METHOD AND APPARATUS FOR DETERMINING OPTICAL DENSITY OF A SOLUTION

Abstract

A method and instrument for determining optical density of a solution is disclosed. A flow cell 1 having at least three light paths (4a, 4b, 4c) is provided (100), wherein each light path has a respective predetermined path length, l. Absorbance readings are taken (400), A, of the solution 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, with 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 he 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 the 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

Claims

1. A method for determining optical density of a solution, the method comprising: providing a flow cell having a solution inlet and a solution outlet, the flow cell comprising at least three light paths arranged such that a direction of flow of a solution from the solution inlet towards the solution outlet passes through each light path, each light path having a respective predetermined path length, l, and adding the sample solution to the sample solution inlet, irradiating the at least three light paths, detecting electromagnetic radiance going through the at least three light paths and taking absorbance readings, A, of the solution at the at least three light paths, for each pair of light paths calculating a slope, αc, by dividing a difference in absorbance reading, ΔA, with a difference in path length, Δl, comparing, the calculated slopes, αc, and a) if the calculated slopes, αc, are the same, using the slope for determining optical density of the solution, or b) if the calculated slopes, αc, are not the same, using the steepest slope of the calculated slopes for determining optical density of the solution, or if the slope of the calculated slopes is in the range of an absorbance reading of 0.01 to 2 then using that slope for determining optical density of the solution.

2. The method of claim 1, wherein the flow cell comprises at least four, at least five or at least six light paths, each light path having a respective predetermined path length, l.

3. The method of claim 1, wherein absorbance readings, A, of the solution from the at least three light paths are taken simultaneously.

4. The method of claim 1, wherein absorbance readings, A, of the solution from the at least three light paths are taken successively.

5. The method of claim 1, wherein the method further comprises a step of calculating a delay time between absorbance readings from the different path lengths.

6. The method of claim 1, wherein when comparing the calculated slopes, αc, and b) if the calculated slopes, αc, are not the same, the slope of the calculated slopes being in the range of an absorbance reading of 0.05-1.5, or 0.2-1, is used for determining optical density of the solution.

7. The method of claim 1, wherein the at least three light paths are irradiated at the same wavelength.

8. The method of claim 1, wherein at least one of the at least three light paths is irradiated at a wavelength which is different from the wavelength used to irradiate the other light path(s).

9. An instrument for determining optical density of a solution, the instrument comprising: a flow cell having a solution inlet and a solution outlet, the flow cell comprising at least three light paths, each light path being arranged such that a direction of flow of a solution from the solution inlet towards the solution outlet passes through each light path, each light path having a respective predetermined path length, l, and a light source arranged to irradiate the at least three light paths, a detector located at an opposite side of the light paths from the light source, the detector arranged to detect electromagnetic radiation going through the light paths from the light source, a data processing means for determining optical density of the solution, wherein the data processing means is arranged to calculate the absorbance, A, of the solution at the at least three light paths, and to calculate a slope, αc, for each pair of light paths by dividing a difference in absorbance reading, ΔA, with a difference in path length, Δl, and comparing the calculated slopes, αc, and a) if the calculated slopes, αc, are the same, the slope is used for determining 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 optical density of the solution, or the slope of the calculated slopes being in the range of an absorbance reading of 0.01 to 2 is used for determining optical density of the solution.

10. The instrument of claim 9, wherein the light source comprises several light units, one for each light path.

11. The instrument of claim 9, wherein the light source is a multiplexing light unit.

12. The instrument of claim 9, wherein the light source is a split light source with one channel for each light path.

13. The instrument of claim 9, wherein the detector comprises several detector units, one for each light path.

14. The instrument of claim 9, wherein the detector is a multiplexing detector unit.

15. The instrument of claim 9, wherein the light paths are arranged at an angle with respect to one another in a spiral arrangement.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIG. 1 shows the principle of slope spectrometry.

[0044] FIG. 2 shows a flow cell with multiple fixed pathlengths for use in an instrument for determining concentration of a sample in solution.

[0045] FIG. 3 shows schematically three irradiated light paths with fixed pathlength.

[0046] FIG. 4 shows schematically a cross section of the flow cell shown in FIG. 2.

[0047] FIG. 5 schematically illustrates a method for determining the concentration of a sample in solution.

[0048] FIG. 6a shows calculated slopes, αc, for each pair of light paths in a flow cell with three light paths. The slopes being calculated by dividing a difference in absorbance reading, ΔA, with a difference in path length, Δl, for each pair of light paths. The calculated slopes being the same.

[0049] FIG. 6b shows calculated slopes, αc, for each pair of light paths in a flow cell with three light paths. The slopes being calculated by dividing a difference in absorbance reading, ΔA, with a difference in path length, Δl, for each pair of light paths. The calculated slopes being different.

[0050] FIG. 6c shows calculated slopes, αc, for each pair of light paths in a flow cell with three light paths. The slopes being calculated by dividing a difference in absorbance reading, ΔA, with a difference in path length, Δl, for each pair of light paths. The calculated slopes being different.

