Supercritical-Fluid Chromatography On-Chip with Two-Photon-Excited

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Supercritical fluid chromatography on-chip with two-photon excited fluorescence detection for high-speed chiral separations Josef J. Heiland, David Geissler, Sebastian K Piendl, Rico Warias, and Detlev Belder Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00726 • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 15, 2019

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Analytical Chemistry


Supercritical fluid chromatography on-chip with two-photon excited fluorescence detection for high-speed chiral separations

Josef J. Heiland†, David Geissler†, Sebastian K. Piendl†, Rico Warias† and Detlev Belder†,* † Institute of Analytical Chemistry, Leipzig University, Linnéstraße 3, 04103 Leipzig, Germany

* Corresponding Author: Detlev Belder E-mail: [email protected]

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Supercritical fluid chromatography on-chip with two-photon excited fluorescence detection for high-speed chiral separations Josef J. Heiland†, David Geissler†, Sebastian K. Piendl†, Rico Warias† and Detlev Belder†,* † Institute of Analytical Chemistry, Leipzig University, Linnéstraße 3, 04103 Leipzig, Germany ABSTRACT: Herein we present the first example of microchip-based supercritical fluid chromatography (SFC). A microfluidic glass chip platform with pressure and temperature control for fast and efficient on-column injection is described. This enabled fast and efficient separation of chiral and achiral compounds within seconds also employing two-photon excitation fluorescence detection. Peak shapes were highly regular and symmetric even for linear flow rates over the packed microchip column in the range of up to 20 mm·s-1.

The application of lab-on-a-chip technology to realize miniaturized chromatographic separation techniques is an active field of research 1–7. The world of chip-based chromatography is full of promises, such as high-speed separations, reduced consumption of sample or chemical waste production and especially the prospect of enhanced system integration. To date, nearly all chromatographic separation techniques have been realized on chip-devices, a development which took off almost 40 years ago with Terry’s first gas chromatographic air analyzer on a silicon wafer 8. Today, gas chromatography 9–11 as well as liquid chromatography 12–21 including variants such as electrochromatography 22–24 or paper chromatography 25– 27 and thin layer chromatography 28 have found chipbased equivalences. Evidently, chip-chromatography has matured over the years as also documented by several reviews 1–7,29–31 and the availability of various commercial products 32–35. There is however still one important missing piece to this story of success - the realization of super critical fluid chromatography (SFC) on-chip. SFC started as a promising separation technology in the 1970s and was thereafter derogatively coined as ‘science fiction chromatography’ 36. Recently, SFC has seen a come-back as an environmentally friendly separation technique for various applications including chiral analysis 37–41. This renaissance of SFC was initiated by the introduction of reliable commercial SFC instruments which used regular packed high-performance liquid chromatography (HPLC) columns, back pressure regulators (BPR), as well as other proven HPLC-technology 42,43. By applying mixtures of supercritical carbon dioxide with polar solvents, like methanol (MeOH), nearly all

stationary phase materials developed for normal-phase or reversed-phase HPLC can be used in SFC, as well as a wide range of dedicated SFC phase materials both for chiral and achiral analysis 44. This allowed for overcoming the limitations of early capillary SFC by widening the application range to more polar compounds which made SFC also attractive for food 45 or pharmaceutical 46 analysis. Taking into account the nearly contemporaneous maturing of chip-chromatography and the revival of supercritical fluid chromatography, chip-SFC seems like the next logical step. Profiting from the low viscosity and fast mass transfer kinetics of supercritical fluids 47, chip-SFC would be very attractive to realize even faster chip chromatographic analysis 48–50 including chiral separations 51–56. A lab-ona-chip SFC approach with seamless system integration of microfluidic functionalities for injection, separation, and detection could elegantly circumvent current technical hurdles and challenges in SFC such as extra column variances 49,57,58. An important aspect in SFC is the reliable thermosetting of parts of the instrument including eluent delivery, sample injector, transfer lines, the separation column and ideally also the detection area. Due to the small footprint and the corresponding low thermal mass of a respective micro-device combined with the prospect of system integration, chip-SFC promises to have a big impact in advancing miniaturized separation science. Taking into account that chip-based liquid chromatography is established, including recent reports on high-temperature chip-HPLC 59, the realization of chip-SFC appears straightforward by simply using


