EP0721580A1 - Procede de detection de substances dans un liquide - Google Patents

Procede de detection de substances dans un liquide

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Publication number
EP0721580A1
EP0721580A1 EP94929070A EP94929070A EP0721580A1 EP 0721580 A1 EP0721580 A1 EP 0721580A1 EP 94929070 A EP94929070 A EP 94929070A EP 94929070 A EP94929070 A EP 94929070A EP 0721580 A1 EP0721580 A1 EP 0721580A1
Authority
EP
European Patent Office
Prior art keywords
refractive index
wavelength
wavelengths
chromatography
substances
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Withdrawn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
EP94929070A
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
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Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP0721580A1 publication Critical patent/EP0721580A1/fr
Withdrawn legal-status Critical Current

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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
    • G01N21/4133—Refractometers, e.g. differential
    • 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

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 ideal detector for liquid chromatography and capillary electrophoresis should (i) measure concentration and thereby be miniaturizable, (ii) exhibit a sufficiently low concentration detection limit ( ⁇ 1 ⁇ M) , (iii) be applicable on 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 presently most popular detection technique in connection with the above mentioned separation methods is UV or visual light absorption.
  • the absorbance though, 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 absorbance methods unsuitable for miniaturization. A consequence of this is that the technique shows poor sensitivity in miniaturized systems.
  • fluorescence measurement measures quantity rather than concentration and would therefore be unsuitable for miniaturization. Due to its very high mass 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 phenomena like stray light, background fluorescence, quenching and variations in the chemical matrix.
  • a third detection alternative is refractometry.
  • This technique is more universally applicable, and may be used 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, since the refractive index measurement is influenced by a number of disturbing phenomena, e.g. temperature and pressure variations. This reduces the attraction of refractometry in capillary electrophoresis and liquid chromatography.
  • the fact that refractometry is a universal (as opposed to selective) technique may be an advantage or a disadvantage in different applications.
  • An advantage of refractometry is the fact that the refractive index of a liquid depends on the concentration of dissolved substances. The technique is thus truly miniaturizable, and it may be applied also to narrow separation columns or capillaries. Further, refractometry is a fast technique, and refractometers are in general simple, robust, and inexpensive instruments.
  • the refractive index was measured at two wavelengths, corresponding to the refractive index maximum and minimum within the anomalous dispersion region of glucose, simultaneously, and the refractive index difference between these two wavelengths was taken as a measure of the concentration of glucose.
  • the advantage was that the measurement was made specific to glucose.
  • Measurement of anomalous dispersion is also described by A. Harming in the international patent application PCT/SE92/00558 as a means to increase the sensitivity of a type of surface plasmon resonance (SPR) and related assays based upon the measurement of chemical interactions on a sensing surface as changes of the refractive index of the surface layer. These changes are caused by the analyte involving or influencing the binding or release of a refractive index enhancing species to or from, respectively, the sensing surface.
  • SPR surface plasmon resonance
  • 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.
  • the index enhancing species may be selected 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.
  • A. Hanning describes a method of determining an analyte in a liquid chromatographic or capillary electrophoretic separation by measuring bulk refractive index, characterized in that the analyte is labeled with a species having a high refractive index at the or at least one measuring wavelength. Further, in the international patent application PCT/SE94/00762, A. Hanning describes a method of determining an analyte in a liquid chromatographic or capillary electrophoretic separation by measuring the displacement of a species in the mobile phase by analyte as a change in refractive index, characterized in that the displaced species have a high refractive index at the or at least one measuring wavelength.
  • the refractive index variation in the vicinity of an absorption peak may be approximated by the negative derivative of the absorptivity with respect to wavelength.
  • the refractive index shows a maximum in the vicinity of the maximum value of the derivative of the absorptivity with respect to wavelength (i.e. at a slightly higher wavelength than the resonance wavelength) , and a minimum in the vicinity of the minimum value of the negative derivative of the absorptivity with respect to wavelength (i.e. at a slightly lower wavelength than the resonance wavelength) .
  • the variation of refractive index with wavelength is called dispersion.
