U.S. patent number 3,869,365 [Application Number 05/423,997] was granted by the patent office on 1975-03-04 for method in counter flow isotachophoresis.
This patent grant is currently assigned to LKG-produkter AB. Invention is credited to Bengt Fritiof Sunden.
United States Patent |
3,869,365 |
Sunden |
March 4, 1975 |
**Please see images for:
( Certificate of Correction ) ** |
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.
Inventors: |
Sunden; Bengt Fritiof (Alvsjo,
SW) |
Assignee: |
LKG-produkter AB (Bromma,
SW)
|
Family
ID: |
20302493 |
Appl.
No.: |
05/423,997 |
Filed: |
December 12, 1973 |
Foreign Application Priority Data
|
|
|
|
|
Dec 19, 1972 [SW] |
|
|
16594/72 |
|
Current U.S.
Class: |
204/549 |
Current CPC
Class: |
G01N
27/44765 (20130101) |
Current International
Class: |
G01N
27/447 (20060101); B01k 005/00 () |
Field of
Search: |
;204/18R,18G,18S,299 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Williams; Howard S.
Assistant Examiner: Prescott; A. C.
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.
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.
* * * * *