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EP0681694A1 - Procede ameliore de detection de l'indice de refraction dans un volume de liquide - Google Patents

Procede ameliore de detection de l'indice de refraction dans un volume de liquide

Info

Publication number
EP0681694A1
EP0681694A1 EP94905892A EP94905892A EP0681694A1 EP 0681694 A1 EP0681694 A1 EP 0681694A1 EP 94905892 A EP94905892 A EP 94905892A EP 94905892 A EP94905892 A EP 94905892A EP 0681694 A1 EP0681694 A1 EP 0681694A1
Authority
EP
European Patent Office
Prior art keywords
refractive index
wavelength
maximum
measurement
measuring
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.)
Withdrawn
Application number
EP94905892A
Other languages
German (de)
English (en)
Inventor
Anders Hanning
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP0681694A1 publication Critical patent/EP0681694A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N2030/621—Detectors specially adapted therefor signal-to-noise ratio
    • G01N2030/625—Detectors specially adapted therefor signal-to-noise ratio by measuring reference material, e.g. carrier without sample
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/84—Preparation of the fraction to be distributed
    • G01N2030/8429—Preparation of the fraction to be distributed adding modificating material
    • G01N2030/8441—Preparation of the fraction to be distributed adding modificating material to modify physical properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
    • G01N21/4133—Refractometers, e.g. differential