  • the wavelength region between the minimum and the maximum in refractive index is called the region of anomalous dispersion, while the wavelength region outside the anomalous region is called the region of normal dispersion.
  • the dispersion has different signs in the normal and anomalous regions, respectively.
  • a simplified description of the relation between absorption and refractive index, based on a local approximation, is given in C. F. Bohren, D. R. Huffman, "Absorption and scattering of light by small particles", Wiley, New York, 1983, Chapter 9. With this local approximation as a starting point, it may be shown that the refractive index difference between the maximum and the minimum is proportional to the maximum value of the absorption coefficient.
  • the present invention provides a method of determining the concentration of substances in a liquid, characterized in that the substances are first separated through liquid chromatography or capillary electrophoresis, and subsequently detected by monitoring the refractive index of the liquid flow at more than one wavelength, and the detection step comprises quantitative evaluation of the variation with wavelength of the refractive index.
  • the method puts no restrictions on the type of analytes, but the highest sensitivity is obtained for analytes having a high absorptivity close to the measurement wavelengths.
  • the method may be used in the infrared, visible, or ultraviolet wavelength regions, but the highest sensitivity is obtained if the measurement wavelengths are chosen in the vicinity of a region in which the analytes have a high absorptivity.
  • the method puts no restrictions on the chromatographic or electrophoretic separation mode that is utilized for the separation.
  • Separation modes include, but are not restricted to, reversed-phase chromatography, adsorption chromatography, ion chromatography, ion pairing chromatography, size exclusion chromatography, affinity chromatography, capillary zone electrophoresis, capillary ion electrophoresis, micellar electrokinetic capillary chromatography (MECC) , isotachophoresis, capillary gel electrophoresis, and capillary isoelectric focusing.
  • MECC micellar electrokinetic capillary chromatography
  • isotachophoresis capillary gel electrophoresis
  • capillary isoelectric focusing Other separation modes conceivable for the purposes of the invention will be apparent to the skilled person.
  • the method puts no restrictions on the type of refractometer used for the measurement.
  • Refractometer types include, but are not restricted to, deflection refractometers, interferometers, Fresnel refractometers, surface plasmon resonance refractometers, and optical waveguide refractometers.
  • Other refractometers conceivable for the purposes of the invention will be apparent to the skilled person.
  • van Heuvelen does not realize that it is practically impossible to determine the concentration of one single substance in a complex mixture of several other, identified or unidentified, substances simply by measuring the refractive index difference at two wavelengths.
  • van Heuvelen utilizes two specific wavelengths in order to determine one specific substance, so the wavelengths are specific to that substance.
  • two specific wavelengths would have to be identified for each substance, which is both theoretically and practically impossible.
  • two, or more, fixed wavelengths are utilized, so the wavelengths are not specific to any single substance. All the eluting substances will be detected, although with varying sensitivities (some substances may even show close to zero sensitivity) .
  • van Heuvelen is explicitly limited to the region of anomalous dispersion
  • van Heuvelen's method is based on the fact that the dispersion has different signs in the normal and anomalous regions, respectively.
  • the present concept is not limited to the anomalous region, and does not put any demands on the sign of the dispersion. It is the magnitude, whether positive or negative, of the dispersion that is monitored, so the present method may be applied in the normal region as well as in the anomalous region.
  • the present inventive concept constitutes a significantly improved method.
  • Gauglitz measures the absolute value of the refractive index at several wavelengths simultaneously, but he does not at all utilize, or even mention the possibility to utilize, the dispersion (in the wide meaning variation of refractive index with wavelength, like e.g. difference, differential, or derivative of the refractive index with respect to wavelength) for quantitative purposes.
  • the dispersion in the wide meaning variation of refractive index with wavelength, like e.g. difference, differential, or derivative of the refractive index with respect to wavelength.
  • the use of the dispersion for quantitative purposes is not even implicitly apparent to Gauglitz.
  • the present method does not utilize labeling of the analyte.
  • the present method does not utilize detection of a substance that is being displaced by the analyte.
  • the variation of the refractive index with wavelength is related to the concentration of analyte in the liquid.
  • the refractive index is monitored at two different wavelengths, and the measured refractive index difference is related to the concentration of analyte in the liquid.
  • the refractive index difference is linearly related to the concentration, as stated in equation (1) above.
