JP4113945B2 - Capillary electrophoretic adsorption / desorption chromatography and apparatus therefor - Google Patents

Description

ここでＢは外部磁場（Ｔ）、ｉは電流（Ａ）、Ｖは微粒子の体積（ｍ³）、Ｓはキャピラリの断面積（ｍ^２）、σ_ｆとσ_ｐは、それぞれ、媒体の伝導率（Ｓｍ^−１）および液中微粒子の電気二重層に起因する微粒子の表面伝導率（Ｓｍ^−１）である。さらに、(1)式と粘性抵抗力の式から微粒子の電磁泳動速度ｖ_ＥＭＰは下式のように表される。
【数２】

ここでｒは粒子半径（ｍ）、ηは媒体の粘度（Ｐａｓ）、Ｃ_Ｗはキャピラリ内壁の抵抗係数である。このように電磁泳動力は粒子の体積、電流および外部磁場に、泳動速度は粒子半径の二乗、電流および外部磁場に比例する。
【００１３】
図１は、キャピラリ内壁に吸着した微粒子に供給される電流と時間との関係を示すグラフである。図に示すように、キャピラリ内壁に吸着した微粒子を時間ｔ_ｉの間に電流値ｉ_ｍａｘまで増加する波形の電流を印加し電磁泳動力により脱着を行うと、電流値が増加するにつれて電磁泳動力が増大してゆき、電磁泳動力が吸着力Ｆ_Ａに対して
【数３】

となったときに微粒子の脱着が起こる。微粒子が脱着したときの電流値を脱着電流ｉ_Ｄとする。
【００１４】
図２は、キャピラリフロー系において微粒子が脱着してから吸着するまでの移動距離ｄを示した概念図である。図に示すように、キャピラリ内壁に吸着している粒子が電磁泳動力によって壁から脱着し、電磁泳動によってキャピラリを横切っている間、フローによって移動する。図１に示した波形の電流により、粒子は脱着電流ｉＤのときに脱着し、泳動し始める。泳動しているときの速度変化が小さいとすると、半径ｒの粒子が内径ｗのキャピラリを電磁泳動により横断するのに要する時間ｔ_ｗは、
【数４】

となる。ここでkは(2)式の電流と半径以外の項をまとめた係数である。粒子は時間ｔ_ｗの間フローによって移動するので、フロー速度をｖ_ｆとすると移動距離ｄは、
【数５】

となる。(5)式で表したように、移動距離ｄは粒子の半径と脱着電流に依存すると考えられる。そのため個々の粒子の性質により移動距離ｄは異なる。
【００１５】
図３は、キャピラリフロー系において2種類の微粒子が吸脱着を繰り返した場合のそれぞれの移動距離を示す概念図である。図の上部と下部の各キャピラリ内の白丸および黒丸で粒子の移動の様子を模式的に示すように、微粒子の性質（粒径、内壁との相互作用など）に応じて一回の吸脱着の移動距離ｄが異なれば、吸脱着を繰り返すことによって、同じ流速であっても一定距離を進むのに要する時間に差ができ、一定のキャピラリ長における保持時間が異なる。このときの粒子の保持時間は、
【数６】

