Miniaturized planar columns in novel support media for liquid phase analysis

Miniaturized planar column devices are described for use in liquid phase analysis, the devices comprising microstructures fabricated by laser ablation in a variety of novel support substrates. Devices formed according to the invention include associated laser-ablated features required for function, such as analyte detection means and fluid communication means. Miniaturized columns constructed under the invention find use in any analysis system performed on either small and/or macromolecular solutes in the liquid phase and may employ chromatographic and/or electrophoretic separation means.

TECHNICAL FIELD 
The present invention relates generally to miniaturized planar column 
technology for liquid phase analysis, and more particularly to fabrication 
of microstructures in novel separation support media using laser ablation 
techniques. The microstructures produced under the present invention find 
use in any analysis system which is performed on either small and/or 
macromolecular solutes in the liquid phase and which may employ 
chromatographic or electrophoretic means of separation, or a combination 
of both. 
BACKGROUND OF THE INVENTION 
In sample analysis instrumentation, and especially in separation systems 
such as liquid chromatography and capillary electrophoresis systems, 
smaller dimensions will generally result in improved performance 
characteristics and at the same time result in reduced production and 
analysis costs. In this regard, miniaturized separation systems provide 
more effective system design, result in lower overhead due to decreased 
instrumentation sizing and additionally enable increased speed of 
analysis, decreased sample and solvent consumption and the possibility of 
increased detection efficiency. 
Accordingly, several approaches towards miniaturization for liquid phase 
analysis have developed in the art; the conventional approach using drawn 
fused-silica capillary, and an evolving approach using silicon 
micromachining. What is currently thought of as conventional in 
miniaturization technology is generally any step toward reduction in size 
of the analysis system. 
In conventional miniaturized technology the instrumentation has not been 
reduced in size; rather, it is the separation compartment size which has 
been significantly reduced. As an example, micro-column liquid 
chromatography (.mu.LC) has been described wherein columns with diameters 
of 100-200 .mu.m are employed as compared to prior column diameters of 
around 4.6 mm. 
Another approach towards miniaturization has been the use of capillary 
electrophoresis (CE) which entails a separation technique carried out in 
capillaries 25-100 .mu.m in diameter. CE has been demonstrated to be 
useful as a method for the separation of small solutes. J. Chromatog. 
218:209 (1981); Analytical Chemistry 53:1298 (1981). In contrast, 
polyacrylamide gel electrophoresis was originally carried out in tubes 1 
mm in diameter. Both of the above described "conventional" miniaturization 
technologies (.mu.LC and CE) represent a first significant step toward 
reducing the size of the chemical portion of a liquid phase analytical 
system. However, even though experimentation with such conventional 
miniaturized devices has helped to verify the advantages of 
miniaturization in principal, there nevertheless remain several major 
problems inherent in those technologies. 
For example, there remains substantial detection limitations in 
conventional capillary electrophoresis technology. For example, in CE, 
optical detection is generally performed on-column by a single-pass 
detection technique wherein electromagnetic energy is passed through the 
sample, the light beam travelling normal to the capillary axis and 
crossing the capillary only a single time. Accordingly, in conventional CE 
systems, the detection path length is inherently limited by the diameter 
of the capillary. 
Given Beer's law, which relates absorbance to the pathlength through the 
following relationship: 
EQU A=.epsilon.*b*C 
where: 
A=the absorbance 
.epsilon.=the molar absorptivity, (l/m,cm) 
b=pathlength (cm) 
C=concentration (m/l) 
it can be readily understood that the absorbance (A) of a sample in a 25 
.mu.m capillary would be a factor of 400.times.less than it would be in a 
conventional 1 cm pathlength cell as typically used in UV/Vis 
spectroscopy. 
In light of this significant detection limitation, there have been a number 
of attempts employed in the prior art to extend detection pathlengths, and 
hence the sensitivity of the analysis in CE systems. In U.S. Pat. No. 
5,061,361 to Gordon, there has been described an approach entailing 
micro-manipulation of the capillary flow-cell to form a bubble at the 
point of detection. In U.S. Pat. No. 5,141,548 to Chervet, the use of a 
Z-shaped configuration in the capillary, with detection performed across 
the extended portion of the Z has been described. Yet another approach has 
sought to increase the detection pathlength by detecting along the major 
axis of the capillary (axial-beam detection). Xi et al., Analytical 
Chemistry 62:1580 (1990). 
In U.S. Pat. No. 5,273,633 to Wang, a further approach to increased 
detection pathlengths in CE has been described where a reflecting surface 
exterior of the capillary is provided, the subject system further 
including an incident window and an exit window downstream of the incident 
window. Under Wang, light entering the incident window passes through a 
section of the capillary by multiple internal reflections before passing 
through the exit window where it is detected, the subject multiple 
internal reflections yielding an effective increase in pathlength. While 
each of the aforementioned approaches has addressed the issue of extending 
the pathlength, each approach is limited in that it entails engineering 
the capillary after-the-fact or otherwise increasing the cost of the 
analysis. 
A second major drawback in the current approach to miniaturization involves 
the chemical activity and chemical instability of silicon dioxide 
(SiO.sub.2) substrates, such as silica, quartz or glass, which are 
commonly used in both CE and .mu.LC systems. More particularly, silicon 
dioxide substrates are characterized as high energy surfaces, in that such 
materials interact irreversibly with a number of compounds and strongly 
adsorb many compounds, most notably bases. The use of silicon dioxide 
materials in separation systems is further restricted due to the chemical 
instability of those substrates, as the dissolution of SiO.sub.2 materials 
increases in basic conditions (at pH's greater than 7.0) due to the 
general weakness of the Si--O--Si bond. 
To avoid the problems arising from the inherent chemical activity of 
silicon dioxide materials, prior separation systems have attempted 
chemical modifications to the inner silica surface of capillary walls. In 
general, such post-formation modifications are difficult as they require 
the provision of an interfacial layer to bond a desired surface treatment 
to the capillary surface, using, for example, silylating agents to create 
Si--O--Si--C bonds. Although such modifications may decrease the 
irreversible adsorption of solute molecules by the capillary surfaces, 
these systems still suffer from the chemical instability of Si--O--Si 
bonds at pH's above 7.0. Accordingly, chemical instability in SiO.sub.2 
materials remains a major problem. 
However, despite the recognized shortcomings with the chemistry of 
SiO.sub.2 substrates, those materials are still used in separation systems 
due to their desirable optical properties. In this regard, potential 
substitute materials which exhibit superior chemical properties compared 
to silicon dioxide materials are generally limited in that they are also 
highly adsorbing in the UV region, where detection is important. 