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supercritical fluids instead of traditional solvents. This is however technically demanding as reflected by the few examples in the literature on using such kind of exotic fluids in microfluidic devices. Most reports focused on fundamental aspects of supercritical fluids for microfluidic application areas such as extraction or droplet microfluidics 60–68 and very recently, also pH studies of aqueous mixtures in contact with dense CO2 in a glass chip 69. The realization of chip-based SFC including the technically demanding task to utilize an integrated microfluidic device with complex functional channel networks at SFC-conditions is however still an unsolved challenge. In this contribution, we aim to address this yet unexplored area in lab-on-a-chip technology. We present a first proof-of-concept study including the development of a packed column chip-SFC setup. The approach was validated with the example of fast chiral separations using on-column one- and two-photon excited fluorescence detection. EXPERIMENTAL PART Chemicals and materials. Solvents like n-heptane, acetonitrile, MeOH, ethanol and 2-propanol (i-PrOH), were of gradient grade and purchased from VWR International (Radnor, PA, USA). Pressurized N45 carbon dioxide (CO2) was obtained from Air Liquide (Paris, FRA). Racemic napropamide (99%) and both enantiomers of 1-(9-anthryl)-2,2,2-trifluoroethanol (98%), 7-amino-4-methyl-coumarin (coumarin-120 - 99%), 2,2-dimethoxy-2phenylacetophenone (99%), 3-(trimethoxysilyl)propyl methacrylate (98%), butyl acrylate (99%), 1,3-butanediol diacrylate (98%) hydrochloric acid (37%), sulfuric acid (95-98%) and acetic acid (99.5%), fluoranthene (98%), benzo(k)fluoranthene (99%), benzo(a)anthracene (99%) were purchased from Sigma-Aldrich (Taufkirchen, GER). High-purity water was obtained from a Smart2Pure purifying system (18.2 MΩ·cm, TKA Wasseraufarbeitungssysteme, Niederelbert, GER). Before use, stock solutions were filtered (0.22 µm polytetrafluoroethylene filter) and stored at 4°C before dilution to desired concentrations. Chiral stationary phase materials (CSP) Chiralpak IB-5 (fully porous, 5 µm particle diameter (dp)) and IC-3 (fully porous, 3 µm dp) were provided by Chiral Technologies Europe (Illkirch, FRA). ProntoSIL 120-3-C18 SH stationary phase material (fully porous, 3 µm dp) was obtained from BISCHOFF Analysentechnik u. -geräte GmbH (Leonberg, GER). General microfluidic parts and tubings were obtained from VICI (Schenkon, SUI) and Upchurch Scientific (IDEX Health & Science, Oak Harbor, WA, USA). Microchip layout and fabrication. The microfluidic glass chips were produced by iX-factory (Micronit, Dortmund, GER) according to our designs using common

