Capillary detector cartridge for electrophoresis.

BACKGROUND OF THE INVENTION

0001This invention relates to electrophoresis. More specifically, it relates to capillary electro­phoresis where a capillary is disposed within a cart­ridge and the electrophoresis occurs within the capillary of the cartridge.

SUMMARY OF THE PRIOR ART

0002Electrophoresis is fundamentally the movement of charged particles within an applied electric field. Capillary electrophoresis is known. In this type of electrophoresis, a capillary is first filled with an electrolyte. Thereafter, sample is injected at one end of the capillary. A detector is registered to a point on the capillary typically adjacent to the other end of the capillary distant from the sample. An electro­potential is applied to the capillary between the respective distal ends thereof. This electropotential is commonly applied by dipping each distal end of the capillary in a separate vial of electrolyte, with each vial being at a different electric potential.

0003With the electric potential applied, two separate flow effects occur.

0004The first of these flow effects is a gross sample flow effect. The sample moves as a mass into the capillary that was previously filled with electro­lyte. Flow throughout the entire column results.

0005The second of these flow effects is the electrophoretic flow. This causes the constituents of the sample having differing electric charge to move relative to the main stream of fluid within the capillary.

0006Classification of the sample is usually detected at a detector. The detector can either measure the electrical resistance across the capillary or alter­nately take an optical measurement, for example, absorp­tion or fluorescence. Likewise, measurements can include the change in the optical index of refraction (commonly referred to as Schlieren). Mass spectroscopy and other related techniques may as well be used.

0007Capillary electrophoresis has heretofore been a relatively difficult laboratory procedure. The cap­illary has to be individually cut, mechanically con­nected to the rest of the system and positioned with respect to a detector. Undue handling of the fragile capillary results.

0008Positioning of the capillary to the detector has been other than trivial. Specifically, capillaries are usually 50 microns inside diameter and 350 microns outside diameter.

0009These capillaries are usually protected by a protective coating. This coating first has to be removed; removal techniques include flame, heating, or chemical etching. Thereafter, the capillary has to be registered to some detector components at the section where the coating is removed. Since the capillary is very small, registration is other than trivial.

0010Sample has to be individually injected to the capillary. Two methods for such injection are known.

0011The first method requires the use of electric charge. In this method the capillary is first filled with electrolyte, an electric potential is applied from an end of a capillary immersed in sample to the oppo­site end of the capillary. The charge causes sample to be drawn into the capillary.

0012Unfortunately, with this method, the sample undergoes some classification as it enters into the capillary due to presence of the forces of electro­phoresis. Hence, for many samples, insertion of the sample by electric charge is not desirable.

0013Additionally, sample can undergo hydrodynamic flow injection. In such injection, one end of the two ends of a capillary is raised above the other distal end of the capillary. Since the capillary has already been filled with electrolyte, flow occurs. By the ex­pedient of immersing the raised end of the capillary in sample, flow of the sample into the capillary can occur.

0014Unfortunately, such a system constrains the geometry of the mechanism to which the capillary is held. For the cartridge system of holding a capillary being proposed herein, such injection is difficult. Further, trying to control the amount of sample fluid introduced precisely and reproducibility into the cap­illary is difficult.

0015It is further required that the capillary be cooled. This cooling is required because the small capillary is subject to electrical resistance heating during the period of time electrophoresis potential is applied. A small current under high voltage flowing in the capillary generates heat. There are two reasons for cooling the capillary from electrical resistance heating.

0016First, where the cartridge can become appreciably heated the classification can be seriously degraded. Such degradation occurs by heat induced dif­fusion of the so-called bands of differently charged particles migrating through the capillary at different speeds. Simply stated, the heat and its resultant dif­fusion degrade and destroy the very classification result it is the purpose of electrophoresis to create in the first place.

0017Second, the heating can occur to the point where the electrolyte in the capillary vaporizes. In this case, the continuity of the electric field across the capillary is destroyed. Moreover, damage to the capillary can occur.