で表される。ここでｌはキャピラリ有効長である。(6)式のように、保持時間は脱着電流ｉ_Ｄおよび粒子半径ｒに依存すると考えられ、これらの違いにより微粒子の分離が可能となる。
【００１６】
図４は、本発明によるキャピラリ電磁泳動吸脱着クロマトグラフィ装置で用いる電磁泳動用キャピラリフローセル部のキャピラリセルを示す概略図である。図に示すように、キャピラリセル１０は、内径100×100μｍ、長さ2cmの正方形フューズドシリカのキャピラリ１２（Polymicro Technologies社製）を具え、この両端を熱収縮チューブ１４を用いてＰＴＦＥチューブ１６につなぎ合わせたものである。
【００１７】
図５は、図４に示したキャピラリセルを含む電磁泳動用キャピラリフローセル部を示す概略図である。この図はキャピラリフローセルの全体図であり、図に示すように、キャピラリフローセル部１００は、キャピラリセル１１０を具え、この両端に三方コネクタ１２０を接続し、三方コネクタ１２０の残りの一方からＡｇ｜ＡｇＣｌ電極１３０を挿入し、他方に送液用ＰＴＦＥチューブ１４０を接続したものである。Ａｇ｜ＡｇＣｌ電極は、銅線を塩化カリウム水溶液中で電気分解することにより作製さいた。その手順は、銅線を電源の＋極につなぎ、白金線を−極につないで、その2本の金属線を１Ｍ塩化カリウム水溶液に浸す。それから、５Ｖで５分間電流を流すと銅線の先端に塩化銀が生成してＡｇ｜ＡｇＣｌ電極が完成する。ここで、磁場発生装置（図示しない）から生じた均一な磁場は図の上下方向に生じ、一方、電極から生じた電流ｉの流れる方向は図に示すように、磁場と実質的に直交するように左右方向に交互に反転する。
【００１８】
図６は、本発明によるキャピラリ電磁泳動吸脱着クロマトグラフィ装置の全体を示す概略図である。図に示すように、キャピラリ電磁泳動吸脱着クロマトグラフィ装置２００は、磁場発生手段として、液体ヘリウムが不要な無冷媒型超伝導磁石２２０（JMT社製、JMTD-10T100HH1）を具える。この超伝導磁石２２０は、直径100mm、長さ950mmの室温ボアを持ち、上フランジ面から560mm±5mmに０〜１０Ｔの均一磁場を作り出すことができ、磁場の方向は図中の「矢印Ｂ」で示すように重力方向（下方）である。このように、磁場がキャピラリの長手方向（即ち電流の流れる方向）に対して直角となるように、装置を配置する。また、本クロマトグラフィ装置２００は、前記均一磁場内のセルホルダに固定されたキャピラリフローセル２３０、ＰＣ２４０、ファンクションシンセサイザー２５０（NF社製）、アンプ２６０（TREC社製、MODEL610-C）、デジタルマルチメータ２７０（IWATSU社製電流計、VOAC7512）、ビデオレコーダ２８０、モニタ２９０、シリンジポンプ３００（Harvard Apparatus社製、pump II）光源３１０（オリンパス社製、MODEL ILV）、CCDカメラ３２０（ELMO社製、ME421）、および光学顕微鏡３３０をも具える。ＰＣ２４０には、「任意波形作成ソフトウェア0105（NF社製）」が導入されており、これを用いて所望の印加電流波形を作成し、ファンクションシンセサイザー２５０に波形を送り電圧を出力し、アンプ２６０により電圧を増幅し、セル中の試料液に印加し、デジタルマルチメータ２７０により電流を測定する。キャピラリフローセル２３０に含まれるＰＴＦＥチューブの一端を内容量１００μｌのシリンジ（HAMILTON社製、GASTIGHT#1701）に接続し、シリンジポンプ３００により微粒子が含まれる試料液を送液する。光源３１０を用いて下方から光を入れ、上方から１０倍の対物レンズの付いた光学顕微鏡３３０で観測し、ＣＣＤカメラ３２０で微粒子の泳動挙動を撮影してこれをモニタ２９０に出力し、ビデオ２８０で録画する。磁場が均一になるように、超伝導磁石のボア内に入れる装置は、全て非磁性のアクリル、アルミ、或いは真鍮を用いて作製してある。
【００１９】
本発明によるキャピラリ電磁泳動吸脱着クロマトグラフィ装置を用いて、その性能および効果を確認するために、2種類の粒径が異なる微粒子を含む溶液を用いて分離実験を行った。
試料
試料液としては、粒径２０μｍ、１０μｍの2種類のポリスチレン粒子を、0.01%の界面活性剤Triton X-100を含む１Ｍ塩化カリウム溶液に分散させたものを使用した。このときの媒体の密度は、ポリスチレンの密度（1.05gcm^-3）とほぼ一致している。
【００２０】
ポリスチレン粒子の保持時間の測定
本実験では、本発明によるキャピラリ電磁泳動吸脱着クロマトグラフィ装置に直径２００μｍの円形シリカキャピラリを使用した。本装置において、キャピラリ有効長１ｍｍでのポリスチレン粒子の保持時間の測定を行った。図１に示した波形の電流をｉ_ｍａｘ＝２０００μＡに、ｔ_ｉ＝２ｓに設定して、試料液に電流を供給した。試料液のフロー速度が４０〜１００μＬ／ｈの範囲内においてポリスチレン粒子の挙動を観察し、ポリスチレン粒子がキャピラリ中を１ｍｍ移動するのにかかる時間をモニタ上で測定し、ポリスチレン粒子の保持時間を求めた。
【００２１】
測定結果
図７は、種類の粒径のポリスチレン粒子の保持時間と流速との関係を示すグラフである。測定条件は、ｉ_ｍａｘ＝２０００μＡ、ｔ_ｉ＝２ｓに設定し、粒径１０μｍ、および２０μｍ、直径２００μｍの円形シリカキャピラリでの有効長１ｍｍである。このような条件において、流速即ちフロー速度を変化させて、保持時間を測定した。どのフロー速度においても、粒径１０μｍの粒子の保持時間の方が、粒径２０μｍよりも小さくなった。クロマトグラフ法の分離能Ｒは物質の保持時間を用いて次式で表される。
【数７】

