WO1995006239A1

WO1995006239A1 - An improved method of detecting substances in a liquid

Info

Publication number: WO1995006239A1
Application number: PCT/SE1994/000762
Authority: WO
Inventors: Anders Hanning
Original assignee: Anders Hanning
Priority date: 1993-08-24
Filing date: 1994-08-22
Publication date: 1995-03-02
Also published as: AU7548894A; SE501663C2; EP0715712A1; SE9302723D0; SE9302723L

Abstract

In 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, the displaced species have a high refractive index at the or at least one measuring wavelength.

Description

AN IMPROVED METHOD OF DETECTING SUBSTANCES IN A LIQUID

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. Detection methods for these separation methods are based on measurement of some physical property, which property differs between the analyte and the mobile phase or the carrier electrolyte. (The term "mobile phase" is strictly correct only in connection with liquid chromatography, while the corresponding strictly correct term in connection with capillary electrophoresis is "carrier electrolyte", but hereinafter "mobile phase" is used as a common term for both these cases.) Such properties may e.g. be absorptivity, fluorescence, or electrochemical activity.

The most common detection mode is direct detection. In this mode, the measured signal originates from the analyte per εe, while the contribution from the mobile phase is assumed to be negligible, or at least constant. Examples are measurement of the absorbance of the analyte at a wavelength where the mobile phase does not absorb and measurement of the fluorescence of a fluorescent analyte in a non-fluorescent mobile phase. In one variation of the direct detection mode, the analyte may be subject to derivatization, i.e. labeled with a molecule ("label") possessing the desired physical property. One example is labeling of a non-fluorescent analyte with a fluorescent label, followed by measurement of the fluorescence of the labeled species.

An alternate detection mode is indirect detection. The key to this mode is that the analyte displaces a species in the mobile phase in the eluted band. The displacement may be based on mechanisms such as conservation of charge, ion pairing, or volume displacement, or on any mechanism by which the injected analytes compete with or perturb the concentration of a species in the mobile phase. The measured signal is derived from the displaced species rather than from the analyte itself. The displacement causes a change in the signal because the concentration of the displaced species is lower in the eluted bands when compared with its steady-state concentration. One example is the displacement of coloured mobile phase additive ions by colourless analyte ions in ion chromatography. This displacement may be monitored by measuring the absorption of the eluting liquid at some suitable wavelength. Indirect detection methods are well established, and are described e.g. by E. S. Yeung and W. G. Kuhr in Analytical Chemistry 1991, 63(5), 275A.

Indirect detection methods are most commonly used in the separation and determination of ions, i.e. in ion capillary electrophoresis and ion chromatography. However, indirect detection may also be used for the determination of non-ionic species and in connection with other separation modes, like e.g. reversed-phase chromatography and micellar electrokinetic capillary chromatography. Indirect detection has some specific advantages as compared to direct detection. Indirect detection offers universal detection for all analytes sharing the same displacement mode, and in some cases there may even exist a single calibration curve for that group of analytes.

Further, there is no need for derivatization in order to convert the analyte of interest into a species that gives a response at the detector. Because the analytes are not chemically altered, collection and further studies are facilitated. On the other hand, the sensitivity of indirect methods is in general not quite as high as that of the corresponding direct methods.

The ideal detector for liquid chromatography and capillary electrophoresis, for example, should (i) measure concentration and thereby be miniaturizable, (ii) exhibit a sufficiently low concentration detection limit (<l μM) , (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. For a detector working in the indirect mode, the detection limit depends on inter alia the dynamic reserve, which is the ability to measure a small change on top of a large signal, and is equal to the signal-to-noise ratio of the background signal.

Indirect detection may be used in conjunction with measurement of light absorption. One example of this is given by G. Bondoux, P. Jandik and . R. Jones in Journal of Chromatography 1992, 602, 79. An apparent disadvantage of the absorption technique, however, is the poor sensitivity. Further, 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.

Indirect detection may also be used in conjunction with fluorescence measurement. Also this technique measures quantity rather than concentration and would therefore, in principle, 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 phenomena like stray light, background fluorescence, quenching and chemical matrix. Miniaturized indirect fluorescence detection most often utilizes laser excitation, and severe demands are put on the intensity stability of the laser in order to obtain a satisfactory dynamic reserve.

