Method and apparatus for determining the optical concentration of a solution

Abstract

A method and apparatus for determining the optical density of a solution is disclosed. A flow cell 1 is provided (100) having at least three optical paths 4a, 4b, 4c, each having a predetermined path length l. Absorbance readings A of the solution are read (400) in the at least three optical paths 4a, 4b, 4c. For each pair of optical paths, a slope αc is calculated (500) by dividing the difference in absorbance readings ΔA by the difference in path length Δl. The calculated slopes αc are compared (600), and (a) if the calculated slopes αc are the same, the slopes are used to determine the optical density of the solution (700), or (b) if the calculated slopes αc are not the same, the steepest of the calculated slopes is used to determine the optical density of the solution (701a), or the calculated slopes that are in the range of 0.01 to 2 absorbance readings are used to determine the optical density of the solution (701b).

Description

Technical Field

[0005]

[0001] This document relates to a method and 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 exiting 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 having a concentration c, such as a protein, DNA, or RNA, the light transmitted through the sample will follow a relational expression 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. <00​​​​​​​​​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 sample concentration to fall within the linear range of the instrument's absorbance detection. Examples of various such systems with variable path lengths are shown in Patent Documents 1, 2, and 3. Using a system with a variable path length, the slope of the absorbance curve obtained when plotted against the path length is a direct measure of the sample substance concentration. Multiple path lengths are measured, and the slope is calculated sequentially, resulting in one absorbance value per scan cycle. There is no need to know the absolute path length. While systems with variable path lengths offer many advantages, such systems may experience problems due to the mechanical adjustment of the path length. Furthermore, there is a delay between measurements at different path lengths, which can lead to inaccurate results with slow response times and narrow peaks.

[0006] Instead of using a system with variable path length to expand the linear dynamic range of an absorbance detector, there is a system that uses a fixed multi-path-length flow cell. See, for example, Patent Document 4. In this system, 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 absorbance of the reference path.

[0007] Such methods require knowing the exact path length of each path. Furthermore, it can be difficult to determine whether short path lengths are still within the system's linear dynamic range. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] U.S. Patent No. 6,747,740 [Patent Document 2] U.S. Patent No. 6,188,474 [Patent Document 3] U.S. Patent No. 7,808,641 [Patent Document 4] U.S. Patent No. 5,214,593 [Overview of the project] [Problems that the invention aims to solve]

[0009] The object of this disclosure is 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 attached independent claims is provided. Non-limiting embodiments arise from the dependent claims, the attached drawings, and the following description. [Means for solving the problem]

[0010] According to a first embodiment, a method for determining the optical density of a solution is provided. The method includes the step of providing a flow cell having a solution inlet and a solution outlet, the flow cell comprising at least three optical paths, the direction of the flow of the solution from the solution inlet to the solution outlet passing through each optical path, and each optical path having a predetermined path length l. The solution is added to the solution inlet. At least three optical paths are irradiated, and electromagnetic radiation passing through at least three optical paths is detected to read the absorbance reading A of the solution in at least three optical paths. For each pair of optical paths, the slope αc is calculated by dividing the difference in absorbance readings ΔA by the difference in path lengths Δl. The calculated slopes αc are then compared. (a) If the calculated slopes αc are the same, the slope is used to determine the optical density of the solution; or (b) If the calculated slopes αc are not the same, the steepest of the calculated slopes is used to determine the optical density of the solution; or the slope among the calculated slopes that falls within the absorbance range of 0.01 to 2 is used to determine the optical density of the solution.

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

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

[0013] At least two of the path lengths must fall within the linear dynamic range of the absorbance readings. The linear dynamic range is the range of sample concentrations over which the absorbance readings are linear. By plotting the absorbance reading responses of different sample concentrations against a rated concentration, a straight line across the dynamic linear concentration range is obtained.

[0014] The Beer-Lambert law is expressed as A = αlc, where A is the absorbance to be measured, 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 for use in gradient spectroscopy as follows: 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 path lengths are considered to be within the linear dynamic range of the absorbance readings. This indicates that all 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 lowest value is close to the LOD, and therefore a shallower slope is obtained. If the calculated slopes are not the same, a slope within the absorbance range of 0.01 to 2 can be used instead to determine the optical concentration of the solution.

[0017] If the calculated slopes are not the same, the first step is to determine which slope is steeper. Then, as a first alternative embodiment, the steepest slope can be used to determine the OD. The validity of the data can be verified using absolute values. Alternatively, as a second alternative embodiment, absolute values ​​can be used to determine the OD. Determining the OD based only on the steepest slope may result in a more uncertain OD determination than using absolute values.

