Method for measuring absorptivity of substance in solution and measuring device therefor

Abstract

Disclosed is a method for measuring the light absorptivity of a substance in a solution in a measurement cell, the method comprising the steps of: emitting (S1) a first light beam from a light source towards a beam splitter; splitting (S3) the first light beam by a beam splitter into a signal light and a reference light; modulating (S5) the signal light; modulating the reference light; providing (S9) the measurement cell such that the signal light (31; 31 ') through the measurement cell; detecting (S11) a signal in the detector, the signal being a combined signal strength of the signal light and the reference light detected by the detector; a synchronous detection (S15) of the detected signal is performed in order to reconstruct the intensities of the signal light and the reference light from the combined signal detected by the detector, said synchronous detection being based on the modulation performed on the signal light and the reference light. A measuring device for performing the method is also disclosed.

Description

Technical Field

[0001] The present invention relates to a method and a measuring device for measuring the absorption rate of substances in a solution. Background Art

[0002] Many substances absorb ultraviolet or visible light due to their chemical composition. The absorption of light by substances has been used for many years as a basis for detecting the presence of such substances and measuring the concentration of such substances. The concentration of a substance can be determined using the Beer Lambert Law: A = Ebc Where: A is the light absorption rate; E is the molar extinction coefficient with the unit of L mol -1 cm -1 ; b is the optical path length of the sample defined in cm; and c is the concentration of the compound in the solution expressed in mol -1 .

[0003] The UV region can be considered to consist of light in the region of wavelengths from 10 nm to 400 nm, and light with wavelengths from 180 nm to 300 nm is called "deep UV". Most analytical instruments for detecting substances that absorb in the deep ultraviolet (UV) region use a mercury lamp, a deuterium lamp, or a xenon flash lamp as a light source. An example of such an instrument is a flow cell, in which a solution containing one or more UV-absorbing substances is placed between a UV light source (e.g., a mercury lamp) and a UV detector (e.g., a photomultiplier tube, a photodiode, or a phototransistor), and the change in the intensity of the UV light reaching the detector is related to the concentration of the UV-absorbing substances in the solution.

[0004] The detection of proteins, nucleic acids, and peptides is extremely important in many industries including environmental science, biological science, and chemical science. Proteins mainly have two absorption peaks in the deep UV region, a very strong absorption band with a maximum at approximately 190 nm (where the peptide bond absorbs), and another weaker peak at approximately 280 nm due to the light absorption by aromatic amino acids (e.g., tyrosine, tryptophan, and phenylalanine).

[0005] WO2007 / 062800 and WO2013 / 178770 describe the use of UV LEDs as light sources for the analysis of the concentration of substances in liquid samples.

[0006] The light introduced from the UV LED to the measuring device is split into a reference light beam directly provided to the detector and a signal light beam provided through the sample and then to the detector. The detected signal light beam is greatly amplified and compared with the amplified form of the detected reference light beam. Problems may exist in these types of systems related to drift because high detector amplification means that any minor inequality in performance in the channel caused by, for example, temperature fluctuations or humidity changes will result in drift in the measured absorption signal. Systems for measuring the change in absorption rate over time in a flowing sample are naturally more sensitive to drift in the system compared to systems that make short measurements on a static sample. SUMMARY OF THE INVENTION

[0007] An object of the present invention is to provide an improved method and measuring device for measuring the light absorption rate of a substance in a solution in a measurement cell.

[0008] Another object of the present invention is to provide a robust and reliable method and measuring device for measuring the light absorption rate of a substance in a solution in a measurement cell.

[0009] This is achieved in the method and measuring device according to the independent claims.

[0010] Thus, only one detector is used to simultaneously measure both the reference light beam and the signal light beam, which means that any drift caused by using two separate detectors is eliminated.

[0011] According to the present invention, a method is for measuring the light absorption rate of a substance in a solution in a measurement cell, the method comprising the following steps: - Emitting a first light beam from a light source towards a beam splitter; - Splitting the first light beam into a signal light beam and a reference light beam by the beam splitter; - Modulating the signal light beam; - Modulating the reference light beam; - Providing a measurement cell such that the signal light beam passes through the measurement cell; - Detecting a signal in a detector, the signal being the combined signal intensity of the signal light beam and the reference light beam detected by the detector; - Performing synchronous detection of the detected signal in order to reconstruct the intensities of the signal light beam and the reference light beam from the combined signal detected by the detector, the synchronous detection being based on the modulation performed on the signal light beam and the reference light beam.