Still another detection technique is refractometry. However, since all substances possess a certain refractive index, the contribution from the analyte and from the mobile phase, respectively, can not be distinguished, but rather the weighted sum of the different contributions gives rise to the measured refractive index. Consequently, there can be no distinction between direct and indirect detection in conjunction with classical refractometry, and therefore the use of indirect refractometric detection has never been reported. Moreover, the sensitivity and signal to noise ratio of presently available refractometric techniques are not satisfactory, which reduces the attraction of refractometry in capillary electrophoresis and liquid chromatography. The noise is to a large extent caused by temporal variations of the temperature, pressure, and composition of the eluting liquid.

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. In regions of high transparency, the refractive index slowly decreases with increasing wavelength (normal dispersion) . In the vicinity of high absorbance, i.e. at resonance wavelengths, however, the refractive index varies heavily with wavelength, a phenomenon called anomalous dispersion. In this region the refractive index is roughly a function of the negative derivative of the absorptivity (extinction coefficient) with respect to wavelength. Thus, at a slightly higher wavelength than the resonance 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 has a minimum. The relation between the absorptivity and the refractive index of a substance is described in a more stringent way by the Kramers-Kronig equations. Measurement of the anomalous part of the dispersion in interference refractometry to obtain more spectral information and increase the signal to noise ratio is described by Gauglitz G. et al., Anal. Chem. 1988, 60, 2609. Measurement of anomalous dispersion is also described 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. More particularly, 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.

A method of determining an analyte in a fluid by measuring bulk refractive index, in which the analyte is labeled with a species having a high refractive index at the or at least one measuring wavelength, is described in copending Swedish patent application 9300231-9. More particular, the labelling species has a high variation of the refractive index with wavelength, and measurement of this variation, e.g. as the difference in refractive index at two different wavelengths, provides a means of determining the concentration of the labeled species. Since this differential refractive index originates specifically from the labeling species, while the contribution from the mobile phase to the differential refractive index is negligible (or at least constant) , this patent application describes a direct refractometric detection method.

In accordance with the present invention, it has now been found that the basic physical principles of the detection methods in the above-mentioned Swedish application, relating to the direct refractometric detection of labeled analytes, also may be favourably applied to indirect detection. In accordance with the invention, this is accomplished by using a mobile phase containing a species that is displaced by the analytes in the eluted bands, which species have a high refractive index, like e.g. a dye. By utilizing the heavy dependence of the displaced species' refractive index on the wavelength in the anomalous dispersion region, which dependence has been generally described by Gauglitz G. et al., supra, for absorbing substances in liquid solutions, the performance of refractometric detectors, and especially the sensitivity, the selectivity, and the noise level, may be improved to a considerable degree.

In its broadest aspect the present invention therefore provides a method of determining an analyte in a liquid chromatographic or capillary electrophoretic separation by measuring refractive index, which method is characterized in that the analyte displaces a species in the mobile phase, said species having a high refractive index at the or at least one measuring wavelength.

The method puts no restrictions on the chromatographic or electrophoretic separation mode that is utilized, as long as the analyte is able to influence the concentration, i.e. displace, a species in the mobile phase. Separation modes include, but are not restricted to, ion capillary electrophoresis, capillary zone electrophoresis, micellar electrokinetic capillary chromatography, isotachophoresis, ion chromatography, ion pairing chromatography, and reversed-phase chromatography. Other separation modes conceivable for the purposes of the invention will be apparent to the skilled person. The method puts no restrictions on the analyte to be determined as long as it is able to displace a species from the mobile phase. Similarly, except for the demand for high refractive index, the method puts no restrictions on the displaced species. For the refractive index measurement per se, conventional refractometers may be used. Examples of such refractometers include 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.

For the purposes of the present invention, the displaced species must absorb light in the ultraviolet, visible, or infrared regions. Indirect detection methods utilizing light absorbing displaced species are well established, compare e.g. Bondoux, G. et al., supra. Since there is a universally valid relationship between the absorptivity and the refractive index of a substance; any species that may be used for indirect absorption detection may also be used for the purposes of the present invention. Exemplary species are organic dyes of the azine, thiazine, oxazine, cyanine, merocyanine, styryl, triphenylmethane, chlorophyll and phthalocyanine types, or simpler species like e.g. tungstate, vanadate, chromate, arsenate, molybdate, thiosulfate, iodide, bromide, nitrate, benzoate, phtalate, or trimesate ions.

