CA2127977C - Method and apparatus for performing and universally detecting capillary isoelectric focusing without mobilization using concentration gradient imaging systems - Google Patents
Method and apparatus for performing and universally detecting capillary isoelectric focusing without mobilization using concentration gradient imaging systems Download PDFInfo
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- B01D57/00—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C
- B01D57/02—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C by electrophoresis
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- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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Abstract
The separations resulting from capillary electrophoresis performed in a microbore capillary tube (35) are detected on-line by focusing a light beam (63) in the form of a line or sheet of light (63) on the capillary passage (36) in which the separations (55a-c) take place so that the width of the sheet of light encompasses the length of the passage in which separations of interest are expelled to take plan. The separations form concentration gradients (16) in the capillary passage (36) encompassed by the light beam and cause refraction of portions of the light beam and variations in the intensity of the light beam due to such refraction.
Alternately, the separated components (55a-d) may absorb light of certain frequencies so if the light beam includes light of the certain frequencies, variation in the intensity of the light beam is caused by such absorbance. The variation in the intensity of light along the width of the light beam after passage through the sample (125) is sensed and is indicative of the concentration gradients (160, 161). By using a reagent (125) which reacts with an analyte not having an isoelectric point to form a product having an isoelectric point (100, 101), such analyze may be detected and measured by isoelectric focusing techniques.
Alternately, the separated components (55a-d) may absorb light of certain frequencies so if the light beam includes light of the certain frequencies, variation in the intensity of the light beam is caused by such absorbance. The variation in the intensity of light along the width of the light beam after passage through the sample (125) is sensed and is indicative of the concentration gradients (160, 161). By using a reagent (125) which reacts with an analyte not having an isoelectric point to form a product having an isoelectric point (100, 101), such analyze may be detected and measured by isoelectric focusing techniques.
Description
93/14389 ~ ''~ PCT/US93/00234 METHOD AND APPi~RATUS FOR PERFORMING AND UNIVERSALLY
DETECTING CAPILLARY ISOELECTRIC FOCUSING WITHOUT
MOBILIZATIODT USIN(= CONCENTRATION GRADIENT IMAGING SYSTEMS
Background of the Invention Fie d: The invention is in the field of capillary electrophoresis, detection methods for capillary electrophorEasis a:nd apparatus based on such methods, and on Schlieren optics.
State of the Art: It has been known for some time that a refractive index gradient such as produced by a concentration gradient in a fluid such as a gas, liquid, or supercrii~ical fluid, will cause deflection of light passed through t:he gradient. The optical method of observing anal mea~;uring the deflection of light caused by refractive index gradient fields is generally referred to as Schliere:n optics. In the past, Schlieren images resulting from light deflections have been recorded on photographic plates and the plates then analyzed for light intensity d:istrib~ution using densitometers. Recently, evaluation of the photographic images has been done by computer. These methods are useful in studying plasmas where very complicated toroidal and parabolic shapes are generated.
United ;States Letters Patent No. 4,547,071 discloses a sensor for :measuring density gradients in a nonhomogeneous fluid sample using Schlieren optics. In such sensor, a laser light beam is directed through a sample chamber and is moved along said chamber. A
quadrant light position sensor located on the opposite side of the chamber detects the deflection of the laser light beam as it is moved through the sample. The amount of deflection indicates the density gradient at any point in the sample. Rather than moving the laser beam along the sample chamber" the beam can be held constant and the sample chamber with sample therein moved. However, moving a laser and detector together in relation to a sample WO 93/14389 ~,1 ~ PCT/US93/00234 chamber and keeping the laser beam focused on the sample chamber, even a relatively large chamber, is difficult, as is moving a sample chamber through the laser beam so as to keep the laser beam properly focused. Trying to do either with a small capillary sample chamber is very difficult and impractical.
My U.S. Patent ~Nos. 4,784,494, 4,940,333 and 4,993,832 show detectors that can be used to detect concentration and thermal gradients in very small samples.
The detectors utilize a light source to generate one or two probe beams of light that pass through the sample having the gradient to be detected and the deflection of the probe beam or beams is measured on a beam position detector. Various light sources may be used to generate the probe beam or beams, such as a laser or light emitting diode (LED). These detectors, however, are designed generally to be used where the gradients to be detected move through the probe beam or beams of light.
It is also known that certain components of a sample may absorb light of certain frequencies and that the concentration of such components may be measured under some conditions by measuring the amount of light from a beam of light passing through the sample that is absorbed by the sample. Such absorption detection is also generally done by passing the sample through the light beam of the detector.
Capillary electrophoresis has become an important separation method in bioanalytical chemistry. Separation and detection of very small amounts of biological samples, about pL - nL volumes, can be achieved with capillary electrophoresis. This is generally not possible with more conventional methods of separation, even with high performance liquid chromatography. There are several capillary electrophoresis separation methods in use for different kinds of samples. They include capillary zone electrophoresis, moving boundary capillary electrophoresis, capillary isotachophoresis, and capillary WHO 93Jtd~9 PCIYtJSl3~O0i~1 isosimctric focusing. Capillary some eleotrophorosis, aoving boundary caplliary elsotrophorwis, and isotaohophoresis ail have the advantage that the sample mops through a capillary sample separation chamber during the a~eparatioa so can be used trith the detectors of ay acted prior patents. Capillary sons alectrophoresiss and aoving boundary capillary sieatxophorea~is are dynsaic processes vhara separation occurs at nn instant in tire and then the ~eones iaa~ediatelp begin to diffuse and o disperse. The diffusion takes place as the samples move through the sample chaaber to the detector. This wakes deteatian of the various,sones more difficult and less aaaurate than li~ay by desir~d_ Isotachophoresi: has the advantage that the sores stay relatively sharp as the saaple moves ttrraugh tba capillary, but isotachaphorasis is w difficult process to ~rorlc with.
Isoalectric focusing has been eaploy~d for separation of sample co~on~nts bassd on differences in their isoelectric points. Recently, the development of ao capillary vlactrophorssi~s techniques has geriersted interest in praforming the isoalactrio focusing in capillaries, since ellici~nt dissipation of Joule heat from s io-:00 ptt diameter capillary eliainates cxmveation alfeats which occur in larger sawple ohasbera and enables highly aftioient separations. Capillaries v3th microbores, i.e., with very wall inner diameter, also require only small amts of sample, which is desirable for analysis of biological matsrials, such ae mox~ocloaal antibodies and other proteins. The capi7.lary isoalactria 3o foausit~g process involves establishing an electrical field between the ends o! the capillary and establishing a stable pH gradient inside the capillary using a mixture of asopholytes. Jrt the arms time, an ampholytic analyte, such as a protein, moves along this pH gradient and is focused at the point where the pH is equivalent to its isoalectric pofnt. 'fhe migration then ceases. Thus, a stationary condition is reached and saintained in the capillary. During this separation process, narrow Gaussian bands are generated with high peak concentrations which results in high separation resolution of the analytes. In order to detect the now focused analytes with available detectors, the focused zones must be moved through a stationary detector which is usually located at one end of the capillary. Thus, the focusing of the sample in the capillary is followed by a mobilization process. The commonly used mobilization process requires addition of salt to the electrolyte at one end of the capillary. The salt causes changes in pH at that end of the capillary. Because of this pH shift, the analytes focused in the capillary are no longer at their isoelectric points and will consequently move or migrate toward the end of the capillary and will pass through the detector. During the mobilization process, distortion of the focused zones and loss in resolution are unavoidable.
Further, the mobilization process also takes at least about 15 minutes compared to the about five minutes required for the focusing itself using commonly available isoelectric focusing systems. Since the detection is necessary, the required mobilization makes the isoelectric focusing a relatively slow separation method thought to have little advantage compared to other capillary electrophoretic techniques. Therefore, it is necessary to develop an on-line detection method to eliminate the mobilization step and thereby improve the speed and performance of detection using the isoelectric focusing separation technique.
Several on-line scanning spectroscopic and radiometric detection methods have been developed for electrophoresis performed on slabs. However, such methods cannot be satisfactorily used with electrophoresis carried out in microbore capillaries because of their small size.
Recently, there have been attempts made to continuously monitor capillary isoelectric focusing separation. In one instance, photographs were taken of the focusing of blue J 93/14389 ~ ~ ~ ~~ PCT/US93/00234 dye stained proteins inside a 0.4 - 0.6 mm i.d. capillary, and the photographs used to study the zones of proteins.
However, this technique requires labeling of the proteins and can not gave good quantitative information. In 5 another instance, the separation in the capillary was monitored using chemical electrodes spaced along the length of the saimple chamber. Although a complicated 100 chemical electrode array was used, the resolution obtained in these experiments was very poor.
With currently available capillary electrophoresis equipment, the capillary tube is generally about a meter in length and each end must be manually positioned in a container holding solute or sample to be separated. The longer the capi7.lary tube, the longer the time necessary for a sample to move through the tube. When a new sample is to be separat:ed, one end of the capillary tube has to be moved to another container which contains the new sample. A:Lso, as the ends of the capillary are moved from container to container, the electrodes necessary for operation of the system must also be moved. Since voltages up to about lOKV are required to operate the system, the person moving the capillary tube and electrodes from container to container may easily come into dangerous contact with the electrodes.
One of the ;most promising applications for capillary electrophoresis is for routine analysis in research laboratories, ph<irmaceutical manufacturing facilities, and hospitals. However, in many cases, relatively rapid separation and accurate detection of samples is required, because the feedback of the analyzed data is essential for observing eaffectiveness of a therapy, adjusting drug doses in treatment of patients in hospitals, or controlling process conditions in industrial manufacturing. Also, since different methods of capillary electrophoresis apply to different types of samples and situations, it would be convenient to be able to run different methods on the same instrument. It is impossible for current commercial WO 9~111~! PCPII,lS~3~01Z~
DETECTING CAPILLARY ISOELECTRIC FOCUSING WITHOUT
MOBILIZATIODT USIN(= CONCENTRATION GRADIENT IMAGING SYSTEMS
Background of the Invention Fie d: The invention is in the field of capillary electrophoresis, detection methods for capillary electrophorEasis a:nd apparatus based on such methods, and on Schlieren optics.
State of the Art: It has been known for some time that a refractive index gradient such as produced by a concentration gradient in a fluid such as a gas, liquid, or supercrii~ical fluid, will cause deflection of light passed through t:he gradient. The optical method of observing anal mea~;uring the deflection of light caused by refractive index gradient fields is generally referred to as Schliere:n optics. In the past, Schlieren images resulting from light deflections have been recorded on photographic plates and the plates then analyzed for light intensity d:istrib~ution using densitometers. Recently, evaluation of the photographic images has been done by computer. These methods are useful in studying plasmas where very complicated toroidal and parabolic shapes are generated.
United ;States Letters Patent No. 4,547,071 discloses a sensor for :measuring density gradients in a nonhomogeneous fluid sample using Schlieren optics. In such sensor, a laser light beam is directed through a sample chamber and is moved along said chamber. A
quadrant light position sensor located on the opposite side of the chamber detects the deflection of the laser light beam as it is moved through the sample. The amount of deflection indicates the density gradient at any point in the sample. Rather than moving the laser beam along the sample chamber" the beam can be held constant and the sample chamber with sample therein moved. However, moving a laser and detector together in relation to a sample WO 93/14389 ~,1 ~ PCT/US93/00234 chamber and keeping the laser beam focused on the sample chamber, even a relatively large chamber, is difficult, as is moving a sample chamber through the laser beam so as to keep the laser beam properly focused. Trying to do either with a small capillary sample chamber is very difficult and impractical.
My U.S. Patent ~Nos. 4,784,494, 4,940,333 and 4,993,832 show detectors that can be used to detect concentration and thermal gradients in very small samples.
The detectors utilize a light source to generate one or two probe beams of light that pass through the sample having the gradient to be detected and the deflection of the probe beam or beams is measured on a beam position detector. Various light sources may be used to generate the probe beam or beams, such as a laser or light emitting diode (LED). These detectors, however, are designed generally to be used where the gradients to be detected move through the probe beam or beams of light.
It is also known that certain components of a sample may absorb light of certain frequencies and that the concentration of such components may be measured under some conditions by measuring the amount of light from a beam of light passing through the sample that is absorbed by the sample. Such absorption detection is also generally done by passing the sample through the light beam of the detector.
Capillary electrophoresis has become an important separation method in bioanalytical chemistry. Separation and detection of very small amounts of biological samples, about pL - nL volumes, can be achieved with capillary electrophoresis. This is generally not possible with more conventional methods of separation, even with high performance liquid chromatography. There are several capillary electrophoresis separation methods in use for different kinds of samples. They include capillary zone electrophoresis, moving boundary capillary electrophoresis, capillary isotachophoresis, and capillary WHO 93Jtd~9 PCIYtJSl3~O0i~1 isosimctric focusing. Capillary some eleotrophorosis, aoving boundary caplliary elsotrophorwis, and isotaohophoresis ail have the advantage that the sample mops through a capillary sample separation chamber during the a~eparatioa so can be used trith the detectors of ay acted prior patents. Capillary sons alectrophoresiss and aoving boundary capillary sieatxophorea~is are dynsaic processes vhara separation occurs at nn instant in tire and then the ~eones iaa~ediatelp begin to diffuse and o disperse. The diffusion takes place as the samples move through the sample chaaber to the detector. This wakes deteatian of the various,sones more difficult and less aaaurate than li~ay by desir~d_ Isotachophoresi: has the advantage that the sores stay relatively sharp as the saaple moves ttrraugh tba capillary, but isotachaphorasis is w difficult process to ~rorlc with.
Isoalectric focusing has been eaploy~d for separation of sample co~on~nts bassd on differences in their isoelectric points. Recently, the development of ao capillary vlactrophorssi~s techniques has geriersted interest in praforming the isoalactrio focusing in capillaries, since ellici~nt dissipation of Joule heat from s io-:00 ptt diameter capillary eliainates cxmveation alfeats which occur in larger sawple ohasbera and enables highly aftioient separations. Capillaries v3th microbores, i.e., with very wall inner diameter, also require only small amts of sample, which is desirable for analysis of biological matsrials, such ae mox~ocloaal antibodies and other proteins. The capi7.lary isoalactria 3o foausit~g process involves establishing an electrical field between the ends o! the capillary and establishing a stable pH gradient inside the capillary using a mixture of asopholytes. Jrt the arms time, an ampholytic analyte, such as a protein, moves along this pH gradient and is focused at the point where the pH is equivalent to its isoalectric pofnt. 'fhe migration then ceases. Thus, a stationary condition is reached and saintained in the capillary. During this separation process, narrow Gaussian bands are generated with high peak concentrations which results in high separation resolution of the analytes. In order to detect the now focused analytes with available detectors, the focused zones must be moved through a stationary detector which is usually located at one end of the capillary. Thus, the focusing of the sample in the capillary is followed by a mobilization process. The commonly used mobilization process requires addition of salt to the electrolyte at one end of the capillary. The salt causes changes in pH at that end of the capillary. Because of this pH shift, the analytes focused in the capillary are no longer at their isoelectric points and will consequently move or migrate toward the end of the capillary and will pass through the detector. During the mobilization process, distortion of the focused zones and loss in resolution are unavoidable.
Further, the mobilization process also takes at least about 15 minutes compared to the about five minutes required for the focusing itself using commonly available isoelectric focusing systems. Since the detection is necessary, the required mobilization makes the isoelectric focusing a relatively slow separation method thought to have little advantage compared to other capillary electrophoretic techniques. Therefore, it is necessary to develop an on-line detection method to eliminate the mobilization step and thereby improve the speed and performance of detection using the isoelectric focusing separation technique.
Several on-line scanning spectroscopic and radiometric detection methods have been developed for electrophoresis performed on slabs. However, such methods cannot be satisfactorily used with electrophoresis carried out in microbore capillaries because of their small size.
Recently, there have been attempts made to continuously monitor capillary isoelectric focusing separation. In one instance, photographs were taken of the focusing of blue J 93/14389 ~ ~ ~ ~~ PCT/US93/00234 dye stained proteins inside a 0.4 - 0.6 mm i.d. capillary, and the photographs used to study the zones of proteins.
However, this technique requires labeling of the proteins and can not gave good quantitative information. In 5 another instance, the separation in the capillary was monitored using chemical electrodes spaced along the length of the saimple chamber. Although a complicated 100 chemical electrode array was used, the resolution obtained in these experiments was very poor.