[0012] Also according to the present invention, a measuring device is arranged for measuring the absorption rate of a substance in a solution in a measurement cell of the measuring device, wherein the measuring device comprises: - A light source for emitting a first light beam; - A beam splitter arranged such that the first light beam is split into a signal light beam and a reference light beam by the beam splitter; - A measurement cell positioned such that the signal light beam will pass through the measurement cell; - A first signal modulation device arranged to modulate the signal light beam; and - A second signal modulation device arranged to modulate the reference light beam; and - A detector arranged to detect the signal light beam when the signal light beam has been modulated and has passed through the measurement cell and also to detect the reference light beam when the reference light beam has been modulated, wherein the detector comprises or is connected to a processing device configured to perform synchronous detection of the detected signals in order to reconstruct the intensities of the signal light beam and the reference light beam from the combined signal detected by the detector, said synchronous detection being based on the modulation performed on the signal light beam and the reference light beam.

[0013] The signal light beam and the reference light beam can be detected simultaneously in the same detector.

[0014] This is possible because of the modulation of the signals, and thereby the change in absorbance over time in a flowing sample can be measured.

[0015] In one embodiment of the invention, the step of modulating the signal light beam comprises modulating the signal light beam at a first frequency, and the step of modulating the reference light beam comprises modulating the reference light beam at a second frequency different from the first frequency. Thus the two signals can be distinguished in the detector.

[0016] In one embodiment of the invention, the steps of modulating the signal light beam and the reference light beam comprise generating a sinusoidal modulation or a square wave modulation for both the signal light beam and the reference light beam.[[ID=I9]]

[0017] In one embodiment of the invention, the measurement cell is a flow-through cell.

[0018] In one embodiment of the invention, the method further comprises the step of changing the direction of one or both of the signal light beam and the reference light beam using at least one light direction changing device such that both the signal light beam and the reference light beam can be detected by the same detector.

[0019] In one embodiment of the invention, the step of modulating one of the signal light beam or the reference light beam comprises controlling a light source.

[0020] In one embodiment of the invention, the measuring device further comprises at least one light direction changing device arranged to change the direction of the reference light beam or the signal light beam such that they can be detected by the same detector.

[0021] In one embodiment of the invention, the first signal modulation device and the second signal modulation device are arranged to modulate the signals at different frequencies.

[0022] In one embodiment of the present invention, the first signal modulation device is a chopper, a shutter, a movable mirror, a tuning fork, or an adjustable gray filter arranged somewhere in the optical path of the signal light.

[0023] In one embodiment of the present invention, the second signal modulation device is a chopper, an optical shutter, a movable mirror, a tuning fork or an adjustable gray filter arranged somewhere in the optical path of the reference light.

[0024] In one embodiment of the present invention, one of the first signal modulation device or the second signal modulation device is a device for controlling a light source.

[0025] In one embodiment of the present invention, the beam splitter is an asymmetric beam splitter that splits the first light beam into a signal beam and a reference beam, wherein a larger portion of the first light beam is directed to the signal beam than to the reference beam. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A prior art measuring device is shown schematically.

[0027] FIG. 2 a schematically shows a measuring device according to an embodiment of the present invention.

[0028] FIG2 b schematically shows a measuring device according to another embodiment of the present invention.

[0029] Figure 3 is a flow chart of a method according to one embodiment of the present invention. DETAILED DESCRIPTION

[0030] Figure 1 A prior art measuring device 1 for measuring the absorbance of a substance in a solution arranged in a measuring cell 3 is schematically shown. The measuring device 1 comprises a light source 5 that emits a first light ray 7. Furthermore, the measuring device comprises a beam splitter 9 that is arranged in the measuring device so that the first light ray 7 is split into a signal light ray 11 and a reference light ray 13 by the beam splitter 9. The measuring device 1 further comprises a measuring cell 3 that is positioned so that the signal light ray 11 will pass through the solution in the measuring cell 3. Furthermore, the measuring device 1 comprises: a first detector 15 that is arranged to detect the signal light ray 11 when the signal light ray 11 has passed through the measuring cell 3; and a second detector 17 that is arranged to detect the reference light ray 13. From the detected reference value and signal value, the absorbance of the sample can be determined by the formula A=log 10 The signal value is calculated using the formula (reference value / signal value). As discussed above, drift is a problem in these types of systems, especially when making long, continuous measurements, such as in liquid chromatography. For example, small fluctuations in temperature can affect two different detector channels unequally, thereby causing drift and inaccurate measurements.