In order to avoid some of the substantial limitations present in 
conventional .mu.LC and CE techniques, and in order to enable even greater 
reduction in separation system sizes, there has been a trend towards 
providing planarized systems having capillary separation microstructures. 
In this regard, production of miniaturized separation systems involving 
fabrication of microstructures in silicon by micromachining or 
microlithographic techniques has been described. See, e.g. Fan et al., 
Anal. Chem. 66(1):177-184 (1994); Manz et al., Adv. in Chrom. 33:1-66 
(1993); Harrison et al., Sens. Actuators, B B10(2): 107-116 (1993); Manz 
et al., Trends Anal. Chem. 10(5): 144-149 (1991); and Manz et al., Sensors 
and Actuators B (Chemical) B1(1-6) :249-255 (1990) . 
The use of micromachining techniques to fabricate separation systems in 
silicon provides the practical benefit of enabling mass production of such 
systems. In this regard, a number of established techniques developed by 
the microelectronics industry involving micromachining of planar 
materials, such as silicon, exist and provide a useful and well accepted 
approach to miniaturization. Examples of the use of such micromachining 
techniques to produce miniaturized separation devices on silicon or 
borosilicate glass chips can be found in U.S. Pat. No. 5,194,133 to Clark 
et al.; U.S. Pat. No. 5,132,012 to Miura et al.; in U.S. Pat. No. 
4,908,112 to Pace; and in U.S. Pat. No. 4,891,120 to Sethi et al. 
Micromachining silicon substrates to form miniaturized separation systems 
generally involves a combination of film deposition, photolithography, 
etching and bonding techniques to fabricate a wide array of three 
dimensional structures. Silicon provides a useful substrate in this regard 
since it exhibits high strength and hardness characteristics and can be 
micromachined to provide structures having dimensions in the order of a 
few micrometers. 
Although silicon micromachining has been useful in the fabrication of 
miniaturized systems on a single surface, there are significant 
disadvantages to the use of this approach in creating the analysis device 
portion of a miniaturized separation system. 
Initially, silicon micromachining is not amenable to producing a high 
degree of alignment between two etched or machined pieces. This has a 
negative impact on the symmetry and shape of a separation channel formed 
by micromachining, which in turn may impact separation efficiency. 
Secondly, sealing of micromachined silicon surfaces is generally carried 
out using adhesives which may be prone to attack by separation conditions 
imposed by liquid phase analyses. Furthermore, under oxidizing conditions, 
a silica surface is formed on the silicon chip substrate. In this regard, 
silicon micromachining is also fundamentally limited by the chemistry of 
SiO.sub.2. Accordingly, there has remained a need for an improved 
miniaturized separation system which is able to avoid the inherent 
shortcomings of conventional miniaturization and silicon micromachining 
techniques. 
SUMMARY OF THE INVENTION 
The present invention relates to a miniaturized planar column device for 
use in a liquid phase analysis system. It is a primary object of the 
present invention to provide a miniaturized column device laser-ablated in 
a substantially planar substrate, wherein said substrate is comprised of a 
material selected to avoid the inherent chemical activity and pH 
instability encountered with silicon and prior silicon dioxide-based 
device substrates. 
The present invention is also related to the provision of detection means 
engineered into a miniaturized planar column device whereby enhanced 
on-column analysis or detection of components in a liquid sample is 
enabled. It is further contemplated under the invention to provide a 
column device for liquid phase analysis having detection means designed 
into the device in significantly compact form as compared to conventional 
technology. In one particular aspect of the present invention, it is 
contemplated to provide optical detection means ablated in a miniaturized 
planar column device and having a substantially enhanced detection 
pathlength. 
It is a further related object of the present invention to provide a device 
featuring improved means for liquid handling, including sample injection, 
and to provide a miniaturized column device with means to interface with a 
variety of external liquid reservoirs. Specifically contemplated herein is 
a system design which allows a variety of injection methods to be readily 
adapted to the planar structure, such as pressure injection, hydrodynamic 
injection or electrokinetic injection. 
It is yet a further related object of the present invention to provide a 
miniaturized total chemical analysis system (.mu.-TAS) fully contained on 
a single, planar surface. In this regard, a miniaturized system according 
to the present invention is capable of performing complex sample handling, 
separation, and detection methods with reduced technician manipulation or 
interaction. Accordingly, the subject invention finds potential 
application in monitoring and/or analysis of components in industrial 
chemical, biological, biochemical and medical processes and the like. 
A particular advantage of the present invention is the use of processes 
other than silicon micromachining techniques or etching techniques to 
create miniaturized columns in a wide variety of polymeric and ceramic 
substrates having desirable attributes for an analysis portion of a 
separation system. More specifically, it is contemplated herein to provide 
a miniaturized planar column device by ablating component microstructures 
in a substrate using laser radiation. In one preferred embodiment, a 
miniaturized column device is formed by providing two substantially planar 
halves having microstructures laser-ablated thereon, which, when the two 
halves are folded upon each other, define a separation compartment 
featuring enhanced symmetry and axial alignment. 
Use of laser ablation techniques to form miniaturized devices according to 
the present invention affords several advantages over prior etching and 
micromachining techniques used to form systems in silicon or silicon 
dioxide materials. Initially, the capability of applying rigid 
computerized control over laser ablation processes allows microstructure 
formation to be executed with great precision, thereby enabling a 
heightened degree of alignment in structures formed by component parts. 
The laser ablation process also avoids problems encountered with 
microlithographic isotropic etching techniques which may undercut masking 
during etching, giving rise to asymmetrical structures having curved side 
walls and flat bottoms. 
Laser ablation further enables the creation of microstructures with greatly 
reduced component size. In this regard, microstructures formed according 
to the invention are capable of having aspect ratios several orders of 
magnitude higher than possible using prior etching techniques, thereby 
providing enhanced separation capabilities in such devices. The use of 
laser-ablation processes to form microstructures in substrates such as 
polymers increases ease of fabrication and lowers per-unit manufacturing 
costs in the subject devices as compared to prior approaches such as 
micromachining devices in silicon. In this regard, devices formed under 
the invention in low-cost polymer substrates have the added feature of 
being capable of use as substantially disposable miniaturized column 
units. 
In another aspect of the instant invention, laser-ablation in planar 
substrates allows for the formation of microstructures of almost any 
geometry or shape. This feature not only enables the formation of complex 
device configurations, but further allows for integration of sample 
preparation, sample injection, post-column reaction and detection means in 
a miniaturized total analysis system of greatly reduced overall 
dimensions. 
The compactness of the analysis portion in a device produced under to the 
present invention, in conjunction with the feature that integral functions 
such as injection, sample handling and detection may be specifically 
engineered into the subject device to provide a .mu.-TAS device, further 
allows for integrated design of system hardware to achieve a greatly 
reduced system footprint. 