With currently available capillary electrophoresis equipment, the capillary tube is generally about a meter in length and each end must be manually positioned in a container holding solute or sample to be separated. The longer the capi7.lary tube, the longer the time necessary for a sample to move through the tube. When a new sample is to be separat:ed, one end of the capillary tube has to be moved to another container which contains the new sample. A:Lso, as the ends of the capillary are moved from container to container, the electrodes necessary for operation of the system must also be moved. Since voltages up to about lOKV are required to operate the system, the person moving the capillary tube and electrodes from container to container may easily come into dangerous contact with the electrodes.
One of the ;most promising applications for capillary electrophoresis is for routine analysis in research laboratories, ph<irmaceutical manufacturing facilities, and hospitals. However, in many cases, relatively rapid separation and accurate detection of samples is required, because the feedback of the analyzed data is essential for observing eaffectiveness of a therapy, adjusting drug doses in treatment of patients in hospitals, or controlling process conditions in industrial manufacturing. Also, since different methods of capillary electrophoresis apply to different types of samples and situations, it would be convenient to be able to run different methods on the same instrument. It is impossible for current commercial WO 9~111~! PCPII,lS~3~01Z~
capillary electrophoresis instruwenta to change lro~n one separation sethaa tv another. 8aoh instrument and capillary cartridge is designed !or a particular typo of separation, e.g., for capillary isotachophoresis, or for aoving boundary capillary e~lectrophorasis. ~1 !urther probles is that current oosssroially available capillary electrophoresis iirstruaents lack sensitive, universal, and i:~axpsnsive detectors. Rlthongh conventional absorption spectrophotometxio de~teators can be universal, they are xo not sensitive snouqt~ for capillary alactrophoresis using narrow capillaries, and as expensive sonoehromator is required. The finorosetric detevtors in use nvt only heed expensive lasers and photomultipliers but also require fluorescent derivatization !or most anaiytec. The is comserdal capillary electrop~doresis instrursnts with such detectors era usually expensive and large devices.
Also, it is sometimes desirable to separate and identify salapla ccmpotients or detatsine if such coasponerita era present in a sample, for caoasponents which do not lend ~fl themselves to separation by eleatrophoretic techniques.
Thus, other more complicated detectors and detection a~etbods are required to detect these oampo=rents.
' S~s~r o! the inyention Z$ According to one aspect of the invention there is provided a capillary electrophoresis system which includes a light transmitting capillary tube having a capillary passage therethrough is which separation of components of . a sample take place, the capillary passage having a diameter. Means is provided for introducing a sample to be separated into the capillary passage.
Also provided is means for creating a separation of a sample by electrophoresis within the capillary passage along a predetermined length of the capillary passage, the predetermined length being substantially greater than the diameter of -6a-the capillary passage. This system also includes a light source for generating a light beam and means for forming the light beam into a sheet of light having a width at least equal to the predetermine8 length of the capillary passage and a height no greater than the diameter of the capillary passage and for directing the sheet of light through the capillary passage so that the light passes through the predetermined length of the capillary passage, said predetermined length being such as to include separations of interest present in the capillary passage. Detectors means located in the path of the sheet of light after passing through the capillary passage for detecting the intensity of light at various positions along the predetermined length of the capillary passage and to provide an output representative of the light intensity at the various positions along the predetermined length of the capillary passage. The intensity of the light at various positions along the predetermined length of the capillary passage is indicative of the sample separation along the predetermined length of the capillary passage.
~t has been found that concentration gradient detention is uniquely suited for use as a detection means and method for detecting the various bands of components as separated by capillary isoelectric focusing techniques. It has also been found that isoelectric focusing can be effectively accomplished in relatively short capillary tubes, such as capillary tubes with about ten centimeters overall length, and that the actual separations take place within an eves shorter portion of such tubes.
Furthers it has been found that a light beam can be generated as a sheet or line of light, i.e., a beam of predetermined width with very small height dimension, that can be focused on the capillary passage in J 93/14389 ~ ~ ~ ~ ~ ~ ~~ ~ PCT/US93/00234 the capillary tube and the separated sample therein so as to extend along the capillary passage through the portion containing the separated sample. The light within the light beam is deflected by the concentration gradients in the sample along the width of the light beam to produce variations in the intensity of the light beam along its width which are representative of the concentration gradients established in the capillary passage by the isoelectric focusing. By monitoring the light beam after passing through lthe capillary tube and sample therein, the separation of i~he sample can be easily and quickly determined.
Rather than using a light beam to detect concentration gr<~dients directly through the deflection of portions oi° the beam, the frequency of light in the beam may be chosen so that light is absorbed by certain components of the sample to be separated. In such instance, light within the light beam is absorbed by certain components in the sample along the width of the light beam to again produce variations in the intensity of the light beam along its width which are representative of the concentration of the certain sample components in the capillary passages that have been concentrated or separated by the isoelectric focusing. Again, by monitoring the light beam after passing through the capillary tube and sample therein, the separation of the sample can be easily and quickly detez~mined. It should also be noted that the concentration and the concentration gradient are related in that t:he concentration gradient is the second derivative of they concentration. Thus, a measurement of concentration is indicative of the concentration gradients, and visa versa.
In a preferred embodiment of the invention, the intensity of the light beam after passing through the sample is m~onitor~ed by a light detector in the form of a photodiode array extending along the portion of the capillary tube where the expected separations take place.
WO 93/14389 21 ~~, r~ J ~ PCT/US93/00234 ..
The photodiode array is made up of individual sensing elements of a size small enough to be able to resolve and detect the differences in light intensity caused by the refraction of the light passing through the concentration gradients in the sample. With such an arrangement, the light source, detector, and sample chamber, i.e., capillary passage, are all fixed relative to one another to maintain accurate light beam focusing and detection, yet the measurements can be made with a stationary sample separation in the capillary. It is not necessary to move the sample through the light beam to obtain a measurement.
The entire sample of interest is comprehended by the light beam passing through the sample. Further, in the preferred embodiment where the concentration gradients are measured directly, the light beam is generated by an inexpensive He-Ne laser with a cylindrical lens to convert the beam from the laser into a sheet of light and to focus the sheet onto the capillary passage. Various other means of producing the sheet of light could be used, however.
In an alternate preferred embodiment of the invention, the intensity of the light beam after passing through the sample is monitored by a light detector in the form of a single photodiode having a narrow sensing area compared to the width of the light beam to be detected so that the detector senses only a small portion of the beam at any one time. The photodiode is mounted for movement along the sample chamber where the expected separations take place. By moving the photodiode along the sample chamber through the beam, the diode measures the light intensity at the particular location it moves through and thereby provides an output proportional to the intensity of the beam at each point the photodiode moves through.
Since the light beam is not moved with the detector, i.e., the sample chamber and the light beam remain fixed in relation to one another, the problem with maintaining alignment of the light beam and sample chamber is not present.
l 93/14389 - ~ ~ ~ ~ ~ r' ~ PCT/US93/00234 A simple and easy to use capillary electrophoresis apparatus c:an be constructed with a first reservoir and a second reservoir connected and held together with a sheet of glass, such as a microscope slide. A capillary tube secured to the :Aide extends between the reservoirs so that the capillary passage through the capillary tube extends between and connects the two reservoirs.
Electrodes are inserted into the reservoirs and the reservoirs are configured to easily accept a tube extending from a syringe to enable liquid to be easily added to or withdrawn from a reservoir. Thus, the solute or sample can lde easily changed in a reservoir by withdrawing it from the reservoir with the syringe and adding new solute: or sample, or mixture, with a syringe.
In such apparatus, the capillary passage will usually be about ten centimeaers long and have a capillary diameter of between ten and one-hundred microns. The reservoirs have small capacity so that only small sample volumes are needed. The vohume of the reservoir, however, will be larger than the volume of the capillary so that enough sample will be present to fill the capillary. With such an arrangemE~nt, any method of electrophoresis may be used.
It is not necessary to use a different apparatus for each different method of electrophoresis.
A method of the invention allows electrophoretic separation, particularly using the isoelectric focusing technique, for various sample components not otherwise subject to such separation. The method involves selecting a reagent capable of separation and having a high degree of specificity for the component to be detected. The reagent is .added to the sample, preferably in an amount sufficient t:o inte:ract with all of the sample component to be detected that might be in the sample. The reagent has its own isoe~lectr:ic point and the product of reagent and component has a different isoelectric point. Thus, with isoelectric focussing, the reagent in the sample, and the product of reagent: and component in the sample, will each WO 93/14389 r PCT/US93/00234 212'797 l 10 be separated to form different bands in the sample which can be detected by the detector of the invention.
The Drawings The best mode presently contemplated for carrying out the invention is shown in the accompanying drawings in which:
Fig. 1 is schematic representation of a light beam passing through a gradient;
Fig. 2, a schematic representation of a light beam passing through a sample chamber, showing the deflection angle produced by the presence of a refractive index gradient in the chamber;
Fig. 3, a second schematic representation of a light beam passing through a sample chamber with a concentration gradient;
Fig. 4, a schematic representation of a light beam passing through a sample chamber similar to Fig. 2, but showing a wide beam with a gradient in part of the beam;
Fig. 5, a further schematic representation of a wide light beam passing through a sample chamber with a concentration gradient in a portion of the beam;
Fig. 6, a vertical section through the capillary passage of an electrophoresis apparatus of the invention;
Fig. 7, a fragmentary horizontal section taken on the line 7-7 of Fig. 6, but showing the light source, lenses, and detector in top plan view;
Fig. 8 a fragmentary vertical section taken through a capillary tube showing sample component separation resulting from isoelectric focusing;
Fig. 9, a vertical section through a light beam formed by the apparatus of the invention;
Fig. 10, a fragmentary vertical section taken on the line 10-10 of Fig. 7;
Fig. 11, a fragmentary top plan view of an alternate detector of the invention;
212"1977 Fig. 12, a block diagram of a detector of the invention;
Fig. 13, a curve representing the light intensity profile of a probe light beam passing through a sample separated by isoe:lectric focusing in the apparatus of the invention a.nd produced as the output signal by a moving detector;
Fig. 14, a .curve representing the integrals of the curve of Fig. 13;
l0 Fig. 15, three curves representing the light intensity profilea of a probe light beam passing through a sample during separation by isoelectric focusing in the apparatus c>f the invention and produced as the output signals by a moving detector;
Fig. 16, i:hree curves representing the light intensity profiles of a probe light beam passing through a sample during separation by isoelectric focusing in the apparatus o~f the invention and produced as the output signal by a diode array detector;
Fig. :17, a fragmentary vertical section of an alternate sample receiving reservoir usable with the apparatus o:E Figs. 6 and 7;
Fig. 18, a schematic representation of light intensity pnofile~s showing separations obtained through a method of the invention;
Fig. 1.9, a curve representing the concentration profile of a sample separated in the apparatus of the invention a:nd mobilized to move through a detector and produced as the output signal by the detector;
Fig. 20, curves showing the concentration gradient profiles of the same sample separated using capillary zone electrophorsais and moving boundary capillary electrophoresis;
Fig. 2~., a curve showing the concentration gradient profile for the same sample as used for Fig. 20, showing the sample separaited by isoelectric focusing;
WO 93/14389 '~ 1 '~ ~ ~ r~ ~ PCT/US93/00234 _ 12 Fig. 22, a curve representing the light intensity profile of a probe light beam passing through a hemoglobin sample during separation by isoelectric focusing in the apparatus of the invention wherein the curve represents concentration of sample components; and Fig. 23, a curve representing the light intensity profile of a probe light beam passing through a hemoglobin sample similar to that for Fig. 22, wherein the curve represents the concentration gradients of the sample components.
Detailed Description of the Preferred Embodiment It is well known that light passing through a refractive index gradient in a solution is deflected. The physical reason for light deflection when passing through this gradient lies in the relationship between the refractive index and light propagation velocity.
Different parts of the light advance to a different degree with time, which generates the phase shift. Thus, as shown in Fig. 1, during a given time period t+dt, light at the top of a light beam indicated by arrow 10 which is passing through a solution with a refractive index of n+dn will travel a distance of D1. The light at the bottom of the light beam indicated by arrow ll which is passing through a solution with a refractive index of n will travel a distance D2. This results in a tilt of the light wavefront and since light travels perpendicular to the wavefront, the light beam is tilted as illustrated. In Fig. 1, D2 is greater than D1 resulting in an upward tilt, but depending upon the values of n and n+dn, the tilt could be downward.
The light path through the refractive index gradient can be calculated by using the Fermat principle that the light path through the medium is such that the time necessary for its traversal is minimum. The relationship between the angle of deflection, 8, and the refractive ~3 index gradient normal t0 the light propnQation dn/dx and path length through this gradient, D, can bs written as tan 8 = sink (D/n)(dn/dx)~
(D/n) (dn/dx) + (dn/dx) ° (D'/na3 : ) + (dn/dx) s ' 8 (Ds/ns51 ) + .. .
where n is the refractive index of the tedium. In situations where the sensor ox the invention will be used, D and 8 are small. ws can then approximate:
Also, it is sometimes desirable to separate and identify salapla ccmpotients or detatsine if such coasponerita era present in a sample, for caoasponents which do not lend ~fl themselves to separation by eleatrophoretic techniques.
Thus, other more complicated detectors and detection a~etbods are required to detect these oampo=rents.
' S~s~r o! the inyention Z$ According to one aspect of the invention there is provided a capillary electrophoresis system which includes a light transmitting capillary tube having a capillary passage therethrough is which separation of components of . a sample take place, the capillary passage having a diameter. Means is provided for introducing a sample to be separated into the capillary passage.
Also provided is means for creating a separation of a sample by electrophoresis within the capillary passage along a predetermined length of the capillary passage, the predetermined length being substantially greater than the diameter of -6a-the capillary passage. This system also includes a light source for generating a light beam and means for forming the light beam into a sheet of light having a width at least equal to the predetermine8 length of the capillary passage and a height no greater than the diameter of the capillary passage and for directing the sheet of light through the capillary passage so that the light passes through the predetermined length of the capillary passage, said predetermined length being such as to include separations of interest present in the capillary passage. Detectors means located in the path of the sheet of light after passing through the capillary passage for detecting the intensity of light at various positions along the predetermined length of the capillary passage and to provide an output representative of the light intensity at the various positions along the predetermined length of the capillary passage. The intensity of the light at various positions along the predetermined length of the capillary passage is indicative of the sample separation along the predetermined length of the capillary passage.
~t has been found that concentration gradient detention is uniquely suited for use as a detection means and method for detecting the various bands of components as separated by capillary isoelectric focusing techniques. It has also been found that isoelectric focusing can be effectively accomplished in relatively short capillary tubes, such as capillary tubes with about ten centimeters overall length, and that the actual separations take place within an eves shorter portion of such tubes.
Furthers it has been found that a light beam can be generated as a sheet or line of light, i.e., a beam of predetermined width with very small height dimension, that can be focused on the capillary passage in J 93/14389 ~ ~ ~ ~ ~ ~ ~~ ~ PCT/US93/00234 the capillary tube and the separated sample therein so as to extend along the capillary passage through the portion containing the separated sample. The light within the light beam is deflected by the concentration gradients in the sample along the width of the light beam to produce variations in the intensity of the light beam along its width which are representative of the concentration gradients established in the capillary passage by the isoelectric focusing. By monitoring the light beam after passing through lthe capillary tube and sample therein, the separation of i~he sample can be easily and quickly determined.
Rather than using a light beam to detect concentration gr<~dients directly through the deflection of portions oi° the beam, the frequency of light in the beam may be chosen so that light is absorbed by certain components of the sample to be separated. In such instance, light within the light beam is absorbed by certain components in the sample along the width of the light beam to again produce variations in the intensity of the light beam along its width which are representative of the concentration of the certain sample components in the capillary passages that have been concentrated or separated by the isoelectric focusing. Again, by monitoring the light beam after passing through the capillary tube and sample therein, the separation of the sample can be easily and quickly detez~mined. It should also be noted that the concentration and the concentration gradient are related in that t:he concentration gradient is the second derivative of they concentration. Thus, a measurement of concentration is indicative of the concentration gradients, and visa versa.
In a preferred embodiment of the invention, the intensity of the light beam after passing through the sample is m~onitor~ed by a light detector in the form of a photodiode array extending along the portion of the capillary tube where the expected separations take place.
