Method and apparatus for determining optical density of solution

Abstract

To provide an improved method and apparatus for determining optical density of a solution.SOLUTION: A method and an instrument for determining optical density of a solution are disclosed. A flow cell 1 having at least three light paths 4a, 4b, 4c is provided (100), where each light path has a respective predetermined path length, l. Absorbance readings, A, of the solution are taken (400) at the at least three light paths 4a, 4b, 4c. For each pair of light paths, a slope, αc, is calculated (500) by dividing a difference in absorbance reading, ΔA, by a difference in path length, Δl. The calculated slopes, αc, are compared (600), and (a) if the calculated slopes, αc, are the same, the slope is used for determining (700) optical density of the solution, or (b) if the calculated slopes, αc, are not the same, the steepest slope of the calculated slopes is used for determining (701a) optical density of the solution, or a slope of the calculated slopes being in the range of an absorbance reading of 0.01 to 2 is used for determining (701b) optical density of the solution.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] This document relates to a method and an apparatus for determining the optical concentration of a solution.

Background Art

[0002] In spectrophotometry, a sample substance under study is placed in a transparent container (cuvette or flow cell). Electromagnetic radiation of a known wavelength λ (i.e., ultraviolet, infrared, visible light, etc.) and intensity is incident on one side of the cuvette. A detector for measuring the intensity of the emerging light is placed on the opposite side of the cuvette. The length that the light travels through the sample is the distance d. For a sample substance consisting of a single and homogeneous substance with a concentration c, such as a protein, DNA, or RNA, the light transmitted through the sample will follow a relationship known as Beer's law, A = εcl. Here, A is the absorbance, ε is the absorption or attenuation coefficient (usually constant at a given wavelength), c is the concentration of the sample, and l is the path length of the light through the sample.

[0003] Such a system is a so-called fixed path length spectrophotometry system. In order to measure absorbance values within the linear range of the instrument, it is often necessary to dilute the sample. However, when used as a detector in a chromatography or filtration system where a monitor needs to continuously measure absorbance over a long period of time, dilution is often not possible.

[0004] One solution to the dilution problem is to reduce the path length when measuring absorbance. By reducing the measurement path length, the volume of the sample can be reduced. With the decrease in the path length, the measured absorption also decreases proportionally to the decrease in the path length. If the concentration of the sample is outside the linear range of the spectrophotometer, it may still be necessary to dilute the sample, or another cuvette with an even shorter path length may be required, after which an accurate absorbance reading can be obtained.

[0005] Spectrophotometers coupled to flow cells with variable path lengths have become a widely used technique for determining the concentration of sample substances with a wide dynamic range, thereby reducing the need to adjust the concentration of the sample to be within the linear range of absorbance detection of the instrument. Examples of various such systems with variable path lengths are shown in Patent Document 1, Patent Document 2, and Patent Document 3. When using a system with a variable path length and plotting against the path length, the slope of the absorbance curve obtained is a direct measure of the sample substance concentration. Multiple path lengths are measured and the slope is continuously calculated, resulting in one absorbance value per scan cycle. There is no need to know the absolute path length. Although there are many advantages to systems with variable path lengths, such systems may experience problems due to mechanical adjustment of the path length. Furthermore, there is a delay between measurements at different path lengths, which can result in slow response times and inaccurate results at narrow peaks.

[0006] Instead of using a system with a variable path length to expand the linear dynamic range of an absorbance detector, there is a system that uses a fixed multi - optical path length flow cell. See, for example, Patent Document 4. There, when developing the relative absorbance for a sample that exceeds its linear dynamic range, the absorbance of the reference beam in a relatively short reference path is multiplied by the ratio of the absorbance of the sample beam in a relatively long sample path to the reference path absorbance.

[0007] In such an approach, it is necessary to accurately know the path length of each path. Furthermore, it can be difficult to determine whether the short path length is still within the linear dynamic range of the system.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0009] It is an object of the present disclosure to provide an improved or at least alternative method and apparatus for determining the optical concentration of a solution. Accordingly, an invention as defined by the appended independent patent claims is provided. Non-limiting embodiments will emerge from the dependent claims, the accompanying drawings, and the following description.

