JPS628746B2 - - Google Patents
Info
- Publication number
- JPS628746B2 JPS628746B2 JP55042425A JP4242580A JPS628746B2 JP S628746 B2 JPS628746 B2 JP S628746B2 JP 55042425 A JP55042425 A JP 55042425A JP 4242580 A JP4242580 A JP 4242580A JP S628746 B2 JPS628746 B2 JP S628746B2
- Authority
- JP
- Japan
- Prior art keywords
- potential gradient
- component
- gradient value
- value
- sample
- 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.)
- Expired
Links
- 239000007788 liquid Substances 0.000 claims description 26
- 238000001962 electrophoresis Methods 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 7
- 238000004451 qualitative analysis Methods 0.000 claims description 6
- 238000003672 processing method Methods 0.000 claims description 4
- 150000002500 ions Chemical class 0.000 description 24
- 238000004458 analytical method Methods 0.000 description 5
- 238000002218 isotachophoresis Methods 0.000 description 5
- 230000007423 decrease Effects 0.000 description 4
- 230000037230 mobility Effects 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000001649 capillary isotachophoresis Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 239000008151 electrolyte solution Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/4473—Arrangements for investigating the separated zones, e.g. localising zones by electric means
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
この発明は、電気泳動分析装置、特に細管式等
速電気泳動分析装置における分析データの処理法
に関するものである。
細管式等速電気泳動分析法は周知のごとく、細
管（キヤピラリチユーブ）の端に試料のどの成分
イオンよりも移動度の大きいイオン（リーデイン
グイオン）を含む電解液（リーデイング液）と、
試料のどの成分イオンよりも移動度の小さいイオ
ン（ターミナルイオン）を含む電解液（ターミナ
ル液）とで境界面をつくり、この境界面にたとえ
ば移動度の異なる成分を含む混合試料を導入し、
電気泳動を開始させると、各試料成分のイオンは
移動度の大きさの順に配列するように分離が進行
し、完全に分離した状態では、分離されたイオン
は単一成分イオンのみを含んだゾーンとなり、互
いに明確な境界面を保持しながら各ゾーン（バン
ド）は、イオン量で決まる一定の巾をもつて等速
度で移動するようになる。この場合、各ゾーンに
はそれぞれ違つた電位勾配が形成されているので
この電位勾配値を検出して各ゾーンの境界面を知
り、定性分析を行なつていた。
このように電位勾配値から定性分析をおこなう
には成分の電位勾配値が明確な階段信号で示され
るエレクトロフエログラムを得る必要がある。し
かしながら、実際のエレクトロフエログラムで
は、成分によつては電位勾配値が距離（時間）と
もに減少方向に連続的に変化するものがあり、特
に多種類の成分を検出しようとする場合には、こ
の現象が避けられず正確な成分の同定が困難であ
つた。
この発明は上記の不都合を解消するために、等
速電気泳動分析における新しいデータ処理法を開
発せんとするもので、以下図について詳述する。
第１図は細管式等速電気泳動分析装置の基本原理
を示すもので、図において１はターミナル液電極
槽、２はリーデイング液電極槽でそれぞれの槽中
のターミナル液３リーデイング液４には電源５に
接続された電極６，７が挿入されている。８は両
電極槽間に設けられている細管式泳動管（キヤピ