Definitions

  • the present invention relates to refractometric detection of a substance or substances in a liquid chromatographic or capillary electrophoretic separation.
  • Capillary electrophoresis and liquid chromatography are modern and well-established separation methods with very high performance.
  • the presently most popular detection technique in such separations is UV or visual absorption.
  • An apparent disadvantage of this technique is the poor sensitivity.
  • the absorbance is proportional to the amount of absorbing substance in the path of the light ray rather than to the concentration of the absorbing substance which makes the method unsuitable for miniaturization.
  • fluorescence measurement measures quantity rather than concentration and would therefore be unsuitable for miniaturization. Due to its very high sensitivity, the fluorescence method is, however, in practice miniaturizable. On the other hand, the instrumentation is complicated and expensive and the method is also sensitive to disturbing phenomenons like stray light, background fluorescence, quenching and chemical matrix.
  • Still another and generally applicable alternative is refractometry, such bulk refractive index measurement being applicable also when the substances of interest neither absorb nor fluoresce.
  • the sensitivity and signal to noise ratio of presently available refractometric detectors are, however, not satisfactory, which reduces the attraction of refractometry in capillary electrophoresis and liquid chromatography.
  • the ideal detector for liquid chromatography and capillary electrophoresis should (i) measure concentration and thereby be miniaturizable, (ii) exhibit a sufficiently low concentration detection limit ( ⁇ l ⁇ M) , (iii) be applicable to a column to avoid zone broadening in couplings or special detection cells, (iv) be fast so that the time constant of the detection will not cause a reduced resolution, and (v) be simple, robust and inexpensive.
  • the refractive index of a substance varies with wavelength throughout the electromagnetic spectrum, this variation being called refractive index dispersion, or simply dispersion. The latter is intimately related to the degree to which radiation is absorbed.
  • the refractive index slowly decreases with increasing wavelength (normal dispersion) .
  • the refractive index varies heavily with wavelength, a phenomenon called anomalous dispersion.
  • the refractive index is roughly a function of the negative derivative of the absorptivity (extinction coefficient) with respect to wavelength.
  • the refractive index reaches a maximum, i.e. where the negative derivative of the absorptivity has its maximum, and at a slightly lower wavelength than the resonance wavelength the refractive index reaches a minimum.
  • the sensitivity is increased by matching the measurement wavelength with the absorptivity maximum of the refractive index enhancing species used in the particular assays, preferably a dye or chromophoric molecule, and specifically such that the measurement wavelength substantially corresponds to the maximum of the negative derivative of the absorptivity with respect to the wavelength. This may be accomplished either by selecting the index enhancing species to conform with the measuring wavelength of a particular instrument or application, or by selecting the measuring wavelength to conform with a specific index enhancing species.
  • the present invention therefore provides a method of determining an analyte in a liquid chromatographic or capillary electrophoretic flow by measuring bulk refractive index, which method is characterized in that the analyte is labeled with a species having a high refractive index at the or at least one measuring wavelength.
  • the method puts no restrictions on the analyte to be determined as long as it can be provided with a label as defined above.
  • refractometry techniques may be broadly divided into (i) transmission techniques, and (ii) reflection techniques.
  • Transmission based refractometers include (i) refractometers utilizing deflection techniques and (ii) refractometers utilizing interferometric techniques.
  • a deflection cell is a simple prism through which a ray of light is directed. For a homogeneous liquid, the deflection of the light ray is dependent on the difference in refractive index between the liquid and the cell wall.
  • An advantage of the prism technique is that the deflection of the ray, for a constant refractive index of the liquid, is only dependent on the top angle of the prism and not on the optical path through the cell.
  • the prism cell can therefore, in principle, be miniaturized to any desired size without any loss of sensitivity.
  • the sensitivity can be doubled through back reflection through the prism cell.
  • Interferometric techniques measure the difference in optical path, caused by different refractive indices, between a sample cell and a reference cell. This difference in optical paths is proportional to the total optical path, and relatively long detector cells will therefore be required for adequate precision to be obtained.
  • the instrumentation is rather complex, including a polarizer, a beam splitter, a beam recombiner and a phase analyzer.
  • Reflection techniques measure the difference in refractive index between two materials, i.e. the liquid in the cell and the cell wall, at a reflecting interface.
  • reflection-based refractometers which may make them less attractive for the present purposes, is the fact that wall effects such as contamination or preferential adsorption can strongly influence the signal so that the detected refractive index may not always be representative of the bulk properties. Radial variations of concentration may also be caused by Joule heating effects in a capillary electrophoretic column.
  • Exemplary of reflection-based refractometers are Fresnel detectors and SPR detectors.
