SE500817C2 - Methods to determine the concentration of an analyte in a liquid chromatographic or capillary electrophoretic flow by measuring the refractive index of the liquid - Google Patents

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

15 20 30 35 500 817 2 column) to avoid bandwidth in couplings or special detection cells, (iv) be fast so that the time constant of the detector does not cause degraded resolution and (v) be simple, robust and inexpensive.

The refractive index of a substance varies with the wavelength over the entire electromagnetic spectrum. This variation is called refractive index dispersion or simply dispersion. The dispersion is closely linked to the degree to which radiation is absorbed. In wavelength ranges with high transmittance, the refractive index decreases slowly with increasing wavelength within the visible and near infrared range (normal dispersion). In the vicinity of areas with high absorbance, i.e. at resonant wavelengths, however, the refractive index varies greatly with the wavelength, a phenomenon called anomalous dispersion. In this range, the refractive index is roughly a function of the negative derivatives of the absorbance (extinction coefficient) with respect to the wavelength. Thus, at a slightly higher wavelength than the resonant path length, the refractive index reaches a maximum, that is, where the negative derivatives of the absorbance have their maximum, and at a slightly lower wavelength than the resonant wavelength, the refractive index reaches a minimum. This relationship between absorptivity and refractive index of a substance is described in a more stringent way by Kramers-Kronig's equations.

Measurement of the anomalous portion of the dispersion by interference refractometry to obtain more spectral information and to increase the signal-to-noise ratio has been described by G. Gauglitz et al., Anal. Chem. 1988, 59, 2609-2612.

Measurement of the anomalous dispersion is also described in the international patent application PCT / SE92 / 00558 as a way to increase the sensitivity of a type of surface plasmon resonance (SPR) measurement and of similar measurement methods, which are based on measuring chemical interactions on a sensor surface in the form of measuring changes in refractive index in the surface layer. These changes are caused by the analyte causing or affecting the binding or release of a refractive index raising substance to 8,500 817 to and from the sensor surface, respectively. More specifically, the sensitivity is increased by matching the measurement path length with the absorptivity maximum of the refractive index enhancing substance used in the specific measurement method, preferably a dye or a chromophore molecule, and in particular so that the measurement path length is at or near the maximum in the negative length of the absorbance wavelength. This can be accomplished either by selecting a refractive index enhancing substance suitable for the measurement path length of a specific instrument or application, or by selecting a measurement path length suitable for a specific refractive index enhancing substance.

In accordance with the present invention, it has now been found that the basic physical principles of the detection methods of the above-mentioned PCT application, which relates to optical detection of binding of biomolecules to solid surfaces, can also be advantageously applied to the detection of liquid phase substances, to examples in capillary-electrophoretic or liquid chromatographic flow. In accordance with the invention, this is achieved by labeling or derivatizing the analytes to be detected in the liquid phase with substances with a high refractive index, for example strong organic dyes. By exploiting the strong dependence on wavelength of the refractive index of this label in areas of anomalous dispersion, which dependence has been described in general terms by G. Gauglitz et al. (see above), which applies to highly absorbent substances in liquid solution, the performance of refractometric detectors, and especially when the limit of detection or sensitivity, can be considerably increased.

In its broadest sense, therefore, the present invention provides a method for measuring the concentration of a substance in a liquid chromatographic or capillary electrophoretic flow by measuring the bulk refractive index, which method is characterized in that the analyte is labeled with a substance with high refractive index at the measuring path length or at at least one of the measuring path lengths.

The method does not impose any restrictions on the type of analyte, only it can be provided with a label as described above.

For refractive index measurement itself, conventional refractometers can be used. A brief description of currently available refractometers is therefore provided.

These refractometric methods can be roughly divided into (i) transmission methods and (ii) reflection methods.

An advantageous feature of transmission methods lies in the fact that they measure a kind of averaged refractive index over the entire liquid volume in the detection cell.

Local variations in the concentration of different substances, for example due to wall effects, do not give a large error in the measured refractive index. Transmission-based refractometers include (i) refractometers that use deflection methods and (ii) refractometers that use interference methods.

An example of a deflection cell is a simple, liquid-filled prism, through which a light beam is passed. For a homogeneous liquid, the deflection of the light beam depends on the difference in refractive index between the liquid and the cell wall.

An advantage of the prism method (compared to other transmission methods) is the fact that the deflection of the light beam, at constant refractive index of the liquid, depends only on the peak angle of the prism and not on the optical wavelength through the cell. The prism cell can therefore in principle be miniaturized to any size without the sensitivity deteriorating. The sensitivity can be doubled by ether reflection through the prism cell.

