JPH04175655A - Capillary electrophoresis device - Google Patents
Capillary electrophoresis deviceInfo
- Publication number
- JPH04175655A JPH04175655A JP2302395A JP30239590A JPH04175655A JP H04175655 A JPH04175655 A JP H04175655A JP 2302395 A JP2302395 A JP 2302395A JP 30239590 A JP30239590 A JP 30239590A JP H04175655 A JPH04175655 A JP H04175655A
- Authority
- JP
- Japan
- Prior art keywords
- capillary
- temperature
- measuring
- hollow
- absorbance
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000005251 capillar electrophoresis Methods 0.000 title claims abstract description 18
- 238000002835 absorbance Methods 0.000 claims abstract description 11
- 238000001962 electrophoresis Methods 0.000 claims description 17
- 230000005684 electric field Effects 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims 2
- 238000009529 body temperature measurement Methods 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 10
- 102000004169 proteins and genes Human genes 0.000 abstract description 8
- 108090000623 proteins and genes Proteins 0.000 abstract description 8
- 239000000243 solution Substances 0.000 abstract description 8
- 239000000872 buffer Substances 0.000 abstract description 3
- 239000011521 glass Substances 0.000 abstract description 3
- 238000005259 measurement Methods 0.000 abstract description 3
- 238000004458 analytical method Methods 0.000 abstract description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 abstract 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 abstract 2
- 239000000049 pigment Substances 0.000 abstract 2
- 102000004506 Blood Proteins Human genes 0.000 abstract 1
- 108010017384 Blood Proteins Proteins 0.000 abstract 1
- 239000003638 chemical reducing agent Substances 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract 1
- 239000000975 dye Substances 0.000 description 14
- 238000013508 migration Methods 0.000 description 8
- 230000005012 migration Effects 0.000 description 8
- 102000004338 Transferrin Human genes 0.000 description 4
- 108090000901 Transferrin Proteins 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 239000012581 transferrin Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000003792 electrolyte Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000001997 free-flow electrophoresis Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
Landscapes
- Measuring Temperature Or Quantity Of Heat (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明はペプチド、蛋白質および核酸などの分離分析法
であるキャピラリ電気泳動法（ＣＺＥ）の再現性、信頼
性の向上法に関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for improving the reproducibility and reliability of capillary electrophoresis (CZE), which is a method for separating and analyzing peptides, proteins, nucleic acids, etc.
従来のキャピラリ電気泳動法（ＣＺＥ法）はアプリティ
カルケミストリー５９巻、１０２１頁から１０２７頁（
１９８７年（Ａｎａｌ、Ｃｈｅｍ、 ５９ 、 ｐｐ１
０２１−１０２７ （１９８７））において論ぜられて
いる。すなわち、内径５０μｍ程度のガラス製中空細管
（キャピラリ）中に電解質水溶液を満たし、細管の両端
を電解質水溶液に浸す。細管の一端に分析したい試料を
注入した後、両端の間に数万ボルト程度の高電圧を付与
し試料を電気泳動分離する。細管の中間に適当な検出器
、例えば、紫外領域での吸光度計測器、を設け、細管中
を通過する物質の濃度を計測すれば試料を分析できる。The conventional capillary electrophoresis method (CZE method) is described in Applicable Chemistry Vol. 59, pp. 1021 to 1027 (
1987 (Anal, Chem, 59, pp1
021-1027 (1987)). That is, a glass hollow capillary having an inner diameter of about 50 μm is filled with an electrolyte aqueous solution, and both ends of the capillary are immersed in the electrolyte aqueous solution. After injecting the sample to be analyzed into one end of the tube, a high voltage of tens of thousands of volts is applied between both ends to electrophoretically separate the sample. A sample can be analyzed by providing a suitable detector, such as an absorbance meter in the ultraviolet region, in the middle of the capillary and measuring the concentration of the substance passing through the capillary.
高電圧の付与によりジュール熱が発生するので細管は、
一般に、恒温槽中に置かれ、細管の冷却が試みられてい
る。The application of high voltage generates Joule heat, so the thin tube
Generally, attempts are made to cool the tube by placing it in a constant temperature bath.
