Method and apparatus for determining optical density of a solution

Abstract

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

Description

Technical Field

[0001] This document relates to methods and apparatus for determining the optical density of a solution. Background Technology

[0002] In spectrophotometry, the sample to be studied is placed in a transparent container (cuvette or flow cell). Electromagnetic radiation of known wavelength λ (i.e., ultraviolet, infrared, visible light, etc.) and intensity are incident on one side of the cuvette. A detector measuring the intensity of the exiting light is placed on the opposite side of the cuvette. The length of light traveling through the sample is d. For a sample composed of a single homogeneous substance with concentration c (e.g., protein, DNA, or RNA), the light transmitted through the sample follows a relationship known as Beer's Law: Where A is the absorbance. is the absorbance or extinction coefficient (which is usually constant at a given wavelength), c is the concentration of the sample, and It is the path length of the light passing through the sample.

[0003] Such a system is called a fixed path length spectrophotometric system. It typically requires sample dilution to measure absorbance values ​​that fall within the instrument's linear range. However, when used as a detector in a chromatography or filtration system, dilution is often not feasible because the monitor needs to continuously measure absorbance over long periods.

[0004] One solution to the dilution problem is to reduce the path length in absorbance measurements. By reducing the measurement path length, the sample volume can be reduced. The reduction in path length also proportionally reduces the measured absorbance. If the sample concentration is outside the linear range of the spectrophotometer, the sample may still need to be diluted before an accurate absorbance reading can be obtained, or an additional tube with an even shorter path length may be required.

[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 sample concentration to fall within the linear range of the instrument's absorbance detection. Examples of different such systems with variable path lengths are shown in US6747740, US6188474, and US7808641. Using a system with variable path lengths, the slope of the absorbance curve obtained when plotted relative to the path length is a direct measure of the sample substance concentration. Measuring multiple path lengths and continuously calculating the slope makes it possible to obtain an absorbance value per scan cycle. There is no need to know the absolute path length. While there are many advantages to systems with variable path lengths, such systems can suffer from problems due to the mechanical adjustment of the path length. There is also a delay between measurements at different path lengths, which can cause slow response times and incorrect results for narrow peaks.

[0006] As an alternative to using a system with variable path length to extend the linear dynamic range of an absorbance detector, there are systems using a fixed multipath flow cell, see, for example, US5214593, in which the relative absorbance of a sample exceeding its linear dynamic range is the ratio of the absorbance of the reference beam in the shorter reference path to the absorbance of the sample beam in the longer sample path to the absorbance of the reference path.

[0007] Using this method, the path length of the corresponding path needs to be completely known. Furthermore, it may be difficult to determine whether the short path length is still within the linear dynamic range of the system. Summary of the Invention

[0008] The purpose of this disclosure is to provide an improved, or at least alternative, method and apparatus for determining the optical density of a solution. Therefore, the invention is provided as defined by the appended independent claims. Non-limiting embodiments emerge from the dependent claims, the drawings, and the following description.

[0009] According to a first aspect, a method for determining the optical density of a solution is provided. The method includes providing a flow cell having a solution inlet and a solution outlet, the flow cell including at least three optical paths arranged such that the solution flows through each optical path in a flow direction from the solution inlet to the solution outlet, wherein each optical path has a corresponding predetermined path length. The solution is added to the solution inlet. At least three optical paths are illuminated, and the electromagnetic radiation passing through at least three optical paths is detected, and the absorbance readings A of the solution at at least three optical paths are acquired. For each pair of optical paths, the difference in absorbance readings is used to... Difference from path length Calculate the slope by division Then, compare the calculated slopes. If the slope calculated in a) If the slope is the same, then the slope is used to determine the optical density of the solution, or if the slope calculated in b) is... If they are not the same, the steepest slope of the calculated slope is used to determine the optical density of the solution, or the slope of the calculated slope in the range of absorbance readings from 0.01 to 2 is used to determine the optical density of the solution.

[0010] Absorbance readings are obtained using a light source positioned on one side of the optical path and a detector positioned on the opposite side of the optical path in at least three optical paths. The light used can be from the visible, near-infrared, or ultraviolet spectra.

[0011] Select a path with a predetermined length based on the expected optical density (OD) range. There are at least three optical paths.

[0012] At least two of the path lengths must be within the linear dynamic range of the absorbance readings. The linear dynamic range is the range of sample concentrations within which the absorbance readings are linear. The response of absorbance readings to different analyte concentrations relative to the nominal concentration should yield a linear response within the dynamic linear concentration range.