Means for Solving the Problems

[0010] According to a first aspect, a method for determining the optical concentration of a solution is provided. The method includes providing a flow cell having a solution inlet and a solution outlet, the flow cell being arranged such that the direction of flow of the solution from the solution inlet to the solution outlet passes through each optical path, and each optical path has a predefined path length l. The solution is added to the solution inlet. At least three optical paths are irradiated, the electromagnetic radiation passing through the at least three optical paths is detected, and the absorbance indication A of the solution in the at least three optical paths is read. For each pair of optical paths, a slope αc is calculated by dividing the difference ΔA in the absorbance indication by the difference Δl in the path length. Then, the calculated slopes αc are compared. (a) If the calculated slopes αc are the same, the slope is used to determine the optical concentration of the solution, or (b) if the calculated slopes αc are not the same, the steepest slope among the calculated slopes is used to determine the optical concentration of the solution, or a slope within the range of absorbance indications of 0.01 to 2 among the calculated slopes is used to determine the optical concentration of the solution.

[0011] The absorbance reading is taken in at least three optical paths using a light source disposed on a first side of the optical path and a detector disposed on the opposite side of the optical path. The light used may be light in the visible spectrum, near-infrared spectrum, or ultraviolet spectrum.

[0012] At least three optical paths having a predefined path length l are selected based on the range of the expected optical density OD.

[0013] At least two of the path lengths need to be within the linear dynamic range of the absorbance reading. The linear dynamic range is the range of sample concentrations for which the absorbance reading is linear. By plotting the response of the absorbance reading to the concentrations of different analytes relative to a reference concentration, a straight line over the dynamic linear concentration range will result.

[0014] Beer-Lambert's law is expressed as A = αlc, where A is the measured absorbance, α is the molar extinction coefficient, l is the path length, and c is the concentration of the sample. This equation can then be rearranged as follows for use in slope spectrometry. A / l = αc

[0015] If the calculated slopes are the same, the slope αc is used to determine the optical concentration of the solution. In this case, all the path lengths are considered to be within the linear dynamic range of the absorbance reading. This indicates that all the path lengths are within the acceptable range (defined as the absorbance range between the limit of detection (LOD) and the linear dynamic range (usually a linear limit of 2 AU)).

[0016] If the calculated slopes are not the same, the steepest of the calculated slopes can be used to determine the optical concentration of the solution. The steepest slope is the most accurate slope. At very low concentrations, in this case, the minimum value is close to the LOD, and thus a shallower slope is obtained. If the calculated slopes are not the same, a slope within the range of absorbance readings from 0.01 to 2 of the calculated slopes can be alternatively used to determine the optical concentration of the solution.

[0017] If the calculated slopes are not the same, the first step would be to examine which slope is steeper. Then, as a first alternative embodiment, the steepest slope can be used to determine the OD. The absolute value can be used to verify the validity of the data. Alternatively, as a second alternative embodiment, the absolute value can be used for the determination of the OD. By performing the OD determination based only on the steepest slope, the OD determination may be more uncertain than using the absolute value.

[0018] Determining the optical density (OD) of a solution, as used herein, means determining the OD of the solution and any particles suspended therein.

[0019] Once the optical density of a solution has been determined, and the molar absorptivity E of the substance is already known, the concentration of the solution can be determined by using the Beer-Lambert law (as defined above). This can be done manually or using a computer. Alternatively, the concentration of the solution can be determined by using a dose-response curve previously created for the solution or the substance suspended therein at a given wavelength, such as 280 nm, or multiple response curves generated at different wavelengths can be used. In some applications, what is important is, for example, the change in absorbance during the period of separating proteins in a chromatograph column, and for that purpose, it is not necessary to determine the concentration of the substance. In that case, it is not necessary to know the molar absorptivity (E). By switching to a second less-absorbed light, better resolution of the rate of change of absorption and thus approaching the maximum or minimum value of the resulting concentration value can be achieved, and when the absorbance reaches a threshold value, it is also possible to more closely monitor this change in absorbance by using light of two frequencies.

