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Technical Note
Micro-machined Fused Silica LCW Capillary Flow Cell Karsten G. Kraiczek, John T. Mannion, Susan Post, Andriy Tsupryk, Varun Raghunathan, Reid Alyn Brennen, and Roland Zengerle Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03219 • Publication Date (Web): 21 Dec 2015 Downloaded from http://pubs.acs.org on December 23, 2015
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Micro-machined Fused Silica LCW Capillary Flow Cell K. G. Kraiczek,*,†,§ J. Mannion,‡ S. Post,‡ A. Tsupryk,‡ V. Raghunathan,‡ R. Brennen,‡ and R. Zengerle§ †
Agilent Technologies, Hewlett-Packard Str.8, D 76337 Waldbronn, Germany
‡
Agilent Technologies, 5301 Stevens Creek Blvd., Santa Clara, CA 95051, USA
§
IMTEK – Department of Microsystems Engineering, University of Freiburg, D-79110 Freiburg, Germany
*
To whom correspondence should be addressed. E-mail :
[email protected] Abstract A planar, chip-based flow cell for UV-VIS absorbance detection in HPLC is presented. The device features a micro-fabricated free-standing liquid core waveguide (LCW) capillary detection tube of long path length that is based on total internal reflection. We report on the linearity and calibration slope characteristics of lithographically produced LCWs with different interior/exterior geometries. 3D ray tracing was indispensable in modeling behavior in the more demanding geometries: multipath behavior may be intrinsic to these waveguides with consequent non-linearity. Fortunately, non-linearity in lithographically easy-to-produce waveguide geometries (such as with a flat, concave exterior and a round interior) is not as detrimental as might be initially expected. Experimental performance is predictably affected by the attainable surface quality of the LCW and efficient and reproducible coupling of the input light into the LCW.
Introduction Liquid core waveguide (LCW) cells for optical detection in analytical instrumentation have been the subject of applied research for decades and have found widespread use in absorption,1-9 fluorescence1014
and Raman15-17 spectrometry. Applications have been reviewed by Dallas and Dasgupta18 while the
pros and cons of various designs have been reviewed variously by Altkorn et al.19, by Schmidt20, and by Hawkins21. LCWs serve as high sensitivity, long-path-length detection cells with small but welldefined sample volumes, and are easily coupled to light sources and photodetectors.8,18 LCW technologies include metal or metal-coated, polymer or polymer-coated, and uncoated glass capillary tubes.
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LCWs based on total internal reflection (TIR) have two sub-types depending on their light-guiding properties.4,6,18 Type-I designs are true LCWs, where TIR occurs at the interface between the liquid and a wall material of lower refractive index (RI), such as Teflon AF.18,22 Thus, the light propagates only through the liquid. In Type-II designs, TIR occurs at the external surface of a transparent wall material confining the liquid and, at the internal surface, the light splits into a transmitted and a reflected portion. Thus, unlike Type-I designs, the light propagates through both the liquid and the wall material, reducing net interaction with the liquid core.3,6 In keeping with existing literature, however, we also refer to Type-II designs as LCW. Cylindrical Type-I and Type-II capillary waveguides have been characterized theoretically and experimentally with respect to coupling efficiencies and guiding properties,3-5 and the Lambert-Beer linearity6 of the detection signal. For absorbance detection, both design concepts were found to be comparable in key performance characteristics such as sensitivity and signal linearity.6 An aspect of LCW cells in high performance liquid chromatography (HPLC) that has not been much discussed in the literature is their relative immunity to RI effects when appropriately designed. RI effects have always been a concern in HPLC absorbance detection with traditional flow cells and flow cell optics,23 and are best minimized by using tapered beam flow cell designs.24 The numerical aperture of a LCW varies with the refractive index of the liquid. This can cause changes in light transmission of up to 10%, and can lead to unacceptable baseline deviations. However, to minimize RI effects, including noise induced by changes in RI, the numerical aperture of the LCW must be at least matched or, better, limited by other components of the optics, e.g., by the use of optical fibers to couple the light in and out. Efficient light ray mixing within an LCW further helps minimize noise induced by changes in RI. Because of their unique properties, inter alia the ease of increasing the path length of the cell, LCW cells are replacing traditional cells in HPLC absorbance detection. However, solvent absorption in the low UV (< 250 nm), and the desire for a large linear dynamic range (LDR), limits practical path lengths to < 100 mm.25 Robustness concerns18,19 associated with uncoated glass/silica capillary waveguides are solved by permanently encapsulating the waveguide in a suitable cell housing. Advances in column separation efficiencies have put higher demands on optical signal detection. The use of sub-two-micron or core-shell packing materials in narrow, short columns (e.g., 1.0 or 2.1 mm ID with 50 mm length) and in capillary columns (0.3–0.5 mm ID) results in very small peak volumes, requiring very small detection volumes to preserve the chromatographic resolution. Desired cell volumes are in the range of 0.2–1 µL for narrow columns and 0.02–0.06 µL for capillary columns.25 The requirement of very narrow bore LCW flow cells (e.g., 100–200 µm ID or even smaller) with ACS Paragon Plus Environment