上記従来技術では細管内の温度が計測されていないので
、得られた分析結果がいかなる温度でのデータが不明で
あり、結果の厳密な理解で不可能である。たとえ細管を
恒温槽内に置いても、付与した高電圧により細管的温度
は設定した恒温槽の温度よりも相当に上昇するという問
題がある。In the above-mentioned conventional technology, the temperature inside the capillary is not measured, so it is unclear at what temperature the obtained analysis results are obtained, and it is impossible to strictly understand the results. Even if the capillary tube is placed in a constant temperature bath, there is a problem in that the temperature of the capillary tube rises considerably higher than the temperature of the thermostatic bath due to the high voltage applied.
ＣＺＥ法のような無担体泳動法では一般に物質の泳動速
度は１℃の温度変化に対して４パ一セント位変化する。In a carrier-free electrophoresis method such as the CZE method, the migration speed of a substance generally changes by about 4 percent for a temperature change of 1°C.
したがって、泳動速度を計測したとき同時に泳動液の温
度も計測しておかないと５得られた泳動速度を他の測定
結果と比較できないことになり、したがって、泳動速度
の値から当該物質を同定することもむずかしくなる。Therefore, if the temperature of the electrophoresis solution is not measured at the same time when the electrophoresis speed is measured, the obtained electrophoresis speed cannot be compared with other measurement results.Therefore, the substance in question cannot be identified from the electrophoresis speed value. It also becomes difficult.
本発明は泳動速度の計測時に泳動媒体の温度も同時に計
測する手段を提供することを目的としている。An object of the present invention is to provide a means for simultaneously measuring the temperature of a migration medium when measuring the migration speed.
上記目的を達成するために本発明では細管内にサーモク
ロミック色素、すなわち、温度により光吸収スペクトル
の変化する色素の溶液を注入し、当該色素の吸光度を計
ることにより細管内の温度を計測した。In order to achieve the above object, in the present invention, a thermochromic dye, that is, a solution of a dye whose light absorption spectrum changes depending on temperature, is injected into a capillary, and the temperature inside the capillary is measured by measuring the absorbance of the dye.
ＣＺＥ法における細管は内径が２５〜１００μｍ程度と
非常に細く、その内部の温度を計るには、サーモクロミ
ックの色素の溶液のような液状の温度検知器を用いるの
が最良である。これにより、泳動分離している物質の妨
害とならずに細管内部の温度を計測できる。他の、例え
ばサーミスタのような温度検知器は、その径をＣＺＥ用
細管程度に小さくすることも困難であるが、たとえ小さ
くできたとしても、サーミスタの存在それ自体が泳動分
離の妨害となるので用いることはできない。The tube used in the CZE method is very thin, with an inner diameter of about 25 to 100 μm, and in order to measure the temperature inside the tube, it is best to use a liquid temperature sensor such as a thermochromic dye solution. Thereby, the temperature inside the capillary can be measured without interfering with the substances being electrophoretically separated. It is difficult to reduce the diameter of other temperature sensors such as thermistors to the size of CZE tubes, but even if they could be made smaller, the presence of the thermistor itself would interfere with electrophoretic separation. It cannot be used.
以下１本発明の一実施例を第１図および第２図により説
明する。まず第２図にて、内径１００μｍのガラス製中
空細管１に３１ｍＭの濃度でｐ Ｈ７、０のリン酸バッ
ファを満たし、一端を減圧装置２に接続し、他端をサー
モクロミック色素。An embodiment of the present invention will be described below with reference to FIGS. 1 and 2. First, in FIG. 2, a hollow glass tube 1 with an inner diameter of 100 μm is filled with a phosphate buffer of pH 7.0 at a concentration of 31 mM, one end is connected to a pressure reducing device 2, and the other end is filled with a thermochromic dye.
