Patent Publication Number: US-2023152283-A1

Title: Light-guiding flow cell technologies

Description:
RELATED APPLICATION 
     This application is a non-provisional patent application claiming priority to U.S. Provisional Patent Application No. 63/280,889, filed Nov. 18, 2021, titled “Light-Guiding Flow Cell Technologies for Absorbance Detection,” which is incorporated herein by reference in its entirety. 
    
    
     FIELD OF THE INVENTION 
     The disclosed technology relates generally to liquid chromatography systems. More particularly, the technology relates to a flow cell for liquid chromatography. 
     BACKGROUND 
     In liquid chromatography systems, a fluidic sample is pumped through a column tube, then to a detector flow cell where the sample flows through the beam path of an optical device so that components of the sample, for example, varying concentrations of specific analyte molecules within a chromatographic band, can be identified. Conventional flow cells may contribute to analyte loss, which can compromise the chromatographic efficiency of the flow cells. Conventional flow cell light-guiding technology also has a high manufacturing burden when forming sub-assemblies of a detector. 
     SUMMARY 
     In one aspect, a flow cell for a liquid chromatography detector comprises a substrate formed of a glass material; a fluidic channel extending through the substrate; and at least one gas filled region formed in the substrate along at least a portion of a length of the fluidic channel, wherein a portion of the glass material separates the fluidic channel and the gas filled region, and wherein an interface between the at least one gas filled region and the portion of the glass material separating the fluidic channel and the at least one gas filled region enables total internal reflection of light propagating along the fluidic channel. 
     The at least one gas filled region may include a first air gap extending along one side of the fluidic channel and a second air gap extending along another side of the fluidic channel. The first and second air gaps may extend in a same longitudinal direction as a direction of extension of the fluidic channel through the substrate. 
     The at least one gas filled region may include a third air gap above the fluidic channel and a fourth air gap below the fluidic channel. The third and fourth air gaps extend may in a same longitudinal direction as a direction of extension of the fluidic channel through the substrate. 
     A first end of the fluidic channel may receive the light, which may propagate to a second end of the fluidic channel where it is directed to a detector. 
     The flow cell of claim  4  may further comprise a mirror at the first end and the second end of the fluidic channel. 
     The mirror may be integrated into the substrate at one or both of the first end and the second end. 
     The flow cell may further comprise a reflective coating about a portion of the fluidic channel. 
     The at least one gas filled region may include air. 
     In another aspect, a flow cell for a liquid chromatography detector comprises a substrate formed of a glass material; a fluidic channel extending through the substrate; and a reflective coating about a portion of the fluidic channel, wherein the reflective coating enables an internal reflection of light propagating along the fluidic channel. 
     The reflective coating may be external to the fluidic channel. 
     The reflective coating may be internal to the fluidic channel. 
     A portion of the glass material may separate the fluidic channel and the reflective coating. 
     The flow cell may further comprise first and second gas filled regions adjacent to and parallel the fluidic channel. 
     The first and second gas filled regions may include at least a portion of the reflective coating. 
     The flow cell may further comprise a third gas filled region above the fluidic channel and a fourth gas filled region below the fluidic channel, the third and fourth gas filled regions extending in a same longitudinal direction as a direction of extension of the fluidic channel through the substrate. 
     The reflective coating may include a mirror integrated in the substrate at an input and an output of the fluidic channel. 
     In another aspect, a flow cell for a liquid chromatography detector comprises a substrate formed of a glass material; a fluidic channel extending through the substrate, the fluidic channel having an input and an output; and an integrated mirror at the input and/or the output, wherein the mirror enables an internal reflection of light propagating along the fluidic channel. 
     The flow cell may further comprise at least one gas filled region adjacent to and parallel to the fluidic channel. A portion of the glass material may separate the fluidic channel and the at least one gas filled region. An interface may be between the at least one gas filled region and the portion of the glass material separating the fluidic channel and the at least one gas filled region enables an internal reflection of light propagating along the fluidic channel. 
     At least one gas filled region may include first and second air gaps. The first air gap may extend along one side of the fluidic channel and the second air gap may extend along another side of the fluidic channel. 
