan Scientific and Medical, Melbourne, Australia). SEM images were taken using an Analytical UHR Schottky Emission Scanning Electron Microscope SU-70 (Hitachi, Tokyo, Japan). For sample loading onto the TLC plates, a Hamilton 10 µL glass syringe (Hamilton Company, Nevada, USA) was used, with sample volumes of 0.5 µL for each solute and mixture spots. For characterization of separations, and generation of intensity profiles, ImageJ (National Institutes of Health, Bethesda, Maryland, USA) was used. GC-MS analysis of the printing resin. Helium was used as carrier gas at a constant flow throughout the analysis, set at 2mL/min. Injector temperature was set at 250 °C, and injections were carried out in split mode, with a split ratio of 20. The oven temperature program was set as follows: 50 °C to 330 °C (hold 4 min) at 5

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°C/min. MS transfer line and ion source temperature were set to 250 °C and 220 °C, respectively. Mass spectra were acquired in the mass range 50 to 500 m/z (10 Hz and 5000 µ/s scan speed). Fabrication of 3D printed plates. PolyJet3D printing technique uses a photopolymer (Veroclear) which is jetted using linearly arranged nozzles to form the separation chip. Micro droplets are sprayed onto the printer build surface and polymerized, using an integrated UV light source for initiation. A support material was also present, which was removed following the printing process. The physical structure of the resulting substrate was a layered deposition of 16 µm polymer, with a depth of 17.3 µm ± 2.4 µm, n =12. All TLC plates were fabricated with Veroclear RGD810 build material, and SUP707™ water-soluble support. Plates were soaked in water to dissolve the support material for up to 6 h, before drying at ambient conditions for 24 h, prior to use. TLC chips with 3 different separation plate thicknesses were fabricated, by changing the width in the XY plane yielding, 100 µm, 150 µm, and 200 µm thick plates. Each device measured 64 mm in length, providing an effective separation channel length of 53 mm. A schematic representation of the printed chip is shown in Figure 1. While the channels were not designed as features in the CAD drawing, hence the flat plate surfaces shown in Figure 1 (A), the successive 16 µm layers fabricated in Z-axis formed micro channels. An illustrative representation of these channels is shown in Figure 1 (B). Of particular note is the fact that the channels were formed on both sides of the plates, however, there was no observed fluidic connection between channels on opposite sides of the chip. In addition, samples were only loaded on the side with barriers, regarded as the ‘top’ side of the chip. Further details of the CAD design can be found in the (.stl) file within the Supplementary Information (SI), and technical drawing shown in SI Figure S1.

Figure 1. Design of 3D printed TLC chip (A), illustration of TLC chip showing 4 separation ‘plates’ with designated sample loading slots. Separation channels were formed by controlling the thickness of the chip in the XY-plane, and the PolyJet technology pressing 16 µm layers on-top of one another in the Z-plane. The chip was orientated on the 64 mm long side, parallel to the X-axis, which formed separation channels running the length of the chip. (B) illustrates the chip design looking through the X-axis, which shows the thickness of the chip (200 µm) and the respective lengths of a (11.9 µm ± 2.9 µm), b (17.3 µm ± 2.4 µm), and c (17.3 µm ± 1.7 µm). SD, n = 12 from SEM images. Spectroscopic analysis of resin and printed substrate. Analysis was performed on the liquid precursor resin, as well as the printed and washed substrate. Both the monomeric raw material (as supplied direct from the manufacturer), and the printed material were subjected to IR analysis, by means of a diamond ATR interface. The resulting spectra are shown in SI Figure S2. The liquid