ラリチユーブ）で９の試料導入部からリーデイン
グ液・ターミナル液の境界面に導入された試料を
泳動させるものである。
この泳動されて各ゾーンに分離された試料成分
は検出器１０で電位勾配値が検出され、この電位
勾配値にともなう検出信号は演算部１１で演算さ
れて例えばエレクトロフエログラムとして表示さ
れる。なお１２は恒温槽である。
第２図は上記のエレクトロフエログラムの一例
で縦軸に電位勾配値・横軸に距離（時間）をとつ
たものである。この図においてはHa，Hb，Hcは
それぞれ試料中の成分の電位勾配値・Hlはリー
デイング液の電位勾配値、Htはターミナル液の
電位勾配値であり、リーデイングイオンとターミ
ナルイオンとの電位勾配の差に対するリーデイン
グイオンと成分イオンとの電位勾配値の差の比に
よつて定義される下記の定性指標値PU（ポテン
シヤルユニツト値）、および成分イオンの電位勾
配値に対するリーデイングイオンの電位勾配値の
比によつて定義された下記の定性指標比PR（ポ
テンシヤル比）によつて成分の同定をおこなう。
なお図中Ａ，Ｂ，Ｃはそれぞれ各試料成分を示
し、いま目的の試料成分をＢとすると上記のPU
値およびPR値は次式で示される。
PU＝Ｈｂ−Ｈｌ／Ｈｔ−Ｈｌ
PR＝Ｈｌ／Ｈｂ
しかしながら、第２図に示したごときエレクト
ロフエログラムのように各階段の電位勾配値が距
離（時間）に対し一定、すなわち水平である場合
は各成分の電位勾配値Ha，Hb……およびターミ
ナル液の電位勾配値Htはそのまま用いることが
できるが第３図のごとく、それぞれの電位勾配値
が距離（時間）とともに減少するものがあると上
式によつては正確なPU値PR値がえられず成分の
同定が不確実になるおそれがある。
この発明は、この第３図のような場合であつて
も正確な電位勾配値が得られるためのデータ処理
方法であつて、目的成分の直前の他の成分の電位
勾配値からの立ち上り高さをもつてこの目的成分
の他成分からの電位勾配値の高さの差とし、リー
デイング液の電位勾配値に、目的成分および目的
成分に至るまでの各成分ごとの上記の差成分を加
えた値を、目的成分の電位勾配値とすることを特
徴とするものである。
第３図のエレクトロフエログラムにおいてHl
はリーデイング液（リーデイングイオン）の電位
勾配値で例えば試料成分Ａのフエログラムは図の
ようにリーデイングイオンの電位勾配値から立ち
上り最大値Ａを示し、ついで若干減少した値
A′から再び立ち上り試料成分Ｂとしての最大値
Ｂを示し、再び減少した値B′から立ち上る……
等々でこのフエログラムは示されている。
なお、Ｔはターミナルイオンの立ち上りの最大
値である。
このフエログラムにおいて従来の方法にあつて
はHa，Hb……を試料成分Ａ，Ｂ……の電位勾配
値とみなして上記のPU値、PR値を算出してい
た。しかし、一般に電位勾配値は水平で不変であ
るとして算出するこの従来の方法にあつては、こ
の第３図のように変化する電位勾配置では、正確
な定性分析が不可能であつた。そこでこの理由を
考察した結果、従来にあつては第３図のフエログ
ラム上、Ａ―A′，Ｂ―B′，Ｃ―C′……等の値を
無視していることにその原因が存在していたこと
が判明した。よつて、この発明においては上記の
無視した値をPU値、PR値の算出式に加えたもの
で、例えば試料成分Ｂについては次の算出式とな
るのである（なお式中の符号は、図中の符号で示
した値に対応する）。
Ｂ試料成分の電位勾配値 ｌ＋ａ＋ｂ
Ｂ試料成分のPU値 PU＝
（ｌ＋ａ＋ｂ）−ｌ／（ｌ＋ａ＋ｂ＋ｃ＋ｔ）−ｌ
＝ａ＋ｂ／ａ＋ｂ＋ｃ＋ｔ
Ｂ試料成分のPR値 PR＝ｌ／ｌ＋ａ＋ｂ
すなわち、ａ＋ｂを成分Ｂとリーデイングイオ
ン（液）との電位勾配値の差とし、ターミナルイ
オン（液）の電位勾配値はリーデイングイオン
（液）の電位勾配値に、リーデイングイオン
（液）とターミナルイオン（液）との間に検出さ
れた他の成分の電位勾配値からの立上り高さ、
ａ，ｂ，ｃ……を加えたことになる。この結果、
各信号の下つた値（従来無視していた値）を加え
補正したことになるのである。
次に、この発明の方法を実際の分析データにあ
てはめてみる。
第４図が、次の分析条件で有機物を分析したと
きのエレクトロフエログラムである。
The present invention relates to a method for processing analytical data in an electrophoretic analyzer, particularly in a capillary isotachophoretic analyzer. As is well known, in capillary isotachophoresis analysis, an electrolytic solution (leading solution) containing ions (leading ions) with higher mobility than any component ion in the sample is placed at the end of a capillary tube.