By the present invention, inherent weaknesses existing in prior approaches 
to liquid phase separation device miniaturization, and problems in using 
silicon micromachining techniques to form miniaturized column devices have 
been addressed. Accordingly, the present invention discloses a 
miniaturized column device capable of performing a variety of liquid phase 
analyses on a wide array of liquid samples.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Before the invention is described in detail, it is to be understood that 
this invention is not limited to the particular component parts of the 
devices described or process steps of the methods described as such 
devices and methods may vary. It is also to be understood that the 
terminology used herein is for purposes of describing particular 
embodiments only, and is not intended to be limiting. 
In this specification and in the claims which follow, reference will be 
made to a number of terms which shall be defined to have the following 
meanings: 
The term "substrate" is used herein to refer to any material which is 
UV-adsorbing, capable of being laser-ablated and which is not silicon or a 
silicon dioxide material such as quartz, fused silica or glass 
(borosilicates). Accordingly, it is contemplated under the present 
invention to form miniaturized column devices in suitable "substrates" 
such as laser ablatable polymers (including polyimides and the like) and 
in laser ablatable ceramics (including aluminum oxides and the like). 
The term "liquid phase analysis" is used to refer to any analysis which is 
done on either small and/or macromolecular solutes in the liquid phase. 
Accordingly, "liquid phase analysis" as used herein includes 
chromatographic separations, electrophoretic separations, and 
electrochromatographic separations. 
In this regard, "chromatographic" processes generally comprise preferential 
separations of components, and include reverse-phase, hydrophobic 
interaction, ion exchange, molecular sieve chromatography and like 
methods. 
"Electrophoretic" separations refers to the migration of particles or 
macromolecules having a net electric charge where said migration is 
influenced by an electric field. Accordingly electrophoretic separations 
contemplated under the invention include separations performed in columns 
packed with gels (such as polyacrylamide, agarose and combinations 
thereof) as well as separations performed in solution. 
"Electrochromatographic" separations refers to combinations of 
electrophoretic and chromatographic techniques. 
The term "motive force" is used to refer to any means for inducing movement 
of a sample along a column in a liquid phase analysis, and includes 
application of an electric potential across any portion of the column, 
application of a pressure differential across any portion of the column or 
any combination thereof. 
The term "surface treatment+ is used to refer to preparation or 
modification of the surface of a microchannel which will be in contact 
with a sample during separation, whereby the separation characteristics of 
the device are altered or otherwise enhanced. Accordingly, "surface 
treatment+ as used herein includes: physical surface adsorptions; covalent 
bonding of selected moieties to functional groups on the surface of 
microchannel substrates (such as to amine, hydroxyl or carboxylic acid 
groups on condensation polymers); methods of coating surfaces, including 
dynamic deactivation of channel surfaces (such as by adding surfactants to 
media), polymer grafting to the surface of channel substrates (such as 
polystyrene or divinyl-benzene) and thin-film deposition of materials such 
as diamond or sapphire to microchannel substrates. 
The term "laser ablation" is used to refer to a machining process using a 
high-energy photon laser such as an excimer laser to ablate features in a 
suitable substrate. The excimer laser can be, for example, of the F.sub.2, 
ArF, KrCl, KrF, or XeCl type. 
In general, any substrate which is UV absorbing provides a suitable 
substrate in which one may laser ablate features. Accordingly, under the 
present invention, microstructures of selected configurations can be 
formed by imaging a lithographic mask onto a suitable substrate, such as a 
polymer or ceramic material, and then laser ablating the substrate with 
laser light in areas that are unprotected by the lithographic mask. 
In laser ablation, short pulses of intense ultraviolet light are absorbed 
in a thin surface layer of material within about 1 .mu.m or less of the 
surface. Preferred pulse energies are greater than about 100 millijoules 
per square centimeter and pulse durations are shorter than about 1 
microsecond. Under these conditions, the intense ultraviolet light 
photo-dissociates the chemical bonds in the material. Furthermore, the 
absorbed ultraviolet energy is concentrated in such a small volume of 
material that it rapidly heats the dissociated fragments and ejects them 
away from the surface of the material. Because these processes occur so 
quickly, there is no time for heat to propagate to the surrounding 
material. As a result, the surrounding region is not melted or otherwise 
damaged, and the perimeter of ablated features can replicate the shape of 
the incident optical beam with precision on the scale of about one 
micrometer. 
Although laser ablation has been described herein using an excimer laser, 
it is to be understood that other ultraviolet light sources with 
substantially the same optical wavelength and energy density may be used 
to accomplish the ablation process. Preferably, the wavelength of such an 
ultraviolet light source will lie in the 150 nm to 400 nm range to allow 
high absorption in the substrate to be ablated. Furthermore, the energy 
density should be greater than about 100 millijoules per square centimeter 
with a pulse length shorter than about 1 microsecond to achieve rapid 
ejection of ablated material with essentially no heating of the 
surrounding remaining material. Laser ablation techniques, such as those 
described above, have been described in the art. Znotins, T. A., et al., 
Laser Focus Electro Optics, (1987) pp. 54-70; U.S. Pat. Nos. 5,291,226 and 
5,305,015 to Schantz et al. 
The term "injection molding" is used to refer to a process for molding 
plastic or nonplastic ceramic shapes by injecting a measured quantity of a 
molten plastic or ceramic substrate into dies (or molds). In one 
embodiment of the present invention, miniaturized column devices may be 
produced using injection molding. 
More particularly, it is contemplated to form a mold or die of a 
miniaturized column device wherein excimer laser-ablation is used to 
define an original microstructure pattern in a suitable polymer substrate. 
The microstructure thus formed may then be coated by a very thin metal 
layer and electroplated (such as by galvano forming) with a metal such as 
nickel to provide a carrier. When the metal carrier is separated from the 
original polymer, an mold insert (or tooling) is provided having the 
negative structure of the polymer. Accordingly, multiple replicas of the 
ablated microstructure pattern may be made in suitable polymer or ceramic 
substrates using injection molding techniques well known in the art. 
The term "LIGA process" is used to refer to a process for fabricating 
microstructures having high aspect ratios and increased structural 
precision using synchrotron radiation lithography, galvanoforming, and 
plastic molding. Under a LIGA process, radiation sensitive plastics are 
lithographically irradiated at high energy radiation using a synchrotron 
source to create desired microstructures (such as channels, ports, 
apertures and micro-alignment means), thereby forming a primary template. 
The primary template is then filled with a metal by electrodeposition 
techniques. The metal structure thus formed comprises a mold insert for 
the fabrication of secondary plastic templates which take the place of the 
primary template. In this manner highly accurate replicas of the original 
microstructures may be formed in a variety of substrates using injection 
or reactive injection molding techniques. The LIGA process has been 
described by Becker, E. W., et al., Microelectric Engineering 4 (1986) pp. 
