JPH05126797A - On-capillary gap junction for detecting fluorescence in capillary electrophoresis - Google Patents

Abstract

PURPOSE: To realize quick assembling for manufacturing a device and mix a reagent without significant dispersion or reduction by setting two capillaries separated by a gap and filling the gap with a buffer so as to couple them functionally. CONSTITUTION: Capillaries 513 and 514 are pressed into the body 51 of a gap junction reactor 50 by means of fitting parts 53 and 54, and while magnifying the cross point 511 of a groove 510 by a leans 52, a gap 512 is adjusted to a selected value (1 to 100μm in general) and a buffer is filled therein. The electric boundary line between the capillaries 513 and 514 eliminates substantially expansion of solute zone while crossing the gap 512, so that a sample liquid flows over the gap 512 into the capillary 514 and is separated electrically, and its luminescent element is detected. When a pair of capillaries or plastic tubes 519 and 520 are provided additionally, flexibility of the device such as fitting of luminescent indicator, addition of buffer, removal of drain, etc., can be further improved.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION This invention relates generally to capillary electrophoresis, and more particularly to the use of capillaries suitable for containing electric field lines to contain a sample stream and having a gap that minimizes the spread of solute zones.

[0002]

Prior Art and Problems to be Solved by the Invention FIG.
FIG. 1 shows an apparatus for electrophoretic separation and electrochromatography. The first buffer solution 11 is contained in a container such as a beaker 13 and the second buffer solution 12 is contained in a second container such as a beaker 14. Capillary 15 inlet end 19
In the beaker 13 and the exit end 110 of the capillary 15 in the beaker 14. High voltage electrode immersed in the first buffer solution
A voltage source 16 having a ground electrode 18 immersed in a second buffer solution 17
Causes a voltage difference of about 5 to 30 kV between these solutions.
This voltage difference causes a current through the capillary 15 of the order of 1 to 150 μA. The capillary 15 has an inner diameter of about 2 to 200 μm and a length generally in the range of 5 cm to 2 meters. Typical diameter range for capillaries is 2-200 μm
However, other diameters can be used.

FIG. 2 shows a small portion of the capillary 15 in detail. The inner cavity 20 of the capillary 15
It is filled with a conductive liquid called "supporting electrolyte". The inner surface of the wall 21 of the capillary 15 consists of silane and silicic acid group 22 (in this example, positive but other supporting electrolyte and wall 21).
, Which can be negative), which leaves excess positively charged ions 23 in the body 24 of the supporting electrolyte near the wall. The voltage source 16 has an electric field E for driving the positively charged liquid body 24 with respect to the cathode of the voltage source 16.
(→) occurs. In addition, positively charged particles are driven to the cathode and negatively charged particles such as particles 25 are driven to the anode of voltage source 16. By attaching a vacuum to the exit end 110 of the capillary 15 or by immersing the entrance end of the capillary in a glass bottle containing the sample and temporarily turning on the electric field to draw some of this sample into the capillary. , The sample is loaded into the capillary 15. The inlet end 19 of the capillary is then re-immersed in the beaker 13 and the electric field is turned on to extract sample ions from the beaker 13 through the capillary 15.

Many biological molecules are amphoteric, and the pH of the supporting electrolyte can be selected to tune the charge signature of selected sample components. Due to this ability to regulate the charge of the sample components, the regulation of the charge of the sample components allows for the separation of the sample components. However, in the separation of biological molecules, they have a selected pore size to separate the components of the sample chosen as the primary separation method, as the change in size and shape is greater than that of charge. It is advantageous to fill the internal cavity 20 of the capillary 15 with a gel.

Detection of sample components can be accomplished by a variety of mechanisms including UV absorption, fluorescence, refractive index, conductivity or electrochemical detection. This is accomplished by making measurements of the electrolyte quality, typically while in the capillary 15 or as it emerges from the exit end 110 of the capillary 15. The detection of fluid in the capillary is generally preferred because the detection cell 111 is part of the capillary and is therefore simple, and typically avoids the solute zone expansion that occurs when the sample emerges from the outlet end 110.