WO 93/14389 21 ~~, r~ J ~ PCT/US93/00234 ..
The photodiode array is made up of individual sensing elements of a size small enough to be able to resolve and detect the differences in light intensity caused by the refraction of the light passing through the concentration gradients in the sample. With such an arrangement, the light source, detector, and sample chamber, i.e., capillary passage, are all fixed relative to one another to maintain accurate light beam focusing and detection, yet the measurements can be made with a stationary sample separation in the capillary. It is not necessary to move the sample through the light beam to obtain a measurement.
The entire sample of interest is comprehended by the light beam passing through the sample. Further, in the preferred embodiment where the concentration gradients are measured directly, the light beam is generated by an inexpensive He-Ne laser with a cylindrical lens to convert the beam from the laser into a sheet of light and to focus the sheet onto the capillary passage. Various other means of producing the sheet of light could be used, however.
In an alternate preferred embodiment of the invention, the intensity of the light beam after passing through the sample is monitored by a light detector in the form of a single photodiode having a narrow sensing area compared to the width of the light beam to be detected so that the detector senses only a small portion of the beam at any one time. The photodiode is mounted for movement along the sample chamber where the expected separations take place. By moving the photodiode along the sample chamber through the beam, the diode measures the light intensity at the particular location it moves through and thereby provides an output proportional to the intensity of the beam at each point the photodiode moves through.
Since the light beam is not moved with the detector, i.e., the sample chamber and the light beam remain fixed in relation to one another, the problem with maintaining alignment of the light beam and sample chamber is not present.
l 93/14389 - ~ ~ ~ ~ ~ r' ~ PCT/US93/00234 A simple and easy to use capillary electrophoresis apparatus c:an be constructed with a first reservoir and a second reservoir connected and held together with a sheet of glass, such as a microscope slide. A capillary tube secured to the :Aide extends between the reservoirs so that the capillary passage through the capillary tube extends between and connects the two reservoirs.
Electrodes are inserted into the reservoirs and the reservoirs are configured to easily accept a tube extending from a syringe to enable liquid to be easily added to or withdrawn from a reservoir. Thus, the solute or sample can lde easily changed in a reservoir by withdrawing it from the reservoir with the syringe and adding new solute: or sample, or mixture, with a syringe.
In such apparatus, the capillary passage will usually be about ten centimeaers long and have a capillary diameter of between ten and one-hundred microns. The reservoirs have small capacity so that only small sample volumes are needed. The vohume of the reservoir, however, will be larger than the volume of the capillary so that enough sample will be present to fill the capillary. With such an arrangemE~nt, any method of electrophoresis may be used.
It is not necessary to use a different apparatus for each different method of electrophoresis.
A method of the invention allows electrophoretic separation, particularly using the isoelectric focusing technique, for various sample components not otherwise subject to such separation. The method involves selecting a reagent capable of separation and having a high degree of specificity for the component to be detected. The reagent is .added to the sample, preferably in an amount sufficient t:o inte:ract with all of the sample component to be detected that might be in the sample. The reagent has its own isoe~lectr:ic point and the product of reagent and component has a different isoelectric point. Thus, with isoelectric focussing, the reagent in the sample, and the product of reagent: and component in the sample, will each WO 93/14389 r PCT/US93/00234 212'797 l 10 be separated to form different bands in the sample which can be detected by the detector of the invention.
The Drawings The best mode presently contemplated for carrying out the invention is shown in the accompanying drawings in which:
Fig. 1 is schematic representation of a light beam passing through a gradient;
Fig. 2, a schematic representation of a light beam passing through a sample chamber, showing the deflection angle produced by the presence of a refractive index gradient in the chamber;
Fig. 3, a second schematic representation of a light beam passing through a sample chamber with a concentration gradient;
Fig. 4, a schematic representation of a light beam passing through a sample chamber similar to Fig. 2, but showing a wide beam with a gradient in part of the beam;
Fig. 5, a further schematic representation of a wide light beam passing through a sample chamber with a concentration gradient in a portion of the beam;
Fig. 6, a vertical section through the capillary passage of an electrophoresis apparatus of the invention;
Fig. 7, a fragmentary horizontal section taken on the line 7-7 of Fig. 6, but showing the light source, lenses, and detector in top plan view;
Fig. 8 a fragmentary vertical section taken through a capillary tube showing sample component separation resulting from isoelectric focusing;
Fig. 9, a vertical section through a light beam formed by the apparatus of the invention;
Fig. 10, a fragmentary vertical section taken on the line 10-10 of Fig. 7;
Fig. 11, a fragmentary top plan view of an alternate detector of the invention;
212"1977 Fig. 12, a block diagram of a detector of the invention;
Fig. 13, a curve representing the light intensity profile of a probe light beam passing through a sample separated by isoe:lectric focusing in the apparatus of the invention a.nd produced as the output signal by a moving detector;
Fig. 14, a .curve representing the integrals of the curve of Fig. 13;
l0 Fig. 15, three curves representing the light intensity profilea of a probe light beam passing through a sample during separation by isoelectric focusing in the apparatus c>f the invention and produced as the output signals by a moving detector;
Fig. 16, i:hree curves representing the light intensity profiles of a probe light beam passing through a sample during separation by isoelectric focusing in the apparatus o~f the invention and produced as the output signal by a diode array detector;
Fig. :17, a fragmentary vertical section of an alternate sample receiving reservoir usable with the apparatus o:E Figs. 6 and 7;
Fig. 18, a schematic representation of light intensity pnofile~s showing separations obtained through a method of the invention;
Fig. 1.9, a curve representing the concentration profile of a sample separated in the apparatus of the invention a:nd mobilized to move through a detector and produced as the output signal by the detector;
Fig. 20, curves showing the concentration gradient profiles of the same sample separated using capillary zone electrophorsais and moving boundary capillary electrophoresis;
Fig. 2~., a curve showing the concentration gradient profile for the same sample as used for Fig. 20, showing the sample separaited by isoelectric focusing;
WO 93/14389 '~ 1 '~ ~ ~ r~ ~ PCT/US93/00234 _ 12 Fig. 22, a curve representing the light intensity profile of a probe light beam passing through a hemoglobin sample during separation by isoelectric focusing in the apparatus of the invention wherein the curve represents concentration of sample components; and Fig. 23, a curve representing the light intensity profile of a probe light beam passing through a hemoglobin sample similar to that for Fig. 22, wherein the curve represents the concentration gradients of the sample components.
Detailed Description of the Preferred Embodiment It is well known that light passing through a refractive index gradient in a solution is deflected. The physical reason for light deflection when passing through this gradient lies in the relationship between the refractive index and light propagation velocity.
Different parts of the light advance to a different degree with time, which generates the phase shift. Thus, as shown in Fig. 1, during a given time period t+dt, light at the top of a light beam indicated by arrow 10 which is passing through a solution with a refractive index of n+dn will travel a distance of D1. The light at the bottom of the light beam indicated by arrow ll which is passing through a solution with a refractive index of n will travel a distance D2. This results in a tilt of the light wavefront and since light travels perpendicular to the wavefront, the light beam is tilted as illustrated. In Fig. 1, D2 is greater than D1 resulting in an upward tilt, but depending upon the values of n and n+dn, the tilt could be downward.
The light path through the refractive index gradient can be calculated by using the Fermat principle that the light path through the medium is such that the time necessary for its traversal is minimum. The relationship between the angle of deflection, 8, and the refractive ~3 index gradient normal t0 the light propnQation dn/dx and path length through this gradient, D, can bs written as tan 8 = sink (D/n)(dn/dx)~
(D/n) (dn/dx) + (dn/dx) ° (D'/na3 : ) + (dn/dx) s ' 8 (Ds/ns51 ) + .. .
where n is the refractive index of the tedium. In situations where the sensor ox the invention will be used, D and 8 are small. ws can then approximate:
8 = (D/n)(dn/dx) to Fig. 2 illustrates the detection principle behind this method. With a nonunifora distribution of a solute in the sample ohambex shown schematically between sample chamber walls 12 and 13 giving a sample chamber distance D, a concentration gradient is established. This gradient 15 forces the corresponding refractive index gradient dn/dx R
(dn/dc)(da/dx), which then tilts or deflects the propagating light beam by angle 9 ~ (b/n) (dn/dc)(dc/dx).
This deflection cat? be measured by measuring the position of the light beam on the position detector 14. The ~o information produced during the measurewent of the concnntration gradient relates to the universal property of the solute -- refractive index n. Consequently, the concentration gradient produced by any solute that has a different n than the solvent will be detected by noting a . 25 deflection or tilt in the light beaa.
Fig. 3 shows the same principle as Fig. 2, but illustrates it somewhat differently. Thus, if a concentration gradient represented by line 16 exists in a sample in a sample chamber defined by walls 12 and 13, a 30 probe beam o! light 1~ directed through the sample will be deflected as indicated above by an angle 6. This causes the position of the beam to cove on the suxface pf the ' position sensor 14 as indicated above.
Fig. 4 is similnr to Fig. 2, but shows a wide light 15 bean with a concentration gradient 18 within the width of the light bean. Thus, rather than the light beam being uniformly deflected as shown in Figs. 1, Z, and 3, a portion of the light beam 19, on one aide of the gradient 18, which does not pass through a gradient, passes xtraight through the sample to detector 14. portion 20 of the light bean passes through the gradient 18 and is defleoted by angle A as shorn by the broken arrows, and falls onto detector 14 partially overlapping portion 19.
Where the overlap ocGUrs on the detector, indicated ~xt 22, the light striking detector 14 is brighter than in non overlapping areas. Portion 21 of the light beans on the other side of the gradient 18, again passes straight through the sable and onto detector 14. As indicated in Fig. 4, there will be an area Z3 Where little or no light will fall. Thus, the single light beam after passing through a sale with one or~aore gradients therein wi7.l have varying intensity indicating the gradients present in the sample. A gradient in the saapie will generally result in a bright spot followed by a spot of very little intensity, or, if just measuring intensity, a level representing the light passing straight through the ao sa~ople, an increased or positive signal (the increased intensity), followed by decreased or negative signal (tbe decreased intensity), followed again by the level representing the light passing etxaight through the sample.
.25 If the detector 14 is broken down into many small detectors, such as an array of detectors, each detector detecting the light from a s=ell portion of the beam, a comparison or scanning o! the individual detectors will produce an output signal representative of the intensity 30 of the light beam falling on the array along its length.
Alternatively, a single, small detector which detects only a portion of the light beam could be positioned to be roved through the beam along its width and measure the light intensity as it is moved.
Fig. 5 ahorrvs the same principle as Fig. 4, but relates it more directly to a s~rstem of the invention.
Thus, again, tire light beam say be considered as many 93/14389 21 ~'~ (~ ~ ~ PCT/US93/00234 parallel light rays 25. A gaussian refractive index gradient produced by a similar gaussian concentration gradient as, would appear in a sample in a sample chamber is represented schematically by curve 26. Line 27 5 represents both t:he plane of the sample chamber where the light beam passes through the sample and an axis indicating length along the sample chamber for the refractive index gradient curve 26. Axis n represents the refractive index of the sample within the sample chambAr.
10 Line 28 represents the detector plane and also an axis representing the intensity of light passing straight through th~~ sam:ple chamber when no refractive index . gradient is present. Curve 29 represents the intensity I
of the light striking the detector plane. When the light 15 beam passes; through the sample chamber, the individual light rays :?5 are refracted and bent out of their original path upon e~ncounitering the refractive index gradient 26 produced by a concentration gradient inside the sample chamber. If the intensity of each light ray is constant, equalling I,p, the relative changes of the light intensity on the detector plane can be given by: .
~~_ L j ° ~ ~Ia9M~1 ~=L.d 1 den _~ 1 dn~~.
~o ° a~ ~~ dxz M~ ~
Here, x is the direction along the sample chamber, z is the direction along the light beam, n is the refractive index inside the sample chamber, d is the diameter ~f the sample chamber, and L is the distance between sample chamber and the detector plane. In this equation, [1/n(x)] dn/dx corresponds to the light beam deflection angle, and :is small. Its high power in the second term of the equation can be neglected, compared with the first term, then:
~- ~ d=n~
I° M~ dxz which shows that the relative changes of the probe beam intensity on the detector plane are proportional to the second derivative of the refractive index inside the capillary. The relationship between the magnitude of the refractive index change and the sample's concentration is approximately linear. Hence, the relative changes of probe beam intensity on the detector plane are also expected to be proportional to the second derivative of the sample's concentration inside the capillary.
Figs. 6 and 7 show a capillary electrophoresis apparatus of the invention. As shown, a capillary tube 35, preferably of glass, has a capillary passage 36 therein. Open containers 37 and 38, forming reservoirs 39 and 40, respectively, are secured to the ends of capillary . tube 35 so that capillary passage 36 communicates with reservoirs 39 and 40. The assembly is held together as a unit by a member 41 to which the capillary tube 35 and containers 37 and 38 are secured. If the capillary tube is glass, it is preferred that member 41 also be glass and that the capillary be secured thereto by epoxy so that no refractive interface is formed between the two. The containers 37 and 38 may be of any suitable material, such as polyethylene, and secured to member 41 by any suitable adhesive, such as epoxy. Containers 37 and 38 may be provided with tops 42 and 43, respectively, which hold electrodes 44 and 45 in position in reservoirs 39 and 40, respectively. Tubes 46 and 47 extend through tops 42 and 43, respectively, to open near the bottom of reservoirs 39 and 40, respectively. Fittings 48 and 49 on the tops of tubes 46 and 47 where they pass through the respective tops are adapted to receive tubes from a source of, or receptacle for, liquid to be added to, or withdrawn from, the reservoir. Such means may conveniently take the form of a syringe such as shown schematically as 50 with a tube 51 to connect it to the appropriate fitting 48 or 49. The syringe may be manually operated or motor driven, or various other types of pumps or delivery systems could be used. Of course, electrodes 44 and 45 and tubes 46 and 47 could be positioned in the reservoirs by various other _ 21279'7 means and containers 37 and 38 could remain with open tops. An advantage of providing containers 37 and 38 with tops, and providing that the tops seal such containers, is that the pres.:ure in the containers can then be controlled. Thus, a syringe or other delivery system can be used to pressurize a reservoir to force liquid into the capillary passage, or can be used to draw a partial vacuum in a reservoir to draw liquid from the opposite reservoir into the capillary passage. The unit described may be mounted on a base 52, if desired.
For tlae unit described, the capillary tube may vary in length as dE~sired, but tube lengths between three centimeter:a and fifteen centimeters have been found satisfactory for various types of capillary electrophesis.
Further, the diameter of the capillary passage may also vary as de:aired, with diameters of between 10 ~cm and 100 ~cm having been found satisfactory. Either round or square capillary passages may be used, but square passages have been found particularly suitable for use with the detector and detection mEahod of the invention. When a square capillary i.s used, diameter of the capillary refers to the length of a side of the square. The size of the reservoir is not critical, although the volume of a reservoir must be large enough so that the capillary passage can be filled by the particular method being used to fill the capillary. In addition, the electrodes must be in contact with liquid in t:he reservoir. Thus, the volume of the reservoirs will generally be larger than the volume of the capillary passage:extending between them. For example, in the config~u.ration shown in Figs. 6 and 7, reservoirs each having a volume of about 0.2 milliliters has been found satisfactory for various size capillary passages, such as a fifteen centimeaer long passage of 20 ~,m diameter.
The electrodles 44 and 45 are connected to a source of high voltage (not shown) by wires 53 and 54, respectively.
The voltage: will preferably be in the range of between 5 KV and 10 KV, depending upon the type of separation being WO 93/14389 ~ PCT/US93/00234 2,12 9~
used. Generally, higher voltages will result in faster separation times, but the voltage is limited by the current flow through the sample in the capillary passage which generates heat in the sample. The heat generation must be kept below the level of heat that is readily dissipated through the capillary tube or the capillary tube may explode. The curtent flow is generally monitored by monitoring current flow in an electrode, generally the current flowing from the cathode. Such monitoring may be with an ampmeter, not shown.
The electrophoresis apparatus shown allows electrophoretic separation using all of the various known electrophoresis separation techniques. These techniques are capillary zone electrophoresis, moving boundary capillary electrophoresis, capillary isotachophoresis, and capillary isoelectric focusing. While with current electrophoresis equipment, separate equipment is needed for each technique, the apparatus described is truly universal in that any of the techniques can be practiced.
