Detecting bubbles in fluid path

Abstract

To provide a method of detecting bubbles in a fluid path, in particular one in an analytical device (e.g., HPLC).SOLUTION: A method of detecting bubbles, in particular gas bubbles, in an analytical device is provided, the method comprising: i) detecting an electromagnetic signal 110 responding to a provided flow / pressure signal in a fluid path, and ii) determining the presence of bubbles in the fluid path based on the detected electromagnetic signal 110. Iii) The electromagnetic signal responding to the flow / pressure signal is an output of a detector, such as a fluorescence detector, of an analytic device.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] Field of the Disclosure The present disclosure relates to a method for detecting air bubbles in a fluid path, particularly a fluid path of an analytical instrument (e.g., HPLC). The method includes detecting an electromagnetic signal in the fluid path responsive to a flow / pressure signal provided thereto, and determining the presence of an air bubble in the fluid path based on the detected electromagnetic signal. The present disclosure also relates to an apparatus including a pressurizing device, an electromagnetic signal detector, and, for example, a control device configured to perform the method. Furthermore, the present disclosure relates to an analytical instrument, particularly a chromatographic instrument such as a High Performance Liquid Chromatography (HPLC) instrument, including the instrument and an analytical region. [Background technology]

[0002] An analytical device is provided for analyzing a sample, such as for performing a chromatographic separation of the sample.

[0003] For example, with respect to liquid separations in a chromatography system, a mobile phase containing a sample fluid (e.g., a chemical or biological mixture) having compounds to be separated is passed through a stationary phase (such as a chromatography column packing) so that the various compounds of the sample fluid are separated and can then be identified.

[0004] Typically, a mobile phase, consisting of one or more solvents, is pumped under high pressure through a chromatography column, which typically contains a packing medium (also called a packing agent or stationary phase). As a sample is carried through the column by the fluid flow, different compounds, each with different affinities for the packing medium, pass through the column at different rates. Compounds with greater affinity for the stationary phase pass through the column more slowly than those with less affinity, and this speed difference results in the compounds being separated from each other as they pass through the column. The stationary phase is typically subjected to mechanical forces, particularly generated by a hydraulic pump that pumps the mobile phase from an upstream connection of the column to a downstream connection of the column. As a result of the flow, relatively high-pressure droplets are generated across the column, depending on the physical properties of the stationary and mobile phases.

[0005] The mobile phase with the compounds to be separated exits the column and passes through a detector, which detects and / or identifies the molecules, for example, by spectrophotometric measurements. A two-dimensional plot of detector measurements versus elution time or volume, known as a chromatogram, may be made, from which compounds may be identified. For each compound, the chromatogram exhibits a distinct curve feature, also referred to as a "peak."

[0006] In preparative chromatography systems, the mobile phase liquid is typically provided at a controlled flow rate (e.g., in the range of 1 mL / min to several thousand mL / min, e.g., in analytical-scale preparative LC in the range of 1-5 mL / min and in preparative-scale in the range of 4-200 mL / min) and at a pressure in the range of tens to hundreds of bar (e.g., 20-600 bar).

[0007] In High Performance Liquid Chromatography (HPLC), the liquid as the mobile phase usually needs to be provided at a very controlled flow rate (e.g., in the range of microliters / minute to milliliters / minute) and at high pressures (typically 20-100 MPa, 200-1000 bar, and currently up to over 200 MPa, 2000 bar) where the compressibility of the liquid becomes readily apparent.

[0008] Such analytical devices generally must analyze samples in a highly accurate manner. Thus, all sources of interference with measurement quality must be identified and eliminated. A particular problem with accurate measurements can be found in the presence of gas bubbles in the fluid path. For example, air bubbles can form in the liquid solvent and interfere with the measurement. Such problems can be particularly problematic when gas bubbles are located at the detector of the analytical device, thereby directly interfering with the measurement results and ultimately damaging the instrument.

[0009] There are various sources of gas bubbles within the flow path of analytical instruments such as HPLC. Frequent bubble sources may include dissolved air, the solvent itself, leakage, air trapped during installation, etc. For example, gas can form bubbles in the flow path, and the radius of the bubbles may depend on the pressure of the surrounding fluid and its viscosity.

[0010] Analytical instruments such as HPLC generally employ detector flow cells, consisting of a detection volume that is often larger in diameter compared to the analytical instrument capillary, and which constitutes a compromise between optical constraints and sensitivity on the one hand, and minimal added dispersion on the other. However, gas bubbles can easily become trapped in this detection volume, which constitutes a temporary location for further smooth bubble expansion.

[0011] The presence of such bubbles, especially in the detection volume (of the flow cell), can affect optical detection in many ways: reduction of the effective detector (flow cell) volume, static deflection of the normal ray path, dynamic deflection of the normal ray path, reflections at phase transitions (particularly problematic for fluorescence detection), pressure-dependent breathing of bubbles (visibility of pump strokes), any movement of bubbles in the detector flow cell volume caused by turbulence and subsequent modulation of the optical path, etc. The aforementioned effects may, for example, affect / result in quantification errors, noise, or pressure ripples.