ここでｔ_１およびｔ_２は、それぞれ物質１および物質２の保持時間である。この分離能Ｒが１以上であるとき、そのクロマトグラフ法によって物質１、２の分離が可能となる。本測定の場合は、分離能Ｒ＝ｔ_２０／ｔ_１０＝３．１となり、ポリスチレン粒子を粒径によって分離することが可能であることが示された。また、この実験において、吸脱着の際のポリスチレン粒子の脱着電流ｉ_Ｄは、粒径１０、２０μｍともに約１０００μＡであった。図７中の曲線はこの脱着電流を用いて(6)式から描いた理論曲線である。破線が粒径２０μｍ、実線が粒径１０μｍを表しており、測定値と理論値とはほぼ一致していることが判明した。このように、(6)式から粒子の保持時間を予測することが可能であり、保持時間から微粒子を同定することが可能である。
この実施例では、電流の正、負の切替が２秒としてるので、フロー速度が４０μL/hの場合、２０マイクロメートルのポリスチレン微粒子で５５回、１０マイクロメートルの微粒子で２０回程度、１ｍｍのキャピラリ内で吸脱着を行なったことになる。
【００２２】
本発明による装置や方法は、微粒子の分離法や分離装置として、環境および保健分野、機能性素材の製造分野、食品・衛生分野などで広く利用され得るものである。
本明細書では、様々な実施態様で本発明の原理を説明してきたが、本発明は上述した実施例に限定されず幾多の変形および修正を施すことが可能であり、これら変形および修正されたものも本発明に含まれることを理解されたい。
【図面の簡単な説明】
【図１】 図１は、キャピラリ内壁に吸着した微粒子に供給される電流と時間との関係を示すグラフである。
【図２】 キャピラリフロー系において微粒子が脱着してから吸着するまでの移動距離ｄを示した概念図である。
【図３】 キャピラリフロー系において2種類の微粒子が吸脱着を繰り返した場合のそれぞれの移動距離を示す概念図である。
【図４】 本発明によるキャピラリ電磁泳動吸脱着クロマトグラフィ装置で用いるキャピラリセルを示す概略図である。
【図５】 図４に示したキャピラリセルを含む電磁泳動用キャピラリフローセル部を示す概略図である。
【図６】 本発明によるキャピラリ電磁泳動吸脱着クロマトグラフィ装置の全体を示す概略図である。
【図７】 2種類の粒径のポリスチレン粒子の保持時間と流速との関係を示すグラフである。
【符号の説明】
２００ キャピラリ電磁泳動吸脱着クロマトグラフィ装置２００
２２０ 超伝導磁石
２３０ キャピラリフローセル
２４０ ＰＣ
２５０ ファンクションシンセサイザー
２６０ アンプ
２７０ デジタルマルチメータ
２８０ ビデオレコーダ
２９０ モニタ
３００ シリンジポンプ
３１０ 光源
３２０ CCDカメラ
３３０ 光学顕微鏡[0001]
BACKGROUND OF THE INVENTION
The present invention relates to capillary electrophoresis adsorption / desorption chromatography and an apparatus therefor.
[0002]
[Prior art]
Biotechnology, molecular biology, in the field of clinical tests, blood cells, subcellular particles, although the separation of such proteins are required, the micrometer level size for the fine particles, such as by conventional electromagnetic electrophoresis and chromatography graphic Separation was impossible. Currently, cell sorters, or flow cytometers, are commercially available and used as a method for separating fine particles at the cell level. However, in this conventional technique, it is necessary to label each cell with a selective fluorescent labeling reagent. When using such cells, there is a problem that it is necessary to remove the labeling reagent.
As for the electrophoretic migration of fine particles in liquid, there is “electrophoretic analysis method and apparatus for fine particles in liquid” (refer to Patent Document 1) by Watanabe et al. Is a basic principle that clarifies the fundamental behavior of electrophoretic movement of fine particles in liquid, but because it is a separation method / apparatus that uses only the electrophoretic movement of individual fine particles in the medium, the efficiency and speed of separation is high. There was a problem of being slow.
[0003]
[Patent Document 1]
JP 2000-105218 A (paragraph 0005, FIG. 1)
[0004]
[Problems to be solved by the invention]
An object of the present invention is to provide a capillary electrophoretic adsorption / desorption chromatography for separating, purifying, and analyzing fine particles in a solution and the apparatus for solving the above-mentioned problems.
[0005]
[Means for Solving the Problems]
Capillary electrophoretic adsorption / desorption chromatography according to the present invention comprises:
Flowing a solution containing fine particles into the capillary;
By applying a magnetic field to the solution flowing in the capillary and flowing a current so as to be substantially orthogonal to the magnetic field, the microparticles contained in the solution are electrophoresed, and the reversal of the polarity of the current is repeated (for example, Reversing the microparticles contained in the solution with the inner wall of the capillary by, for example, reversing at a predetermined cycle),
Moving the microparticles by flow;
It is characterized by including.
According to the present invention, the electromagnetic particles are electrophoresed using the electromagnetic buoyancy of the individual fine particles in the medium, and the interaction between the fine particle surface and the capillary inner wall inherent to the individual fine particles is utilized. The separation efficiency and separation speed can be significantly improved. According to the present invention, in electrophoretic electrophoresis, in a normal model, it is considered that the moving speed changes according to the size of the particle size of the fine particles, so that the fine particles having different particle sizes can be separated on this principle. . Furthermore, since the present invention also utilizes the interaction between the capillary material and the fine particles, the interaction between the capillary material and the fine particles (which requires a large amount of energy for adsorption / desorption with the capillary inner wall) When fine particles having a small effect are mixed, the sample can be separated and purified at a very high speed and with high efficiency. In addition, the separation efficiency and separation speed of the fine particles can be further improved by appropriately selecting the capillary material according to the fine particles to be separated. In the present invention, the label reagent required in the cell sorter of the prior art is unnecessary, so that the separation of the label reagent is unnecessary, and the separated microparticles coming out from the capillary are used immediately without any treatment. Is possible.