In the UV absorption measurement, the UV beam passes through the capillary 15 to a photodetector for recording the absorption spectrum of the sample. Capillary 15 is typically a detection cell that does not interfere with the transmission of light through the capillary.
Has a polyamide protective coating that burns out in the 111 area. Generally, capillaries are typically tubes with an inner diameter of 1 to 700 microns and an outer diameter of 0.16 centimeters.

In order to maximize the signal to noise ratio of the measurement, it is important that almost all of the UV light passes through the electrolyte. For this purpose, it is necessary to concentrate the UV light beam on the capillary 15 and make the beam diameter smaller than the inner diameter of the capillary 15. Due to the small diameter of the capillaries,
Precise adjustment of the UV beam is required. Since the capillary wall is curved and thicker than the inner diameter of the capillary,
A slight misalignment of the light beam can cause the light beam to bend, thereby highlighting this misalignment. This means that the beam passing outside the flow of electrolytic solution will be significantly subdivided.

In fluorescence detection, a fluorescent "label" is attached to the sample molecule. The label is attached before or after the electrophoretic separation of the components. However, since the label can change the charge state at the attachment position, which affects the electrophoretic separation, it is preferable to attach the label after the separation so as not to disturb the separation process. This is especially important for samples with various label attachment positions. Also, some labels, such as o-phthaldialdehyde (OPA) used to label amino groups, degrade at a rate that is amino acid dependent. It is therefore advantageous to label the sample just before entering the detection cell 111.

Post-capillary reactors suitable for introducing a fluorescent label immediately before the fluorescence detector are described in the publication (Donald J. Rose and James.
W. Jorgenson, Post-capillary fluorescence detector in capillary zone electrophoresis with o-phthaldialdehyde, Journal of Chromatography, 447 (1988) 117-131). The reactor is shown in FIG. 3 and the test apparatus incorporating the reactor is shown in FIG. This reactor 30 is a capillary
It is in the form of a T-tube 41 having a set of three ferrules 31-33 that accommodate the outlet end 110 of 15, the inlet end 34 of the reaction capillary 35 and the end 36 of the reagent capillary 37.

For high resolution, it is important that the sample solute zone be as narrow as possible. In order to avoid the spread of these zones, the outer diameter De of the electrophoresis capillary 15 is
It is smaller than the inner diameter d _r of the reaction capillary 35 so that the capillary 15 can be inserted into the capillary 35 at a short distance. The small diameter of the capillaries requires the use of a microscope for this insertion process. The labeling reagent reservoir 42 is
Hydrostatic pressure at the end 34 of the reaction capillary 35 is at a height Δh above the reservoirs 13 and 14 so as to push the labeling reagent.

In another example, the exit end 110 of the electrophoretic capillary 15 is dipped in a corrosive liquid to reduce its outer diameter.
This is done to reduce the spread of the zone caused by turbulent flow of reagents beyond the blunt outlet end 110 of the electrophoretic capillary 15. The need to use a microscope to insert this end into the reaction capillary 35, the time required to carefully etch the exit end 110, and the brittleness of this etched end are due to the manufacturing assembly of the reactor. Make the time quite long.

[0012] Stephen et al. (On-line connector for microcolumn: Application to on-column o-phthaldialdehyde derivatization of amino acids separated by capillary zone electrophoresis, Anal. Chem., 2625-2629, 60 volumes).
(1988)) show a microcolumn connector that uses a laser to create a pair of aligned holes through opposite sides of a 75 μm diameter capillary. A pair of smaller diameter capillaries are inserted into this hole to create inlet and outlet channels for fluid from the capillary zone electrophoresis (CZE) device. The chemical fluid flows into a 75 μm capillary and draws a CZE flow into the smaller diameter capillaries to attach fluorescent labels to the chemical separated by, for example, CZE.