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sufficient path length (e.g., 10 mm) encourage micro-fabricated cells. Further, microfabrication allows the implementation of designs that integrate multiple flow cells, in series or parallel, in a small area. Type-I micro-fabricated LCW channels coated with Teflon AF have been demonstrated by Manor et al.26 in glass and by Datta et al.27 in silicon. Other than being smaller, they differ in two aspects from standard capillary LCWs. First, the cross-sectional geometry of micro-fabricated channels is no longer circular. This depends on the properties of the substrate material and the micro-fabrication process used. For example, these channels have a flat bathtub-like cross-section for a wet-etched glass channel26 or a hexagon-like cross-section for a KOH-etched silicon channel.27 This is a minor drawback for this type of micro-fabricated Type-I waveguides. Secondly, the deposition and secure adhesion of an internal coating in a long, narrow channel is a greater practical problem. The objective of this work is to investigate micro-fabricated small-volume LCW flow cells for UVdetection in capillary-scale HPLC. Specifically, we examine a Type-II flow cell concept that does not require any coating to provide TIR and allows for a large range of flow cell sizes.
Materials and Methods Design Considerations and Fabrication Process For a Type-II, micro-machined, low-volume, long-path-length cell, requirements on design, materials, surface finish and fabrication methods include: Transparency of the material in the UV-Vis, chemical inertness, and processability by standard MEMS techniques. Fused silica (FS) meets these properties, and is readily available as thin wafers with an arithmetic average surface roughness (Ra) < 2 nm. The surface roughness Ra required for a micro-machined free-standing TIR detection tube is less than 10 nm to reach the surface quality of drawn FS capillaries used in conventional TIR flow cell designs. The ratio of the wall thickness to the inner diameter of the detection tube has to be larger than 1:10 to achieve a minimum pressure tolerance of 20 bar. Figure 1 shows the waveguide geometries investigated in this work.
Figure 1 near here. The Round/Round geometry (a) is the performance benchmark, but difficult to fabricate lithographically. All the other geometries can be shaped by isotropic wet etching (d, e) or anisotropic dry etching (b) or a combination (c). Dry etching leads to Ra significantly worse than 10 nm. In a properly configured wet etching scheme, the etching surfaces parallel to the polished wafer surface should retain their surface quality. From this point, although (d) may look unattractive, it uses the most straightforward fabrication, using only wet etching. ACS Paragon Plus Environment
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Figure 2 near here. Liu has fabricated separation channels for DNA sequencing of circular cross section in glass by bonding two hemi-cylindrical channels.28 Our fabrication of a free-standing Type-II LCW also begins with two high-quality fused-silica wafers (Figure 2), etched identically and then bonded face to face to form the channels. A series of photolithography and hydrofluoric (HF) acid etch steps are used to generate a pattern of trenches of different depths that later become the inner surfaces and outer side walls of the suspended LCW tube. The identically structured wafers are then cleaned, aligned, and bonded together with the etched faces in contact, forming long tubular cavities. After bonding, the bulk material above and below the tubular cavities is etched away and the different cavity diameters, in combination with a precisely timed etch, results in a free-standing Concave/Round LCW tube as shown in Fig.1d. With an appropriate machining allowance, this geometry can be improved rapidly in the early phase of an ‘over-etching’. Here, the concave side wall radius increases and thus reduces the overhang and blunts the sharp edges. For more details refer to Supporting Information (A). The Octagon/Round geometry, shown in Fig.1e, can be obtained by adding additional lithographic and etch steps that clip off the overhanging edges. Post-processing includes dicing, side polishing and fluidic port drilling. Ray Tracing in LCWs Existing analytical solutions and simulations are mostly for cylindrical LCWs and are based on meridional (2D) ray approximations.3-6 Accurate modeling of a cylindrical Type-II LCW already represents a significant hurdle6 compared to a Type-I LCW, primarily because the light rays split at each liquid/solid interface. Three-dimensional (3D) ray tracing was found to be indispensable in modeling the more demanding waveguide geometries where an analytical solution may not be straightforward. The calculation mode needs to be selected based on the choice of a non-absorbing or absorbing liquid, the desired accuracy, and permissible computing time. It is easy to understand that 3D ray tracing in ray split mode best reflects reality for a Type-II LCW. In ray split mode, each child ray carries the fraction of energy corresponding to the reflection and transmission coefficients of the interface.29 The problem with ray splitting is that the number of traced rays grows exponentially, and a large number of source (or parent) rays are required for accurate results. One parent ray can split into many thousands of child rays, resulting in a formidable computation time. A fast and accurate alternative in situations near the limit of detection (LOD, near-zero absorbance), however, is the refraction-only mode, where only the refracted ray is traced, and contains all of the energy. ACS Paragon Plus Environment