Interferometric methods measure the difference in optical wavelength, caused by difference in refractive index, between a sample cell and a reference cell. This difference in optical path length is proportional to the total optical wavelength, and rather long detection cells are therefore required to achieve sufficiently good precision.

The instrumentation is quite complex and includes a polarizer, a beam splitter, an inverted beam splitter that reconnects the sub-beams and a phase analyzer.

Reflection methods measure the difference in refractive index between two materials, such as the liquid in the cell and the cell wall, at a reflective interface. However, reflection-based refractometers have a disadvantage that makes them less attractive for the purposes intended here, namely the fact that wall effects, such as contamination or selective adsorption, can strongly affect the measurement signal, so that the measured refractive index is not always representative of the entire liquid volume. Radial concentration variations can also be caused by the development of Joule heat in capillary electrophoresis columns. Examples of reflection-based refractometers are Fresnel and SPR detectors.

The Fresnel detector measures the refractive index as the change in intensity of light reflected or transmitted at a dielectric interface, which change is due to the change in reflectance or transmittance caused by the changed refractive index in the liquid. Since the reflectance is independent of the length of the cell, the Fresnel detector can in principle be miniaturized.

The SPR (surface plasmon resonance) surface is based on the phenomenon that SPR causes the intensity of a reflected light beam to have a distinct minimum at a certain reflection angle, which means that the SPR measurement includes a position determination (or a relative intensity determination). . For a more detailed description of SPR and its applications in analytical contexts, reference may be made, for example, to WO 90/05295 and WO 90/05305. A disadvantage of SPR in the present context is the fact that it is mainly a surface-sensitive technique, where the total measuring depth from the interface is about 1 μm. Surface contamination and selective adsorption can therefore affect the measured signal to a considerable extent. Furthermore, the SPR detector requires very high path length stability and reproducibility of the light source, since the measured minimum intensity angle depends not only on the refractive index, but also on the wavelength itself.

The types of refractometers mentioned above are, of course, merely exemplary, and other types of refractometers which are likely to be used for the purposes of the invention will be apparent to those skilled in the art.

It will be readily appreciated that the refractometer types described above can be readily applied to parallelepipedic columns or capillaries. The refractive index inside round columns or capillaries, on the other hand, can be measured, for example, by analyzing the interference pattern generated by a laser beam, described by A. E. Bruno et al., Anal. Chem. 1991, _51, 2689-2697. methods that use optical guides are also of great refractometric interest in this context due to the small dimensions of optical fibers.

For the purposes of the present invention, the refractive index enhancing substance is or preferably includes a dye or a chromophore molecule.

Derivatization methods for labeling molecules with chromophores are well established. Such methods are used, for example, 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). Of course, the dyes used for refractive index labeling need not be fluorescent, so in principle any dye that can be linked to an analyte molecule by a chemical bond can be used. Examples of suitable dyes are those of the azine, thiazine, oxazine, cyanine, merocyanine, styryl, triphenylmethane, chlorophyll and phthalocyanine types.

In a variant of the invented method, the measurement is performed at a single wavelength at or near the refractive index maximum of the market substance, i.e. at or near the maximum in the negative derivatives of the absorptivity with respect to the wavelength. Thus, in accordance with this variant of the invention, the measurement should be performed at or as close to the maximum of the negative derivative of the absorptivity with respect to the wavelength as possible. If the measurement path length is selected on the high wavelength side of the maximum for the negative derivative of the absorbance with respect to the wavelength, the distance between the measurement wavelength and said maximum should preferably be less than 100 nm (corresponding to a possible sensitivity increase of at least about 5 times by mass, depending on absorptivity), and more preferably less than 50 nm (corresponding to a possible increase in sensitivity by at least about 10 times on a mass basis, depending on the absorptivity).

If the measurement wavelength is selected on the low wavelength side of the maximum of the negative derivative of the absorptivity with respect to the wavelength, the measurement wavelength must be very close to said maximum, since the refractive index decreases again as one approaches the wavelength of the absorptivity maximum.

Since the label substance should have a high refractive index, the absorptivity (extinction coefficient) of the label substance of the analyte in this case should be as high as possible, preferably higher than about 20 lg'1cm'1, more preferably higher than about 50 lg'1cm'1, and especially preferably higher than about 100 lg'1cm'1.

By a suitable choice of label and measuring wavelength, a very high refractive index can be obtained. The specific measurement wavelength to be selected for a specific label or probe, or vice versa, will, of course, depend on, inter alia, the specific probe and can be readily determined by those skilled in the art once he has become aware of the present invention.