In one aspect of the inventive method, 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 displaced species.

In accordance with this aspect of the invention, 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. If the measurement wavelength is chosen on the high wavelength side of 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, and more preferably less than 50 nm. If 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. Since the displaced species should have a high refractive index, the absorptivity (extinction coefficient) of the displaced species should in this case be as high as possible, preferably higher than about 20 lg^'^-cm^"1, more preferably higher than about 50 lg^"1cm^"1, and especially higher than about 100 lg^"1cm^"1.

As discussed above, this aspect may not, in a strict sense, be classified as an indirect method. However, in every band where the analyte displaces the mobile phase species, the refractive index changes, except in cases where the analyte has exactly the same refractive index as the displaced species. In particular, by choosing a displaced species with sufficiently high refractive index, every analyte band will cause a decrease in refractive index.

By proper selection of the displaced species and the measurement wavelength a very high refractive index may be obtained, and so the sensitivity may be significantly enhanced as compared to classical refractometry. Which specific measuring wavelength to choose for a specific displaced species, or vice versa, will, of course, depend on inter alia the particular species and may readily be established by the skilled person once he has had knowledge of the present invention. In another aspect, the measurement comprises determining the refractive index variation of the mobile phase with wavelength for a number of discrete wavelengths or for a continuous range of wavelengths, this variation being representative of the concentration of the displaced species.

In one, and presently preferred embodiment of this aspect, the measurement is performed as a differential measurement at two or more wavelengths. Of course, in this embodiment, the different measurements at the respective wavelengths will have to be performed substantially simultaneously or in a rapid succession.

In the specific case of measurement at two different wavelengths, 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 displaced 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 displaced species.

Measuring at more than two discrete wavelengths (for one and the same displaced species) 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.

Measuring the refractive index simultaneously or in rapid succession at a great number of wavelengths will produce a refractive index spectrum. In such a case 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. As mentioned above, it is also within the present inventive concept to measure the refractive index variation for a continuous wavelength range, covering the anomalous dispersion range of the displaced species.

In the preferred differential or dual wavelength measurement described above, the same considerations concerning the wavelength selection ranges with regard to the position of the maximum refractive index are, of course, applicable as for the single wavelength measurement discussed above. In this case the requirements on the refractive index of the displaced species are not as high as in the single wavelength measurement. The absorptivity (extinction coefficient) of the displaced species should, however, preferably be higher than about 5 lg'^-cm"^"1, and more preferably higher than about 10 lg^-1cm^_1.

Instead of performing the low refractive index measurement within the refractive index minimum plateau, it can be performed on the high wavelength side of the refractive index maximum. Since the refractive index decreases with wavelength in this region, a large refractive index difference can be obtained if the wavelength difference is sufficiently large. In this mode, the sensitivity enhancement will not be quite as large as if the low refractive index measurement is made near the refractive index minimum.

An important advantage of refractometry is that it measures concentration instead of quantity and therefore is truly miniaturizable, depending, of course, on the refractometer technique chosen. 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.

In comparison with conventional refractometry, the method of the present invention will have a substantially increased sensitivity. Using the differential mode will also reduce noise due to temporal variations in temperature, pressure, composition, etc., of the eluting liquid and make the measured signal specific with respect to the displaced species, which is the definition of an indirect method. 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.

It is readily understood that the criteria for an ideal detector for liquid chromatography and capillary electrophoresis given further above will be met for a refractometer used with the differential mode of the method of the invention. Thus, concentration is measured and miniaturization permitting on-column detection is therefore possible. Further, the detection limit is sufficiently low and the detection is, as is refractometry in general, very fast, the time constant of the detection thus giving no contribution to zone broadening. Finally, the detector can (depending, of course, on the specific refractometer principle chosen) be made simple, robust and inexpensive. A detector designed for measurements in accordance with the present invention will permit measurements in both a "single wavelength mode" and a "dual wavelength mode". Thus, 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. In parallel therewith, a dual wavelength measurement of the displaced species may be made. This will give a selective, indirect monitoring of displacing analytes and a substantially increased sensitivity. A universal and a selective monitoring may thus be made simultaneously in one and the same cell.