[0020] In this method, when using a flow cell with a variable path length, there is no risk of moving parts / repositioning problems or leakage. Also, due to the simpler configuration, there is less risk of creating stagnant zones that are difficult to clean. The flow cell used has a plurality of predefined fixed path lengths. In this method, a fast response time is obtained, and it is possible to determine which slope is correct for the determination of the optical density to be based on. If the path length is scanned, it may take several seconds to obtain all the values, and if the concentration changes during that time, the indication will not be accurate. With simultaneous measurements, the values can be obtained at a high frequency.

[0021] The flow cell can comprise at least 4, at least 5, or at least 6 optical paths, each optical path having a predefined path length l respectively.

[0022] By using more optical paths, a larger dynamic range and better data for evaluating which slopes are within the linear dynamic range and which are outside can be obtained. It may also be easier to find a range that is typically within a preferred measurement range between 0.3 AU and 1.5 AU. This is a range that is well above the detection limit (noise level) and where there is still sufficient light reaching the detector (1.5 AU = 3% reaches the detector).

[0023] The absorbance indication A of the sample solution can be read simultaneously from at least three optical paths.

[0024] The timing is directly linked to the flow rate of the solution, along with the flow rate and tube diameter. It is possible to calculate when zones with a certain absorbance reach different optical paths, and then the information can be used to improve the temporal resolution of the measurement. This is commonly done in chromatographic equipment to align results from different sensors (UV, conductivity, pH).

[0025] The absorbance indication A of the solution from at least three optical paths can instead be read continuously.

[0026] The method can further include the step of calculating the delay time between absorbance indications from different path lengths.

[0027] This additional step can be used, for example, when analyzing a solution with a rapid change in absorbance characteristics.

[0028] When comparing the calculated slope αc, (b) if the calculated slopes αc are not the same, the slope within the range of absorbance indications from 0.05 to 1.5 or 0.2 to 1 of the calculated slopes can be used to determine the optical concentration of the solution.

[0029] In one embodiment, at least three optical paths can be irradiated with the same wavelength.

[0030] By using the same wavelength, it is possible to determine the optical density (OD) of an unknown solution and any particles suspended therein.

[0031] In another embodiment, at least one of the at least three optical paths can be irradiated with a wavelength different from the wavelength used to irradiate the other optical paths.

[0032] Such a method can be used, for example, when the optical density (OD) of a solution with a known extinction coefficient is analyzed. When comparing the slopes from different optical paths irradiated with different wavelengths, the differences in the wavelengths used must be corrected.

[0033] Alternatively, different wavelengths can be used during the optimization phase of the method. To identify the optimal wavelength for the irradiation of all optical paths, the optical paths are irradiated with different wavelengths. If the wavelength used for the irradiation of an optical path results in an optical path that is saturated (and thus excluded from the OD determination), this wavelength is not used as the wavelength for irradiating the optical path.

[0034] According to a second aspect, an apparatus for determining the optical concentration of a solution is provided, the apparatus having a flow cell with a solution inlet and a solution outlet, the flow cell comprising at least three optical paths, the direction of flow of the solution from the solution inlet to the solution outlet passing through each optical path, and each optical path being arranged such that each has a predefined path length l. A light source is arranged to irradiate at least three optical paths. A detector is placed on the opposite side of the light source from the optical path, and is arranged such that the detector detects electromagnetic radiation passing through the optical path from the light source. Data processing means are arranged to determine the optical concentration of the solution. The data processing means calculates the absorbance indication A of the solution for at least three optical paths, calculates the slope αc for each pair of optical paths by dividing the difference ΔA in the absorbance indications by the difference Δl in the path lengths, compares the calculated slopes αc, and (a) if the calculated slopes αc are the same, the slope is used to determine the optical concentration of the solution, or (b) if the calculated slopes αc are not the same, the steepest calculated slope is used to determine the optical concentration of the solution, or slopes within the range of absorbance indications of 0.01 to 2 of the calculated slopes are arranged to be used to determine the optical concentration of the solution.