  • the refractive index is continuously monitored at more than two discrete wavelengths, or in a continuous wavelength interval.
  • the quantification may again be performed as a simple difference between two values, or the quantification procedure may comprise e.g.
  • the differentiation, derivatization, or integration of the obtained refractive index spectrum, or the quantification procedure may include any of the common techniques, including multivariate techniques, for quantitative evaluation of spectra, like e.g. principal component analysis (PCA) , partial least squares (PLS) and factor analysis.
  • PCA principal component analysis
  • PLS partial least squares
  • the information concerning the variation of the refractive index with wavelength may be used to determine the structure of or to identify the analytes.
  • the refractive index is measured at two wavelengths, and the information concerning the sign and magnitude of the dispersion is used for qualitative analysis. This is analogous to measurement of the absorption at a single wavelength, which also gives a limited structural information.
  • the refractive index is continuously monitored at more than two discrete wavelengths, or in a continuous wavelength interval.
  • the spectral information may also in this case be used to determine the structure of or to identify the analytes. A large number of well-established methods for the evaluation of spectra may be used for the qualitative analysis in this case, e.g.
  • a lamp with several emission lines, or with a broad emission spectrum in combination with a monochromator (sometimes termed a polychromator, since all the different wavelengths are allowed to pass through the sample) and a diode array or CCD detector.
  • a monochromator sometimes termed a polychromator, since all the different wavelengths are allowed to pass through the sample
  • CCD detector diode array
  • the present inventive concept has a number of advantages as compared to conventional single wavelength refractometry.
  • a "snapshot” instead of measuring absolute refractive index, the influence of the above mentioned sources of noise, like e.g. temperature and pressure variations, and mechanical vibrations, will decrease, leading to increased signal-to- noise ratio and improved sensitivity.
  • Measurement at several wavelengths gives spectral information, that may be used for purposes of qualitative analysis.
  • the degree of universal versus selective detection may be controlled through the choice of measurement wavelengths.
  • all the advantages of conventional refractometry remain: miniaturizability, rapidity, simplicity, robustness, and low cost.
  • Conventional single wavelength refractometry may also be performed with a refractometer designed for multi- wavelength measurements. Single wavelength and multi- wavelength measurements may thus be performed simultaneously in one single detection cell.
  • Fig. 1 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.
  • Both lasers l and 2 were of diode type with collimating optics.
  • Laser l 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 & Hoyer) and was driven by a second voltage unit, Diode Laser DL 25 Control Unit (Spindler & Hoyer) .
  • the two lasers 1, 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 that 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) , driven by a Panasonic Power Supply WV- CD52, was mounted at the other end of plate 9 at a distance of about 0.8 m (varying a little between the test series to be described) from prism cuvette 3 to be vertically adjustable by a micrometer screw. A bent black steel hood was placed over the CCD camera to screen stray light. The CCD camera picture was projected to TV screen 5 (Electrohome 10"), to which was taped a transparent cross- ruled pattern (OH film with a 1.4 X enlarged millimeter paper copied on it) . The transparent cross-ruled pattern was used for measuring distances on the TV screen 5 by counting squares in the pattern.
  • the lasers 1 and 2 were adjusted laterally and vertically and the brass tube laterally such that nice and symmetrical light pictures (1-1.5 mm spots) of the two respective laser beams were obtained above each other on the TV screen.
  • a low laser intensity, a high contrast on the screen and an exposure time of 1/4 second were used. Then the brightness was adjusted until a sharp picture for the eye was obtained, and finally the diaphragm was adjusted until a sharp picture was obtained on the photograph.
  • the position of the spots was determined by taking a photograph of the TV screen 5, and then counting the checks in a microscope to determine the positions of the left and right edges, respectively, of the spots, and the center of each spot was assumed to be halfway between them.
  • Liquid handling system
  • the liquid handling system used is shown in Fig. 2 and consisted of a pump 11 of peristaltic type (Pi, Pharmacia LKB Biotechnology AB, Uppsala, Sweden) .
  • the pump 11 was connected, on one hand, to a sample reservoir 12 via a tube 13, and, on the other hand, to a manual turn valve 14 via a tube 15.
  • a tube 16 connected valve 14 with a drain.
  • a tube 19 connected cuvette 3 with a cuvette drain.
  • Manual valve 14 permitted liquid to be pumped either to the drain or to cuvette 3.
  • Tween® 20 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
  • the refractive index spectrum for 1 mM 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 spectrographically 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.