[0012] Therefore, the presence of bubbles in the flow path of an analytical device can cause significant drawbacks in terms of the quality of the analysis. Conventionally, bubble detection can be based on the fact that bubbles usually have low wavelength dependency, which can be found by evaluating the spectrum. However, conventional methods require additional efforts (e.g., bubble detectors) and at the same time provide rather weak criteria for the presence of bubbles. Summary of the Invention

[0013] Disclosure Overview There is a need to efficiently and reliably detect the presence of air bubbles in a fluid path (in an analytical device). This problem is solved by the independent claims. Further embodiments are given by the dependent claims.

[0014] According to one aspect of the present invention, a method for detecting bubbles (particularly gas bubbles such as air bubbles) in an analytical device (e.g., HPLC) (particularly a detection volume) is described, the method comprising:

[0015] i) Detecting an electromagnetic signal (particularly an optical signal) in a fluid path (particularly a (fluorescence) detector of an analytical device) in response to a provided flow / pressure signal (e.g. provided by a pressurizing device such as a pump).

[0016] ii) determining the presence of air bubbles in said fluid path based on detected electromagnetic signals (particularly since air bubbles respond to pressure signals by contracting and expanding);

[0017] According to a further aspect of the present invention, an apparatus is described that includes: i) a pressurizing device (particularly a pump or metering device) configured to provide a flow / pressure signal to a fluid path; ii) an electromagnetic signal detector (e.g., a fluorescence detector) configured to detect an electromagnetic signal in the fluid path responsive to the provided pressure signal; and iii) a control device configured to determine the presence of an air bubble in the fluid path based on the detected electromagnetic signal.

[0018] According to a further aspect of the present invention, an analytical device is described, which comprises the device described above and an analytical region, in particular a chromatographic region, coupled to the device and configured to analyze a fluid sample.

[0019] In the context of this specification, the term "analytical device" may refer to a device particularly suitable for performing an analysis of a sample. In one example, an analytical device is used to analyze (characterize) a sample by separation (e.g., chromatography) of the sample. In the context of this specification, the term "chromatographic device" may refer to a device particularly suitable for performing a chromatographic analysis suitable for analyzing a sample, such as for performing a chromatographic separation of the sample. Examples of analytical devices may include liquid chromatography (LC) devices, particularly high-performance liquid chromatography (HPLC) devices or ultra-high-performance liquid chromatography (UHPLC) devices, electrophoresis systems, microfluidic devices, cell sorters (e.g., FACS - Fluorescence Activated Cell Sorting), or spectrophotometers. In one embodiment, the analytical device includes an (optical) detection device that is coupled or can be coupled to a pressure source.

[0020] In the context of this application, the term "fluid sample" particularly refers to any liquid and / or gaseous medium to be analyzed, and may also include solid particles, depending on the context. Such a fluid sample may contain multiple fractions of molecules or particles to be separated, e.g., small mass molecules or large mass biomolecules such as proteins. Separation of a fluid sample into fractions may involve specific separation criteria (e.g., mass, volume, chemical properties, etc.) according to which the separation is performed.

[0021] In the context of this application, the term "mobile phase" may specifically refer to any liquid and / or gaseous medium that may serve as a fluid carrier for a fluid sample during separation. A mobile phase may be a solvent or solvent composition (e.g., consisting of water and an organic solvent such as ethanol or acetonitrile). In an isocratic separation mode of a liquid chromatography instrument, the mobile phase may have a constant composition over time. However, in a gradient mode, the composition of the mobile phase may change over time, particularly to desorb fractions of the fluid sample previously adsorbed on the stationary phase of the separation unit.

[0022] In the context of this application, the term "fluid / solvent driver" (or pumping device) may particularly refer to an entity capable of driving a fluid (i.e., a liquid and / or gas, optionally including solid particles), in particular a fluid sample and / or a mobile phase. For example, the fluid driver may be a pump (e.g., embodied as a piston pump or a peristaltic pump) or another high-pressure source. For example, the fluid driver may be a high-pressure pump capable of driving a fluid at a pressure of at least 500 bar. Additionally or alternatively, the movement of the mobile phase may be induced by electrostatic forces. In further embodiments, a metering device may be used as the pressurizing / pumping device. In further embodiments, a piezoelectric element may be used to generate a pressure change in the detection cell, and even to eventually remove air bubbles.

[0023] In the context of this application, the term "sample separation unit" may particularly refer to a fluidic element configured to pass a fluid sample and separate the fluid sample into different molecular or particle groups upon introduction of the fluid sample into the separation unit. An example of a separation unit is a liquid chromatography column, which is capable of capturing or retarding and selectively releasing different fractions of a fluid sample.