[0051] FIG. 6d shows calculated slopes, αc, for each pair of light paths in a flow cell with three light paths. The slopes being calculated by dividing a difference in absorbance reading, ΔA, with a difference in path length, Δl, for each pair of light paths. The calculated slopes being different.

DETAILED DESCRIPTION

[0052] In FIG. 5 is schematically illustrated a method for determining optical density of a solution by means of spectrophotometry. The solution may e.g. contain proteins, DNA or RNA, consisting of a single homogeneous substance with a concentration c. Determining optical density (OD) of a solution is here meant determining the OD of the solution and any particles suspended therein. The method comprises a step of providing 100 a flow cell 1 comprising a solution inlet 2 and a solution outlet 3. In FIG. 2 is shown such a flow cell 1 with three light paths 4a, 4b, 4c (the number of light paths may be more than three, such as four, five, six or more), wherein the flow cell 1 may be used in an instrument (not illustrated) for determining the optical density of a solution or sample(s)/particle(s) in the solution. In FIG. 4 is shown a cross section of the flow cell in FIG. 2. In FIG. 3 is schematically illustrated three irradiated light paths 4a, 4b, 4c with fixed pathlength.

[0053] The light paths 4a, 4b, 4c may be constituted by separate units with a respective predetermined fixed pathlength, l, replaceable mountable in the flow cell 1. Such a unit may be e.g. be a cuvette. Alternatively, the light paths 4a, 4b, 4c may be arranged in the flow cell as illustrated in FIG. 4. The light paths/units with predetermined path lengths, l, are chosen based on the expected optical density, OD, 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 readings. The linear dynamic range being the range of sample concentrations over which the absorbance readings are linear. Plotting the response of absorbance readings of different analyte concentrations against nominal concentration should give a straight line over the dynamic linear concentration range.

[0054] In a second step 200, the solution is added to the solution inlet 2. The light paths 4a, 4b, 4c are arranged such that a direction of flow F of a solution added to the flow cell at the solution inlet 2 on its way towards the solution outlet 3 passes through each light path 4a, 4b, 4c. The at least three different light paths 4a, 4b, 4c may be arranged in the flow cell 1 in a direction substantially perpendicular to the direction of flow F of the solution flowing from the inlet 2 towards the outlet 3 of the flow cell 1, as illustrated in FIG. 3. Other directions of the flow F than perpendicular to the light paths are possible as long as the liquid in the optical path is not forming a stagnant zone and as long as the liquid/solution fills the entire volume of the light path 4a, 4b, 4c. For example, at least part of the flow could be parallel with the light path. If the light path is short, special measures may have to be taken to adjust the flow F of the solution by for example redirecting part of the solution past the light path. The solution/liquid should also be homogenously mixed such that the concentration of particle(s) in the solution is substantially the same in all the at least three light paths 4a, 4b, 4c, when irradiating the light paths.

[0055] As illustrated in FIGS. 3 and 4 the light paths 4a, 4b, 4c should be arranged such that light irradiating one light path does not irradiate a separate neighbouring light path, i.e. there is no “crosstalk” between the light paths. Hence, as illustrated in FIGS. 3 and 4, the light paths 4a, 4b, 4c could be arranged substantially parallel to each other or they may be arranged in any other way in relation to each other as long as there is no crosstalk between light paths. Alternatively, the light paths 4a, 4b, 4c could be arranged at an angle (e.g. 90 degrees) to each other with a minor distance between light paths in the z-direction, forming a spiral arrangement. Such an arrangement of light paths would be more compact than an arrangement with substantially parallel light paths, minimizing flow cell dead volume.

[0056] The at least three light paths 4a, 4b, 4c may be irradiated 300 at the same wavelength, simultaneously or successively, using a light source 5a, 5b, 5c arranged at a first side of the light path 4a, 4b, 4c. Alternatively, at least one of the at least three light paths 4a, 4b, 4c may be irradiated at a wavelength which is different from the wavelength used to irradiate the other light path(s).

[0057] The light used may be light in the visible spectrum, in the near infrared spectrum or in the ultraviolet spectrum. Electromagnetic radiation going through the light paths is detected by a detector 6a, 6b, 6c arranged at an opposite side of the light paths from the light source and absorbance readings, A, of the solution taken 400 at the at least three light paths.

[0058] Absorbance measures the amount of attenuation, or intensity loss, when light passes through the sample solution. OD measures the amount of attenuation per centimetre pathlength and this value is then directly related to concentration. Scattered light is most often very small compared to absorbance.

[0059] The light source 5a, 5b, 5c may comprise several light units, one for each light path 4a, 4b, 4c, as illustrated in FIG. 4. The light source 5a, 5b, 5c may be a multiplexing light unit for fast switching between different light paths. Alternatively, the light source 5a, 5b, 5c may be a split light source with one channel for each light path.

[0060] The detector 6a, 6b, 6c may comprise several detector units, one for each light path 4a, 4b, 4c, as illustrated in FIG. 4. The detector 6a, 6b, 6c may be a multiplexing detector unit.

[0061] For each pair of light paths 4a, 4b, 4c a slope, αc, is calculated 500 by dividing a difference in absorbance reading, ΔA, with a difference in path length, Δl.

