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Temperature gradient elution and superheated eluents in chip-HPLC Josef J. Heiland, Carsten Lotter, Volkmar Stein, Laura Mauritz, and Detlev Belder Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00142 • Publication Date (Web): 27 Feb 2017 Downloaded from http://pubs.acs.org on March 1, 2017

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Analytical Chemistry


Temperature gradient elution and superheated eluents in chip-HPLC

Josef J. Heilanda, Carsten Lottera, Volkmar Steinb, Laura Mauritza and Detlev Beldera,*

a

Institute of Analytical Chemistry, University of Leipzig, Linnéstraße 3, D-04103 Leipzig, Germany b Fraunhofer ICT-IMM, Carl-Zeiss-Str. 18-20, D-55129 Mainz, Germany * Corresponding Author: Detlev Belder Institute of Analytical Chemistry Department of Chemistry and Mineralogy University of Leipzig Linnéstraße 3 D-04103 Leipzig Germany Tel.: +49 (0) 341 / 97 36-091, Fax: +49 (0) 341 / 97 36-115, E-mail: [email protected]

Author List: Josef Johann Heiland, Carsten Lotter, Volkmar Stein, Laura Mauritz, Detlev Belder

ACS Paragon Plus Environment


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Temperature gradient elution and superheated eluents in chip-HPLC Josef J. Heilanda, Carsten Lottera, Volkmar Steinb, Laura Mauritza and Detlev Beldera,* a b

Institute of Analytical Chemistry, University of Leipzig, Linnéstraße 3, 04103 Leipzig, Germany Fraunhofer ICT-IMM, Carl-Zeiss-Str. 18-20, 55129 Mainz, Germany

ABSTRACT: Utilizing temperature as an active parame-

ter for optimization in chip-based liquid chromatography is an important step towards high-speed and highefficiency separations on the microscale. A device including a low thermal mass micro thermostat and a microfluidic glass chip as central elements were designed and evaluated for maximal heating performance of up to 4.7°C·s-1 at up to 200°C. With this enabling technology, high-speed separations in temperature gradient mode were performed both in common reversed-phase eluents and environmental friendly ethanol-based alternatives.

Working at elevated separation temperatures is an attractive way to increase the kinetic performance of chromatographic systems.1 The benefits of hightemperature high-performance liquid chromatography (HT-HPLC) have been reviewed extensively in the past.2–6 Column temperature affects both mass transfer kinetics as well as solvent elution strength.6,7 At increased temperatures, the eluent viscosity decreases3,5 allowing for operation at higher linear flow rates without compromising separation efficiency,3,8,9 as shown by the so-called “flattening-out” of the van Deemter curve.10 In conjunction with the advent of high-temperature stable phase material,8,11 elevated temperatures enable to use higher linear flow rates or smaller particle diameters for a given maximum system pressure limit,12–14 leading to high-speed separations.1 In an effort to attain maximum efficiency at highest analysis speeds, ever so smaller stationary phase material is utilized. This comes at the expense of system backpressure and detrimental frictional heating.15 Whilst the former aspect is partially overcome by applying shorter columns or by engineering more potent pumping systems (currently standing at a commercial limit of about 1500 bar), the latter aspect might call for a change of paradigm. One viable solution to minimize strong extracolumn band-broadening effects, especially when working with column IDs smaller than 1 mm, may be the development towards integrated, powerful analytical platforms,16 since also for state-of-the-art (U)HPLC

instrumentation can struggle with capillary column dimensions.17,18 With conventional column diameters, friction forces originating from hydraulic resistance19,20 heat the column. This leads to the formation of radial and axial temperature gradients causing peak deformation during separations. Thus, efforts to insulate the column to work in steady-state temperature regimes have to be undertaken.21–23 Due to low thermal masses,24,25 superior temperature control and very fast response times, µm-sized columns enable for highly efficient separations with heated eluents. Furthermore, thermal mismatch conditions26 due to insufficient eluent preheating17,27 can be neglected in microchip and capillary column dimensions. Accordingly, microfluidics28 is very attractive to fully explore the potential of temperature-controlled liquid chromatography.29,30 Chip-HPLC has the unique advantage of seamless integration of different functionalities on a single device31– 35 In previous work, we have developed a chip-based HPLC device based on glass chips and commercial particulate phase materials.36–38 Moreover, by using a direct on-column injection strategy39 and high-pressure stable interface technology,40 we recently could successfully establish a multi-dimensional HPLC system41 on-chip as well as the seamless integration of a continuous-flow (CF) micro reactor and HPLC with mass spectrometric (MS) detection on a single chip device.42 Due to the abovementioned merits, column thermostatting in capillary43 and chip-HPLC44 has already attracted some scientific attention. Shih et al.45,46 reported on a LC chip-device based on Parylene working at temperatures up to 100°C, with heating rates of 3.6°C·min-1. The achieved separation performance of this medium pressure (about 40 bar) device was, however, rather moderate (600 plates on a 8 mm separation column). The aim of the current work was to explore the true potential of high-temperature chip-HLPC building up on our previous work on realizing high-pressure and highperformance HPLC in integrated glass chips. Accordingly, the current contribution focusses on the development