ポリ（３−トコシルチオフェン）の塩化メチレン溶液の
入った容器３に入れ、減圧操作によりサーモクコミック
色素溶液を少量細管に注入する。次に、容器３の内容を
分析対象、例えば血清蛋白トランスフェリンを含む上記
リン酸バッファに入れ変え、ふたたび減圧操作により蛋
白質水溶液を少量細管に注入する。この細管を第１図の
キャピラリ電気泳動装置に設置する。つまり、リン酸バ
ッファ１２の入った電極槽１３と５の間の細管１を設定
する。この際、サーモクロミック色素および蛋白質を注
入した細管の端を正極４の側電極槽１３に配置し、他端
を負極６の側電極槽５に配する。７は電気泳動用の高圧
電源を示し、１１は恒温槽を示す。細管の中間に２２０
ｎｍおよび５００ｎｍの２つの波長で細管の吸光度を計
測する検出器８を配する。２２０ｎｍの波長の光は蛋白
質の濃度を測定するためであり、５００ｎｍの光はサー
モクロミック色素の吸光度、すなわち、温度を計測する
ためである。検出器の出力は分離図形（電気泳動図）を
記録するための装置９および細管的温度の表示装置１０
に導かれる。細管的温度の信号を記録計９に導いて記録
計」二に温度を表示してもよいことは勿論である。サー
モクロミック色素の吸光度を温度に換算するには、電気
泳動する前にあらかじめ細管を色素溶液で満たし、細管
を温度既知の恒温槽に入れて吸光度を電圧をかけずに計
測して温度と吸光度の関係を調へておけばよい。さらに
、恒温槽の温度制御器１５に上記検出器８で検出される
サーモクロミック色素の吸光度の値を温度検出値として
入力し、１５により検出値が一定になるように恒温槽の
温度を制御する植成とすれば、電気泳動中もキャピラリ
内の温度を一定に保つことができる。It is placed in a container 3 containing a methylene chloride solution of poly(3-tocosylthiophene), and a small amount of the thermocomic dye solution is injected into the capillary by vacuum operation. Next, the contents of the container 3 are replaced with the above-mentioned phosphate buffer containing the object to be analyzed, for example, the serum protein transferrin, and a small amount of the protein aqueous solution is injected into the capillary by a vacuum operation again. This thin tube is installed in the capillary electrophoresis apparatus shown in FIG. That is, the thin tube 1 is set between the electrode baths 13 and 5 containing the phosphate buffer 12. At this time, the end of the thin tube into which the thermochromic dye and protein have been injected is placed in the side electrode tank 13 of the positive electrode 4, and the other end is placed in the side electrode tank 5 of the negative electrode 6. 7 indicates a high voltage power supply for electrophoresis, and 11 indicates a constant temperature bath. 220 in the middle of the tubule
A detector 8 is provided to measure the absorbance of the capillary at two wavelengths: nm and 500 nm. The light with a wavelength of 220 nm is used to measure the concentration of protein, and the light with a wavelength of 500 nm is used to measure the absorbance of the thermochromic dye, that is, the temperature. The output of the detector is a device 9 for recording the separation pattern (electropherogram) and a device 10 for displaying the tubular temperature.
guided by. Of course, the capillary temperature signal may be guided to the recorder 9 and the temperature may be displayed on the recorder 2. To convert the absorbance of a thermochromic dye into temperature, before electrophoresis, fill a capillary with a dye solution, place the capillary in a constant temperature bath with a known temperature, measure the absorbance without applying any voltage, and calculate the difference between temperature and absorbance. All you have to do is check your relationships. Further, the absorbance value of the thermochromic dye detected by the detector 8 is inputted to the temperature controller 15 of the thermostatic chamber as a temperature detection value, and the temperature of the thermostatic chamber is controlled by the temperature controller 15 so that the detected value is constant. If implanted, the temperature inside the capillary can be kept constant even during electrophoresis.