[0062] The principle of slope spectroscopy is illustrated in FIG. 1. The Beer-Lambert law is expressed as A=αlc, where A is the measured absorbance, α is the molar absorption coefficient, l is the pathlength, and c is the sample concentration. This equation can then be rearranged for use with slope spectroscopy: A/l=αc.

[0063] Regression coefficient analysis may be used to calculate quality data for the method. For measurements comparing slope and pathlength, a linear regression equation may be written as A=ml +b, where m is the slope of the regression line, and b is the y-intercept. Dimensional equality then allows for replacement of the left-hand side of the second equation above with the slope term from the third equation, yielding the following: m=αc. That resulting equation is referred to as the slope spectroscopy equation. At very low sample concentrations noise may be limiting by giving shallower slopes.

[0064] Thereafter the calculated slopes, αc, are compared 600. If a) the calculated slopes, αc, are the same, see FIG. 4a, the slope is used for determining 700 the optical density (OD) of the solution/sample in solution. In this case all path lengths are considered to be within the linear dynamic range of the absorbance readings.

[0065] If b) the calculated slopes, αc, are not the same, see FIGS. 4b-4d, the steepest slope of the calculated slopes may be used for determining 701a the OD of the solution. As an alternative, when the calculated slopes, αc, are not the same, the slope of the calculated slopes being in the range of an absorbance reading of 0.01 to 2, or 0.05-1.0, or 0.2-1, may be used for determining 701b optical density of the solution.

[0066] If the calculated slopes are not the same, a first step would be to check which slope is steeper. Then the steepest slope may be used for determining the OD, as in the first alternative. The absolute values could be used to verify the validity of the data. Alternatively, as in the second alternative, the absolute values could be used for the determination of the OD. To base the OD determination on the steepest slope only could give a more uncertain OD determination than using the absolute values.

[0067] In FIG. 4b is shown a case wherein the shortest pathlength is below the limit of detection. In FIG. 4c is shown a case wherein the longest pathlength is saturated, while the two shorter pathlengths have an absorbance of 0.2-0.1 AU. In FIG. 4d is shown a case wherein the shortest pathlength is being saturated, while the longer pathlengths have an absorbance of 0.2-1.5. A pathlength being saturated should be excluded from OD determination.

[0068] When the flow cell comprises more than three different light paths, each light path having a respective predetermined path length, l, a slope, αc, is calculated 500 for each pair of light paths by dividing a difference in absorbance reading, ΔA, with a difference in path length, Δl. If there are e.g. five different light paths, the number of slopes calculated is four. These slopes are then compared 600 as described above and if a) the calculated slopes, αc, are the same the slope is used for determining 700 the optical density of the solution.

[0069] If b) the calculated slopes, αc, are not the same, see FIGS. 4b-4d, the steepest slope of the calculated slopes may be used for determining 701a the OD of the solution.

[0070] As an alternative, when the calculated slopes, αc, are not the same, the slope of the calculated slopes being in the range of an absorbance reading of 0.01 to 2 may be used for determining 701b optical density of the solution. Using more light paths will give a larger dynamic range and better data to assess which slope is within the linear dynamic range and which are outside.

[0071] When the at least three light paths 4a, 4b, 4c are irradiated at the same wavelength it is possible to determine the OD of an unknown solution and any particles suspended therein.

[0072] When at least one of the at least three light paths 4a, 4b, 4c is irradiated at a wavelength which is different from the wavelength used to irradiate the other light path(s) OD of a solution with known absorbance coefficient may be analysed. When comparing slopes from different light paths irradiated at different wavelengths, the difference in wavelength used has to be corrected for.

[0073] Alternatively, different wavelengths may be used in the method during an optimization phase. The light paths are irradiated at different wavelengths to identify an optimal wavelength for irradiation of all light paths 4a, 4b, 4c. If a wavelength used for irradiation of a light path results in the light path being saturated (and, hence, excluded from OD determination), this wavelength will not be used as the wavelength for irradiating the light paths.

[0074] Data processing means 7 (FIG. 2) may be arranged to take the absorbance readings 400 of a solution at the at least three light paths 4a, 4b, 4c, calculate 500 a slope, αc, for each pair of light paths 4a, 4b, 4c by dividing a difference in absorbance reading, ΔA, with a difference in path length, Δl, comparing 600 the calculated slopes, αc, and determining 700, 701a, 701b optical density of the solution (as described above).

[0075] Absorbance readings, A, of the sample solution may be taken simultaneously from the at least three light paths 4a, 4b, 4c. It is, however, not necessary to do measurements simultaneously. Absorbance measurements of the different light paths 4a, 4b, 4c can be done sequentially if the flow rate/movement of the solution between locations for different light paths is known and can be corrected for. This will improve response time.

[0076] The method may further comprise a step of calculating a delay time between absorbance readings from the different path lengths 4a, 4b, 4c. This extra step may be used for example when analysing (sample) solutions with fast changes in absorbance properties. With the knowledge of solution flow rate and tubing diameter, the transit time of a sample peak between the different locations of the different optical paths may be calculated and can be used to align the result from these optical paths.