  • the Fresnel detector measures the refractive index as a change in intensity of reflected or transmitted light at a dielectric interface due to the change in reflectivity or transmittance caused by a refractive index change in the liquid. Since the reflectivity is independent of the cell length, the Fresnel detector may, in principle, be miniaturized.
  • the SPR detector is based upon the phenomenon that SPR causes the intensity of a reflected light ray to show a distinct minimum at a certain angle, the determination of SPR therefore involving a position measurement (or a relative intensity measurement) .
  • a position measurement or a relative intensity measurement
  • SPR For a more detailed description of SPR and its application in analytical contexts it may, for example, be referred to WO 90/05295 and WO 90/05305.
  • One drawback of SPR in the present context is that it primarily is a surface technique, the total measurement depth from the surface being about 1 ⁇ m. Surface contamination and preferential adsorption may therefore influence the signal to a considerable degree.
  • the SPR detector further requires very high wavelength stability and reproducibility of the light source, since the minimum angle to be determined does not depend only on the refractive index but also on the wavelength per se.
  • the above mentioned refractometers are, of course, only examples, and other refractometers conceivable for the purposes of the invention will be apparent to the skilled person. It is readily understood that the above described refractometer types may conveniently be applied to flat columns or capillaries.
  • the refractive index inside round columns or capillaries may e.g. be measured by analyzing the interference pattern generated by a laser beam, as described by A. E. Bruno et al. , Anal.
  • the labelling species preferably is or includes a dye or chromophoric molecule.
  • Derivatization techniques for labeling molecules with chromophores are well established. Such techniques are e.g. used to label molecules with fluorophores in connection with fluorescence detection (e.g. as described by Y. Ohkura and H. Nohta in "Advances in Chromatography", Volume 29, J.C. Giddings, E. Grushka, P.R. Brown (Eds.), Marcel Dekker, New York, 1989, Chap. 5) .
  • the dyes used for refractive index labeling need not, of course, be fluorescent, so in principle any dye that can be attached to an analyte molecule by a chemical bond may be used.
  • Exemplary dyes are of the azine, thiazine, oxazine, cyanine, merocyanine, styryl, triphenylmethane, chlorophyll and phthalocyanine types.
  • the measurement is performed at a single wavelength at or near the refractive index maximum, i.e. at or near the maximum of the negative derivative of the absorptivity with respect to wavelength of the labelling species.
  • the measurement should thus be performed at, or as close as possible to the maximum of the negative derivative of the absorptivity with respect to wavelength.
  • the distance between the measurement wavelength and said maximum should preferably be less than 100 nm (corresponding to a possible enhancement of at least about 5 times, on a mass basis, depending on the absorptivity) , and more preferably less than 50 nm (corresponding to a possible enhancement of at least about 10 times, on a mass basis, depending on the absorptivity) .
  • the measurement wavelength is chosen on the low wavelength side of the maximum of the negative derivative of the absorptivity with respect to wavelength, the measurement wavelength must be very close to said maximum, since the refractive index again decreases when the wavelength of the absorptivity maximum is approached.
  • the labeling species should have a high refractive index, the absorptivity (extinction coefficient) of the analyte labeling species should in this case be as high as possible, preferably higher than about 20 lg "1 cm “1 , more preferably higher than about 50 lg "1 cm “1 , and especially higher than about 100 lg "1 cm “1 .
  • the measurement comprises determining the refractive index variation of the label with wavelength for a number of discrete wavelengths or for a continuous range of wavelengths, this variation being representative of the concentration of the labelled species.
  • the measurement is performed as a differential measurement at two or more wavelengths.
  • the different measurements at the respective wavelengths will have to be performed substantially simultaneously or in a rapid succession.
  • one measuring wavelength is preferably selected (as in the case of the single wavelength measurement described above) at or near the refractive index maximum, i.e. at or near the maximum of the negative derivative of the absorptivity with respect to wavelength of the labelling species.
  • the other measuring wavelength should preferably be at or near the refractive index minimum plateau (in the anomalous region of the dispersion curve, i.e. refractive index vs. wavelength, the dispersion curve exhibits a minimum plateau rather than a defined dip) , or stated otherwise, in the vicinity of the maximum of the derivative of the absorptivity with respect to wavelength of the labeling species.
  • Measuring at more than two discrete wavelengths will provide more information about the dispersion, and thereby a more robust interpretation of the detected signal, and the noise may be reduced by averaging the measurement results obtained.
  • the determined refractive index variation may be based upon measurement of the area under the spectrum graph rather than on the difference between the refractive indices at pairs of discrete wavelengths.
  • the absorptivity (extinction coefficient) of the analyte labeling species should, however, preferably be higher than about 10 lg "1 cm “1 , and more preferably higher than about 20 lg "1 cm “1 .