[0006]
Further, the capillary electrophoretic adsorption / desorption chromatography according to the present invention comprises:
Furthermore, the step of detecting the fine particles in the capillary using a microscope (such as an optical microscope, a fluorescence microscope, a microscope system for measuring light absorption / scattering),
Is also included.
According to the present invention, it is possible to observe the movement of nanometer-order microparticles that cannot be observed with the naked eye. since it to observe, etc. conditions and migration speed, can be separated more efficiently rapidly microparticles to understand the behavior of particles in more detail.
[0007]
Further, the capillary electrophoretic adsorption / desorption chromatography according to the present invention comprises:
The capillary cross-sectional shape is a square, a rectangle, an ellipse, or a circle.
For example, when a capillary having a square or rectangular cross section is used, it is suitable for observing an electrophoretic model in which adsorption and desorption are repeated by going back and forth between two planes where fine particles face each other. When a capillary with a circular cross section is used, fine particles are attracted by an external force and move on the axis of the diameter, which is suitable for adsorption / desorption chromatography. As described above, various capillaries of various shapes and materials can be used depending on the application.
[0008]
Further, the capillary electrophoretic adsorption / desorption chromatography according to the present invention comprises:
The inner wall of the capillary is subjected to a surface treatment,
It is characterized by that.
In the present invention, the interaction between the particle and the inner wall is a very important parameter, and the desorption current changes according to this interaction. It is thus possible to modify the surface to design chemical, selective or functional group specific “interactions”. Such surface treatment of the inner wall of the capillary is very effective for separation and analysis of various cells and proteins. That is, in addition to the physical adsorption force of the van der Waals force, various chemical adsorption forces can be added to increase the selectivity by subjecting the inner wall of the capillary to a surface treatment. Various surface treatments such as chemical treatment, physical treatment, and electrical treatment can be considered. For example, hydrophilic treatment, non-hydrophilic treatment, or various substances (metal, chemical substance, etc.) Is applied, vapor-deposited or etched to form a film, or the surface is modified. In this way, the interaction with fine particles (ie, desorption current) can be changed by applying a surface treatment suitable for fine particles desired to be separated to glass or various plastics used as ordinary capillaries. Therefore, it is possible to improve the separation efficiency of fine particles, and to separate fine particles that cannot be separated by ordinary materials.
[0009]
A capillary electrophoretic adsorption / desorption chromatography device according to the present invention comprises:
Capillary,
Magnetic field generating means;
Current supply means;
Means for flowing a solution containing fine particles in the capillary;
Applying a magnetic field to the solution flowing in the capillary using a magnetic field generating means, and causing a current to flow substantially perpendicular to the magnetic field using an electric current supply means, thereby causing the microparticles contained in the solution to be electrophoresed, Furthermore, means for adsorbing and desorbing the microparticles contained in the solution with the inner wall of the capillary by repeatedly reversing the polarity of the current;
It is characterized by including.
In addition, the capillary electrophoretic adsorption / desorption chromatography device according to the present invention comprises:
Furthermore, a microscope for detecting the fine particles in the capillary,
Is also included.
In addition, the capillary electrophoretic adsorption / desorption chromatography device according to the present invention comprises:
The capillary cross-sectional shape is a square, a rectangle, an ellipse, or a circle.
Further, the capillary electrophoretic adsorption / desorption chromatography device according to the present invention comprises:
The inner wall of the capillary is subjected to a surface treatment,
It is characterized by that.
[0010]
