Method In Counter Flow Isotachophoresis

Abstract

A method for fixing the sample in a certain position in a counter flow isotachophoresis column comprises the adjustment of voltage and counterflow values to a constant at which the power from the electric field and the power from the counterflow compensate each other at the desired position of the sample.

Description

The present invention refers to a method in counter flow isotachophoresis, more precisely a method for stabilizing the sample in a pre-determined position in such isotachophoresis.

In counter flow isotachophoresis a separation of an ionized sample comprising ions of a certain polarity takes place by introducing the sample in a column arranged between two electrodes, a leading electrolyte, comprising ions of the same polarity but having a higher mobility than the sample, being introduced in the column between the sample and the electrode towards which said ions migrate when a voltage is applied to the electrodes, and a terminating electrolyte comprising ions of said polarity having a lower mobility than that of the sample ions being introduced between the sample and the other electrode, the leading electrolyte being supplied to the column under pressure so as to bring the electrolyte to flow in a direction towards the sample. Isotachophoresis is described more in detail, e.g. in Analytica Chemica Acta 38 (1967) pages 233-237 under the name of "Displacement electrophoresis" and is also described in the Swedish Pat. No. 340,376 and corresponding U.S. Pat. No. 3,705,845. As appears from these publications conventional isotachophoresis suffers from the drawback that if the ion concentrations are low and the differences in mobility of the sample ions are small, a very long column is required which means that in order to obtain sufficient field strength in the column, very high voltages are necessary. The length of the column could, however, be reduced significantly, if one applies a so called counter flow of the leading electrolyte, i.e. this electrolyte is pumped in a direction opposite that of the sample ions. (See e.g. Preets and Pfeifer, Analytica Chemica Acta 38 (1967) pages 225-260.) For in such isotachophoresis it is possible to obtain a separation without moving the boundary between sample and the leading electrolyte in the column. By reducing the length of the column, the required field strength could thereby be obtained by using considerably lower voltages. The problem is, however, to choose the amplitude of the counter flow and the electrical current in the column in such a way that the sample is in a substantially fixed position until the separation is completed and an equilibrium has taken place. In order to solve these problems, one could observe the border between leading electrolyte and the first zone of the sample and continuously, manually adjust the counter flow in order to keep this boundary in a fixed position. This means, however, that the apparatus must be manually controlled during the complete separation, and furthermore, it is difficult manually to provide the very small changes of the counter flow which are required not to disturb the separation. From the Swedish Pat. No. 340,376 and said U.S. Pat. 3,705,845 it is, furthermore, known to automate the adjustment procedure by using a detector which detects said boundary, and when the boundary moves compensates this movement by changing the amplitude of the counter flow. The drawback of this method is that it requires an extra detector and electronic circuitry for controlling the counter flow, which means that the apparatus will be quite expensive. Furthermore, it is required that the boundary is sharply defined since this boundary is the parameter from which the regulation is based. Since counter flow isotachophoresis is mainly used when the components of the sample are difficult to separate this boundary will, at least in the beginning, be rather diffuse which makes the control uncertain.

It is an object of the present invention to provide a method for automatic control of the counter flow and the voltage across the column so as to keep the sample in a fixed position without the requirement of any extra detector and appertaining electronic circuitry.

The invention will now be described in detail, reference being made to the enclosed drawing in which:

FIG. 1 schematically shows the process of ion separation in isotachophoresis,

FIG. 2 shows an apparatus for carrying out the method according to the invention, and

FIG. 3 by means of diagram explains the working principle of the apparatus according to FIG. 2.