0018It is also required that remote ends of the capillary be electrically isolated. Where remote ends of the capillary come in either contact or close prox­imity, arcing with destruction of both the capillary and the electrophoresis in progress can occur

SUMMARY OF THE INVENTION

0019A capillary for electrophoresis is disposed in a cartridge body. The cartridge body is cooled and provides a serpentine path for the capillary. The cap­illary is disposed over the serpentine path and pro­trudes vertically downward from the bottom of the car­tridge at two remote spaced apart locations at the bottom of the cartridge. These two remote spaced apart locations dispose the protruding capillary ends at the same eleva­tion to restrict flow within the capillary to flow induced by electrical force or fluid pressure injection. These distal ends are sealed about their periphery with respect to the cartridge where they exit the cartridge. The distal ends protrude downwardly a sufficient distance from the bottom of the cartridge body to be able to become immersed in an electrolyte for filling the cap­illary (preferably at a high pressure on one end of the capillary -- about 10 psi), a sample for charging the capillary (at a low measure pressure at one end of the capillary -- about 1 psi) or an electrolyte at an elec­trical connection for causing electrophoresis in the capillary (with the same pressure at both ends of the capillary). One portion of the capillary adjacent to an end is registered to detector components defined by the cartridge. In one preferred embodiment the capil­lary is placed within a hollow cartridge body, spiral wound about a mandrel and cooled by a coolant, which can be forced circulated to dissipate by convection heat generated during the electrophoresis. An alter­nate construction includes a boron nitride block having a serpentine depression. A capillary is threaded to the required length in the serpentine depression, insu­lated at points of crossover to prevent undesired arcing, and pasted to the block with water soluble boron nitride paste. In both cases, the cartridge provides an imme­diately interchangeable site for electrophoresis which can conveniently be utilized for automatic electrolyte and sample injection, automated electrophoresis and automated detection without special fittings connected to the capillary. Interchanging of the cartridge permits rapid change of length, detection mode and capillaries merely by the substitution of cartridges.

Other Objects, Features and Advantages

0020An object of this invention is to provide a detector cartridge for capillary electrophoresis in which the capillary is precut to a desired length, threaded over a compact serpentine path, and positioned to detector apparatus for insertion to and use in cap­illary electrophoresis apparatus. Accordingly, an en­closed cartridge is utilized. The cartridge defines a serpentine path for the capillary used in the electro­phoresis. A precut length of capillary is positioned within the cartridge. The capillary at one end is positioned for detection through detector components in the cartridge. The capillary at its two distal ends protrudes from the bottom surface of the cartridge at two spaced apart, downwardly depending locations. Each capillary end depends a sufficient distance to enable injection of electrolyte or sample in communication with vials having electrolyte with the required electro­potential therein. The periphery of the capillary is sealed to each cartridge at the exit.

0021An advantage of this cartridge is that it may be readily interchanged. Differing capillaries can readily be supplied by differing cartridges. Further, handling of the delicate capillary is eliminated.

0022A further advantage of the cartridge is that differing lengths of capillary are easily accommodated to the same size cartridge. By the expedient of winding the serpentine path back upon itself, increased lengths of capillaries can all be accommodated within the same size cartridge.

0023An additional advantage is that the capillary can be registered for detection adjacent to one of the two distal ends at the time the cartridge is fabricated. Consequently, the technician running samples through the electrophoresis no longer need worry about the registration of capillary to the detector.

0024Yet another advantage of the cartridge is that it readily accommodates all types of detection currently utilized with electrophoresis. Resistance detection, light absorption, fluorescent detection, Schlieren effect detection and mass spectroscopy can all be used. Such detectors can be positioned at one end of the capillary with the detection scheme interrupt­ing the required cooling of the capillary over a minimal length.

0025An additional advantage is that portion of the detection scheme built into the cartridge can be held to a minimum. Detection apparatus utilized out­side the cartridge can have most of the required com­plicated instrumentation affixed exterior of the car­tridge. Consequently, the construction of the detector apparatus with respect to the cartridge remains simple. Complexities of detection are reserved for apparatus outside of the cartridge itself.