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resin was placed upon an ATR platform, and the spectrum was obtained. Following fabrication, the printed substrate was washed several times in water, and following the removal of support material, was placed upon an ATR platform for analysis. The spectra were obtained in the range of 400 cm-1 to 4, 000 cm-1. Using a laser of 830 nm, Raman spectra were obtained for the monomeric raw material only, due to diffraction of the laser light upon the printed material. The resulting spectrum is shown in SI Figure S3. Operation details for both the IR and Raman analysis can be found above, under “instrumentation”. Fluorescamine derivatisation of biomolecules. A solution of 3 mg/mL fluorescamine was prepared in DMSO, and mixed. Standards of proteins, myoglobin and lysozyme were prepared to a concentration of 0.1 mg/mL. For each biomolecule, a ratio of 3:1 protein to fluorescamine (vol/vol) was prepared. The vials were protected from light, and left to react at room temperature for 15 min. For the separation mixture, 40 µL of myoglobin (0.1 mg/mL) was mixed with 40 µL lysozyme (0.1 mg/mL).

The polarity of the printed substrate was determined using contact angle measurements. Through contact angle measurements with deionised water, an angle of 62.1°± 1.62° (CA left = 59.214, right = 61.406) was observed, indicating a hydrophilic nature to the Veroclear material (see SI Figure S6). Surface roughness was measured to have an Ra of 1.34 µm, which would suggest that the surface roughness should not interfere with the wettability of the polymer36. The fabricated chips and separation plates were also studied under scanning electron microscopy (Figure 2). The printed layers formed the boundary for each separation channel. It was observed that a by-product of the printing process produced some polymer cross-linking, producing a connected network across the interstitial spaces between each printed layer (detailed in Figure 2 (B) and (C)), resulting in pseudo flow-through pores, akin to porous polymer stationary phases, thus indirectly providing greater contact area with potential for solute interaction.

TLC chromatographic separations. TLC chromatographic separations were performed using a polymeric sealable specimen cup. For the separation of water soluble dyes, and fluorescent dyes, a mobile phase of 20 mM NaOH was placed into the specimen cup. A volume of 1.5 mL was required for each separation. For the separation of fluorescently tagged proteins, namely myoglobin (pI 6.8, 7.2) and lysozyme (pI 11.35), a mobile phase of 10 mM NaOH was used, with a mobile phase volume of 1.5 mL. For proteins, sample spots were dried prior to separation, and visualised under an inhouse prepared UV LED array (λ= 395 nm), and photographed. In all cases, photographic images of the separation medium were recorded using a Canon 60D DSLR camera. Digital photographs were processed in two steps. Firstly, the raw photographic image was post-processed using Photoshop CS 6, while in the second step, measurements were recorded from processed images, using ImageJ software. Results and discussion Material characterisation. Veroclear printing resin contains a number of propriety components, as indicated by the manufacturer, such as an acrylic monomer, acrylic oligomer, photo-initiator, acrylic acid ester, and other un-named components. The resulting FTIR spectra for the non-polymerised resin indicated a material of mixed polarity, comprising of amides (N-H and C=O stretches), as well as some alkene groups, esters, and aromatic stretches (see SI Figure S2, SI Figure S3, and Table S1 for details and analysis). The printed substrate also demonstrated these functional groups, and stretches that are characteristic for their polymerised counterparts. The identification of functional groups within the material proved to be rather complex using FTIR and Raman spectroscopy alone, and enhanced structural data was achieved using GC-MS approaches. From the literature28,29, and GC-MS analysis of the resin (SI Figure S4), the following molecules were identified, such as the photo-initiator (Irgacure 184)30, a photo curable diluent (isobornyl acrylate)31, an acrylic monomer (4-acryloylmorpholine), a crosslinker (glycerol propoxylate (1PO/OH) triacrylate)32, a monomer of low shrinkage and high refractive index (tricyclodecane dimethanoldiacrylate)33, and a cyclic ketone, which is used as a precursor in the production of polymers and nylon (cyclohexanone)34,35. The structures of the identified monomers are shown in SI Figure S5. The polymerised substrate is rich in carbonyl groups, esters, amides, aliphatic chain groups, and aromatic groups, resulting in a net negative surface charge.