wet-etching, fusion-bonding and powder-blasting methods. The chips were made of borosilicate glass (Borofloat-33®) and were diced to dimensions of 45 × 10 mm from a 1.1 mm thick wafer. Two structured glass slides were combined to form a microchip with a thickness of 2.2 mm. The chip layout has been discussed in detail elsewhere 15. Briefly, a 35 mm column compartment (semi-circular cross-section of 90 µm max. width) is connected via an injection cross to powderblasted fluid access cones in the top glass slide. All etched channel structures are present in the bottom slide. The column was packed according to a slurry packing procedure described in the supporting information and elsewhere 70. The five mg·ml-1 (in MeOH) slurry was pressed into the column compartment under ultrasonication. Both ends of the column channel are tapered by a two-step etching process and additionally covered by a small porous polymer frit. A similar photopolymerization process was used to seal the packing channel off by the formation of a defined wall-anchored, fluid-tight polymer plug. Fluorescence detection. An inverted epi-fluorescence microscope (IX70, Olympus, Hamburg, GER) and a 40fold magnification objective (NA=0.6, LUCPlanFLN; Olympus) was used for epi-fluorescence detection. Furthermore, a high-pressure mercury vapor lamp (Mercury Short Arc HBO 103 W/2, OSRAM, Augsburg, GER) as excitation source, a dichroic mirror (380 nm, AHF Analysentechnik, Tübingen, GER) and a band-pass filter (350/50, AHF Analysentechnik) were used. Fluorescence emission was filtered (long-pass filter >390 nm, AHF Analysentechnik) and collected by a photomultiplier tube (Hamamatsu Photonics, Herrsching am Ammersee, GER) at 500 mV. As excitation source for the two-photon absorption process, a frequency-doubled ps-pulsed Nd:YVO4 laser (Cougar, TimeBandwidth Products, Lumentum, Milpitas, CA, USA) at 532 nm wavelength and 20 MHz repetition rate was used. After passing several lenses and optics, the excitation light was guided into a microscope system based on a MicroTime200 platform (PicoQuant, Berlin, GER) filling the back aperture of a 40x LUCPlanFLN objective (Olympus). The focused laser beam excited the analytes right after the chromatographic column, and the fluorescence emission was collected by the same objective. The emission was separated from excitation light by a dichroic 532 nm short pass mirror (AHF Analysentechnik) before passing several guiding optics through a DUG11 emission filter (Schott, Mainz, GER) and hitting a photomultiplier tube (PMA 165-N-M, PicoQuant). For data acquisition SymPhoTime software (PicoQuant) was used, applying a binning of 150 ms. A more detailed description of the optical setup is given elsewhere 71. Microchip operation. Exact temperature control of the eluent and the chip column was achieved using a low



thermal mass contact thermostat 59. The whole setup was placed on top of an inverted microscope to facilitate onchip fluorescence detection as well as imaging of the fluidic processes during method development. For this purpose, a steel housing frame was designed to bring all fluidic components into close proximity at a minimal footprint. Pre-column valving, pressure metering, and back-pressure regulation were located on an adjustable sidearm on a custom metal enclosure close to the thermostat. Precise backpressure control for all fluidic paths was achieved by implementing backpressure regulators and pressure gauges (HPLC inline pressure meter, Duratec Analysentechnik, Hockenheim, GER) pre- and post-column. To adjust the backpressure of each fluid flow path, both pre- and post-column, combinations of fixed (500 and 1000 psi BPR Assembly SST, Upchurch Scientific) and adjustable (BPR2 and BPR3, VICI Jour) BPR in series were used as indicated (Figure S5). Other post-column fluidics encompassed a pressure meter and a shut-off valve. Valve switching was automated using a Chromatography Data Station (Clarity, DataApex, Prague, CZE), which also logged the pressure sensor data. Detailed schematic drawings and photographs of the whole setup including the fluidic circuitry, the arrangement of various pumps and valves are shown in the supporting information. RESULTS AND DISCUSSION For the development of this first chip-SFC system, we built upon our previous work on chip-HPLC using high pressure resistant microfluidic glass chips with incorporated packed columns 19,59,72. The chips contain a 35 mm long column compartment and a microfluidic channel cross functionality which works as sample injector and flow splitter 15. A simplified scheme displaying the fluid flows and the injection process is shown in Figure 1.

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Figure 1| Setup and working principle of the SFC-chip approach. A schematic representation of the fluid flows at the column head injection cross during sample A) injection and B) elution. C) Simplified microfluidic layout of the chip (top view) and the main external fluidic elements like pumps, backpressure regulation (BPR), valving and pressure metering. A detailed setup is given in the supporting information.

