Slope spectroscopy without reference signal

Abstract

Techniques and apparatus for improving absorbance measurement based on a variable path length measurement (VPT) instrument architecture are provided. [Solution] The method comprises the steps of: determining whether the fluctuation in the intensity of the probe radiation satisfies a stability criterion; defining a first path length L1 of the probe radiation passing through the fluid sample by directing the probe radiation to pass through the probe when the probe is positioned at a first position; measuring the transmission intensity I1 of the probe radiation after it has passed through the fluid sample when the probe is positioned at a first position; defining a second path length L2 of the probe radiation passing through the fluid sample by directing the probe radiation to pass through the probe when the probe is positioned at a second position; measuring the transmission intensity I2 of the probe radiation after it has passed through the fluid sample when the probe is positioned at a second position; and, if the stability criterion is met, determining the concentration C of the material in the fluid sample based on L1, I1, L2, and I2.

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application claims priority under Section 119 of the United States Patent Act to U.S. Provisional Application No. 63 / 343,357, filed on 18 May 2022, entitled “NO-REF-SIGNAL SLOPE SPECTROSCOPIC MEASUREMENT,” the entire application of which is incorporated herein by reference for all purposes.

[0002] [Technical field] Embodiments of this disclosure generally relate to spectroscopic analysis, and more specifically to solution analysis using a light source coupled to a variable path length measurement system. [Background technology]

[0003] Absorption spectroscopy is used to measure the composition and / or properties of materials in any phase, gas, liquid, or solid. For example, the optical absorption spectrum of a liquid substance can be measured to determine the concentration or other properties of the type of substance of interest in the liquid medium. The absorption spectrum can provide a distribution of optical attenuation (due to absorbance) as a function of the wavelength of light. In known spectrophotometers, the sample substance under study is placed in a transparent container, thereby allowing electromagnetic radiation (light) of known wavelength λ (i.e., ultraviolet, infrared, visible light, etc.) and intensity I to pass through the transparent container and then be measured using a suitable detector.

[0004] In known ultraviolet (UV) / visible spectrophotometers, a container such as a standard cuvette is used, which may have a path length of standard cm, and incident light is conducted through this path length in the liquid containing the substance being measured. For a sample consisting of a single homogeneous substance with concentration c, the light transmitted through the sample follows the relationship known as Beer's law: A = εCL, where A is the absorbance (also known as the optical density (OD) of the sample at wavelength λ, where OD = -log of the ratio of transmitted light to incident light), ε is the extinction coefficient or extinction coefficient (usually constant at a given wavelength), C is the concentration of the sample, and L is the path length of light passing through the sample. Therefore, in principle, information regarding the concentration of a homogeneous substance can be determined based on the recorded light intensity of the signal passing through the sample container. However, under some circumstances, determining the concentration in such an apparatus can be difficult. In many cases, the compound of interest in the solution is highly concentrated. For example, certain biological samples such as proteins, DNA, or RNA are often isolated at concentrations outside the linear range of the spectrophotometer when their absorbance is measured. Therefore, dilution of the sample is often necessary to measure absorbance values ​​within the instrument's linear range. This frequently requires multiple dilutions, resulting in both dilution errors and the removal of diluted samples for any downstream applications. Thus, it is useful to collect existing samples without knowing the possible concentrations and to measure the absorbance of these samples without dilution. One resulting feature common to these known ultraviolet (UV) / visible spectrophotometers is the ability to determine the path length L with high accuracy, thereby enabling accurate concentration measurements.

[0005] To address these challenges, techniques based on variable path length spectrophotometers have recently been developed. This type of spectroscopic system can generally employ known light sources, such as those based on ultraviolet / visible spectrophotometers. Light from the ultraviolet / visible spectrophotometer is then directed to a special probe in an analytical instrument configured to dynamically change the path length L within a special sample chamber during absorbance measurement. Thus, the intensity of transmitted radiation generated from the light source of the ultraviolet / visible spectrophotometer is detected after passing through the sample chamber, while the movement of the probe varies the path length L through multiple different positions. This results in a series of measurements that produce different values ​​of A for each different value of l, without requiring knowledge of any specific path length L to determine the concentration C.

[0006] While such variable path length spectroscopy can be performed through a production system and adapted for in-line measurement of samples, the equipment required for such measurement scenarios may require enormous installation effort and an excessive amount of space. For example, a UV / Vis spectrophotometer system used as a light source may occupy several cubic feet of space and weigh approximately tens of kilograms. Furthermore, determination A generally requires that multiple intensity measurements may be necessary for each sample measurement taken at a given path length L.