35-56. Descriptions of numerous polymer substrates which may be injection 
molded using LIGA templates, and which are suitable substrates in the 
practice of the subject invention, may be found in "Contemporary Polymer 
Chemistry", Allcock H R and Lampe, F. W. (Prentice-Hall, Inc.) New Jersey 
(1981). 
Accordingly, the invention concerns formation of miniaturized column 
devices using laser ablation in a suitable substrate. It is also 
contemplated to form column devices according to the invention using 
injection molding techniques wherein the original microstructure has been 
formed by an excimer laser ablation process, or where the original 
microstructure has been formed using a LIGA process. 
More particularly, microstructures such as separation compartments, 
injection means, detection means and micro-alignment means may be formed 
in a planar substrate by excimer laser ablation. A frequency multiplied 
YAG laser may also be used in place of the excimer laser. In such a case, 
a complex microstructure pattern useful for practicing the invention may 
be formed on a suitable polymeric or ceramic substrate by combining a 
masking process with a laser ablation means, such as in a step-and-repeat 
process, where such processes would be readily understood by one of 
ordinary skill in the art. 
In the practice of the invention, a preferred substrate comprises a 
polyimide material such as those available under the trademarks 
Kapton.RTM. or Upilex.RTM. from DuPont (Wilmington, Del.), although the 
particular substrate selected may comprise any other suitable polymer or 
ceramic substrate. Polymer materials particularly contemplated herein 
include materials selected from the following classes: polyimide, 
polycarbonate, polyester, polyamide, polyether, polyolefin, or mixtures 
thereof. Further, the polymer material selected may be produced in long 
strips on a reel, and, optional sprocket holes along the sides of the 
material may be provided to accurately and securely transport the 
substrate through a step-and-repeat process. 
Under the invention, the selected polymer material is transported to a 
laser processing chamber and laser-ablated in a pattern defined by one or 
more masks using laser radiation. In a preferred embodiment, such masks 
define all of the ablated features for an extended area of the material, 
for example encompassing multiple apertures (including inlet and outlet 
ports), micro-alignment means and separation chambers. 
Alternatively, patterns such as the aperture pattern, the separation 
channel pattern, etc., may be placed side by side on a common mask 
substrate which is substantially larger than the laser beam. Such patterns 
may then be moved sequentially into the beam. In other contemplated 
production methods, one or more masks may be used to form apertures 
through the substrate, and another mask and laser energy level (and/or 
number of laser shots) may be used to define separation channels which are 
only formed through a portion of the thickness of the substrate. The 
masking material used in such masks will preferably be highly reflecting 
at the laser wavelength, consisting of, for example, a multilayer 
dielectric material or a metal such as aluminum. 
The laser ablation system employed in the invention generally includes beam 
delivery optics, alignment optics, a high precision and high speed mask 
shuttle system, and a processing chamber including mechanism for handling 
and positioning the material. In a preferred embodiment, the laser system 
uses a projection mask configuration wherein a precision lens interposed 
between the mask and the substrate projects the excimer laser light onto 
the substrate in the image of the pattern defined on the mask. 
It will be readily apparent to one of ordinary skill in the art that laser 
ablation may be used to form miniaturized separation channels and 
apertures in a wide variety of geometries. Any geometry which does not 
include undercutting may be provided using ablation techniques, such as 
modulation of laser light intensity across the substrate, stepping the 
beam across the surface or stepping the fluence and number of pulses 
applied to each location to control corresponding depth. Further, 
laser-ablated channels or chambers produced according to the invention are 
easily fabricated having ratios of channel depth to channel width which 
are much greater than previously possible using etching techniques such as 
silicon micromachining. Such aspect ratios can easily exceed unity, and 
may even reach to 10. 
In a preferred embodiment of the invention, channels of a semi-circular 
cross section are laser ablated by controlling exposure intensity or by 
making multiple exposures with the beam being reoriented between each 
exposure. Accordingly, when a corresponding semi-circular channel is 
aligned with a channel thus formed, a separation chamber of highly 
symmetrical circular cross-section is defined which may be desirable for 
enhanced fluid flow through the separation device. 
As a final step in laser ablation processes contemplated by the invention, 
a cleaning step is performed wherein the laser-ablated portion of the 
substrate is positioned under a cleaning station. At the cleaning station, 
debris from the laser ablation are removed according to standard industry 
practice. 
As will be appreciated by those working in the field of liquid phase 
analysis devices, the above-described method may be used to produce a wide 
variety of miniaturized devices. One such device is represented in FIG. 1 
where a particular embodiment of a miniaturized column device is generally 
indicated at 2. Generally, miniaturized column 2 is formed in a selected 
substrate 4 using laser ablation techniques. The substrate 4 generally 
comprises first and second substantially planar opposing surfaces 
indicated at 6 and 8 respectively, and is selected from a material other 
than silicon which is UV absorbing and, accordingly, laser-ablatable. 
In a particular embodiment of the invention, the miniaturized column device 
2 comprises a column structure ablated on a chip, which, in the practice 
of the invention may be a machinable form of the plastic polyimide such as 
Vespel.RTM.. It is particularly contemplated in the invention to use such 
a polyimide substrate as, based on considerable experience with the 
shortcomings of fused silica and research into alternatives thereof, 
polyimides have proved to be a highly desirable substrate material for the 
analysis portion of a liquid phase separation system. 
In this regard, it has been demonstrated that polyimides exhibit low 
sorptive properties towards proteins, which are known to be particularly 
difficult to analyze in prior silicon dioxide-based separation systems. 
Successful demonstrations of separations with this difficult class of 
solutes typically ensures that separation of other classes of solutes will 
be not be problematic. Further, since polyimide is a condensation polymer, 
it is possible to chemically bond groups to the surface which may provide 
a variety of desirable surface properties, depending on the target 
analysis. Unlike prior silicon dioxide based systems, these bonds to the 
polymeric substrate demonstrate pH stability in the basic region (pH 
9-10). 
Referring now to FIGS. 1-3, the substrate 4 has a microchannel 10 
laser-ablated in a first planar surface 6. It will be readily appreciated 
that, although the microchannel 10 has been represented in a generally 
extended form, microchannels formed under the invention may be ablated in 
a large variety of configurations, such as in a straight, serpentine, 
spiral, or any tortuous path desired. Further, as described in greater 
detail above, the microchannel 10 may be formed in a wide variety of 
channel geometries including semi-circular, rectangular, rhomboid, and the 
like, and the channels may be formed in a wide range of aspect ratios. It 
is also noted that a device having a plurality of microchannels 
laser-ablated thereon falls within the spirit of the present invention. 