     The flow cell may further comprise a third air gap above the fluidic channel and a fourth air gap below the fluidic channel, the third and fourth air gaps extending in a same longitudinal direction as a direction of extension of the fluidic channel through the substrate. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The above and further advantages of this invention may be better understood by referring to the following description in conjunction with the accompanying drawings, in which like numerals indicate like structural elements and features in the various figures. For clarity, not every element may be labeled in every figure. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. 
         FIG.  1    is a perspective view of an example of a flow cell. 
         FIG.  2    is a cross-sectional view of an example of an air-glass interface of a flow cell. 
         FIG.  3    is a schematic view of a liquid chromatography system including another example of a flow cell. 
         FIG.  4    is a schematic view of a liquid chromatography system including another example of a flow cell. 
         FIG.  5    is a schematic view of a liquid chromatography system including another example of a flow cell. 
         FIG.  6    is a schematic view of a liquid chromatography system including another example of a flow cell. 
         FIG.  7    is a perspective view of another example of a flow cell. 
         FIG.  8    illustrates the flow cell of  FIG.  7    transmitting light by total internal reflection (TIR) during an operation. 
         FIG.  9    is a front view of another example of a flow cell. 
         FIGS.  10 A- 10 E  are cross-sectional front views of other examples of a flow cell. 
         FIG.  11    is a flowchart representation of an example of a method for forming a light guiding flow cell. 
         FIG.  12    is a flowchart representation of an example of another method for forming a light guiding flow cell. 
     
    
    
     DETAILED DESCRIPTION 
     Reference in the specification to an embodiment or example means that a particular feature, structure, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the teaching. References to a particular embodiment or example within the specification do not necessarily all refer to the same embodiment or example. 
     The present teaching will now be described in detail with reference to exemplary embodiments or examples thereof as shown in the accompanying drawings. While the present teaching is described in conjunction with various embodiments and examples, it is not intended that the present teaching be limited to such embodiments and examples. On the contrary, the present teaching encompasses various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art. Those of ordinary skill having access to the teaching herein will recognize additional implementations, modifications, and embodiments, as well as other fields of use, which are within the scope of the present disclosure as described herein. 
     In brief overview, embodiments and examples disclosed herein rely on modern glass manufacturing and glass bonding technologies so that reflection-improving and analyte loss-reducing structures can be formed in a glass substrate for directing light in a chromatography application. Glass can be compatible with biopolymers and can be produced robustly, while reducing the manufacturing burden. A light-fluid interface can be formed in the glass material itself that provides both desirable light throughout and chromatographic efficiency. The interface may include a combination of air gaps, integrated mirrors, and/or reflective materials such as metal formed in the glass material for providing light guidance through flow cells. Therefore, the light can be detected by a single channel detector or a broadband light source such as a spectrometer, but not limited thereto. 
     A flow cell formed according to the foregoing offers benefits over conventional flow cell light-guiding technology, for example, flow cells formed of a Teflon™ AF (amorphous fluoropolymer) material. However, Teflon™ AF requires performance tradeoffs to implement. A main reason for developing the inventive concept is that Teflon™ based light-guiding flow cells display poor performance in aqueous SEC and IEX applications due to surface interaction with large biomolecules, such as recovery loss and peak tailing. In addition, Teflon™ AF is subject to contamination and fouling and involves a more complicated manufacturing process. In particular, much of the manufacturing burden is related to the tolerance requirement associated with the small volume of the flow cell and a significant number of manual assembly steps. Conventional glass capillary light-guiding flow cells offer a better throughout than Teflon™ AF cells. However, glass capillary flow cells involve manufacturing complexities, especially with respect to fluidic coupling efficiencies and can also have difficulty satisfying certain design requirements such as pressure ratings. 
       FIG.  1    is a perspective view of an example of a flow cell  100 . The flow cell  100  can be formed by a semiconductor wafer fabrication process, for example, a CMOS process involving isotropic/anisotropic etching, laser subtractive processing, and the like, described below but not limited thereto. A plurality of flow cells  100 , e.g., 64 cells, may be fabricated from a single wafer. 