Figure 2. Scanning electron microscopic (SEM) images of 3D printed TLC chips of 200 µm thickness. (A) Substrate of separation chip showing parallel micro channels (11.9 µm ± 2.9 µm, n = 12) corresponding to the printer head orientation, taken at x300 magni-



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fication. (B) Micro channels formed by 16 µm layers pressed together leaving 17.3 µm ± 2.4 µm, n = 12, deep channels (x1300)). (C) Enhanced magnification (x7000) of printed substrate highlighting varying micro structures found throughout the substrate, here an 8 µm void was formed with a bridge of 1.2 µm. Optimisation of the printed substrate. The dimensions of the printed substrate were investigated in two approaches, firstly the thickness of the layer which generated the separation channels, and secondly the orientation of the separation channels within the printed chip. The direction of flow for the separation is dependent on the direction of printing. Both directions of printing were investigated, with the resulting chip produced by either, printing the chip parallel to the print head (X-axis) or perpendicular to the print head (Z-axis). This produced flow through channels of different orientations, whilst the design of the plate remained the same. In the first instance, layers of varying thickness were printed, from 50 µm to 200 µm. Printed layers < 100 µm thick were not stable, and produced filaments/fibres in lieu of a solid, connected substrate. The 100 µm thick devices provided a stable structure, however, the separation channels were not uniform in distribution, i.e. the width of printed layer, and thus the separation channels, were heterogeneous. Planar chips of 100, 150, and 200 µm thicknesses all exhibited the pseudo crosslinking i.e. bridge like features between the printed layers (SI Figure S7).One of the limiting factors in the use of the thinner devices (i.e. 100 µm thick) was the mechanical breakdown and cracking of the polymer, which was observed as the polymer dried, and shrank. The printing of layers and separation channels in the 200 µm printed substrate demonstrated enhanced linear homogeneity of the channels, relative to the thinner substrates, which would produce a similar flow profile throughout each separation channel. From these points, the optimum thickness was deemed to be 200 µm, which was used for the remaining experiments. The substrate could be used multiple times, once washed and dried, with little to no mechanical failures. Even when a temperature of 37 °C was used to dry the printed substrates, the polymer structure remained intact.On the second approach, the orientation of the final substrate channels was investigated, by printing the device parallel to the print head (X-axis) or perpendicular to the print head (Zaxis). For chips printed in line with the X-axis, the separation was complete in approximately 10-60 min, depending on the separation and application. Conversely, on the chip printed in line with the Zaxis, the solute bands spread not only upwards through the channels, but also along the horizontal channel geometry, contaminating adjacent sample spots. The solute spots did eventually progress through the horizontal channelled chip, however, the time required was in excess of 3 days. Application of planar substrates to chromatographic separations. Separation of phenol red and bromothymol blue. Preliminary experiments were performed using model solutes, such as water soluble dyes, phenol red (PHR) and bromothymol blue (BTB) shown in Figure 3. When ionised, the dyes display red and blue colours, respectively. At neutral pH, both dyes appear yellow. To ensure the ionisation of the solutes, a basic mobile phase was prepared with a 20 mM concentration of NaOH. For a simple comparison, all separations were simultaneously performed on cellulose paper as well as the printed substrate. Sample loading, i.e. spot sizes in TLC are generally from 1- 4 mm in diameter, with 1- 5µL sample volume used, however, in this application, 0.5 µL of sample was applied to both the printed substrate and cellulose paper. For the water soluble dye mixture, a solution of 0.5 mg/mL was prepared in mobile phase, and was subsequently loaded onto the 3D printed chip. Successful separations were obtained in 60 min (Figure 3), where only the BTB was retained by the stationary phase. The separation selectivity observed, suggested an overall negative charge

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on the printed substrate, which was also inferred by material characterisation. An additional benefit of using the 3D printed substrate is that it can be washed and re-used. In this example, the separation was repeated four times upon the single chip. The retardation factor (RF) for the PHR (unretained) sample was 0.90 ± 0.05 (n = 16), while the RF for BTB was approximately 0.34 ± 0.08 (n = 8). The standard deviation values were less than 0.09 in both instances, with a % RSD of 22.5 % for BTB, and 5.47 % for PHR. Whilst these values appear to be significant, they reflect a change of ~0.1 units in R F.