As shown in these references, it is desirable to have a mechanism for exposing the CZE stream to other chemicals for derivation without compromising the separation of the constituent (peak) zones of the CZE stream. Unfortunately, both of these connectors require relatively fragile connector components to be aligned and assembled under the microscope, which is relatively time consuming and time consuming to manufacture.

[0014]

According to a preferred embodiment, a junction reactor is shown to be particularly useful for mixing reagent buffers on capillaries using electrophoresis and micellar chromatographic separations. .. This reactor functionally connects two capillaries separated by a buffer filled gap. The reactor is capable of mixing reagents through a gap without significant dispersion or degradation in electrophoresis and micellar separation. Also, two such as open tubular coated capillaries and gel-filled capillaries.
Capillaries of three different functional types can be used in succession, which allows the separation capabilities of these two types of processes to be successively applied to a given sample.

In this reactor, the outlet end of the first capillary is vertically connected to be substantially collinear with the inlet end of the second capillary, and the two ends are separated by a small gap. To do. A portion of these two capillaries in this junction is immersed in a buffer solution that can supply the reagents to be mixed using electrophoresis or micellar separation.

The flow velocities of the fluids in the two capillaries can be adjusted so that adjusted amounts of fluid can be drawn into the second capillaries. Adjusting the flow rate involves a number of parameters such as the inner diameter of the capillaries, the pressure drop across each capillary, the coating on the inside of the capillaries, the filling of one or both capillaries with packing material, and the potential drop and length of each capillary It can be adjusted. In contrast, in capillary zone electrophoresis, the flow F _eo is mainly determined by the applied voltage, the ionic strength of the buffer solution, the viscosity of the buffer solution, and the inner surface area of the capillary wall.

Reagents can be selected to attach the fluorescent label to the sample components so that the sample components can be detected fluorescently. The on-capillary nature of this reaction is advantageous for several reasons. The sample components can be labeled immediately in front of the fluorescence detector so that the time between the attachment of the label and the fluorescence measurement on the decaying label before reaching the detector is short. Also, the presence of the label can significantly effect the electrophoresis or micellar separation process.
Therefore, it is advantageous to minimize the distance that the labeled sample travels before reaching the detector.

For electrophoretic separation, between the buffer at the first inlet end of these two capillaries and the buffer at the second outlet end of these two capillaries,
A voltage difference V ₁ is applied. This voltage difference causes electrophoretic migration of ions within the two capillaries that can be used to electrophoretically separate the components of the sample supplied to the inlet end of the first capillary.

A buffer-filled gap can also be used as part of an improved mechanism for illuminating the sample liquid with a light beam in the detection of absorbance and fluorescence. In the case of absorbance detection, the first optical fiber is
Light is directed to the second optical fiber through this gap,
The second optical fiber directs this light to the photodetector. For fluorescence detection, light passing through the buffer-filled gap avoids light scattering by the surface of the sample-carrying capillary, which is common in the prior art. By eliminating this source of scatter, the mechanism reduces background noise, which significantly increases the signal-to-noise ratio for such measurements.

[0020]

Experimental results show that the gap does not significantly widen the sample constituent zone. An electric field line from the outlet end of the first capillary to the inlet end of the second capillary constrains the radial flow of sample in the gap,
The extent of the solute zone as it traverses the gap can be substantially eliminated.

[0021]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below with reference to the accompanying drawings. However, it is not limited to this. FIG. 5 shows an on-capillary interstitial junction reactor 50 that is particularly useful for capillary electrophoresis and micellar electrokinetic chromatography. This reactor is made of transparent, non-conductive material such as plexiglass or polymethylpentene, width 2.5 cm, height 2.5 cm, and thickness 1
It consists of a cm body 51, a lens 52 and a pair of plastic fittings 53 and 54. Since polymethylpentene is particularly transparent and chemically inert, it is selected and used as a body material. This allows the user to see inside the reactor and avoid reaction of the reagents with the body passing through the reactor.