The main challenge is the design of a microfluidic circuitry capable of reliably operating with multiple high pressures microstreams of greatly differing compressibility. A general layout of the major components to this setup is outlined in Figure 1C. An indepth schematic of the whole experimental setup is presented in the supporting information. For the delivery of dense CO2-based eluents, we utilized a commercial binary SFC-Pump (Agilent 1260 Infinity SFC, Agilent Technologies, Santa Clara, CA, USA) equipped with MeOH as the modifier. To introduce the liquid sample to the column head, a standard HPLC-pump (G1310B, isocratic Agilent 1260 pump) operated in normal-phase mode with n-heptane and fitted with an outlet check-valve was used. At the injection cross (Figure 1A), two microstreams are intersected to form a small sample zone on the column head by infusion. The sample is propagated by a n-heptane stream, whereas a small CO2/MeOH stream (pinch stream) is used to keep the eluent channel free of sample during this procedure. By switching the microfluidic valves in elution position (Figure 1B), the eluent CO2/MeOH stream is directed towards the chip column. In order to achieve the goal of fast analysis speeds, the injection process was performed in an automated way with software control. An optimized protocol leads to 9 s total injection time for sample transfer and infusion. This standard injection method was developed using fluorescence imaging at the injection cross with dye samples (coumarin-120) for parameter optimization. For this efficient injection method 73, it is necessary to use two CO2/MeOH streams (pinch and eluent). On the other hand, when working with multiple compressible microstreams and rapidly changing fluid paths, the setup has to be designed in a way, that switching valves has minimal impact on the fluid dynamics in the system. For this purpose, the SFC-pump was modified to yield two fluid streams. This was achieved by a custom modification to the SFC-mass spectrometry (MS) connection kit (G4309-68715, Agilent Tech.) adding restriction capillaries and check valves as schematically shown in Figure S7. With this altered setup, it was possible to extract two defined microstreams at a set composition and pressure delivered by a single common SFC-pump, designed for much higher flowrates. This was achieved by splitting the main flow (0.5 to 1 ml·min-1, 150 bar outlet pressure, 0-30% modifier content) coming from the pump head twice in a row to yield the eluent microstream (after the first split) and the auxiliary pinch microstream (secondly, after the check valve). The split


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ratio of main and side stream was roughly adjusted to be 2:1 by restriction tubing lengths. A check valve was inserted into the fluid path of the auxiliary flow to minimize feedback to the main eluent stream during valve switching processes. Furthermore, the setup was designed as such, that all microstreams are pressurized independently of the valve positions, to minimize wear on the column and ensure fast response times. The process was monitored by pressure meters before and after the column. After method development, the following procedure was established to realize first chip-SFC measurements. Prior to operating in SFC-mode, the whole system including the microfluidic device was purged with MeOH. Thereafter, it was brought to and above SFC working pressure to ensure fluid tightness and two fluid streams of equal composition were taken from the splitter at 150 bar working pressure. Also, the previously developed chip-to-tube interfaces using special clamps, proved to be compatible with the SFC-conditions, assuring stable and leakage-free chip-to-world interfacing with negligible band broadening 72. The critical phase parameters of CO2-streams are highly dependent on the added modifier content 74 and thus, today most SFC separations in practice are actually carried out at subcritical conditions despite the name. However, exact temperature control of the separation column is essential for reliable operation. Microfluidic systems benefit from small thermal masses and in turn rapid thermal equilibration. Based on our recent work on thermal-gradient chip-HPLC 59, we applied a custom microcolumn thermostat to encompass the whole chip column (see red zone Figure 1C). This infinite heat reservoir counters possible decompression cooling effects 75 like peak distortion or clogging due to ice formation. Similar measures were adopted for designing the remaining microfluidic circuitry by using stainless-steel tubing and parts were necessary for rapid heat exchange. Critical components, like the main BPR of the microfluidic system, were immersed as a whole in a fluid bath (i-PrOH) for thermal equilibration. Furthermore, a mobile phase temperature conditioner (Caloratherm, Selerity Tech., Salt Lake City, UT, USA) set to 50°C was used as an infinite heat reservoir at the common outlet of all microstreams to prevent ice formation. A photograph of the realized laboratory set-up is shown in the supporting information (Figure S1). In order to facilitate method development, we expanded the setup to utilize the same chromatographic system in SFC as well as in HPLC mode by the introduction of a manual switching valve. This allowed to regularly purge the system with conventional solvents to test the principal functionality of the whole fluidic and chromatographic setup. After realization of the setup and successful method development using fluorescent dyes, we applied the approach for the analysis of enantiomers as one of the