This is an advantage because in some instances, it may be advantageous to evaluate -a sample using two or more of the methods since different methods rely on different properties of the sample. For example, capillary zone electrophoresis and moving boundary capillary electrophoresis separate sample components based on differences in the mobility of each component.
Isoelectric focusing separates components based on the different isoelectric point of each component.
Capillary zone electrophoresis generally will require only three steps using the apparatus of the invention.
The first is filling the capillary passage with buffer solution. Generally, both reservoirs will be filled with buffer solution. The capillary passage can be filled hydrodynamically by filling one reservoir to a higher level than the other and allowing the buffer from the higher filled reservoir to run into the other reservoir allowing their levels to equalize. Alternatively, if a ry ) 93/14389 - ~ ~ ~ ~ ~ ~ ~ PCT/US93/00234 reservoir is fully enclosed, the reservoir can be pressurized) to cause the buffer to flow into the capillary passage. With buffer in both the reservoir and the capillary passage, the buffer is then removed from the anodic reservoir, i.e., the reservoir having the anode or positive e7.ectrode therein, and is replaced with sample solution. The high voltage is then turned on and the sample is drawn into the capillary electrokinetically, i.e., by action of the voltage across the capillary passage. The length of the sample plug drawn into the capillary i.s controlled by the time the sample solution remains in the reservoir and the voltage across the electrodes. When the desired sample plug is introduced into the capillary passage, the sample solution is removed from the reservoir and buffer returned to the reservoir.
The voltage remains across the electrodes. Sample separation and movement through the capillary passage continues under t:he influence of the voltage. After the sample has moved through the passage, the voltage can be disconnected.
Operation in the moving boundary capillary electrophoresis mode is similar. Initially, both reservoirs and the capillary are filled with buffer. In this method, the high voltage can be applied at this point. Th.e buffer is then removed from the anodic reservoir a:nd replaced with sample solution. The sample solution is drawn into the capillary passage electrokinetically. When the desired amount of sample is present in 'the capillary passage, the sample solution is withdrawn from the reservoir and the buffer again placed in the reservoir. This whole process is easily automated by using two syringe pumps controlled by a computer. Each pump could have its own tube, equivalent to tube 46 shown in Figs. 6 and 7, extending into the reservoir, or the pumps could be va:lved through an automatically controlled valve to a :jingle tube in the reservoir.
WO 93/ 14389 ~ ~ ~ ~ ~ ~ ~ PCT/US93/00234 With capillary isotachophoresis, a first or leading electrolyte is introduced hydrodynamically into the capillary passage. This is followed by hydrodynamic introduction of a sample plug into the capillary passage.
5 This would be achieved by removing the leading electrolyte from the anodic reservoir and filling the reservoir with sample to a higher level than the liquid in the cathodic reservoir. When the desired sample plug flows into the capillary passage, the sample is removed and the second or 10 tailing electrolyte is introduced into the anodic reservoir and into the capillary passage. Thus, the capillary passage includes a sample plug between leading and tailing electrolytes. The high voltage is then connected between the electrodes to perform the 15 separation.
With each of the three separation modes described above, i.e., capillary zone electrophoresis, moving boundary capillary electrophoresis, and capillary isotachophoresis, as the separation takes place, the 20 sample naturally migrates through the capillary passage from the anodic end to the cathodic end.
For capillary isoelectric focusing using the described apparatus, the sample, which includes the sample to be separated as well as carrier ampholytes, is introduced into the capillary passage. This may be done hydrodynamically or by placing the sample solution under pressure in one reservoir. After the sample is introduced into the passage, the anodic reservoir is filled with an analyte and the cathodic reservoir filled with a catholyte. The high do voltage is then connected. Under the influence of the electric f field, the ampholytes are arranged by their isoelectric points in order of increasing isoelectric points from anode to cathode. The sample components will migrate to the point in the capillary passage where their isoelectric points are equivalent to the pH established by the ampholytes, to create narrow zones of the sample components. As shown in 2 ~ 2 7 9 7 7 PCT/US93/00234 Fig. 8 components 55a, 55b, 55c are focused into scarp narrow zones in the solute solution 55d within the capillary passage of capillary tube 56. When separation is complete, the separation remains as long as the voltage remains. The separation process can be monitored by monitoring the current f low through the capillary passage .
The current flow will reach a minimum and remain stable at that minimum when separation has been achieved. The actual separation will take place usually in about four to seven minutes.
As indicated, the isoelectric focusing separates the sample components into very narrow bands. These narrow bands forms very high concentration gradients at their boundaries. Thus, the isoelectric focusing forms very high concentration gradients in the capillary even for low concentrations of sample components. The detection of concentration gradients, rather than detection of concentration, is uniquely applicable to isoelectric focussing and re:~ults in high sensitivity of detection and high resolution" The ratio of the sensitivities of detectors based on concentration gradient detection and detectors based on concentration detection may be expressed as a function of the zone or boundary width a'x:
where Q'x is the standard deviation of the concentration distribution in the zone or boundary which is Gaussian.
The equation predicts that the sensitivity of a concentration gradient detector increases more quickly with a decreasing zone width than does the sensitivity of detectors based on concentration detection, and that the concentration gradient detector is more suitable than the detectors x>ased on concentration detection for isoelectric focusing 'which has self-concentration and focusing properties ,.
WO 93/14389 ~ 1 ~ ~ ~ ~ PCT/US93/00234 If it is desired to mobilize the sample as focused in the capillary passage, the catholyte is withdrawn from the cathodic reservoir and replaced with a solution of different pH. This causes the separated sample to migrate through the passage.
Various concentration gradient detectors can be used with the electrophoresis apparatus described. A single light beam focused through the capillary passage at one end of the passage to measure the deflection of the light beam as the concentration gradients caused by the electrophoretic separation pass through the light beam has been found satisfactory and to have much better resolution than other prior art detectors. This is because the concentration gradient rather than merely concentration is being measured. However, a detector which can measure the separations along the length of the relevant portion of the capillary passage and does not require movement or migration of the separated sample in the capillary passage is preferred for detection when using isoelectric focusing which sets up a stationary separation in the passage since such detector does not require mobilization of the separated sample. By detecting a sample separated by isoelectric focusing without having to mobilize the separated sample, the results are obtained much more quickly. While the focusing itself generally takes between about four and seven minutes, the mobilization usually takes between an additional fifteen to forty-five minutes. By monitoring and detecting the stationary sample, results are obtained as rapidly as the focusing occurs. Further, excellent high resolution is maintained between sample components because this resolution is not lost or distorted through mobilization.
The basic concept of the detectors of the invention is to focus a light beam which comprehends the entire length of interest of the capillary tube in which separations occur onto the capillary tube so that it passes through the sample along the length of the sample oontaining the separations of interest. This may range frog a two cm le»gth along the capillary tube to ten or mots eenti~ters. The intensity of the beam after passing through the sample is sensed or detected in a way that the variations in intensity along the width o! the bees which has pas~xed through the desired length of sample is determined. Thus, the width of tare beam has to be broken down into iany wall segments, each indfvidnally sensed or detected, in order to provide the desired output. In io , preferred forms of the imertion, a wide but thin light beam 59, Fig. 9 is generated and Focused onto the capillary passage so that the vtidth of the bsal~ w will . coaprehend the length along the capillary passage where separations of interest have taken place, and so that the height of the beam h is preferably smaller than the diaaeter o! the capillary passage. Thus, the height h of the bsato shown in Fig. 9 is somewhat exaggerated !or purposes of illustration. There are several ways such a light beam say be generated including the use of lenses 30 and the use of masks, i.s., an opaque sheet o! material with a slit tdsrsirv. Further, the light !or the light bsaa say originate from a laser, a light cititting diode (LED), a laser diode, or an LED or laser diode array.
Preferred detectors of the invention are shown in ,25 pig's. ?, and 10-1~. As shown, a light source 60, such as a lasex, or a laser diode, with appropriate lenses as needed, is arranged to direct a light beam sl, of circular cross section, toward capillary passage 36. ~ cylindrical lens s2 in the path of light beam 61 focuses the circular 9o beau 61 into a thin bean or sheet of light 63 with a width apprvxi.~sataly equal to the width or the circular bean.
This sheet of light is aligned~with and focused onto capillary passage 36. I! the width of the beaa is shorter than the entire length of the capillary passage, the beau 35 is set to pass through the length of the passage where it is expected that separations of interest will take place.
The beam 63 ritay pass directly through the capillary passage to a detector, or tray peas through the capillary passage to lens 64 vJhich expand the beam 63 before it reaabes the detector. The expanded laeam is labeled 65.
Such expansion can provide increased resolution of detection, particularly where the inareaents of detection are not as saall as desired.
J The preferred form of the invetion utilises two alternate detecting or sensing techniqaea. one is single detector, such as photodiode 66, ~oounted to be aoved linearly along the width of bean 65 to sense the inten*ity of light in light beaa 65 as a function of the linear position of photodiode 66. Since the light beam being sensed is very thin and the changes in intensity occur in substantially a single dirsction, i.e.;
along the . 15 width of the bear, accurate alignment of photodiode i with the beam from the standpoint of height of the beam is 1 not necessary.
?hatodiode 66 may be moved along the width of beala s3 in a variety of ways, and the way moveaent is obtained is o not critical. llny apparatus for moving the photodiode say be used as long ag the linear position of the diode can be kept track of. This may merely be a means that once started, will ~rnrs~thraugh tire entire length o!
travel of ' interest at a constant rate. An sxa~ple of apparatus for " 25 moving diode 6s is shown in Figs. ? and s0. a track 70 is mounted on base 52 arid receives slidably therein a carriage aaseably ?1. Carriage asseably ?1 includes Teflone ar similar plastic bearinge~ ?2 which ride against track 70 to reducs friction and a threaded aeaber 73 which 30 accepts a threaded shaft 74 therethrough. ~1 platform ?5 with bracket 76 and photodiade 65 haunted thereon is seaurad to carriage 91. Shaft ~4, Fig. ?, is coupled through sleeve ?e to the output shaft ?9 of stepper motor 80. As threaded shaft 74 is rotated by stepper rotor 80, 3S carriage 71, platform 95, and photodiode 66 move along track ?o. The direction of travel depends upon the direction of rotation of the stepper motor at~d the speed of travel depends upon the speed of rotation of the stepper motor. The speed of rotation, direction, and amount of rotation of a stepper motor may be accurately controlled in known manner so the rotation of the motor 5 and travel of photodiode 66 through the width of light beam 65 can be accurately monitored and controlled.
During a scan of photodiode 66 along the width of light beam 65, photodiode 66 will produce an output signal proportiona:L to the light intensity of beam 65 striking 10 photodiode 66 and, thus, a signal proportional to the light inten:aity air all points in the light beam along the path of travel of the photodiode. If it is desired to .further limit tlhe width of the segment sensed by photodiode 66, a shield 81 may be mounted in front of 15 photodiode E>6 with a slit 82, Fig. 7, therein of desired width. The output. of the photodiode is through leads 83.
The detector. electronics used would be standard and well known.
An alternate detector is shown in Fig. 11 and 20 replaces the:moving photodiode detector of Figs. 7 and 10.
The detector- of F:ig. 11 is a photodiode array 85 mounted in the path of light beam 63 after passage through the sample or in the path of expanded light beam 65. Many one dimensional or two dimensional photodiode arrays could be .25 used, with the cost of the array balanced against the desired resolution. The preferred arrays have very small sensing elements spaced along the width of the array. For example, arrays o~° charge coupled devices which are used in television and video cameras having sensing elements one one-thousandth of an inch in size are readily available, but thE: larger of such arrays and arrays of a grade with all elements working, are expensive. Arrays with even ::mallet sensing elements are also becoming available. Such .arrays may be obtained with as many as 1024 elementa in one dimension. Thus, the width of the array and the width of the light beam comprehended thereby would be broken down into 1024 individually sensed ~.~o~~~~ns. The electronics for scanning an array are standard and well known. The connections between the electronics and the array are through wires represented schematically as 86.
An overall block diagram of a detector system is shown in Fig. 12. The light source is represented by block 90 and includes the necessary components, such as a laser and lenses, to produce the necessary sheet of light, indicated by broken line 91. Light beam 91 passes through the capillary sample chamber 92 to the detector 93 which measures the intensity profile of the beam after passing through the sample. The output of the detector goes to any necessary interface electronics 94 which processes the signals from the detector and sends them to computer 95.
Signals from the computer needed to control the detector, such as scanning signals or motor control signals, or other control signals, are sent from computer 95, through interface electronics 94, to detector 93. With such a system, the intensity information obtained can be displayed in real time on the computer monitor and stored in memory for later display or processing. The computer 95 can also operate through interface electronics 96 to operate any reagent pumps, voltage supplies, or other equipment, indicated as block 97, to automate the . 25 capillary electrophoresis separations conducted in the capillary sample passage. This can allow coordination between the conditions of the capillary electrophoresis taking place in the sample chamber based upon the detected results.
In a prototype of the detector, a 100 ~cm diameter, 6.5 cm long square capillary passage was used in the apparatus shown for the isoelectric focusing. The capillary tube was glass and was glued with epoxy between two glass slides. The capillary passage walls were coated with non-crosslinked acrylamide to eliminate electroosmosis. The containers forming the reservoirs were polyethylene. The apparatus was mounted on a two-axis stage so the tilt angles in the borisental and vertical planes were adjustable to aid in alignment of the probe light beam with the capillary passage. A Ha-Ne laser manufactured by UniphaseT" of Sea Jose, California was toted to generate the probe light beam. The bean from the laser was expanded to a 2 cn diameter beam and was then focused into the capillary passage by a 6 am fooal length aylindrival lens mounted on a three-axis stage. The cylindrical lens produced a sheet of light of about x om 1o in width. After passage through the capillary passage, the 2 ant beam was expanded to a ZO cm beam in the detector plane by a 25 mat local length lens mounted behind the capillaxy tube. In,thist way, 1 cm width in the detector plane corresponded to a 1 mm length of ,the capillary passage. This sakes it easier to aaasurs the intensity profile of the bean. In so~te experiments, a single photodfade with a shield with 0.1 bm slit therein was mounted on a one-axis stage driven by the aoving part o!
a mode 3418 syringe pump trade by 4rion ~tessarah, Inc. of Massachusetts. This was located so the photodiode scanned in the detector plane. The scanning distance of the photodiade so mounted vas about 150 mat which corresponded to a 15 man length of the cetpillary passage. In other experi.msnts, a one-dimensional, 128 element photodiode a5 array was used to atonitor the intensity a! the beam in the detector plane. This array was able to monitor a 3 MAm length of the capillary passage. The whole system was mounted on a vibration isolation table. The data obtained by the detectors was collected through an IBM''" DACA board in a PC-AT personal computer, using the ASYSTm software supplied by Asyst Software Technology, Znc., Rochester, New York.
~In the tests of the systems, all chemicals ears reagent grads, and solutions were prepared using deionized eater. 10 mM HzPO, and 20 m!1 HaON were used as the anolyte and catholyte, respectively. NaOIM solution Bras degassed before use, by sparging with helium. Samples used include WO 9x111389 ~YUS93/007,34 ~~-chymotrypsin (tYPe =I, Sigma), phosphorylase b (Sigma) and ovalbumin (grade V, Sigma). semplss were ~al.xed with ampholyte (pharmalyte pN 3 - io, sierra) solution Ior a final concentration o! 2t nmpholyte. solutions ware filtered using o.2 pm pore sire cellulose acetate filters (Sartorius'~', Gottingen, Germany). The sample concentration introduced into the capillary ranged from 0.3sg/mL to 1 The sample was introduced into the capillary passage 1o by pressure generated by a syringe. A plug of 1x agarose gol i» the reservoir of the anadic end of capillary (prepared in the anolyte, 10 mM H~p04) was used to avoid hydrodynamic flow in the 100 um i.d. capillary. After introduction of the sample into the capillary passage, a 5 1cV do voltage was applied arid current passing through the capillazy was monitored to follow the focusing process. Typically, the current dropped from 15 ~cA to about 2.5 ~cA in 4-~ min, and then became stable for hours.
this minimum current flow indicates the isoelectric focusing process has been completed.