Fittings 53 and 54 are available from Optimize Technology and are available from Head 5
5, thread 56, and this fitting in hole 58 in body 51
Includes a tapered end 57 that acts as a ferrule when secured to. The hole 58 is threaded to accommodate the threads 56 of the fitting and has a tapered end 59 for pressing against the end 57 of the fitting to act as a ferrule. A hole 58 and a set of 0.16 cm diameter channels 510 are machined into the body 51. A small gap 51 between a pair of capillaries 513 and 514 inserted by fittings 53 and 54, respectively.
A lens 52 for enlarging the intersection 511 of these grooves is glued to the side of the body 51 so that 2 can be adjusted. Gap 51
The length of 2 is generally in the range 1 to 400 microns, preferably in the range 20 to 50 microns.

Each capillary 513 and 514 has an outer diameter of 375
It is micron and the inner diameter can be selected in the range of 2 to 200 microns. Each of these capillaries has an outer diameter
Teflon tube 515 with 0.16 cm and 275 micron inner diameter
So that each Teflon tube fits snugly around the capillary. An overhanging tool, such as a needle, is pushed into the end of the Teflon tube so that it can be pushed through the smaller Teflon tube, causing a slight overhang. The Teflon is sufficiently resilient to allow even larger diameter capillaries to slide easily through the Teflon tube so that it extends about 1-2 mm beyond the other end of the tube.

The Teflon jacket portion of each capillary is pushed in through the 0.16 cm bore 516 through the connecting parts connected next, and the mounting parts are slightly fixed to the body 51,
Fit the overhanging end 57 against the capillary so that it is airtight. The Teflon jacket capillaries extend into the groove 510 as these fittings are screwed into the holes 58. A small amount of Teflon sleeve friction allows the capillaries 513 and 514 to be pushed further through the fittings 53 and 54, respectively, and the gap 512 adjusted to a user-selected value, typically in the range of 1-100 microns. Hold the magnifying glass in front of the lens 52 during the process of adjusting the gap,
The image of the intersection 511 of the groove is magnified sufficiently so that the desired gap can be selected.

A reactor having only fittings 53 and 54 can be used to combine the test liquid which is transferred via the capillary 513 to the capillary 514. However, additional fittings and connecting holes are included in the reactor 50 so that air bubbles can be removed from the intersection 511 and the test liquid can flow past the gap 512 and past the capillary 514. In the embodiment of FIG. 5, two additional holes 58 and fittings 51 are provided.
An additional pair of capillaries or plastic tubing 519 and 520 with a diameter of 0.16 cm containing 7 and 518 are connected to the reactor. "Capillary" generally has an inner diameter of 1 to 700
A tube in the micron range with an outer diameter of 0.16 cm. The utility of these additional capillaries is shown in FIG.
This figure shows an electrophoretic device utilizing an on-capillary gap junction for fluorescence detection.

The inlet end of the capillary 513 is slightly dipped in the sample solution so as to be drawn into a small amount of the sample solution, and then the inlet end is dipped in the anode buffer solution of the buffer reservoir 61.
A positive voltage from the high voltage source 62 is applied to the anodic buffer solution by an electrode 63 which is also immersed in the anodic buffer solution. The outlet end of the capillary 514 is dipped into the cathode buffer solution in the grounded buffer solution reservoir 64. This applied voltage produces an electrophoretic flow of buffer molecules filled in capillaries 513 and 514, separating the different components of the sample solution. Capillary 51
4 passes through a fluorescence detector 60 for fluorescence detection of the electrically separated components of the sample liquid.

It is necessary to attach a fluorescent label to the components so that these components can be detected by fluorescence. Since this label generally has a short decay time and interferes with electrophoretic separation, it is convenient to attach the label directly in front of the fluorescence detector. This is done by including an additional capillary 519 connected to a reagent reservoir 65 containing a reagent capable of reacting with the sample liquid for attaching the fluorescent label. Since the fluorescence detector is close to the reactor 50 (on the order of 5-8 centimeters), only a very small amount of fluorescence depletion occurs before the labeled sample passes through the fluorescence detector.