main SFC applications. For this purpose, we utilized a chip which we packed with commercially available, enantioselective Chiralpak IB-5 material as stationary phase. The chip separation temperature was set to 30°C with the microcolumn thermostat, in accordance with literate reports to gain optimal resolution in chiral SFC 76. This temperature range can be regarded as a compromise between achieving close to critical parameters and for optimal chiral recognition at typically lower temperatures. Pirkle’s alcohol (2,2,2-trifluoro-1-(9-anthryl)ethanol) was chosen as a test analyte in a 1:2 enantiomeric ratio 77. A representative high-speed separation is displayed in Figure 2. As standard method development parameters, 20 vol% MeOH was added at an inlet pressure of 150 bar and a column outlet pressure of 100 bar. The resulting conditions allowed for continuous operation of the system at linear velocities approaching 20 mm·s-1 without any apparent leakages or column defects. Both enantiomers were baseline separated using Chiralpak IB-5 material in 12 s. For detection purposes, we used an acquisition rate of 25 Hz corresponding to about 50 or more data points per peak at the expected peak widths (1-2 s) to prevent undersampling.

Figure 2| High-speed enantioseparations with chip-SFC at u ~ 20 mm·s-1. Overlay of three sequential injections (red, blue and black trace) of a 1:2 enantiomeric ratio sample. The resulting peak area ratio was 0.50 (R) to 1.00 (S) with plate numbers (N·m-1) of about 9500 at a reduced plate height (h) of about 20 and the retention times (6.170.10) s and (9.840.10) s, respectively. Parameter: chip separation, 35 mm column, fully-porous 5 µm dp material, Chiralpak IB-5, u: 19.31 mm·s-1, eluent: CO2/MeOH (80/20 vol%) at an inlet pressure of 150 bar, column outlet pressure: 100 bar, T = 30°C, detection epi-fluorescence, ex: (35025) nm, em: >380 nm, 40x objective, 25 Hz acquisition rate, sample: 33.0 µg·ml-1 (R)-Pirkle’s alcohol and 66.0 µg·ml-1 (S)Pirkle’s alcohol in n-heptane. t0 marks the column dead time.

The set of experiments revealed a high reproducibility as well as excellent peak shapes obtained from high-speed separations, as demonstrated by the exemplary overlay of three sequential injections. For the peaks displayed in



Figure 2 values for peak asymmetry near the theoretical optimum (total asymmetry = 1.15 to 1.18 compared to ideal Gaussian peaks at 1.00, at 10% peak height 78) are derived. The highly regular shape of the eluting peaks is a measure for the efficiency of the sample injection and the overall separation bed quality. Even at maximum column permeability (dp 5 μm), the sample zone formed on the column head is efficiently eluted over the particulate bed without distortion despite very high linear velocities (u) of 19.3 mm·s-1. An important aspect of working with super- and subcritical fluids is the control of pressure and thus fluid density throughout the system. To this end, various back pressure regulators and pressure meters were included in the fluidic circuitry of the chip periphery. A key parameter is the pressure drop over the column, as indicated by the simplified schematic in Figure 1 and displayed in more detail in Figure S7. Especially for highspeed separations with only several seconds of total runtime, heterogeneities in the pressure difference may lead to peak distortions and should be kept minimal.