[0013] The Beer-Lambert law is expressed as Where A is the measured absorbance. It is the molar absorption coefficient. Where is the path length and c is the sample concentration. This equation can then be rearranged for use with slope spectroscopy: .

[0014] If the calculated slopes are the same, then the slope Used to determine the optical density 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 detection limit (LOD) and the linear dynamic range (typically the linearity limit of 2 AU).

[0015] If the calculated slopes are not identical, the steepest slope can be used to determine the optical density of the solution. The steepest slope is the most accurate slope. For very low concentrations, the lowest value is close to the LOD, thus producing a shallower slope. If the calculated slopes are not identical, the slope of the calculated slope within the range of absorbance readings from 0.01 to 2 can alternatively be used to determine the optical density of the solution.

[0016] If the calculated slopes are not the same, the first step is to check which slope is steeper. The steepest slope can then be used to determine the OD, as in the first alternative. Absolute values ​​may be used to test the validity of the data. Alternatively, as in the second alternative, absolute values ​​may be used to determine the OD. Compared to using absolute values, determining the OD based solely on the steepest slope may yield a more uncertain OD determination.

[0017] Determining the optical density (OD) of a solution here means determining the OD of the solution and any particles suspended in it.

[0018] Once the optical density of the solution has been determined, the concentration of the solution can be determined using Beer-Lambert's law (as defined above), where the molar absorbance E of the substance is known. This can be done manually or using a computer. Alternatively, the concentration of the solution can be determined using a dose-response curve previously generated for the solution or the substance suspended therein at a given wavelength (e.g., 280 nm), or by using multiple response curves generated at different wavelengths. In some applications, such as during the separation of proteins in a chromatographic column, the change in absorbance is of interest, and therefore there is no need to determine the concentration of the substance. In that case, the molar absorbance (E) need not be known. Using two frequencies of light also allows this change in absorbance to be monitored more closely when the absorbance reaches a threshold, where switching to a second, less absorbent light provides better resolution of the rate of change of absorbance and thus closer to the maximum or minimum concentration value.

[0019] This method eliminates the moving parts / repositioning issues and leakage risks associated with flow cells using variable path lengths. Furthermore, due to its simpler construction, it carries a lower risk of creating stagnant areas that are difficult to clean. The flow cell used comprises multiple predetermined fixed path lengths. This method achieves a fast response time and makes it possible to determine which slope is correctly used as the basis for optical density determination. If the path length is scanned, it may take several seconds to obtain all values, and if the concentration changes during that time, the readings will be inaccurate. Simultaneous measurement allows for the acquisition of values ​​at a high frequency.

[0020] The flow cell may include at least four, at least five, or at least six optical paths, each with a corresponding predetermined path length. .

[0021] Using more optical paths will provide a greater dynamic range and better data to assess which slopes are within the linear dynamic range and which are outside it. It may also be easier to find the range within the preferred range of measurements, which is typically between 0.3 AU and 1.5 AU. This is well above the detection limit (noise level) and there is still enough light to reach the detector (1.5 AU = 3% reaching the detector).

[0022] It can simultaneously acquire absorbance readings A of sample solutions from at least three optical paths.

[0023] Timing is directly related to solution flow rate and is directly related to flow rate and tube diameter. It allows calculation of when a region with a certain absorbance reaches different light paths, and this information can then be used to increase the temporal resolution of the measurement. This is typically performed in chromatographic equipment to align results from different sensors (UV, conductivity, pH).

[0024] Alternatively, absorbance readings A of the solution can be acquired consecutively from at least three optical paths.

[0025] The method may further include the following step: calculating the time delay between absorbance readings from different path lengths.

[0026] For example, this additional step can be used when analyzing solutions with rapidly changing absorbance properties.

[0027] When comparing the calculated slopes When, and if the slope calculated in b) If they are not the same, the slope of the calculated slope, which is in the range of absorbance readings of 0.05-1.5 or 0.2-1, can be used to determine the optical density of the solution.

[0028] In one embodiment, at least three optical paths can be illuminated with the same wavelength.

[0029] Using the same wavelength, it is possible to determine the optical density (OD) of an unknown solution and any particles suspended in it.

[0030] In another embodiment, at least one of the three optical paths may be illuminated at a wavelength different from the wavelength used to illuminate the other (one or more) optical paths.

