WO1996024045A1

WO1996024045A1 - Method, apparatus, and use of apparatus, for optical characterization of liquids

Info

Publication number: WO1996024045A1
Application number: PCT/SE1996/000087
Authority: WO
Inventors: Anders Hanning
Original assignee: Anders Hanning
Priority date: 1995-02-01
Filing date: 1996-01-26
Publication date: 1996-08-08
Also published as: SE9500356D0

Abstract

A method for optical characterization of a liquid, comprising that light is brought to interact with the liquid, and that said interaction is detected, characterized in that the two characteristic optical properties, the refractive index and the absorbance of the liquid are determined at more than one wavelength, and an apparatus to carry out said method, comprising light source, sample cell, and light detector, characterized in that it comprises only one sample cell, preferably flow cell, for the determination of both refractive index and absorbance.

Description

METHOD AND APPARATUS FOR OPTICAL CHARACTERIZATION

OF LIQUIDS

The present invention relates to a method and to an apparatus for the optical characterization of liquids, referring to determination of the two characteristic optical properties, the refractive index and the absorbance, of the liquids. In particular, the invention relates to optical characterization of flowing liquids, and even more particularly to flowing liquids in connection with chemical analysis by means of liquid chromatography or capillary electrophoresis. Such optical characterization constitutes a means of performing detection and determination of the concentration of dissolved substances ("analytes") in connection with the above mentioned chemical analysis methods. In the subsequent discussion, the account is mainly concentrated on liquid chromatography detection, but the invention may equally well be applied to capillary electrophoresis or to the characterization of other kinds of liquids, flowing or static.

There exist a number of well-established methods and apparatuses, refractometers, for the determination of the refractive index of different substances, including liquids. Some examples of refractometers are prism refractometers (deflection refractometers) , Fresnel refractometers, surface plasmon resonance refractometers, interference refractometers, and different kinds of refractometers utilizing optical waveguides. Different kinds of refractometers, in particular prism and Fresnel refractometers, are often utilized in connection with chemical analysis, and particularly in connection with liquid chromatography detection. Most chromatography refractometers measures the refractive index either at one single wavelength, or as an average over a wider wavelength range. Both these methods involve a number of disadvantages: limited signal level, high noise level caused be temperature variations and other phenomena, and low selectivity. On the other hand, refractometry has several specific advantages in connection with chromatography detection, e.g. the possibility of universal detection (detection of all kinds of substances) , and the fact that the sample cell can be miniaturized without any 5 decrease of the signal level. In the Swedish patent applications 9300231-9, 9302723-3, and 9303149-0, A. Hannmg describes different methods of performing chromatographic refractive index detection at more than one wavelength, which brings about several advantages, e.g.

10 increased signal level, decreased noise level, and controllable selectivity, while the above mentioned general advantages of refractometric detection are preserved.

Methods to determine the absorbance of substances, including liquids, are also well established, ana will not

15 ce further discussed. Measurement of absorbance is the most common detection method m connection with liquid cnromatograpny. This method has several advantages, like e.g. high signal level, robustness, and fairly controllable selectivity. The main αisadvantages are tne neeα of a

20 cnromophore in the detected molecule, which implies that absorbance detection is not universal, and the fact that tne signal level decreases upon miniaturization of tne sample cell, since the absorbance signal is linearly related to the optical length of the sample cell.

25 To be strict, it should be noted that the absoroance (dimensionless quantity) is a measure of how large a fraction of the light that is absorbed in a certain, fixed experiment. The light absorbing ability of a certain optical material, e.g. a liquid, is, on the other hand,

M> αescribed by the optical property absorption coefficient (dimension: length^" ) . The product of the sample thickness and its absorption coefficient equals the aosorbance. Since "absorbance" is a well-established term within analytical chemistry, this term is henceforth used. It is more correct

^■?5 tnough, from a physical point of view, to use the term "absorption coefficient" of a liquid. Henceforth, the expression "determination of the absorbance" refers to determination of the aosoroance, the absorption coefficient, a quantity proportional or approximately proportional to the absorbance or the absorption coefficient, or a change in any of these quantities. Likewise, the expression "determination of the 5 refractive index" henceforth refers to determination of the refractive index, a quantity proportional or approximately proportional to the refractive index, or a change in any of these quantities.