Figure 3. Retention studies of a mixture of dyes, bromothymol blue (BTB), and phenol red (PHR), on the cellulose paper (A), and on 3D printed plate (B). The sample spot was 0.5 µL in each case. Two samples of PHR were also included on both separation media for reference and reproducibility (for 3D print). Mobile phase consisted of 900 µL H2O, 100 µL 0.2 M NaOH. Development time 20


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min (A), 60 min (B). (C) Processed photograph with the background removed showing the separated dyes only. (D) Chromatogram of the 3D printed plate separation from (C). The initial separation indicated that some of the BTB was not retained by the stationary phase, however, in subsequent experiments the dye was retained, repeatedly. This indicated that some support material remained in the polymer interstices until after the first chromatographic separation. Manufacturer’s notes indicated that a wash of 0.2 % NaOH would be useful in the removal of the support material, however, it was discovered that in the preparation of substrates for microfluidics, the base may have a negative effect on the substrate characteristics if washed for extended periods. The presence of dilute NaOH in the mobile phase facilitated the removal of the support material, as evidenced following the first separation where repeatability was improved (see SI Figure S8). For comparative purposes, both separations were also performed on a cellulose substrate of similar length. Band separation was not completed on the cellulose substrate in the case of PHR and BTB (see Figure 3 (A)), when used under the same conditions. The printed substrate has provided enhanced separation ability when compared to cellulose. In addition, solvent movement through the plates of the 3D printed device was found to be at least twice as fast as that observed with the cellulose separation substrate (see SI Figure S9). Separation of fluorescent dyes. The separation of fluorescent dyes was also performed under the same conditions, using another printed separation chip. Rhodamine 6G, rhodamine B, and fluorescein (positive, neutral and negative, respectively, at basic pH), were loaded onto a printed TLC chip. After 20 min of development time in a saturated system, using the same mobile phase conditions, fluorescein had travelled the length of the separation plates, along with the solvent front, i.e. was unretained, as shown in Figure 4. Rhodamine 6G demonstrated an RF value of 0, whilst the individual standards of rhodamine B and fluorescein demonstrated RF values of 0.14 ± 0.03, and 0.81 ± 0.04, respectively. The separation was repeated on three different 3D printed chips. Each standard was included in each chip (2 of 4 plates of the chip), along with two plates dedicated to the separation of the mixtures (mix 1, and mix 2). The RF values show little variation, with the RF for the rhodamine B standard, at 0.07 ± 0.03 (n = 3, in all figures reported here), and with the fluorescein standard at 0.59 ± 0.17. The average values for each solute in the separation of the mixtures were also measured. The RF values for rhodamine B (mix 1) was 0.10 ± 0.01, and for mix 2, 0.10 ± 0.01, demonstrating very little variation in RF between the standard plate, and the mixture plates. The RF values for fluorescein were also measured for each mixture (mix 1, and mix 2); for fluorescein in mix 1 the RF value was 0.65 ± 0.13, and for mix 2, RF of 0.75 ± 0.06. Small variations in the local availability of solvent, and thus force of capillary action, may have caused the variation in RF, as well as minute differences in the printed substrate channels. When compared to the cellulose substrate, it was observed that rhodamine B was highly retained within the 3D printed chip (SI Figure S10). The RF values for rhodamine B were three times higher with the cellulose substrate, at 0.35 ± 0.52 (n = 3), while for fluorescein they were similar to the 3D printed chip, with RF values of 0.68 ± 0.03. The mobility of fluorescein provides further evidence of the surface functionality and charge. Thus, whilst both materials demonstrate similar surface functionality, the 3D printed device demonstrated greater resolution and greater retention of rhodamine B, when compared to the cellulose substrate (SI Figure S10). This may

be attributed to the controlled capillary structures, and the crosslinking observed between the channels, which may enhance the surface area of each channel with the separation plate.