[0031] Figure 2a schematically shows a measuring device 21 according to an embodiment of the present invention. The measuring device 21 is arranged to measure the absorption rate of a substance in a solution provided in a measuring cell 23 of the measuring device. The measuring device 21 includes a light source 25 that emits a first light beam 27. The light source can be, for example, an LED or any other suitable type of lamp. The measuring device also includes a beam splitter 29 that is arranged such that the first light beam 27 is split into a signal ray 31 and a reference ray 33 by the beam splitter 29. The beam splitter 29 can be, for example, a semi-transparent mirror or two side-by-side optical fibers. The measuring device 21 also includes a measuring cell 23 that is positioned such that the signal ray 31 will pass through the solution in the measuring cell 23. The measuring cell 23 can be a static measuring cell that includes a sample to be measured, but it can also be a flow-through cell through which the sample flows during continuous measurement.

[0032] In addition, the measuring device 21 includes a first signal modulation device 35 arranged to modulate the signal ray 31 and a second signal modulation device 37 arranged to modulate the reference ray 33. The first signal modulation device 35 is positioned between the beam splitter 29 and the measuring cell 23 in this embodiment. However, it can also be positioned after the measuring cell 23 and before a detector 39 (which is also provided in the measuring device 21). According to the present invention, the detector 39 is arranged to detect the signal ray 31 when the signal ray 31 has been modulated and passed through the measuring cell 23 and to also detect the reference ray 33 when the reference ray 33 has been modulated. In the embodiment shown here, the detector 39 is arranged such that the signal ray 31 is detected by the detector 39 without any additional redirection. The reference ray 33 is instead redirected by a light direction changing device 41 in this embodiment such that the reference ray 33 is detected by the detector 39 after being redirected by the light direction changing device 41 and modulated by the second signal modulation device 37. The light direction changing device can be a mirror.

[0033] The second signal modulation device 37 is shown here as being provided after the light direction changing device 41, but it can also be provided between the beam splitter 29 and the light direction changing device 41. Of course, the light direction changing device 41 can instead be provided in the signal light path and the detector 39 in the path of the reference ray, i.e., the direction of the reference ray 33 is directly after the beam splitter 29. More than one light direction changing device can also be provided in the measuring device in order to direct the signal ray and the reference ray to the same detector in a suitable manner. Another alternative (which would not require a light direction changing device at all) could have two slightly divergent rays from the light source impinge on a detector that is large enough to receive both of them (or by guiding the light in optical fibers). The alternative with optical fibers is shown in Figure 2b. See further description below.

[0034] With the measuring device according to the invention, both the signal beam 31 and the reference beam 33 are detected by the same detector 39. The detector 39 includes or is connected to a processing device 43 which is configured to perform synchronous detection of the detected signals in order to reconstruct the intensities of the signal beam 31 and the reference beam 33 from the combined signal detected by the detector 39. This is achieved by multiplying the detector signals by the modulation signals respectively and then low-pass filtering the results. In this way, two different signals are reconstructed and the absorption can be calculated from them. The processing device 43 can be constituted entirely by hardware, entirely by software or as a combination of both. With this measuring device, the signal beam 31 and the reference beam 33 can be detected simultaneously by the same detector 39.

[0035] The detector 39 typically also includes a photodiode, an amplifier and an AD converter (not shown in the figure). In addition, an optical device is typically provided between the light source 25 and the beam splitter 29. However, this is not shown in the figure. This optical device can include a collimating lens, an aperture and possibly also a filter.

[0036] For example, the first signal modulation device 35 and the second signal modulation device 37 can be choppers, shutters, movable mirrors, tuning forks or adjustable grey filters. They can be separate devices or provided in the same device (if the design of the measuring device and the distance between the signal beam and the reference beam allow that). The first signal modulation device 35 and the second signal modulation device 37 will modulate the signals at different frequencies so that they can be distinguished in the detector. Modulation of the light beam produces amplitude modulation at the set frequencies. In one example, the reference beam can be modulated at a frequency somewhere in the range from 100 Hz to a few kHz, and the signal beam is modulated at a frequency that is at least 30% different from the first frequency. The frequencies need to be chosen such that they do not interfere with other frequencies or their harmonics in the system.

[0037] The amplitude modulation can be sinusoidal modulation, square-wave modulation or any other suitable waveform.

[0038] In another embodiment of the invention, one of the first signal modulation device or the second signal modulation device 35, 37 is instead a device for controlling the light source 25. Controlling the light source 25 (such as, for example, turning it on and off at a specific frequency) will of course affect both the signal beam and the reference beam, but in combination with one of the signal modulation devices for the signal beam or the reference beam, the two signals can still be distinguished from each other correctly in the detector (if the measured signal is adjusted by adding the difference between the "light on" / "modulator on" and "light on" / "modulator off" values to each "light off" / "modulator on" part of the signal).