     The flow cell  100  is constructed and arranged to include a combination of fluidic channel diameter, cross-sectional shape, and turn/corner configurations providing an optimal chromatographic performance by reducing the dispersion of an analyte peak passing through the flow cell. In doing so, the flow cell  100  comprises a substrate  102 . In some embodiments, the substrate  102  may be formed of two sub-plates  111 ,  112  that are bonded to each other. The sub-plates  111 ,  112  can be formed of silica, fused silica, quartz, and/or glass-related material that permits the sub-plates  111 ,  112  to be bonded together to form the substrate  102  having a fluidic channel  104  and at least one air gap  106 A,  106 B (generally,  106 ). Other materials may include transparent materials, ceramics, and/or polymers. In some embodiments, an intermediate layer (not shown), for example, formed of boron phosphorous glass or the like, may be positioned between the sub-plates  111 ,  112  to allow for fusion bonding during manufacturing. In other embodiments, the substrate  102  includes a unitary body formed of glass, silica, fused silica, quartz, and/or related material and. The material forming the substrate  102  is transparent within a transmitted light wavelength of interest. The substrate material also permits high-pressure operation, is compatible with a wide range of analytes and mobile phase components, and can be functionalized, for example, via chemical vapor deposition or other type of deposition of organic layers where glass is more amenable to coating process than Teflon AF. 
     The channel and air gaps  106  are formed by an etching process, for example, wet etch, isotropic, and/or anisotropic etching. In one embodiment, each sub-plate  111 ,  112  may include a portion of the fluidic channel  104  and air gaps  106  and when coupled together form the complete periphery of the fluidic channel  104  and air gap(s)  106 . In another embodiment, the entire fluidic channel  104  and air gap(s)  106  are formed in a single unitary (unbonded) substrate  102 , for example. Accordingly, embodiments of the present inventive concept allow for air gaps, or other regions in a glass substrate that encompass gas such as air, or an aerogel, vacuum, or material proximal to and/or about some or all the fluidic channel. The air-glass interface provides a combination of refractive indices to provide total internal reflection. 
     The fluidic channel  104  is constructed and arranged to allow light to interact with a fluidic sample flowing through the central lumen of the fluidic channel  104 . Although reference is made to light, the embodiments herein may pertain to optical energy, photons, or related features of light energy. In some embodiments, the light includes photons ranging from 180-900 nm. An optical fiber (not shown) can be coupled to an interface at the inlet and outlet of the channel  104 . In some embodiments, the fiber is a multi-mode fiber (MMF) having a numeral aperture of 0.28 but not limited thereto, a core size of 240 μm but not limited thereto, and/or a wavelength range of 190-400 nm but not limited thereto. Other fiber configurations, ranges, and structures may equally apply depending on optical and mechanical design parameters of the apparatus, such as NA, core diameter, fiber coupling efficiency, light-guide size, geometry, length, wall thickness, and surface roughness requirements. For example, end surfaces of a fiber can be sealed with a transparent material such as a window or lens. 
     As shown in  FIG.  1   , the air gap(s)  106  can extend in a same direction as the fluidic channel  104 . The fluidic channel  104  and air gap(s)  106  can have a geometry, shape, dimension, and/or other configuration parameter that complies with the application in which the flow cell  100  is used, for example, shown in  FIGS.  7  and  9   . Although the term air gap is referred to in  FIG.  1   , equivalent terms may equally apply such as voids or regions. In addition, air gaps  106  may be at least partially filled with gases, liquid, and/or solids other than air. For example, a gas filled region may be an air gap, but not limited thereto. In this example, the gas filled region may be partially or fully filled with gas. 
     In another embodiment, as shown in  FIG.  2   , a flow cell  200  includes an air gap  206  that extends about a periphery of the fluidic channel  204 , rather than on both sides of the fluidic channel  204  is described in  FIG.  1   . The air gap  206  is separated from the fluidic channel  204  by a glass region  203  by a region of separation having a sufficient thickness to provide stability to the flow cell structure. 
     In another embodiment, as shown in  FIG.  3   , a flow cell  350  includes air gaps  306 A,  306 B (generally,  306 ) that are offset to accommodate the contours of a fluidic channel  304 . The air gaps  306  can be parallel to a main section of the fluidic channel  304  that extends along a length of the glass substrate  302 , e.g., the fluidic channel  304  including inlet and outlet sections extending from the ends of the main section of the fluidic channel  304  as shown in  FIG.  3   . Each end interface at the inlet and outlet sections can include an optical fiber (not shown) as the light transmitting passage through the channel  304 . 