Figure 3| Visualization of the differential pressure drop over the column during a chip-SFC separation. The sharp increase in the first run-time second is due a re-equilibration event caused by valve switching for injection. The column is kept under pressure at all times, only the pressure meter is switched into the main flow. The slight increase in differential pressure around 5 s is related to an n-heptane plug used as a sample carrier fluid, passing through restriction capillaries (Figure S5u) after the chip flow splitter. Parameters: see Figure 2.

Figure 3 exemplarily shows the pressure drop over the column during such a high-speed separation. The pressure difference is calculated from two sensors close to the column inlet and outlet within the microfluidic circuitry. The initial sharp increase in pressure difference within the first second is based on the valve switching process during injection. The extent of the pressure drop, however, is most likely far less distinct, since the column is always kept under constant pressure, but the sensor is switched into the stream (see pressure sensor before BPR1 in Figure S5). Other than that, there is a slight pressure peak of about 0.3 bar around 5 s runtime. This is due to the more viscous n-heptane rich fluid passing

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through restriction capillaries, but the small dimensions and high linear flow rates enable the system to rapidly reach a steady pressure state after only about 8 s. With the current setup, maximum inlet pressures of up to 160 bar were applied for separations. The microfluidic chips themselves are capable of continuously withstanding 250 – 300 bar, as used for the slurry packing procedure. The outlet pressure was modulated between 100 and 130 bar to achieve the desired linear velocities between 5 and 20 mm·s-1 over the 35 mm long chip columns. For future investigations with longer chip columns and sub2 µm/core-shell stationary phase material bigger pressure drops will be applied, but even sub-2 µm particles are not expected to create a necessary pressure drop over the chip column in excess of 250 bar. 36

Figure 4| Achiral separations of three PAHs at different modifier contents. Mean reduced plate heights were between 16 (20 vol% modifier) and 23 (pure CO2). Parameter: chip separation, 35 mm column, fully-porous 3 µm dp material, ProntoSIL 120-3C18 SH, u: 10.7 mm·s-1, eluent: CO2/MeOH with the corresponding modifier content (20, 5, 0 vol%) at an inlet pressure of 160 bar, column outlet pressure: 120 bar, T = 30°C, detection: see Figure 2, sample: 10.0 µg·ml-1 fluoranthene (F, k = 3.1 for 20 vol% MeOH), 10.0 µg·ml-1 benzo(a)anthracence (BaA, k = 4.5 for 20 vol% MeOH) and 1.0 µg·ml-1 benzo(k)fluoranthene (BkF, k = 7.4 for 20 vol% MeOH) in MeOH/H2O (80/20 vol%).

An interesting application aspect of SFC is its orthogonality with HPLC. Besides specialized SFC phase material, the two techniques may be performed mostly on the same columns and instrumentation, as long as


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compression compatibility related detector noise is addressed. This technology convergence is also reflected by major instrument manufactures providing platforms capable of operating in HPLC as well as in SFC-mode (unified or convergence chromatography 79,80). While SFC requires a more complex instrumental base accompanied with higher entry cost, it is not as much limited by maximum column pressure drop as current (U)HPLC technology in the context of sub-2 µm stationary phase material. Furthermore, SFC offers other benefits like mild on-line extraction or alternative detection techniques like electron impact ionization mass spectrometry or flame ionization (FID) when utilizing pure CO2 eluents. To study the properties of chip-SFC in more detail a set of experiments was performed at varying experimental conditions. For this purpose, a series of polycyclic aromatic hydrocarbons (PAHs) separations were performed on chips packed with standard 3 µm dp C18 stationary phase material 81. In these experiments, we varied the amount of methanol contents in the CO2 mobile phase. The obtained chromatograms using 100 vol% CO2, 95 vol% CO2 and 80 vol% CO2 with the respective modifier content are compared in Figure 4. The best separation was obtained with 20 vol% modifier content. But even with pure CO2 eluents, a baseline separation within roughly 1.5 min was possible. Taking into account that we used suboptimal pre-existing SFC and HPLC peripherals for this proof-of-concept study, it is understandable that the achieved reduced plate heights (h) around 15-25 are far from optimal 49. This rather moderate efficiency can be explained by the very small overall column volume (about 100 nL), and the comparatively large injection volume. This is evident from the peak shapes of fast eluting compounds which show fronting distortions, as seen in Figure 4. A possible way to address this issue would be a `solvent-less` injection in form of an in-line solid phase extraction 36.