Additional capillaries, such as capillaries 520, and valve 66 are connected to reactor 50 to make the electrophoretic device more flexible. For example, the capillary 519 is connected to an additional buffer reservoir 68 and valve 67 is actuated to apply the additional reagent to the sample or the additional buffer for diluting the sample. Valve 66 is also connected to a waste reservoir to draw waste liquid from reactor 50. A vacuum pump 610 or a reservoir 68 below reactor 50 may provide a negative head pressure on the liquid in reservoir 69. In this way, the reactor 50 can apply buffers and reagents to the sample solution on-capillary, facilitating the electrophoretic separation and detection steps.

The heater member 521, which is connected to a remote power source (not shown in the figure), is generally used to accelerate the reaction.
Used to raise junction temperature to ~ 30 degrees. Many of the reactions are very temperature dependent, and even slight changes in temperature can significantly affect the reaction rate. This is particularly advantageous for reactions that attach a fluorescent label, as the fluorescent detector is located only a short distance from the intersection 511, resulting in a minimal reduction in fluorescence before the labeled sample reaches the detector.

The reactor 50 can also be used to perform spectrophotometry of the sample solution. For absorbance measurement, this involves attaching a pair of optical fibers 71 and 72 (as shown in FIG. 7) to a capillary 519 via fittings 517 and 518.
It can be done by inserting instead of 520. The optical fiber 71 has an input end connected to a light source, supplies an optical beam 73 through a gap 512 to the fiber 72 and sends this light to a photodetector. This structure has the advantage of reduced light scattering compared to other optical devices where the optical beam passes through the curved side of the capillary, such as fluorescence detector 60. In fluorescence measurements, the optical beam is projected through lens 52 into gap 512, again reducing light scattering, as compared to a device that projects light through the capillary side. This is of particular importance for fluorescence measurements, as it reduces the light intensity of the fluorescence by far, compared to the intensity of the optical beam. The scattered light from the solute molecule to the fluorescence emission (first signal) serves to reduce the sensitivity of the overall fluorescence measurement.

[0031] Figure 8 shows that the electroosmotic flow rate F _e01 capillary 513 as smaller than the electroosmotic flow rate F _e02 capillary 514 can select the parameters of the device. Therefore, in addition to liquid from capillary 513, add buffer or reagent from tube 517 to the fluid arrow.
_Pull out into capillary 514 as indicated by 81 (F _reagent ). In this state, a large number of parameters can be selected. That is, (1) the inner diameter of the capillary 514 is made larger than that of the capillary 513, and the inner surface area of the capillary 514 is large.
3 except that an electroosmotic flow rate F _e02 larger than the electroosmotic flow rate F _e01 generated in 3 is generated.
514 is the same. (2) Bundle the capillaries 513 more closely than the capillaries 514. Or (3) Capillary 51
The fluid in 7 builds up the pressure at intersection 511 more than without the fluid injection from capillary 517. It is important that reactor 50 does not significantly reduce the resolution of the electrophoretic separation. From the experimental results, this case was actually confirmed.
Electric field line 91 (shown in FIG. 9) extending from the exit end of capillary 513 to the entrance end of capillary 514.
Pushes the sample component ions existing in the capillary 513 into the capillary 514 through the gap 512.

FIGS. 10A, 10B, 11A and 11B show the broadening of the sample peak zone caused by introducing a gap into the electrophoresis path. These figures show that the peak does not decrease significantly when an intentional lateral offset is introduced between the capillaries at intersection 511. Figure 10A shows the electrophoretic data for a single electrophoretic capillary with an inner diameter of 100 microns, and Figure 10B was carefully folded to create a 125 micron gap.
Shows electrophoretic data for the same capillary after coupling into reactor 50 with a 50 micron lateral strip.
FIG. 11A shows the electrophoretic data for a single electrophoretic capillary with an inner diameter of 50 microns, and FIG. 11B after careful folding and coupling into the reactor 50 with a 20 micron deliberate lateral strip. The electrophoretic data of the same capillary is shown. Although it has a significant effect on the baseline signal, the three main peaks remain quite distinct and the main peaks are broadened only by a factor of about 4.