An interface is created with an electrolytic solution (terminal solution) containing ions (terminal ions) with lower mobility than any of the component ions in the sample, and a mixed sample containing components with different mobilities is introduced into this interface, for example.
When electrophoresis is started, separation progresses in such a way that the ions of each sample component are arranged in the order of their mobility, and in a state of complete separation, the separated ions form a zone containing only single component ions. Thus, each zone (band) moves at a constant speed with a constant width determined by the amount of ions while maintaining a clear boundary surface with each other. In this case, since different potential gradients are formed in each zone, the boundary surfaces of each zone are known by detecting the potential gradient values and qualitative analysis is performed. In order to perform qualitative analysis from potential gradient values in this way, it is necessary to obtain an electropherogram in which the potential gradient values of the components are shown as clear step signals. However, in an actual electropherogram, the potential gradient value of some components may change continuously in a decreasing direction with distance (time), and this is especially true when trying to detect many types of components. This phenomenon was unavoidable and it was difficult to accurately identify the components. This invention aims to develop a new data processing method in isotachophoresis analysis in order to eliminate the above-mentioned disadvantages, and will be described in detail below with reference to the figures.
Figure 1 shows the basic principle of a capillary type isotachophoresis analyzer. In the figure, 1 is a terminal liquid electrode tank, 2 is a leading liquid electrode tank, and terminal liquid 3 and leading liquid 4 in each tank are powered by a power supply. Electrodes 6, 7 connected to 5 are inserted. Reference numeral 8 denotes a capillary electrophoresis tube provided between both electrode tanks, which allows the sample introduced from the sample introduction section 9 to the interface between the leading liquid and the terminal liquid to migrate. A potential gradient value of the electrophoresed and separated sample components into each zone is detected by a detector 10, and a detection signal accompanying this potential gradient value is computed by a calculation unit 11 and displayed as, for example, an electropherogram. Note that 12 is a constant temperature bath. FIG. 2 is an example of the electropherogram described above, in which the vertical axis represents the potential gradient value and the horizontal axis represents distance (time). In this figure, Ha, Hb, and Hc are the potential gradient values of the components in the sample, Hl is the potential gradient value of the leading liquid, and Ht is the potential gradient value of the terminal liquid. The following qualitative index value PU (potential unit value) is defined by the ratio of the difference in potential gradient value between the leading ion and component ion to the difference, and the ratio of the potential gradient value of the leading ion to the potential gradient value of the component ion. The components are identified using the following qualitative index ratio PR (potential ratio) defined by:
In the figure, A, B, and C indicate each sample component, and if the target sample component is B, the above PU
The value and PR value are shown by the following formula. PU=Hb-Hl/Ht-Hl PR=Hl/Hb However, when the potential gradient value of each step is constant with respect to distance (time), that is, horizontal, as in the electropherogram shown in Figure 2, The potential gradient values Ha, Hb... of each component and the potential gradient value Ht of the terminal liquid can be used as they are, but as shown in Figure 3, if there is a potential gradient value that decreases with distance (time), Depending on the formula, accurate PU and PR values may not be obtained and component identification may become uncertain. The present invention is a data processing method for obtaining accurate potential gradient values even in the case shown in FIG. Let be the difference in the height of the potential gradient value of this target component from other components, and add the above difference components for the target component and each component up to the target component to the potential gradient value of the leading liquid. is the potential gradient value of the target component. In the electropherogram of Figure 3, Hl
is the potential gradient value of the leading liquid (leading ion). For example, in the ferrogram of sample component A, as shown in the figure, the potential gradient value of the leading ion rises to the maximum value A, and then the value decreases slightly.
