HU227140B1 - Method and apparatus for determining the absorbtion of weakly absorbing and/or scattering samples - Google Patents

Abstract

Determination of absorption of weakly absorbing and/or scattering liquid sample involves transmitting light of light source into sphere from sample to a detector through a light conducting unit, generating a homogeneous diffuse light for illuminating the sample inside a measuring body and correcting the measured absorbance spectrum using a mathematical model. The measuring body has a wall with a reflecting inner surface and an opening for receiving the sample and an opening or surface not coated with reflecting lining associated with a light source. The absorbance spectrum is measured by illuminating the sample inside the hollow body using the light source and then measuring absorbance spectrum of light received from the sample. An independent claim is included for an apparatus for determining the absorption of weakly absorbing and/or scattering liquid samples.

Description

The present invention relates to a method and apparatus for determining the absorption of poorly absorbent and / or light scattering liquid samples.

In particular, the present invention relates to a method for determining the absorption of poorly absorbent and / or light scattering liquid samples, wherein:

- the sample is filled into a hollow measuring container with a reflecting surface with a permeable aperture and an aperture or non-reflecting surface associated with a light source,

illuminate the sample in the hollow vessel with the light of the light source, and

- measure the absorption spectrum of the sample.

The invention further relates to an apparatus for determining the absorption of poorly absorbent and / or light scattering liquid samples having a hollow measuring cup with a reflective surface, having at least one sample opening aperture and an aperture or non-reflective surface / window. for illuminating a hollow body and transmitting light exiting the hollow body, wherein the aperture or surface / window not covered by the reflecting layer is associated with a light source and a transmitted light detector.

Conventionally, spectrophotometry is a convenient and reliable method for measuring medium absorbing optically pure samples. However, if the sample is only slightly absorbent or is not optically clear, the lack of sensitivity or loss of light scattering will result in a loss of measurement accuracy and will not be able to determine absorption. Important applications such as determination of chlorophyll in the fresh water absorption where borne capable of photosynthesis particles chlorophyll concentration (c) is generally C = 10 ^-9 M ^_1 where klorofillmolekula absorption coefficient ε = 10 ⁵ M ^_1 cm ^_1 and 1 cm light path (I) results in an absorption value of Α = εοΙ = 10 ^-4 , which is much lower than the sensitivity threshold of a conventional spectrophotometer. Another important application is the detection of weekly allowed or forbidden transitions in quantum mechanics and the study of flash or chemically induced absorption transients in dissolved systems.

For low absorbing samples, the only solution at a given concentration is to increase the optical path length of the measuring beam passing through the sample. Except for very long sample holders, which are generally incompatible with modern spectrophotometers, the optical path length can be increased by multipath cuvettes. In fact, such a solution is described by Haggquist GW and Naqvi KR: A Simple Method for Increasing Effective Path Length by Laser Kinetic Spectroscopy, [Review of Scientific Instruments, 65 (7), pp. 2188-2189, July 1994], The authors proposed a rectangular cuvette of 1 cm basic edge length. which is coated on the outside with a reflective layer in the geometric arrangement shown in Figure 3. With this arrangement, the optical path length can be increased up to ten times. However, the technical limitation is that the light beam is reflected from both the mirror and non-mirror surfaces, and the absorption of the cell walls results in unwanted attenuation.

Conventionally, the absorption spectrum of turbid samples is determined by placing a cuvette containing the sample in an Ulbricht integrating sphere, the inner surface of which is coated with a diffuse reflective layer. In this arrangement, a portion of the transmitted and scattered light reaches a detector after multiple reflections (Figure 1). The absorption spectrum of the sample can be determined by measuring the intensity of the detected light as a function of the wavelength of the meter beam. A photometer sphere employing this measurement principle can be found, for example, in U.S. Patent No. 4,310,246. This procedure eliminates measurement errors due to absorption spectral scattering, but is only applicable to samples with moderate absorption.

A major step forward has been the introduction of a spherical cuvette, the outer surface of which, with the exception of a gauge inlet and outlet, and a filling / pouring aperture, is coated with reflective or diffuse reflective material, thereby increasing sensitivity by up to 100 times could be eliminated [I. Ketskeméty, L. Kozma, A method for the spectrophotometric investigation of absorption and fluorescence emission in the media of extremely high peak absorption, Acta Phys. Acad. Sci. Hung. 29, 331-339 (1970) and I. Ketskeméty, L. Kozma, E. Farkas, Absorption and Fluorescence Spectra and Yield of Highly Diluted Solutions, Acta Phys. Chem., Szeged, 16, 7-14, 1970] However, as noted by Ketskeméty and Kozma, repetitive total reflection on the wall leads to leakage at the light inlet and outlet. In addition, as other studies have shown, this improvement has resulted in distortions in the shape of the absorption spectrum measured.

