WO1994017393A1

WO1994017393A1 - An improved method of liquid bulk refractive index detection

Info

Publication number: WO1994017393A1
Application number: PCT/SE1994/000045
Authority: WO
Inventors: Anders Hanning
Original assignee: Anders Hanning
Priority date: 1993-01-27
Filing date: 1994-01-21
Publication date: 1994-08-04
Also published as: SE500817C2; SE9300231L; EP0681694A1; SE9300231D0; AU5981794A

Abstract

In a method of determining an analyte in a liquid chromatographic or capillary electrophoretic separation by measuring bulk refractive index, the analyte is labeled with a species having a high refractive index at the or at least one measuring wavelength.

Description

AN IMPROVED METHOD OF LIQUID BULK REFRACTIVE INDEX

DETECTION

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, 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.

An alternative technique is fluorescence measurement. Also this technique 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, 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. 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 reaches 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. 2609-2612. 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 (SP ) 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.

In accordance with the present invention, it has now been found that the basic physical principles of the detection methods in the above-mentioned PCT application, relating to the optical detection of the binding of biomolecules to solid surfaces, also may be favourably applied to the detection of substances in a liquid bulk, e.g. in a capillary electrophoresis or liquid chromatography flow. In accordance with the invention, this is accomplished by labelling or tagging the analytes to be detected in the bulk by species having a high refractive index, such as strong organic dyes. By utilizing the heavy dependence of the labeling species refractive index on the wavelength in the anomalous dispersion region, which dependence has been generally described by Gauglitz G. et al., supra, for highly absorbing substances in liquid solutions, the performance of refractometric detectors, and especially the detection limit, i.e. the sensitivity, 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 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.

For the refractive index measurement per se, conventional refractometers may be used. A brief description of currently available refractometers will therefore be given.

These refractometry techniques may be broadly divided into (i) transmission techniques, and (ii) reflection techniques.

An advantageous feature of transmission techniques resides in the fact that they measure some kind of average refractive index over the total bulk of the liquid in the detector cell. Local variations in the concentration of different species, e.g. due to wall effects, do not decisively affect the detected refractive index. Transmission based refractometers include (i) refractometers utilizing deflection techniques and (ii) refractometers utilizing interferometric techniques. One example of 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 (as compared to other transmission techniques) 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.

However, a disadvantage of 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) . 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 μm. 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, on the other hand, may e.g. be measured by analyzing the interference pattern generated by a laser beam, as described by A. E. Bruno et al. , Anal.

Chem. 1991, 63, 2689-2697. Optical waveguide refractometric techniques are also of great interest due to the small size of optical fibers.

For the purposes of the present invention, 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.

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 labelling 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 (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) . 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 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^"1cm^"1, more preferably higher than about 50 lg^"1cm^"1, and especially higher than about 100 lg^"1cm^"1.

By proper selection of the labelling species and the measurement wavelength a very high refractive index may be obtained. Which specific measuring wavelength to choose for a specific label or tag, or vice versa, will, of course, depend on inter alia the particular label 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 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.

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 for the case of determining an analyte in a liquid flow, 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 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 (for one and the same refractive labelling 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 labelling 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 labelling species are not as high as in the single wavelength measurement. The absorptivity (extinction coefficient) of the analyte labeling species should, however, preferably be higher than about 10 lg^"1cm^"1, and more preferably higher than about 20 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, 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.

An important advantage of refractometry is that the refractive index depends on concentration (instead of on quantity) , and therefore the technique is truly miniaturizable, depending, of course, on the specific refractometer type 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 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.

It is readily understood that the criteria for an ideal detector for liquid chromatography and capillary refractometry 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 peak 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 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.

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. 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; 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 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 δ. Hoyer) . The two lasers l, 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 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 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 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) , driven by a Panasonic Power Supply WV- CD52, was mounted at the other end of plate 9 at a distance

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 M 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 660 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 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.

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 which 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

l. 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.

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

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

4. The method according to claim 1 or 3, characterized in that the bulk refractive index is measured at two different wavelengths, the measured refractive index difference being related to the concentration of the analyte in the fluid.

5. The method according to any one of claims 1 to 4, characterized in that the or one measuring wavelength is selected at or near the refractive index maximum of said labelling 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 labelling 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 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 of the refractive index with wavelength being representative of the concentration of the analyte.

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.

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

12. The method according to any one of claims l to ll, characterized in that said refractive index determination is based upon prism deflection, Fresnel detector, surface plasmon resonance (SPR) , or interferometry.