0026A further object of this invention is to seal the capillary around its periphery at its spaced apart exits from the cartridge. With seal of the periphery of the capillary to the cartridge bottom depending car­tridge capillaries at their spaced apart distal ends can be immersed in liquid within the vials. The re­quired filling of the capillaries with electrolyte and injection of sample can occur. Only the vials need be manipulated, sealed and pressurized with respect to the cartridge.

0027An advantage of the seal of the capillary periphery to the cartridge bottom is that the surface of the cartridge adjacent to the depending capillaries can be used as a surface against which a pressure seal can be formed. Consequently, by immersing the end of the capillary in a vial of either electrolyte or sample and sealing the vial with respect to the bottom of the cartridge, pressure injection of the electrolyte or sample is possible. Specifically, and once the seal of a vial is made, application of pressure to the inside of the sealed vial causes entrance of fluid into the capillary under pressure.

0028An advantage of this aspect of the invention is that the cartridge intimately participates in the injection of sample. Handling of the capillary during such injection is not required. Pressure injection to an electrophoresis capillary is vastly simplified.

0029A further object to this invention is to utilize the cartridge to maintain the capillary at the correct temperature for electrophoresis. According to a first embodiment of this invention. the cartridge is provided with a hollow interior. The hollow cartridge is filled with coolant. When the cartridge is in use, cooling of the capillary against the electric resis­tance heating caused by the electrophoresis potential occurs. This cooling occurs by the resultant convection of heat generated to the coolant.

0030An advantage of this aspect is that provision for the circulation of coolant may be made. Consequently, the temperature of its capillary may be maintained at predictable limits during capillary electrophoresis.

0031According to a second and preferred embodiment of this invention, the cartridge is fabricated from a plate of highly thermally conductive material. Such a material can include a plate of boron nitride. This boron nitride is configured from one side of the plate with a serpentine groove. The capillary is threaded into this serpentine groove. Once the capillary is threaded and in place in the serpentine groove, it is in turn "pasted" into the groove. Such pasting prefer­ably occurs using a water soluble boron nitride paste. The paste is molded in place, dries, and forms an inte­grally cooling media from the capillary to the plate.

0032An advantage of this aspect is that when the cartridge is in use, cooling of the capillary against electric resistance heating can occur. Simply stated, the boron nitride cartridge is registered at at least one side to a "heat sink" -- such as a pure aluminum block of metal. This block of metal is preferably cooled. During electrophoresis, resistance heating is mitigated by the conduction of heat generated in the capillary through the plate of boron nitride and into the adjacent heat sink.

0033The advantage of this second embodiment of the invention is that the resultant heat transfer by conduction to the boron nitride has been found to be superior even to the convection. Assuming a 1.5 Kv/cm electrical potential in a 50 micron inside diameter capillary and 200-400 microamps of current, heat transfer in the solid boron carbide cartridge to temperature levels of only 10°C difference from operating ambient temperature can be maintained. Such heat transfer can be compared to heat transfer of a capillary by a fully fluorinated coolant where capillary temperature differential is maintained at 25°C relative to operating ambient temperature.

BRIEF DESCRIPTION OF THE DRAWINGS

0034Other objects, features and advantages of this invention will become more apparent after referring to the following specification and attached drawings in which: <ul id="ul0001" list-style="none"><li>Fig. 1 is a perspective view partially broken away of a capillary electrophoresis cartridge having a capillary depend from the cartridge at both distal ends and showing an interior mandrel for the winding of the capillary with the cartridge here containing coolant, preferably forced circulated for cooling of the capillary;</li><li>Fig. 2 is a detail of the cartridge adjacent to the detector taken along lines 2-2 of Fig. 1;</li><li>Fig. 3 is a front elevation of an alternate embodiment of the cartridge showing a solid block of conductive material configured with a serpentine path for the introduction of a capillary;</li><li>Fig. 4A is a bottom elevation of the cartridge of Fig. 3;</li><li>Fig. 4B is a perspective of the cartridge of Fig. 4A placed within a holder with the periphery of the capillary sealed to the holder at its point of dependency from the holder;</li><li>Fig. 5 is a detail of the channel in the cartridge of Fig. 3;</li><li>Fig. 6 is a detail of the channel adjacent to the detector of the cartridge of Fig. 3;</li><li>Fig. 7 is a perspective view of the cartridge of Fig. 3 utilized in an electrophoresis environment with the cartridge shown registered to a heat sink and the depending capillaries immersed in vials having either solvent or sample.</li><li>Figs. 8A and 8B are respective front and rear perspective elevations of the presently preferred car­tridge of this invention illustrating in exploded views the components of the cartridge and their relative placement; and,</li><li>Fig. 9 is a detail illustrating how a seal of the capillary can be effected in the embodiment of Figs. 8A and 8B.</li></ul>