[0039] In one embodiment of the present invention, the beam splitter 29 may be an asymmetric beam splitter, where a larger portion of the first beam 27 is directed into the signal beam 31 compared to the reference beam 33. This can be useful because a larger portion of the dynamic range of the detector is then used for the signal beam.

[0040] FIG. 2b schematically shows a measuring device 21' according to another embodiment of the present invention. In this embodiment, the beam splitter 29' is provided as two optical fibers. The first beam 27' (light from the light source 25') is split into a signal beam 31' transmitted in the first optical fiber and a reference beam 33' transmitted in the second optical fiber. The signal beam 31' is modulated in the first signal modulation device 35'. Before the signal beam 31' is detected in the detector 39', the signal beam 31' will pass through the solution in the measuring cell 23'. Before the reference beam 33' is detected in the same detector 39', the reference beam 33' is modulated in the second signal modulation device 37'. Most of the details of this embodiment are the same as those in the embodiment described above and will not be further described here.

[0041] Figure 3 is a flowchart of a method for measuring the light absorption rate of a substance in a solution in a measuring cell 23 according to an embodiment of the present invention. Also referring to FIG. 2, the method includes the steps described below: S1: Transmit a first beam 27 from the light source 25 towards the beam splitter 29.

[0042] S3: Split the first beam 27 into a signal beam 31 and a reference beam 33 by the beam splitter 29.

[0043] S5: Modulate the signal beam 31 using the first signal modulation device 35.

[0044] S7: Modulate the reference beam 33 using the second signal modulation device 37. However, if the distance and configuration between the signal beam and the reference beam allow, the first signal modulation device and the second signal modulation device 35, 37 can be made into the same device.

[0045] In one embodiment of the present invention, the step of modulating the signal beam includes modulating the signal beam at a first frequency, and the step of modulating the reference beam includes modulating the reference beam at a second frequency different from the first frequency. In addition, one possibility of modulating the signal beam and the reference beam is to generate amplitude modulation for both the signal beam and the reference beam. The amplitude modulation waveform can be sinusoidal, square wave, or any suitable waveform.

[0046] S9: Provide the measuring cell 23 such that the signal beam 31 passes through the measuring cell.

[0047] S11: Detect a signal in detector 39, which is the combined signal intensity of signal light ray 31 and reference light ray 33 detected by detector 39.

[0048] S15: Perform synchronous detection of the detected signal so as to reconstruct the intensities of signal light ray 31 and reference light ray 33 from the combined signal detected by detector 39 based on the modulation performed on signal light ray 31 and reference light ray 33.

[0049] This method can be used to simultaneously detect signal light ray 31 in detector 39 and detect reference light ray 33 in the same detector 39.

[0050] In one embodiment, the method further comprises the step of: using at least one light direction changing device 41 to change the direction of one or both of signal light ray 31 and reference light ray 33 such that both the signal light ray and the reference light ray can be detected by the same detector 39. Another alternative can use optical fibers as discussed above.

[0051] In an application where a sample flows through a measurement cell, the measurement cell is called a flow cell. For example, this can be in a chromatography system where it is necessary to continuously measure the concentration of substances in the flow. In an example of a chromatography system, the protein concentration can be measured in the effluent from a chromatography column. The effluent from the chromatography column is arranged to pass through the flow cell of the measuring device according to the present invention. The long and continuous measurement of a flowing sample is more sensitive to drift in the system compared to when measuring on a static sample. Thus, the method and measuring device according to this invention are particularly suitable for measuring the absorbance of substances in a solution disposed in a flow cell.

Claims

1. A method for measuring the light absorption rate of a substance in a solution in a measurement cell (23; 23'), the measurement cell (23; 23') being included in a chromatography system, the method comprising the following steps: - Emitting (S1) a first light beam (27; 27') from a light source (25; 25') towards a beam splitter (29; 29'); - Splitting (S3) the first light beam (27; 27') into a signal light ray (31; 31') and a reference light ray (33; 33') by means of the beam splitter (29; 29'); - Modulating (S5) the signal light ray (31; 31'); - Modulating (S7) the reference light ray (33; 33'); - Providing (S9) the measurement cell (23; 23') such that the signal light ray (31; 31') passes through the measurement cell (23; 23'); - Detecting (S11) a signal in a detector (39; 39') by directly providing the signal light ray (31; 31') and the reference light ray (33; 33') to the detector (39; 39'), wherein the signal has a combined signal intensity of the signal light ray (31; 31') and the reference light ray (33; 33') detected by the detector (39; 39'); - Performing synchronous detection (S15) of the detected signal in order to reconstruct the intensities of the signal light ray (31; 31') and the reference light ray (33; 33') from the combined signal detected by the detector (39; 39'), the synchronous detection being based on the modulation performed on the signal light ray and the reference light ray.