[0031] For example, such a method can be used to analyze the optical density (OD) of a solution with a known absorbance coefficient. When comparing the slopes of different light paths illuminated at different wavelengths, the difference in wavelengths used must be corrected.

[0032] Alternatively, different wavelengths can be used in the method during the optimization phase. The optical path is illuminated with different wavelengths to identify the optimal wavelength for illuminating the entire optical path. If a wavelength used to illuminate the optical path causes saturation (and is therefore excluded from the OD determination), that wavelength will not be used for illuminating the optical path.

[0033] According to a second aspect, an instrument for determining the optical density of a solution is provided, the instrument comprising a flow cell having a solution inlet and a solution outlet, the flow cell comprising at least three optical paths, each optical path being arranged such that the solution flows through each optical path in a flow direction from the solution inlet to the solution outlet, each optical path having a corresponding predetermined path length. A light source is arranged to illuminate at least three optical paths. A detector is located on the side of each optical path opposite to the light source, and the detector is arranged to detect electromagnetic radiation from the light source through the optical paths. A data processing unit is arranged to determine the optical density of the solution. The data processing unit is arranged to: calculate the absorbance A of the solution at at least three optical paths; and for each pair of optical paths, calculate the difference in absorbance readings. Difference from path length Calculate the slope by division And compare the calculated slopes. and a) if the calculated slope If the slope is the same, then the slope is used to determine the optical density of the solution, or b) if the calculated slope If they are not the same, the steepest slope of the calculated slope is used to determine the optical density of the solution, or the slope of the calculated slope in the range of absorbance readings from 0.01 to 2 is used to determine the optical density of the solution.

[0034] The light source may include several optical units, one optical unit for each optical path.

[0035] The light source can be a multiplexed light unit.

[0036] Multiplexed optical units can be arranged for rapid switching between different optical paths.

[0037] The light source can be a split light source, where each optical path has one channel.

[0038] Such a light source can include a beam splitter. For high-quality data, a reference detector can also be present. The beam splitter is used to separate the light, thus compensating for variations in the light source intensity.

[0039] The detector may include several detector units, one detector unit for each optical path.

[0040] The detector can be a multiplexed detector unit.

[0041] In this case, the electromagnetic radiation detected by the optical path is not detected simultaneously, but sequentially at a high speed (much higher than the speed required for the monitoring process (less than 1 second cycle time)). Attached Figure Description

[0042] Figure 1 This illustrates the principle of slope spectroscopy.

[0043] Figure 2 A flow cell with multiple fixed path lengths is shown for use in an instrument for determining the concentration of a sample in a solution.

[0044] Figure 3 The diagram illustrates three illumination light paths with fixed path lengths.

[0045] Figure 4 It is shown in the diagram. Figure 2 The cross-section of the flow cell is shown.

[0046] Figure 5 The diagram illustrates a method for determining the concentration of a sample in a solution.

[0047] Figure 6a The calculated slope is shown for each pair of optical paths in a flow cell with three optical paths. For each optical path pair, the difference in absorbance readings is used to... Difference from path length The slope is calculated by division. The calculated slopes are the same.

[0048] Figure 6b The calculated slope is shown for each pair of optical paths in a flow cell with three optical paths. For each optical path pair, the difference in absorbance readings is used to... Difference from path length The slope is calculated by division. The calculated slopes are different.

[0049] Figure 6c The calculated slope is shown for each pair of optical paths in a flow cell with three optical paths. For each optical path pair, the difference in absorbance readings is used to... Difference from path length The slope is calculated by division. The calculated slopes are different.

[0050] Figure 6d The calculated slope is shown for each pair of optical paths in a flow cell with three optical paths. For each optical path pair, the difference in absorbance readings is used to... Difference from path length The slope is calculated by division. The calculated slopes are different. Detailed Implementation

[0051] exist Figure 5 The schematic diagram illustrates a method for determining the optical density of a solution by spectrophotometry. The solution may, for example, contain a single homogeneous substance, such as protein, DNA, or RNA, having a concentration c. Determining the optical density (OD) of the solution here means determining the OD of the solution and any particles suspended therein. The method includes the step of providing a 100-cell flow cell 1, which includes a solution inlet 2 and a solution outlet 3. Figure 2 The diagram shows a flow cell 1 with three optical paths 4a, 4b, and 4c (the number of optical paths can be more than three, for example, four, five, six, or more), wherein the flow cell 1 can be used in an instrument for determining the optical density of a solution or one or more samples / particles in a solution. Figure 4 What is shown is Figure 2 The cross-section of the flow cell. Figure 3 The diagram shows three illumination paths 4a, 4b, and 4c with fixed path lengths.