Referring particularly to FIGS. 1 and 4, a cover plate 12 is arranged over 
said first planar surface 6 and, in combination with the laser-ablated 
microchannel 10, forms an elongate separation compartment 14. Cover plate 
12 may be formed from any suitable substrate such as polyimide, the 
selection of the substrate only being limited by avoidance of undesirable 
separation surfaces such as silicon or silicon dioxide materials. 
Under the invention, cover plate 12 may be fixably aligned over the first 
planar surface 6 to form a liquid-tight separation compartment by using 
pressure sealing techniques, by using external means to urge the pieces 
together (such as clips, tension springs or associated clamping apparatus) 
or by using adhesives well known in the art of bonding polymers, ceramics 
and the like. 
Referring to FIGS. 1-3, a particular embodiment of the invention is shown 
wherein cover plate 12 further comprises apertures ablated therein. In 
this regard, a first aperture communicates with the separation compartment 
14 at a first end 16 thereof to form an inlet port 18 enabling the passage 
of fluid from an external source into said separation compartment. A 
second aperture communicates with the separation compartment 14 at a 
second end 20 thereof to form an outlet port 22 enabling passage of fluid 
from the separation compartment to an external receptacle. Accordingly, a 
miniaturized column device is formed having a flow path extending from the 
first end 16 of the separation compartment and passing to the second end 
20 thereof, whereby liquid phase analysis of samples may be carried out 
using techniques well known in the art. 
Referring still to FIGS. 1-3, a particular embodiment of the invention is 
shown comprising sample introduction means laser-ablated into both the 
substrate 4 and cover plate 12. An internally ablated by-pass channel 24 
is formed in substrate 4, said channel 24 being disposed near the first 
end 16 of the separation compartment. Two additional apertures 26 and 28 
are formed in cover plate 12 and are arranged to cooperate with first and 
second ends (indicated at 30 and 32 respectively) of the by-pass channel 
24. In this manner, a sample being held in an external reservoir may be 
introduced into by-pass channel 24 to form a sample plug of a known volume 
(defined by the dimensions of the channel 24). The sample plug thus formed 
may then be introduced into the first end 16 of the separation compartment 
14 via inlet port 18 by communicating external mechanical valving with 
said inlet port and laser-ablated apertures 26 and 28 and flushing 
solution through the by-pass channel 24 into the separation compartment. 
It is noted that the ablated by-pass channel 24 and apertures 26 and 28 
further enable a wide variety of sample introduction techniques to be 
practiced under the invention. Particularly, having a by-pass channel 
which is not connected to the separation compartment allows a user to 
flush a sample through the by-pass channel without experiencing sample 
carry-over or column contamination. As will be appreciated by one of 
ordinary skill in the art after reading this specification, one such 
sample introduction technique may be effected by butt-coupling an 
associated rotor to a stator (not shown) on the external surface of a 
miniaturized column where the rotor selectively interfaces external tubing 
and fluid sources with inlet port 18 and apertures 26 and 28, allowing a 
sample to be flushed from the by-pass channel 24 into external tubing from 
which the sample may then be introduced into the column via inlet port 18 
for liquid phase analysis thereof. In this regard, a miniaturized column 
device formed in a polyimide substrate enables a ceramic rotor, pressed to 
the device using tensioned force (to form a liquid-tight seal), to still 
rotate between selected aperture positions on the device due to the 
friction characteristics of the two materials. 
Accordingly, in the practice of the invention, external hardware provides 
the mechanical valving necessary for communication of a miniaturized 
column device to different external liquid reservoirs, such as an 
electrolyte solution, flush solution or the sample via laser-ablated holes 
designed into the cover plate 12. This feature allows a variety of 
injection methods to be adapted to a miniaturized planar column device 
constructed according to the invention, including pressure injection, 
hydrodynamic injection or electrokinetic injection. In the particular 
embodiment of FIGS. 1-3, it is contemplated that external valving and 
injection means communicate with the separation device by butt-coupling to 
the laser-ablated apertures, however, any other suitable methods of 
connection known in the art may easily be adapted to the invention. 
Further, it is noted that numerous other sample introduction and fluid 
interfacing designs may be practiced and still fall within the spirit of 
the subject invention. 
Also under the invention, a wide variety of means for applying a motive 
force along the length of the separation compartment 14 may be associated 
with the subject device. In this regard, a pressure differential or 
electric potential may be applied along the entire length of the 
separation compartment by interfacing motive means with inlet port 18 and 
outlet port 22 
The use of substrates such as polyimides in the construction of 
miniaturized columns under the invention allows the possibility of using 
refractive-index (RI) detection to detect separated analytes of interest 
passing through the subject columns. In this regard, the provision of an 
associated laser diode which emits radiation at a wavelength where 
polyimide is "transparent" (such as at&gt;450 nm) allows for a detection 
setup where no additional features need to be ablated in the column 
devices. 
Referring now to FIGS. 2 and 3, in a preferred embodiment of the invention, 
detection means may be ablated into the substrate 4 and cover plate 12, 
where said detection means is disposed substantially downstream of the 
first end 16 of the separation compartment 14. More particularly, an 
aperture 34 may be ablated through substrate 4 to communicate with the 
separation compartment 14. A corresponding aperture 36 may be likewise 
formed in cover plate 12, and arranged so that it will be in co-axial 
alignment with aperture 34 when the cover plate is affixed to the 
substrate to form the separation compartment 14. In this manner, 
electrodes (not shown) may be connected to the miniaturized column device 
via the apertures 34 and 36 to detect separated analytes of interest 
passing through the separation compartment by electrochemical detection 
techniques. 
Referring to FIG. 5, a further contemplated embodiment of the invention, 
indicated at 2' is shown comprising a preferred detection means indicated 
generally at 42. More particularly, a first transparent sheet 38 is 
provided wherein the cover plate 12 is interposed between said first 
transparent sheet and substrate 4. A second transparent sheet 40 is also 
provided wherein the second sheet is disposed over the second planar 
surface 8 of the substrate 4. In this manner, detection means 42 allows 
optical detection of separated analytes passing through separation 
compartment 14 via transmission of radiation orthogonal to the major axis 
of the separation compartment (and, accordingly, orthogonal to the 
direction of electro-osmotic flow in an electrophoretic separation). 
Further, in the practice of the invention, the transparent sheets may 
comprise materials such as quartz, diamond, sapphire, fused silica or any 
other suitable substrate which enables light transmission therethrough. 
The subject transparent sheets may be formed with just enough surface area 
to cover and seal the detection apertures 34 and 36, or said sheets may be 
sized to cover up to the entire surface area of the column device. In this 
regard, additional structural rigidity may be provided to a column device 
formed in a particularly thin substrate film, such as a thin-film 
polyimide substrate, by employing a substantially coplanar sheet of, for 
example, fused silica. 