[0007] This disclosure provides further information regarding these and other considerations. [Overview of the project]

[0008] In one embodiment, a method for determining the concentration of a material may include the step of determining whether the variation in the intensity of probe radiation emitted by the light source of an absorbance spectroscopy system satisfies a stability criterion. The method may further include the step of directing probe radiation to pass through the probe when the probe is positioned at a first position, defining a first path length L1 of the probe radiation passing through a fluid sample, and measuring the transmission intensity I1 of the probe radiation after it has passed through the fluid sample when the probe is positioned at the first position. The method may also include the step of directing probe radiation to pass through the probe when the probe is positioned at a second position, defining a second path length L2 of the probe radiation passing through a fluid sample, and measuring the transmission intensity I2 of the probe radiation after it has passed through the fluid sample when the probe is positioned at the second position. If the stability criterion is met, the method may additionally include the step of determining the concentration C of the material in the fluid sample based on L1, I1, L2, and I2.

[0009] In another embodiment, a non-temporary computer-readable storage medium is provided that stores computer-readable program code executable by a processor for determining whether fluctuations in the intensity of probe radiation emitted by a light source of an absorbance spectroscopy system meet a stability criterion; when the probe is positioned at a first location, the light source directs the probe radiation to pass through the probe, defining a first path length L1 of the probe radiation passing through a fluid sample; receiving the transmission intensity I1 of the probe radiation after it has passed through the fluid sample; when the probe is positioned at a second location, the light source directs the probe radiation to pass through the probe, defining a second path length L2 of the probe radiation passing through a fluid sample; receiving the transmission intensity I2 of the probe radiation after it has passed through the fluid sample; and, if the fluctuations in intensity meet a stability criterion, determining the concentration C of the material in the fluid sample based on L1, I1, L2, and I2.

[0010] In a further embodiment, a measuring device is provided comprising a light source for generating a probe signal; and a measuring instrument for receiving the probe signal. The measuring instrument has a sample container for accommodating a fluid sample, the sample container including a container wall, and a probe configured to direct the probe signal through the sample container, where the probe is movable along the probe direction with respect to the container wall so as to vary the path length L of the probe signal through the fluid sample. The measuring device may also have a detector arranged to receive the probe signal after passing through the container wall; and a control system. The control system determines whether fluctuations in the intensity of the probe radiation emitted by the light source meet a stability criterion; and, if the fluctuations in intensity meet the stability criterion, may be configured to calculate the concentration C of a material in the fluid sample based on the change in intensity of the probe signal measured as a function of the change in path length L. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The accompanying drawings illustrate preferred embodiments of the disclosed method devised heretofore for practical use of its principles.

[0012] [[ID=I0]] [Figure 1A] An absorption spectroscopic apparatus according to an embodiment of the present disclosure is shown.

[0013] [Figure 1B] An absorption spectroscopic apparatus according to an embodiment of the present disclosure is shown.

[0014] [Figure 1C] A configuration is provided showing a general geometry for determining the concentration of a substance according to the principle of slope spectroscopy. [[ID=I6]]

[0015] [Figure 2] An absorption spectroscopic apparatus according to an embodiment of the present disclosure is shown.

[0016] [Figure 3] Another absorption spectroscopic apparatus according to an embodiment of the present disclosure is shown.

[0017] [Figure 4] Shows an exemplary absorption spectrum according to an embodiment of the present disclosure.

[0018] [Figure 5] Presents an exemplary process flow.

[0019] [Figure 6] Presents another exemplary process flow.

Mode for Carrying Out the Invention

[0020] According to an embodiment of the present disclosure, a technique and an apparatus for improving absorbance measurement based on a variable-pathlength-measurement (VPT) apparatus architecture are provided. This embodiment particularly provides a rational and dynamic approach for determining the concentration of a material in a fluid sample. In the approach of this embodiment, while varying the path length of radiation passing through the fluid sample, a plurality of intensity measurement values recorded when the radiation passes through the fluid sample are employed. As detailed below and in contrast to known absorbance spectroscopy techniques, this embodiment determines the change in absorbance of the fluid sample and thus the concentration C of the material in the fluid sample without the need to perform a reference signal measurement.

[0021] Figure 1A shows an absorption spectrometer, referred to as System 100, according to an embodiment of the present disclosure. System 100 may comprise a miniature light source 102, a measuring instrument 110 coupled to the miniature light source 102, and a detector 112 positioned next to the measuring instrument 110. The miniature light source 102 may have a light-emitting diode (LED) that generates radiation 104 at a target wavelength in the UV to IR range, and specifically in the range of 190 nm to 1100 nm or 250 nm to 1000 nm, according to various non-limiting embodiments. In some examples, the miniature light source 102 may represent a single LED or an array of LEDs that emit radiation at a single wavelength. In other embodiments, multiple LEDs may be provided, where a given LED emits radiation at a different wavelength than another LED.