[0052] Optical paths 4a, 4b, and 4c can be composed of individual units, each having a predetermined fixed path length. This unit is replaceable and installable within flow cell 1. Such a unit could be, for example, a test tube. Alternatively, optical paths 4a, 4b, and 4c could be as follows: Figure 4 They are arranged in the flow cell as illustrated in the figure. A predetermined path length is selected based on the expected optical density (OD) range of the solution to be measured. The optical path / unit. At least two of the path lengths need to be within the linear dynamic range of the absorbance readings. The linear dynamic range is the range of sample concentrations within which the absorbance readings are linear. The response of absorbance readings to different analyte concentrations relative to the nominal concentration should give a linear response within the dynamic linear concentration range.

[0053] In the second step 200, a solution is added to the solution inlet 2. Optical paths 4a, 4b, and 4c are arranged such that the flow direction F of the solution added to the flow cell at the solution inlet 2 toward the solution outlet 3 passes through each optical path 4a, 4b, and 4c. At least three different optical paths 4a, 4b, and 4c may 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, as shown below. Figure 3As illustrated in the diagram. Other flow directions F, different from those perpendicular to the light path, are possible, provided that the liquid in the light path does not form stagnant regions and that the liquid / solution fills the entire volume of light paths 4a, 4b, and 4c. For example, at least a portion of the flow may be parallel to the light path. If the light path is short, special measures may have to be taken to adjust the flow F of the solution, for example, by redirecting a portion of the solution passing through the light path. The solution / liquid should also be homogeneously mixed such that the concentration of one or more particles in the solution is substantially the same in all at least three light paths 4a, 4b, and 4c when the light path is illuminated.

[0054] like Figure 3 and Figure 4 As illustrated in the diagram, optical paths 4a, 4b, and 4c should be arranged such that light illuminating one optical path does not illuminate any adjacent optical path, i.e., there is no "crosstalk" between optical paths. Therefore, as shown... Figure 3 and Figure 4 As illustrated, optical paths 4a, 4b, and 4c can be arranged substantially parallel to each other, or they can be arranged in any other manner 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 with the minimum distance between them in the z-direction, thus forming a spiral arrangement. Such an arrangement of optical paths will be more compact than an arrangement with substantially parallel optical paths, thereby minimizing the dead volume of the flow cell.

[0055] Light sources 5a, 5b, and 5c can be used to simultaneously or sequentially illuminate at least three optical paths 4a, 4b, and 4c at the same wavelength, said light sources being arranged on a first side of optical paths 4a, 4b, and 4c. Alternatively, at least one of the at least three optical paths 4a, 4b, and 4c can be illuminated at a wavelength different from the wavelength used to illuminate the other(one or more) optical paths.

[0056] The light used can be from the visible, near-infrared, or ultraviolet spectra. Electromagnetic radiation through the optical path is detected by detectors 6a, 6b, and 6c, which are arranged on the side of the optical path opposite to the light source, and the absorbance reading A of the solution is acquired in at least three optical paths.

[0057] Absorbance measures the amount of light attenuation or intensity loss as it passes through a sample solution. OD measures the attenuation per centimeter of path length, and this value is directly related to concentration. Scattered light is usually (most often) very small compared to absorbance.

[0058] Light sources 5a, 5b, and 5c may include several optical units, with one optical unit for each optical path 4a, 4b, and 4c, such as... Figure 4As illustrated in the diagram. Light sources 5a, 5b, and 5c can be multiplexed optical units used for rapid switching between different optical paths. Alternatively, light sources 5a, 5b, and 5c can be split light sources, with one channel for each optical path.

[0059] Detectors 6a, 6b, and 6c may include several detector units, with one detector unit for each optical path 4a, 4b, and 4c, such as... Figure 4 As shown in the diagram. Detectors 6a, 6b, and 6c can be multiplexed detector units.

[0060] For each pair of optical paths 4a, 4b, and 4c, the difference in absorbance readings is used... Difference from path length Divide to calculate the 500 slope .

[0061] The principle of slope spectroscopy is... Figure 1 The diagram in the middle shows the Beer-Lambert law, expressed as follows: Where A is the measured absorbance. It is the molar absorption coefficient. Where is the path length and c is the sample concentration. This equation can then be rearranged for use with slope spectroscopy: .