0035Referring to Fig. 1, cartridge C is a container approximately 4 inches by 4 inches by 1 inch thick. The cartridge is fabricated of a dielectric material. A dielectric material, such as a commercial plastic may be used.

0036The cartridge consists of two main sections. These two main sections are bonded or fastened together to create an inner chamber 20 that holds the capillary. As will hereinafter be pointed out, this inner chamber also contains a fluid to maintain a constant temperature within the capillary.

0037Inner portion 14 comprises the main body of cartridge C. Outer portion 16 of the cartridge C in­cludes a coolant inlet 22 and a coolant outlet 24. The coolant can be a fluid such as distilled water or a completely fluorinated hydrocarbon. Completely fluo­rinated hydrocarbons are preferred over distilled water because contamination of the hydrocarbons does not as easily generate undesired conductivity of the coolant.

0038It will be understood that coolant circulation interior of the cartridge is not required; it is only preferred.

0039The capillary tube is threaded from a first aperture 30 tapped for a thread seal 32 past a bobbin 34 and onto a mandrel 36. Dependent upon the length of the capillary tube to be used, several spiral winds around mandrel 36 can occur. Capillaries of lengths from 20 cm to 100 cm have been used. Typically, the capillary tube exits mandrel 36 and thereafter passes through a seal 38 to a detector volume D. At detector volume D the capillary again passes through a seal 39 outwardly through aperture 40.

0040Once the capillary is threaded and in place, male threaded seals 32 are provided in apertures 30, 40. These seals cause a liquid tight penetration bet­ween the exterior of the capillary and the cartridge. As will hereinafter become apparent, these seals enable the cartridge to participate in the pressure injection of fluid to the capillary.

0041It is important to note that capillary Q at its distal ends 31 and 41 protrudes downwardly below the bottom 44 of the cartridge. To protect the pro­truding capillary from damage, two protective and de­tachably removable flaps 42 on either side of the car­tridge can be utilized. These protective flaps each depending down from one side of the cartridge, prevent breakage of the protruding capillary.

0042Mandrel 36, like the cartridge itself, is made of dielectric material, such as a commercial plastic. It is important that the mandrel separate the coils of the capillary. For example, it is preferred that the sides of the mandrel are threaded with threads 47. These threads 47 prevent two portions of the capillary with greatly varying electrical potentials from con­tacting each other. Should such contact occur, the difference in electrical potential could cause an arc between respective capillary portions. Such an arc would destroy the capillary as well as destroy electro­phoresis in process.

0043Some attention can be directed to the detector area D.

0044In one embodiment used for UV absorption detection, a plate 60 is provided. Plate 60 has a V groove 62, capillary Q is registered to the V groove 62. The plate also is provided with a pin hole, 63 registered to the groove. In practice this groove and pin hole have been fabricated using chemical milling technology so that registration of the capillary to the aperture is accomplished by laying the capillary in the groove. This pin hole enables light to pass from one side of the cartridge to the other side of the cartridge.

0045When registration of the capillary tube to the pinhole occurs, clamping of the capillary to the plate 60 occurs.

0046It should be understood that the portion of the capillary tube adjacent to the detector D typically has the protective coating such as a polyimide coating removed. This removal of the coating enables the silica of the capillary to be exposed for direct optical detection.

0047It should be understood that the detector opening in the cartridge is kept as small as possible. This is to minimize the length of the capillary that is not in contact with the coolant and therefore not cooled.