Fig. 13 shorts the beau intensity profile detected by the single photodiode scanned across the probe beam which passes through the part of the capillary located 3.5 - 5 - cm distance from the anodic end. Two sharp, high peaks 100 dad 101 are observed in the probe beam intensity profile shown in Fig. 13, which correspond to the positions expecteA fox the focused phosphorylase b (isoelectric point 6.3), and ovalbumin (isoeleCtric point 4.7), respectively. The concentrations of analytes are 3D about 1 mg/mL each. This result demonstrates that the focused proteins inside the capillary can be detected by this simple imaging system. The signal peak 100 corresponding to focused zone of phosphoryla$e b and its integrals are illustrated in Fig, i4, which clearly shows the second derivative characteristic of the detected signal loo. since this imaging system is an on-line detector, the isoelectric focusing process itself can be O 93/14389 _ 212 7 9 7 7 PCT/US93/00234 monitored. Fig. 15 shows the focusing process of phosphorylase b and ovalbumin. The concentrations of the samples area 0.5 ~mg/mL, which corresponds to 3.4 pmole of phosphorylase b and 7.2 pmole ovalbumin injected into the 6.5 cm lone square capillary. At the beginning (0 Min.) of the focusing, as shown by curve a in Fig. 15, no sharp peaks are observed. The detected signals are the probe beam intensity profile after it passes through the capillary. Many low peaks in Fig. 15 are generated by refractive index defects in the capillary wall or coating materials i.n the inner wall of the capillary, and their positions do not. change with the time, which can be observed in. curves .a, b, and c of Fig. 15. In curves b and c, two aecond derivative peaks 102 and 103 appear and become higher with longer focusing time, which correspond to focused ;phosphorylase b and ovalbumin. In addition to these two high peaks, other small peaks can be observed and become higher with the time in the curves b and c of Fig. 15. Those peaks are associated with the minor components in the: samples. It should be mentioned that the concentration gradients generated by the components of carrier ampholytea can also be detected because of the universal nature of the detector. The refractive index fluctuations produced by the carrier ampholytes can be noticed in 'the integral of the detected signals shown in Fig. 14. lHowever the second derivative nature of the imaging detector effectively reduces the amplitudes of low frequency broad signals generated by the wide bands of the carrier ampholytea. As shown in Fig. 13, high signal peaks can only be observed for high concentration gradients produced at the boundaries of narrow protein zones.
Figs. 7.3 and 15 were obtained with the moving single photodiode detector. Fig. 16 shows the focusing process for a «-chymotrypsin sample and covers a 3 mm length of the capillary passage. The curves of Fig. 16 were obtained using the 128 element linear photodiode array WO 93/14389 212 g~'~ PCT/US93/00234 described above. This clearly shows the practicality of using sensor arrays in the detector of the invention.
Further, the results obtained show that the detectors of the invention not only detect the focused analytes in the 5 sample, but can also be used for monitoring and studying the dynamics of the isoelectric focusing process inside the capillary passage.
Fig. 17 shows a second embodiment of a sample chamber for the electrophoresis apparatus of the invention. Some 10 sample solutions, such as blood serum samples, contain a high level of salts. This makes the sample have a high degree of electrical conductivity. If such a sample was introduced directly into a capillary passage, the voltage applied for the separation would cause excessive current 15 flow through the sample, excessive heating, and could result in explosion of the capillary tube. Thus, preparation of a sample to remove the salts therefrom is necessary prior to subjecting it to capillary electrophoresis separation. The apparatus shown in Fig.
20 17 can be used to prepare the sample for electrophoresis as part of the overall procedure.
The modification of the apparatus as shown in Fig. 17 is to provide a container 120 forming the sample reservoir 121 inside a second container 122. Sample container 120 25 is made of a porous membrane material such as a cellulose acetate membrane material. Alternately, container 120 could be a combination of porous membrane material and other material, such, for example, as a polyethylene container with a porous membrane bottom. Container 122 is 30 made of normal non-porous material such as polyethylene or glass. The capillary tube 123 passes through the wall of container 122 in a sealed manner and is secured in sample container 120 so that capillary passage 124 communicates with sample reservoir 121. Generally, the illustrated construction will be needed for only one of the reservoirs as shown in the apparatus of Fig. 6, usually the anodic reservoir. The sample 125 is introduced into sample 7 93/14389 ~ , ~- ~ 1,~,; PCT/US93/00234 reservoir 1.21, but will not initially flow into capillary tube 124. Water, which may also contain the desired ampholytes, is placed as solution 126 in container 122 so as to surround a portion of container 120. It is preferable to continually flow the water-ampholytes solution through container 122 so for that purpose an inlet tube 127 f~.~om a source of water-ampholyte solution may be provided to introduce such solution to container 122 while an outlet 128 may be provided so that solution may flow from container 122. With sample 125 in sample reservoir 7.21, the salts therein will pass through the porous membrane into the water solution 126 in container 122. Simultaneously, if the water also contains ampholytes, the ampholytes will pass through the membrane into the sample. Thus, the sample can be prepared by the removal of salts and the addition of ampholytes while in the sample reservoir. The sample 125 will remain in sample reservoir 121 for the time required to exchange the salts from the sample to the water through the membrane, and to exchange the ampholytes from the water solution to the sample. When the sample is properly prepared, the sample will be moved into the capillary passage such as through pre:asurizing the sample reservoir 121. The sample reservoir will include a sealed top 129 to allow pressurization oi: the reservoir to cause the sample to flow into the capillary passage, the electrode 130, and tube 131, a;s shown in the apparatus of Fig. 6.
The invention also includes a method of determining the presence or concentration of a particular component that does not have an isoelectric point so would not normally be focused and detected by capillary isoelectric focusing. The method includes the steps of adding a reagent hav~.ng a urell-def fined isoelectric point and having a high degree of specificity toward the component to be detected to a sample which may have the component therein, under conditions where the reagent and component will form a combination or complex of the two which will have an '~~,~ ~~~~~
", _ , , - , PGTiU ~ 9 3 / 0 0 2 3 4 03 Recd ~'~t/Ft~ 0 Z ~,U~ 1993.
isoelectric point different from the isoelectric point of the reagent itself, and then detecting the presence of the complex through iaoelectric focusing.
The meahod is useful in instances where it is desirable to detect the presence of a component, for example a toxin, in a sample, such as a body fluid, but the toxin may not have an isoelectric point so would not normally bas separated and detected by isoelectric focusing. In the method of the inventi~, a reagent having a high dE:gree of specificity toward a target component or analyte is used. The reagent is a substance which has a well-defined isoelectric point and forms a sharp band inside the capillary which may be easily detected through capillary isoelectric focusing and the detectors of the invention as a second derivative of the Gaussian. This :is shown by signal 135 in the curves labeled a, b, and .c in Fig. 18. The reagent, for example, may be proteain which is an antibody for the toxin being looked for o~r could be a computer designed and laboratory synthesized organic molecule, for example, a synthetic cavity ligand. The component being looked for can be a toxin or other mo7.ecule which generally will not have an isoelectric point:. The reagent, R, is picked to specifically and strongly interact or react chemically with the target component or analyte A, to form, for example, the: product:
R + A = RA
The product of the: chemical process, RA, has a different isoelectric point and therefore focuses in a different part of the capillary then the reagent R. Thus, the product is i.ndicat:ed as peak 1-36 in curve b of Fig.
18.
In addition, the hE:ight of the corresponding signal 136 is proportionally related to the amount of the target component present. The reagent can also be ~ie~= .-ned to react with several target analytes to produce several different products. In such instance, several corresponding signals 136, 137, and 138 may be obtained as SUBSTITUTE SHEET
.. 212977 ~ _ .. .
pCT~~.~~ 93 /00 23~+
33 03 Rec'c~ ~'~fT/~ '~ 0 2 ~~.~JG 1995 shown in curve c of Fig. 18. It will usually be desirable to ensure that there is an excess of reagent added to the sample so that all. of the component present in the sample will react with reagent. In this way, the signal representing the component-reagent product, i.e., peaks 136, I37, or :138, will be proportional to the concentration of component in the sample. The presence of a peak 135 for the reagent as well as a peak for the product will. indicate this excess.
l0 Because: reagents having very high specificity for components of a sample being looked for can be produced to therefore provide accurate detection and concentration information, the method described using the simple and relatively inexpensive detector of the invention has the potential to replace many tedious analytical procedures and expensive instrumentation.
As mentioned previously, the electrophoresis separating apparatus of the invention could be used with a detector ;simila:r to those shown in my prior patents having a beam located at one end of the capillary passage and mobilizing 'the sample separation obtained by isoelectric focusing so that it flows through the capillary and the: detector beam. Fig. 19 shows the results obtained using capillary electrophoresis apparatus similar to that shown but with a laser diode as the light source for a light beam passed through one end of a 20 ~,m capillary passage in a 12 cm long capillary tube. The sample used contained 120 fmol of human hemoglobin indicated by peak 140, myoglobin indicated by peak 141, 270 fmol of human carbonic anhydrase indicated by peak 142, 240 fmo~l of bovine carbonic anhydrase indicated by peak 143, and 410 fmol of 8-lactoglobulin indicated by peak 144. The con<:entration of the sample was 0.2 mg/mL.
Figs. 20 and ai show the difference in separation of a sample b:y capillary zone electrophoresis, moving boundary capillary electrophoresis, and isoelectric focusing. :Figs. 20 and 21 show electropherograms of ~sUBSTITUT~ SHEE1°
WO 93114389 PC'F/US91l90I34 avalbu~ain separated by the three separation methods. The ovalbumin was purified by the manufacturer with slab cone electrophoresis whiatr is based ot~ a sample s mobility differences. l~s expected, tt~e electropherogram of capillary zone electrophoresis separation, curve a in Fig.
20, shows only one peak, 150. The elsctropherogram of moving boundary capillary electrophoresis separation, curve b in Pig. 20, also shaves one peak, lxl, since it is based on the same separation principle, a difference in component mobility, as capillary zone electrophoresis.
However, the electropharogra~a of the same sa~rple separated by isoelectric focusing, Pig. 21, shows more than four peeks. The highest peak 15Z, corresponds to ovalbumin, and other peaks can be attributed to impurities or minor components in the sample which have almost the same mobilities as that of ovalbumin, but different iroelectric points from that of avalbumin. xhe electropharagrams or curves shown were obtained using the apparatus with detector at the end thereof and mobilizing the sample 2o separation obtained by isoslectric focusing. This shows the significant difference and improvement in separation and sensitivity obtained by using isoelectric focusing.
The detector of the invention cnn also be used as an absorbanca imaging detector to detect concentration of separated sample components directly rather than x~easuring concentration gradients. The only difference in such case, is the nature of the light beam which provides light of frequencies to be absorbed by sample components. The light beam may be generated by ttn incoherent light source such as a halogen lamp or may be generated by a laser ox variable frequency laser. If from an incoherent light source the light is preferably filtered to limit it to the desired frequencies. The components o! the sample as separated along the capillary passage will absorb some of the light so the light intRnslty will vary along the width of the light beam. The detector, as indicated above, detects the variation in light intensity along the width of the light beam.
In an example of the absorbance imaging detector, the light beam source was a halogen lamp. The light from the 5 lamp was collected by a paraboloidal ref lector which reflected light onto a mirror. The light beam was filtered by a color filter which was transparent in 400 nm to 600 nm wavele:ngth range. The light beam was then focused into a 200 ~m wide slit which was focused onto a 10 200 ~cm dia.meter capillary tube. The image of the capillary illuminated by the light beam was projected by a 10 cm focal length lens onto a 1024 pixel or sensing element charge-coupled device (CCD) such as a S3903-1024Q
made by Hamamatsu,, Hamamatsu City, Japan, which had a 25-15 mm X 0.5-mm sensing area. The changes in the light beam intensity profile due to the refractive gradient inside the capillary was eliminated by focusing the image of the capillary onto the. detector plane, i.e., the sensor. The data was collected by an IBM DACA board, in a PC-AT
20 personal computer. An averaging method was applied to reduce the :random noise. - For each measurement, the CCD
was scanned ten times in 1 second and these scans were averaged. The background image which was recorded before the focusing voltage was turned on, was first subtracted 25 from the sample images, and the sample images were then normalized by 'the background image to eliminate fluctuation: created by the source beam intensity distribution. ~, human hemoglobin sample from Sigma Chemical Co., containing 75% methemoglobin, balanced 30 primarily 'with oxyhemoglobin, was separated using isoelectric focusing. The electropherogram obtained is shown in Fig. 22. Peaks 160 and 161 correspond to two variants of hemoglobin; methemoglobin (pl 7.2, comprises 75% of the ;sample) and oxyhemoglobin (pl 7.0, comprises 35 less than 2_°°>% of 'the sample), respectively. Other peaks may correspond to other variants of hemoglobin. The 1Ab 93114989 PCT/US93/ODZ~t r~ssulta show effective detection through the absorbance .
detection.
Figure Z3 shows the eleatropherogram of the sa~ae hemoglobin sample es tbat in ~'ig. Z2, which is focused in a 100 pm diameter capillary and detected using concentxatioa gradient iaaging. The resolution of the concentration gradient imaging shown in Fig. 23 is better than that of the absorbance imaging due to the second derivative nature of the concentration gradient imaging l0 which eliminates broad bands and fluctuations.
Further, when using an incoharet~t light source for the absorbance . detection, use of narrow capillaries is limited.
For absorbance imaging using narr~ capillaries, such ae capillaries with loo ~m diaaater or lass, an adjustable i5 wavelength laser is necessary, and !or use in detecting proteins, an adjustable wavelength laser working in the W
range is necessary since most proteins only have ahsorbanca bands in the tN range. Thus, the concentration gxadient imaging appears most practical fax small diaseter Zo aapillsry tubes. However, for larger diameter capi7.lary tubes, the absorbance imaging can produce excalient results.
iihereas this invention is here illustrated and described with reference to e~obodimeats thereof presently Z5 contemplated as the best mode of carrying out such invention in actual practice, it is to be understood that various changes clay be made in adapting the invention to different embodiments without departing from the broader inventive concepts disclosed herein and comprehended by 3Q the claims that follow.
(dn/dc)(da/dx), which then tilts or deflects the propagating light beam by angle 9 ~ (b/n) (dn/dc)(dc/dx).
This deflection cat? be measured by measuring the position of the light beam on the position detector 14. The ~o information produced during the measurewent of the concnntration gradient relates to the universal property of the solute -- refractive index n. Consequently, the concentration gradient produced by any solute that has a different n than the solvent will be detected by noting a . 25 deflection or tilt in the light beaa.
Fig. 3 shows the same principle as Fig. 2, but illustrates it somewhat differently. Thus, if a concentration gradient represented by line 16 exists in a sample in a sample chamber defined by walls 12 and 13, a 30 probe beam o! light 1~ directed through the sample will be deflected as indicated above by an angle 6. This causes the position of the beam to cove on the suxface pf the ' position sensor 14 as indicated above.
Fig. 4 is similnr to Fig. 2, but shows a wide light 15 bean with a concentration gradient 18 within the width of the light bean. Thus, rather than the light beam being uniformly deflected as shown in Figs. 1, Z, and 3, a portion of the light beam 19, on one aide of the gradient 18, which does not pass through a gradient, passes xtraight through the sample to detector 14. portion 20 of the light bean passes through the gradient 18 and is defleoted by angle A as shorn by the broken arrows, and falls onto detector 14 partially overlapping portion 19.
Where the overlap ocGUrs on the detector, indicated ~xt 22, the light striking detector 14 is brighter than in non overlapping areas. Portion 21 of the light beans on the other side of the gradient 18, again passes straight through the sable and onto detector 14. As indicated in Fig. 4, there will be an area Z3 Where little or no light will fall. Thus, the single light beam after passing through a sale with one or~aore gradients therein wi7.l have varying intensity indicating the gradients present in the sample. A gradient in the saapie will generally result in a bright spot followed by a spot of very little intensity, or, if just measuring intensity, a level representing the light passing straight through the ao sa~ople, an increased or positive signal (the increased intensity), followed by decreased or negative signal (tbe decreased intensity), followed again by the level representing the light passing etxaight through the sample.
.25 If the detector 14 is broken down into many small detectors, such as an array of detectors, each detector detecting the light from a s=ell portion of the beam, a comparison or scanning o! the individual detectors will produce an output signal representative of the intensity 30 of the light beam falling on the array along its length.
Alternatively, a single, small detector which detects only a portion of the light beam could be positioned to be roved through the beam along its width and measure the light intensity as it is moved.
