US3705845A

US3705845A - Method in counterflow isotachophoresis

Info

Publication number: US3705845A
Application number: US148657A
Authority: US
Inventors: Frans Everaerts
Original assignee: LKB Produkter AB
Current assignee: Pfizer Health AB
Priority date: 1970-06-02
Filing date: 1971-06-01
Publication date: 1972-12-12
Anticipated expiration: 1989-12-12
Also published as: FR2093980A1; DE2127391A1; GB1333796A; FR2093980B1; DE2127391B2

Abstract

METHOD IN COUNTERFLOW ISOTACHOPHORESIS. INTO A COLUMN PROVIDED WITH TWO ELECTRODES A SAMPLE COMPRISING IONS OF DIFFERENT MOBILITY TO BE SEPARATED IS INTRODUCED TOGETHER WITH A FIRST ELECTROLYTE BETWEEN THE SAMPLE AND ONE OF THE ELECTRODES AND A SECOND ELECTROLYTE BETWEEN THE SAMPLE AND THE OTHER ELECTRODE. THESE FIRST AND SECOND ELECTROLYTES COMPRISE IONS OF HIGHER AND LOWER MOBILITY RESPECTIVELY THAN THE IONS OF SAID SAMPLE. A VOLTAGE IS APPLIED TO THE ELECTRODES AND A PRESSURE IS APPLIED TO ONE OF SAID ELECTROLYTES. A BOUNDARY BETWEEN ZONES OF IONS HAVING DIFFERENT MOBILITIES IS DETECTED AND FROM THIS DETECTION

A CONTROL SIGNAL IS GENERATED, SAID CONTROL SIGNAL CONTROLLING SAID PRESSURE AND/OR SAID VOLTAGE.

Description

FIG. 1a

RIG 1' F. EVERAERTS METHOD IN COUNTERFLOW ISOTACHOPHORESIS Filed June 1, 1971 DecQ 12, 1972 FIG. 2

United States Extent 3,705,845 METHOD IN COUNTERFLOW ISOTACHOPHORESIS Frans Everaerts, Weert, Netherlands, assignor to LKB- Produkter AB, Bromma, Sweden Filed June 1, 1971, Ser. No. 148,657 Int. Cl. B01k 5/00 US. Cl. 204-180 R 4 Claims ABSTRACT OF THE DISCLOSURE Method in counterflow isotachophoresis. Into a column provided with two electrodes a sample comprising ions of diiferent mobility to be separated is introduced together with a first electrolyte between the sample and one of the electrodes and a second electrolyte between the sample and the other electrode. These first and second electrolytes comprise ions of higher and lower mobility respectively than the ions of said sample. A voltage is applied to the electrodes and a pressure is applied to one of said electrolytes. A boundary between zones of ions having different mobilities is detected and from this detection a control signal is generated, said control signal controlling said pressure and/or said voltage.

The present invention relates to a method in counterflow isotachophoresis, in which a sample, containing ions of the same polarity which are to be separated, is introduced into a column arranged between two electrodes, at which in that part of the column which is situated between the sample and that electrode towards which the ions in question are migrating when a voltage is applied to the electrodes, a leading electrolyte is introduced containing ions of still the same polarity but with a higher mobility than that of the sample ions, and between the sample and the other electrode a terminating electrolyte is introduced containing ions of still the same polarity but with a lower mobility than that of the sample ions, and that the above mentioned leading electrolyte is supplied to the column under a pressure so that the electrolyte is made flowing towards the sample.

Isotachophoresis (Greek: iso=equal, tacho=speed) is an electrophoretic separatiofi method which is more closely described in e.g. Anal. Chim. Acta 38 (1967) 23 3-237, under the name of Displacement Electrophoresis. Isotachophoresis is principally carried out in that way that a sample containing a number of different ions with the same polarity is introduced into some type of column in which the sample is located between two electrolytes, containing ions of the same polarity as those ions in the sample which are going to be separated, whereat one of the electrolytes contains ions with a higher mobility than that of the sample ions and the other electrolyte contains ions with a lower mobility than that of the sample ions. The electrolyte containing ions with a higher mobility, the so called leading electrolyte, is at that introduced in that part of the column which is connected to the electrode, towards which the sample ions are migrating, and the electrolyte with the slower ions, the so called terminating electrolyte, is introduced at the other electrode. If a voltage is then applied to the electrodes, the ions will be migrating in the column, at which the ions of the sample will be separated according to their mobility. The course thus obtained will be closer explained below.