[0035] The light source can comprise a plurality of light emitting units, one for each optical path.

[0036] The light source can be a multiplexed light emitting unit.

[0037] The multiplexing unit can be arranged for fast switching between different optical paths.

[0038] The light source can be a split light source having one channel for each optical path.

[0039] Such a light source can include a beam splitter. For high-quality data, a reference detector may be present. The light is split by the beam splitter so as to cancel out variations in the light source intensity.

[0040] The detector can comprise a plurality of detector units, one for each optical path.

[0041] The detector may be a multiplexed detector unit.

[0042] In such a case, the electromagnetic radiation detected through the optical path is not detected simultaneously, but is detected sequentially at a high speed (much faster than required to monitor the process (shorter than a cycle time of 1 second)).

Brief Description of the Drawings

[0043]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6a

Figure 6b

Figure 6c

Figure 6d

DETAILED DESCRIPTION OF THE INVENTION

[0044] FIG. 5 schematically illustrates a method for determining the optical concentration of a solution by spectrophotometry. The solution can consist of a single and homogeneous substance having a concentration c and can contain, for example, proteins, DNA, or RNA. Determining the optical density (OD) of the solution means, in this specification, determining the OD of the solution and any particles suspended therein. The method includes step 100 of providing a flow cell 1 having a solution inlet 2 and a solution outlet 3. FIG. 2 shows such a flow cell 1 having three optical paths 4a, 4b, 4c (the number of optical paths can be more than 3, such as 4, 5, 6, or more), and the flow cell 1 can be used in an apparatus (not shown) for determining the optical concentration of a solution or a sample / particle in the solution. FIG. 4 shows a cross-section of the flow cell in FIG. 2. FIG. 3 schematically illustrates three irradiation optical paths 4a, 4b, 4c having a fixed path length.

[0045] The optical paths 4a, 4b, 4c can be constituted by separate units each having a predefined fixed path length l that can be interchangeably mounted in the flow cell 1. Such a unit can be, for example, a cuvette. Alternatively, the optical paths 4a, 4b, 4c can be arranged in the flow cell as shown in FIG. 4. The optical path / unit having a predefined path length l is selected based on the expected optical density OD of the range of the solution to be measured. At least two of the path lengths need to be within the linear dynamic range of the absorbance reading. The linear dynamic range is the range of concentrations of a sample for which the absorbance reading is linear. By plotting the response of the absorbance reading to the concentrations of different specimens with respect to the rated concentration, a straight line over the dynamic linear concentration range will result.

[0046] In the second step 200, a solution is added to the solution inlet 2. The flow direction F of the solution added to the flow cell at the solution inlet 2 on the way towards the solution outlet 3 is such that the optical paths 4a, 4b, 4c are arranged to pass through each of the optical paths 4a, 4b, 4c. As shown in FIG. 3, at least three different optical paths 4a, 4b, 4c can be arranged in the flow cell 1 in a direction substantially perpendicular to the flow direction F of the solution flowing from the inlet 2 to the outlet 3 of the flow cell 1. As long as the liquid in the optical path does not form a stagnant zone and the entire volume of the optical paths 4a, 4b, 4c is filled with the liquid / solution, other flow directions F other than perpendicular to the optical path are possible. For example, at least a part of the flow may be parallel to the optical path. If the optical path is short, for example, special measures may have to be taken to adjust the solution flow F, such as by changing the direction of the portion of the solution that has passed through the optical path. When irradiating the optical path, the solution / liquid must still be mixed homogeneously so that the concentration of particles in the solution is approximately the same for all of the at least three optical paths 4a, 4b, 4c.

