Chromatographic separation device

A chromatographic separation device comprises a body 2 of a semiconductor material which body has a longitudinal channel 1 formed in a surface thereof, the channel 1 being capable of containing a predetermined volume of a liquid or solid material for a chromatography test or separation procedure, the channel carrying at least one electrode 6 positioned intermediate the channel ends. The semiconductor body may additionally support an electronic or optical sensor 8 arranged in line with said channel 1 to provide an integrated detection system.

This invention relates to a chromatographic separation device. It relates 
particularly to the provision of such a device that can be used for a 
variety of applications such as electrolysis, chromatography, 
electrophoresis and the study of electrokinetic phenomena. 
The development of separation technology has brought with it the need to be 
able to work accurately with very small test samples and possibly to 
provide a separation cell that can be integrated with a detection system. 
The signals from an integrated sensor will be less likely to be influenced 
by noise or leakage as compared with those of a discrete sensor. The 
sensing element would thus make use of a non-specific technique such as 
conductivity, optical (absorbance, refractive index) or electrochemical 
properties or, more likely, a suitable combination of these. 
The present invention was devised to provide a separation device that was 
capable of being manufactured in a miniature form to assist the 
microseparation and detection of biochemical and other chemical species. 
According to the invention, there is provided a chromatographic separation 
device comprising a body of a semiconductor material which body has a 
longitudinal channel formed in a surface thereof, the channel being 
capable of containing a predetermined volume of a liquid or solid material 
for a chromatographic test or separation procedure, the channel carrying 
at least one electrode positioned intermediate the channel ends. 
Preferably, an open side of the said channel is closed by a cover plate. 
The channel may be formed by an integrated circuit technique such as 
photolithography and micromachining. Alternatively, the channel may be 
formed by a micromechanical machining technique such as electromechanical 
sawing. 
The body of semiconductor material may be a silicon wafer. The separation 
device may further carry an electronic or optical sensor element which is 
located in line with the channel. The body of semiconductor material may 
be provided with two or more of the longitudinal channels, the channels 
being located in a mutually parallel arrangement.

The construction of the chromatographic separation device of the invention 
begins with the preparation of a slice of semiconductor material and in 
the embodiment about to be described this was a silicon wafer. For 
convenience in use of the separation device some of the test slices were 
formed with two channels located in a mutually parallel arrangement. 
The formation of very narrow channels in silicon wafer material can be 
achieved by using the well-developed integrated circuit technology of 
photolithography, micromachining or micromechanical machining. 
Micromachining utilises the controlled etching characteristics of silicon, 
and involves anisotropic and isotropic wet and/or dry etching. Anisotropic 
etchants, which are also known as orientation-dependent or 
crystallographic etchants, etch silicon at different rates in different 
directions in the crystal lattice; they can form a variety of well-defined 
shapes with sharp edges and corners. Typical examples include hot alkaline 
solutions such as aqueous potassium hydroxide (KOH) or sodium hydroxide 
and a mixture of ethylendiamine, pyrocatechol and water known as EDP. Dry 
etching techniques such as reactive ion etching and argon ion beam milling 
can also be employed to perform anisotropic etching. Isotropic etchants, 
on the other hand, etch the silicon crystal at the same rate in all 
directions and generally produce rounded shapes. Typical examples include 
mixtures of hydrofluoric, nitric and acetic acids known as HNA. 
The fabrication of the channel structures by micromachining involved the 
following main steps: formation of a layer of silicon dioxide on the 
silicon wafer body by a standard thermal oxidation process; definition of 
patterns on the oxidised surface by using standard photoresist and 
photolithography processes; removal of oxide by wet or dry etching where 
channels were to be formed thus exposing the silicon surface; removal of 
photoresist and etching the silicon body in places where it was 
unprotected by the oxide mask. In structures requiring long etching times, 
silicon nitride was generally employed as a masking material in place of 
silicon oxide (SiO.sub.2). In certain cases, gold and chromium metals 
might alternatively be employed for this purpose. 