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and evaluation of a column thermostatting approach enabling high-temperature liquid chromatography in superheated eluents at working pressures of up to 350 bar. EXPERIMENTAL PART Chemicals and materials. Gradient grade solvents acetone, acetonitrile, chloroform, ethanol, n-heptane, nhexane, methanol, 2-propanol were purchased from VWR International (Radnor, USA). Formic acid (LCMS grade, 98%), 7-amino-4-methyl-coumarin (99%), anthracene (97%), fluoranthene (98%), benzo[k]fluoranthene (99%), benzo[a]anthracene (99%), benzathrone (90%), 3-aminofluoranthene (90%), 3(trimethoxysilyl)propyl methacrylate (98%), 2,2dimethoxy-2-phenylacetophenone (99%), trichloro(1H,1H,2H,2H-perfluorooctyl)silane (97%), 1,3butanediol diacrylate (98%), butyl acrylate (99%) were purchased from Sigma-Aldrich (Taufkirchen, Germany). A Smart2Pure purifying system was used to obtain highpurity water (18.2 MΩ·cm, TKA Wasseraufarbeitungsysteme, Niederelbert, Germany). High-temperature stable XBridge C18 BEH 3.5 µm and 2.5 µm (Waters Corp., Milford, USA) phase material was used for column generation.11,47 Filtered (0.22 µm polytetrafluoroethylene filter) stock solutions were stored at 4°C and diluted to desired concentrations prior to use. Microchip layout and fabrication. The microfluidic HPLC chips were produced according to our designs and specifications by iX-factory (Dortmund, Germany) utilizing common photolithographic, wet-etching, powderblasting and fusing-bonding methods. The finished chips (made of BOROFLOAT®33) were sliced to dimensions of 45 mm × 10 mm at a thickness of 2.2 mm (see figure 1 top right). The bottom glass plate includes the etched fluidic channels comprising of a 35 mm long column segment (semicircular cross-section at a width of 90 µm and a depth of 40 µm), a channel cross used for injection purposes and featuring as an on-chip flow splitter, a packing channel intersecting the column compartment half-way through and a post-column T-junction. This adds a make-up flow functionality (not used in this work) to facilitate chip-MS coupling in future studies. Furthermore, powder-blasted fluidic connection ports were integrated into the top plate. The basic functionality and the column manufacturing process were described in detail previously.38,48 Briefly, a modified slurry packing procedure was applied for column generation via the packing channel. The slurry (5 mg·ml-1) was prepared in 30/70 v/v acetone/n-hexane to minimize particle agglomeration.49 The particles are retained by photopolymerized porous polymer monoliths generated over the particle retaining elements at both sides of the column chamber. The packed column was sealed dead-