In FIG. 1, reference 1 denotes a column in which an anode 5 and a cathode 4 are arranged. It is, furthermore, presumed that the sample to be separated is introduced in the part of the column denoted S, the sample comprising two different anions C.sub.1 .sup.- and C.sub.2 .sup.- of which C.sub.1 .sup.- is supposed to have a higher mobility than C.sub.2 .sup.-. The part of the column denoted L is filled with the above described leading electrolyte which consists of anions A.sup.- having a higher mobility than all anions in the sample. The part of the column T closed to the cathode is filled with an electrolyte comprising an anion B.sup.- having a mobility which is lower than that of the anions in the sample. When a direct voltage is supplied to the electrodes 4 and 5, the anions will migrate towards the anode 5. Because of the different mobility of the anions a zonewise and stepwise growing voltage gradient will be obtained across the zones L, S, and T, respectively. The voltage gradient across the zone S will, however, imply that the ions within this zone are separated according to their mobility so that the ions C.sub.1 .sup.-, which have the higher mobility, are located close to the leading electrolyte and the ions C.sub.2 .sup.- with the lower mobility are located close to the terminating electrolyte. When a voltage is supplied to the column, the anions of the sample will thus be separated and after the separation, the different zones of the column will migrate towards the anode 5 with a velocity which is dependent upon the mobility of the ion A.sup.-, a zonewise growing potential being obtained across the column. The thus formed zones will be very stable, since if an anion from one zone e.g. diffuses from its original zone into a zone in front of this zone, the anion will due to the lower potential gradient in the zone in front obtain a reduced velocity and be brought back into its original zone. In the same way an anion which diffuses into a zone behind its original zone will be brought back to its original zone because of the higher voltage gradient in the zone behind. In order to detect the different zones and their lengths one preferably uses the stepwise growing potential. One could e.g. measure the stepwise rising temperature at the outside of the column or directly measure the potential in one or several points of the column. It is, of course, also possible by means of electrodes to measure the conductivity of the zones which pass a certain point and so far as the separated substances are UV-absorbing, one could also in a conventional way measure the UV-absorption.

As mentioned above, it is, however, a drawback of the above described separation method that, especially when the difference in mobility between the different ions of the sample is small, a fairly long column is required for the separation which means that in order to obtain a sufficient field strength a very high voltage must be applied to the electrodes which involves complicated design and safety problems. The length of the column could, however, be considerably reduced if, during the separation, leading electrolyte is supplied to the column as a counter flow. The amplitude of the counter flow could then preferably be chosen so as to keep the boundary between the zones L and S in a fixed position. As mentioned above, the control of the counter flow is either carried out manually by means of observations of the zone boundary and by increasing or reducing the counter flow pressure when this boundary moves or the control is carried out by regulating the counter flow automatically by means of a special detector which senses the zone boundary and in dependence of movements of this boundary controls the counter flow pressure. This will, however, require that the zone boundary is well defined which is not normally the case in low sample concentrations and small differences in mobility. It is, therefore, an object of the present invention to provide a method in which the zone boundary automatically is brought to a fixed position without the requirements of any controlling detector.

In FIG. 2 there is shown schematically an apparatus for carrying out counter flow isotachophoresis according to the invention. In FIG. 2, reference 1 denotes a separation column in which the sample can be introduced via an input port 3 between a terminating and a leading electrolyte T and L, respectively. A voltage is applied to the column and the electrolyte by means of a voltage supply 2 which is connected to electrodes 4 and 5, respectively, whereby the sample S migrates into the column. The power supply 2 is designed in such a way that either an adjustable constant voltage or an adjustable constant current can be applied to the column. The power supply is, furthermore, provided with a current differentiator which indicates the variations of the current with respect to time. The vessel containing leading electrolyte is separated from the column by means of a semi-permeable membrane 14 in order to make it possible to flow the column without effecting the contents of the vessel. The column is, furthermore, provided with an input port 13 from which a counter flow of leading electrolyte can be generated by means of a syringe 15 driven by a motor 16. The column is further provided with a first detector 10 for detection of the separated zones. This detector is connected to a plotter 12 via an amplifier 11. The apparatus is, furthermore, provided with a second detector 7 which could be used for stabilizing the locations of the zones. This detector is in a corresponding manner connected to a plotter 9 via an amplifier 8.