The essential advantage of isotachophoresis in comparison with conventional electrophoresis is that substantially smaller amounts of sample can be separated and that very sharp boundaries are achieved between the separated zones of sample, the zones of sample thus not be- Patented Dec. 12, 1972 ing extended during the course of separation as in conventional electrophoresis. A disadvantage is, however, that a long column is to be used in working with very small ions concentrations and in working with sample ions with very slight diiference in mobility, whereby very high potentials are required to give sufficient field strength in the column. The length of the column might, however, be considerably reduced if a counterflow of the leading electrolyte is utilized, i.e. the leading electrolyte is pumped against the migration direction of the sample ions. (See e.g. Preetz and Preifer, Anal. Chim. Acta 38 (1967 255- 260.) In that case a separation can be achieved without moving in the column the boundary between the sample and the leading electrolyte. By shortening the column the requisite field strength can be obtained by considerably lower potentials. The problem is, however, to choose the counterfiow and the electric current in the column in such a way that one of the zones, which are obtained when the sample is separated, is mainly immobile until the separation is completed and until an equivalence state has ensued. To solve this problem is hitherto proceeded in that way that the boundary between the leading electrolyte and the first zone of the sample is observated and the counterflow is being adjusted e.g. by means of a vertically adjustable reservoir, connected to the column and containing the leading electrolyte, as soon as the boundary has any tendency to move in either direction. This, however, brings about that the device must be kept under observation during all the separation course and it is further difiicult manually to accomplish the exceedingly small variations of the counterfiow which are necessary in order that the separation should not be disturbed. Thus the purpose of the present invention is to provide a method in counterflow isotachophoresis in which those disadvantages are eliminated.

The invention will now be closer explained with reference to the accompanying drawing in which FIGS. la and 1b schematically show how the different ions of a sample are separated, and

FIG. 2, also schematically, shows a device for accomplishing the method according to the invention.

In FIGS. 1a and lb the number 1 denotes a column in which an anode 5 and a cathode 4 are introduced. It is further assumed that the sample which is to be separated is introduced into that part of the column which is designated with S, and that the sample consists of salts containing two diiferent anions C and C of which C is assumed to have a higher mobility than C and a common cation R+. The part of the column which is designated L is filled with the above mentioned leading electrolyte, which consists of anions A with a higher mobility than all the anions of the sample and a cation, designated R+, suitably common with the sample. The part T of the column which is turned towards the cathode is filled with an electrolyte which contains an anion B-, the mobility of which is lower than of all anions of the sample, and a cation R+, common with the leading electrolyte. When a direct current voltage is applied to the

electrodes

4 and 5 the cations will migrate towards the cathode 4 and the anions will migrate towards the anode 5. As a result of the different mobilities of the anions a zonewise stepwise increasing potential gradient will then be obtained over the zones L, S, and T respectively. The potential gradient over the zone S will, however, lead to that the anions in the zone will be separated according to their mobility, so that those ions C," with the higher mobility will locate. themselves nearest to the leading electrolyte, and those ions C having the lower mobility will locate themselves nearest to the zone T. Provided that a mainly constant current is forced through the column, the anions of the sample will thus be separated and after the separation the ditferent zones in the column will migrate stable since an anion which e.g. is ditfusing into an ahead lying zone with a lower potential gradient will get a lower speed so that it will be brought back to its original zone. In the same way an anion which is diflusing into a rear zone because of the potential gradient present in that zone will be brought back into its original zone. A very good self-stabilizing of the Zone boundaries is thus achieved. In order to detect the different zones and their lengths the stepwise increasing potential gradient is suitably utilized. For example the correspondingly stepwise increasing temperature on the outer surface of the column 5 could be measured or the potential at one or more points on the column could be directly measured. Of course it is also possible, by means of the electrode, to measure the conductivity of the zones passing a certain point, and so far the separated substances are UV-absorbing, the UV- absorption could also be measured in conventional manner.

As has been mentioned in the introduction it is, however, a disadvantage of the separation method described above, that a rather long column is required for the separation, especially when the difference is small between the mobilities of the different ions in the sample, why in order to achieve the requisite field strengths a very high voltage must be applied to the electrodes with accompanying problems of construction and of safety. The

length of the column can, however, be considerably reduced, if leading electrolyte is continuously pumped to the column during the separation. The magnitude of the counterflow can then suitably be chosen in a way that the boundary between the zones L and S principally is taining leading electrolyte has been raised or lowered.

This procedure has, however, the drawback that the sample must be kept under permanent observation and further an incautious action on the liquid level might bring about that the magnitude of the counterflow is altered in such a way that the separation is seriously 4.5

disturbed. The purpose of the present invention is therefore to provide a procedure by means of which the counterflow always is given such a value that a certain zone boundary is occupying a fixed position. The invention will now be more closely explained with reference to FIG.

2, which schematically shows a device for performing the procedure according to the invention.