volume free by a defined polymer plug generated via laser-assisted photo-polymerization.38 Microcolumn thermostat. The microcolumn thermostat was designed for rapid temperature gradient performance at minimal heat capacity. To this end, two cylindircal high-performance microheating elements (20 W / 24 V, diameter 2.8 mm, length 40 mm, no. 305541, Türk+Hillinger, Tuttlingen, Germany) pressed into cubic aluminum heat dissipating structures were symmetrically built to enclose the column compartment of the chip in a sandwich-like heating structure from both sides (see figure 2 insert). Elastic heat-conductive foil (3 W·mK-1, 0.5 mm thickness, no. SB-V0-3, DETAKTA, Norderstedt, Germany) acted as heat mediator on both sides of the chip. Each heating element was regulated individually by two Pt100 temperature sensors (no. S651PDY24A, Telemeter Electronic, Donauwörth, Germany) placed between heat conductive foil and the aluminum heat spreader in a specially fit grooving. A third Pt100 sensor recorded the ambient temperature in the proximity of the column thermostat. The body of the column thermostat was built from polyether ether ketone (PEEK) designed for minimal heat capacity (max. continuous working temperature: 260°C). An optional axial 40 mm compact fan (no. 414 F, ebm-papst, St. Georgen, Germany) was included to increase cooling rates. A custom-built LabVIEW software and control in conjunction with a 24 V DC lab power supply (no. FSP 2410, Voltcraft, Conrad Electronic, Hirschau, Germany) was used for heater operation. For chromatographic operation in isothermal mode, the chip column (made of hightemperature stable phase material) was thermally equilibrated for 120 s for each temperature before the runs. Fluorescence detection. Epi-fluorescence detection and imaging was performed with an inverted epifluorescence microscope (IX70, Olympus Deutschland, Hamburg, Germany) and a 40-fold magnification objective (NA=0.6, LUCPlanFLN; Olympus Deutschland). The setup further included a high-pressure mercury vapor lamp (Mercury Short Arc HBO 103 W/2, OSRAM, Augsburg, Germany) as excitation source, a dichroic mirror (380 nm, AHF Analysentechnik) and a band-pass filter (350/50, AHF Analysentechnik, Tübingen Germany). Filtered fluorescent light (long-pass filter >390 nm, AHF Analysentechnik) was collected by a photomultiplier tube (Hamamatsu Photonics Deutschland, Herrsching am Ammersee, Germany) set to 500 mV control voltage. The setup was controlled and data was evaluated using Clarity (DataApex, Prague, Czech Republic). The reproducible positioning of the chip was achieved by two integrated CCD bullet cameras (KPC-VBN190PHB HRes, KJL-Sicherheitssysteme, Hamburg, Germany) in combination with two multi-axis translation stages (T12XYZ/M and PY005/M, Thorlabs, Munich, Germany).



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Microchip operation. The operating principle of the chromatography system implies two different fluid flow situations (injection and elution). The basic principle has been described in detail elsewhere.40 Compared to the systems we used in previous works, a key modification required for high-temperature chip-chromatography in superheated eluents is the addition of two back-pressure regulators (BPR, 500 psi no. P-765, Upchurch Scientific, IDEX Health & Science, Oak Harbor, USA) both on pre- and post-column side (see figure 1). Also two pressure meters (HPLC inline pressure meter, Duratec Analysentechnik, Hockenheim, Germany) were included to monitor the pressure drop over the column and to prevent phase transitions on the column. The microfluidic circuitry is operated in an inter-coordinated valve switching pattern with three HPLC pumps to create a small sample plug on the column inlet at the injection cross in injection mode, whilst keeping the channel for the elution fluid flow free of sample. After sample transfer, the system is switched to elution mode, where only the elution pump fluid flow is directed to the column head. The injection cross acts as an integrated flow splitter facilitating the use of cost-efficient standard HPLCpump equipment.

Figure 1. Microchip layout and microfluidic circuitry with highlighted main functionalities pumping (mid right), valving (mid), pre-column (lower right) and post-column backpressure stabilization (left). The chip layout is described in a schematic drawing (upper left) and photograph (upper right). All microfluidic connections were realized with PEEK 360 µm outer diameter (OD) capillary tubings unless otherwise noted.

Further components were a flow sensor (Liqui-Flow mini, series LM-02, Bronkhorst High-Tech, Ruurlo, Netherlands) as well as a 10-port and a 6-port valve (CHEMINERT C72MPKH-4670D and C72MPKH4676D, 100 µm bore, VICI, Schenkon, Switzerland). The microchip was interfaced with other fluidic parts via home-built steel-clamps40 in connection with PEEK capillary tubing (inner diameter between 50 µm and 125 µm, VICI, Schenkon). The detailed tubing dimen-

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sions, as depicted in figure 1, were: a) 90 cm (length) – 100 µm (inner diameter - ID) 1/16 inch (outer diameter OD) PEEK, b) 70 cm – 75 µm 1/16 inch PEEK, c) 70 cm – 100 µm 1/16 inch PEEK, d) 10 cm – 75 µm, e) 15 cm – 75 µm, f) 20 cm – 75 µm, g) 10 cm – 50 µm, h) 15 cm – 50 µm, i) 20 cm – 50 µm, j) 30 cm – 50 µm, k) 50 cm – 50 µm, l) 75 cm – 50 µm, m) 10 cm – 100 µm, n) 10 cm – 125 µm and 10 cm – 120 µm 1/32 inch SST, o) 40 cm – 125 µm loop volume: 4.9 µl, p) 70 cm – 170 µm 1/32 inch SST. Microfluidic connections d) to n) were realized with PEEK 360 µm OD capillary tubing unless otherwise noted. RESULTS AND DISCUSSION To explore the possibilities of high-temperature liquid chromatography for chip-based separation technology, we have developed a micro column thermostat to provide high heating and cooling rates. The thermostat is mainly constructed from PEEK. The microfluidic chip is encompassed by two independent thermostatting units with direct thermal contact to the glass surface. The design is capable of thermostatting at a maximum heating rate of 4.7 C·s-1 from 30°C to 200°C. Heating rates were reported in the range of several °C·min-1 for conventional columns12,50,51 or up to few centigrade per second for capillary columns.43 The highest heating rates of up to 30°C·s-1 have be obtained by resistive capillary heating.52–55 While such resistive heating concepts would also be very attractive to increase the heating rates for chip devices, we utilized an external heating element for this proof-of-concept study. Therefore, the chip-integrated 35 mm long HPLC column compartment is in contact within 1.1 mm on both sides from the heaters ensuring direct heat transfer (see figure 2 insert).