     The air-glass interface illustrated in  FIGS.  1 - 3    includes a combination of different refractive indices to provide a desirable total internal reflection (TIR), which can only occur when light travels from a higher index of refraction to a medium or lower index of refraction. Light guiding via total internal reflection may be achieved by forming circumferential air gaps around the central fluidic channel. For example, as shown in  FIG.  1   , the ends of the air gaps  106  can be smooth and rounded to contribute to total internal reflection. In another example, as shown in  FIG.  2   , the circumferential air gap  206  about the channel  204  can contribute to TIR. Other factors that provide for total internal reflection may include surface characteristics such as roughness, wall thickness, air gap dimensions, and so on. In other embodiments, TIR occurs at a wall of the flow cell, for example, in cases where a reflective material abuts the exterior wall as shown in  FIGS.  10 A and  10 D . 
       FIG.  4    shows a view of a liquid chromatography system  400  employing an example of a flow cell. In some embodiments, the flow cell may be formed on a substrate  452  or the like, for example, similar to a glass substrate described with reference to  FIGS.  1 - 3   . 
     In some embodiments, the flow cell integrates one or more high-reflection mirrors  462  within the substrate  452  to decrease the flow cell volume by incorporating a folded-path design. In other embodiments, as shown, the mirror  462  is a separate structure adjacent the flow cell for optically communicating with the flow cell. In other embodiments, as shown in  FIG.  3   , a flow cell  300  can include a combination of air gaps  306  and integrated mirror  361 . In other embodiments, as shown in  FIG.  5   , an integrated mirror  561  is at a bottom region of a flow cell  500  where a V-shaped fluidic channel  504  changes direction and a minimal amount of glass is present between the fluidic channel  504  and the integrated mirror  561 . In particular, the fluidic channel  504  has a first straight portion  504 A that extends in a direction of light rays output from a UV source  521  and a second straight portion  504 B that extends in a direction of light rays reflected from the integrated mirror  561 . In other embodiments, the substrate can be coated with a reflective material such as an ultraviolet (UV)-enhanced aluminum (Al) coating or other metallic coatings or other reflective surfaces. A reflective coating may be up to at least 100 nm, but not limited thereto. Such high reflective surfaces directly onto the substrate can enable reflection at the internal or external walls. One application of the integrated mirrors/reflective coatings is to provide multiple light passes through a single length of the fluidic channel. These configurations in  FIGS.  3 - 6    can result in at least double the path length per unit of flow cell volume. These configurations can increase absorbance (i.e. signal to noise ratio) while reducing the volume of the cell (i.e. reducing peak dispersion). The high reflective surface of the flow cells of  FIGS.  3 - 6   , respectively, can generally reflect more than 80% of the incident energy. 
     Referring again to  FIG.  3   , the liquid chromatography system  300  includes a light source  321 , a beamsplitter  322 , and a detector  323 . The light source  321  is constructed and arranged to direct external light rays so that they enter the flow cell  350  at or near the input, and more specifically, a first bend near the input in the fluidic channel  304 . For example, the light source  302  can be a broadband LED light source. In some embodiments, the light source  302  can a laser-based light source. The fluidic channel  304  extends through the flow cell body, in particular, the glass substrate  302 , and includes inlet and outlet ends and an elongated section between the inlet and outlet ends. The inlet and outlet ends may each have an interface coupled or integrated thereto. The elongated section may connect the inlet and outlet ends by bends extending on both sides of the flow cell  350 . The integrated mirror  361  may be proximal to the second bend. The light source  321  can direct the light rays through the first bend to the elongated section, then through the second bend to the integrated mirror  361  which reflects the light to the beamsplitter  322  where they are detected by the detector  323  or the like and analyzed, for example, providing information about, the solutes contained in the sample, for example. The detector  323  can be a single channel detector or a broadband light source such as a spectrometer, but not limited thereto. 