Abstract

Procédé d'évaluation de la concentration de substances dans un liquide consistant tout d'abord à les isoler par chromatographie en phase liquide ou par électrophorèse capillaire, puis à les détecter en surveillant l'indice de réfraction du courant liquide sur plus d'une longueur d'onde, l'étape de détection comportant l'évaluation quantitative des variations de l'indice de réfraction en fonction des longueurs d'onde. Ladite évaluation quantitative peut se faire par le calcul de la différence, de la différentielle ou de la dérivée de l'indice de réfraction en fonction de la longueur d'onde, ou par intégration, analyse des principaux composants, méthode des moindres carrés partiels, analyse factorielle ou lissage des courbes.
EP94929070A 1993-09-28 1994-09-26 Procede de detection de substances dans un liquide Withdrawn EP0721580A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
SE9303149A SE503289C2 (sv) 1993-09-28 1993-09-28 Sätt att bestämma koncentrationen av ämnen i en vätska
SE9303149 1993-09-28
PCT/SE1994/000887 WO1995009355A1 (fr) 1993-09-28 1994-09-26 Procede de detection de substances dans un liquide

Publications (1)

Publication Number Publication Date
EP0721580A1 true EP0721580A1 (fr) 1996-07-17

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EP94929070A Withdrawn EP0721580A1 (fr) 1993-09-28 1994-09-26 Procede de detection de substances dans un liquide

Country Status (5)

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EP (1) EP0721580A1 (fr)
JP (1) JPH09503064A (fr)
AU (1) AU7825994A (fr)
SE (1) SE503289C2 (fr)
WO (1) WO1995009355A1 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19540456C2 (de) * 1995-10-30 1997-10-09 Buschmann Johannes Verfahren zur Messung der Glukosekonzentration in einer Flüssigkeit und Verwendung des Verfahrens
US5870185A (en) * 1996-10-21 1999-02-09 C.F.C. Technology, Inc. Apparatus and method for fluid analysis
US7200494B2 (en) 2001-10-30 2007-04-03 Hitachi, Ltd. Method and apparatus for chromatographic data processing
US6748333B1 (en) 1999-09-27 2004-06-08 Hitachi, Ltd. Method and apparatus for chromatographic data processing, and chromatograph
JP2004016609A (ja) 2002-06-19 2004-01-22 Omron Healthcare Co Ltd 体液成分濃度測定方法及び体液成分濃度測定装置
GB0415882D0 (en) * 2004-07-15 2004-08-18 Univ Southampton Optical sensors
US10156520B2 (en) * 2013-06-07 2018-12-18 Malvern Panalytical Limited Array based sample characterization
CN112147098B (zh) * 2020-11-03 2024-01-16 安徽大学 一种基于反常色散效应的气体种类与浓度检测系统
CN117214125B (zh) * 2023-11-09 2024-01-26 南京盛略科技有限公司 一种基于检测光纤的液体成分检测系统及方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL66127A (en) * 1982-06-24 1987-11-30 Israel State Method and apparatus for measuring the index of refraction of fluids
US4704029A (en) * 1985-12-26 1987-11-03 Research Corporation Blood glucose monitor

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9509355A1 *

Also Published As

Publication number Publication date
SE9303149D0 (sv) 1993-09-28
SE503289C2 (sv) 1996-05-13
AU7825994A (en) 1995-04-18
WO1995009355A1 (fr) 1995-04-06
JPH09503064A (ja) 1997-03-25
SE9303149L (sv) 1995-03-29

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