It follows from the foregoing remarks that rectangular multipath cuvettes and Ketskeméty hollow spheres are the most suitable for high sensitivity spectrophotometric measurements.

The object of the present invention is to eliminate the remaining faults in these systems and to provide a correction method which enables the real absorption spectrum to be determined and the measurement procedures to be fitted to modern spetrophotometers, laser equipment and to enable flash photometry and transient spectroscopy. other forms.

According to the present invention, there is provided an improved process wherein

- transferring the light of the light source from the light source into the measuring vessel and from the absorbent and / or light scattering sample to the detector,

generating diffuse light to illuminate the sample inside the vessel, and

correcting the measured absorption spectrum with a correction function.

HU 227 140 Β1

In a preferred embodiment, the correction function (mathematical model) takes into account the geometry and material characteristics of the measuring cell and the absorbance of the sample.

In this context, the invention also provides an apparatus for determining the absorption of poorly absorbent and / or light scattering liquid samples, wherein:

- using a light guide device between the light source or detector and the aperture or transparent window,

- applying a dielectric coating on the reflective layer on the wall of the hollow vessel, and

applying a diffuser to the hollow measuring vessel end of the light guide device.

In a preferred embodiment, the hollow measuring vessel is comprised of two portions bordered by a spherical wall with a reflecting layer and having at least one aperture suitable for filling a sample and for transmitting light into the hollow body and for collecting light from the hollow body. The two parts are joined together to form a hollow body surrounded by a uniform spherical surface.

Alternatively, the light guide device is connected to the hollow measuring cup at the sample loading / unloading opening.

In the apparatus according to the invention, the light guide device connected to the sample loading / unloading opening in the hollow measuring vessel may also be a bifurcated light guide.

According to the invention, the hollow measuring vessel may also be a cuvette having substantially flat side walls and front walls, wherein at least two opposite side walls are provided with a reflecting surface. The cuvette has an opening for loading and evacuating the sample and at least one non-reflective window for illuminating the hollow body and collecting light from the hollow body. A light-guide device is connected to the window and connected to a light source and a detector.

The invention will now be described in more detail with reference to exemplary embodiments of the accompanying drawings, in which:

Figure 1 is a schematic drawing of a conventional integrating sphere, a

Figure 2 is a schematic drawing of an integrating sphere according to the invention, a

Figure 3 is a schematic diagram of a multipath cuvette according to the invention, a

Figure 4 is a schematic diagram illustrating the assembly of parts of an integrating sphere implementing the measurement method of the present invention;

Figure 5 is a schematic diagram of three possible single beam photometer arrangements, a

Figure 6 is a schematic drawing of a dual beam photometer arrangement, a

Figure 7 is a schematic drawing of a dual wavelength photometer array and

Figure 8 is a schematic diagram of a dual beam photometer arrangement with a light chopper and a monochromator.

The modification of the conventional arrangement of the IS hollow measuring vessel shown in Figure 1, such as an integrating sphere (e.g., the sphere used by Ketskeméty et al., Previously described in detail), is shown in Figure 2. As a result of the improvement, the conventional sphere used by Ketskeméty et al. Is provided with an appropriate coating on the outer or inner surface which forms a diffuse or reflective reflective surface. According to the invention, a dielectric protective layer is disposed on the reflective layer on the outer or inner surface of the hollow body wall. The reflective layer may be any reflective metal such as silver or aluminum. For example, the dielectric layer may be a composite layer of two dielectric layers having different refractive indexes. The two layers can be used in pairs of alternating layers, where the number of pairs is up to ten or, if necessary, up to 100. The use of inner surface layers results in greater sensitivity, but due to direct and continuous contact with the liquid sample, the protective layer must be adequately protected against mechanical and chemical corrosion. When using external coatings, the sensitivity is lower due to the losses caused by the wall of the hollow measuring vessel, but only mechanical protection is required because the coating is not constantly in contact with chemicals.

With these devices, in the case of an internal reflecting surface, undesirable optical effects (absorption, reflection, refraction, polarization distortion, etc.) can be eliminated at the wall of the measuring vessel.