     Referring again to  FIG.  4   , the liquid chromatography system  400  includes a light source  421 , a beamsplitter  422 , a detector  423 , which may be similar to or the same as the  300  includes a light source  321 , a beamsplitter  322 , and a detector  323  of  FIG.  3   . In addition, the system  400  may include optical transfer elements  431 ,  432  such as lenses, concave mirrors, and the like, which may be co-located with the light source  421 , beamsplitter  422 , and detector  423  at a proximal end of the channel  404 . The reflective coating or mirror  462  may be at a distal end of the channel  404 . The light beam(s) extend through the elongated straight portion of the fluidic channel  404  to the mirror  462 , which reflects the light through the fluidic channel  404  to the beamsplitter  422  where they are detected by the detector  423  or the like and analyzed. 
     The liquid chromatography system  600  is similar to the system  400  of  FIG.  4   , except that a first optical element  631  is at the proximal end of the channel  604  and a second optical element  632  and detector  623  are at a distal end of the channel  604 . Other details of the operation of the liquid chromatography system  600  are not repeated for brevity. 
     Referring again to  FIG.  5   , the liquid chromatography system  400  also includes a light source  521  and a detector  523 . Here, the light source  521  can direct the light rays through the first straight section  504 A of the fluidic channel they are detected by the detector  523  or the like and analyzed. 
       FIG.  7    is a perspective view of another example of a flow cell  700 . The flow cell  700  can be formed by an anisotropic etching process, but is not limited thereto. The flow cell  700  in some examples can be implemented in a liquid chromatography system, for example, shown in  FIGS.  3 - 6   . 
     In some embodiments, as shown in  FIG.  7   , the flow cell  700  includes a square fluidic channel  704 . The flow cell  700  includes a first square air gap  706 A on a first side of the fluidic channel  704  and a second square gap  706 B on a second side of the fluidic channel  704  opposite the first air gap  706 A. The channel  704  and square air gaps  706 A,  706 B (generally,  706 ) can have a same dimension, for example, 280 micron width. The flow cell  700  may have a path length of 1-10 mm but not limited thereto. 
     In addition, the flow cell  700  includes a first rectangular air gap  707 A above the fluidic channel  704  and a second rectangular air gap  707 B below the fluidic channel  704 . The quadrilateral; e.g., rectangular, air gaps  707 A,  707 B (generally,  707 ) can have a same dimension, for example, 520 micron width and 260 micron height. The channel  704  may have a length of up to 10 mm, or longer but not limited thereto. The thickness of the glass walls of the substrate  704  may be 100 microns or more, but not limited thereto. 
     As shown in  FIG.  8   , the flow cell  700  can transmit light in a manner such that the flow cell  70  provides TIR at the glass-air interfaces. The arrangement of air gaps  706 ,  707  in combination with the geometry of the channel  704  can minimize or eliminate a loss of light through the channel  704 . Accordingly, transmission is well maintained in a microchannel  704  fabricated via anisotropic etching. In some embodiments, a laser subtractive method is applied, where the illuminated area has an etch rate that is faster than the non-illuminated area permitting an etching of arbitrary shapes within the bulk of the substrate. 
     As previously described, the channel  704  may have a quadrilateral geometry, e.g., square or rectangular shape, formed by anisotropic etching. Other shapes may equally apply, such as but not limited to trapezoidal, triangular, elliptical, and so on. The channel  704  formed in this manner may have different transmission characteristics, for example, improved transmission, as compared to a channel having rounded corners. Nevertheless, as shown in  FIG.  9   , a flow cell  900  may have circular e.g., elliptical or other rounded, channels  904  and air gaps  906 A,  906 B (generally,  906 ),  907 A, and  907 B (generally,  907 ) formed by wet isotropic etching. In this example, the channel  904  and adjacent air gaps  906  may have a same dimension, for example, 380 micron width and rounded corners. The air gaps  907  above and below the channel  904  may have a same dimension, e.g., a width of 1100 microns. 