さて、電気泳動を始めるに先立って、細管、検出器２両
電極槽を恒温槽１１の中に入れ２６℃に保って１両電極
４，６間に２２０ボルト／ ｃｍの電界強度を付与した
ところ、３．２０分後にサーモクロミック色素が検出器
に到達し、温度４２℃の値が計測された。恒温槽の設定
温度よりも１６℃もジュール熱によって細管内の温度が
上昇していることがわかった。９．３４ 分後にはトラ
ンスフェリンが検出器に到達した。サーモクロミック色
素の到達時間、すなわち、保持時間から、細管内の電気
滲透流速は４２°Ｃで２．２０×１Ｏ−２ｃｘ＋Ｔ ／
ボルト・分と計算され、トランスフェリンの保持間から
この蛋白の泳動速度は４２℃で７．５４Ｘ１０−’ｃｎ
／ボルト・分と計算された。Now, before starting electrophoresis, the capillary tube, detector, and two electrode baths were placed in a constant temperature bath 11, kept at 26°C, and an electric field strength of 220 volts/cm was applied between the two electrodes 4 and 6. , 3. After 20 minutes, the thermochromic dye reached the detector and a temperature of 42°C was measured. It was found that the temperature inside the tube was 16°C higher than the set temperature of the constant temperature bath due to Joule heat. Transferrin reached the detector after 9.34 minutes. From the arrival time of the thermochromic dye, that is, the retention time, the electropermeation flow rate in the capillary is 2.20×1O−2cx+T/at 42°C.
The migration speed of this protein was calculated as volt-minutes and the retention time of transferrin was 7.54 x 10-'cn at 42°C.
/volt-minute.
以上、本発明によれば恒温槽の設定温度に比して細管内
の温度は大幅に上昇していることがわかった、また、泳
動温度と泳動速度を組みにして同時に計測することがで
きるようになり、計測された泳動速度のデータの信頼性
が高まった。恒温槽の温度を変化させてトランスフェリ
ンの泳動速度の温度依存性を求めたところ、１℃の温度
上昇により約３．５％速度が上昇することがわかった。As described above, it has been found that according to the present invention, the temperature inside the thin tube increases significantly compared to the set temperature of the thermostatic chamber. This increased the reliability of the measured migration speed data. When the temperature dependence of transferrin migration speed was determined by changing the temperature of the constant temperature bath, it was found that the speed increased by about 3.5% with a temperature increase of 1°C.
この数値からも細管内温度の計測の重要性は明らかであ
る。細管内温度を計測しない場合、泳動速度は恒温槽の
設定温度で計測されたものとみなされ、たとえば細管内
温度と恒温槽温度の差が１０℃あれば泳動速度は約３５
％もの誤差を含むことになる。The importance of measuring the temperature inside the tube is clear from this value. If the temperature inside the capillary tube is not measured, the migration speed is assumed to be measured at the set temperature of the thermostatic chamber. For example, if the difference between the temperature inside the capillary tube and the temperature of the thermostatic chamber is 10°C, the migration speed will be approximately 35°C.
This will include an error of as much as %.
また、細管内温度の計測は泳動速度計測の信頼性を上昇
させるのみでなく、泳動電圧の最適化にも有効である。Furthermore, measuring the temperature inside the capillary not only increases the reliability of electrophoresis velocity measurement but also is effective for optimizing the electrophoresis voltage.
一般に、泳動電圧を高めるとＣＺＥの分離能は上昇され
るが、これは温度上昇が抑制された場合のみである。実
際には細管内温度が上昇してしまうので、用いうる泳動
電圧には限界がある。例えば、内径１００μｍの細管に
３１ｍＭのリン酸バッファを満たして用いた場合、恒温
槽温度を３０″Ｃに設定すると、用いつる最高電界強度
は約３００ボルト／―となる。これ以上の電界強度では
細管内のバッファ温度は沸点近くとなり乱流が生じ、正
常な泳動図が得られなくなった。In general, increasing the electrophoresis voltage increases the resolution of CZE, but only if the temperature increase is suppressed. In reality, the temperature inside the capillary increases, so there is a limit to the electrophoresis voltage that can be used. For example, when using a thin tube with an inner diameter of 100 μm filled with 31 mM phosphate buffer and setting the constant temperature bath temperature to 30''C, the maximum electric field strength to be used will be approximately 300 volts/-.If the electric field strength is higher than this, The buffer temperature within the tube was close to the boiling point, causing turbulence and making it impossible to obtain a normal electrophoretogram.
また、高温で泳動すると蛋白質が変性し、泳動速度が変
化する可能性がある。Furthermore, electrophoresis at high temperatures may denature proteins and change the electrophoresis speed.
なお、本発明に用いうるサーモクロミック色素としては
ポリ（３−アルキルチオフェン）類、および、スピロピ
ラン類などがある。Note that thermochromic dyes that can be used in the present invention include poly(3-alkylthiophene)s and spiropyrans.