Of the above mentioned demands on chromatography lϋ detection methods, the demands on high signal level and low noise level are easy to understand, since these properties make possible tne determination of low concentrations of analyte. The ability to miniaturize the analytical system is also important, since miniaturization implies better

15 separation of different analytes, faster analyses, and lower consumption of sample and solvents. Controllable selectivity is important, too, since different analytical problems put different demands on the selectivity: sometimes it is desirable to detect all substances

20 universally, sometimes it is desirable to detect a group of substances selectively, and sometimes it is desirable to detect one single substance specifically.

The two detection methods refractometry and absoroance detection complement each other, and each alternative has

25 its specific advantages and disadvantages with respect to different chemical analysis problems. To make use of the advantages of both alternatives m connection with liquid chromatography, two detectors are often connected in series. However, this serial connection brings about

30 certain disadvantages, mainly zone broadening and impaired separation efficiency caused by the increased system dead volume, but also increased instrumental complexity and increased cost due to the use of two detectors. Alternatively, one kind of detector is used at a time, and

^5 the detector is exchanged dependent on which detector is best suited for the specific analytical problem at hand. This procedure involves an appreciable amount of extra work, and tne specific disaαvantages of the detector used for the moment is not avoided.

The present invention is based on the idea of determining both refractive index and absorbance at more than one wavelength, preferably in one single detector.

Both kinds of measurements are actually based on a similar optical experimental setup: light is emitted from a light source, interacts with the liquid sample, preferably in a sample cell, and is detected by a light detector. If one single flow cell is utilized for both refractive index detection and absorbance detection, the problems with zone broadening and impaired separation efficiency, caused by increased dead volume, are avoided. If one single optical setup (light source, flow cell, light detector) is utilize for both detection methods, tne instrumental complexity an high cost of using two separate detectors are avoided. By measuring both refractive index and absorbance at more tha one wavelength, the advantages of both methods are exploited, and the complementary information, that the two methods yield, makes possible a more complete optical characterization of the analyzed liquid.

Thus, in one aspect, the present invention provides a method for optical characterization of a liquid, comprising that light is brought to interact with the liquid, and that said interaction is detected, that m its oroadest sense is characterized in that the refractive index and the absorbance of the liquid are determined at more than one wavelength. The optical characterization may, in different embodiments, take place at two, or more than two, discrete wavelengths, or over a continuous wavelength range. More special embodiments are characterized in that the liquid is a flowing liquid, and in particular a flowing liquid in connection with liquid chromatography or capillary electrophoresis. In another aspect, the present invention provides an apparatus, comprising light source, sample cell, and light detector, for the determination of the refractive index and the absorbance of a liquid at more than one wavelength, which in its widest sense is characterized in that it comprises only one sample cell, preferably flow cell, for the determination of both refractive index and absorbance. More special embodiments are characterized m that the apparatus moreover comprises only one light source, or one set of light sources, and only one light detector, or one set of light detectors, for the determination of both refractive index and absorbance.