The use of the apparatus according to FIG. 2 will now be described in detail, reference being made to the diagrams in FIG. 3a-c. FIG. 3a shows the current through a column according to FIG. 2 when a constant current VO is applied to the electrodes as a function of time, and furthermore, the position of the sample S in the column 1 during the process is indicated. When the voltage is applied the sample S is supposed to be located at the upper end of the column. The column is thereby completely filled with the leading electrolyte, i.e. the electrolyte having the highest conductivity. During the process the sample will then under separation of its components migrate downwards along the column. Provided that a constant voltage VO is applied to the column, the current will then successively decrease as the contents of leading electrolyte of the column gradually decreases whereas the contents of terminating electrolyte, i.e. a component having a lower conductivity will increase. When the sample reaches the bottom end of the column, the column will be completely filled with terminating electrolyte and the current will have a constant value. At a certain point of time t0 the current will thus have a certain value IO which defines the position SO of the sample in the column. If now in the column one of a number of different counter flows Cf1, Cf2, and Cf3 where Cf1 > Cf2 > Cf3 are generated, the corresponding current diagrams will turn out as shown in FIG. 3b. For a certain position of the sample S10, S20, and S30, respectively for the different counter flows, the counter flow will compensate the forward driving effect and the current through the column will thus be constant. If the voltage VO is increased to a value Vl > VO, these positions of the sample will be moved downwards along the column, i.e. the voltage will drive the sample further into the column. If thus, for a certain sample and certain leading and terminating electrolytes, the counter flow and the voltage for which the sample will be fixed in a certain position are known, this voltage- and counter flow values could be adjusted at the beginning of the experiment and the sample will then migrate to the pre-determined position and be fixed in this position because of the appearing equilibrium.

Very often these exact parameters are known. If this is not the case, one could use the fact that in equilibrium the current is constant to make the necessary adjustments. Thereby the sample is transfered, without any counter flow applied, a suitable distance into the column whereafter counter flow and voltage are varied so as to obtain a constant current which could be detected from the current differential meter of the voltage supply 2. The position of the sample could thereby either be determined ocularly or by means of a suitable detector, e.g. a thermodetector, a potential detector, or a conductive detector (Ref. 7 in FIG. 2).

The essential principle of the invention is thus that by applying a column constant counter flow and a constant current to the column it is possible to fix the sample at an equilibrium where the current through the column is constant. In order to choose the suitable values for counter flow and voltage one could thereby use a current differential detector or some other conventional detector located along the column. According to the invention one will thus obtain a process where it is very simple and unexpensive to fix the sample in a pre-determined position during an arbitrary time.

Claims

We claim:

1. Method in counter flow isotachophoresis of the type wherein a sample comprising ions of the same polarity to be separated is introduced into a column provided with first and second electrodes, with a first electrolyte between the sample and said first electrode and a second electrolyte between the sample ans said second electrode, the first and second electrolytes comprising ions of higher and lower mobilities respectively than the ions of the sample and an electrical potential is applied between said first and second electrodes having a polarity such that the sample ions will tend to migrate towards said first electrode and a pressure difference is applied between said first and second electrolytes, the improvement which includes the steps of:

independently adjusting the value of said electrical potential and the value of said pressure difference to produce with the aid of a current differential detector an electrical current flow having a constant value such that the influence of said constant current flow equalizes the adjusted opposing counter flow of electrolyte to maintain the sample at a desired position in the column.

2. Method according to claim 1, characterized in, that the adjustment of said values is made by using a current differerential detector, the values being chosen so as to make the derivative of the electrical current zero.

3. Method according to claim 1, characterized in, that the values of the counter flow and the voltage are adjusted by means of an indication means located along the column.

4. Method according to claim 3, characterized in, that the indication means consists of a thermodetector.

5. Method according to claim 3, characterized in, that the indication means consists of a UV-detector.

6. Method according to claim 3, characterized in, that the indication means consists of a conductivity detector.

7. Method according to claim 3, characterized in, that the indication means consists of a potential detector.

8. The method according to claim 1, which includes the step of introducing the sample into the column in the absence of a counter flow of electrolyte.