[0022] The measuring instrument 110 is configured to contain a fluid sample containing the material of the substance to be measured, and details of the modification of the measuring instrument 110 are discussed below. The detector 112 is configured to detect the intensity I of radiation transmitted through a given fluid sample contained in the measuring instrument 110, and this radiation is shown as attenuated radiation 111. According to the Lambert-Beer law shown in Equation 1 below, the concentration C of the material in the sample can be determined as A / eL, where A is the absorbance and e is the molar extinction coefficient.

number

[0023] Next, A is log 10 The result is determined as (I0 / I), where I0 is the intensity of radiation 104 and I is the intensity of attenuated radiation 111. To measure the value of I0, the system 100 further includes a reference detector 106 for receiving a portion of the radiation 104 before it is conducted through the measuring instrument 110. This parameter is used to directly calculate the absorbance according to Equation 2, which is the formula for calculating absorbance.

number

[0024] Therefore, in a given measurement instance, absorbance A is determined when detector 112 measures I based on attenuated radiation 111, while reference detector 106 measures I0. According to the slope spectroscopy approach, Lambert-Beer's law can be rewritten as A / L,=e C, and further extended to DA / DL,=e C, where entities DA / DL are considered to be slope parameters m. During operation, system 100 operates according to the principles of slope spectroscopy to determine the change in absorbance A as a function of change in path length L, by varying the path length L over which radiation 104 travels, and thus directly determining the value of C for a given substance given knowledge of e for that substance.

[0025] The details of the deformation operation of the measuring instrument 110 are discussed below with reference to Figures 2 and 3. In short, the measuring instrument 110 employs a movable optical probe (as shown in Figure 2, which will be discussed in more detail below) to vary the path length L of radiation 104 passing through a given fluid sample (not shown in Figure 1A, but see fluid sample 211 in Figure 2) present in the measuring instrument 110. Since the intensity of attenuated radiation 111 changes with the change in path length L, the change in absorbance A as a function of the change in path length L can be directly determined using the change in I as a function of the change in path length L. Therefore, by employing the system 100 with knowledge of I0 given by the reference detector 106, and by varying the path length of radiation 104 (to determine Dl) as the radiation 104 passes through the measuring instrument 110, and detecting the change in the intensity of attenuated radiation 111 (to determine DA), the concentration C of the material in the fluid sample can be easily determined.

[0026] Figure 1C provides a configuration 180 showing details of the geometric arrangement for determining the concentration of a substance according to the principle of slope spectroscopy. In configuration 180, a light source, such as a small light source 102, directs radiation 104 through a sample 182, such as a fluid sample. The sample 182 attenuates or absorbs a portion of the radiation 104, so that the attenuated radiation, such as attenuated radiation 111, generally exhibits an intensity I lower than the intensity I0 of radiation 104. A beam splitter 184 or similar device is provided to direct a portion of the radiation 104 to a reference detector 106 without passing it through the sample 182 in order to record the value of I0. As described above, within the measuring instrument 110, a movable probe can vary the path length L, while simultaneously measuring the change in absorbance A. For each value of L, the measured value of the intensity I of the attenuated radiation 111 is recorded, and the measured value of the intensity I0 of radiation 104 is also recorded. In this way, DA is determined for a given change in path length Dl as follows.

number

[0027] Figure 1B shows an absorption spectrometer, designated as System 150. This system can operate similarly to System 100 to determine the concentration C of a substance in the measuring instrument 110, where similar components are labeled identically. The difference is that System 150 employs a light source 152, which can be a broad-spectrum light source such as a known ultraviolet / visible / infrared (UV / vis / IR) absorption spectrometer, where radiation 160 represents light that can be generated over a broad emission spectrum at intervals of several seconds or minutes. Similar to System 100, radiation 160 can be directed to a reference detector 156 to measure I0, while attenuated radiation 161 is measured at detector 112 after passing through the fluid sample in the measuring instrument 110.

[0028] In both embodiments of Figure 1A and Figure 1B, a control system 130 is provided to facilitate a rational and improved operation of each absorbance spectroscopy measurement. Briefly, the control system 130 may comprise a non-temporary computer-readable storage medium having instructions that, when executed using an electronic processor, perform one or more of the operations described below. The control system 130 may comprise various components, including dedicated electronic controllers, communication interface routines, or algorithms, for controlling the operation of various components of system 100 or system 150. The control system 130 may control the operation of system 100 or system 150, including the operation of the system in different operating modes. In the “standard” slope spectroscopy mode, the concentration C is determined by determining DA for each value of L using measured values ​​of I and I0, as described above. In the “no reference signal” mode or NRS slope spectroscopy mode, system 100 or system 150 may operate to determine the concentration C without measuring I0, as described below. The NRS mode in slope spectroscopy offers greater flexibility and speed for measurements, as well as potentially higher accuracy.