Anisotropic wet etching was performed mainly on silicon wafers with two 
alternative types of crystal surface orientations namely &lt;100&gt; and &lt;110&gt;. 
Etching along {110} planes was quite rapid compared with {100} planes. The 
attack along a {111} plane was extremely slow, if it occurred at all, by 
the action of anistropic etchants. A variety of channels was formed by 
controlling orientation, shape and size of the oxide openings on the 
surface of these wafers, and etching silicon with standard anisotropic 
etchants mentioned earlier. When etching with these etchants, proper mask 
alignment with specific crystallographic axes of the wafer was considered 
of utmost importance if the required structures were to be precise. Some 
typical cross-sectional profiles of channels etched in this way in silicon 
for utilising in chromatographic and electrophoretic devices are described 
below. 
As shown in FIG. 1, V-shaped channels 1 were formed in a silicon wafer body 
2 by an alkaline etchant (KOH) acting through rectangular openings in an 
oxide mask 3 oriented along the &lt;110&gt; direction of a &lt;100&gt; wafer with 
{111} side walls. Etching was stopped in the early stages to produce the 
structure shown in FIG. 2. In the formation of narrow channels, the depth 
needed to be closely controlled by the width of the openings in the oxide 
etch mask. For producing deeper structures (greater than 50 microns), 
silicon nitride was employed as the masking material. Electrochemical 
etching was also employed to reduce the problem of undercutting the mask. 
In a &lt;110&gt; oriented wafer, two sets of {111} planes are aligned 
perpendicular to the (110) surface plane although not to each other. Long, 
deep and closely spaced channels, with vertical wall {111} side 
terminations (FIG. 3), were etched by potassium hydroxide reagent in a 
&lt;110&gt; silicon wafer; the etching ratio in the &lt;110&gt; to &lt;111&gt; direction was 
very high (this ratio being about 400 to 1). 
An isotropic etchant (HNA), with continuous agitation, was employed to 
produce the channel structures shown in FIG. 4. The HNA etch can be 
employed to produce a variety of channel structures such as serpentine, 
spiral, etc. the alignments of which are independent of crystallographic 
orientations of the silicon wafer material. A plan view of a typical 
serpentine etched structure is shown in FIG. 5. 
Although rarely used for etching silicon, the technique of micromechanical 
machining has been found to be suitable as an alternative process for 
forming the channels. It mainly involves electromechanical sawing with 
very fine diamond blades under controlled conditions to produce nearly 
rectangular, closely spaced channels in the silicon. The shapes formed are 
independent of crystallographic orientation of the silicon wafer. A 
typical sawed cross section (75 microns .times.200 microns) is shown in 
FIG. 6. 
The metal contact electrodes required in the fabrication of electrophoretic 
devices and for the detection/sensing of species by conductivity and other 
electrochemical measurements were generally deposited by a standard 
multimetal sputter deposition process. Four alternative metallisation 
schemes namely titanium/gold, titanium/platinum/gold, chromium/gold and 
chromium/platinum/gold were employed for this purpose. The thickness of 
each of the metals titanium, platinum and chromium was usually around one 
hundred nanometres whereas that of gold varied between 1 micron to 3 
microns depending upon particular design and application. Metal contact 
patterns were produced either by using standard photoresist, 
photolithography, metal etching and resist float-off technique or by 
depositing metals directly through contact ceramic or metal masks. A 
variety of etched and sawed column structures were electroded; one typical 
cross section view of such a structure is given in FIG. 7. 
As depicted in FIG. 7, the silicon wafer body 2 carries a silicon oxide 
mask 3 having a thickness of about 0.5 microns and above the channel 1 an 
electrode 6 area has been deposited. The electrode 6 area covers the two 
sides of the channel 1 and it also descends down the side walls of the 
channel and across the channel bottom. The presence of electrode material 
in the channel is not required because it reduces the cross-sectional area 
of the channel. Accordingly, this excess electrode material was removed by 
etching or by the electromechanical sawing operation to leave only the 
portions of electrode 6 which lie on top of the oxide mask 3. 