[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 concentration of a solution is determined, and the molar absorbance 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 a 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 by using multiple reaction curves generated at different wavelengths. In some applications, what matters is the change in absorbance during the period of separating proteins in a chromatographic column, for example, and for this purpose, it is not necessary to determine the concentration of the substance. In that case, it is not necessary to know the molar absorbance (E). It is also possible to monitor this change in absorbance in more detail by using light of two frequencies, when the absorbance reaches a threshold, which can be achieved by switching to a second, less absorbent light, thereby achieving better resolution of the rate of change in absorption and consequently approaching the maximum or minimum value of the concentration.

[0020] This method eliminates the risk of moving parts / relocation problems or leaks when using a flow cell with variable path length. Furthermore, the simpler configuration reduces the risk of creating stagnant zones that are difficult to clean. The flow cell used has multiple predetermined fixed path lengths. This method provides a fast response time and allows for determining which slope is correct for determining optical density. When the path length is scanned, it may take several seconds to obtain all values, and if the density changes during that time, the reading will be inaccurate. Simultaneous measurement allows for obtaining values ​​at a high frequency.

[0021] The flow cell may have at least four, at least five, or at least six optical paths, each having a predetermined path length l.

[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 are obtained. It may also be easy to find a range that is usually 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 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 simultaneously read 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 a zone with a certain absorbance reaches different optical paths, and then the information can be used to improve the temporal resolution of the measurement. This is generally done in chromatographic equipment to align the 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 of 0.05 - 1.5 or 0.2 - 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 within it.

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

[0032] Such a method can be used, for example, when analyzing the optical density (OD) of a solution having a known absorbance coefficient. When comparing the gradients from different optical paths irradiated at different wavelengths, the difference in 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 illuminating the entire optical path, the optical path is irradiated with different wavelengths. If the wavelength used for illuminating the optical path results in a saturated path (and therefore excluded from the OD determination), this wavelength is not used for illuminating the optical path.

[0034] According to a second embodiment, an apparatus for determining the optical density of a solution is provided, the apparatus having a flow cell having a solution inlet and a solution outlet, the flow cell having at least three optical paths, the direction of the solution flow from the solution inlet to the solution outlet passing through each optical path, and each optical path having a predetermined path length l. A light source is arranged to illuminate at least three optical paths. A detector is placed on the opposite side of the optical path from the light source and is arranged to detect electromagnetic radiation from the light source through the optical path. Data processing means are arranged to determine the optical density of the solution. The data processing means calculates the absorbance reading A of the solution in at least three optical paths, calculates the slope αc for each pair of optical paths by dividing the difference in absorbance readings ΔA by the difference in path lengths Δl, and compares the calculated slopes αc, such that (a) if the calculated slopes αc are the same, the slope is used to determine the optical density 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 density of the solution, or the slope among the calculated slopes that falls within the absorbance reading range of 0.01 to 2 is used to determine the optical density of the solution.

[0035] The light source can have multiple light-emitting units, one for each optical path.

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

[0037] Multiplexing units can be used for fast switching between different optical paths.

[0038] The light source may be a split-type light source having one channel in each optical path.

[0039] Such a light source may include a beam splitter. A reference detector may also be present for high-quality data. The light is split by the beam splitter to compensate for variations in the light source intensity.

[0040] The detector can be equipped with multiple detector units, one for each optical path.

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

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

[0043] [Figure 1] This diagram illustrates the principle of gradient spectroscopy. [Figure 2] This figure shows a flow cell with multiple fixed path lengths, used in an instrument for determining the concentration of a sample in a solution. [Figure 3] This diagram schematically shows three irradiation light paths with fixed path lengths. [Figure 4] Figure 2 is a schematic cross-sectional view of the flow cell shown. [Figure 5] This diagram schematically illustrates a method for determining the concentration of a sample in a solution. [Figure 6a] This figure shows the calculated slope αc for each pair of optical paths in a flow cell with three optical paths. The slope is calculated for each pair of optical paths by dividing the difference in absorbance readings ΔA by the difference in path lengths Δl. The calculated slopes are the same. [Figure 6b] This figure shows the calculated slope αc for each pair of optical paths in a flow cell with three optical paths. The slope is calculated for each pair of optical paths by dividing the difference in absorbance readings ΔA by the difference in path lengths Δl. The calculated slopes are different. [Figure 6c] This figure shows the calculated slope αc for each pair of optical paths in a flow cell with three optical paths. The slope is calculated for each pair of optical paths by dividing the difference in absorbance readings ΔA by the difference in path lengths Δl. The calculated slopes are different. [Figure 6d]This figure shows the calculated slope αc for each pair of optical paths in a flow cell with three optical paths. The slope is calculated for each pair of optical paths by dividing the difference in absorbance readings ΔA by the difference in path lengths Δl. The calculated slopes are different. [Modes for carrying out the invention]