The principle of the present invention is that when a uniform magnetic field is applied to an electrolyte solution containing fine particles, and an electric current is passed so as to be substantially orthogonal thereto, an electromagnetic force acts on the solution as a medium, and this is regarded as “buoyancy” for the fine particles. It works to cause "migration" of fine particles. That is, the present invention has developed a new method and apparatus for separating fine particles in a flow solution using electromagnetic force. An electrolyte solution containing fine particles is caused to flow through a glass capillary placed in a uniform magnetic field, and an alternating current having a controlled waveform is applied to the capillary so as to be substantially perpendicular to the magnetic field lines. Due to the electromagnetic buoyancy generated at this time, the moving fine particles are repeatedly adsorbed and desorbed from the inner wall of the capillary to achieve chromatographic separation. According to the present invention, a new separation and analysis method can be performed even at a single particle level of environmental fine particles and biological fine particles that cannot be separated by a normal method.
As described above, the feature of the present invention is to control the adsorption / desorption behavior to the inner wall of the container by giving a moving force based on the electromagnetic buoyancy to the fine particles in the medium flowing in the capillary. That is, by switching the direction of the current, adsorption and desorption can be repeated, and the velocity of the fine particles flowing out from the capillary can be a characteristic value for each fine particle. By appropriately setting the flow rate of the medium, the magnitude of the current and its periodic pattern, and the direction, the chromatographic separation performance by multi-stage adsorption / desorption can be optimized.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.
Capillary electrophoretic adsorption / desorption chromatography realized by the present invention applies electrophoresis of fine particles in liquid to the capillary flow system, and the electrophoretic force acting on the fine particles and the adsorption / desorption of fine particles on the inner wall of the capillary Is a technique for separating and purifying fine particles. Electrophoresis is a phenomenon in which fine particles migrate in a direction perpendicular to an electric field and a magnetic field by applying a uniform electric field and a uniform magnetic field perpendicular to the electrolyte solution in which the fine particles are dispersed. In electrophoresis, if the polarity of the current is changed, the direction in which the electrophoretic force acts changes according to the direction of the current, and the direction of migration of the fine particles is reversed. In the capillary flow system, when the electrophoresis is performed while changing the polarity of the current, the microparticles migrate in the medium in the capillary, and when they reach the inner wall of the capillary, they are pressed against the inner wall of the capillary by the electrophoretic force and adsorbed. Thereafter, when the polarity of the current changes and exceeds a specific desorption current, the microparticles are desorbed from the inner wall of the capillary by the electrophoretic force and migrate toward the inner wall on the opposite side. That is, the microparticles repeat a cycle of “migration → adsorption → desorption → migration”. At this time, if the flow speed is not fast enough to push away the adsorbed fine particles, the fine particles are flowed by the flow only during migration and migrate in a zigzag manner in the capillaries. The distance carried by the flow during one cycle from desorption to adsorption increases as the time across the capillary increases, that is, as the electrophoretic velocity decreases. If this distance is different for each fine particle, the difference widens by repeated adsorption and desorption, and the outflow time from the capillary varies depending on the type of fine particle. More difference in elution time, because the particles to separate and analyze the basic concept of adsorption chromatography.
[0012]
Fine particle separation model Next, in capillary flow system electrophoresis used in the present invention, a fine particle separation model using adsorption / desorption is prepared as follows.
In electrophoretic migration, both the electromagnetic force F _EMW (Electromagnetophoretic weight) acting on the particle itself and the electromagnetic buoyancy F _EMB (Electromagnetophoretic Buoyancy) opposite to the electromagnetic force acting on the medium act on the fine particles in the liquid. The electrophoretic force F _EMP acting on the medium fine particles is the sum of these two forces and is expressed by the following equation.
[Expression 1]

