US3912609A - Method at isotachophoretical separation to detect spectrophotometrically zone boundaries obtained - Google Patents

Method at isotachophoretical separation to detect spectrophotometrically zone boundaries obtained Download PDF

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US3912609A
US3912609A US432836A US43283674A US3912609A US 3912609 A US3912609 A US 3912609A US 432836 A US432836 A US 432836A US 43283674 A US43283674 A US 43283674A US 3912609 A US3912609 A US 3912609A
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column
separation
isotachophoretical
ions
detection
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Tord Lennart Arlinger
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Pfizer Health AB
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LKB Produkter AB
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    • 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
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
    • 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
    • G01N27/44747—Composition of gel or of carrier mixture

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  • ABSTRACT A method for spectrophotometrically detecting con- [30] Foreign Application i it Data stituents in an isotachophoretical column, consists in employing a counter-iron whose molar absorptivities Jan. 15, 1973 Sweden 7300492 differ i hin the range between acidic and basic con- [52] U S Cl 204/180 204/299 ditions existing in the column. [51] BOIK 5/00 2 Claims, 10 Drawing Figures T S L I A. V AI A R+ 4- 4- R C R B C; A"
  • At isotachphoresis a separation of an ionized sample containing ions of a certain polarity is carried out in that way that the sample is introduced into a column, arranged between two electrodes, a leading electrolyte being introduced into that part of the column which is present between the sample and the electrode towards which said ions are migrating when a voltage is applied to the electrodes, said leading electrolyte containing ions of the same polarity but with higher'mobility than the sample ions, and a terminating electrolyte being introduced into that part of the column which is present between the sample and the other electrode, said terminating electrolyte containing ions of said polarity with lower mobility than those, of the sample ions.
  • ion species having opposite polarity a so-called counter-ion.
  • the counter-ion suitably has buffering properties. Isotachophoresis is more closely described e.g. in Analytica Chemica Acta 38 (1967) pp 233-237, termed Displacement Electrophoresis and in the patent specification (corresponding to Swedish Pat. No. 340.376).
  • an isotachophoretical separation of ions sharp boundaries between the zones formed by the ions are obtained.
  • some kind of detector is usually arranged at the column for detection of the zone boundaries obtained.
  • An object of this detection is to indicate when sharp zone boundaries have been formed between all sample zones, indicating completed separation.
  • Another purpose of such a detection is to govern a'counter flow, utilized in several cases, in order that the zone boundary between leading electrolyte andsample mixture is kept stationary in the column as is described in the above mentioned patent specification.
  • the detection methods which have come to practice are principally thermal detection, based on the fact that the heat emission is different in the different zones and is increasing in the direction from leading towards terminating electrolyte, and spectrophotometrical detection of the different zones.
  • the latter 'method provides faster measu'rementthan the first one, which necessarily have to work with a time lag of about 5 seconds.
  • the spectrophotometrical detection allows a far greater resolution than the thermal detection, more exactly some SO-lOO times greater.
  • a prerequisite of the spectrophotometrical detection is however that the various separated ion species show absorbance. The number of substances which show absorbance within the visible spectrum is very small.
  • the purpose of the present invention is to provide for spectrophotometrical detection of zones of substances, which do not show absorbance, and to allow the greater rapidity and greater resolution characteristic of spectrophotometrical detection as compared to thermal detection.
  • FIG. 1 schematically shows a column prior to an isotachophoretical separation
  • FIG. 2 shows the same column after achieved equilibrium
  • FIG. 3 shows schematically the electrical field strength E along the column
  • FIG. 4 shows the concentration of the different anions at equilibrium
  • FIG. 5 shows the concentration of the counter-ion R along the column, i I
  • FIG. 6 shows schematically the pH-course along the column
  • FIGS. 7a-9 show detector curves of isotachophoretical separations, shown as examples.
  • FIGS. 1 and 2 is denoted by 1, a column in which an anode 2 and a cathode 3 are introduced.
  • FIG. l is denoted by 1, a column in which an anode 2 and a cathode 3 are introduced.
  • the sample to be separated is introduced in that part of y the column which is denoted by S, the sample consisting of salts containing three different anions C C and C of which C is assumed to have greater mobility than C which in its turn is assumed to have greater mobility than C