[0047] As shown in FIGS. 3 and 4, the optical paths 4a, 4b, 4c must be arranged such that the light irradiating one optical path does not irradiate an adjacent separate optical path, i.e., there is no "crosstalk" between the optical paths. Thus, as shown in FIGS. 3 and 4, the optical paths 4a, 4b, 4c may be arranged substantially parallel to each other, or they can be arranged in any other way relative to each other as long as there is no crosstalk between the optical paths. Alternatively, the optical paths 4a, 4b, 4c can be arranged at an angle (e.g., 90 degrees) relative to each other, forming a helical arrangement having a small distance between the optical paths in the z direction. Such an arrangement of the optical paths is smaller than an arrangement having substantially parallel optical paths and will minimize the dead volume of the flow cell.

[0048] At least three optical paths 4a, 4b, 4c can be irradiated simultaneously or continuously at the same wavelength using light sources 5a, 5b, 5c arranged on the first side of the optical paths 4a, 4b, 4c (300). Alternatively, at least one of the at least three optical paths 4a, 4b, 4c can be irradiated at a wavelength different from the wavelength used to irradiate the other optical paths.

[0049] The light used may be light in the visible spectrum, near-infrared spectrum, or ultraviolet spectrum. The electromagnetic radiation passing through the optical path is detected by detectors 6a, 6b, 6c arranged on the opposite side from the light source of the optical path, and the indication A of the absorbance of the solution in at least three optical paths is read (400).

[0050] Absorbance measures the amount of attenuation or the decrease in intensity when light passes through the sample solution. OD measures the amount of attenuation per centimeter path length, and this value is directly related to the concentration here. Scattered light is almost always very small compared to absorbance.

[0051] The light sources 5a, 5b, 5c can each comprise a plurality of light-emitting units for each optical path 4a, 4b, 4c, as shown in FIG. 4. The light sources 5a, 5b, 5c may be multiplexed light-emitting units for fast switching between different optical paths. Alternatively, the light sources 5a, 5b, 5c may be split-type light sources each having one channel for each optical path.

[0052] The detectors 6a, 6b, 6c can each comprise a plurality of detector units for each optical path 4a, 4b, 4c, as shown in FIG. 4. The detectors 6a, 6b, 6c may be multiplexed detector units.

[0053] For each pair of the optical paths 4a, 4b, 4c, the slope αc is calculated by dividing the difference ΔA in the indication of absorbance by the difference Δl in path length (500).

[0054] The principle of slope spectrometry is illustrated in Fig. 1. The Beer-Lambert law is expressed as A = αlc, where A is the measured absorbance, α is the molar absorptivity, l is the path length, and c is the concentration of the sample. This equation can then be rearranged as follows for use in slope spectrometry. A / l = αc

[0055] Regression coefficient analysis can be used to calculate the quality data for the method. For measurements comparing the slope and the path length, the linear regression equation can be written as A = ml + b, where m is the slope of the regression line and b is the y-intercept. Then, by the dimensional equation, the left side of the second equation above can be replaced with the slope term from the third equation, resulting in the following equation. m = αc The resulting equation is called the slope spectrometry equation. At very low sample concentrations, noise can be limited by giving a shallower slope.

[0056] Thereafter, the calculated slope αc is compared (600). (a) If the calculated slopes αc are the same, refer to Fig. 4a, and the slope is used to determine the optical density (OD) of the sample in the solution / solution (700). In this case, it is considered that the entire path length is within the linear dynamic range of the absorbance indication.

[0057] (b) If the calculated slopes αc are not the same, refer to Figs. 4b - 4d, and the steepest slope among the calculated slopes can be used to determine the OD of the solution (701a). Alternatively, if the calculated slopes αc are not the same, a slope within the range of absorbance indications of 0.01 - 2, or 0.05 - 1.0, or 0.2 - 1 among the calculated slopes can be used to determine the optical concentration of the solution (701b).

[0058] If the calculated slopes are not the same, the first step is to examine which slope is steeper. Then, as a first alternative embodiment, the steepest slope can be used to determine OD. The absolute value can be used to verify the validity of the data. Alternatively, as a second alternative embodiment, the absolute value can be used for the determination of OD. By performing OD determination based only on the steepest slope, OD determination may become more uncertain than using the absolute value.