[0062] Regression coefficient analysis can be used to calculate the quality data used in the method. For comparing measurements of slope and path length, the linear regression equation can be written as... , where m is the slope of the regression line and b is the y-intercept. Dimensional equality allows the slope term from the third equation to be substituted for the left side of the second equation above, resulting in the following equation: The resulting equation is called the slope spectral equation. At very low sample concentrations, noise can be limited by providing a shallower slope.

[0063] Then, compare the slopes calculated by 600. If the slope calculated in a) Same, see Figure 6a The slope is then used to determine the optical density (OD) of the sample in 700 solutions / solution. In this case, all path lengths are considered to be within the linear dynamic range of the absorbance readings.

[0064] If the slope calculated in b) They are different, see below. Figures 6b-6d The steepest slope calculated can then be used to determine the OD of the 701a solution. Alternatively, when the calculated slope... When they are not the same, the slope of the calculated slope, which is in the range of absorbance readings of 0.01 to 2 or 0.05-1.0 or 0.2-1, can be used to determine the optical density of the 701b solution.

[0065] If the calculated slopes are not the same, the first step is to check which slope is steeper. The steepest slope can then be used to determine the OD, as in the first alternative. Absolute values ​​may be used to test the validity of the data. Alternatively, as in the second alternative, absolute values ​​may be used to determine the OD. Compared to using absolute values, determining the OD based solely on the steepest slope may yield a more uncertain OD determination.

[0066] exist Figure 6b The diagram shows cases where the shortest path length is below the detection limit. Figure 6c The diagram shows the case where the longest path length is saturated, while the two shorter path lengths have absorbance of 0.2–0.1 AU. Figure 6d The diagram shows the cases where the shortest path length is saturated, while longer path lengths have an absorbance of 0.2–1.5. Saturated path lengths should be excluded from OD determination.

[0067] When the flow cell includes more than three different optical paths (each optical path has a corresponding predetermined path length) When, for each pair of optical paths, the difference in absorbance readings is used... Difference from path length Divide to calculate the slope of 500 If, for example, there are five different optical paths, then the number of slopes calculated is four. These slopes are then compared as described above, and if a) the slope calculated... If they are the same, the slope is used to determine the optical density of the 700 solution.

[0068] If the slope calculated in b) They are different, see below. Figures 6b-6d The steepest slope calculated can be used to determine the OD of the 701a solution.

[0069] As an alternative, when the calculated slope When they are not identical, the calculated slope, falling within the range of absorbance readings from 0.01 to 2, can be used to determine the optical density of the 701b solution. Using more optical paths will give a larger dynamic range and better data to assess which slopes are within the linear dynamic range and which are outside it.

[0070] When at least three optical paths 4a, 4b, and 4c are illuminated with the same wavelength, it is possible to determine the OD of an unknown solution and any particles suspended therein.

[0071] The OD of a solution with a known absorbance coefficient can be analyzed when at least one of three optical paths 4a, 4b, 4c is irradiated with a wavelength different from the wavelength used to irradiate the other optical paths(s). When comparing the slopes from different optical paths irradiated at different wavelengths, the difference in wavelengths used must be corrected.

[0072] Alternatively, different wavelengths may be used in the method during the optimization phase. The optical paths are illuminated with different wavelengths to identify the optimal wavelengths for illuminating all optical paths 4a, 4b, and 4c. If a wavelength used to illuminate the optical path causes saturation (and is therefore excluded from the OD determination), that wavelength will not be used for illuminating the optical path.

[0073] Data processing unit 7 ( Figure 2 It can be arranged to: acquire absorbance readings 400 of the solution at at least three optical paths 4a, 4b, and 4c; for each pair of optical paths 4a, 4b, and 4c, by measuring the difference in absorbance readings... Difference from path length Divide to calculate the 500 slope Compare the slopes calculated by 600. And determine the optical densities of solutions 700, 701a, and 701b (as described above).

[0074] The absorbance readings A of the sample solution can be obtained simultaneously from at least three optical paths 4a, 4b, and 4c. However, simultaneous measurements are not necessary. If the flow rate / movement of the solution between the positions used for different optical paths is known and can be corrected, absorbance measurements for different optical paths 4a, 4b, and 4c can be performed sequentially. This will improve the response time.