  • That part of the column which is denoted by L is filled with the above mentioned leading electrolyte, which consists of anions A, having greater mobility than all anions in the sample.
  • That part of the column which is nearest to the cathode, T is filled with an electrolyte containing an anion B having a mobility which is smaller than those of all anions in the sample.
  • a so-called counter-ion R which suitably has buffering properties.
  • a direct voltage is applied to the electrodes 2 and 3 the anions will migrate towards the anode 2.
  • the electrical field strength over the zones L, S and T, respectively will increase stepwise over the respective zones. This will however bring about that the anions present in the zone S will be separated according to their mobilities, so that the ions C, having the greater mobility will form a zone nearest to the leading electrolyte, followed by a zone consisting of C and finally by a zone consisting of C; next to the terminating electrolyte, which is shown in FIG. 2.
  • the electrical field strength will increase stepwise.
  • concentration stage there is a concentration stage between the different anions at the different zone boundaries. This is shown in FIG. 4.
  • the counterion should suitably have buffering properties. If so, the total concentration of the counter-ions will show considerably smaller stages at the different zone boundaries, as is hinted in FIG. 5. Also the pH is changing at the different zoneboundaries, e.g. as is shown schematically in FIG. 6.
  • a counter-ion R having buffering properties, the counter-ion being chosen in that way that its molar absorptivities at acid and basic conditions, respectively, differ at a wave-length, suitable for measurement.
  • the pH- course shown in FIG. 6 then will bring about an absorbance course along the column. 7
  • FIGS. 7a and 7b are illustrated a separation of five anions by isotachophoresis.
  • FIG. 7a illustrates a separation according to the prior art
  • FIG. 7b shows the result of a separation according to the present invention.
  • FIGS. 7a and 7b shows from top to bottom detector readings from a thermal detector, a differential thermal detector and a spectrophotometrical detector, respectively.
  • a certain section corresponding to a certain ion is denoted by a number corresponding to that ion.
  • the leading electrolyte, denoted by l is 0.01M Cl
  • the terminating ion, denoted by 7 is capronate.
  • Five ions are separated, 2 C10 3 oxalate, 4 tartrate, 5 citrate, 6 acetate.
  • the counter-ion is 0.0465M B-alanine, while peak in-FIG, 7a between sample components 5 and 6 is due to a contamination in the sample.
  • FIG. 8 Another'separation is shown as an example in FIG. 8.
  • the figure shows from top to bottom a curve from a spectrophotometrical detector at 254 nm and a curve from a thermal detector.
  • the system to be separated in the example of FIG. 8 is as leading electrolyte 0.01M (CH;,) NCI in methanol, saturated with sulfanilic acid and adjusted to pH 4.4 (as shown by an ordinary calomel-KCI electrode containing water) with (CH NOH.
  • 'Iemiinating electrolyte is 0.2M zinc acetate in methanol, and counter-ion is used sulfanilic acid.
  • FIG. 9 relates to a separation of the same ions as in the example of .FIG. 8, in a methanolic system.
  • the leading electrolyte is 0.0089M NaCl 0.0007M NaOF COCH in methanol, saturated with sulfanilic acid and with apH of 5.0 (as measured ,with an ordinary calomel-KCI electrodecontaining water).
  • the terminating electrolyte and the counter-ion are the same as in the example of FIG. 8.
  • the spectrophotometrical reading is made at 254 nm.
  • FIGS. 7a and 7b shows an isotachophoretical separation in a water. system
  • FIGS. 8 and 9 show separations in methanolic systems.
  • FIGS. and 7b also shows separation and detection according to the present invention, of anions
  • FIGS. 8 and 9 show separations and de: tection according to the present invention, of cations.
  • a counter-ioncould be chosen which is showing different molar absorptivities at acid and basic conditions at some wave-lengths within the UV- range and thus allow spectrophotometrical detection within the UV-range of substances which are not UV- absorbing
  • the method according to the present invention could be used aswell for spectrophotometrical detection of sample zones, some of which show absorbance.
  • the invention could often advantageously be used also for detection of sample zones after an isotachophoretical separationof the sample mixture, where each sample :component is showing absorbance atsome wave-length, vbut where the spectrophotometrical detection. according to the present invention, e. g. with still anothersuitably chosen wave-length, will give a considerably more clear-cut result.
  • the improvement which comprises the step of adding to the column an electrolyte containing counter-ions whose molar absorptivities within the spectral range employed differ in accordance with the pH values.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
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US432836A 1973-01-15 1974-01-14 Method at isotachophoretical separation to detect spectrophotometrically zone boundaries obtained Expired - Lifetime US3912609A (en)