12A and 12B show the first with an inner diameter of 50 microns.
FIG. 6 shows the effect of the size of the gap when the outlet end of the capillary is connected to the inlet end of a second capillary with an inner diameter of 100 microns in the reactor 50. Due to the difference in inner diameter, some of the buffer solution at the intersection 511 is drawn into the second capillary along with the flow of sample liquid from the first capillary. In FIG. 12A, there are 50 capillaries.
There are micron gaps and 25 micron pieces. In FIG. 12B, the gap is increased to 400 microns and the resulting peak is reduced by a factor of about 1/2 in amplitude and has a peak half width of 2
It shows that it is increasing by the factor of.

FIGS. 13A and 13B show the results of comparing the effect of height of buffer reservoir 68 with buffer reservoirs 61 and 64. In Figure 13A, these three buffer reservoirs were at the same height. In FIG. 13B, reservoir 68 was 5 cm higher than reservoirs 61 and 64. The additional pressure in the reactor 50 will reduce the flow rate of the sample through the capillary 514 and increase the dilution of the sample as it passes through the junction. this is,
The peak height in FIG. 13B will be significantly reduced compared to FIG. 13A. In these figures, the upper trace is for the fluorescence detector and the lower trace is for the UV absorption detector. FIG. 13C shows the results when reservoir 68 is 2 cm lower than the other two reservoirs.

Figures 14A and 14B show the use of an electrophoretically separated tryptophan and histidine orthophthalaldehyde (OPA) post column induction reactor 50. These figures show the UV absorption data for these samples. In FIG. 14A, the UV light wavelength is 200 nm,
In FIG. 14B, the UV light wavelength was 230 nm. The tryptophan and histidine peaks are shown in both figures.

FIGS. 15A and 15B show OPA-induced kinetics,
The effect of increasing the temperature by 10 ° C. is shown for the concomitant increase in fluorescence. These figures show a reactor temperature of 30 ° C (Fig. 13
It shows a significantly stronger fluorescence peak when held at 40 ° C. (FIG. 13B) than when held in A).

[Brief description of drawings]

FIG. 1 shows a conventional apparatus for electrophoretic separation chromatography.

2 is a detailed view of a cross section of a capillary of the device of FIG.

FIG. 3 shows a prior art T-shaped reactor with a first capillary extending into a second capillary.

FIG. 4 shows the use of the reactor of FIG. 3 in an electrophoretic separation device.

FIG. 5A shows an on-capillary interstitial junction reactor that is particularly useful for capillary electrophoresis and micellar electrokinetic chromatography. B is an enlarged view of the gap junction reactor showing in detail a portion of the reactor adjacent to the gap junction.

FIG. 6 is a view showing a capillary zone electrophoresis apparatus using the reactor of FIG.

FIG. 7 shows the use of the reactor for optical detection by the gap between the capillaries.

[8] the first capillary is a diagram showing the entrainment of fluid bath when with small electroosmotic flow rate F _e01 than the electroosmotic flow rate F _e02 of the second capillary.

9 is a diagram showing the electric field and fluid flow lines across the gap in the reactor of FIG.

FIG. 10A shows electrophoretic data for a single 50 micron ID electrophoretic capillary, B folds carefully and with a 20 micron intentional lateral strip. It is a figure which shows the electrophoresis data in the case of the same capillary after connecting inside.

FIG. 11A shows a gap with a capillary of 50 microns.
Diagram showing fluorescence data with a 25 micron strip,
B is a diagram showing fluorescence data when the capillaries have a gap of 400 microns.