The cross-sectional view of the wafer body 2 then appears as in FIG. 8, and 
a plan view is given in FIG. 9. A view of the wafer body with the cover 
plate 7 in place is given in FIG. 10. 
FIG. 11 gives a plan view of a practical construction in which a circular 
silicon wafer body 2 having a diameter of three inches forms a substrate 
for four channels 1 each having a width about 75 microns and a depth of 
about 200 microns. Each channel 1 has two electrodes 6, the two electrodes 
being spaced longitudinally along the length of the channel. At the end of 
each channel 1 there is provided an electronic or optical sensor 8, the 
four sensors being mounted as an array on the surface of the wafer body 2. 
The resulting open channel structures formed by the abovementioned 
techniques in silicon wafer material were covered over with a Pyrex 
(Registered Trade Mark) glass cover plate which was merely placed over the 
open side of the channels or in alternative embodiments was bonded into 
place anodically or with an adhesive. This produced an enclosed capillary 
channel structure the dimensions of which could be varied from a fraction 
of a micron to many microns (about 300 microns). The minimum dimensions of 
the channels which can be employed in chromatographic devices will be 
limited by the `molecular size` of the species under detection whereas in 
the case of electrophoretic devices these will be mainly controlled by the 
thickness of the double layer of the species. The nonaqueous systems such 
as proteins and macromolecules are most likely to have larger values of 
the double layer thickness (a few orders of magnitude) compared with 
aqueous systems about 0.5 nanometers. 
Use of the chromatographic separation device of the invention is 
illustrated by the following Examples: EXAMPLE 1 Chromatography in 
Microchannels and Detection on a Silicon Wafer 
In a sample of the device corresponding to that shown in FIG. 11, the 
channels (width about 75 microns, depth about 200 microns) were filled 
with a 1.5% agarose solution, immobilised and held in place by covering 
with a glass cover plate. The operation of filling the channel was 
effected by conventional methods such as diffusion and the application of 
high pressures. The wafer was placed in a beaker containing a small 
quantity of the enzyme beta-galactosidase in sodium phosphate buffer 
solution. The device was then left standing overnight in a vertical 
position with the lower channel ends just dipping in the solution. The 
channels were subsequently analysed for enzyme activity using 
4-methylumbellifyl-beta-D-galactopyranoside, which is enzymatically 
cleaved to form a fluorescent product. Under ultraviolet irradiation the 
channels were seen to fluoresce and it was concluded that the enzyme had 
migrated up the channels. 
To detect the species near the `exit` end of the channels by conductivity 
measurements, the above experiment was repeated using sodium phosphate 
buffer solutions of various concentrations. Agarose was immobilised in the 
channels as before. The devices were allowed to stand upright in a small 
volume of sodium phosphate buffer solution, and the conductance between 
the two metal pad electrodes was then measured. The results are shown in 
the graph of FIG. 12, where the vertical axis measures conductance (in 
microsiemens) and the horizontal axis measures concentration (in 
millimoles) of the relevant sodium phosphate buffer solution. It can be 
seen that the steady state conductance is linearly related to the ionic 
strength of the buffer solution. 
These two experiments demonstrate that it is possible for biochemical or 
other chemical species to migrate along these microscopic channels and to 
be detected fluoroscopically or for the concentration of the migrating 
chemical to be determined quantitatively by the conductance measurements 
when using an integrated sensor. 