The method of the invention will now be illustrated in the following non-limiting Example, reference also being made to the accompanying drawings wherein: 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; and

Fig. 4 is a plot of laser spot distances vs. HITC concentrations.

EXAMPLE Differential refractometer 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 1 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. At the point of intersection of the two laser beams, 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 1 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 μl 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 latter was photographed with camera 6 (Polaroid® 600 SE) , mounted on a tripod, at distance of about 0.5 m using a Polaroid® 611 Video Image Recording Film. The enlargement from CCD camera 4 to TV screen 5 was about 30 X.

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. As a general strategy, 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 ll 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. Via a 0.4 mm cannula 17, a tube 18 connected valve 14 with flow cuvette 3 described in connection with Fig. l. 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.

During the measurements to be described below, liquid was pumped through the cuvette at about 23 μl/min. When the liquid was changed the valve 14 was turned to drain, and the flow was increased ten times to purge the pump tubes 13 and 15. Then the flow was decreased again, the valve was set in cuvette position and liquid was pumped for at least 15 minutes through the cuvette (due to the dead volume of the cannula 17) until measurements were conducted. Preparation of dye solutions

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 μm 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 μM 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 μm filter. A series dilution to seven different concentrations of HITC was performed by diluting different volumes of the HITC stock solution with the citrate/ethanol buffer to 25 ml:

HITC (ml) HITC cone. (μM) 25 500

12.5 250

6.25 125

3.125 62.5

1.563 ' 31.25 0.781 15.63

0 0

Measuring of refractive index

The refractive index spectrum for l mM HITC is shown in Fig. 3 (solid line: theoretically calculated curve, crosses: experimental data) . As appears therefrom, 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 6,60 nm is on the refractive index minimum plateau.

With reference to the above described apparatus (Figs, l and 2), 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.

For each dye concentration, two photographs were taken with an interval of a couple of minutes. The distance between the two laser light spots was estimated as described above by counting squares. The results are presented in Figure 4 that shows the average values of the spot distance in millimeters as a function of the HITC concentration. The relation is rectilinear with a very good fitting, the determination coefficient being 0.9992. The slope of the straight line, i.e. the sensitivity with regard to HITC concentration, is 3.1 μm/μM, or in angular units, 0.00024 °/μM, or, in refractive index units (RIU) , 2.0 μRIU/μM. The above described experiments clearly demonstrate the feasibility of the present inventive concept.

The invention is, of course, not restricted to the above specially described embodiments, but many changes and modifications may be made without departing from the general inventive concept as defined in the following claims. CLAIMS

1. 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.

2. The method according to claim 1, characterized in that the refractive index is measured at a single wavelength.

3. The method according to claim l or 2, characterized in that the displaced species has a high variation of the refractive index with wavelength.

4. The method according to claim l or 3, characterized in that the refractive index is measured at two different wavelengths, the measured refractive index difference being related to the concentration of the displaced species.

5. The method according to any one of claims l to 4, characterized in that the or one measuring wavelength is selected at or near the refractive index maximum of said displaced species.

6. The method according to claim 4 or 5, characterized in that one measuring wavelength is selected at or near the refractive index minimum plateau of said displaced species on the low wavelength side of the refractive index maximum.

7. The method according to claim 4 or 5, characterized in that one measuring wavelength is selected on the high wavelength side of the refractive index maximum.

8. The method according to any one of claims 5 to 7, characterized in that when the or one measurement

Claims

wavelength is on the high wavelength side of the refractive index maximum, the distance between the measurement wavelength and said maximum is less than 100 nm, more preferably less than 50 nm, and that when said measurement wavelength is on the low wavelength side, the measurement wavelength is close to said maximum.

9. The method according to any one of claims 4 to 8, characterized in that said determination comprises determining the refractive index at more than two wavelengths, the variation with wavelength of the refractive index being representative of the concentration of the displaced species.

10. The method according to claim 9, characterized in that said determination comprises determining the variation of the refractive index with wavelength for a continuous range of wavelengths.

11. The method according to any one of claims l to 10, characterized in that said displaced species comprises a chromophore or dye.