[0059] Figure 4b shows a case where the shortest path length is below the detection limit. Figure 4c shows a case where the longest path length saturates while two shorter path lengths have absorbances of 0.2 - 0.1 AU. Figure 4d shows a case where the shortest path length saturates while a longer path length has an absorbance of 0.2 - 1.5. The saturated path lengths must be excluded from the OD determination.

[0060] When the flow cell has more than three different optical paths, each with a predefined path length l, for each pair of optical paths, the slope αc is calculated (500) by dividing the difference in absorbance readings ΔA by the difference in path lengths Δl. For example, if there are five different optical paths, the number of calculated slopes is four. These slopes are then compared as described above (600), and (a) if the calculated slopes αc are the same, the slope is used to determine the optical concentration of the solution (700).

[0061] (b) If the calculated slopes αc are not the same, referring to Figures 4b - 4d, the steepest of the calculated slopes can be used to determine the OD of the solution (701a).

[0062] Alternatively, if the calculated slopes αc are not the same, a slope within the absorbance indication range of 0.01 to 2 of the calculated slopes can be used to determine the optical concentration of the solution (701b). By using more optical paths, a larger dynamic range and better data can be obtained, and it will be possible to evaluate which slopes are within the linear dynamic range and which are outside.

[0063] When at least three optical paths 4a, 4b, 4c are irradiated with the same wavelength, it is possible to determine the OD of an unknown solution and any particles suspended therein.

[0064] When at least one of at least three optical paths 4a, 4b, 4c is irradiated with a wavelength different from the wavelength used to irradiate the other optical paths, it is possible to analyze the OD of a solution having a known absorbance coefficient. When comparing the slopes from different optical paths irradiated with different wavelengths, the difference in the wavelengths used must be corrected.

[0065] Alternatively, in the method, different wavelengths can be used during the optimization phase. To identify the optimal wavelength for the irradiation of the entire optical paths 4a, 4b, 4c, the optical paths are irradiated with different wavelengths. If the wavelength used for the irradiation of the optical path results in an optical path that is saturated (and thus excluded from the OD determination), this wavelength is not used as the wavelength for irradiating the optical path.

[0066] The data processing means 7 (Figure 2) can be arranged to read the absorbance indications of the solution in at least three optical paths 4a, 4b, 4c (400), calculate the slope αc for each pair of the optical paths 4a, 4b, 4c by dividing the difference ΔA in the absorbance indications by the difference Δl in the path lengths (500), compare the calculated slopes αc (600), and determine the optical concentration of the solution (700, 701a, 701b) as described above.

[0067] The indication A of the absorbance of the sample solution can be read simultaneously from at least three optical paths 4a, 4b, 4c. However, it is not necessary to perform the measurements simultaneously. If the flow / movement of the solution between the locations of the different optical paths is known and can be corrected, the absorbance measurements of the different optical paths 4a, 4b, 4c can be performed sequentially. This will improve the response time.

[0068] The method can further include the step of calculating the delay time between the indications of absorbance from different path lengths 4a, 4b, 4c. This additional step can be used, for example, when analyzing a (sample) solution having a rapid change in absorbance characteristics. When the flow rate of the solution and the tube diameter are known, the transit time of the sample peak between different locations of the different optical paths can be calculated and used to align the results from these optical paths.

Explanation of symbols

[0069] 1 Flow cell 2 Solution inlet 3 Solution outlet 4a Optical path 4b Optical path 4c Optical path 5a Light source 5b Light source 5c Light source 6a Detector 6b Detector 6c Detector 7 Data processing means