[0044] Figure 5 schematically illustrates a method for determining the optical density of a solution by spectrophotometry. The solution may consist of a single, homogeneous substance having a concentration c, such as a protein, DNA, or RNA. Determining the optical density (OD) of a solution means 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. Figure 2 shows such a flow cell 1 having three optical paths 4a, 4b, and 4c (the number of optical paths may be more than 3, such as 4, 5, 6, or more), and the flow cell 1 can be used in an instrument (not shown) for determining the optical density of a solution or a sample / particle in a solution. Figure 4 shows a cross-section of the flow cell in Figure 2. Figure 3 schematically illustrates three irradiation optical paths 4a, 4b, and 4c having fixed path lengths.

[0045] The optical paths 4a, 4b, and 4c can be composed of separate units, each having a predetermined fixed path length l, which are interchangeably mounted in the flow cell 1. Such units may be, for example, cuvettes. Alternatively, the optical paths 4a, 4b, and 4c can be arranged in the flow cell as shown in Figure 4. The optical paths / units with predetermined path lengths l are selected based on the expected optical concentration OD of the range of solutions to be measured. At least two of the path lengths must be within the linear dynamic range of absorbance readings. The linear dynamic range is the range of sample concentrations over which the absorbance readings are linear. By plotting the absorbance reading responses of different sample concentrations against a rated concentration, a straight line across the dynamic linear concentration range is obtained.

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

[0047] As illustrated in Figures 3 and 4, optical paths 4a, 4b, and 4c must be arranged such that light illuminating one optical path does not illuminate another adjacent optical path, i.e., there is no "crosstalk" between optical paths. Therefore, as illustrated in Figures 3 and 4, optical paths 4a, 4b, and 4c may be arranged approximately 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, optical paths 4a, 4b, and 4c may be arranged at an angle (e.g., 90 degrees) to each other, forming a helical arrangement with a small distance between optical paths in the z direction. Such an arrangement of optical paths is smaller than an arrangement with approximately parallel optical paths, thus minimizing the ineffective volume of the flow cell.

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

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

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

[0051] As shown in Figure 4, the light sources 5a, 5b, and 5c may be equipped with multiple light-emitting units, one for each optical path 4a, 4b, and 4c. The light sources 5a, 5b, and 5c may be multiplexed light-emitting units for fast switching between different optical paths. Alternatively, the light sources 5a, 5b, and 5c may be split-type light sources having one channel for each optical path.

[0052] As shown in Figure 4, detectors 6a, 6b, and 6c may consist of multiple detector units, one for each optical path 4a, 4b, and 4c. Detectors 6a, 6b, and 6c may be multiplexed detector units.

[0053] The slope αc is calculated by dividing the difference in absorbance readings ΔA between each pair of optical paths 4a, 4b, and 4c by the difference in path lengths Δl (500).

[0054] The principle of gradient spectroscopy is illustrated in Figure 1. The Beer-Lambert law is expressed as A = αlc, where A is the absorbance to be measured, 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 for use in gradient spectroscopy as follows: A / l = αc

[0055] Regression coefficient analysis can be used to calculate quality data about a method. For measurements comparing slope and 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, using the dimensional equation, it becomes possible to replace the left side of the above second equation with the slope term from the third equation, yielding the following equation: m=αc The resulting equation is called the slope spectroscopy equation. At very low sample concentrations, noise can be limited by applying a shallower slope.

[0056] Subsequently, the calculated slopes αc are compared (600). (a) If the calculated slopes αc are the same, refer to Figure 4a, and the slope is used to determine the optical density (OD) of the solution / sample in the solution (700). In this case, it is assumed that all path lengths are within the linear dynamic range of the absorbance readings.

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

[0058] If the calculated slopes are not the same, the first step is to determine which slope is steeper. Then, as a first alternative embodiment, the steepest slope can be used to determine the OD. The validity of the data can be verified using absolute values. Alternatively, as a second alternative embodiment, absolute values ​​can be used to determine the OD. Determining the OD based only on the steepest slope may result in a more uncertain OD determination than using absolute values.

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

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

[0061] (b) If the calculated slopes αc are not the same, refer to Figures 4b to 4d and use the steepest of the calculated slopes to determine the OD of the solution (701a).