Figure 4. 3D printed TLC chip showing separation of fluorescent dyes, namely rhodamine 6G (Rho 6G), rhodamine B (Rho B), and fluorescein (Flu). (A) Photograph showing the chip with separated dyes indicating their respective charges at basic pH. (B) Processed photograph with the background removed showing the separated dyes only. (C) Generated chromatogram from the processed image shown in (B). Separation conditions as described in Figure 3. Separation of fluorescently tagged proteins. For a routine type analysis, a number of proteins were screened using a variety of mobile phase conditions. The presence of various functional groups within the printed substrate resulted in the need for a detection system other than ninhydrin, which is regularly used for the detection of amino groups in biomolecules, post-separation. Ultimately, two proteins were fluorescently tagged with fluorescamine, namely myoglobin (pI 6.8, 7.2) and lysozyme (pI 11.35).



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produce, the plates can be washed and dried, and used towards additional experiments.

ASSOCIATED CONTENT Supporting Information The Supporting Information includes the SolidWorks drawing used in the chip design, characterization of the material (GCMS) and the resulting structures found, a brief description of the spectroscopic methods of analysis (IR, Raman), contact angle measurements, the effect of plate thickness upon the polymer structure by SEM, repeatability of the separation using a single chip multiple times, and a plot of the liquid front versus development time in both cellulose and the printed chip, with similar dimensions. The Supporting Information is available free of charge on the ACS Publications website. Supplementary Information (SI) (PDF format) AUTHOR INFORMATION

Figure 5. 3D printed TLC chip showing the separation of fluorescamine tagged proteins, myoglobin, and lysozyme. (A) Processed photograph, with the background removed showing the fluorescent proteins. (B) Generated chromatogram from the processed image shown in (A). Separation conditions; mobile phase 10 mMNaOH, 1.5 mL mobile phase. Protein concentration 1 mg/mLin phosphate buffered saline. Sample spot, 1 µL. Detection performed on a dry chip using an in-house prepared UV LED array (λ395 nm). Separation run time 10 min. The design of the printed device resulted in the ability to rapidly screen mobile phases during method development, as both individual standards and mixtures could be run on a single chip, before washing and re-using the substrate, once dried. Using a mobile phase of 10 mM NaOH, the protein samples could be resolved, using a separation time of 10 min, and were visualised under a 395 nm light source (Figure 5). The separation mechanism was driven by the difference in the pI’s of the proteins, under these conditions. Lysozyme was retained upon the plate, with an RF value of 0.03 ± 0.01 (15 % RSD, n = 3), whilst myoglobin demonstrated migration along the plate, with an RF value of 0.71 ± 0.07 (9.1 % RSD), as shown in Figure 5.

Corresponding Author * Prof. Brett Paull, ARC Centre of Excellence for Electromaterials Science, School of Physical Sciences, University of Tasmania, Sandy Bay, Hobart 7001, Tasmania, Australia. Author Contributions All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT Dr. J. M. Cabot at ACES, is thanked for his assistance and knowledge in fluorescent tagging of proteins. Dr.T. Rodemann at the Central Science laboratory, is acknowledged for his help with Raman Spectroscopy. This study is supported by ARC Centre of Excellence for Electromaterials Science (ACES) (Grant CE140100012). Australian Research Council Discovery Grant DP 140104323.

Conclusions. This work demonstrates, for the first time, that a 3D printed substrate for chromatographic applications can be produced in a single-step approach, using already existing 3D printing technology. This work also reports the use of inherent micro-architectures towards separation, with no additional modifications required prior to use. The substrate, albeit planar in nature, provided a resolved separation of model solutes, ranging from visible dyes to fluorescently tagged proteins. The printed plates were manufactured in-house, over a short period of time (approx. 1 h, for 10 chips), at low cost (ca. USD 3.5 per chip), providing reproducible, reusable, solid substrates, suitable for TLC type separations. The substrate outperformed cellulose of equal length, under the same conditions, providing small, portable and robust substrates for solute separation. In addition to the benefits of being inexpensive to


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