0048The reader will understand that the particular detector scheme utilized at D may vary. In the particular detector scheme shown, a protruding cylinder 80 registers the cartridge to detector apparatus (not shown). A detector aperture 82 holds a fiber optic 86 to abut the capillary Q. Light passing through the fiber optic is incident on the capillary. This light fluoresces the bands of material isolated by electrophoresis. Light passes out of the detection region outer housing 16 to a detector (not shown).

0049Detector aperture D can also be the light entrance for light passing through the capillary for absorption. In such case, light will pass out of the cartridge at detector aperture D in portion 14 of the housing.

0050Likewise, conventional electric resistance measurements can be made across the capillary. In such measurement, changing electrical resistance is measured across the capillary due to passage of classified material under electrophoresis. Similarly, measurements based upon differing optical index of refraction of the ad­vancing classified components (Schlieren) as well as mass spectroscopy can be used.

0051It should be noted that the length of the capillary in the vicinity of the detector aperture D is held to a minimum. This is because coolant may not be disposed around the capillary. Consequently undesired heating may occur if the noncoolant exposed portion of the capillary is not maintained at an absolute minimum.

0052It will be seen that the detector is kept as close as possible to the distal end 41 of the capillary. This is to maximize the percentage length of the capillary that is used for separation. This also minimizes the voltage that needs to be applied to the entire capillary to obtain the required potential gradient in the portion of the capillary before the detector.

0053Having set forth an embodiment of this invention with respect to Figs. 1 and 2, an alternate embodiment can be shown with respect to Figs. 3-7.

0054Referring to Fig. 3 a cartridge C′ made from a block of boron nitride is illustrated. This block is approximately 4 inches by 4 inches by 3/16th of an inch thick.

0055The block includes a serpentine path S. Serpentine path S includes multiple serpentine channels configured in any desired pattern in the block. Two constraints must be followed in the serpentine path S.

0056Referring to the curvature 100 in the upper portion of cartridge C′, the radius of curvature must be restricted to a radius wherein the capillary and its polyimide coating do not break or tear.

0057Second, at channels such as cross channel 102, where the channels cross over one from another, a channel of increased depth must be placed. Further, and after threading a capillary Q in a channel of in­creased depth, a dielectric coating is required. The dielectric coating is applied before a second remote portion of the capillary Q is threaded to the crossover point. This dielectric coating prevents the electro­potential from capillaries having remote spaced apart positions in the serpentine path from arcing and destroying the desired electrophoresis.

0058Whereas a normal channel is 34/1000ths of an inch, a deep channel may be in the order of 64/1000ths of an inch to accommodate both the overlying capillaries and the required electrical insulation placed therebetween.

0059Referring to Fig. 5, a capillary channel having a capillary Q is illustrated at a typical detail of the serpentine channel S. The channel is wider at the bottom than at the top. This configuration is accommodated by having an interior groove opening at an angle of 60°. This 60° opening includes 30° departure from the vertical at each of the channel side walls. This channel is uniformly configured throughout the serpentine path S with the exception of the detector aperture D′.

0060Referring to Fig. 4A, it can be seen that the detector aperture D′ is configured with a square sec­tioned channel. This channel extends from the detector aperture D′ to the point where the capillary Q depends from the cartridge C′.

0061Once the capillary Q is laid in the serpentine channel to the desired length, (usually in the range of 20 to 100 cm), a boron nitride paste is applied uniformly over the channel of the serpentine path S. Boron nitride paste, which is typically water soluble, dries in place leaving a solid block. This solid block typically has a high thermal conductivity. This high thermal conduc­tivity has been found more efficient in keeping the block cool from resistance heating encountered in the electrophoresis. (See upper right hand corner of Fig. 7)

0062A word can be made about the detector aperture D′. Specifically, and in this embodiment, it is no longer necessary to seal the capillary Q as it crosses the aperture D′.

0063Further, capillary Q in crossing aperture D′ is cooled at both sides immediately adjacent to the point of detection. Superior cooling results.

0064Referring to Fig. 4B, sealing the distal ends of the capillary to the cartridge is accomplished by inserting the boron nitride block into a plastic case 143 with apertures 140 along the bottom surface 141. As before, to effect the seal of the capillary to the bottom surface, sealant is inserted into openings 142 which communicate to apertures 140, filling the apertures.