The expression "sample cell" is used m a wide sense, as a means of physically defining the volume element of the liquid that is being characterized, and covers on column detection as well. In liquid chromatography and capillary electrophoresis, on column detection means tr.at part of the separation column as such is being used for tne detection, and that no separate, external detection cell is being used. The expression "comprises only one sample cell" means that only one single cell is being used for a certain measurement or a certain analysis, but this does not exclude tnat the sample cell may be exchangeα between different measurements or different analyses. Neither is it excluded, that the apparatus, besides a sample cell, may comprise a reference cell, which is discussed in greater detail below. Neither is it excluded, that the apparatus may comprise one, or more than one, other kind of sample cell, that are being used for other purposes, e.g., electrochemical detection. The expression "one set of light sources" means that a number of different lamps may be used simultaneously or in sequence in the apparatus, but that the same set of lamps is being used for both the refractive index and the absorbance measurement. An analogous example is the use of two different lamps, e.g. one deuterium lamp and one tungsten lamp, to cover different wavelength regions m some absorbance detectors. The expression "one set of light detectors" means that more than one light detector may be used simultaneously, but that the same set of light detectors is used for both the refractive index measurement and the absorbance measurement. As an example, a number of photodiodes or diode arrays may be mounted at different positions within the apparatus. The expression "one light detector, or one set of light detectors" also covers the case with detectors consisting of a number of discrete light sensitive regions, like e.g. diode arrays and charge transfer devices.

The expression "determined over a continuous wavelength range" refers to the determination of refractive index and absorbance as functions of wavelength over this range. This is in contrast to determination of the average of these quantities over a wavelength range.

In one embodiment of the invention, the refractive mαex and the aosorbance are determined simultaneously, i.e. m principle are the same set of quanta of light utilized for both determinations. In another eπiDodiment, the two determinations are performed sequentially at different points in time. In order to obtain refractive index and absorbance chromatograms, respectively, that correspond to each other, it is required that both quantities are determined within a time frame that is appreciably shorter than the chromatographic peak width, e.g. one tenth thereof, corresponding to e.g. a few tenths of a second.

In one embodiment, the light interacts with the same, single volume element of the liquid at the determination of both refractive index and absorbance. This is, for example, the case when the sample volume is physically defined by the same, single sample cell and the determinations are performed simultaneously. A sample cell, though, is not necessary m order to physically define a volume element of a liquid. Such a volume element may, e.g., be defined by the evanescent field from an optical waveguide. In a sequential determination, the same volume element may be analyzed if the liquid is static. If the liquid is flowing, partially or totally different volume elements may be analyzed in a sequential determination. Just like in the preceding paragraph, in order to obtain corresponding refractive index and absorbance chromatograms, it is required that both quantities are determined within a volume difference that is appreciably smaller than the volume corresponding to the chromatographic peak width.

In a preferred embodiment, the sample cell is a prism cell filled with liquid. The light is transmitted through the cell. The refractive index determination comprises determination of the deflection of the light, while the absorbance determination comprises determination of the intensity of the light. A few advantages of this embodiment are the instrumental and conceptual simplicity, since refractive index and absorbance are determined as two, mutually independent phenomena: the position of the light beam on the detector yields the refractive index, while the intensity of the light beam yields the absorbance. At high absorbance values, non-linear effects may appear. This is partly due to the fact that the optical pathlength is not constant across the prism cell (which influences the absorbance measurement), and partly due to the fact that high absorbance influences the symmetry of the light spot (which influences the position determination, i.e. the refractive index measurement) . However, at the low absorbance values generally encountered in connection with liquid chromatography, these non-linear effects may be neglected.

The invention is not limited, however, to prism deflection refracto etry. Other conceivable kinds of refractometers are Fresnel refractometers (the intensities of the primary reflected beam and the transmitted-reflected beam, e.g., may be compared), interferometers (the intensity and phase shift of two beams, e.g., may be compared), or refractometers based on optical waveguides. Other conceivable kinds of refractometers are apparent to the skilled person.

The invention is not limited to any particular light wavelength range, but the optical measurement may be performed within one or more than one of the wavelength ranges UV (ultra violet), VIS (visible light), NIR (near infra red) , and IR (infra red) . The invention is not limited to any particular separation mechanism or separation mode in connection with liquid chromatography or capillary electrophoresis detection. Conceivable separation modes include, but are 5 not limited to, reversed phase chromatography, adsorption chromatography, ion exchange chromatography, ion pair chromatography, size exclusion chromatography, affinity chromatography, capillary zone electrophoresis, capillary ion electrophoresis, micellar electrokmetic capillary 10 chromatography, isotachophoresis, capillary gel electrophoresis, and capillary isoelectric focusing. Other conceivable chromatographic or electrcphoretic separation modes are apparent to the skilled person.