[0029] According to embodiments of the present disclosure, the NRS slope spectroscopy mode may be used or initiated routinely, or initiated when certain stability criteria for operating the absorbance spectroscopy system are met, in which case Equation 3B for determining A may be simplified. The stability criteria may be met, for example, when the variation in absorbance intensity is below a threshold, as will be discussed further below. According to Equation 3B (see above) outlined for absorbance calculations using known slope spectroscopy approaches, the change in absorbance DA between a first instance t1 (corresponding to a first path length L1) and a second instance t2 (corresponding to a second path length L2) is given by the parameter

number

[0030] However, the inventors believe that under certain circumstances, item

number

Number

Number

[0031] The determination of the value when the instability of the incident intensity is low enough to enable measurements using the new NRS slope spectroscopy mode can be determined according to the application. However, generally, in a situation where I0 varies very slightly over a given time, this variation of the incident intensity can be specified as ±α%. Accordingly, the term alpha is

Number

number

[0032] Therefore, depending on the application, an upper limit may be set for the maximum value of 'a' to determine when the NRS slope spectroscopy mode should be adopted. For example, for absorbance measurements regulated under the United States Pharmacopeia (USP) guidelines for the operation of UV-Vis spectrophotometers, the USP requires that the absorbance deviation be less than ±0.01. Therefore, for slope spectroscopy measurements performed in accordance with the USP guidelines, 0.01 > log(1+α), This means that |α| < 2.33%. Therefore, in some embodiments, the stability criterion may be met if the absorbance deviation is less than a certain value such as ±0.03, ±0.02, or ±0.01. In the latter case, the stability criterion corresponds equivalently to |α| < 2.33%, where a can be defined by the formula described herein.

[0033] In one example, a slope spectrometer including an LED light source, generally configured according to the embodiment shown in Figure 1A, was used to determine the light source stability. Over a specific test period, the maximum I max and minimum I min The incident intensity was recorded along with the following results.

number

[0034] Therefore, in the above example, the value of a is well below the upper limit of 2.33% set by the USP, and the use of NRS slope spectroscopy may be appropriate.

[0035] The use of NRS slope spectroscopy offers advantages for determining material concentrations in fluid samples, including the ability to measure concentrations more accurately, more quickly, and in a more dynamic manner. Figure 2 shows an absorption spectrometer, shown as System 200, according to an embodiment of the present disclosure. System 200 may comprise a miniature light source 102, a variation of a measuring instrument 110 coupled to the miniature light source 102, and a detector 112 positioned next to the measuring instrument 110. The miniature light source 102 may have one or more light-emitting diodes for generating radiation 210 at one or more target wavelengths, as discussed above with respect to Figure 1A.

[0036] In this variation, the measuring instrument 110 has a movable probe 208, which may be an optical fiber, a fibrette, or a bundle of fibers, configured to conduct radiation 202 into a sample chamber container 215 containing a fluid sample 211 containing a material of interest whose concentration C is to be measured. The radiation 210 is directed to enter and pass through the movable probe 208 along the probe axis 206. As shown in Figure 2, the movable probe 208 can be moved parallel to the container wall 212 of the sample container 210 to change the path length L of the radiation. In particular, the path length L represents the distance between the probe tip 208A and the lower part of the container wall 212. Therefore, the value of L corresponds to the distance that the radiation can travel through the sample 182 when the fluid sample is placed inside the sample container 215. Note that a window that is transparent to the radiation 210 can be provided to conduct the radiation out of the sample container 215, resulting in attenuated radiation 220, whose intensity I can be detected by the detector 222. Preferred examples of the detector 112 include, among others, photomultiplier tubes, photodiodes, avalanche photodiodes, charge-coupled devices (CCDs), and multiplier CCDs. Although shown positioned in the line of sight relative to the probe 172, in various embodiments the detector 112 can be integrated into the measuring instrument 110B or remotely positioned by operably coupling the detector 112 to an optical emitter (not shown) capable of carrying electromagnetic radiation traveling through the sample to the detector. The optical emitter may be fused silica, glass, plastic, or any transparent material suitable for the wavelength range of the electromagnetic source and detector. The optical emitter may be formed of a single fiber or multiple fibers, which may have different diameters depending on the use of the measuring instrument 110. In various non-limiting embodiments, the fiber diameters are in the range of about 0.005 mm to about 20.0 mm.

[0037] To facilitate concentration measurements using an approach where DA / DL is equal to eC, a drive component (not shown separately) may be a motor that moves the probe tip 208A in parallel along the probe axis 206. The drive component may provide continuous motion or may be configured to vary the path length L in precise steps. In various non-limiting embodiments, preferred examples of the drive component include a stepping motor, a servo motor, a piezo motor, an electric motor, and a magnetic motor, or any device that can be controlled to provide a variable path length L through the sample. In some embodiments of incremental or step-like motion, the movable probe 208 is moved in increments ranging from 0.2 μm to 1 cm relative to the sample container 215, more specifically, in increments ranging from 1 μm to 50 μm. In other embodiments, the movable probe 208 may be moved continuously to continuously vary L.