In another variant of the invention, the measurement comprises determining the variation of the refractive index of the label with wavelength, at a number of discrete wavelengths or over a continuous wavelength range, where this variation represents the concentration of the label.

In one embodiment, which is the presently preferred one, of this variant, the measurement is performed as a differential measurement at two or more wavelengths.

Of course, in order to in this embodiment determine the content of an analyte in a liquid flow, the different measurements at the respective wavelength must be performed practically simultaneously or in a very fast sequence.

In the particular case of measurement at two different wavelengths, a measurement path length is preferably selected at or near the refractive index maximum of the labeled substance (as in the case of measurement at one wavelength, as described above), i.e. at or near the maximum in the negative derivative of the absorbance with respect to the wavelength . The second measurement wavelength should preferably be at or near the minimum plateau of the refractive index (in the anomalous region of the dispersion curve, i.e. the refractive index versus wavelength, the dispersion curve shows a minimum plateau rather than a well-defined slump), or in other words, in the vicinity of the wavelength.

Measurement at more than two discrete wavelengths (using one and the same refractive index marker substance) results in more information about the dispersion, which allows a more robust interpretation of the detected signal and a reduction of the noise by averaging over the obtained measured values.

Measurement of refractive index simultaneously or in rapid succession at a large number of wavelengths results in a refractive index spectrum. In such a case, the determination of the refractive index variation can be based on measuring the area under the spectral curve rather than on the difference in refractive index between pairs of discrete wavelengths.

In the presently invented method, as mentioned above, it also includes measuring the refractive index variation over a continuous wavelength range, which covers the anomalous dispersion region of the label.

In the preferred differential measurement or two-wavelength measurement, which is described above, of course, the same views apply to the choice of measurement wavelengths relative to the position of the refractive index maximum as in the case of measurement at one wavelength, as discussed above. 10 lS 20 30 35 500 817 In this case, the requirements for the refractive index of the marking substance are not as high as when measuring at a wavelength. However, the absorbance (extinction coefficient) of the label of the analyte should preferably be higher than about 10 g / cm 1, and more preferably higher than about 1 g -1 cm -1.

Instead of performing the measurement of low refractive index on the minimum plateau of the refractive index, 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 large enough. In this case, the increase in probability will not be quite as great as in the case that the measurement of low refractive index is made close to the refractive index minimum, but the reduction of the noise and the increase of the selectivity will still be obtained.

It should be noted that when this differential measurement embodiment is used, only one detector cell is required for all the mentioned refractometer types except for the interferometer.

An important advantage of refractometry is that the refractive index is dependent on concentration instead of the amount of substance, and the technology is therefore truly miniaturizable, but of course depending on which type of refractometer is chosen. The measured signal will be independent of the size of the detection volume, and the detection volume can therefore, in principle, be miniaturized to any degree.

For example, the technology allows direct detection on column.

Compared to conventional refractometry, the present invented method has considerably higher sensitivity.

By using the differential variant, the noise caused by variations in temperature, pressure, flow, etc. is also reduced, and the measured signal becomes specific with respect to the labeled molecules. In this latter case, no reference flow or reference cell is needed, since the measurement at two wavelengths is self-compensating, as described above. It will be readily appreciated that the criteria for an ideal liquid chromatography and capillary electrophoresis detector set forth above will be met by a refractometer using the differential variant of the method embodied in the invention. Thus, it is the concentration that is measured, which enables miniaturization that allows on column detection. Furthermore, the detection limit is sufficiently low, and the measurement is, as for refractometry in general, very fast, so that the time constant of the detection does not make any contribution to the bandwidth. Finally, the detector (of course depending on the specific refractometer type chosen) can be made simple, robust and inexpensive.

A detector designed to make measurements in accordance with the present invention will be able to perform measurements according to both the "single wavelength variant" and the "two wavelength variant". Thus, a classical refractive index monitoring at a single wavelength can be performed, which detects all substances, absorbs or fluoresces At the same time, a two-wavelength measurement of color-labeled analytes can be made, which provides a selective monitoring of labeled analytes and a significantly increased sensitivity, so a universal and a selective monitoring can be performed simultaneously in one and the same cell.

The method of the present invention will now be non-limiting. The Example, including those not illustrated in the following, also referring to the accompanying figures, of which: Figure 1 is a sketchy drawing of the experimental setup used in the Example; Figure 2 is a sketchy drawing of the liquid handling system used in the Example; Figure 3 is a graph showing the refractive index spectrum of the dye HITC used in the Example; and l Figure 4 is a graph showing distances between laser light spots as a function of HITC concentration. 10 20 30 35 500 8b1.7 11 EXAMPLE 2. :: I. JJ E I l An experimental differential refractometer was constructed as sketched in Figure 1.