EXAMPLE 2 
A Chromatographic Separation and Sensing Device 
To construct a practical chromatographic device as shown in FIG. 13, a 
portion of the silicon wafer body 2 measuring 7.5mm .times.65mm and being 
provided with rectangular channels 1 (dimensions: 100 microns .times.200 
microns) was mounted using an epoxy resin adhesive onto a an alumina strip 
8. The alumina strip 8 measured 13mm 33 50 mm. In turn, the strip 8 was 
epoxy resin bonded to a gold plated electronics mounting package 9 having 
insulated pins 11 for making electrical connections. The chromatographic 
device was then bonded to the pins 11 by means of gold wires 12 attached 
to the electrode 6 areas. 
In tests carried out on the separation and sensing properties of this 
device, the channels were filled with a variety of chromatographic media 
for the resolution of gases and volatiles by gas chromatography and a 
variety of other materials by liquid chromatography. Thus, chromatographic 
media comprising styrene/divinylbenzene ion exchange resins and acrylic 
carboxylic, tertiary amino and chelating resins were formed in situ by 
polymerisation onto an allyldimethyl chlorosilane-activated channel. These 
media were exploited for the resolution of a variety of charged metallic, 
organic and proteinaceous species. Similarly, silica or alumina adsorbents 
comprising oxidised silicon or aluminium channels were used for the 
micro-chromatography of complex lipids, fatty acids, steroids, alkaloids, 
phenols, hydrocarbons, dicarboxylic acids, amino acids, esters, peroxides, 
aldehydes, alcohols and nucleic acids. 
Proteins and enzymes were resolved by gel filtration media comprising 
sephadexes, agaroses, acrylics or porous silicas, by ion exchange on 
carbohydrate-based or acrylic exchanges, hydrophobic adsorbents such as 
phenyl or alkyl-agaroses. Selective adsorption of individual proteins or 
groups of proteins was achieved by microaffinity chromatography on 
immobilised dyes, lectins, chelating ligands, protein A, boronate, 
heparin, nucleotides, immunoligands, oligonucleotides and nucleic acids, 
and appropriate chiral phases for the resolution of isomeric and chiral 
compounds. 
EXAMPLE 3 
An Electrophoretic Separation and Sensing Device 
In a similar construction to that used for the chromatographic device of 
Example 2, an electrophoretic device as depicted in FIG. 14 was built. For 
the electrophoretic device, the electrode 6 areas needed to be provided at 
both ends of the channels 1, consequently the pins 11 and the gold wires 
12 were similarly required to be located at both the ends of the channels 
1. 
In tests carried out on the separation and sensing properties of the 
device, it was found that the positioning of the electrodes at the distal 
ends of the channel 1 filled with an appropriate electrophoretic medium 
permitted miniaturised electrophoresis to be performed within minutes. For 
example, electrophoresis in agarose gel resolved proteins according to 
charge, whilst polyacrylamide gel electrophoresis was found to impose a 
sieving effect additional to the charge separation. Such a miniaturised 
device containing agarose or acrylamide gels could be used to resolve 
proteins, enzymes and isoenzymes in normal and abnormal serum samples. 
Similarly, agarose and polyacrylamide gel electrophoresis may be used for 
the resolution of nucleic acids and oligonucleotides and the device finds 
particular application in DNA restriction fragment analysis, DNA 
sequencing and probe analysis. These supports are also applicable to a 
variety of immunological, electrofocussing and affinity techniques. 
The chromatographic separation device of the invention is proposed to be 
employed to study the electrokinetic or zeta potential, which is involved 
in electro-osmosis, electrophoresis and allied phenomena. THe zeta 
potential is the potential between the fixed and freely mobile part of the 
double layer and for a certain class of electrolytes it has been generally 
reported to control the electrophoretic mobility. A knowledge of these 
parameters such as the zeta potential of certain ionic species under 
examination may be of help to predict the response speed of these ions for 
detection. 
The foregoing description of embodiments of the invention has been given by 
way of example only and a number of modifications may be made without 
departing from the scope of the invention as defined in the appended 
claims. For instance, in some applications it may be possible to use the 
separation device without need for a cover plate over the open side of the 
channel.