[0038] System 200 further comprises a reference detector 204, which may function similarly to the reference detector 106 for measuring the incident intensity I0 of radiation 210, as generally discussed above. In this embodiment, system 200 may also comprise a control system 130. Various inputs to the control system 130 may include I0, L, and I. In one example, information regarding L may be transmitted from component 214, which may be a motor assembly, a sensor, or another component providing positional information. In some implementations, the control system 130 may determine that the intensity fluctuations meet a stability criterion, thereby allowing system 200 to operate in an NRS slope spectroscopy mode in which the position of the movable probe changes through several different positions. Since it is not necessary to record I0, at each position of the probe, only the values ​​of L and I of the attenuated radiation 220 are recorded. Thus, a slope parameter m equal to DA / DL, or equivalently equal to eC, is given by m =

number

[0039] Figure 3 shows an absorbance spectrometer, shown as System 250, according to an embodiment of the present disclosure. System 250 may be considered a variation of System 200 discussed above, with similar components being labeled the same. The difference is that the measuring instrument 256 includes a sample chamber container 260, which includes an inlet port 262 for receiving a fluid sample 211 and an outlet port 264 for guiding the fluid sample 211 out of the sample chamber container 260. Thus, the measuring system 110A may be used to couple with the indicated processing system 270 to provide a dynamic measurement of the concentration C of the material in the fluid sample as the fluid sample passes through the measuring instrument 256. Thus, the processing system 270 may represent any suitable system that generates the fluid sample to be measured, such as a chromatography system, a protein purification system, a filtration system, or other fluid processing system. Therefore, System 250 provides an architecture for dynamically measuring concentrations in a fluid sample when the fluid sample being measured is fluid and can fluctuate, such as the concentration C of the material being measured. The advantage offered by this approach is that, under conditions where the intensity fluctuation of the light source falls below the threshold condition or a threshold meaning the threshold, it is not necessary to measure I0 to determine C, and therefore, as given by equation (4), it is simply necessary to record the measured values ​​of the transmitted intensity I in conjunction with the movement of the probe 208 used to vary L.

[0040] To further illustrate the determination of concentration C using an embodiment of the LED light source, Figure 4 shows an exemplary absorption spectrum according to an embodiment of the present disclosure. In this example, the graph in Figure 4 shows the detected radiant intensity as a function of wavelength in the near-ultraviolet region. Three spectra are shown, spectrum 402, spectrum 404, and spectrum 406, each consisting of a single peak, and these represent the detected intensity of UV light emitted from the LED light source.

[0041] Therefore, spectrum 402 presents data collected in a first instance where the path length of the radiation is directed to pass through a probe positioned at a first location, defining the path length L1 through the fluid sample. Similarly, spectrum 404 presents data collected in a second instance where the path length of the radiation is directed to pass through a probe positioned at a second location, defining the path length L2 through the fluid sample. Spectrum 406 presents data collected in a third instance where the path length of the radiation is directed to pass through a probe positioned at a third location, defining the path length L3 through the fluid sample. Assuming that the concentration C is equal to DA / (DLe) for the time frame between the first and second instances, C can be directly derived by determining the intensity difference between the intensity I2 in spectrum 404 and the intensity I1 in spectrum 402. This is because DL is simply L2-L1, and DA is simply logI1-logI2 under conditions where the light source intensity variability is below an acceptable threshold. Similarly, C can be directly derived by determining the intensity difference between intensity I3 of spectrum 406 and intensity I2 of spectrum 404 for the time frame represented between the second and third instances.

[0042] This NRS slope spectroscopy approach can be easily extended, for example, to record multiple different measurements of I without measuring I0 at multiple different probe positions in order to more accurately determine the concentration. In other words, I1 and L1 are recorded at the first probe position, I2 and L2 are recorded at the second probe position, I3 and L3 are recorded at the third probe position, and so on. In some implementations, the determination of C may be performed in the following manner, where C = (DA / DL) / e according to the Lambert-Beer law. The intensity data I1, I2, I3 are converted to absorbance data A (equivalent to logI) by the data used to determine logI1, logI2, logI3, etc. Linear regression is performed based on a set of data plotted as a function of L for three or more probe positions to determine a regression line whose slope is proportional to = (DA / DL). In this case, DA and DL are, for example, L1, I1, L n , and logI n Rather than the exact value, it is determined from the logI and L values ​​at each end of the regression line. In this way, the calculated concentration C may reflect the true value more accurately than the concentration determined from a set of intensity and path length measurements performed at just two probe locations.

[0043] Furthermore, since the incident intensity measurements are not recorded for different probe positions, in embodiments of small LED light sources, the overall duration of a set of intensity measurements sufficient to determine C can be reduced to just a few seconds.

[0044] Figure 5 shows a process flow 500 according to an embodiment of the present disclosure. In block 502, the variation in the intensity I0 of the incident probe radiation from the light source is measured. In some embodiments, the light source may be a known spectrometer, such as an ultraviolet / visible or ultraviolet / visible / infrared spectrometer, configured to produce radiation over a wide range of wavelengths. In other embodiments, the light source may be a miniature LED light source based on a single LED type emitting radiation over a narrow wavelength range, or a miniature LED light source based on multiple different LEDs emitting radiation over multiple different narrow wavelength ranges. The variation in intensity may be specified as ±α% and may be measured over a given period of time, such as a predetermined interval of several seconds or up to several minutes.