The 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 the diode type with collimating optics. Laser 1 had a wavelength of 660 nm (more precisely 658.5 nm) (Melles-Griot) and was powered by a power supply (Mascot Electronics Type 719). The second laser 2 had a wavelength of 780 nm (Spindler & Hoyer) and was powered by a second power supply, the Diode Laser DL 25 Control Unit (Spindles & Hoyer). The two lasers 1 and 2 were mounted at right angles to each other on a steel plate 7.

At the intersection of the laser beams, a blackened brass tube (not shown; inner diameter 20 mm) was screwed into plate 7. The brass tube had slits, in which a filter 8 (Melles-Griot Short wavelength pass filter) with a cutoff wavelength of approximately 700 nm at a 45 ° angle to beam directions. This filter 8 transmitted the 660 nm beam from laser 1 but reflected the 780 nm beam from laser 2, with the resultant effect that the two beams were allowed to coincide. A hole with a diameter of 1 mm served as a starting gap.

Flow cuvette 3, a commercial cuvette with two prism cells (consisting of two 45 ° prism cells, 1.5 x 7 mm, volume 8 pl, with the hypotenuses facing each other) intended for a refractive index detector for liquid chromatography (Pharmacia LKB Biotechnology AB, Uppsala, Sweden), was then screwed to the outside of the brass tube adjacent to its exit gap. Only one of the two prism cells in cuvette 3 was used, and connected through tubing (not shown) to a simple fluid handling system described below (the other prism cell remained empty). steel plate 7 was anchored with a bolt 10 at one end of an aluminum plate 9, and was thus rotatable. CCD camera 4 (Panasonic WV-CD50), which was driven by a power supply 10 20 30 35 'light images (diameter 1-1.5 mm on the CCD camera) 500 817 12 Panasonic WV-CD52, was mounted on the other end of plate 9 at a distance of about 0.8 m (slightly varying between the different test series described below) from flow cuvette 3, and was adjustable vertically by means of a micrometer screw. A curved, black steel cover was placed over the CCD camera to shield from stray light.

The image from the CCD camera was projected on a TV screen 5 (Electrohome 10 "), on which a transparent grid was taped (overhead film on which a millimeter paper with 1.4 X magnification was copied). This transparent grid was used to measure distances on TV screen 5. by frame counting.The TV screen was photographed with a camera 6 (Polaroidc) 600 SE) on a tripod, at a distance of about 0.5 m and with film PolaroidC> 611 Video Image Recording Film. 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 so that neat, symmetrical from the two lasers were obtained just above each other vertically on the TV screen. As a general strategy, low laser intensity, high screen contrast and 1/4 second exposure time were used. Thereafter, the brightness of the screen was adjusted until a sharp image was obtained on the screen, and finally the aperture was adjusted until a sharp image was obtained on the photograph.

The position of the light spots was determined by taking a photograph of a TV screen 5, and then by grating in a microscope determining the position of the left and right edges of the light spots, respectively, and then assuming that the center of each spot was between the two edges.

The fluid management system, shown in Figure 2, consisted of a peristaltic pump 11 (P1, Pharmacia LKB Biotechnology AB, Uppsala, Sweden). On one side, pump 11 was connected to a sample container 12 via a hose 13, and on the other side to a manual rotary valve 14 via a hose 15. A hose 16 connected valve 14 to the sludge. Via a 0.4 m cannula 10 15 20 30 35 n SÛÛ 817 17, a hose 18 connected valve 14 with flow cuvette 3, which was described in connection with figure 1. A hose 19 connected cuvette 3 with a cuvette slide. The manual valve 14 made it possible to pump liquid either to the sludge or to the cuvette 3.

During the measurements, described below, liquid was pumped at about 23 pl / min through the flow cuvette. when changing fluid, valve 14 was turned to sludge, and the flow was increased tenfold to clear the pump hoses 13 and 15. Then the flow was lowered again, the valve was turned to cuvette position and liquid was pumped for at least 15 minutes through the cuvette (due to the dead volume in cannula 17). Citrate buffer (pH 3, 0.1 M citrate, 0.4 M NaCl, 0.05% TweenC> 20) was prepared by dissolving 21 g of citric acid (M&B pa) and 23 g of sodium chloride (Merck pa) in 1000 ml of deionized water. 5 ml of TweenC) 20 (Calbiochem 655206, 10%, protein grade) was added. 4 M sodium hydroxide (p.a.) was added to adjust the pH from 1.95 to 3.00, and the solution was filtered through a 0.22 μm filter. 500 ml of the citrate buffer was then mixed with 500 ml of spectrographically pure ethanol and homogenized by sonication for a few minutes.