Accordingly, the above described optical detection means 42 enables 
adaptation of a variety of external optical detection means to 
miniaturized columns constructed according to the invention. Further, 
sealing of the transparent sheets 38 and 40 to the miniaturized column 
device 2' is readily enabled, for example, when substrate 4 and cover 
plate 12 are formed in polyimide materials which include a layer of a 
thermal adhesive form of polyimide, since it is known that 
quartz/Kapton.RTM. bonds formed using such adhesives are very resilient. 
Sealing of other preferred transparent sheet materials, such as diamond, 
sapphire or fused-silica to the subject device may be accomplished using 
adhesion techniques well known in the art. 
The possibility of detecting with radiation over a range of electromagnetic 
wavelengths offers a variety of spectrophotometric detection techniques to 
be interfaced with a miniaturized column according to the invention, 
including UV/Vis, fluorescence, refractive index (RI) and Raman. 
Furthermore, as will be readily appreciated, the use of optical detection 
means comprising apertures ablated into the substrate and cover plate 
provides great control over the effective detection pathlength in a 
miniaturized column device constructed under the invention. In this 
regard, the detection pathlength will be substantially equal to the 
combined thickness of the substrate 4 and the cover plate 12, and 
detection pathlengths of up to 250 .mu.m are readily obtainable using the 
subject detection means 42 in thin-film substrates such as polyimides. 
Referring now to FIG. 6, it can be seen that apertures 34 and 36 provide an 
enlarged volume in separation compartment 14 at the point of intersection 
with the detection means 42, where that volume will be proportional to the 
combined thickness of substrate 4 and cover plate 12. In this manner, 
sample plugs passing through separation compartment 14 may be subject to 
untoward distortion as the plug is influenced by the increased compartment 
volume in the detection area, especially where the combined thickness of 
the substrate and cover plate exceeds about 250 .mu.m, thereby possibly 
reducing separation efficiency in the device. 
Accordingly, in the present invention wherein detection pathlengths 
exceeding 250 .mu.m are desired, an alternative device embodiment may be 
provided having laser-ablated features on two opposing surfaces of a 
substrate. More particularly, in FIGS. 7 and 8, a further embodiment of a 
miniaturized column device constructed under the invention is generally 
indicated at 52. The miniaturized column comprises a substrate 54 having 
first and second substantially planar opposing surfaces respectively 
indicated at 56 and 58. The substrate 54 has a first microchannel 60 laser 
ablated in the first planar surface 56 and a second microchannel 62 laser 
ablated in the second planar surface 58, wherein said microchannels may be 
provided in a wide variety of geometries, configurations and aspect ratios 
as described in greater detail above. 
The miniaturized column device of FIGS. 7 and 8 further comprises first and 
second cover plates, indicated at 64 and 66 respectively, which, in 
combination with the first and second microchannels 60 and 62, define 
first and second elongate separation compartments when substrate 54 is 
sandwiched between said first and second cover plates. 
Referring still to FIGS. 7 and 8, a plurality of apertures may be 
laser-ablated in the device to provide an extended separation compartment, 
and further to establish fluid communication means. More particularly, a 
conduit means 72, comprising a laser ablated aperture in substrate 54 
having an axis which is orthogonal to said first and second planar 
surfaces 56 and 58, communicates a distal end 74 of said first 
microchannel 60 with a first end 76 of said second microchannel 62 to form 
an extended separation compartment. 
Further, an aperture 68, laser ablated in the first cover plate 64, enables 
fluid communication with the first microchannel 60, and a second aperture 
70, laser ablated in the second cover plate 66, enables fluid 
communication with the second microchannel 62. As will be readily 
appreciated, when said aperture 68 is used as an inlet port, and said 
second aperture 70 is used as an outlet port, a miniaturized column device 
is provided having a flow path extending along the combined length of said 
first and second microchannels 60 and 62. 
In the embodiment of the invention as shown in FIGS. 7 and 8, a wide 
variety of sample introduction means may be employed, such as those 
described in detail above. External hardware may also be interfaced to the 
subject device to provide liquid handling capabilities, and a variety of 
means for applying a motive force along the length of the separation 
compartment may be associated with the device, such as by interfacing 
motive means with the first and/or second apertures 68 and 70 as described 
in greater detail above. 
Additionally, detection means may easily be included in the subject 
embodiment. In this regard, a first aperture 78 may be laser ablated in 
the first cover plate 64, and a second aperture 80 may likewise be formed 
in the second cover plate 66 such that said first and second apertures 
will be in co-axial alignment with conduit means 72 when substrate 54 is 
sandwiched between said first and second cover plates. Accordingly, 
detection of analytes in a separated sample passing through the conduit 
means is easily enabled, such as by connecting electrodes to the 
miniaturized column via apertures 78 and 80 and detecting using 
electrochemical techniques. 
However, a key feature of the laser-ablated conduit means 72 in the 
invention is the ability to provide an extended optical detection 
pathlength of up to 1 mm, or greater, without experiencing untoward sample 
plug distortion due to increased separation compartment volumes at the 
point of detection. Referring to FIGS. 7-9, first and second transparent 
sheets, indicated at 82 and 84 respectively, may be provided such that the 
first cover plate 64 is interposed between said first transparent sheet 
and the first planar surface 56, and the second cover plate 66 is 
interposed between said second transparent sheet and the second planar 
surface 58. Under the invention, transparent sheets 82 and 84 may be 
selected from materials such as quartz crystal, fused silica, diamond, 
sapphire and the like. Further, said transparent sheets may be provided 
having just enough surface area to cover and seal the apertures 78 and 80, 
or said sheets may be sized to cover up to the entire surface area of the 
column device. As described in greater detail above, this feature allows 
additional structural rigidity to be provided to a column device formed in 
a particularly thin substrate. 
As best shown in FIG. 9, the subject arrangement allows optical detection 
of sample analytes passing through the miniaturized column device to be 
carried out along an optical detection pathlength 86 corresponding to the 
major axis of the conduit means 72. As will be readily appreciated, the 
optical detection pathlength 86 is substantially determined by the 
thickness of the substrate 54, and, accordingly, a great deal of 
flexibility in tailoring a miniaturized column device having .mu.-meter 
column dimensions and optical pathlengths of up to 1 mm or greater is 
thereby enabled under the instant invention. In this manner, a wide 
variety of associated optical detection devices may be interfaced with a 
miniaturized column constructed under the invention, and detection of 
analytes in samples passing through the conduit means 72 may be carried 
out using UV/Vis, fluorescence, refractive index (RI), Raman and like 
spectrophotometric techniques. 