[0045] In determination block 504, a determination is made as to whether the variation in the intensity of I0 is below a threshold. The threshold may be set based on a standard for measuring a given class of material, a standard for operating a given type or apparatus, or on any preferred criterion. In some non-limiting embodiments, the threshold for variation in I0 may be ±0.03, ±0.02, or ±0.01. In the latter case, the stability criterion corresponds equivalently to |α|<2.33%, where a may be defined by the formula described herein.

[0046] If so, the flow proceeds to block 506.

[0047] In block 506, when the probe is positioned in a first location, probe radiation from the light source is directed to pass through the probe. The probe may be an optical fiber, a fibret, a bundle of fibers, or other suitable structure adapted to conduct probe radiation. At the first probe position, the probe tip may be positioned near or within the fluid sample, and the first probe position acts to define a first path length L1 of the probe radiation passing through the fluid sample. In particular, the path length L1 may represent the distance between the probe tip and the wall of the sample container or other vessel housing the fluid sample.

[0048] In block 508, with the probe positioned in a first location, a procedure is performed to measure the transmission intensity I1 of the probe radiation after it has passed through a fluid sample. The transmission intensity can be measured by any suitable detector, such as an electron detector, which is adapted to detect radiation across the wavelength range of the probe radiation.

[0049] In block 510, when the probe is positioned at a second location, probe radiation from the light source is directed to pass through the probe. The second probe position may define a second path length L2 of the probe radiation passing through the fluid sample.

[0050] In block 512, when the probe is positioned in a second location, a procedure is performed to measure the transmission intensity I2 of the probe radiation after it has passed through a fluid sample.

[0051] In block 514, a procedure is performed to determine the concentration C of the material in the fluid sample based on L1, logI1, L2, and logI2. For example, the concentration C can be calculated according to the Lambert-Beer law as C=(DA / DL) / e, where DL is given by |L1-L2| and DA is given by logI1-logI2.

[0052] If it is determined in decision block 504 that the variation in I0 is not below the threshold, the flow proceeds to block 516. In block 516, when the probe is positioned at the first position, the incident intensity I of the probe radiation before it passes through the fluid sample to be measured is... 01 An operation to measure is performed. This measurement may represent, for example, the first measurement in a series of absorbance measurements for a material in a fluid sample.

[0053] In this situation, the flow proceeds to block 506A, which is generally executed according to block 506 described earlier.

[0054] After block 506A, the flow proceeds to block 518, where the transmission intensity I1 of the probe radiation after passing through the fluid sample is measured with the probe positioned in the first position. Note that the operations of blocks 516, 518, and 506A can essentially be performed simultaneously.

[0055] Next, the flow proceeds to block 520, and when the probe is positioned in the second position, the incident intensity I of the probe radiation before it passes through the fluid sample. 02 The operation to measure is performed.

[0056] In this situation, the flow proceeds to block 510A, which is generally executed according to block 510 as described earlier. After block 510A, the flow proceeds to block 522, where the transmission intensity I2 of the probe radiation after passing through the fluid sample is measured, with the probe positioned in the second location. Note that the operations of blocks 520, 522, and 510A can essentially be performed simultaneously.

[0057] Next, the flow proceeds to block 524, L1, I1, L2, I2, I 01 and I 02 Based on this, an operation is performed to determine the concentration C of the material in the fluid sample. For example, the concentration C can be calculated according to the Lambert-Beer law as C = (DA / DL) / e, where DL is given by |L1-L2| and DA is logI1-logI 2.+ log(I 01 / I 02 It is given by ).

[0058] Figure 6 shows another process flow 600 according to an embodiment of the present disclosure. In block 602, in the first instance, when the probe is positioned at a first position p1, an operation is performed to direct probe radiation through the probe and define a first path length L1 of the probe radiation through the fluid sample. Probe radiation can be generated by light from a known spectrometer, such as an ultraviolet / visible or ultraviolet / visible / infrared spectrometer, configured to generate radiation over a wide range of wavelengths. In other embodiments, the light source may be a miniature LED light source based on a single LED type emitting radiation over a narrow wavelength range, or a miniature LED light source based on multiple different LEDs emitting radiation over multiple different narrow wavelength ranges.

[0059] In block 604, in the first instance, the transmission intensity I1 of the probe radiation after passing through the fluid sample is measured.

[0060] In block 606, when probes are positioned at multiple additional locations p2, p3, ... in multiple additional instances, the probe radiation is directed to pass through the probes, and multiple additional path lengths L2, L3 are defined for the probe radiation passing through the fluid sample.

[0061] In block 608, the operation is performed to measure the transmission intensities I2, I3, ... of the probe radiation after it has passed through the fluid sample at multiple additional distances.