Fig. 5 ahorrvs the same principle as Fig. 4, but relates it more directly to a s~rstem of the invention.
Thus, again, tire light beam say be considered as many 93/14389 21 ~'~ (~ ~ ~ PCT/US93/00234 parallel light rays 25. A gaussian refractive index gradient produced by a similar gaussian concentration gradient as, would appear in a sample in a sample chamber is represented schematically by curve 26. Line 27 5 represents both t:he plane of the sample chamber where the light beam passes through the sample and an axis indicating length along the sample chamber for the refractive index gradient curve 26. Axis n represents the refractive index of the sample within the sample chambAr.
10 Line 28 represents the detector plane and also an axis representing the intensity of light passing straight through th~~ sam:ple chamber when no refractive index . gradient is present. Curve 29 represents the intensity I
of the light striking the detector plane. When the light 15 beam passes; through the sample chamber, the individual light rays :?5 are refracted and bent out of their original path upon e~ncounitering the refractive index gradient 26 produced by a concentration gradient inside the sample chamber. If the intensity of each light ray is constant, equalling I,p, the relative changes of the light intensity on the detector plane can be given by: .
~~_ L j ° ~ ~Ia9M~1 ~=L.d 1 den _~ 1 dn~~.
~o ° a~ ~~ dxz M~ ~
Here, x is the direction along the sample chamber, z is the direction along the light beam, n is the refractive index inside the sample chamber, d is the diameter ~f the sample chamber, and L is the distance between sample chamber and the detector plane. In this equation, [1/n(x)] dn/dx corresponds to the light beam deflection angle, and :is small. Its high power in the second term of the equation can be neglected, compared with the first term, then:
~- ~ d=n~
I° M~ dxz which shows that the relative changes of the probe beam intensity on the detector plane are proportional to the second derivative of the refractive index inside the capillary. The relationship between the magnitude of the refractive index change and the sample's concentration is approximately linear. Hence, the relative changes of probe beam intensity on the detector plane are also expected to be proportional to the second derivative of the sample's concentration inside the capillary.
Figs. 6 and 7 show a capillary electrophoresis apparatus of the invention. As shown, a capillary tube 35, preferably of glass, has a capillary passage 36 therein. Open containers 37 and 38, forming reservoirs 39 and 40, respectively, are secured to the ends of capillary . tube 35 so that capillary passage 36 communicates with reservoirs 39 and 40. The assembly is held together as a unit by a member 41 to which the capillary tube 35 and containers 37 and 38 are secured. If the capillary tube is glass, it is preferred that member 41 also be glass and that the capillary be secured thereto by epoxy so that no refractive interface is formed between the two. The containers 37 and 38 may be of any suitable material, such as polyethylene, and secured to member 41 by any suitable adhesive, such as epoxy. Containers 37 and 38 may be provided with tops 42 and 43, respectively, which hold electrodes 44 and 45 in position in reservoirs 39 and 40, respectively. Tubes 46 and 47 extend through tops 42 and 43, respectively, to open near the bottom of reservoirs 39 and 40, respectively. Fittings 48 and 49 on the tops of tubes 46 and 47 where they pass through the respective tops are adapted to receive tubes from a source of, or receptacle for, liquid to be added to, or withdrawn from, the reservoir. Such means may conveniently take the form of a syringe such as shown schematically as 50 with a tube 51 to connect it to the appropriate fitting 48 or 49. The syringe may be manually operated or motor driven, or various other types of pumps or delivery systems could be used. Of course, electrodes 44 and 45 and tubes 46 and 47 could be positioned in the reservoirs by various other _ 21279'7 means and containers 37 and 38 could remain with open tops. An advantage of providing containers 37 and 38 with tops, and providing that the tops seal such containers, is that the pres.:ure in the containers can then be controlled. Thus, a syringe or other delivery system can be used to pressurize a reservoir to force liquid into the capillary passage, or can be used to draw a partial vacuum in a reservoir to draw liquid from the opposite reservoir into the capillary passage. The unit described may be mounted on a base 52, if desired.
For tlae unit described, the capillary tube may vary in length as dE~sired, but tube lengths between three centimeter:a and fifteen centimeters have been found satisfactory for various types of capillary electrophesis.
Further, the diameter of the capillary passage may also vary as de:aired, with diameters of between 10 ~cm and 100 ~cm having been found satisfactory. Either round or square capillary passages may be used, but square passages have been found particularly suitable for use with the detector and detection mEahod of the invention. When a square capillary i.s used, diameter of the capillary refers to the length of a side of the square. The size of the reservoir is not critical, although the volume of a reservoir must be large enough so that the capillary passage can be filled by the particular method being used to fill the capillary. In addition, the electrodes must be in contact with liquid in t:he reservoir. Thus, the volume of the reservoirs will generally be larger than the volume of the capillary passage:extending between them. For example, in the config~u.ration shown in Figs. 6 and 7, reservoirs each having a volume of about 0.2 milliliters has been found satisfactory for various size capillary passages, such as a fifteen centimeaer long passage of 20 ~,m diameter.
The electrodles 44 and 45 are connected to a source of high voltage (not shown) by wires 53 and 54, respectively.
The voltage: will preferably be in the range of between 5 KV and 10 KV, depending upon the type of separation being WO 93/14389 ~ PCT/US93/00234 2,12 9~
used. Generally, higher voltages will result in faster separation times, but the voltage is limited by the current flow through the sample in the capillary passage which generates heat in the sample. The heat generation must be kept below the level of heat that is readily dissipated through the capillary tube or the capillary tube may explode. The curtent flow is generally monitored by monitoring current flow in an electrode, generally the current flowing from the cathode. Such monitoring may be with an ampmeter, not shown.
The electrophoresis apparatus shown allows electrophoretic separation using all of the various known electrophoresis separation techniques. These techniques are capillary zone electrophoresis, moving boundary capillary electrophoresis, capillary isotachophoresis, and capillary isoelectric focusing. While with current electrophoresis equipment, separate equipment is needed for each technique, the apparatus described is truly universal in that any of the techniques can be practiced.
This is an advantage because in some instances, it may be advantageous to evaluate -a sample using two or more of the methods since different methods rely on different properties of the sample. For example, capillary zone electrophoresis and moving boundary capillary electrophoresis separate sample components based on differences in the mobility of each component.
Isoelectric focusing separates components based on the different isoelectric point of each component.
Capillary zone electrophoresis generally will require only three steps using the apparatus of the invention.
The first is filling the capillary passage with buffer solution. Generally, both reservoirs will be filled with buffer solution. The capillary passage can be filled hydrodynamically by filling one reservoir to a higher level than the other and allowing the buffer from the higher filled reservoir to run into the other reservoir allowing their levels to equalize. Alternatively, if a ry ) 93/14389 - ~ ~ ~ ~ ~ ~ ~ PCT/US93/00234 reservoir is fully enclosed, the reservoir can be pressurized) to cause the buffer to flow into the capillary passage. With buffer in both the reservoir and the capillary passage, the buffer is then removed from the anodic reservoir, i.e., the reservoir having the anode or positive e7.ectrode therein, and is replaced with sample solution. The high voltage is then turned on and the sample is drawn into the capillary electrokinetically, i.e., by action of the voltage across the capillary passage. The length of the sample plug drawn into the capillary i.s controlled by the time the sample solution remains in the reservoir and the voltage across the electrodes. When the desired sample plug is introduced into the capillary passage, the sample solution is removed from the reservoir and buffer returned to the reservoir.
The voltage remains across the electrodes. Sample separation and movement through the capillary passage continues under t:he influence of the voltage. After the sample has moved through the passage, the voltage can be disconnected.
Operation in the moving boundary capillary electrophoresis mode is similar. Initially, both reservoirs and the capillary are filled with buffer. In this method, the high voltage can be applied at this point. Th.e buffer is then removed from the anodic reservoir a:nd replaced with sample solution. The sample solution is drawn into the capillary passage electrokinetically. When the desired amount of sample is present in 'the capillary passage, the sample solution is withdrawn from the reservoir and the buffer again placed in the reservoir. This whole process is easily automated by using two syringe pumps controlled by a computer. Each pump could have its own tube, equivalent to tube 46 shown in Figs. 6 and 7, extending into the reservoir, or the pumps could be va:lved through an automatically controlled valve to a :jingle tube in the reservoir.
WO 93/ 14389 ~ ~ ~ ~ ~ ~ ~ PCT/US93/00234 With capillary isotachophoresis, a first or leading electrolyte is introduced hydrodynamically into the capillary passage. This is followed by hydrodynamic introduction of a sample plug into the capillary passage.
5 This would be achieved by removing the leading electrolyte from the anodic reservoir and filling the reservoir with sample to a higher level than the liquid in the cathodic reservoir. When the desired sample plug flows into the capillary passage, the sample is removed and the second or 10 tailing electrolyte is introduced into the anodic reservoir and into the capillary passage. Thus, the capillary passage includes a sample plug between leading and tailing electrolytes. The high voltage is then connected between the electrodes to perform the 15 separation.
With each of the three separation modes described above, i.e., capillary zone electrophoresis, moving boundary capillary electrophoresis, and capillary isotachophoresis, as the separation takes place, the 20 sample naturally migrates through the capillary passage from the anodic end to the cathodic end.
For capillary isoelectric focusing using the described apparatus, the sample, which includes the sample to be separated as well as carrier ampholytes, is introduced into the capillary passage. This may be done hydrodynamically or by placing the sample solution under pressure in one reservoir. After the sample is introduced into the passage, the anodic reservoir is filled with an analyte and the cathodic reservoir filled with a catholyte. The high do voltage is then connected. Under the influence of the electric f field, the ampholytes are arranged by their isoelectric points in order of increasing isoelectric points from anode to cathode. The sample components will migrate to the point in the capillary passage where their isoelectric points are equivalent to the pH established by the ampholytes, to create narrow zones of the sample components. As shown in 2 ~ 2 7 9 7 7 PCT/US93/00234 Fig. 8 components 55a, 55b, 55c are focused into scarp narrow zones in the solute solution 55d within the capillary passage of capillary tube 56. When separation is complete, the separation remains as long as the voltage remains. The separation process can be monitored by monitoring the current f low through the capillary passage .
The current flow will reach a minimum and remain stable at that minimum when separation has been achieved. The actual separation will take place usually in about four to seven minutes.
As indicated, the isoelectric focusing separates the sample components into very narrow bands. These narrow bands forms very high concentration gradients at their boundaries. Thus, the isoelectric focusing forms very high concentration gradients in the capillary even for low concentrations of sample components. The detection of concentration gradients, rather than detection of concentration, is uniquely applicable to isoelectric focussing and re:~ults in high sensitivity of detection and high resolution" The ratio of the sensitivities of detectors based on concentration gradient detection and detectors based on concentration detection may be expressed as a function of the zone or boundary width a'x:
where Q'x is the standard deviation of the concentration distribution in the zone or boundary which is Gaussian.
The equation predicts that the sensitivity of a concentration gradient detector increases more quickly with a decreasing zone width than does the sensitivity of detectors based on concentration detection, and that the concentration gradient detector is more suitable than the detectors x>ased on concentration detection for isoelectric focusing 'which has self-concentration and focusing properties ,.
WO 93/14389 ~ 1 ~ ~ ~ ~ PCT/US93/00234 If it is desired to mobilize the sample as focused in the capillary passage, the catholyte is withdrawn from the cathodic reservoir and replaced with a solution of different pH. This causes the separated sample to migrate through the passage.
Various concentration gradient detectors can be used with the electrophoresis apparatus described. A single light beam focused through the capillary passage at one end of the passage to measure the deflection of the light beam as the concentration gradients caused by the electrophoretic separation pass through the light beam has been found satisfactory and to have much better resolution than other prior art detectors. This is because the concentration gradient rather than merely concentration is being measured. However, a detector which can measure the separations along the length of the relevant portion of the capillary passage and does not require movement or migration of the separated sample in the capillary passage is preferred for detection when using isoelectric focusing which sets up a stationary separation in the passage since such detector does not require mobilization of the separated sample. By detecting a sample separated by isoelectric focusing without having to mobilize the separated sample, the results are obtained much more quickly. While the focusing itself generally takes between about four and seven minutes, the mobilization usually takes between an additional fifteen to forty-five minutes. By monitoring and detecting the stationary sample, results are obtained as rapidly as the focusing occurs. Further, excellent high resolution is maintained between sample components because this resolution is not lost or distorted through mobilization.
The basic concept of the detectors of the invention is to focus a light beam which comprehends the entire length of interest of the capillary tube in which separations occur onto the capillary tube so that it passes through the sample along the length of the sample oontaining the separations of interest. This may range frog a two cm le»gth along the capillary tube to ten or mots eenti~ters. The intensity of the beam after passing through the sample is sensed or detected in a way that the variations in intensity along the width o! the bees which has pas~xed through the desired length of sample is determined. Thus, the width of tare beam has to be broken down into iany wall segments, each indfvidnally sensed or detected, in order to provide the desired output. In io , preferred forms of the imertion, a wide but thin light beam 59, Fig. 9 is generated and Focused onto the capillary passage so that the vtidth of the bsal~ w will . coaprehend the length along the capillary passage where separations of interest have taken place, and so that the height of the beam h is preferably smaller than the diaaeter o! the capillary passage. Thus, the height h of the bsato shown in Fig. 9 is somewhat exaggerated !or purposes of illustration. There are several ways such a light beam say be generated including the use of lenses 30 and the use of masks, i.s., an opaque sheet o! material with a slit tdsrsirv. Further, the light !or the light bsaa say originate from a laser, a light cititting diode (LED), a laser diode, or an LED or laser diode array.
Preferred detectors of the invention are shown in ,25 pig's. ?, and 10-1~. As shown, a light source 60, such as a lasex, or a laser diode, with appropriate lenses as needed, is arranged to direct a light beam sl, of circular cross section, toward capillary passage 36. ~ cylindrical lens s2 in the path of light beam 61 focuses the circular 9o beau 61 into a thin bean or sheet of light 63 with a width apprvxi.~sataly equal to the width or the circular bean.
This sheet of light is aligned~with and focused onto capillary passage 36. I! the width of the beaa is shorter than the entire length of the capillary passage, the beau 35 is set to pass through the length of the passage where it is expected that separations of interest will take place.
The beam 63 ritay pass directly through the capillary passage to a detector, or tray peas through the capillary passage to lens 64 vJhich expand the beam 63 before it reaabes the detector. The expanded laeam is labeled 65.
Such expansion can provide increased resolution of detection, particularly where the inareaents of detection are not as saall as desired.
J The preferred form of the invetion utilises two alternate detecting or sensing techniqaea. one is single detector, such as photodiode 66, ~oounted to be aoved linearly along the width of bean 65 to sense the inten*ity of light in light beaa 65 as a function of the linear position of photodiode 66. Since the light beam being sensed is very thin and the changes in intensity occur in substantially a single dirsction, i.e.;
along the . 15 width of the bear, accurate alignment of photodiode i with the beam from the standpoint of height of the beam is 1 not necessary.
?hatodiode 66 may be moved along the width of beala s3 in a variety of ways, and the way moveaent is obtained is o not critical. llny apparatus for moving the photodiode say be used as long ag the linear position of the diode can be kept track of. This may merely be a means that once started, will ~rnrs~thraugh tire entire length o!
travel of ' interest at a constant rate. An sxa~ple of apparatus for " 25 moving diode 6s is shown in Figs. ? and s0. a track 70 is mounted on base 52 arid receives slidably therein a carriage aaseably ?1. Carriage asseably ?1 includes Teflone ar similar plastic bearinge~ ?2 which ride against track 70 to reducs friction and a threaded aeaber 73 which 30 accepts a threaded shaft 74 therethrough. ~1 platform ?5 with bracket 76 and photodiade 65 haunted thereon is seaurad to carriage 91. Shaft ~4, Fig. ?, is coupled through sleeve ?e to the output shaft ?9 of stepper motor 80. As threaded shaft 74 is rotated by stepper rotor 80, 3S carriage 71, platform 95, and photodiode 66 move along track ?o. The direction of travel depends upon the direction of rotation of the stepper motor at~d the speed of travel depends upon the speed of rotation of the stepper motor. The speed of rotation, direction, and amount of rotation of a stepper motor may be accurately controlled in known manner so the rotation of the motor 5 and travel of photodiode 66 through the width of light beam 65 can be accurately monitored and controlled.