[0062] Alternatively, if the calculated slopes αc are not the same, a slope within the absorbance range of 0.01 to 2 among the calculated slopes can be used to determine the optical concentration of the solution (701b). Using more optical paths yields a larger dynamic range and better data, allowing for evaluation of which slopes are within the linear dynamic range and which are outside of it.

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

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

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

[0066] The data processing means 7 (Figure 2) can be configured to read the absorbance readings of the solution in at least three optical paths 4a, 4b, and 4c (400), calculate the slope αc for each pair of optical paths 4a, 4b, and 4c by dividing the difference ΔA of the absorbance readings by the difference Δl of 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 absorbance reading A of the sample solution can be read simultaneously from at least three optical paths 4a, 4b, and 4c. However, simultaneous measurements are not necessary. If the flow rate / motion of the solution between different optical path locations is known and can be compensated for, absorbance measurements at different optical paths 4a, 4b, and 4c can be performed sequentially. This improves the response time.

[0068] The method may further include a step of calculating the delay time between absorbance readings from different path lengths 4a, 4b, and 4c. This additional step can be used, for example, when analyzing a (sample) solution that has a rapid change in absorbance characteristics. Knowing the flow rate of the solution and the tube diameter, the transition time of the sample peak between different locations in different optical paths can be calculated and used to reconcile the results from these optical paths. [Explanation of Symbols]

[0069] 1 flow cell 2 Solution Inlet 3 Solution outlet 4a light path 4b light 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, A step (100) of providing a flow cell (1) having a solution inlet (2) and a solution outlet (3), wherein the flow cell (1) comprises at least three optical paths (4a, 4b, 4c) such that the direction (F) of the flow of solution from the solution inlet (2) to the solution outlet (3) passes through each optical path, and each optical path (4a, 4b, 4c) has a predetermined different path length l; The steps include adding the aforementioned solution to the solution inlet (2) (200), The step (300) of irradiating at least three optical paths (4a, 4b, 4c), Step (400): Detect electromagnetic radiation passing through the at least three optical paths (4a, 4b, 4c) and read the absorbance A of the solution in the at least three optical paths (4a, 4b, 4c); Step (500): Calculate the slope αc for each pair of optical paths (4a, 4b, 4c) by dividing the difference in absorbance readings ΔA by the difference in path lengths Δl; Step (600) of comparing the calculated slope αc; If the calculated slope αc is the same, the step (700) of using the slope to determine the optical concentration of the solution, If the calculated slopes αc are not the same, the step (701a) of using the steepest slope among the calculated slopes to determine the optical density of the solution; A method characterized by including the following.

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 predetermined path length l.

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

4. The method according to claim 1 or 2, wherein the absorbance index A of the solution from 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 the step of calculating the delay time between absorbance readings 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 with the same wavelength (300).

7. The method according to any one of claims 1 to 5, wherein at least one of the 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 instrument for determining the optical concentration of a solution, A flow cell (1) having a solution inlet (2) and a solution outlet (3), wherein the flow cell (1) comprises at least three optical paths (4a, 4b, 4c), 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) is arranged such that each has a predetermined different path length l. A light source (5a, 5b, 5c) is arranged to illuminate the at least three optical paths (4a, 4b, 4c), Detectors (6a, 6b, 6c) are placed on the opposite side of the optical path (4a, 4b, 4c) from the light sources (5a, 5b, 5c), and are arranged to detect electromagnetic radiation that has passed from the light sources (5a, 5b, 5c) through the optical path (4a, 4b, 4c). A data processing means (7) for determining the optical density of the solution, wherein the absorbance index A of the solution is calculated for at least three optical paths (4a, 4b, 4c), the slope αc for each pair of optical paths (4a, 4b, 4c) is calculated by dividing the difference in absorbance index ΔA by the difference in path length Δl, the calculated slopes αc are compared, and (a) if the calculated slopes αc are the same, the slope is used to determine the optical density 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 density of the solution. A device characterized by having the following features.

9. The apparatus according to claim 8, wherein the light sources (5a, 5b, 5c) include one or more light-emitting units in each optical path (4a, 4b, 4c).

10. The apparatus according to claim 8, wherein the light source (5a, 5b, 5c) is a multiplexed light-emitting unit.

11. The apparatus according to claim 8 or 10, wherein the light sources (5a, 5b, 5c) are split-type light sources having one channel in each optical path (4a, 4b, 4c).

12. The apparatus according to any one of claims 8 to 11, wherein the detectors (6a, 6b, 6c) include one detector unit for each optical path (4a, 4b, 4c).

13. The apparatus according to any one of claims 8 to 11, wherein the detectors (6a, 6b, 6c) are multiplexed detector units.

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

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