  • the sensitivity enhancement will not be quite as large as if the low refractive index measurement is made near the refractive index minimum, but the noise reduction and the increased selectivity will still be obtained. It is to be noted that when using this differential measuring embodiment, only one detector cell will be required for all mentioned refractometers except the interferometer.
  • the measuring signal will be independent of the size of the detection volume, and the detection volume may thus, in principle, be miniaturized to the extent desired. It will therefore, for example, readily permit on-column detection.
  • the method of the present invention will have a substantially increased sensitivity.
  • Using the differential mode will also reduce noise due to variations in temperature, pressure, flow, etc., and make the measured signal specific with respect to the labeled molecules. In the latter case no reference flow or cell will be required as the dual wavelength measurement is self-compensating as has been described above.
  • a classical refractive index monitoring may be performed at a single wavelength, detecting all species, including those which do not absorb or fluoresce or are electrochemically active.
  • a dual wavelength measurement of dye-labelled analytes may be made. This will give a selective monitoring of labelled analytes and a substantially increased sensitivity. A universal and a selective monitoring may thus be made simultaneously in one and the same cell.
  • Fig. l is a schematic diagram showing the experimental set-up used in the Example
  • Fig. 2 is a schematic diagram showing the liquid handling system used in the Example
  • Fig. 3 is a diagram showing the refractive index spectrum for the dye HITC used in the Example.
  • Fig. 4 is a plot of laser spot distances vs. HITC concentrations.
  • Fig. 1 An experimental differential refractometer instrument was constructed as schematically illustrated in Fig. 1. This instrument consisted of two lasers 1 and 2, respectively, a prism-shaped flow cuvette 3, a CCD camera 4, a TV screen 5 and a Polaroid® camera 6.
  • Both lasers l and 2 were of diode type with collimating optics.
  • Laser 1 had a wavelength of 660 nm (more precisely 658.5 nm) (Melles-Griot) and was driven by a voltage unit (Mascot Electronics Type 719) .
  • the other laser 2 had a wavelength of 780 nm (Spindler s_ Hoyer) and was driven by a second voltage unit, Diode Laser DL 25 Control Unit (Spindler ⁇ . Hoyer) .
  • the two lasers l, 2 were mounted at right angles to each other on a steel plate 7.
  • a blackened brass tube (not shown; inner diameter 20 mm) was fastened to plate 7.
  • the brass tube had slits in which a short wavelength pass filter 8 (Melles-Griot) having a cut- off at about 700 nm was mounted at an angle of 45° to the beam directions.
  • This filter 8 transmitted the 660 nm beam of laser l but reflected the 780 nm beam of laser 2 with the resulting effect that the two beams were made to coincide.
  • An aperture of l mm diameter served as exit slit.
  • Flow cuvette 3 a commercial dual prism cell cuvette (consisting of two 45° prism cells, 1.5 x 7 mm, 8 ⁇ l volume, with their hypotenuses applied against each other) for a liquid chromatography refractive index detector (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) , was then screwed to the outside of the brass tube in connection to the exit slit thereof. Only one of the two prism cells of cuvette 3 was used and connected by tubes (not shown) to a simple liquid handling system which will be described below (the other cell remained empty) . Steel plate 7 was turnably mounted to an aluminium plate 9 at one end thereof by a screw bolt 10.
  • CCD camera 4 Panasonic WV-CD50
  • a Panasonic Power Supply WV- CD52 was mounted at the other end of plate 9 at a distance
  • a citrate buffer (pH 3, 0.1 M citrate, 0.4 M NaCl, 0.05% Tween® 20) was prepared by dissolving 21 g of citric acid (M & B p.a.) and 23 g of NaCl (Merck p.a.) in 1000 ml of purified water. 5 ml of Tween® 20 (Calbiochem 655206, 10%, protein grade) were added. 4 M NaOH (p.a.) was added to adjust the pH from 1.95 to 3.00, and the mixture was filtered through a 0.22 ⁇ m filter. 500 ml of the citrate buffer were then mixed with 500 ml of spectrographically pure ethanol and homogenized with ultrasonic sound for a couple of minutes.
  • a 500 ⁇ M stock solution of the dye HITC (1, 1' , 3, 3, 3 ' , 3 ' -hexamethylindotricarbocyanine) was then prepared by mixing 14 mg of HITC iodide (Sigma H0387, 94% purity, M ⁇ 537 g/mol,; Sigma Chemical Co., St. Louis, Mo., U.S.A.) with 50 ml of the above prepared citrate/ethanol buffer. After homogenization with ultrasonic sound for a couple of minutes, the mixture was filtered through a 0.45 ⁇ m filter.
  • the refractive index spectrum for l M HITC is shown in Fig. 3 (solid line: theoretically calculated curve, crosses: experimental data) .
  • the measuring wavelength 780 nm (laser 2) is in the peak region of the refractive index on the high wavelength side thereof, whereas the second measuring wavelength 660 nm is on the refractive index minimum plateau.
  • the positioning of the liquid-filled cuvette cell (3) was adjusted by filling the cell with ethanol/water 50/50 (purified water and spectrophotographically pure ethanol) by means of a syringe and turning the steel plate (7) until the two laser light beams were centered above each other on the CCD camera.
  • the distance between the cuvette and the CCD camera was 73 cm.
  • Refractive index measurements were then performed for the different HITC concentrations described above, the liquid handling being carried out as described above under "Liquid handling system" .
  • the laser intensity, TV brightness and camera exposure and diaphragm settings were adjusted for each different dye concentration to obtain sharp pictures of the light spots.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Molecular Biology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