A 500 pM stock solution of the dye HITC (1,1 ', 3,3,3', 3'-hexamethylindotricarbocyanine) was then prepared by dissolving 14 mg of HITC iodide (Sigma H0387, 94% purity, molecular weight 537 g / mol; Sigma Chemical Co., St. Louis, Mb., USA) in 50 ml of the citrate / ethanol buffer prepared above. After homogenization with ultrasound for a few minutes, the solution was filtered through a 0.45 μm filter.

A dilution series to seven different HITC concentrations before measurement. was made by diluting different volumes of the HITC stock solution to 25 ml with citrate / ethanol buffer: HlIC_imll HlIQ; kQn§l_luML 25 soo 12.5 zso 6.25 izs 3.125 62.5 1.563 31.25 5Û0 817 14 0.778 15.63 O 0 in. V I n.. 1 The refractive index spectrum for 1 mM HITC is shown in Figure 3 S (solid line: theoretically calculated curve, cross: experimental data). As can be seen, the measurement wavelength 780 nm (laser 2) is close to the maximum in the refractive index, on its high wavelength side, while the other measurement wavelength 660 nm (laser 1) is on the minimum plate of the refractive index.

Referring to the apparatus described above (Figures 1 and 2), the position of the flow cuvette (3) was adjusted by filling it with ethanol / water 50/50 (deionized water and spectrographically pure ethanol) using a syringe and then turning the stand plate ( 7) until the two laser light spots were centered on top of each other on the CCD camera. The distance between the flow cuvette and the CCD camera was 73 cm.

Then, refractive index measurements were performed for each of the different HITC concentrations, which are described above, the liquid handling taking place as described above under "Liquid handling system". The laser intensities, TV screen brightness, and camera exposure time and aperture were adjusted for each dye concentration to obtain sharp images of the light spots. For each dye concentration, two photographs were taken every few minutes. The distance between the two laser light spots was estimated as described above by square counting. The results are presented in Figure 4, which shows the mean value of the light spot distance in millimeters as a function of HITC concentration. The relationship is rectilinear with a very good degree of fit, the coefficient of determination is 0.9992. The slope of the straight line, i.e. the sensitivity with respect to HITC concentration, is 3.1 μm / pM, or in angular units 0.00024 ° / pM, or in refractive index units (RIU) 2.0 pRIU / μM.

The experiments described above clearly demonstrate the feasibility of the present inventive method. The invention is of course not limited to the embodiments specifically described above, but a number of changes and modifications may be made without departing from the general, invented method as defined in the following claims.

Claims (12)

10 20 30 35 500 817 10 BAIENIKBY A method of determining the concentration of an analyte in a liquid chromatographic or capillary electrophoretic flow by measuring the refractive index of the liquid, characterized in that the analyte is labeled with a substance having a high refractive index at the measuring wavelength or at at least one of the measuring path lengths. Method according to Claim 1, characterized in that the refractive index of the liquid is measured at a single wavelength. A method according to claim 1 or 2, characterized in that the refractive index of the market substance varies greatly with wavelength. A method according to claim 1 or 3, characterized in that the refractive index of the liquid is measured at two different wavelengths, wherein the measured refractive index difference is a measure of the concentration of analyte in the liquid. Method according to any one of claims 1-4, characterized in that the single or one measuring wall length is selected at or in the vicinity of the refractive index maximum of said label substance (Figure 3). Method according to claim 4 or 5, characterized in that a measuring path length is selected at or near said label 'refractive index' minimum plate on the path length side of the refractive index maximum (Figure 3). Method according to Claim 4 or 5, characterized in that a measuring path length is selected on the high path side if the refractive index maximum (Figure 3). A method according to any one of claims 5-7, characterized in that when the single or one measuring path length is on the high path length side of the refractive index maximum, then the distance 10 500 817 between the measuring path length and said maximum is less than 100 nm, more preferably less than 50 nm, and that when said measuring path length is on the path length side, the measuring path length is close to said maximum. A method according to any one of claims 4-8, characterized in that said determination comprises measuring the refractive index at more than two wavelengths, the refractive index variation with wavelength being a measure of the concentration of analyte. A method according to claim 9, characterized in that said determination comprises measuring the refractive index variation with wavelength over a continuous wavelength range. A method according to any one of claims 1-10, characterized in that said label substance comprises a chromophore or a dye. A method according to any one of claims 1-11, characterized in that said refractive index measurement is based on prism deflection, Fresnel detector, surface plasmon resonance or interferometry.