It rises again from A', shows the maximum value B as sample component B, and rises again from the decreased value B'...
This ferrogram is shown in and so on. Note that T is the maximum value of the terminal ion rise. In this ferrogram, in the conventional method, the above-mentioned PU value and PR value were calculated by regarding Ha, Hb, . . . as potential gradient values of sample components A, B, . However, in this conventional method, which generally calculates the potential gradient value on the assumption that it is horizontal and unchanging, accurate qualitative analysis is not possible when the potential gradient position changes as shown in FIG. After considering the reason for this, we found that the reason for this is that in the past, values such as A-A', B-B', C-C', etc. were ignored on the ferogram shown in Figure 3. It turned out that he had. Therefore, in this invention, the above-mentioned ignored values are added to the calculation formula for the PU value and PR value, and for example, for sample component B, the calculation formula is as follows (note that the symbols in the formula are as shown in the figure). (corresponds to the value indicated by the symbol inside). Potential gradient value of B sample component l+a+b PU value of B sample component PU= (l+a+b)-l/(l+a+b+c+t)-l
= a + b / a + b + c + t PR value of sample component B PR = l / l + a + b In other words, a + b is the difference in potential gradient value between component B and the leading ion (liquid), and the potential gradient value of the terminal ion (liquid) is the difference in potential gradient value of the leading ion (liquid). ), the rise height from the potential gradient value of other components detected between the leading ion (liquid) and the terminal ion (liquid),
This means that a, b, c... are added. As a result,
This means that the lowered values of each signal (values that were ignored in the past) are added and corrected. Next, the method of this invention will be applied to actual analytical data. FIG. 4 is an electropherogram obtained when organic substances were analyzed under the following analysis conditions.
【表】【table】
【表】
このエレクトロフエログラムを従来法と本発明
の方法で処理した結果を下表に示す。[Table] The results of processing this electropherogram using the conventional method and the method of the present invention are shown in the table below.
【表】
この表よりわかるようにこの発明法によれば正
常値に近くなる。
この発明の効果は、本来、各成分の電位勾配は
変動しないと考えられているが、実際上は第３図
のように電位勾配が距離（時間）によつて低下す
る成分がある。このような場合でもこの発明の方
法によつて正確な成分の同定がおこなえ、特に多
種の成分の試料の定性分析をおこなう場合では、
リーデイング液とターミナル液の選定は制限され
成分のなかには一定した電位勾配値がえられない
ものができ、正確な定性分析を困難にしていた
が、この発明の等速電気泳動データ処理法によつ
て、上記の場合でも同定が可能になり、また分析
条件の巾が拡大され、等速電気泳動分析法の用途
が拡大することが期待できるものである。
なお、この発明は前記のエレクトロフエログラ
ムの作図による外コンピユータにより解析する場
合にも前記と全く同様の成果がえられるものであ
る。この場合、リーデイングイオン（液）、ター
ミナルイオン（液）および各試料成分の電位勾配
値および他成分からの立上り高さ等々を記憶し順
次読み出して演算処理することにより容易に実
施・実現できるものである。[Table] As can be seen from this table, according to the method of this invention, the values are close to normal. The effect of this invention is originally thought to be that the potential gradient of each component does not change, but in reality, as shown in FIG. 3, there are components whose potential gradient decreases with distance (time). Even in such cases, the method of the present invention allows accurate component identification, especially when performing qualitative analysis of samples containing various components.
The selection of leading and terminal liquids was limited, and some components did not have a constant potential gradient value, making accurate qualitative analysis difficult. However, the isotachophoresis data processing method of this invention Identification becomes possible even in the above cases, and the range of analysis conditions is expanded, so it is expected that the applications of isotachophoresis analysis will be expanded. In addition, the present invention can achieve exactly the same results as described above even when the electropherogram is drawn and analyzed using an external computer. In this case, it can be easily implemented and realized by memorizing the potential gradient values of the leading ion (liquid), terminal ion (liquid), and each sample component, the rise height from other components, etc., and sequentially reading them out and performing arithmetic processing. be.