Figure 2. Photograph of the micro column thermostat (left) with an installed microfluidic chip (A), PEEK housing (B), cooling fan (C) and reference temperature sensor (D). 1€coin for size comparison. Schematic drawing of the 45 mm × 10 mm × 2.2 mm microfluidic chip with visualized contact-heated area (top right). The 35 mm long HPLC column is completely covered. Crosssectional drawing of thermostats’ internals (bottom right) with column channel (a), elastic heat conductive foil (b), temperature sensor (c) and heating element (e) pressed into an aluminium heat spreader (d).


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The two heating circuits are controlled separately by LabVIEW®. The thermostatting performance was determined to be below 1% RSD (30.3±0.3°C and 80.1±0.9°C each for n = 1000 measurement points for set temperatures of 30°C and 80°C – see figure S1). The design allows for a quick, easy and reproducible exchange of chip devices due to internal positioning pins and a sledge mounted design. The top heating element is incorporated in a mobile pilot carriage. With a set screw (top PEEK screw in figure 2) a defined amount of torque is applied to lock the chip into position. To investigate high-temperature conditions in microfluidic chromatography a series of isocratic separations of seven PAHs at different temperatures was conducted. As evident from figure 3, the separation can be significantly accelerated by raising the temperature. The total runtime can be reduced from over 90 s to below 30 s by increasing the separation temperature from 30°C to 80°C, without significant loss in separation efficiency (of N > 115.000·m-1 for the last eluting peak). High linear velocities (over 7 mm·s-1) were explicitly chosen in this study to show that detrimental band-broadening effects due to thermal mismatch conditions,29 resulting from radial temperature gradients and poor eluent preheating, can be neglected.24,25

Figure 3. Isothermal separations (from 30°C to 80°C, ∆t = 10°C or ∆t = 1°C for the insert) at column flow rates between 750 nl·min-1 and 1250 nl·min-1 and a constant primary flow rate of 700 µl·min-1 (65/35% v/v acetonitrile/water). The PAH sample solution (in order of elution) contained 0.5 µg·ml-1 7-amino-4-methylcoumarin (1), 8.0 µg·ml-1 3-aminofluoranthene (2), 70.0 µg·ml-1 benzanthrone (3), 20.0 µg·ml-1 anthracene (4), 15.0 µg·ml-1 fluoranthene (5), 50.0 µg·ml-1 benz[a]anthracene (6) and 15.0 µg·ml-1 benzo[k]fluor-anthene (7) in 40/60% v/v acetonitrile/water. Plate numbers were calculated for the last eluting peak.

graphic runs, which resulted in significant retention time shifts as shown for compound 6. A detailed presentation of this set of chromatograms can be found in the supporting information (see figure S2). During experimental work, no stationary phase degradation was observed, which is in accordance with the findings of Teutenberg et al., who investigated the excellent suitability of the phase material for hightemperature HPLC comprehensively.47 We could even expand the working temperature up to 200°C, to the range of so-called superheated eluents, by adding two backpressure regulators (500 psi) as shown in figure 1 to prevent boiling of the liquid phase.52 At elevated temperatures, the elution strength of water (for reversed-phase mode) increases, which allows for reducing the amount of organic solvents.53 Chromatography in superheated aqueous eluent systems56,57 is, accordingly, a hot topic in the context of socalled green analytical chemistry.58,59 With the further developed system, we were able to perform separations in 99.5/0.5% v/v water/acetonitrile at 180°C as shown in figure S3. However, as fluorescence detection turned out to be challenging due to temperature induced quenching and difficulties to properly dissolve the PAH-analytes, the approach was not further pursued within this work. But, for less hydrophobic samples and alternative detection techniques this concept appears to be very promising. In the context of green chemistry, it is also interesting to use ethanol as eluent instead of acetonitrile or methanol. While ethanol is not a solvent of choice in HPLC due to the rather high viscosity, this issue can be elegantly circumvented when working at elevated temperatures.5,60 As shown in figure 4, we were able to perform high-speed separations in heated ethanol/water mixtures. Although the separation performance in terms of peak shape and plates numbers (about 110.000·m-1 with reduced plate heights of 3.2) is slightly inferior to experiments with acetonitrile-based eluents, it allows an appropriate resolution of most compounds in less than a minute. By temperature programming from 60°C to 150°C the separation can be significantly accelerated. This also leads to improved peak shapes and reduced peak widths quite similar to solvent gradient elution. Such rapid temperature gradients achievable in chipHPLC are also attractive as an alternative to solvent gradients, which are not trivial to implement in microfluidics due to technical challenges in mixing and solvent delivery.