Figure 5| Two-photon excitation fluorescence detection for chip-SFC. Plate numbers (N·m-1) were about 20000 at h  15. Parameter: chip separation, 35 mm column, fully-porous 3 µm dp material, Chiralpak IC-3, u: 10.5 mm·s-1, eluent: CO2/MeOH (85/15 vol%) at an inlet pressure of 150 bar, column outlet pressure: 100 bar, T = 25°C, detection: epi-fluorescence, ex: 532 nm Nd:YVO4 laser, em: (34035) nm, 40x objective, 25 Hz acquisition rate, sample: 20.0 µg·ml-1 rac-napropamide in nheptane.

In this first prove-of-concept in chip-SFC, we applied fluorescence detection at 35025 nm excitation wavelength. Deep-UV fluorescence detection at wavelengths below 300 nm would show a much broader range of application 82,83. To enable label-free detection of aromatic compounds in economical, non-deep-UV transparent materials, like our borosilicate chips, twophoton excitation (TPE) is an interesting alternative 71,84. For this purpose, the chiral herbicide napropamide was chosen as an analyte for chip-SFC with label-free TPE fluorescence detection. Being from the group of acylanilides, it is used for the control of pre-emergent weeds in produce fields 85. It is sold as a racemic formulation, but it has been shown that the (–)-isomer of napropamide is more toxic towards biological factors, like root growth and fresh weight 86. Employing a different chiral selector (Chiralpak IC-3, 3 µm dp), a fast enantiomer separation in less than 20 s of a racemic herbicide standard is displayed in Figure 5. Interestingly, we also observed a clear distortion of the baseline (t0), which can be used as a marker for the system dead time and is normally not the case for epifluorescence detection and removes the need for fluorescent compounds without retention. Blank injections of pure n-heptane have confirmed these findings. The same is true for the standard fluorescence detection in Figure 2, but in this case by a short decrease in the signal intensity. Most likely, the distortions are based on scattering effects due to fast refractive index changes by a passing fluid zone. CONCLUSION In this contribution, we present the first microchipbased SFC. To this end, we have developed a temperature and pressure controlled microfluidic setup for high efficiency, direct on-column sample injection. The setup produced highly regular and symmetric peak shapes even for fast linear flow rates over the packed microfluidic column of about 20 mm·s-1. Employing different optical detection techniques, the setup was tested for fast chiral separations on different commercial CSPs, yielding separation speeds below 20 s. By employing smaller diameter CSPs even higher efficiencies are envisioned. These results show the principal feasibility of performing SFC on glass microchips using a standard SFC-pump and HPLC peripherals. Based on these proof-of-concept results, further research will focus on aspects of



peripheral miniaturization, in analogy to literature reports towards hand-portable chromatography instrumentation 87,88. Furthermore, a miniaturized, split-less eluent delivery system as just recently shown by Andersson et al. 89 would be a promising next step in system maturation. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Detailed instrumentation and microfluidic setup, chip column fabrication, general procedures, effects of column pressure drop and column temperature on retention times.

AUTHOR INFORMATION Corresponding Author * E-Mail: [email protected]; Phone: +49 341 97 36091; Fax: +49 341 97 36115

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors acknowledge Chiral Technologies Europe (especially Dr. Pilar Franco) for providing chiral stationary phase material.

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