Claims

1. A method for determining the optical concentration of a solution, comprising: A step (100) of providing a flow cell (1) having a solution inlet (2) and a solution outlet (3), wherein the flow cell (1) is arranged such that the direction (F) of the solution flow from the solution inlet (2) to the solution outlet (3) passes through each optical path, and each optical path (4a, 4b, 4c) has a predefined path length l, and the step comprises at least three optical paths (4a, 4b, 4c); A step (200) of adding the solution to the solution inlet (2); A step (300) of irradiating the at least three optical paths (4a, 4b, 4c); A step (400) of detecting electromagnetic radiation passing through the at least three optical paths (4a, 4b, 4c) and reading an indication A of the absorbance of the solution in the at least three optical paths (4a, 4b, 4c); A step (500) of calculating a slope αc for each pair of the optical paths (4a, 4b, 4c) by dividing the difference ΔA in the indication of absorbance by the difference Δl in path length; A step (600) of comparing the calculated slopes αc; and (a) When the calculated slopes αc are the same, using the slope for determining the optical concentration of the solution (step 700), or (b) When the calculated slopes αc are within the range of absorbance readings from 0.01 to 2, using the slope for determining the optical concentration of the solution (step 701b); A method characterized by comprising the above steps.

2. The method according to claim 1, wherein the flow cell (1) comprises at least four, at least five, or at least six optical paths (4a, 4b, 4c), and each optical path has a predefined path length l.

3. The method according to claim 1 or 2, wherein the indication A of the absorbance of the solution from the at least three optical paths (4a, 4b, 4c) is read simultaneously.

4. The method according to claim 1 or 2, wherein the indication A of the absorbance of the solution from the at least three optical paths (4a, 4b, 4c) is read continuously.

5. The method according to any one of claims 1 to 4, further comprising a step of calculating a delay time between indications of absorbance from different path lengths.

6. The method according to any one of claims 1 to 5, wherein the at least three optical paths (4a, 4b, 4c) are irradiated at the same wavelength (300).

7. The method according to any one of claims 1 to 5, wherein at least one of the at least three optical paths (4a, 4b, 4c) is irradiated with a wavelength different from the wavelength used to irradiate the other optical paths (300).

8. An apparatus for determining the optical concentration of a solution, comprising a flow cell (1) having a solution inlet (2) and a solution outlet (3), the flow cell (1) comprising at least three optical paths (4a, 4b, 4c), and the direction (F) of the flow of the solution from the solution inlet (2) towards the solution outlet (3) passing through each optical path, and each optical path (4a, 4b, 4c) being arranged such that each optical path has a predefined path length l, the flow cell (1); light sources (5a, 5b, 5c) arranged to irradiate the at least three optical paths (4a, 4b, 4c); detectors (6a, 6b, 6c) placed on the opposite side of the light sources (5a, 5b, 5c) with respect to the optical paths (4a, 4b, 4c), the detectors (6a, 6b, 6c) being arranged to detect electromagnetic radiation that has passed through the optical paths (4a, 4b, 4c) from the light sources (5a, 5b, 5c); data processing means (7) for determining the optical concentration of the solution, the data processing means (7) calculating an absorbance indication A of the solution in the at least three optical paths (4a, 4b, 4c), calculating a slope αc for each pair of optical paths (4a, 4b, 4c) by dividing the difference ΔA in the absorbance indications by the difference Δl in the path lengths, comparing the calculated slopes αc, and (a) if the calculated slopes αc are the same, using the slope to determine the optical concentration of the solution, or (b) if the calculated slopes αc are within a range of absorbance readings from 0.01 to 2, using the slope to determine the optical concentration of the solution; An apparatus, characterized by comprising the above components.

9. The apparatus according to claim 8, wherein the light sources (5a, 5b, 5c) each comprise a plurality of light emitting units, one for each optical path (4a, 4b, 4c).

10. The apparatus according to claim 8 or 9, wherein the light sources (5a, 5b, 5c) are multiplexed light emitting units.

11. The apparatus according to any one of claims 8 to 10, wherein the light sources (5a, 5b, 5c) are split light sources each having one channel for each optical path (4a, 4b, 4c).

12. The device according to any one of claims 8 to 11, wherein the detectors (6a, 6b, 6c) each comprise a plurality of detector units, one for each optical path (4a, 4b, 4c).

13. The device according to any one of claims 8 to 12, wherein the detectors (6a, 6b, 6c) are multiplexed detector units.

14. The device according to any one of claims 8 to 12, wherein the optical paths (4a, 4b, 4c) are arranged at an angle to each other in a helical arrangement.

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