0065Operation of the cartridge C′ can be easily understood with respect to Fig. 7. Typically, cartridge C′ is registered to cooling block 120. Cooling block 120 has sources of coolant 121, 122 communicated to it. The cartridge C′ is registered to one side of block 120, preferably made out of pure aluminum. Efficient cooling along the entire length of the capillary Q occurs. For example, it has been found that the solid state boron nitride block utilized enables resistance heating to be held to a level of 10° above ambient operating temperature instead of 25° for coolant convective cooling of the capillary Q.

0066Referring to Fig. 8A, the interior of the cartridge C˝ is set forth. Cartridge C˝ defines an interior volume V, which volume V contains coolant around capillary Q.

0067Capillary Q is initially threaded through air aperture passing out the bottom of the cartridge (hidden from view). The capillary is then passed a sufficient number of times around a mandrel 136 having helical threads 111. Threads 111 are given sufficient depth and spacing so that remote parts of the capillary do not make electric contact or arc through the capillary during the electrophoretic process. As illustrated in the exploded perspective of Fig. 8A, the mandrel is first wound and thereafter inserted interior of the volume V in the cartridge C.

0068The capillary Q typically is aligned the first wind around mandrel M. After the requisite number of turns to accommodate the intended length of the capillary the capillary passed over exit surface 115. At this surface the capillary gradually turns vertically downward departing exit surface 115 at point 116. Thereafter, the capillary passes across the detector aperture D, along a vertical exit passage 119 and out cartridge bottom 130. The capillary Q is registered to a bar 118 within detector D, this bar having a small central aperture for the passage of light. As will hereinafter be set forth, passage of the light through the cartridge C˝ at detector D allows the classified components to be identified.

0069Assuming that the capillary is completely threaded interior of the cartridge C, an O-ring is thereafter threaded completely around the interior of the volume V. This O-ring in passing over the capillary Q, traps the capillary in place.

0070Once the O-ring is in place, a cover 124 is placed over and sealed in position, enclosing the cartridge. (See Fig. 8B)

0071Turning to the view of Fig. 8B, the rear surface of the cartridge can be seen and understood. Detector D has a concentric and essentially circular gathering surface 125 for aligning optics to the aperture 126. Preferably, this is a V-groove 123 (see broken lines). Similarly, plate 124 has a similar aperture 127. Thus, when plate 124 closes the cartridge, light can pass through the cartridge and through the capillary Q for required detection of classified "bands" of like particles.

0072When circular gathering surface 125 is registered to a V groove, it is required the cartridge C˝ be held level with respect to its bottom surface 130. Accordingly, a second round surface 129 is down­wardly disposed for resting on a leveling bar 131 (see broken lines), this leveling bar being attached to ap­paratus for holding the cartridge (not otherwise shown).

0073With the exception of the indentation of the cartridge to define gathering surface 125 and leveling surface 129, the rear of the cartridge is essentially rectilinear.

0074A Word relating to two conduits 121 and 122 shown in broken lines in Fig. 8A. These conduits 121 and 122 are for the entry and exit of cooling fluid used during the electrophoresis within the cartridge C. It can be seen that the conduits start at the bottom surface of cartridge C˝ and extend into the volume V. Thus, before the electric potential is placed across the distal ends of the capillary Q, the entire volume of the cartridge C˝ can be flooded with coolant. Like­wise, and immediately before cartridges are removed, draining of the inner volume V can likewise occur.

0075Referring to the section illustrated in Fig. 9, the detail of the sealing of the capillary Q can be seen and understood. Capillary Q is threaded out aper­ture 119 of cartridge "C". In such threading it is depended the required distance below the bottom 130 of the cartridge.

0076Immediately before block 124 closes cartridge C˝, capillary Q has sealant forced through apertures 112 and 117. Such sealant, preferably a silicon rubber compound, fills apertures 112 and 117, surrounds the capillary Q, and effects a sealing of the periphery of the capillary Q to the cartridge C. As will hereafter become more apparent, this sealing enables fluid to be forced under pressure up the interior of the capillary Q.