In FIG. 2 the number 1 denotes a column, which is arranged between an anode chamber 3 and a cathode chamber 2. In-the electrode chambers an anode 5 and a cathode 4 are respectively introduced and these are connected to a current source 6. The column is further equipped with a multiway valve 7 by means of which the column could either be connected to the chamber 2 or to a sample injecting means 8. By this injecting means 30 the sample can be introduced into a capillary between the terminating electrolyte, which is introduced into the cathode chamber 2, and the leading electrolyte, which is introduced into the anode chamber 3 and in addition into that part of the column which is situated between the 5 valve and this chamber. That part of the column which is situated at the anode is further suitably equipped with a valve 10 via which leading electrolyte could be supplied or removed. Further, a diaphragm 20 is arranged between the column and the anode chamber 5 in a way that the column could be flushed with liquid without changing the circumstances in the chamber. With those polarities of the

electrodes

4 and 5 which have been chosen in the figure, it is then assumed that the anions of the sample are the subjects of separation. If the sample on the other hand would contain a number of diflerent cations the reverse polarity of the electrodes would of course be chosen. The device is further equipped with means for varying the counterflow of the leading electrolyte. This means consist of a reservoir 11, in which a floater 13 can be moved up and down so that the liquid level in the reservoir and thus the pressure of the counterflow can be varied. A spindle 14 which is supporting the floater is introduced into an electromagnet 15 so that by varying the current in the electromagnet the floater is brought up and down in the reservoir. The governing of the current through the electromagnet is accomplished by means of a detection means 17 connected to the column. The detection organ can be thermocouple but can also consist of any other of the detecting elements which has been stated above in connection to FIGS. la and lb. The thermocouple is connected to an amplifier 18, the output of which in its turn is connected to a governing device 19. This one is in its turn connected to the electromagnet 15. The signal from the thermocouple should then govern the counterflow in order that a zone boundary, suitably the boundary between'the leading electrolyte and the first sample zone, is mainly stationary at the thermocouple. As hinted above there will be obtained a temperature rise at this boundary which temperature rise also will cause a temperature rise on the outer surface of the column. Thus the governing device 19 should principally give an output signal which keeps the floater 13 in a fixed position when the signal from the thermocouple corresponds to a temperature which constitutes the mean value between the temperature in the leading electrolyte and that in the first sample zone. A signal corresponding to a lower temperature should further cause a slow decreasing of the coun terflow by means of raising the floater 13, and a signal corresponding to a higher temperature should correspondingly cause a slow increase of the counterflow, i.e. a lowering of the floater 13. In this connection it should be stated that counterflow governing by means of a vertically adjustable floater of course only is one example of varying the eounterflow. A great advantage of the shown counterflow governing is, however, that very small counterflow alterations, free from pulsation, can be achieved, to a very inconsiderable extent disturbing the separation in the column. However, any pressure generating means could be generally utilized. As appears from the drawing the governing device 19 is also connected to a current source 6, and the purpose of this connection is that by varying the electric current in the column in dependence of the current from the thermocouple a certain zone boundary should be kept in a fixed position. Of course it is also possible to fix the zone boundary by alfecting both the counterflow and the electric current.

We claim: 1. Method in counterfiow isotachophoresis comprising the steps of:

introducing into a column provided with first and second electrodes a sample comprising ions of the same polarity to be separated, a first electrolyte between the sample and said first electrode and a second electrolyte between the sample and said second electrode, said first and second electrolytes comprising ions of higher and lower mobilities respectively than the ions of said sample; applying a voltage between said first and second elec trodes, the voltage having such a polarity which will give the sample ions a tendency to migrate towards said first electrode; applying a pressure difference between said first and second electrolytes; detecting by a detection means a boundary between zones of ions having different mobilities; and obtaining a control signal from said detection means, the. control signal controlling said pressure in order to govern said boundary to move with a desired velocity.

2. Method as recited in claim 1, wherein said velocity is zero.

3. Method in counterflow isotachophoresis comprising the steps of:

introducing into a column provided with first and second electrodes a sample comprising ions of the same polarity to be separated, a first electrolyte between the sample and said first electrode and a second electrolyte between the sample and said second electrode, said first and second electrolytes comprising ions of higher and lower mobilities respectively than the ions of said sample;

applying a voltage between said first and second electrodes, the voltage having such a polarity which will give the sample ions a tendency to migrate towards said first electrode;

applying a pressure difi'erence between said first and second electrolytes;

detecting by a detection means a boundary between zones of ions having different mobilities; and

UNITED STATES PATENTS 2,711,379 6/1955 Rothstein 204180 R X 3,305,471 2/ 1967 Von Miinchhausen et a1.

204-180 R X 3,384,564 5/1968 Ornstein et a1. 204-180 G 3,533,933 10/1970 Strauch 204-180 G JOHN H. MACK, Primary Examiner A. C. PRESCOTT, Assistant Examiner US. Cl. X.R.

UNITED STATES PATENT OFFICE, CERTIFICATE OF CORRECTION Patent No. 3,705, 5 Dated m r 1 19-72 Inventods) Frans Everaerts It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

In the heading, after "Filed June 1, 1971, Ser'. No.

l I8,657" add Claims Priority Swedish Application No.

762 I/7O, filed June 2, l970- Signed and sealed this 8th day of May 1973.

(5L aL) Attest:

ED 5431) 1-5.. FLETGHER,J'R. ROBERT GOTTS'CHALK attesting Officer Commissioner of Patents FORM PO-IOSO (IO-69) USCOMM-DC 60375-p59 U45, GOV ERNMENT PRINTING OFFICE: I969 O366'334 I