The importance of exact column thermostatting, especially in high-speed applications, is exemplarily displayed in the insert of figure 3. In this set of experiments the temperature was stepwise decreased in one degree (50°C to 45°C) steps between the individual chromato-



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cool the chip from 200°C to 60°C within about 150 s (see figure S4 and S5). To further accelerate cooling rates in future designs a Peltier-based cooling-unit43 or cooling side channels are promising.

Figure 4. High-temperature chromatography with ethanol/water based eluents. Separation was achieved in 25 s when applying thermal gradients. Column: 35 mm length, material: 2.5 µm particle diameter, XBridge C18 BEH; primary flow rate: both 150 µl·min-1, eluent pump: 70/30% v/v ethanol/water, pinch pump: 10 µl·min-1 - 60/40% v/v ethanol/water, sample pump: 10 µl·min-1 40/60% v/v acetonitrile/water, separation temperature: bottom 70°C isothermal (N>110.000 m-1), top: temperature gradient from 60°C to 150°C set temperature. Sample solution identical to figure 3.

The potential of this approach is shown in figure 5, where a high-speed solvent gradient separation of PAHs (isothermal at 30°C) is compared with a thermal gradient separation at isocratic conditions. The gradient from 80% to 100% v/v acetonitrile was applied by changing the composition at the binary eluent pump. Method development was facilitated as the pre-column pressure meter can be used to monitor the gradient progression throughout the run.40 While the separation takes 40 seconds in the isothermal solvent gradient elution mode, using temperature programming at isocratic conditions (only 65% v/v acetonitrile) instead the separation can be achieved in less than 20 seconds. Furthermore, method development was much more straightforward for the thermal gradient elution, since no gradient dwell volumes had to be determined beforehand. Still, besides active heating, also cooling rates have to be considered for their impact on overall cycle times. For this purpose, a forced-air cooling fan (see figure°2C) was included in the device setup allowing to

Figure 5. Comparison of an isothermal (30°C) solvent gradient separation (top) and an isocratic temperature gradient separation (bottom). Column: 35 mm length, material: 2.5 µm particle diameter, XBridge C18 BEH; primary flow rate: 300 µl·min-1, sample solution identical to figure 3, only benzo[k]fluor-anthene (7) was spiked to 25 µg·ml-1. Rc: Resolution of critical peak pair.

CONCLUSION Having developed and evaluated an approach for hightemperature programmed chip-based HPLC capable of working at up to 200°C at heating rates of up to 4.7 C·s-1. The system allows to thermostat the column reliably at a precision below 1% RSD. We were able to show the importance of exact column temperature control by depicting the clear shift pattern in retention times for separations carried out at temperatures differing only by 1°C. By combining HT-HPLC and chip-technology, we were able to circumvent thermal mismatch conditions and to benefit from minimal thermal masses for applying rapid temperature gradients. With this approach highspeed chip-chromatography in so-called green eluent systems based on ethanol-water binary mixtures was performed.


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We have explored the possibility of using thermal gradients and super-heated eluents to speed-up separations as an alternative to conventional solvent gradients. In a direct comparison, we were able to reduce the separation time by more than a half with a thermal gradient from 90°C to 180°C. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: (PDF). Precise thermostatting, reproducibility studies, heating and cooling rates, user interface and separations in highly aqueous eluent systems.

AUTHOR INFORMATION Corresponding Author

*Tel.: +49 (0) 341 / 97 - 36091; Fax: +49 (0) 341 / 97 36115; E-mail: [email protected] Funding Sources

We would like to thank the Deutsche Forschungsgemeinschaft (DFG), the Studienstiftung des deutschen Volkes (Josef J. Heiland) and the Arbeitsgemeinschaft industrieller Forschung (AiF) for funding. Notes

The Authors declare no competing financial interest.

ACKNOWLEDGMENT Dr. T. Teutenberg and Dr. S. Wiese from the IUTA Duisburg (Germany) are acknowledged for their valuable support on aspects of high-temperature LC.

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