Accordingly, novel miniaturized column devices have been described which 
are laser ablated into a substrate other than silicon or silicon dioxide 
materials, and which avoid several major problems which have come to be 
associated with prior attempts at providing micro-column devices. The use 
of laser ablation techniques in the practice of the invention enables 
highly symmetrical and accurately defined micro-column devices to be 
fabricated in a wide class of polymeric and ceramic substrates to provide 
a variety of miniaturized liquid-phase analysis systems. In this regard, 
miniaturized columns may be provided which have micro-capillary dimensions 
(ranging from 20-200 .mu.m in diameter) and column detection pathlengths 
of up to 1 mm or greater. This feature has not been attainable in prior 
attempts at miniaturization, such as in capillary electrophoresis, without 
substantial engineering of a device after capillary formation. Further, 
laser ablation of miniaturized columns in inert substrates such as 
polyimides avoids the problems encountered in prior devices formed in 
silicon or silicon dioxide-based materials. Such problems include the 
inherent chemical activity and pH instability of silicon and silicon 
dioxide-based substrates which limits the types of separations capable of 
being performed in those devices. 
In the practice of the invention, miniaturized column devices may be formed 
by laser ablating a set of desired features in a selected substrate using 
a step-and-repeat process to form discrete units. In this regard, it is 
particularly contemplated to laser ablate the subject devices in 
condensation polymer substrates including polyimides, polyamides, 
poly-esters and polycarbonates. Further, the instant invention may be 
practiced using either a laser ablation process or a LIGA process to form 
templates encompassing a set of desired features, whereby multiple copies 
of miniaturized columns may be mass-produced using injection molding 
techniques well known in the art. More particularly, it is contemplated 
herein to form miniaturized columns by injection molding in substrates 
comprised of materials such as the following: polycarbonates; polyesters, 
including poly(ethylene terephthalate) and poly(butylene terephthalate); 
polyamides, (such as nylons); polyethers, including polyformaldehyde and 
poly(phenylene sulfide); polyimides, such as Kapton.RTM. and Upilex.RTM.; 
polyolefin compounds, including ABS polymers, Kel-F copolymers, 
poly(methyl methacrylate), poly(styrene-butadiene) copolymers, 
poly(tetrafluoroethylene), poly(ethylenevinyl acetate) copolymers, 
poly(N-vinylcarbazole) and polystyrene. 
Laser ablation of microchannels in the surfaces of the above-described 
substrates has the added feature of enabling a wide variety of surface 
treatments to be applied to the microchannels before formation of the 
separation compartment. That is, the open configuration of laser-ablated 
microchannels produced using the method of the invention enables a number 
of surface treatments or modifications to be performed which are not 
possible in closed format constructions, such as in prior 
microcapillaries. More specifically, laser ablation in condensation 
polymer substrates provides microchannels with surfaces featuring 
functional groups, such as carboxyl groups, hydroxyl groups and amine 
groups, thereby enabling chemical bonding of selected species to the 
surface of the subject microchannels using techniques well known in the 
art. Other surface treatments enabled by the open configuration of the 
instant devices include surface adsorptions, polymer graftings and thin 
film deposition of materials such as diamond or sapphire to microchannel 
surfaces using masking and deposition techniques and dynamic deactivation 
techniques well known in the art of liquid separations. 
The ability to exert rigid computerized control over the present laser 
ablation processes enables extremely precise microstructure formation, 
which, in turn, enables the formation of miniaturized columns having 
features ablated in two substantially planar components wherein those 
components may be aligned to define a composite separation compartment of 
enhanced symmetry and axial alignment. In this regard, it is contemplated 
to provide a further embodiment of the invention wherein laser ablation is 
used to create two component halves which, when folded or aligned with one 
another, define a single miniaturized column device. 
Referring now to FIG. 10, a miniaturized column for liquid phase analysis 
of a sample is generally indicated at 102. The miniaturized column 102 is 
formed by providing a support body 104 having first and second component 
halves indicated at 106 and 108 respectively. The support body may 
comprise a substantially planar substrate such as a polyimide film which 
is both laser ablatable and flexible so as to enable folding after 
ablation; however, the particular substrate selected is not considered to 
be limiting in the invention. 
The first and second component halves 106 and 108 each have substantially 
planar interior surfaces, indicated at 110 and 112 respectively, wherein 
miniaturized column features may be laser ablated. More particularly, a 
first microchannel pattern 114 is laser ablated in the first planar 
interior surface 110 and a second microchannel pattern 116 is laser 
ablated in the second planar interior surface 112. Under the invention, 
said first and second microchannel patterns are ablated in the support 
body 104 so as to provide the mirror image of each other. 
Referring now to FIGS. 11 and 12, a separation compartment 118, comprising 
an elongate bore defined by the first and second microchannel patterns 114 
and 116 may be formed by aligning (such as by folding) the first and 
second component halves 106 and 108 in facing abutment with each other. In 
the practice of the invention, the first and second component halves may 
be held in fixable alignment with one another to form a liquid-tight 
separation compartment using pressure sealing techniques, such as by 
application of tensioned force, or by use of adhesives well known in the 
art of liquid phase separation devices. It is further contemplated under 
the invention to form first and second microchannels 114 and 116 having 
semi-circular cross-sections whereby alignment of the component halves 
defines a separation compartment 118 having a highly symmetrical circular 
cross-section to enable enhanced fluid flow therethrough; however, as 
discussed above, a wide variety of microchannel geometries are also within 
the spirit of the invention. 
In a further preferred embodiment of the invention, it is particularly 
contemplated to form the support body 104 from a polymer laminate 
substrate comprising a Kapton.RTM. film co-extruded with a thin layer of a 
thermal plastic form of polyimide referred to as KJ and available from 
DuPont (Wilmington, Del.). In this manner, the first and second component 
halves 106 and 108 may be heat sealed together, resulting in a 
liquid-tight weld that has the same chemical properties and, accordingly, 
the same mechanical, electrical and chemical stability, as the bulk 
Kapton.RTM. material. 
Referring now to FIGS. 10-12, the miniaturized column device 102 further 
comprises means for communicating associated external fluid containment 
means (not shown) with the separation compartment 118 to provide a 
liquid-phase separation device. More particularly, a plurality of 
apertures may be laser ablated in the support body 104, wherein said 
apertures extend from at least one exterior surface of the support body 
and communicate with at least one microchannel, said apertures permitting 
the passage of fluid therethrough. In this regard, an inlet port 120 may 
be laser ablated in the first component half 106 and communicate with a 
first end 122 of said first microchannel 114. In the same manner, an 
outlet port 124 may be ablated in the first component half and communicate 
with a second end 126 of said first microchannel 114. 