L'invention concerne un procédé de détermination d'un analyte dans une séparation électrophorétique capillaire ou chromatographique de liquides qui consiste à mesurer l'indice de réfraction dans un volume de liquide, l'analyte étant marqué avec une espèce ayant un indice de réfraction élevé au niveau d'au moins la ou une longueur d'onde de mesure.
EP94905892A 1993-01-27 1994-01-21 Procede ameliore de detection de l'indice de refraction dans un volume de liquide Withdrawn EP0681694A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
SE9300231 1993-01-27
SE9300231A SE500817C2 (sv) 1993-01-27 1993-01-27 Sätt att bestämma koncentrationen av en analyt i ett vätskekromatografiskt eller kapillärelektroforetiskt flöde genom att mäta vätskans brytningsindex
PCT/SE1994/000045 WO1994017393A1 (fr) 1993-01-27 1994-01-21 Procede ameliore de detection de l'indice de refraction dans un volume de liquide

Publications (1)

Publication Number Publication Date
EP0681694A1 true EP0681694A1 (fr) 1995-11-15

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ID=20388665

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Application Number Title Priority Date Filing Date
EP94905892A Withdrawn EP0681694A1 (fr) 1993-01-27 1994-01-21 Procede ameliore de detection de l'indice de refraction dans un volume de liquide

Country Status (4)

Country Link
EP (1) EP0681694A1 (fr)
AU (1) AU5981794A (fr)
SE (1) SE500817C2 (fr)
WO (1) WO1994017393A1 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE9500356D0 (sv) * 1995-02-01 1995-02-01 Anders Hanning Sätt och anordning för optisk karakterisering av vätskor
SE9602788L (sv) * 1996-07-16 1998-01-17 Anders Hanning Förbättrad refraktometrisk metod
DE102009033426A1 (de) * 2009-07-16 2011-02-03 Technische Universität München Elektrophorese-Messvorrichtung und Messverfahren
CN101782515B (zh) * 2010-03-05 2011-07-20 陕西师范大学 基于全反射光阑效应的液体折射率测量方法
CN101776572B (zh) * 2010-03-05 2011-07-20 陕西师范大学 液体折射率ccd实时测量装置及其测量方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4213699A (en) * 1976-02-27 1980-07-22 S.A. Texaco Belgium N.V. Method of measuring low concentrations of a light absorbing component
US4381895A (en) * 1980-02-28 1983-05-03 Biovation, Inc. Method and apparatus for automatic flow-through digital refractometer
US4704029A (en) * 1985-12-26 1987-11-03 Research Corporation Blood glucose monitor
US4952055A (en) * 1988-10-03 1990-08-28 Wyatt Technology Corporation Differential refractometer
SE9200917D0 (sv) * 1991-08-20 1992-03-25 Pharmacia Biosensor Ab Assay method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9417393A1 *

Also Published As

Publication number Publication date
SE9300231L (sv) 1994-07-28
SE9300231D0 (sv) 1993-01-27
AU5981794A (en) 1994-08-15
WO1994017393A1 (fr) 1994-08-04
SE500817C2 (sv) 1994-09-12

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