Another result of this development is the use of an LG light guide (fiber optic cable) between the opening for loading and evacuating the liquid sample in the hollow vessel and the LS light source illuminating the liquid sample in the hollow vessel. Instead of an opening in the hollow vessel for loading and discharging the liquid sample, a transparent surface (window) may be used to illuminate the liquid sample in the hollow vessel. In this case, the illuminating device does not need to be removed while the IS hollow vessel is being loaded or emptied. A second LG light guide is used between a second aperture or transparent surface (window) and a detector. These light guiding devices allow the LS light source and the D detector to be even further away from the measuring cup. In addition, the flexibility of the light guide allows the measuring vessel to be moved relative to the LS light source and D detector. When using a BLG double-ended (bifurcated) conductor (Figures 4-8), instead of two apertures or windows (transparent surface), only one aperture or window is required to connect the common end of the dual-ended BLG. This allows for simpler design and construction and at the same time enhances the efficiency of the apparatus of the invention.

According to the invention, it is important that the light illuminating the liquid sample provides a homogeneous distribution of photon gas within the vessel. To this end,

A DP diffuser is positioned at the light entry point of the 140 Β1 cavity. For example, the diffuser DP is a polygonal prism fixed at the end of the fiber optic cable facing the hollow measuring vessel.

The sensitivity of the absorbance measurements can be increased by up to three orders of magnitude using the integrating sphere of the present invention, wherein the measuring beam suffers multiple reflections inside the measuring vessel. The light used for measurement can be continuous or intermittent (for example, a series of flashes).

The Heggquist and Naqvi MPC multiplier cuvettes may also be further improved by applying a dielectric protective layer to the reflective layer RL on the inner or outer surface of the cuvette (Figure 3). In this way, undesirable optical effects (absorption, reflection, refraction, polarization distortion, etc.) can be eliminated on the inner mirror surface. For non-scattering patterns, much longer light path lengths can be achieved by using a highly collimated light beam, such as an LS light source emitting a laser beam, and by selecting the entrance window, exit window, and angle of incidence. The proper design of the output beam enables the use of polychromators and multi-channel D detectors, which simplifies the use and / or use of the apparatus with commercially available diode-array spectrophotometers or flash light lysis apparatus.

Figure 4 illustrates the steps of assembling portions of a hollow measuring vessel according to the invention. In the first step (on the left) two hemispheres of the same size are shown, one of which has a filling / discharge opening (upper part 1) to which a short loading / discharge tube is connected. Both the upper part 1 and the lower part 2 are provided with a diffuse or reflective reflective layer and a dielectric protective layer on its inner or outer surface. In some embodiments, it may be advantageous to leave one or more windows (transparent part) on the reflecting surface that allow light to pass through.

In the second step (center), the upper and lower parts 1 and 2 are joined with their open sides facing each other and the parts are permanently fixed to each other by gluing, tight sealing or the like. The integrating sphere can then be filled with the liquid sample through the filler orifice.

In the third step (right), a double-ended (bifurcated) BLG light guide is inserted into the sphere. A diffuser prism DP is connected to the fitting (common) end of the BLG light guide to ensure a homogeneous distribution of photon gas in the vessel. On the other side of the double-ended light guide, one end is connected to the LS light source illuminating the liquid sample in the hollow vessel and the other end to the detector D receiving the reflected and / or scattered light. The illumination can be continuous or intermittent (for example, flash series).

5A-5C. Figures 5A show three different single-beam photometer arrangements: 5A. conventional photometer with a monochromator, 5B. conventional photometer with a polychromator and diode array, 5C. conventional photometer with dual monochromator.

5A: The single-beam spectrophotometer has a L source, an associated collimator optic, a dispersion element (prism or diffraction grating), and an aperture or slit to select a small bandwidth, i.e., quasi monochromatic light. The illumination light enters the IS hollow vessel through a BLG light guide. The absolute intensity of the output light is measured by a photodetector D (photoelectron multiplier or photodiode). The photodetector D is connected to a data acquisition and processing unit 5. The data acquisition and processing unit 5 also controls the light source L and the monochromator M. The change in absorbance induced by light can also be measured by using a flashlight or other actinic (e.g., photochemical) illumination, the light of which is passed through an additional 4 light conductors (gray line). For measuring chemically induced changes in absorption, the same loading aperture can be used to inject or inject different reagents.