[0062] In block 610, the operation involves performing a linear regression to determine the slope m of a line for a set of data plotted as a function of L1, L2, L3, where m is determined from the ratio of the change in absorbance to the change in path length. Intensity data I1, I2, etc. are converted to absorbance data by determining logI1, logI2, etc. to determine A1, A2, etc. In other words, a linear regression is performed on a set of data constructed from multiple data points, where the data points represent, for example, A1, L1; A2, L2; A3, L3; and so on. In particular, the linear regression performed in block 610 is used to define a line that best fits the set of data points A, L, where the slope of the fitted line defines the effective value of m.

[0063] In block 612, an operation is performed to determine the concentration C of the material in the fluid sample, where C = m / e, where e is the molar extinction coefficient of the material, and m is determined in the same way as in block 610.

[0064] While this configuration is disclosed with reference to specific embodiments, numerous modifications, alterations, and changes are possible to the described embodiments without departing from the spirit and scope of the disclosed configuration, as defined in the appended claims. Accordingly, this configuration is not limited to the described embodiments and is intended to have the entire scope defined by the following claims and their equivalents.

Claims

1. A method for determining the concentration of a material: The step of determining whether the fluctuations in the intensity of probe radiation emitted by the light source of the absorbance spectroscopy system meet the stability criteria; When the probe is positioned in a first location, the probe radiation is directed to pass through the probe, and the first path length L of the probe radiation passing through the fluid sample containing the material is... １ The stage of defining; When the probe is positioned at the first location, the transmission intensity I of the probe radiation after it has passed through the fluid sample. １ The stage of measurement; When the probe is positioned at the second location, the probe radiation is directed to pass through the probe, and the second path length L of the probe radiation passing through the fluid sample is... ２ The stage of defining; When the probe is positioned at the second location, the transmission intensity I of the probe radiation after it has passed through the fluid sample. ２ The step of measuring; and If the aforementioned stability criteria are met, L １ , I １ , L ２ , and I ２ A step of determining the concentration C of the material in the fluid sample based on the above. A method for providing this.

2. If the aforementioned stability criteria are met, the concentration C is: It is determined that C = (DA / DL) / e, where e is the molar absorption coefficient of the material, and DL is the absolute value of the difference between L １ and L ２ and ΔA = log I1 - log I2. The method according to claim 1

3. The variation in the intensity of the probe radiation is given by a, where a = (I ｍａｘ -I ｍｉｎ ) / I ｍｉｎ And I ｍａｘ is the maximum value of radiation intensity recorded during a given period, and I ｍｉｎ The method according to claim 1, wherein is the minimum value of intensity recorded during the given period.

4. The method according to claim 3, wherein the duration of the given period is 1 second to 100 seconds.

5. The method according to claim 1, wherein, if the stability criteria are met, the incident intensity of the probe radiation before it passes through the fluid sample is not measured at the first position or the second position.

6. The aforementioned transmission intensity I １ and the transmission intensity I ２ This is determined by the measuring instrument of the absorbance spectroscopy system, and the measuring instrument is: The probe has a sample container for containing the fluid sample, the sample container includes a container wall, and the probe has a first path length L that determines the path length of the probe signal passing through the fluid sample. １ From the aforementioned second path length L ２ It is movable along the probe direction relative to the container wall so as to change the state. The method according to claim 1.

7. The stability criterion includes a state of the light source in which the fluctuation of the intensity is below a threshold, and the method, when the fluctuation of the intensity is above the threshold: When the probe is positioned at the first position, the incident intensity I of the probe radiation before it passes through the fluid sample ０１ The stage of measurement; When the probe is positioned at the second location, the incident intensity I of the probe radiation before it passes through the fluid sample ０２ The step of measuring; and L １ , I １ , L ２ , I ２ , I ０１ and I ０２ Based on this, the step of determining the concentration C of the material in the fluid sample. The method according to claim 1, further comprising:

8. C = (DA / DL) / e, where DL is |L １ -L ２ Given by |, DA is logI １ -logI ２ +log(I ０１ / I ０２ The method according to claim 7, as provided by ).

9. The probe is located at the first position in the first instance, and at the second position in the second instance, and the method is: For at least one additional instance, when the probe is positioned at at least one additional location, the probe radiation is directed to pass through the probe, and at least one additional path length L for the probe radiation passing through the fluid sample. ｎ The stage of defining each of them; In the at least one additional instance, the transmission intensity I of the probe radiation after passing through the fluid sample ｎ The step of measuring; and Perform linear regression and A １ A ２ A ｎ ...to L １ , L ２ , L ｎ The step of determining the slope m of the line of the data set plotted as a function of logI, where A is equal to logI. The method according to claim 1, further comprising:

10. In the processor, A procedure for determining whether the intensity fluctuations of probe radiation emitted by the light source of an absorbance spectroscopy system meet stability criteria; When the probe is positioned in the first location, the light source is directed to direct the probe radiation through the probe, and the first path length L of the probe radiation passing through the fluid sample is determined. １ Steps to define; The transmission intensity I of the probe radiation after passing through the fluid sample. １ Procedure for receiving; When the probe is positioned at the second location, the light source is directed to direct the probe radiation through the probe, thereby determining the second path length L of the probe radiation passing through the fluid sample. ２ Steps to define; The transmission intensity I of the probe radiation after passing through the fluid sample. ２ Procedure for receiving; and If the fluctuations in the aforementioned intensity satisfy the stability criteria, L １ , I １ , L ２ , and I ２ Procedure for determining the concentration C of the material in the fluid sample based on the above A computer program designed to execute something.