本発明によれば、ＣＺＥ法における細管内の温度が計れ
るので被分析物質の保持時間の測定の信頼性が高まり、
また、印加電圧の最適化を行なうことが可能となる。According to the present invention, since the temperature inside the capillary in the CZE method can be measured, the reliability of measuring the retention time of the analyte increases,
Furthermore, it becomes possible to optimize the applied voltage.
第１図は本発明の一実施例のＣＺＥ装置の構成図である
。第２図はＣＺＥ用細管の充填法を示す構成図である。
１・・・中空細管、８・・・検出器、４，６・・電極、
５゜１３・・・電極槽、７・・・高圧電源、９・・・記
録計、１０め
￥Ｊ ｉ 図
Ｙ ｚ 団
誠五装置 ・FIG. 1 is a block diagram of a CZE device according to an embodiment of the present invention. FIG. 2 is a configuration diagram showing a method of filling a CZE thin tube. 1... Hollow tube, 8... Detector, 4, 6... Electrode,
5゜13... Electrode tank, 7... High voltage power supply, 9... Recorder, 10th ￥ J i Figure Y z Dan Seigo equipment ・
Claims (1)
、該中空細管の一端に注入された試料を前記ふたつのバ
ッファ溶液槽の間に印加する電界で移動せしめるキャピ
ラリ電気泳動装置において、前記中空細管の内部温度を
計測する温度計測手段を設けたことを特徴とするキャピ
ラリ電気泳動装置。 ２、前記中空細管にはサーモクロミック色素が注入され
、前記温度計測手段は、前記中空細管での前記サーモク
ロミック色素の吸光度変化を検出することを特徴とする
請求項１に記載のキャピラリ電気泳動装置。 ３、前記中空細管は恒温槽の中に設置され、前記温度計
測手段の出力が一定となるよう前記恒温槽の温度を制御
する温度制御器をさらに有することを特徴とする請求項
１に記載のキャピラリ電気泳動装置。[Claims] 1. A capillary electric capillary in which a hollow capillary is installed between two buffer solution tanks, and a sample injected into one end of the hollow capillary is moved by an electric field applied between the two buffer solution tanks. A capillary electrophoresis apparatus, characterized in that the electrophoresis apparatus is provided with a temperature measuring means for measuring the internal temperature of the hollow thin tube. 2. The capillary electrophoresis device according to claim 1, wherein a thermochromic dye is injected into the hollow capillary, and the temperature measuring means detects a change in absorbance of the thermochromic dye in the hollow capillary. . 3. The hollow thin tube is installed in a constant temperature bath, and further includes a temperature controller that controls the temperature of the constant temperature bath so that the output of the temperature measurement means is constant. Capillary electrophoresis device.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2302395A JPH04175655A (en) | 1990-11-09 | 1990-11-09 | Capillary electrophoresis device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2302395A JPH04175655A (en) | 1990-11-09 | 1990-11-09 | Capillary electrophoresis device |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04175655A true JPH04175655A (en) | 1992-06-23 |
Family
ID=17908397
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2302395A Pending JPH04175655A (en) | 1990-11-09 | 1990-11-09 | Capillary electrophoresis device |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04175655A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003166976A (en) * | 2001-12-04 | 2003-06-13 | Hitachi High-Technologies Corp | Multiple-capillary electrophoretic apparatus |
| JP2009168674A (en) * | 2008-01-17 | 2009-07-30 | Olympus Corp | Automated analyzer and method for detecting temperature abnormality of liquid in reaction vessel |
| JP2011501135A (en) * | 2007-10-15 | 2011-01-06 | バイエル・ヘルスケア・エルエルシー | Method and assembly for determining the temperature of a test sensor |
-
1990
- 1990-11-09 JP JP2302395A patent/JPH04175655A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003166976A (en) * | 2001-12-04 | 2003-06-13 | Hitachi High-Technologies Corp | Multiple-capillary electrophoretic apparatus |
| US7459068B2 (en) | 2001-12-04 | 2008-12-02 | Applied Biosystems Inc. | Multi-capillary electrophoresis apparatus |
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