第１図は細管式等速電気泳動分析装置の基本原
理を説明する図、第２図はエレクトロフエログラ
ムの一例を示す図、第３図はこの発明の方法を用
いる成分の電位勾配値が距離（時間）にしたがつ
て減少するエレクトロフエログラムの一例、第４
図は有機酸を分析したときのエレクトロフエログ
ラムである。
１…ターミナル液電極槽、２…リーデイング液
電極槽、３…ターミナル液、４…リーデイング
液、５…電源、６，７…電極、８…キヤピラリー
チユーブ、９…試料導入部、１０…検出器、１１
…演算部。
Fig. 1 is a diagram explaining the basic principle of a capillary isotachophoresis analyzer, Fig. 2 is a diagram showing an example of an electropherogram, and Fig. 3 is a diagram showing the potential gradient value of a component using the method of this invention over a distance. An example of an electropherogram that decreases over time, 4th
The figure shows an electropherogram when an organic acid was analyzed. DESCRIPTION OF SYMBOLS 1... Terminal liquid electrode tank, 2... Leading liquid electrode tank, 3... Terminal liquid, 4... Leading liquid, 5... Power supply, 6, 7... Electrode, 8... Capillary reach tube, 9... Sample introduction part, 10... Detector , 11
...Arithmetic section.
Claims (1)
導入した試料を泳動させ、各試料成分をその易動
度の相違により分離するとともに各試料成分の電
位勾配値を検出し試料の定性分析を行なうものに
おいて、目的成分の直前の他の成分の電位勾配値
からの立ち上り高さをもつて、この目的成分の他
成分からの電位勾配値の高さの差とし、リーデイ
ング液の電位勾配値に、目的成分および目的成分
に至るまでの各成分ごとの上記の差成分を加えた
値を、目的成分の電位勾配値とすることを特徴と
する電気泳動分析データ処理方法。1 In a method that performs qualitative analysis of the sample by electrophoresing the sample introduced at the interface between the leading liquid and the terminal liquid, separating each sample component based on the difference in mobility, and detecting the potential gradient value of each sample component. , the rise height from the potential gradient value of the other component immediately before the target component is taken as the difference in the height of the potential gradient value of this target component from the other component, and the potential gradient value of the leading liquid is An electrophoretic analysis data processing method characterized in that a value obtained by adding the above-mentioned difference components for each component up to the target component is used as a potential gradient value of the target component.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4242580A JPS56138245A (en) | 1980-03-31 | 1980-03-31 | Processing method for analyzed data of cataphoresis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4242580A JPS56138245A (en) | 1980-03-31 | 1980-03-31 | Processing method for analyzed data of cataphoresis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS56138245A JPS56138245A (en) | 1981-10-28 |
| JPS628746B2 true JPS628746B2 (en) | 1987-02-24 |
Family
ID=12635701
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP4242580A Granted JPS56138245A (en) | 1980-03-31 | 1980-03-31 | Processing method for analyzed data of cataphoresis |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS56138245A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63156143U (en) * | 1987-03-31 | 1988-10-13 | ||
| JPH01177641U (en) * | 1988-06-06 | 1989-12-19 | ||
| JPH0226853U (en) * | 1988-08-08 | 1990-02-21 |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5876747A (en) * | 1981-10-30 | 1983-05-09 | Shimadzu Corp | Data processor for equispeed electrophoresis analyzer |
-
1980
- 1980-03-31 JP JP4242580A patent/JPS56138245A/en active Granted
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63156143U (en) * | 1987-03-31 | 1988-10-13 | ||
| JPH01177641U (en) * | 1988-06-06 | 1989-12-19 | ||
| JPH0226853U (en) * | 1988-08-08 | 1990-02-21 |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS56138245A (en) | 1981-10-28 |
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