       FIGS.  10 A- 10 E  are cross-sectional front views of other flow cell configurations having various channel geometries which can affect overall transmission characteristics. As Some or all of the internal or external walls can be coated with a reflective coating. The TIR has better reflectance but is more sensitive to the angle of incidence and can&#39;t guide (reflect) rays below the critical angle. TIR efficiency is also influenced by the cleanliness of the glass-air interface and the scattering properties of this interface. While contamination is not a problem for embedded air gap, it may be of concern for external surfaces. To remediate this issue, the top and bottom surfaces of  FIGS.  7  and  9    can be covered with a reflecting coating to increase the robustness of the device ( FIG.  10 A ). The reflective coatings are also less demanding than TIR in term of surface roughness. For devices where the air gap can&#39;t produce efficient transmission efficiency either due to the geometrical factors or surface scattering properties, the air gaps can be coated prior to the substrate bonding to fully enclose the fluidic volume with a reflective layer at the external walls ( FIG.  10 B ). Alternatively, shown, some or all of the internal or external walls of a flow cell can be coated with a reflective coating. In particular,  FIG.  10 A  illustrates a flow cell  1000 A. Flow cell  1000 A may have a generally square or rectangular channel  1004 , but with two rounded corners  1011 A,  1011 B and two sharp edges  1012 A,  1012 B. Other channel shapes can equally apply that the reflection of the rays at the glass-air interface back to the fluidic channel  1004  with minimal glass propagation. Flow cell  1000 A may also have adjacent air gaps  1006 A,  1006 B, a first reflective coating  1009 A above the channel  1004 , and a second reflective coating  1009 B below the channel  1004 . The second reflective coating  1009 B may be applied directly on the substrate  1002 . After the fluidic channel  1004  and air gaps  1006  are formed in the substrate  1002  by etching or the like, at least one other layer  1008  formed of a layer of silicon or related material may be positioned on a top surface of the substrate  1002  and cover an exposed top region of the substrate  1002  to complete the formation of the channel  1004 . In other embodiments, the layers  1002 ,  1008  both have identical etches to allow for them to bring the ratio of the size of the channel and air gaps to 1:1. The layers  1002 ,  1008  can be attached by anodic bonding or the like. The first reflective coating  1009 A may be applied directly on the intermediate layer  1008 . The abovementioned elements of the flow cell  1000 A may be formed of materials described in other embodiments above, and are not repeated for brevity. 
     During operation, the TIR and reflective coating can reflect the light back toward the center of the fluidic channel  1004  and are exchangeable. The TIR has better reflectance but is more sensitive to the angle of incidence and cannot guide, e.g., reflect, light rays below a critical angle. TIR efficiency is also influenced by the purity or cleanliness of the glass-air interface and the scattering properties of the interface. While contamination is not a problem for the embedded air gaps  1006 , it may be of concern for external surfaces. To remedy this issue, referring again to the configurations shown in  FIGS.  7 - 9   , the top and bottom surfaces can be covered with a reflecting coating to increase the robustness of the flow cell  1000 A. 
     The reflective coatings are generally less demanding than TIR in term of surface roughness. For flow cell devices where the air gap cannot produce efficient transmission efficiency either due to the geometrical factors or surface scattering properties, as shown in  FIG.  10 B , the air gaps can be coated or at least partially filled with a reflective coating  1013 A,  1013 B (generally  1013 ) prior to the substrate bonding to fully enclose the fluidic volume with a reflective layer at the external walls ( FIG.  10 B ). 
     A flow cell  1000 C shown in  FIG.  10 C  includes a reflective coating  1019  that is similar to the reflective coatings  1009 ,  1013  of  FIGS.  10 A and  10 B , respectively. However, the reflective coating  1019  is coated about the interior of the substrate  1022  instead of the exterior of the substrate  1002  of the flow cells  1000 A and  1000 B. Therefore, the perimeter of the channel  2024  is formed by the coating  1019  to fully enclose the fluidic volume with the reflective layer, i.e., coating  1019 . The coating  1019  may be protected from the solvent flowing within the flow cell  1000 C. Many oxides and fluoride protective layers can be selected for this based on their solvent compatibility and overall transmission. The channel  1024  may have a square or rectangular shape as shown, or may have at least two curved corners, for example, shown in  FIGS.  9 ,  10 A and  10 B  but are not limited thereto. Therefore, the channel  1024 , like the fluidic channels of the other flow cells of  FIGS.  10 A- 10 E  may have different shapes, for example, depending on the process used to form the flow cells. 
     A flow cell  1000 D shown in  FIG.  10 D  has a circular configuration. A reflective coating  1029  is about the substrate  1032  so that the glass material of the substrate  1032  is between the reflective coating  1029  and the channel  1032 . 