As is readily apparent, a liquid phase separation device may thereby be 
formed, having a flow path extending from the first end 122 of the 
microchannel 114 to the second end 126 thereof, by communicating fluids 
from an associated source (not shown) through the inlet port 120, passing 
the fluids through the separation compartment 118 formed by the alignment 
of microchannels 114 and 116, and allowing the fluids to exit the 
separation compartment via the outlet port 126. In this manner, a wide 
variety of liquid phase analysis procedures may be carried out in the 
subject miniaturized column device using techniques well known in the art. 
Furthermore, various means for applying a motive force along the length of 
the separation compartment 118, such as a pressure differential or 
electric potential, may be readily interfaced to the column device via the 
inlet and outlet ports, or by interfacing with the separation compartment 
via additional apertures which may be ablated in the support body 104. 
Inlet port 120 may be formed such that a variety of external fluid and/or 
sample introduction means may be readily interfaced with the miniaturized 
column device 102. As discussed in greater detail above, such means 
include external pressure injection, hydrodynamic injection or 
electrokinetic injection mechanisms. 
Referring now to FIGS. 10 and 11, the miniaturized column device 102 
further comprises detection means laser ablated in the support body 104. 
More particularly, a first aperture 128 is ablated in said first component 
half 106 and communicates with the first microchannel 114 at a point near 
the second end 126 thereof. A second aperture 130 is likewise formed in 
said second component half 108 to communicate with the second microchannel 
116. Accordingly, a wide variety of associated detection means may then be 
interfaced to the separation compartment 118 to detect separated analytes 
of interest passing therethrough, such as by connection of electrodes to 
the miniaturized column via the first and second apertures 128 and 130. 
In yet a further preferred embodiment of the invention, an optical 
detection means is provided in the miniaturized column device 102. In this 
regard, first and second apertures 128 and 130 may be ablated in the 
support body 104 such that when the component halves are aligned to form 
the separation compartment 118 said apertures are in co-axial alignment 
with one another, said apertures further having axes orthogonal to the 
plane of said support body. As will be readily appreciated by one of 
ordinary skill in the art, by providing transparent sheets (not shown), 
disposed over the exterior of the support body 104 and covering said first 
and second apertures 128 and 130, a sample passing through separation 
compartment 118 may be analyzed by interfacing spectrophotometric 
detection means with said sample through the transparent sheets using 
techniques well known in the art. The optical detection pathlength may be 
substantially determined by the combined thickness of said first and 
second component halves 106 and 108. In this manner, an optical detection 
pathlength of up to 250 .mu.m is readily provided by ablating the 
miniaturized column device in a 125 .mu.m polymer film. 
Accordingly, there have been described several preferred embodiments of a 
miniaturized column device formed according to the invention by laser 
ablating microstructures on component parts and aligning the components to 
form columns having enhanced symmetries. As described in detail above, 
formation of the subject microchannels in the open configuration enables a 
wide variety of surface treatments and modifications to be applied to the 
interior surfaces of the channels before formation of the separation 
compartment. In this manner, a wide variety of liquid phase analysis 
techniques may be carried out in the composite separation compartments 
thus formed, including chromatographic, electrophoretic and 
electrochromatographic separations. 
In the practice of the invention, it is further contemplated to provide 
optional means for the precise alignment of component support body halves, 
thereby ensuring accurate definition of a composite separation compartment 
formed under the invention. More particularly, in a further preferred 
embodiment of the invention, micro-alignment means are provided to enable 
enhanced alignment of laser-ablated component parts such as microchannels, 
detection apertures and the like. 
Referring now to FIGS. 13 and 14, a miniaturized column device constructed 
according to the present invention is generally indicated at 150 and is 
formed in a flexible substrate 152. The column device comprises first and 
second support body halves, indicated at 154 and 156 respectively, each 
having a substantially planar interior surface indicated at 158 and 160 
respectively. The interior surfaces comprise laser-ablated 
microstructures, generally indicated at 162, where said microstructures 
are arranged to provide the mirror image of one another in the same manner 
as described in greater detail above. 
The accurate alignment of component parts may be enabled by forming a 
miniaturized column device in a flexible substrate 152 having at least one 
fold means, generally indicated at 180, such that a first body half 154 
may be folded to overlie a second body half 156. The fold means 180 may 
comprise a row of spaced-apart perforations ablated in the substrate 152, 
spaced-apart slot-like depressions or apertures ablated so as to extend 
only part way through the substrate, or the like. The perforations or 
depressions may have circular, diamond, hexagonal or other shapes that 
promote hinge formation along a predetermined straight line. 
Accordingly, in the practice of the invention, the fold means 180 allows 
said first and second support body halves 154 and 156 to hingably fold 
upon one another and accurately align composite features defined by said 
microstructures ablated on said first and second planar interior surfaces 
158 and 160. 
It is further contemplated to provide additional micro-alignment means 
formed either by laser ablation or by other methods of fabricating shaped 
pieces well known in the art. More specifically, a plurality of 
laser-ablated apertures (not shown) may be provided in said first and 
second support body halves 154 and 156 where said apertures are so 
arranged such that co-axial alignment thereof enables the precise 
alignment of the support body halves to define composite features such as 
an ablated elongate bore. Alignment may be effected using an external 
apparatus with means (such as pins) for cooperating with said co-axial 
apertures to maintain the body halves in proper alignment with one 
another. 
Referring to FIGS. 13 and 14, in yet another particular embodiment of the 
invention, micro-alignment means may been formed in said first and second 
support body halves 154 and 156 using fabrication techniques well known in 
the art e.g., molding or the like. In this manner, a plurality of 
projections, indicated at 164, 166 and 168, may be formed in said first 
support body half 154. A plurality of depressions, indicated at 170, 172 
and 174, may be formed in said second support body half 156. 
Accordingly, as is readily apparent, the micro-alignment means are 
configured to form corresponding structures with one another, whereby 
projection 164 mates with depression 170, projection 166 mates with 
depression 172, and projection 168 mates with depression 174 when said 
support body halves are aligned in facing abutment with one another. In 
this manner, positive and precise alignment of support body halves 154 and 
156 is enabled, thereby accurately defining composite features defined by 
said laser-ablated microstructures 162. 
As will be readily apparent to one of ordinary skill in the art after 
reading this specification, a wide variety of corresponding 
micro-alignment features may be formed in the subject miniaturized column 
devices without departing from the spirit of the instant invention. Such 
additional features include any combination of holes and/or corresponding 
structures such as grooves and ridges in said component parts where said 
features cooperate to enable precise alignment of the component body 
parts. 
Further, while the present invention has been described with reference to 
specific preferred embodiments, it is understood that the description and 
examples included herein are intended to illustrate and not limit the 
scope of the invention, which is defined by the scope of the appended 
claims.