5B: A similar arrangement as described above. We use wide band illumination. The spectral resolution is provided by a combination of a PCH polychromator and a DA diode array detector on the detection side. Illumination can be continuous or intermittent. In the latter case, the DA diode array detector measures the intensity synchronously with the light output of the flashlight. The necessary synchronization is carried out by the data acquisition and processing unit 5, which is connected to the diode field and the light source L. Light-induced absorption changes can also be measured, for example, by using an additional flash between two control (monitoring) flashes (dashed vertical line). Again, the light is guided to and from the IS hollow vessel using a BLG fiber optic cable.

5C .: With this layout, it is possible to perform three different types of measurements.

(i) Conventional absorption spectra of non-fluorescent samples can be measured by simultaneously scanning the M1 and M2 monochromator on the illumination and detection sides. In this case, the monochromators are set to the same wavelength. As shown, monochromator M1 is positioned between collimator lenses C1 and C2 and monochromator M2 is positioned between collimator lenses C3 and C4.

(ii) The emission spectra of the fluorescent samples can be measured by recording the wavelength of illuminating light (by setting the M1 monochromator) over a defined range where the sample is capable of absorbing light while the M2 monochromator positioned on the detection side scans the wavelength of light emitted by the fluorescent sample.

(iii) The excitation spectrum of the fluorescent samples can be measured by detecting the wavelength of detected light (by setting M2 monochromator) at a specified value, where the sample emits fluorescent light while changing the wavelength of illumination light by scanning a monochromator M1

EN 227 140 Β1, where the sample is capable of absorbing light. The M1 and M2 monochromator and the D detector are connected to 5 data acquisition and processing units.

Figure 6 is a diagram of a dual beam photometer arrangement. In the drawing, the light beam of the light source L passes through the collimator lenses C1, C2 and the monochromator M, then passes to the beam divider B. The beam splitter B divides the measuring beam, which then passes through the sample and a reference beam RB. Since the light beam passing through the sample and the reference path does not merge, two D1 and D2 detectors are needed to measure each intensity. By inserting and adjusting a damping element 3 (an additional integrating sphere or a neutral gray filter) on the reference path, the intensity of the beam of the sample beam SB and the reference beam RB can be adjusted to a similar range. This makes the intensity measurement more reliable, so that the same sensitivity range of the detectors can be applied on the SB and RB reference beams. The change in absorbance induced by light can also be measured by passing a second light-guide cable, for example, a photochemical (actinic) illumination (e.g. a series of flashes) into the measuring vessel (gray line). In this arrangement, the data acquisition and processing unit 5 is connected to the detectors D1, D2 and the light source L.

Figure 7 is a schematic diagram of a dual wavelength photometer array. In this arrangement, the light of the light source L passes through a collimator lens C and is then transmitted to a rotating mirror or chopper CH, which is applied to each of the monochromators M1 and M2, alternating several times per second. The beam of light passing through the monochromators M1 and M2 is combined by the beam junction BC, so that both beams of light are alternately transmitted to the IS hollow vessel containing the sample. Since the measurement is made on the combined avenue, a D detector is sufficient to measure each intensity. The difference in sample absorbance is measured at two selected wavelengths, defined by M1 and M2 monochromators. Dual-wavelength spectroscopy is performed automatically by alternating two incident wavelengths of light. In this case, the wavelength of one beam is fixed to a specific value while the wavelength of the other beam is scanned over a defined wavelength range. The change in absorbance induced by light can also be measured by passing a second light guide cable 4, for example, to actinic illumination (for example, a series of flashes) which produces a photochemical effect (gray line). As with other configurations, the change in absorbance due to the addition of chemical reagents can be measured using an additional dosing / injection opening in the measuring vessel. In this arrangement, the data acquisition and processing unit 5 (analyzer and control unit) is connected to the detector D, the monochromator M1, M2, the rotary mirror CH and the light source L and controls them according to a measuring program.