The apparatus according to the invention may, of l - course, also be used to determine either refractive index or absorbance, or to perform measurements at one single wavelength. Such simple measurements are adequate for many kinds of simpler analysis problems.

A few particular emoodiments of the invention will now 0 be more closely illustrated in the following non-limiting discussion, reference also being made to the accompanying drawings, wherein:

Figure 1 is a schematic illustration of the general optical setup; 5 Figure 2 is a schematic illustration of an embodiment where the measurement is performed at two wavelengths;

Figure 3 is schematic illustration of an embodiment where the measurement is performed at several wavelengths, utilizing fixed optics; 0 Figure 4 is a schematic illustration of an embodiment where the measurement is performed at several wavelengths, utilizing movable optics;

Figure 5 is a schematic illustration of an embodiment utilizing a reference cell. 5 Figure 1 illustrates the general optical setup: light from a light source (1) interacts with the liquid, preferably contained in a sample cell (2), and s detected by the light detector (3) . Figure 2 shows a more detailed, but still schematically simplified, example of an optical setup for measurements at two wavelengths. Light from the light source (1) passes through the sample cell (4), is reflected by the grating (5), and is detected by the set of light detectors (6) . The light source (1) in the figure is supposed to emit polychromatic light, so the grating (5) is necessary in order to select wavelengths. Alternate ways to select wavelengths are, e.g., by means of filters and mirrors. The different wavelengths of tne polychromatic light will, when passing the sample cell (4), be distributed along one dimension in space, since the refractive index of the sample varies with wavelengtn. The grating is oriented in Ξύcn a way, that the spectral separation of tne grating takes place along a dimension, which is orthogonal to the dimension, along which the prism has distributed the wavelengths corresponding to different refractive indices. The set of light detectors (6) in the figure consists of two one-oimensional detectors, i.e. detectors extended along one dimension. The detectors may, e.g., be one- dimensional diode arrays, consisting of a number, e.g. 16, light sensitive diodes arranged m a row. Since the grating distributes the light beams m space, the detectors may be moved, so as to be hit by light cf the desired wavelengths. Alternatively, one or both detectors may be fixed, and the wavelengths may be chosen by rotating the grating around one of its axes. The position of the light spot on the two detectors, respectively, may then be determined by reading out all the diodes, and calculating the exact position of the spot, defined as, e.g., the position of the center of gravity or the maximum intensity of the light spot. The total intensity of the light is obtained by integrating the light intensity over all diodes. Other conceivable kinds of light detectors are, e.g., split diodes, which are small dioαe arrays containing two, closely spaced light sensitive regions, or position sensitive detectors, which are able to directly register tne position of a light spot. Another conceivable variation is an arrangement of a number of discrete photodiodes. When the refractive index of the sample varies heavily, e.g. when using so called graαient elution or when different kinds of liquids are studied, the deflection of the light beam may vary so much, that the light beam does not hit the set of light detectors, and especially so if small light detectors are used. In order to compensate for this, the grating may be rotated arcund its other axis — in this way, the light beam may always be directed onto the light detectors, or onto the optimum position on the detectors. The rotation angle of the grating may be regulated by a feedback signal from tne detectors. The grating, which rotates around two axes, may ce replaced by a grating, which rotates around one axis, plus a rotating mirror, or by a fixed grating plus two rotating mirrors.