During a scan of photodiode 66 along the width of light beam 65, photodiode 66 will produce an output signal proportiona:L to the light intensity of beam 65 striking 10 photodiode 66 and, thus, a signal proportional to the light inten:aity air all points in the light beam along the path of travel of the photodiode. If it is desired to .further limit tlhe width of the segment sensed by photodiode 66, a shield 81 may be mounted in front of 15 photodiode E>6 with a slit 82, Fig. 7, therein of desired width. The output. of the photodiode is through leads 83.
The detector. electronics used would be standard and well known.
An alternate detector is shown in Fig. 11 and 20 replaces the:moving photodiode detector of Figs. 7 and 10.
The detector- of F:ig. 11 is a photodiode array 85 mounted in the path of light beam 63 after passage through the sample or in the path of expanded light beam 65. Many one dimensional or two dimensional photodiode arrays could be .25 used, with the cost of the array balanced against the desired resolution. The preferred arrays have very small sensing elements spaced along the width of the array. For example, arrays o~° charge coupled devices which are used in television and video cameras having sensing elements one one-thousandth of an inch in size are readily available, but thE: larger of such arrays and arrays of a grade with all elements working, are expensive. Arrays with even ::mallet sensing elements are also becoming available. Such .arrays may be obtained with as many as 1024 elementa in one dimension. Thus, the width of the array and the width of the light beam comprehended thereby would be broken down into 1024 individually sensed ~.~o~~~~ns. The electronics for scanning an array are standard and well known. The connections between the electronics and the array are through wires represented schematically as 86.
An overall block diagram of a detector system is shown in Fig. 12. The light source is represented by block 90 and includes the necessary components, such as a laser and lenses, to produce the necessary sheet of light, indicated by broken line 91. Light beam 91 passes through the capillary sample chamber 92 to the detector 93 which measures the intensity profile of the beam after passing through the sample. The output of the detector goes to any necessary interface electronics 94 which processes the signals from the detector and sends them to computer 95.
Signals from the computer needed to control the detector, such as scanning signals or motor control signals, or other control signals, are sent from computer 95, through interface electronics 94, to detector 93. With such a system, the intensity information obtained can be displayed in real time on the computer monitor and stored in memory for later display or processing. The computer 95 can also operate through interface electronics 96 to operate any reagent pumps, voltage supplies, or other equipment, indicated as block 97, to automate the . 25 capillary electrophoresis separations conducted in the capillary sample passage. This can allow coordination between the conditions of the capillary electrophoresis taking place in the sample chamber based upon the detected results.
In a prototype of the detector, a 100 ~cm diameter, 6.5 cm long square capillary passage was used in the apparatus shown for the isoelectric focusing. The capillary tube was glass and was glued with epoxy between two glass slides. The capillary passage walls were coated with non-crosslinked acrylamide to eliminate electroosmosis. The containers forming the reservoirs were polyethylene. The apparatus was mounted on a two-axis stage so the tilt angles in the borisental and vertical planes were adjustable to aid in alignment of the probe light beam with the capillary passage. A Ha-Ne laser manufactured by UniphaseT" of Sea Jose, California was toted to generate the probe light beam. The bean from the laser was expanded to a 2 cn diameter beam and was then focused into the capillary passage by a 6 am fooal length aylindrival lens mounted on a three-axis stage. The cylindrical lens produced a sheet of light of about x om 1o in width. After passage through the capillary passage, the 2 ant beam was expanded to a ZO cm beam in the detector plane by a 25 mat local length lens mounted behind the capillaxy tube. In,thist way, 1 cm width in the detector plane corresponded to a 1 mm length of ,the capillary passage. This sakes it easier to aaasurs the intensity profile of the bean. In so~te experiments, a single photodfade with a shield with 0.1 bm slit therein was mounted on a one-axis stage driven by the aoving part o!
a mode 3418 syringe pump trade by 4rion ~tessarah, Inc. of Massachusetts. This was located so the photodiode scanned in the detector plane. The scanning distance of the photodiade so mounted vas about 150 mat which corresponded to a 15 man length of the cetpillary passage. In other experi.msnts, a one-dimensional, 128 element photodiode a5 array was used to atonitor the intensity a! the beam in the detector plane. This array was able to monitor a 3 MAm length of the capillary passage. The whole system was mounted on a vibration isolation table. The data obtained by the detectors was collected through an IBM''" DACA board in a PC-AT personal computer, using the ASYSTm software supplied by Asyst Software Technology, Znc., Rochester, New York.
~In the tests of the systems, all chemicals ears reagent grads, and solutions were prepared using deionized eater. 10 mM HzPO, and 20 m!1 HaON were used as the anolyte and catholyte, respectively. NaOIM solution Bras degassed before use, by sparging with helium. Samples used include WO 9x111389 ~YUS93/007,34 ~~-chymotrypsin (tYPe =I, Sigma), phosphorylase b (Sigma) and ovalbumin (grade V, Sigma). semplss were ~al.xed with ampholyte (pharmalyte pN 3 - io, sierra) solution Ior a final concentration o! 2t nmpholyte. solutions ware filtered using o.2 pm pore sire cellulose acetate filters (Sartorius'~', Gottingen, Germany). The sample concentration introduced into the capillary ranged from 0.3sg/mL to 1 The sample was introduced into the capillary passage 1o by pressure generated by a syringe. A plug of 1x agarose gol i» the reservoir of the anadic end of capillary (prepared in the anolyte, 10 mM H~p04) was used to avoid hydrodynamic flow in the 100 um i.d. capillary. After introduction of the sample into the capillary passage, a 5 1cV do voltage was applied arid current passing through the capillazy was monitored to follow the focusing process. Typically, the current dropped from 15 ~cA to about 2.5 ~cA in 4-~ min, and then became stable for hours.
this minimum current flow indicates the isoelectric focusing process has been completed.
Fig. 13 shorts the beau intensity profile detected by the single photodiode scanned across the probe beam which passes through the part of the capillary located 3.5 - 5 - cm distance from the anodic end. Two sharp, high peaks 100 dad 101 are observed in the probe beam intensity profile shown in Fig. 13, which correspond to the positions expecteA fox the focused phosphorylase b (isoelectric point 6.3), and ovalbumin (isoeleCtric point 4.7), respectively. The concentrations of analytes are 3D about 1 mg/mL each. This result demonstrates that the focused proteins inside the capillary can be detected by this simple imaging system. The signal peak 100 corresponding to focused zone of phosphoryla$e b and its integrals are illustrated in Fig, i4, which clearly shows the second derivative characteristic of the detected signal loo. since this imaging system is an on-line detector, the isoelectric focusing process itself can be O 93/14389 _ 212 7 9 7 7 PCT/US93/00234 monitored. Fig. 15 shows the focusing process of phosphorylase b and ovalbumin. The concentrations of the samples area 0.5 ~mg/mL, which corresponds to 3.4 pmole of phosphorylase b and 7.2 pmole ovalbumin injected into the 6.5 cm lone square capillary. At the beginning (0 Min.) of the focusing, as shown by curve a in Fig. 15, no sharp peaks are observed. The detected signals are the probe beam intensity profile after it passes through the capillary. Many low peaks in Fig. 15 are generated by refractive index defects in the capillary wall or coating materials i.n the inner wall of the capillary, and their positions do not. change with the time, which can be observed in. curves .a, b, and c of Fig. 15. In curves b and c, two aecond derivative peaks 102 and 103 appear and become higher with longer focusing time, which correspond to focused ;phosphorylase b and ovalbumin. In addition to these two high peaks, other small peaks can be observed and become higher with the time in the curves b and c of Fig. 15. Those peaks are associated with the minor components in the: samples. It should be mentioned that the concentration gradients generated by the components of carrier ampholytea can also be detected because of the universal nature of the detector. The refractive index fluctuations produced by the carrier ampholytes can be noticed in 'the integral of the detected signals shown in Fig. 14. lHowever the second derivative nature of the imaging detector effectively reduces the amplitudes of low frequency broad signals generated by the wide bands of the carrier ampholytea. As shown in Fig. 13, high signal peaks can only be observed for high concentration gradients produced at the boundaries of narrow protein zones.
Figs. 7.3 and 15 were obtained with the moving single photodiode detector. Fig. 16 shows the focusing process for a «-chymotrypsin sample and covers a 3 mm length of the capillary passage. The curves of Fig. 16 were obtained using the 128 element linear photodiode array WO 93/14389 212 g~'~ PCT/US93/00234 described above. This clearly shows the practicality of using sensor arrays in the detector of the invention.
Further, the results obtained show that the detectors of the invention not only detect the focused analytes in the 5 sample, but can also be used for monitoring and studying the dynamics of the isoelectric focusing process inside the capillary passage.
Fig. 17 shows a second embodiment of a sample chamber for the electrophoresis apparatus of the invention. Some 10 sample solutions, such as blood serum samples, contain a high level of salts. This makes the sample have a high degree of electrical conductivity. If such a sample was introduced directly into a capillary passage, the voltage applied for the separation would cause excessive current 15 flow through the sample, excessive heating, and could result in explosion of the capillary tube. Thus, preparation of a sample to remove the salts therefrom is necessary prior to subjecting it to capillary electrophoresis separation. The apparatus shown in Fig.
20 17 can be used to prepare the sample for electrophoresis as part of the overall procedure.
The modification of the apparatus as shown in Fig. 17 is to provide a container 120 forming the sample reservoir 121 inside a second container 122. Sample container 120 25 is made of a porous membrane material such as a cellulose acetate membrane material. Alternately, container 120 could be a combination of porous membrane material and other material, such, for example, as a polyethylene container with a porous membrane bottom. Container 122 is 30 made of normal non-porous material such as polyethylene or glass. The capillary tube 123 passes through the wall of container 122 in a sealed manner and is secured in sample container 120 so that capillary passage 124 communicates with sample reservoir 121. Generally, the illustrated construction will be needed for only one of the reservoirs as shown in the apparatus of Fig. 6, usually the anodic reservoir. The sample 125 is introduced into sample 7 93/14389 ~ , ~- ~ 1,~,; PCT/US93/00234 reservoir 1.21, but will not initially flow into capillary tube 124. Water, which may also contain the desired ampholytes, is placed as solution 126 in container 122 so as to surround a portion of container 120. It is preferable to continually flow the water-ampholytes solution through container 122 so for that purpose an inlet tube 127 f~.~om a source of water-ampholyte solution may be provided to introduce such solution to container 122 while an outlet 128 may be provided so that solution may flow from container 122. With sample 125 in sample reservoir 7.21, the salts therein will pass through the porous membrane into the water solution 126 in container 122. Simultaneously, if the water also contains ampholytes, the ampholytes will pass through the membrane into the sample. Thus, the sample can be prepared by the removal of salts and the addition of ampholytes while in the sample reservoir. The sample 125 will remain in sample reservoir 121 for the time required to exchange the salts from the sample to the water through the membrane, and to exchange the ampholytes from the water solution to the sample. When the sample is properly prepared, the sample will be moved into the capillary passage such as through pre:asurizing the sample reservoir 121. The sample reservoir will include a sealed top 129 to allow pressurization oi: the reservoir to cause the sample to flow into the capillary passage, the electrode 130, and tube 131, a;s shown in the apparatus of Fig. 6.
The invention also includes a method of determining the presence or concentration of a particular component that does not have an isoelectric point so would not normally be focused and detected by capillary isoelectric focusing. The method includes the steps of adding a reagent hav~.ng a urell-def fined isoelectric point and having a high degree of specificity toward the component to be detected to a sample which may have the component therein, under conditions where the reagent and component will form a combination or complex of the two which will have an '~~,~ ~~~~~
", _ , , - , PGTiU ~ 9 3 / 0 0 2 3 4 03 Recd ~'~t/Ft~ 0 Z ~,U~ 1993.
isoelectric point different from the isoelectric point of the reagent itself, and then detecting the presence of the complex through iaoelectric focusing.
The meahod is useful in instances where it is desirable to detect the presence of a component, for example a toxin, in a sample, such as a body fluid, but the toxin may not have an isoelectric point so would not normally bas separated and detected by isoelectric focusing. In the method of the inventi~, a reagent having a high dE:gree of specificity toward a target component or analyte is used. The reagent is a substance which has a well-defined isoelectric point and forms a sharp band inside the capillary which may be easily detected through capillary isoelectric focusing and the detectors of the invention as a second derivative of the Gaussian. This :is shown by signal 135 in the curves labeled a, b, and .c in Fig. 18. The reagent, for example, may be proteain which is an antibody for the toxin being looked for o~r could be a computer designed and laboratory synthesized organic molecule, for example, a synthetic cavity ligand. The component being looked for can be a toxin or other mo7.ecule which generally will not have an isoelectric point:. The reagent, R, is picked to specifically and strongly interact or react chemically with the target component or analyte A, to form, for example, the: product:
R + A = RA
The product of the: chemical process, RA, has a different isoelectric point and therefore focuses in a different part of the capillary then the reagent R. Thus, the product is i.ndicat:ed as peak 1-36 in curve b of Fig.
18.
In addition, the hE:ight of the corresponding signal 136 is proportionally related to the amount of the target component present. The reagent can also be ~ie~= .-ned to react with several target analytes to produce several different products. In such instance, several corresponding signals 136, 137, and 138 may be obtained as SUBSTITUTE SHEET
.. 212977 ~ _ .. .
pCT~~.~~ 93 /00 23~+
33 03 Rec'c~ ~'~fT/~ '~ 0 2 ~~.~JG 1995 shown in curve c of Fig. 18. It will usually be desirable to ensure that there is an excess of reagent added to the sample so that all. of the component present in the sample will react with reagent. In this way, the signal representing the component-reagent product, i.e., peaks 136, I37, or :138, will be proportional to the concentration of component in the sample. The presence of a peak 135 for the reagent as well as a peak for the product will. indicate this excess.
l0 Because: reagents having very high specificity for components of a sample being looked for can be produced to therefore provide accurate detection and concentration information, the method described using the simple and relatively inexpensive detector of the invention has the potential to replace many tedious analytical procedures and expensive instrumentation.
As mentioned previously, the electrophoresis separating apparatus of the invention could be used with a detector ;simila:r to those shown in my prior patents having a beam located at one end of the capillary passage and mobilizing 'the sample separation obtained by isoelectric focusing so that it flows through the capillary and the: detector beam. Fig. 19 shows the results obtained using capillary electrophoresis apparatus similar to that shown but with a laser diode as the light source for a light beam passed through one end of a 20 ~,m capillary passage in a 12 cm long capillary tube. The sample used contained 120 fmol of human hemoglobin indicated by peak 140, myoglobin indicated by peak 141, 270 fmol of human carbonic anhydrase indicated by peak 142, 240 fmo~l of bovine carbonic anhydrase indicated by peak 143, and 410 fmol of 8-lactoglobulin indicated by peak 144. The con<:entration of the sample was 0.2 mg/mL.
Figs. 20 and ai show the difference in separation of a sample b:y capillary zone electrophoresis, moving boundary capillary electrophoresis, and isoelectric focusing. :Figs. 20 and 21 show electropherograms of ~sUBSTITUT~ SHEE1°
WO 93114389 PC'F/US91l90I34 avalbu~ain separated by the three separation methods. The ovalbumin was purified by the manufacturer with slab cone electrophoresis whiatr is based ot~ a sample s mobility differences. l~s expected, tt~e electropherogram of capillary zone electrophoresis separation, curve a in Fig.
20, shows only one peak, 150. The elsctropherogram of moving boundary capillary electrophoresis separation, curve b in Pig. 20, also shaves one peak, lxl, since it is based on the same separation principle, a difference in component mobility, as capillary zone electrophoresis.
However, the electropharogra~a of the same sa~rple separated by isoelectric focusing, Pig. 21, shows more than four peeks. The highest peak 15Z, corresponds to ovalbumin, and other peaks can be attributed to impurities or minor components in the sample which have almost the same mobilities as that of ovalbumin, but different iroelectric points from that of avalbumin. xhe electropharagrams or curves shown were obtained using the apparatus with detector at the end thereof and mobilizing the sample 2o separation obtained by isoslectric focusing. This shows the significant difference and improvement in separation and sensitivity obtained by using isoelectric focusing.