Figure 8 is a schematic diagram of a dual-beam photometer assembly with a rotary chopper CH and a monochromator M. In the dual-beam spectrophotometer, light passes through collimator lenses C1, C2, monochromator M and is rotated by the rotating mirror CH several times per second in the direction of the sample and a damping element 3. The beam of light passing through the sample path SB and the reference path RB is combined by the beam junction BC. Since the measurement is on the unified avenue, a D detector is sufficient to measure the intensity. By insertion and adjustment of a damping element 3 on the reference radius RB, the intensity of the sample radius SB and that of the reference radius RB can be adjusted to approximately the same range. This makes the intensity measurement more reliable, so that the same sensitivity range of the D detectors can be used to measure the intensity of the sample beam and the reference beam. Similarly to other arrangements, a change in absorption caused by light or a chemical can be measured, for example, by passing a second light guide cable 4 (e.g., a gray line) or introducing a chemical reagent into the measuring vessel. In this arrangement, the data acquisition and analyzer unit is connected to detector D1, rotary mirror CH, monochromator M, and light source L.

Since, in the various embodiments, the signal from the sample and transformed by the detector is distorted in the measurement spectrum, it is necessary to correct the distortion during the measurement procedure in order to obtain the correct absorption spectrum.

To this end, a mathematical correction is made in the process of the invention, whereby the empirically determined parameters depend on the geometry and material constant of the measuring cell (cell) and on the absorbance of the sample. We have found that there are at least two simple relationships that allow conversion of measured absorbance to real values.

Let Α * (λ, Ό) be the absorbance measured at wavelength and C the concentration of the liquid sample. It was found that the concentration dependence of the measured absorption can be modeled by the following expression aC

A'fcC) .— (1)

With the following limits:

(2)

By rewriting formula (1)

It follows from formula (4) that

HU 227 140 Β1

For known A * and values, we can determine the true value of A absorption. The real absorption spectra thus calculated, after concentration-dependent normalization, can be overlapped as experimentally verified.

Claims (9)

PATENT CLAIMS 1. A method for determining the absorbance of poorly absorbent and / or light scattering liquid samples, comprising: - the sample is filled into a hollow measuring vessel (IS) having a reflective surface wall having a sample passage aperture and an aperture or non-reflecting surface associated with a light source (L, LS), illuminating the sample in the hollow flask with light from the light source (L, LS), and - measuring the absorbance spectrum of the sample by measuring the change in the intensity of light transmitted, - transferring the light from the light source (L, LS) from the light source (L, LS) to the detector (D) through a light guide (LG, BLG) and from the absorbent and / or light scattering sample, generating diffuse light to illuminate the sample inside the dish, and correcting the measured absorption spectrum with a correction function. 2. The method of claim 1, wherein the sample is illuminated by a flash. 3. The method of claim 1, further comprising adding a chemical to the sample. 4. A method according to any one of claims 1 to 5, characterized in that the correction function includes dependence on the geometry and material constant of the vessel and on the absorption of the sample. 5. Apparatus for determining the absorption of poorly absorbent and / or light scattering liquid samples having a hollow measuring cup (IS) having a reflecting surface, having at least one sample-permeable opening, and an opening or a non-reflecting surface / window in the hollow. for illuminating the body and transmitting light emitted from the hollow body, wherein the aperture or surface / window not covered by the reflecting layer is associated with a light source (L, LS) and a transmitted light detector (D), characterized in that: - a light guide (LG, BLG) is used between the light source (L, LS) or the detector (D) and the aperture or transparent window, - applying a dielectric coating on the reflective layer on the wall of the hollow measuring vessel (IS), and - a diffuser (DP) is used at the end of the light guide (LG, BLG) hollow measuring vessel (IS). Apparatus according to claim 5, characterized in that the hollow measuring vessel (IS) consists of an upper and a lower part (1, 2), bounded by a reflective layer, such as a spherical wall, and on which at least one sample for filling and filling, and an opening for receiving light into the hollow body and for receiving light from the hollow body (LG, BLG), wherein the upper and lower portions (1,2) are joined together to form a uniform hollow body, e.g., bounded by a spherical surface. Apparatus according to claim 6, characterized in that the light guide (LG, BLG) is connected to an opening for filling and filling the sample in the hollow measuring vessel (IS). Apparatus according to claim 7, characterized in that a light-guide double-ended (bifurcated) light-guide (BLG) is connected to the hollow measuring vessel (IS) at an opening suitable for filling and filling the sample. Apparatus according to claim 5, characterized in that the hollow measuring vessel is a path multiplication cuvette (MPC) having a substantially flat side wall and an end wall, wherein at least two opposite side walls are provided with reflecting surfaces (RL); the multiplier (MPC) cuvette has an aperture for loading and / or evacuating the sample and at least one non-reflective window for illuminating the hollow body and collecting light from the hollow body, wherein the light is connected to a light source (LG) LS) and on the other hand it is connected to a detector (D).