Figure 3 shows an example of a similar optical setup for measurements at several wavelengths, utilizing fixed optics. Again is shown how light from the light source (1) passes through the sample cell (4), and is detected oy the set of light detectors (7) . A monochromator (8), which distributes the wavelengths along one dimension, is situated between the sample cell and the set of light αetectors. The light source (1) in the figure is supposed to emit polychromatic light. The monochromator (8) is preferably a grating, or an arrangement of a number cf optical components like gratings, filters, and mirrors. The light detector (7) is a two-dimensional detector, i.e. the detector is extended along two dimensions, like e.g. a two- dimensional diode array or a charge transfer device detector, CTD. A CTD may be either a charge coupled device, CCD, or a charge injection device, CID. The detector s positioned m such a way, that one of its axes coincides with the direction of the wavelength separation of the grating, while its other axis coincides with the direction of the refractive index separation of the prismatic sample cell. In this manner, a graph showing refractive index as a function of wavelength is imaged onto the detector. The intensity of light of a certain wavelength is obtained by integrating all the intensity contributions pertaining to this wavelength. In a similar embodiment, the set of light detectors may consist of a number of one-dimensional detectors. In this manner may, e.g., a number of one- dimensional diode arrays be positioned in such a way, that different wavelengths, after being separated by the grating, hit different diode arrays. Different wavelengths may be selected by moving the diode arrays or by rotating the grating. Figure 4 shows a related way to perform measurements at several wavelengths, utilizing movable optics. Once again, the light detector (9) is of the one-dimensional kind, and the grating (10) distributes the wavelengths along a dimension, which is orthogonal to the long axis of the detector. By quickly turning the grating (10) so that all wavelengths are scanned over the detector (9), while simultaneously reading the detector several times, the result is that one refractive index spectrum and one absorbance spectrum are monitored. Some alternative embodiments are, to utilize a rotating mirror in combination with a fixed grating, or to utilize a fixed grating, and instead move the detector quickly through space. In order to make full use of the advantages of multi-wavelength refractometric measurements, it is essential that the wavelengths are scanned quickly, so that any time dependent sources of noise, like e.g. temperature variations of the sample, will not be able to influence the measurement.

An alternative way to perform measurements at different wavelengths is to utilize a light source with variable wavelength, like e.g. a tunable laser.

The different embodiments to perform optical measurements at two or more than two wavelengths, or over a continuous wavelength range, is not limited to the examples described above. Other conceivable embodiments are apparent to the skilled person.

For certain combinations of liquids and wavelengths, the refractive index may vary heavily with wavelength (or, in other words, the refractive index dispersion may be large) , leading to large variations with wavelength of the deflection in the prism. The result may be, that there is not room for the entire refractive index spectrum on the detector. Figure 5 shows an embodiment that solves this problem. In most liquid chromatographic and capillary electrophoretic analyses, the object is to determine the concentration of one or several analytes dissolved in a liquid. The embodiment m figure 5 utilizes a double prism cell (11) . One of the single prism half cells, the sample cell (12), is filled with liquid with dissolved analyte, wnile the other single prism half cell, the reference cell (13) , is filled with the same liquid, but without dissolved analyte. When the light beam passes the double cell (11), the deflection in the sample cell is compensated by the deflection in the reference cell, so the deflection pertaining to the solvent is cancelled out. Only the refractive index contribution, and the corresponding dispersion contribution, pertaining to the analyte, is monitored by the light detector. The dispersion contribution pertaining to the analyte is m general so small, that it does not cause any problem with accommodating the entire spectrum on the light detector. In some alternative embodiments, the reference cell (13) may be filled with another liquid, than that in which the analyte is dissolved, or the reference cell may be a solid prism. The only demand is, that the material should have about the same dispersion as the liquid, m which the analyte is dissolved, so that the refractive index spectrum can be accommodated on the detector. Reference cells may also be utilized m other refractometric techniques, which is apparent to the skilled person.

In order to increase the precision of tne absorbance measurement, e.g. at time dependent intensity variations of the light source, a device for reference measurement of the intensity of the light source may be utilized. Also in this case may a reference cell (preferably filled with liquid) , through which the reference light beam passes before its intensity is measured, be utilized.