Where B is the external magnetic field (T), i is the current (A), V is the volume of the fine particles (m ³ ), S is the cross-sectional area of the capillary (m ² ), and σ _f and σ _p are the conduction of the medium, respectively. Rate (Sm ⁻¹ ) and surface conductivity (Sm ⁻¹ ) of fine particles resulting from the electric double layer of fine particles in liquid. Furthermore, from the equation (1) and the viscous resistance force, the electrophoretic velocity v _EMP of the fine particles is expressed as the following equation.
[Expression 2]

Here, r is the particle radius (m), η is the viscosity (Pas) of the medium, and _CW is the resistance coefficient of the inner wall of the capillary. Thus, the electrophoretic force is proportional to the particle volume, current and external magnetic field, and the electrophoretic velocity is proportional to the square of the particle radius, current and external magnetic field.
[0013]
FIG. 1 is a graph showing the relationship between the current supplied to the fine particles adsorbed on the inner wall of the capillary and time. As shown in the figure, when the microparticles adsorbed on the inner wall of the capillary are desorbed by the electrophoretic force by applying a current having a waveform that increases to the current value _imax during the time t _i , the electrophoretic force increases as the current value increases. Increases and the electrophoretic force is less than the adsorption force F _A

Then, desorption of fine particles occurs. The current value when fine particles are desorbed and desorption current i _D.
[0014]
FIG. 2 is a conceptual diagram showing the moving distance d from when the fine particles are desorbed to when they are adsorbed in the capillary flow system. As shown in the figure, the particles adsorbed on the inner wall of the capillary are desorbed from the wall by the electrophoretic force, and move by the flow while traversing the capillary by the electrophoretic movement. Due to the current of the waveform shown in FIG. 1, the particles are desorbed at the desorption current iD and begin to migrate. When the speed change is small when on migration, the time t _w of particles of radius r takes the capillary inner diameter w to traverse the electromagnetic Electrophoresis,
[Expression 4]

It becomes. Here, k is a coefficient obtained by summarizing terms other than the current and the radius in equation (2). Since the particles are moved by the flow during the time t _w, the moving distance d between the flow velocity v _f,
[Equation 5]

It becomes. As represented by the equation (5), the moving distance d is considered to depend on the radius of the particles and the desorption current. Therefore, the moving distance d varies depending on the properties of individual particles.
[0015]
FIG. 3 is a conceptual diagram showing the moving distances when two kinds of fine particles repeat adsorption and desorption in the capillary flow system. As shown schematically in the white and black circles in the capillaries at the top and bottom of the figure, the adsorption / desorption can be performed once depending on the properties of the fine particles (particle size, interaction with the inner wall, etc.). If the moving distance d is different, by repeating the adsorption / desorption, the time required to travel a certain distance can be different even at the same flow rate, and the holding time at a certain capillary length is different. The particle retention time at this time is
[Formula 6]