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The LOD in absorbance measurements is directly proportional to the detector noise divided by the detector’s sensitivity. The detector noise is, to a first approximation, inversely proportional to the square root of the number of photons arriving at the photodetector. Similarly, accuracy of calculation in ray tracing is inversely proportional to the square root of the number of parent rays (N) used in the calculations. The standard deviation of a ray-tracing calculation, expressed in absorbance units (AU), is given by the following equation: _ ≅
0.434 √
Calculations near the noise level (< 10-5 AU peak-to-peak) of an HPLC detector would require more than 1011 parent rays for each baseline data point, and would make no sense unless the real refractive index distribution within the LCW is known and considered. Static refractive index effects, e.g., changes in Fresnel losses at the entrance and exit of the LCW, are of the order of 10-4 AU, and would require a minimum of 109 parent rays to be resolved on the baseline of a chromatogram. Between 106 and 104 parent rays are required to calculate one data point in the absorbance range between 0.01 AU and 10 AU respectively. For a cylindrical Type-II LCW (id 200 µm, w = 20 µm, Lz = 10 mm) and 106 parent rays it takes about 2 hours on a Workstation (HP Z620, Intel® Xeon® Processor E5-2643) to calculate 1 data point of a calibration curve in 3D ray tracing in split mode, whereby each child ray — and child rays of child rays — is consequently calculated to its end using a custom in-house developed ray-tracing software. Therefore, the calculation of a complete and accurate calibration curve with sufficient data points can be performed without super-computer levels of processing within a day. Non-Linearity of LCWs and Discussion of Modelling Results Lambert-Beer’s law assumes that the interaction path length with the sample is the same for all light rays. Variability in path length (the multipath effect, not to be confused with multipass cells such as the White cell, which may use multiple reflections but has essentially a single path) results in nonlinearity.30 This generally undesirable characteristic was deliberately intensified by Dasgupta31 to extend the dynamic range of an absorbance detector by using a helical ‘multipath’ LCW flow cell. In contrast to stray light,30 which typically limits the linear range of an HPLC absorbance detector, the absorbance does not saturate because of the multipath effect,32-34 and can be extended by applying corrections in the instrument firmware, as has become common practice in aerosol-based detectors.35 However, in a typical HPLC absorbance detector application (below 3 AU), the inherent non-linearity of a straight LCW is negligible, and is often overlooked.6 The analytical solution for the inherent nonlinearity and the photometric accuracy for a Type-I (or true) LCW, as well as ray-tracing results for ACS Paragon Plus Environment
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Type-I and Type-II cylindrical LCWs, are presented and discussed in detail in the Supporting Information (B). For non-cylindrical LCWs, however, non-linearities may not be negligible. Figure 3 shows calculated linearity curves from 3D ray tracing in split mode for the different waveguide geometries presented in Figure 1. The wall thickness in X and Y (Figure 1) and the inner diameter is the same for all geometries to ensure comparability. If only meridional or orthogonal (2D) ray tracing is used, the results are the same for all geometries; no differences can be distinguished. However, when 3D ray tracing in split mode is carried out, it is readily seen that the Round/Round geometry performs the best, followed by the Square/Square configuration. The inherent non-linearity of the Square/Round, Concave/Round and Octagon/Round geometries may limit the LDR to below what is desired in present-day HPLC detectors. The ‘over-etching’ of the Concave/Round waveguide to reduce the overhang and blunt the edges does not really help to extend the upper limit of the LDR. Simulations of the Square/Round and Concave/Round (at breakthrough of the release etch, in other words: not overetched and side wall with lowest possible radius) are very similar. The Octagon/Round configuration is geometrically the most similar to the Round/Round benchmark, but its linearity is not significantly better than the Square/Round. In summary, the calculated non-linearity is always significant whenever the internal and external shapes are different. Light rays from the liquid core that enter the wall do not always return immediately into the liquid due to TIR at the solid/liquid interface. This occurs whenever the difference in the angle between the surface normal to the internal and external surface becomes too large. Interestingly, this can never happen with a concentric Round/Round geometry, where the light rays are always refracted back into the liquid core regardless of the wall thickness. A detailed discussion of the ray propagation in different waveguide geometries is given in the Supporting Information (C).