2. The method according to claim 1, wherein Simultaneously detecting the signal light ray (31; 31') and the reference light ray (33; 33') in the same detector (39; 39').

3. The method according to claim 1 or 2, characterized in that, The step of modulating (S5) the signal light ray (31; 31') includes modulating the signal light ray at a first frequency, and the step of modulating (S7) the reference light ray (33; 33') includes modulating the reference light ray at a second frequency different from the first frequency.

4. The method according to any one of claims 1 to 3, characterized in that The steps of modulating (S5, S7) the signal light ray (31; 31') and the reference light ray (33; 33') include generating a sine modulation or a square wave modulation for both the signal light ray and the reference light ray.

5. The method according to any one of the preceding claims, characterized in that, The measurement cell (23; 23') is a flow-through cell.

6. The method according to any one of the preceding claims, characterized in that The method further comprises the following step: changing the direction of one or both of the signal light ray (31) and the reference light ray (33) by means of at least one light direction changing device (41) such that both the signal light ray and the reference light ray can be detected by the same detector (39).

7. The method according to any one of the preceding claims, characterized in that, The step of modulating (S5, S7) one of the signal light ray (31; 31') or the reference light ray (33; 33') includes controlling the light source (25; 25').

8. A measuring device (21; 21'), which is used for measuring the absorption rate of a substance in a solution in a measurement cell (23; 23') of the measuring device (21; 21'), the measuring device being included in a chromatography system, wherein the measuring device comprises: - A light source (25; 25'), which emits a first light beam (27; 27')； - A beam splitter (29; 29'), which is arranged such that the first light beam (27; 27') is split by the beam splitter (29; 29') into a signal ray (31; 31') and a reference ray (33; 33'); - The measurement cell (23; 23'), which is positioned such that the signal ray (31; 31') will pass through the measurement cell; - A first signal modulation device (35; 35') arranged to modulate the signal ray (31; 31'); and - A second signal modulation device (37; 37') arranged to modulate the reference ray (33; 33'); and - A detector (39; 39'), which is arranged to directly detect the signal ray (31; 31') when the signal ray (31; 31') has been modulated and passed through the measurement cell (23; 23') and to also directly detect the reference ray (33; 33') when the reference ray (33; 33') has been modulated, wherein the detector (39; 39') includes or is connected to a processing device (43; 43'), the processing device (43; 43') being configured to perform synchronous detection of the detected signals in order to reconstruct the intensities of the signal ray (31; 31') and the reference ray (33; 33') from the combined signal detected by the detector (39; 39'), the synchronous detection being based on the modulation performed on the signal ray and the reference ray.

9. The measuring device (21; 21') according to claim 8, characterized in that, The measuring device (21; 21') further includes at least one light direction changing device (41), which is arranged to change the direction of the reference ray (33; 33') or the signal ray (31; 31') such that they can be detected by the same detector (39; 39').

10. The measuring device according to claim 8 or 9, characterized in that, The measurement cell (23; 23') is a flow cell.

11. The measuring device according to any one of claims 8 to 10, characterized in that, The first signal modulation device (35; 35') and the second signal modulation device (37; 37') are arranged to modulate signals at different frequencies.

12. The measuring device according to any one of claims 8 to 11, characterized in that, The first signal modulation device (35; 35') is a chopper, a light shutter, a movable mirror, a tuning fork or an adjustable grey filter arranged somewhere in the optical path of the signal ray (31; 31').

13. The measuring device according to any one of claims 8-12, characterized in that, The second signal modulation device (37; 37') is a chopper, a light shutter, a movable mirror, a tuning fork or an adjustable grey filter arranged somewhere in the optical path of the reference ray (33; 33').

14. The measuring device according to any one of claims 8-13, characterized in that, One of the first signal modulation device or the second signal modulation device (35; 35', 37; 37') is a device for controlling the light source (25; 25').

15. The measuring device according to any one of claims 8-14, characterized in that, The beam splitter (29; 29') is an asymmetric beam splitter that splits the first light beam (27; 27') into a signal ray (31; 31') and a reference ray (33; 33'), wherein the portion of the first light beam directed to the signal ray is larger than the portion directed to the reference ray.

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