It is represented by Here, l is the effective capillary length. As shown in the equation (6), the holding time is considered to depend on the desorption current i _D and the particle radius r, and separation of fine particles is possible due to these differences.
[0016]
FIG. 4 is a schematic view showing a capillary cell of an electrophoretic capillary flow cell unit used in a capillary electrophoretic adsorption / desorption chromatography device according to the present invention. As shown in the figure, the capillary cell 10 includes a square fused silica capillary 12 (manufactured by Polymicro Technologies) having an inner diameter of 100 × 100 μm and a length of 2 cm, and both ends thereof are attached to a PTFE tube 16 using a heat shrinkable tube 14. It is connected.
[0017]
FIG. 5 is a schematic diagram showing a capillary flow cell unit for electrophoresis including the capillary cell shown in FIG. This figure is an overall view of the capillary flow cell. As shown in the figure, the capillary flow cell unit 100 includes a capillary cell 110, a three-way connector 120 is connected to both ends thereof, and Ag | AgCl from the other one of the three-way connector 120 is connected. The electrode 130 is inserted, and the liquid-feeding PTFE tube 140 is connected to the other. The Ag | AgCl electrode was prepared by electrolyzing a copper wire in an aqueous potassium chloride solution. The procedure is to connect a copper wire to the positive pole of the power source, a platinum wire to the negative pole, and immerse the two metal wires in a 1M aqueous potassium chloride solution. Then, when a current is passed at 5 V for 5 minutes, silver chloride is generated at the tip of the copper wire, and an Ag | AgCl electrode is completed. Here, a uniform magnetic field generated from a magnetic field generator (not shown) is generated in the vertical direction of the figure, while the direction in which the current i generated from the electrode flows is substantially orthogonal to the magnetic field as shown in the figure. Inverted alternately in the horizontal direction.
[0018]
FIG. 6 is a schematic view showing the entire capillary electrophoretic adsorption / desorption chromatography apparatus according to the present invention. As shown in the figure, the capillary electrophoretic adsorption / desorption chromatography device 200 includes a refrigerant-free superconducting magnet 220 (JMTD-10T100HH1 manufactured by JMT) that does not require liquid helium as a magnetic field generating means. This superconducting magnet 220 has a room temperature bore with a diameter of 100 mm and a length of 950 mm, and can create a uniform magnetic field of 0 to 10 T from the upper flange surface to 560 mm ± 5 mm, and the direction of the magnetic field is “arrow B” in the figure. As shown by the direction of gravity (downward). In this way, the apparatus is arranged so that the magnetic field is perpendicular to the longitudinal direction of the capillary (that is, the direction of current flow). The chromatography apparatus 200 includes a capillary flow cell 230, a PC 240, a function synthesizer 250 (manufactured by NF), an amplifier 260 (manufactured by TREC, MODEL610-C), a digital multimeter 270 (fixed to the cell holder in the uniform magnetic field. IWATSU ammeter, VOAC7512), video recorder 280, monitor 290, syringe pump 300 (Harvard Apparatus, pump II) light source 310 (Olympus, MODEL ILV), CCD camera 320 (ELMO, ME421), And an optical microscope 330. In the PC 240, “arbitrary waveform creation software 0105 (manufactured by NF)” is introduced, and a desired applied current waveform is created using this software, and the waveform is sent to the function synthesizer 250 to output a voltage. The voltage is amplified and applied to the sample solution in the cell, and the current is measured by the digital multimeter 270. One end of the PTFE tube included in the capillary flow cell 230 is connected to a syringe (has GASTIGHT # 1701 manufactured by HAMILTON) having an internal volume of 100 μl, and the sample liquid containing fine particles is fed by the syringe pump 300. Light is applied from below using a light source 310, observed from above with an optical microscope 330 with a 10 × objective lens, and the behavior of microparticles is photographed with a CCD camera 320 and output to a monitor 290. Video 280 Record with. All devices placed in the bore of the superconducting magnet are made of non-magnetic acrylic, aluminum, or brass so that the magnetic field is uniform.
[0019]
In order to confirm the performance and effect of the capillary electrophoretic adsorption / desorption chromatography apparatus according to the present invention, a separation experiment was performed using two kinds of solutions containing fine particles having different particle diameters.
Sample The sample solution used was a dispersion of two types of polystyrene particles having a particle size of 20 [mu] m and 10 [mu] m in a 1M potassium chloride solution containing 0.01% surfactant Triton X-100. The density of the medium at this time is almost the same as the density of polystyrene (1.05 gcm ⁻³ ).
[0020]
Measurement of polystyrene particle retention time In this experiment, a circular silica capillary having a diameter of 200 μm was used in the capillary electrophoretic adsorption / desorption chromatography apparatus according to the present invention. In this apparatus, the retention time of polystyrene particles with a capillary effective length of 1 mm was measured. The current having the waveform shown in FIG. 1 was set to i _max = 2000 μA and t _i = 2s, and the current was supplied to the sample solution. The behavior of polystyrene particles is observed when the flow rate of the sample liquid is in the range of 40 to 100 μL / h, the time taken for the polystyrene particles to move 1 mm in the capillary is measured on the monitor, and the retention time of the polystyrene particles is obtained. It was.
[0021]
Measurement result FIG. 7 is a graph showing the relationship between the retention time and flow rate of polystyrene particles of various particle sizes. The measurement conditions are set to i _max = 2000 μA, t _i = 2s, and the effective length is 1 mm for a circular silica capillary having a particle size of 10 μm and 20 μm and a diameter of 200 μm. Under these conditions, the holding time was measured by changing the flow rate, that is, the flow rate. At any flow rate, the retention time of particles having a particle size of 10 μm was smaller than the particle size of 20 μm. The resolution R of the chromatographic method is expressed by the following formula using the retention time of the substance.
[Expression 7]