11. The aforementioned basket C is: The formula C = (DA / DL) / e is determined, where e is the molar extinction coefficient of the material and DL is L １ and L ２ The computer program according to claim 10, wherein ΔA is the absolute value of the difference between and ΔA = logI1 - logI2.

12. The variation in the intensity of the probe radiation is given by v, where v = (I ｍａｘ -I ｍｉｎ ) / I ｍｉｎ And I ｍａｘ is the maximum value of radiation intensity recorded during a given period, and I ｍｉｎ The computer program according to claim 10, wherein v is the minimum intensity recorded during the given period, and the stability criterion is met when v is below a threshold.

13. The aforementioned processor, If the fluctuations in intensity satisfy the stability criteria, the incident intensity I of the probe radiation at the first position before passing through the fluid sample ０１ Without receiving the measurement value, and before passing through the fluid sample, the incident intensity I of the probe radiation at the second position ０２ The computer program according to claim 10, which causes the computer program to perform a procedure for determining the concentration C without receiving the measured value.

14. The aforementioned processor, If the fluctuations in the intensity do not meet the stability criteria: When the probe is positioned at the first position, the incident intensity I of the probe radiation before it passes through the fluid sample ０１ Procedure for receiving; When the probe is positioned at the second location, the incident intensity I of the probe radiation before it passes through the fluid sample ０２ Procedure for receiving; and L １ , I １ , L ２ , I ２ , I ０１ and I ０２ A procedure for determining the concentration C of the material in the fluid sample based on the above. A computer program according to claim 10, which causes to execute

15. The processor is instructed to perform a procedure to determine C by calculating the change in absorbance DA from the first instance to the second instance, where C = (DA / DL) / e, and DL is |L|. １ -L ２ Given by |, DA is logI １ -logI ２ +log(I ０１ / I ０２ The computer program according to claim 14, provided by ).

16. The probe is located at the first position in the first instance and at the second position in the second instance, and the computer program is located at the processor: For at least one additional instance, when the probe is positioned at at least one additional location, the light source is directed to direct the probe radiation through the probe, thereby providing at least one additional path length L for the probe radiation through the fluid sample. ｎ The procedure for defining each of them; In the at least one additional instance, the transmission intensity I of the probe radiation after passing through the fluid sample ｎ Procedure for measuring; and Perform linear regression and A １ A ２ A ｎ ...to L １ , L ２ , L ｎ A procedure for determining the slope m of a line in a set of data plotted as a function of logI, where A is equal to logI. A computer program according to claim 10, which causes to execute

17. A light source for generating probe signals; and Measuring device for receiving the aforementioned probe signal A measuring device comprising: A sample container for containing a fluid sample, the sample container including a container wall; A probe configured to direct the probe signal so that it passes through the sample container, the probe being movable along the probe direction relative to the container wall to change the path length L of the probe signal passing through the fluid sample; A detector positioned to receive the probe signal after it has passed through the container wall; and It is a control system: Determining whether the fluctuations in the intensity of the probe radiation emitted by the light source meet the stability criteria; and If the fluctuations in intensity satisfy the stability criteria, the concentration C of the material in the fluid sample is calculated based on the change in the intensity of the probe signal, which is measured as a function of the change in the path length L. Control system configured in such a way A measuring device having the following features.

18. The aforementioned control system is: When the probe is positioned at the first position, the transmission intensity I of the probe radiation after it has passed through the fluid sample. １ Received; When the probe is positioned at the second position, the transmission intensity I of the probe radiation after it has passed through the fluid sample. ２ Receive; and C is calculated as (DA / DL) / e, where e is the molar extinction coefficient of the material and DL is L １ and L ２ It is the absolute value of the difference between them, and ΔA = logI1 - logI2, where L １ L is the first path length at the first position. ２ This is the second path length at the second position. The measuring device according to claim 17, configured as described above.

19. The control system: If the fluctuation of the intensity satisfies the stability criterion, the incident intensity I of the probe radiation at the first position before it passes through the fluid sample. ０１ Without receiving the measurement value, and before passing through the fluid sample, the incident intensity I of the probe radiation at the second position ０２ The measuring device according to claim 18, configured to determine the concentration C without receiving the measured value.

20. The measuring apparatus according to claim 17, wherein the light source includes a light-emitting diode (LED) for generating radiation at a target wavelength in the range from ultraviolet to infrared.

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