     A flow cell  1000 E shown in  FIG.  10 E  likewise has a circular configuration. Here, a reflective coating  1039  is internal to the glass material of the substrate  1042  so that the reflective coating  1039  forms the perimeter for the channel  1042 . 
       FIG.  11    is a flowchart representation of an example of a method  1100  for forming a light guiding flow cell. In describing the method  1100 , reference can be made to elements of  FIGS.  1 - 10   . In particular, the method  1100  can be applied to form the flow cell  900  of  FIG.  9   . 
     At block  1102 , a masking operation can be performed on two semiconductor wafers. The wafers may be formed of fused silica or the like. At block  1104 , the masked wafers are etched, for example, according to a wet etching operation to form portions of the fluidic channel and air gaps. For example, each wafer may be etched to form a hemispheric half of the channel and air gaps. When the wafers are bonded together (block  1108 ), the channel and air gaps are formed. 
     At block  1106 , an intermediate layer of quartz or the like, for example, boron phosphorus glass may be formed on at least one of the fused silica wafers to allow for fusion bonding of the wafers. Accordingly, at block  1108 , the wafers are bonded together. The wafers having identical etches can be coupled to bring the ratio of the size of the channel and air gaps to 1:1. Here, the intermediate layer, e.g., a boron phosphorous glass layer, may be between the two wafers for fusion bonding. The type of bonding may depend on the materials. For example, low temperature bonding may form a glass-silica interface. High temperature bonding may form a glass-glass interface. 
     At block  1110 , the bonded wafers are masked so that at block  1112  a second etching operation can be formed on the two surfaces, for example, to form additional air gaps above and below the channel, e.g., similar to air gaps  907  shown in  FIG.  9   . 
     At block  1114 , the wafer can be diced or otherwise segmented to form a plurality of flow cells. 
       FIG.  12    is a flowchart representation of an example of a method  1200  for forming a light guiding flow cell. The method  1200  can be applied to form the flow cell  700  of  FIG.  7   . 
     At block  1202 , two semiconductor wafers were exposed to a laser for performing a laser subtractive method. The wafers may be formed of fused silica or the like. At block  1204 , the wafers are etched. For example, one wafer may be etched to form the fluidic channel  704  and air gaps  706 A,  706 B, and  707 B of the flow cell  700  of  FIG.  7   , and the other wafer may be etched to form the air gap  707 A of the flow cell  700  of  FIG.  7   . 
     At block  1206 , a surface polishing operation is performed on the etched interior regions defining the channel and air gaps. At block  1208 , an intermediate layer of quartz or the like, for example, boron phosphorus glass may be formed on the top surfaces of the wafers to allow for fusion bonding of the wafers. Accordingly, at block  1210 , the wafers are bonded together. At block  1212 , the wafer can be diced or otherwise segmented to form a plurality of flow cells, for example, shown in  FIG.  7   . 
     The abovementioned flow cell constructions and manufacturing processes address design limitations of current flow cell manufacturing technologies and can provide for a next generation of flow cells. Embodiments of these flow cells may withstand higher pressure. High pressure operation allows for a single flow cell design for all absorbance detector variants, improved flow cell resilience, and a reduction in the pressure dependence of optical noise, e.g., a greater signal to noise ratio. Embodiments of these flow cells may also improve chromatographic performance. The ability to maintain peak fidelity through the detector reduces dispersion and results in improved signal in both the optical detector and any subsequent mode of detection connected downstream of the optical detector. Embodiments of these flow cells may also improve optical performance by providing a construction which enables total internal reflection and novel reflective surfaces and improves the light throughput and maximizes both signal to noise and can be also leveraged to further reduce chromatographic band broadening. Embodiments of these flow cells may also improve compatibility. For example, replacing Teflon™ AF with glass can result in improved mobile phase and analyte compatibility, which can expand the applicability of such technology to support a broader range of applications and markets. Embodiments of these flow cells may also increase the pressure and temperature reliance which will improve the robustness of the flow cell. 
     While various examples have been shown and described, the description is intended to be exemplary, rather than limiting and it should be understood by those of ordinary skill in the art that various changes in form and detail may be made therein without departing from the scope of the invention as recited in the accompanying claims.