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SE7300492A SE369279B (de) 1973-01-15 1973-01-15

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US (1) US3912609A (de)
JP (1) JPS49105598A (de)
DE (1) DE2401620C3 (de)
FR (1) FR2214126B1 (de)
GB (1) GB1459320A (de)
SE (1) SE369279B (de)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4139440A (en) * 1977-06-20 1979-02-13 Government Of The United States Electrofocusing in buffers
US4295949A (en) * 1979-05-25 1981-10-20 Olympus Optical Co., Ltd. Method for determining boundary points in electrophoresis
US4666577A (en) * 1985-02-07 1987-05-19 Olympus Optical Co., Ltd. Method of recording electrophoretic image pattern
US4666578A (en) * 1985-02-27 1987-05-19 Olympus Optical Co., Ltd. Method of measuring total protein of sample with the aid of electrophoretic image
EP0442315A1 (de) * 1990-01-29 1991-08-21 Waters Investments Limited Verfahren für die Trennung von konischen Arten mittels Kapillar-Elektrophorese
US8414754B1 (en) * 2006-05-31 2013-04-09 The Board Of Trustees Of The Leland Stanford Junior University Electrophoretic sample analysis and approach therefor
US10415030B2 (en) 2016-01-29 2019-09-17 Purigen Biosystems, Inc. Isotachophoresis for purification of nucleic acids
US11041150B2 (en) 2017-08-02 2021-06-22 Purigen Biosystems, Inc. Systems, devices, and methods for isotachophoresis

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2962426A (en) * 1958-09-09 1960-11-29 Cambridge Instr Company Inc Electrochemical method for analyzing materials
US3616456A (en) * 1968-05-10 1971-10-26 Lkb Produkter Ab Device for isoelectric separation of ampholytes
US3705845A (en) * 1970-06-02 1972-12-12 Lkb Produkter Ab Method in counterflow isotachophoresis

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2962426A (en) * 1958-09-09 1960-11-29 Cambridge Instr Company Inc Electrochemical method for analyzing materials
US3616456A (en) * 1968-05-10 1971-10-26 Lkb Produkter Ab Device for isoelectric separation of ampholytes
US3705845A (en) * 1970-06-02 1972-12-12 Lkb Produkter Ab Method in counterflow isotachophoresis

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4139440A (en) * 1977-06-20 1979-02-13 Government Of The United States Electrofocusing in buffers
US4295949A (en) * 1979-05-25 1981-10-20 Olympus Optical Co., Ltd. Method for determining boundary points in electrophoresis
US4666577A (en) * 1985-02-07 1987-05-19 Olympus Optical Co., Ltd. Method of recording electrophoretic image pattern
US4666578A (en) * 1985-02-27 1987-05-19 Olympus Optical Co., Ltd. Method of measuring total protein of sample with the aid of electrophoretic image
EP0442315A1 (de) * 1990-01-29 1991-08-21 Waters Investments Limited Verfahren für die Trennung von konischen Arten mittels Kapillar-Elektrophorese
US8414754B1 (en) * 2006-05-31 2013-04-09 The Board Of Trustees Of The Leland Stanford Junior University Electrophoretic sample analysis and approach therefor
US10415030B2 (en) 2016-01-29 2019-09-17 Purigen Biosystems, Inc. Isotachophoresis for purification of nucleic acids
US10822603B2 (en) 2016-01-29 2020-11-03 Purigen Biosystems, Inc. Isotachophoresis for purification of nucleic acids
US11674132B2 (en) 2016-01-29 2023-06-13 Purigen Biosystems, Inc. Isotachophoresis for purification of nucleic acids
US12006496B2 (en) 2016-01-29 2024-06-11 Purigen Biosystems, Inc. Isotachophoresis for purification of nucleic acids
US11041150B2 (en) 2017-08-02 2021-06-22 Purigen Biosystems, Inc. Systems, devices, and methods for isotachophoresis
US11987789B2 (en) 2017-08-02 2024-05-21 Purigen Biosystems, Inc. Systems, devices, and methods for isotachophoresis

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FR2214126B1 (de) 1980-01-04
DE2401620A1 (de) 1974-07-25
GB1459320A (en) 1976-12-22
SE369279B (de) 1974-08-19
DE2401620B2 (de) 1975-02-20
FR2214126A1 (de) 1974-08-09
DE2401620C3 (de) 1975-10-02
JPS49105598A (de) 1974-10-05

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