Here, t ₁ and t ₂ are retention times of the substance 1 and the substance 2, respectively. When the resolution R is 1 or more, the substances 1 and 2 can be separated by the chromatographic method. In the case of this measurement, the resolution R = t ₂₀ / t ₁₀ = 3.1, indicating that the polystyrene particles can be separated by the particle size. In this experiment, the desorption current i _D of the polystyrene particles during the adsorption / desorption was about 1000 μA for both the particle sizes of 10 and 20 μm. The curve in FIG. 7 is a theoretical curve drawn from equation (6) using this desorption current. The broken line represents a particle size of 20 μm, and the solid line represents a particle size of 10 μm, and it was found that the measured value and the theoretical value almost coincided. As described above, the retention time of the particles can be predicted from the equation (6), and the fine particles can be identified from the retention time.
In this embodiment, since the positive / negative switching of the current is 2 seconds, when the flow rate is 40 μL / h, 55 times with 20 μm polystyrene fine particles, about 20 times with 10 μm fine particles, Adsorption / desorption is performed in the capillary.
[0022]
The apparatus and method according to the present invention can be widely used in the environment and health field, functional material manufacturing field, food and sanitary field, etc., as a fine particle separation method and separation apparatus.
In the present specification, the principle of the present invention has been described in various embodiments. However, the present invention is not limited to the above-described embodiments, and many variations and modifications can be made. It should be understood that these are also included in the present invention.
[Brief description of the drawings]
FIG. 1 is a graph showing the relationship between current supplied to fine particles adsorbed on the inner wall of a capillary and time.
FIG. 2 is a conceptual diagram showing a moving distance d from when a microparticle is desorbed to when it is adsorbed in a capillary flow system.
FIG. 3 is a conceptual diagram showing movement distances when two types of microparticles repeatedly adsorb and desorb in a capillary flow system.
FIG. 4 is a schematic view showing a capillary cell used in a capillary electrophoretic adsorption / desorption chromatography device according to the present invention.
5 is a schematic view showing a capillary flow cell portion for electrophoresis including the capillary cell shown in FIG. 4. FIG.
FIG. 6 is a schematic view showing the entire capillary electrophoretic adsorption / desorption chromatography apparatus according to the present invention.
FIG. 7 is a graph showing the relationship between retention time and flow rate of polystyrene particles having two types of particle sizes.
[Explanation of symbols]
200 Capillary Electrophoresis Adsorption / Desorption Chromatography Apparatus 200
220 Superconducting magnet 230 Capillary flow cell 240 PC
250 Function synthesizer 260 Amplifier 270 Digital multimeter 280 Video recorder 290 Monitor 300 Syringe pump 310 Light source 320 CCD camera 330 Optical microscope

Claims (10)

Capillary electrophoretic adsorption / desorption chromatography,
Flowing a solution containing fine particles into the capillary;
Applying a magnetic field to the solution flowing in the capillary by flowing current so as to be substantially perpendicular to the magnetic field, the fine particles contained in the solution was electromagnetic focusing, by further repeating the polarity inversion of the current A step of adsorbing and desorbing the fine particles contained in the solution with the inner wall of the capillary, and separating and / or purifying the fine particles;
Capillary electrophoretic adsorption / desorption chromatography, comprising: The capillary electrophoretic adsorption / desorption chromatography according to claim 1,
Measuring the retention time of the microparticles in a certain capillary length of the capillary;
Capillary electrophoretic adsorption / desorption chromatography characterized by including. The capillary electrophoretic adsorption / desorption chromatography according to claim 1 or 2,
Furthermore, detecting the fine particles in the capillary using a microscope,
Capillary electrophoretic adsorption / desorption chromatography characterized by including. In capillary electrophoretic adsorption / desorption chromatography according to any one of claims 1 to 3,
The shape of the capillary cross section is a square, a rectangle, an ellipse, or a circle.
Capillary electrophoretic adsorption / desorption chromatography characterized by the above. In the capillary electrophoretic adsorption / desorption chromatography according to any one of claims 1 to 4,
The inner wall of the capillary is subjected to a surface treatment,
Capillary electrophoretic adsorption / desorption chromatography characterized by the above. A capillary electrophoretic adsorption / desorption chromatography device comprising:
Means for flowing a solution containing fine particles in the capillary;
Applying a magnetic field to the solution flowing in the capillary by flowing current so as to be substantially perpendicular to the magnetic field, the fine particles contained in the solution was electromagnetic focusing, by further repeating the polarity inversion of the current Means for adsorbing and desorbing the fine particles contained in the solution with the inner wall of the capillary, and separating and / or purifying the fine particles;
A capillary electrophoretic adsorption / desorption chromatographic apparatus comprising: The capillary electrophoretic adsorption / desorption chromatography device according to claim 6,
Means for measuring the retention time of the microparticles in a certain capillary length of the capillary;
A capillary electrophoretic adsorption / desorption chromatographic apparatus characterized by comprising: The capillary electrophoretic adsorption / desorption chromatography device according to claim 7,
Furthermore, a microscope for detecting the fine particles in the capillary,
A capillary electrophoretic adsorption / desorption chromatographic apparatus characterized by comprising: The capillary electrophoretic adsorption / desorption chromatography device according to any one of claims 6 to 8,
The shape of the capillary cross section is a square, a rectangle, an ellipse, or a circle.
Capillary electrophoretic adsorption / desorption chromatography apparatus characterized by the above. The capillary electrophoretic adsorption / desorption chromatography device according to any one of claims 6 to 9,
The inner wall of the capillary is subjected to a surface treatment,
Capillary electrophoretic adsorption / desorption chromatography apparatus characterized by the above.

Images