Figure 3 near here. The comparison of the sensitivity in the lower absorbance range is, as expected, more favorable. Table 1 summarizes the calculated sensitivity S as the average slope of the linearity curve between 0 and 0.5 AU. The loss in sensitivity compared to the best case scenario is only about -25% for the easy-tofabricate Concave/Round design. However, for an all-wet-etch design, the Octagon/Round is preferable to the simple Concave/Round because the number of total internal reflections at the external surface is lower and thus there are fewer losses due to surface scattering.
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Table 1 near here.
Experimental Section All linearity experiments were performed in accordance with ASTM E 685-93.36 Stock solution 1 = caffeine in water (19.419 mg/L). Stock solution 2 = caffeine in water (194.19 mg/L). Mobile phase 1 = water (%A) and stock solution 1 (%B). Mobile phase 2 = water (%A) and stock solution 2 (%B). Flow rate = 10 µL/min. No column. Temperature of mobile phase = 25 °C. Step gradients for each stock solution were performed starting at %B = 0 to %B = 100 in steps of 10, each step 6 min long. The resulting caffeine step concentration profiles are 0, 10, 20 to 100 µmol/L and 0, 100, 200 to 1000 µmol/L. The absorbance plateaus of each gradient step were evaluated and plotted. The planar Type-II µ-flow cell chip with the Concave/Round LCW was connected to a 1200 Series Capillary-scale Pump (Agilent Technologies) using a custom-built, planar polyimide hydraulic connector board having an interface to a standard finger-tight fit (Upchurch) connected to a 25 µm ID fused silica/PEEK capillary. Light from a deuterium discharge lamp (Heraeus DX 201 ø0.5 mm) was coupled into an optical UV fiber (Polymicro FDPE ø100 µm) by a custom-built ellipsoid mirror. The light emerging from the input fiber was focused through the FS flow cell chip window into the liquid core (ø200 µm) of a Concave/Round LCW using two fused silica ball lenses (ø4 mm and ø3 mm; Edmund Optics) as shown in Figure 4. The light was collected over the entire cross-section at the distal end of the flow cell chip using a UV-grade silica optical fiber (Polymicro FDP ø600 µm) that, in turn, was coupled to an Agilent 1200 series Diode Array Detector. The Concave/Round capillary LCW was ‘over-etched’ and has an inner diameter of 220 µm and is 10 mm long. The wall thickness at the narrowest point in X and Y is 20 µm and the concave side wall radius is 230 µm. For the optical alignment, the µ-flow cell chip was filled with a very high-concentration caffeine solution, and the intensity ratio of the diode array detector output signals at 330 nm to 273 nm was maximized. The signal obtained by direct coupling of the input fiber to the diode array detector was taken as the 100% T reference point.
Figure 4 near here. Experimental Results and Discussions When using a Type-II LCW, it is important to avoid the direct coupling of light rays into the waveguide wall or to prevent light rays that propagate primarily in the wall from reaching the photo ACS Paragon Plus Environment
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detector. Meeting at least one of the two conditions is sufficient to avoid non-linearity of the absorbance signal induced by wall transmission, which is essentially equivalent to stray light. For conventional LCW flow cell designs this is best resolved by introducing an optical fiber directly into the liquid at the entrance of the LCW. Using the same scheme at the exit avoids capturing wallscattered light and simplifies opto-fluidic connections, but light throughput suffers. A detailed discussion of the coupling efficiency and transmission characteristics of a cylindrical Type-II is given in the Supporting Information (E). In the case of the µ-flow cell chip design described in this study, the light was carefully focused into the liquid core by means of spherical lenses as shown in Figure 4.
The good surface quality of the optical grade polished FS wafer (Ra,0° < 2 nm) is maintained in the “0°, horizontal” wet-etched surfaces of the Concave/Round LCW even after a long release etch. This was measured by Atomic Force Microscopy (AFM). However, the roughness of all the curved surfaces changes gradually into a “90°, vertical” roughness pattern that is defined by the resulting line quality of the etched trenches at the wafer surface (bond interface). The surface quality of the fabricated flow cell chips tested came from two different processes. The first had a vertical-surface roughness Ra,90° > 50 nm (hereinafter referred to as SQ1) and the second Ra,90° < 10 nm (improved process, hereinafter referred to as SQ2). Vertical surface roughness was estimated by Scanning Electron Microscopy (SEM). The calculated transmittance for the optics in Figure 4 with a water-filled cell was 25% (not taking into account losses due to surface scattering). The measured transmittance in the range 200–600 nm was