FIG. 12A shows electrophoretic data for a capillary with a 50 micron gap and a 25 micron strip, B.
FIG. 3 is a diagram showing electrophoretic data in which the factor of the spectrum obtained by increasing the gap to 400 μm decreased by half in size and increased by 2 in the half width of the peak.

13A-13C show the effect of height of buffer reservoir 68 on buffer reservoirs 61 and 64.

Figure 14 A and B show the use of reactor 50 for the separation of tryptophan and histidine.

FIG. 15: A and B show the post-column reaction of orthophthalaldehyde with tryptophan and histidine.
It is a figure which shows the change of the fluorescence response produced by the temperature increase of 10 degreeC.

[Explanation of symbols]

 11 ... 1st buffer solution 12 ... 2nd buffer solution 13 ... Beaker 14 ... Beaker 15 ... Capillary 16 ... Voltage source 17 ... High voltage electrode 18 ... Ground electrode 19 ... Inlet end 110 ... Outlet end 111 ... Detection cell 20 ... Inner cavity 21 ... Capillary wall 22 ... Silane and silicic acid group 23 ... Positively charged Ions 24 ... Liquid body 25 ... Particles 30 ... Reactor 31 ... Ferrule 32 ... Ferrule 33 ... Ferrule 34 ... Inlet end 35 ... Reaction capillary 36 ... End 37 ... Reagent capillary 41 ... T-shaped tube 42 ... Labeling reagent reservoir 50 ... On-capillary gap junction reactor 51 ... Body 52 ... Lens 53 ... Mounting part 5 4 ... Attachment parts 55 ... Head 56 ... Thread 57 ... Tapered end 58 ... Hole 59 ... Tapered end 510 ... Groove 511 ... Groove intersection 512・ ・ ・ Gap 513 ・ ・ ・ Capillary 514 ・ ・ ・ Capillary 515 ・ ・ ・ Teflon tube 516 ・ ・ ・ Bore 517 ・ ・ ・ Mounting part 518 ・ ・ ・ Mounting part 519 ・ ・ ・ Plastic tube or capillary 520 ・ ・ ・ Plastic Tube or capillary 521 ... Heater member 60 ... Fluorescence detector 61 ... Buffer solution reservoir 62 ... High voltage source 63 ... Electrode 64 ... Buffer solution reservoir 65 ... Reagent storage Device 66 ... Valve 67 ... Valve 68 ... Reservoir 69 ... Reservoir 610 ... Vacuum pump 71 ... Optical fiber 72 ... Optical Aiba 81 ... fluid arrows 91 ... field lines

Claims (24)