The invention is, of course, not restricted to the embodiments specifically described above, or to the embodiments shown in the figures, 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 for optical characterization of a liquid, comprising that light is brought to interact with the liquid, and that said interaction is detected, characterized in that the two characteristic optical properties, the refractive mαex and the absorbance, of th liquid are determined at more than one wavelength.

2. The method according to claim 1, characterized in that tne refractive index and the absorbance are determined at two, or more than two, discrete wavelengths.

3. The method according to claim 1, characterized in that the refractive index and the absorbance are determined ove a continuous range of wavelengths.

4. The method according to any of claims 1 to 3, characterized in that said liquid is a flowing liquid.

5. The method according to any of claims 1 to 4, characterized in that said liquid is a liquid flow m connection with liquid chromatography or capillary e±ectropnoresis.

6. The method according to any of claims 1 to 5, characterized in that the refractive index and the aosoroance are determined simultaneously.

7. The method according to any of claims 1 to 5, characterized in that the refractive index and the absorbance are determined m sequence.

8. The method according to any of claims 1 to 7, characterized in that the light interacts with the same, single volume element of the liquid at the determination of both refractive index and absorbance.

9. The method according to any of claims 1 to 8, characterized in that the volume element of the liquid, with which the light interacts, is physically defined by a sample cell, preferably a flow cell.

10. The method according to any of claims 1 to 9, characterized in that a prismatic sample cell, preferably flow cell, is used for the optical measurement, and that the refractive index determination comprises determination of the deflection of the lignt, and that tne absorbance determination comprises determination of the intensity of the light.

11. The method according to any of claims 1 to 9, characterized in that the refractive index determination is based on Fresnel refractometry, mterferometry, or optical waveguide refractometry.

12. The method according to any of claims 1 to 11, characterized in that a reference sample, preferably a liquid contained in a reference cell, is utilized at the refractive index determination.

13. The method according to any of claims 1 to 12, characterized in that the optical measurement is performed within one, or more than one, of the wavelength ranges UV, VIS, NIR, or IR.

14. An apparatus to carry out tne method according to any of claims 1 to 9, comprising light source (1), sample cell (2), and light detector (3), characterized in that it comprises only one sample cell (2), preferably flow cell, for the determination of both refractive index and absorbance.

15. The apparatus according to claim 14, characterized in that it comprises only one light source (1), or one set of light sources, for the determination of both refractive index and absorbance.

16. The apparatus according to any of claims 14 to 15, characterized in that it comprises only one light detector (3), or one set of light detectors, for the determination of both refractive index and absorbance.

17. The apparatus according to any of claims 14 to 16, characterized in that it comprises a prismatic sample cell (4) .

18. The apparatus according to any of claims 14 to 17, characterized in that it comprises a movaole device, preferably a revolving mirror or a revolving grating, that directs the light onto, or onto the optimum position on, the light detector at variations in the refractive index of the sample (5, 8, 10) .

19. The apparatus according to any of claims 14 to 18, characterized in that the light detector, or the set of light detectors, comprises one, or more than one, unit of the kind photodiode, split diode, position sensitive detector, diode array, or cπarge transfer device (6, 7, 9) .

20. The apparatus according to any of claims 14 to 19, characterized in that it comprises a device, preferably comprising one, or more than one, grating or filter, to select wavelengths, alternatively that it comprises a device, preferably one, or more than one, grating, to distribute the wavelengths along one dimension, or alternatively that it comprises a device, preferably a revolving grating or a revolving mirror, to scan the wavelengths along one dimension (5, 8, 10) .

21. The apparatus according to any of claims 14 to 20, characterized in that it cc orises a reference cell (13)

22. The apparatus according to any of claims 14 to 21, characterized in that it comprises a device for reference measurement of the intensity of the light source.

23. Use of the apparatus according to any of claims 14 to 22 for optical characterization of a liquid flow in connection with liquid chromatography or capillary electrophoresis, characterized in that the two optical properties, the refractive index and the absorbance, of the liquid are determined simultaneously or in sequence.