[0075] The method may further include the step of calculating the time delay between absorbance readings from different path lengths 4a, 4b, and 4c. This additional step can be used, for example, when analyzing a (sample) solution with rapidly changing absorbance properties. Given the solution flow rate and tube diameter, the transit times of sample peaks at different locations along different optical paths can be calculated and used to align results from these optical paths.

Claims

1. A method for determining the optical density of a solution, the method comprising: Provided (100) a flow cell (1) having a solution inlet (2) and a solution outlet (3), the flow cell (1) comprising at least three optical paths arranged such that the solution flows from the solution inlet (2) toward the solution outlet (3) through each optical path in a flow direction (F), each optical path having a selected corresponding predetermined path length. ,as well as Add (200) of the sample solution to the solution inlet (2). Illuminate (300) the at least three optical paths, Electromagnetic radiation passing through the at least three optical paths is detected, and the absorbance readings A of the solution at the at least three optical paths are obtained (400). For each optical path pair, the difference in absorbance readings is used... Difference from path length Divide to calculate the slope of (500) , Compare the slope calculated by (600) ,as well as a) If the calculated slope If the slopes are the same, then the slopes are used to determine the optical density of the solution (700). b) If the calculated slope If they are not the same, the steepest slope of the calculated slope is used to determine the optical density of the solution in (701a), or if the slope of the calculated slope is in the range of absorbance readings from 0.2 to 2, that slope is used to determine the optical density of the solution in (701b). Each corresponding optical route is composed of a separate unit, which has a corresponding predetermined fixed path length and is replaceable and installable in the flow pool (1).

2. The method as described in claim 1, wherein, The flow cell (1) includes at least four, at least five, or at least six optical paths, each optical path having a corresponding predetermined path length. .

3. The method as described in claim 1 or 2, wherein, Simultaneously, absorbance readings A of the solution from the at least three optical paths are acquired.

4. The method as described in claim 1 or 2, wherein, The absorbance readings A of the solution are acquired consecutively from the at least three optical paths.

5. The method as described in claim 1 or 2, wherein, The method further includes the following step: calculating the time delay between absorbance readings from different path lengths.

6. The method as described in claim 1 or 2, wherein, When comparing the slope calculated by (600) When, and b) if the calculated slope If they are not the same, the slope of the calculated slope, which is in the range of absorbance readings of 0.2-1, is used to determine the optical density of the solution (701b).

7. The method as described in claim 1 or 2, wherein, Illuminate (300) the at least three optical paths with the same wavelength.

8. The method as claimed in claim 1 or 2, wherein, At least one of the at least three optical paths (300) is illuminated with a wavelength different from the wavelength used to illuminate the other one or more optical paths.

9. An instrument for determining the optical density of a solution, the instrument 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, each optical path being arranged such that the solution flows from the solution inlet (2) toward the solution outlet (3) through each optical path in a flow direction (F), each optical path having a selected corresponding predetermined path length. Each corresponding optical route is composed of a separate unit, which has a corresponding predetermined fixed path length and is replaceable and installable in the flow pool (1). A light source, arranged to illuminate the at least three optical paths. A detector, located on the side of the optical path opposite to the light source, is arranged to detect electromagnetic radiation passing through the optical path from the light source. Data processing unit (7), the data processing unit (7) being used to determine the optical density of the solution, wherein the data processing unit (7) is arranged to: calculate the absorbance readings A of the solution at the at least three optical paths; and for each pair of optical paths, by calculating the difference in absorbance readings... Difference from path length Calculate the slope by division And compare the calculated slopes. and a) if the calculated slope If the slope is the same, then the slope is used to determine the optical density of the solution, b) if the calculated slope If they are not the same, the steepest slope of the calculated slope is used to determine the optical density of the solution, or the slope of the calculated slope in the range of absorbance readings from 0.2 to 2 is used to determine the optical density of the solution.

10. The instrument as claimed in claim 9, wherein, The light source comprises several optical units, with one optical unit for each optical path.

11. The instrument as claimed in claim 9 or 10, wherein, The light source is a multiplexed light unit.

12. The instrument as claimed in claim 9 or 10, wherein, The light source is a split light source, with one channel for each optical path.

13. The instrument as described in claim 9 or 10, wherein, The detector comprises several detector units, with one detector unit for each optical path.

14. The instrument as claimed in claim 9 or 10, wherein, The detector is a multiplexed detector unit.

15. The instrument as claimed in claim 9 or 10, wherein, The optical paths are arranged in a spiral configuration at a certain angle to each other.

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