[Claims] 1. (a) Inserting the outlet end of the first capillary into the first end of a first groove extending through the interstitial junction reactor and attaching the capillary to the first fitting. (B) Inserting the inlet end of the second capillary into the second end of the first groove and using this capillary with the second fitting. Attached to the reactor, the first and second fittings are positioned such that the fibers are substantially collinear at the outlet end of the first capillary and the inlet end of the second capillary. And each of these capillaries is inserted into the reactor to a depth such that the outlet end of the first capillary separates from the inlet end of the second capillary by the gap,
These capillaries are not partially overlapping; (c) the two ends and the gap between them are submerged in a first buffer solution; and (d) the exit end of the first capillary to the first end. 2 to create an electric field line that traverses to the entrance end of the capillary,
This creates a non-zero voltage difference between the liquid at the outlet end of the first capillary and the liquid at the inlet end of the second capillary, which causes the sample component zone to widen during passage through the gap. A method of connecting a liquid from the outlet end of the first capillary to the inlet end of the second capillary, the process comprising steps to make the value smaller than zero. 2. The method of claim 1 further comprising the step of: (e) adjusting the temperature of the reactor and thereby adjusting the reaction rate within the reactor. 3. The method of claim 1, wherein the first and second capillaries have different inner diameters. 4. The method of claim 1, wherein the outlet end of the first capillary and the inlet end of the second capillary are separated by a distance that is about the inner diameter of one of the two capillaries. 5. An optical imaging device for enlarging a region including (f) a gap during the step of connecting and aligning the outlet end of the first capillary and the inlet end of the second capillary. The method according to claim 1, wherein the accuracy is increased in selecting a desired distance between these two ends, by calibrating with. 6. The method of claim 1, wherein the voltage across the gap is generated by creating a voltage difference between the liquid at the inlet end of the first capillary and the liquid at the outlet end of the second capillary. .. 7. The method of claim 1, further comprising the step of (g1) imaging the light beam passing through the gap. 8. The method of claim 7, further comprising: (h1) using a first optical fiber to collect a light beam traveling from the gap. 9. Further, before the step (g1), (g0) a second groove that crosses the entrance end of the first optical fiber through the first groove.
, And (g0 ') attach this capillary to the interstitial junction reactor and make a third inlet to orient the first inlet end of the optical fiber to receive light from the interstitial. 9. The method of claim 8 including the steps of using the mounting component of. 10. The step of imaging a light beam through the gap comprises passing the beam from an exit end of a second optical fiber attached to the gap junction reactor by a fourth fitting, The inlet end of the first optical fiber is substantially collinear such that the light beam passes through the gap to the first optical fiber, which makes the method particularly suitable for absorbance measurements. The method according to claim 9. 11. The light is imaged through the gap without significantly illuminating the first or second capillaries, thereby substantially reducing scatter from such capillaries. The method described. 12. The method of claim 8 wherein the entrance end of the first optical fiber is oriented so as to be substantially free of direct light from the light beam. 13. The outlet end of the first capillary is second
A gap junction reactor for connecting the first and second capillaries so that they are substantially collinear with the inlet end of the first capillary, between the first fitting and the second fitting. A body having a first groove leading to; a first capillary outlet end;
Inserted into the first end of the first groove and clamped in the first position by the first fitting; the inlet end of the second capillary is substantially the outlet end and the inlet end. Collinearly inserted into the second end of the first groove and clamped in the second position by the second fitting, each of these capillaries having two ends. The parts are inserted to a depth within this groove so that they are separated by a gap; and so that the power supply produces a voltage difference between the outlet end of the first capillary and the inlet end of the second capillary, A interstitial junction reactor adapted for fluid connection in the first and second capillaries. 14. The interstitial junction reactor of claim 13 further including a heater attached to said body. 15. A gap junction reactor in which the first and second capillaries have different inner diameters. 16. The capillary according to claim 1, wherein the outlet end of the first capillary and the inlet end of the second capillary are separated by a distance about the inner diameter of one capillary.
The gap junction reactor described in 3. 17. The lens of claim 13 further comprising a lens on the side of the reactor for allowing a user to stare through the lens to optically magnify the first groove section. Gap junction reactor. 18. The interstitial junction reactor of claim 13 wherein said body is polymethylpentene, which has a high degree of transparency and chemical inertness. 19. The gap junction reactor of claim 13 further including an optical beam source imaging the gap. 20. The method of claim 13, further comprising a first optical fiber oriented in position within the gap junction reactor for receiving light traveling from the gap between the first and second capillaries. Gap junction reactor. 21. The interstitial junction reactor of claim 20, further comprising a third fitting located relative to the interstitial junction reactor to claim the first optical fiber. 22. The gap junction reactor of claim 21 further comprising a second optical fiber positioned and clamped to the reactor by a fourth fitting to direct the light beam through the gap. 23. The gap of claim 19, wherein the light beams are directed through the gap into the first and second capillaries without substantially overlapping, thereby reducing scattering from the capillaries. Junction reactor. 24. The method of claim 23, further comprising a first optical fiber positioned and oriented within the gap junction reactor to receive light traveling from the gap between the first and second capillaries. Gap Junction Reactor.