The detector of the invention cnn also be used as an absorbanca imaging detector to detect concentration of separated sample components directly rather than x~easuring concentration gradients. The only difference in such case, is the nature of the light beam which provides light of frequencies to be absorbed by sample components. The light beam may be generated by ttn incoherent light source such as a halogen lamp or may be generated by a laser ox variable frequency laser. If from an incoherent light source the light is preferably filtered to limit it to the desired frequencies. The components o! the sample as separated along the capillary passage will absorb some of the light so the light intRnslty will vary along the width of the light beam. The detector, as indicated above, detects the variation in light intensity along the width of the light beam.
In an example of the absorbance imaging detector, the light beam source was a halogen lamp. The light from the 5 lamp was collected by a paraboloidal ref lector which reflected light onto a mirror. The light beam was filtered by a color filter which was transparent in 400 nm to 600 nm wavele:ngth range. The light beam was then focused into a 200 ~m wide slit which was focused onto a 10 200 ~cm dia.meter capillary tube. The image of the capillary illuminated by the light beam was projected by a 10 cm focal length lens onto a 1024 pixel or sensing element charge-coupled device (CCD) such as a S3903-1024Q
made by Hamamatsu,, Hamamatsu City, Japan, which had a 25-15 mm X 0.5-mm sensing area. The changes in the light beam intensity profile due to the refractive gradient inside the capillary was eliminated by focusing the image of the capillary onto the. detector plane, i.e., the sensor. The data was collected by an IBM DACA board, in a PC-AT
20 personal computer. An averaging method was applied to reduce the :random noise. - For each measurement, the CCD
was scanned ten times in 1 second and these scans were averaged. The background image which was recorded before the focusing voltage was turned on, was first subtracted 25 from the sample images, and the sample images were then normalized by 'the background image to eliminate fluctuation: created by the source beam intensity distribution. ~, human hemoglobin sample from Sigma Chemical Co., containing 75% methemoglobin, balanced 30 primarily 'with oxyhemoglobin, was separated using isoelectric focusing. The electropherogram obtained is shown in Fig. 22. Peaks 160 and 161 correspond to two variants of hemoglobin; methemoglobin (pl 7.2, comprises 75% of the ;sample) and oxyhemoglobin (pl 7.0, comprises 35 less than 2_°°>% of 'the sample), respectively. Other peaks may correspond to other variants of hemoglobin. The 1Ab 93114989 PCT/US93/ODZ~t r~ssulta show effective detection through the absorbance .
detection.
Figure Z3 shows the eleatropherogram of the sa~ae hemoglobin sample es tbat in ~'ig. Z2, which is focused in a 100 pm diameter capillary and detected using concentxatioa gradient iaaging. The resolution of the concentration gradient imaging shown in Fig. 23 is better than that of the absorbance imaging due to the second derivative nature of the concentration gradient imaging l0 which eliminates broad bands and fluctuations.
Further, when using an incoharet~t light source for the absorbance . detection, use of narrow capillaries is limited.
For absorbance imaging using narr~ capillaries, such ae capillaries with loo ~m diaaater or lass, an adjustable i5 wavelength laser is necessary, and !or use in detecting proteins, an adjustable wavelength laser working in the W
range is necessary since most proteins only have ahsorbanca bands in the tN range. Thus, the concentration gxadient imaging appears most practical fax small diaseter Zo aapillsry tubes. However, for larger diameter capi7.lary tubes, the absorbance imaging can produce excalient results.
iihereas this invention is here illustrated and described with reference to e~obodimeats thereof presently Z5 contemplated as the best mode of carrying out such invention in actual practice, it is to be understood that various changes clay be made in adapting the invention to different embodiments without departing from the broader inventive concepts disclosed herein and comprehended by 3Q the claims that follow.
Claims (20)
1. A capillary electrophoresis system, comprising:
a light transmitting capillary tube having a capillary passage therethrough in which separation of components of a sample take place, said capillary passage having a diameter;
means for introducing a sample to be separated into the capillary passage;
means for creating a separation of the sample by electrophoresis within the capillary passage along a predetermined length of the capillary passage, said predetermined length of the capillary passage being substantially greater than the diameter of the capillary passage;
a light source for generating a light beam;
means for forming the light beam into a sheet of light having a width at least equal to the predetermined length of the capillary passage and a height no greater than the diameter of the capillary passage and for directing the sheet of light through the capillary passage so that the light passes through the predetermined length of the capillary passage, said predetermined length being such as to include separations of interest present in the capillary passage;
and detectors means located in the path of the sheet of light after passing through the capillary passage for detecting the intensity of light at various positions along the predetermined length of the capillary passage and to provide an output representative of the light intensity at the various positions along the predetermined length of the capillary passage, the intensity of the light at various positions along the predetermined length of the capillary passage being indicative of sample separation along the predetermined length of the capillary passage.
a light transmitting capillary tube having a capillary passage therethrough in which separation of components of a sample take place, said capillary passage having a diameter;
means for introducing a sample to be separated into the capillary passage;
means for creating a separation of the sample by electrophoresis within the capillary passage along a predetermined length of the capillary passage, said predetermined length of the capillary passage being substantially greater than the diameter of the capillary passage;
a light source for generating a light beam;
means for forming the light beam into a sheet of light having a width at least equal to the predetermined length of the capillary passage and a height no greater than the diameter of the capillary passage and for directing the sheet of light through the capillary passage so that the light passes through the predetermined length of the capillary passage, said predetermined length being such as to include separations of interest present in the capillary passage;
and detectors means located in the path of the sheet of light after passing through the capillary passage for detecting the intensity of light at various positions along the predetermined length of the capillary passage and to provide an output representative of the light intensity at the various positions along the predetermined length of the capillary passage, the intensity of the light at various positions along the predetermined length of the capillary passage being indicative of sample separation along the predetermined length of the capillary passage.
2. The capillary electrophoresis system according to claim 1, wherein the diameter of the capillary passage is between and 100 microns.
3. The capillary electrophoresis system according to claim 1, wherein the detector means is an array of light sensors extending along the predetermined length of the passage in fixed position with respect to the passage and positioned with respect to the passage so that light from the sheet of light passing through the passage is focused on the array, whereby individual detectors of the array detect light at the various positions along the predetermined length.
4. The capillary electrophoresis system according to claim 3, wherein the light detecting array is a photodiode array.
5. The capillary electrophoresis system according to claim 1, wherein the detector means includes a detector having a narrow sensing area compared to the width of the light beam and means for moving the detector along the predetermined length of the passage.
6. The capillary electrophoresis system according to claim 5, wherein the detector is a photodiode.
7. The capillary electrophoresis system according to claim 6, wherein a shield is provided between the photodiode and the capillary passage which moves With the photodiode, and an opening in the shield which defines the sensing area of the photodiode.
8. The capillary electrophoresis system according to claim 1, wherein the means for generating a light beam is a laser which generates the light beam and the means for forming the light beam into a sheet of light is a cylindrical lens which shapes the beam.
9. The capillary electrophoresis system according to claim 1, including a lens positioned between the capillary passage and the detector to expand the light beam after passage through the capillary passage.
10. The capillary electrophoresis system according to claim 1, wherein the intensity of the light at various positions along the predetermined length of the passage is indicative of the sample component concentration gradients in the separated sample in the passage.
11. The capillary electrophoresis system according to claim 1, wherein the intensity of the light at various positions along the predetermined length of the passage is indicative of the light absorption of separated sample components in the passage.
12. The capillary electrophoresis system according to claim 11, wherein the light directed through the capillary passage a.s substantially of a frequency to be absorbed by an expected component of the sample.
13. A capillary electrophoresis system, comprising:
a light transmitting capillary tube having a capillary passage therethrough in which separation of components of a sample take place, said capillary passage having a diameter;
a reservoir at each end of the passage in fluid communication with the passage, each reservoir configured to receive an electrode positioned therein and at least one of the reservoirs adapted to cooperate with a means for adding liquid to or withdrawing liquid from the reservoir;
means securing the reservoirs at opposite ends of the capillary tube;
an electrode in each of the reservoirs adapted to be connected to a source of voltage for creating a separation of the sample by electrophoresis within the capillary passage along a predetermined length of the capillary passage, said predetermined length of the capillary passage being substantially greater than the diameter of the capillary passage;
a light source for generating a light beam;
means for forming the light beam into a sheet of light having a width at least equal to the predetermined length of the capillary passage and a height no greater than the diameter of the capillary passage and for directing the sheet of light through the capillary passage so that the light passes through the predetermined length of the capillary passage, said predetermined length being such as to include separations of interest present in the capillary passage;
and detector means located in the path of the sheet of light after passing through the capillary passage for detecting the intensity of light at various positions along the predetermined length of the capillary passage and to provide an output representative of the light intensity at the various positions along the predetermined length of the capillary passage, the intensity of the light at various positions along the predetermined length of the capillary passage being indicative of sample separation along the predetermined length of the capillary passage.
a light transmitting capillary tube having a capillary passage therethrough in which separation of components of a sample take place, said capillary passage having a diameter;
a reservoir at each end of the passage in fluid communication with the passage, each reservoir configured to receive an electrode positioned therein and at least one of the reservoirs adapted to cooperate with a means for adding liquid to or withdrawing liquid from the reservoir;
means securing the reservoirs at opposite ends of the capillary tube;
an electrode in each of the reservoirs adapted to be connected to a source of voltage for creating a separation of the sample by electrophoresis within the capillary passage along a predetermined length of the capillary passage, said predetermined length of the capillary passage being substantially greater than the diameter of the capillary passage;
a light source for generating a light beam;
means for forming the light beam into a sheet of light having a width at least equal to the predetermined length of the capillary passage and a height no greater than the diameter of the capillary passage and for directing the sheet of light through the capillary passage so that the light passes through the predetermined length of the capillary passage, said predetermined length being such as to include separations of interest present in the capillary passage;
and detector means located in the path of the sheet of light after passing through the capillary passage for detecting the intensity of light at various positions along the predetermined length of the capillary passage and to provide an output representative of the light intensity at the various positions along the predetermined length of the capillary passage, the intensity of the light at various positions along the predetermined length of the capillary passage being indicative of sample separation along the predetermined length of the capillary passage.
14. The capillary electrophoresis system according to claim 13, additionally including means cooperable with one of the reservoirs for adding liquid to or withdrawing liquid from the reservoir.
15. The capillary electrophoresis system according to claim 14, wherein the means for adding liquid to or withdrawing liquid from the reservoir is a syringe and a tube secured to the syringe and extending into the reservoir.
16. The capillary electrophoresis system according to claim 13, wherein the capillary passage has a diameter of between and 100 microns.
17. The capillary electrophoresis system according to claim 13, wherein the capillary passage is 10 centimeters long.
18. The capillary electrophoresis system according to claim 13, wherein the means securing the reservoirs at opposite ends of the capillary tube is a piece of glass with the capillary tube secured thereto.
19. A capillary electrophoresis system, comprising:
a light transmitting capillary tube having a capillary passage therethrough in which separation of components of a sample take place, said capillary passage having a diameter;
means for introducing a sample to be separated into the capillary passage;
means for creating a separation of the sample by electrophoresis within the capillary passage along a predetermined length of the capillary passage, said predetermined length of the capillary passage being substantially greater than the diameter of the capillary passage;
a light source for generating a light beam;
means for forming the light beam into a sheet of light having a width at least equal to the predetermined length of the capillary passage and for directing the sheet of light through the capillary tube so that light passes through the predetermined length of the capillary passage, said predetermined length being such as to include separations of interest present in the passage;
and detector means located in the path of the sheet of light after passing through the capillary tube for detecting the intensity of light that has passed through the capillary passage in a portion of the sheet of light no greater in height than the diameter. of the capillary passage at various positions along the predetermined length of the capillary passage and to provide an output representative of the light intensity at the various positions along the predetermined length of the capillary passage, the intensity of the light at various positions along the predetermined length of the capillary passage being indicative of sample separation along the predetermined length of the capillary passage.
a light transmitting capillary tube having a capillary passage therethrough in which separation of components of a sample take place, said capillary passage having a diameter;
means for introducing a sample to be separated into the capillary passage;
means for creating a separation of the sample by electrophoresis within the capillary passage along a predetermined length of the capillary passage, said predetermined length of the capillary passage being substantially greater than the diameter of the capillary passage;
a light source for generating a light beam;
means for forming the light beam into a sheet of light having a width at least equal to the predetermined length of the capillary passage and for directing the sheet of light through the capillary tube so that light passes through the predetermined length of the capillary passage, said predetermined length being such as to include separations of interest present in the passage;
and detector means located in the path of the sheet of light after passing through the capillary tube for detecting the intensity of light that has passed through the capillary passage in a portion of the sheet of light no greater in height than the diameter. of the capillary passage at various positions along the predetermined length of the capillary passage and to provide an output representative of the light intensity at the various positions along the predetermined length of the capillary passage, the intensity of the light at various positions along the predetermined length of the capillary passage being indicative of sample separation along the predetermined length of the capillary passage.
20. A capillary electrophoresis system, comprising:
a light transmitting capillary tube having a capillary passage therethrough in which separation of components of a sample take place, said capillary passage having a diameter;
a reservoir at each end of the passage in fluid communication with the passage, each reservoir configured to receive an electrode positioned therein and at least one of the reservoirs adapted to cooperate with a means for adding liquid to or withdrawing liquid from the reservoir;
means securing the reservoirs at opposite ends of the capillary tube;
an electrode in each of the reservoirs adapted to be connected to a source of voltage for creating a separation of the sample by electrophoresis within the capillary passage along a predetermined length of the capillary passage, said predetermined length of the capillary passage being substantially greater than the diameter of the capillary passage;
a light source for generating a light beam;
means for forming the light beam into a sheet of light having a width at least equal to the predetermined length of the capillary passage and for directing the sheet of light through the capillary tube so that light passes through the predetermined length of the capillary passage, said predetermined length being such as to include separations of interest present in the capillary passage:
and detector means located in the path of the sheet of light after passing through the capillary tube for detecting the intensity of light that has passed through the capillary passage in a portion of the sheet of light no greater in height than the diameter of the capillary passage at various positions along the predetermined length of the capillary passage and to provide an output representative of the light intensity at the various positions along the predetermined length of the capillary passage, the intensity of the light at various positions along the predetermined length of the capillary passage being indicative of sample separation along the predetermined length of the capillary passage.
a light transmitting capillary tube having a capillary passage therethrough in which separation of components of a sample take place, said capillary passage having a diameter;
a reservoir at each end of the passage in fluid communication with the passage, each reservoir configured to receive an electrode positioned therein and at least one of the reservoirs adapted to cooperate with a means for adding liquid to or withdrawing liquid from the reservoir;
means securing the reservoirs at opposite ends of the capillary tube;
an electrode in each of the reservoirs adapted to be connected to a source of voltage for creating a separation of the sample by electrophoresis within the capillary passage along a predetermined length of the capillary passage, said predetermined length of the capillary passage being substantially greater than the diameter of the capillary passage;
a light source for generating a light beam;
means for forming the light beam into a sheet of light having a width at least equal to the predetermined length of the capillary passage and for directing the sheet of light through the capillary tube so that light passes through the predetermined length of the capillary passage, said predetermined length being such as to include separations of interest present in the capillary passage:
and detector means located in the path of the sheet of light after passing through the capillary tube for detecting the intensity of light that has passed through the capillary passage in a portion of the sheet of light no greater in height than the diameter of the capillary passage at various positions along the predetermined length of the capillary passage and to provide an output representative of the light intensity at the various positions along the predetermined length of the capillary passage, the intensity of the light at various positions along the predetermined length of the capillary passage being indicative of sample separation along the predetermined length of the capillary passage.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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US81932592A | 1992-01-13 | 1992-01-13 | |
US819,325 | 1992-01-13 | ||
PCT/US1993/000234 WO1993014389A1 (en) | 1992-01-13 | 1993-01-13 | Method and apparatus for performing and universally detecting capillary isoelectric focusing without mobilization using concentration gradient imaging systems |
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CA2127977A1 CA2127977A1 (en) | 1993-07-22 |
CA2127977C true CA2127977C (en) | 2006-03-28 |
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CA002127977A Expired - Lifetime CA2127977C (en) | 1992-01-13 | 1993-01-13 | Method and apparatus for performing and universally detecting capillary isoelectric focusing without mobilization using concentration gradient imaging systems |
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CA (1) | CA2127977C (en) |
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1993
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