[0024] In the context of this specification, the term "bubbles" may particularly refer to fluid-filled voids (e.g., gas bubbles, especially air bubbles) that may be present in fluids such as a liquid mobile phase and / or a fluid sample to be analyzed. The presence of such bubbles is usually undesirable, as they may lead to several drawbacks in the performance of analytical devices (see above). Furthermore, high-precision instrumentation may be damaged by the bubble(s).

[0025] In the context of this specification, the term "flow / pressure signal" may particularly refer to a signal providing flow / pressure over time and / or related to changes in flow / pressure over time. In one example, the pressure signal may be generated by a pressurizing device such as a pump or a metering device. In another example, the pressure signal may be generated by opening or closing a valve or by connecting / disconnecting a fluid path. In another example, the pressure signal may be provided via a bypass. In a basic embodiment, the pressure signal may include a (short-term) pressure increase, such as a pressure pulse. In further embodiments, the pressure signal may include (continuous) pressure changes over time, e.g., may vary periodically. The pressure signal may arise from the flow signal as a result of a restriction.

[0026] In a preferred embodiment, the pressure signal may include a periodic pressure change, e.g., a series of pressure signals / pulses. These signals may be the same (e.g., same length, same intensity) or different. In one example, the pressure signal is generated in a fluid path in which a bubble to be detected is located. In this manner, a pattern of pressure signals may be applied to the bubble, causing it to contract and expand. In one example, the pressure signal may be detected by a pressure sensor, e.g., in units of bar. In another example, the flow / pressure signal may be detected by a flow (flow rate) sensor, e.g., in units of volume / time (e.g., mL / min).

[0027] In the context of this specification, the term "electromagnetic signal" may particularly refer to a signal detected by an electromagnetic signal detector (e.g., a UV (visible) detector or a fluorescence detector) of an analytical device. In one example, electromagnetic radiation (e.g., (UV) light) may be provided to a sample (e.g., a fluid sample in a mobile phase) in a detector flow cell. Then, for example, the absorption, reflection, scattering, or fluorescence of the sample may be measured as the electromagnetic signal by the electromagnetic signal detector. The electromagnetic signal may be detected in such a measurement, for example, as a peak or peaks in a graph showing detector signal intensity versus time (see, for example, FIG. 2). In one embodiment, the electromagnetic signal may be measured in response to the pressure signal described above. In one embodiment, a bubble may conform to the pressure pattern of the pressure signal, such that the detected electromagnetic signal associated with the bubble may exhibit an equivalent pattern, thereby enabling the presence of the bubble to be detected. In one example, the electromagnetic signal may include a single pulse, and in a further example, the electromagnetic signal may include a series of signals / pulses (e.g., the same pattern as the pressure signal).

[0028] According to exemplary embodiments, the present disclosure may be based on the idea that the presence of air bubbles in a fluid path (in an analytical device) may be detected in an efficient and reliable manner, where a pressure signal (e.g., a series of pressure pulses) is provided to the fluid path, and an electromagnetic signal is then detected, based on which the presence of the air bubbles may be determined. In this regard, the inventors surprisingly discovered that air bubbles in the fluid path (particularly in a detector flow cell) respond to the provided pressure signal with contractions and expansions that can be measured based on the detection of the electromagnetic signal. In this way, a particularly efficient and reliable technique may be provided for detecting air bubbles "on the fly." In this case, it has been found that air bubbles may cause large amounts of light scattering, refraction, and reflection, which results in a strong signal (particularly in fluorescence detection).

[0029] In one embodiment, a pressure signal (e.g., a pressure square wave) is applied to a fluid path using a pressure device (e.g., a pump). The gas bubbles respond with rhythmic contractions and expansions at the same fundamental frequency as the pressure signal (pressure square wave). The presence of such behavior can be determined by measuring the electromagnetic signal. The obtained electromagnetic signal may contain frequency components at the fundamental frequency of the pressure (square wave) and multiples thereof. Therefore, a particularly efficient evaluation of the electromagnetic signal can be performed in the frequency domain. The electromagnetic signal can also be evaluated in the time domain with appropriate filtering or processing algorithms such as the Goertzel algorithm.

[0030] The present disclosure can be implemented simply and directly into existing analytical equipment (e.g., HPLC). For example, pumps and detectors already present in HPLC can be used as is. Thus, in one example, the present disclosure can be implemented as software for an established system only. In another example, hardware can be configured specifically for the present disclosure.

[0031] Illustrative Embodiments In one embodiment, the electromagnetic signal is detected by a detector (particularly an electromagnetic signal detector) of the analytical device. The actual detector may be a detector of a common analytical device (particularly HPLC), such as a fluorescence detector, UV / Vis detector, conductivity detector, refractive index detector, diode array detector, electrochemical detector, etc. This may provide the advantage that no additional hardware / detector is required, thereby saving costs and effort. In fact, an electromagnetic signal detector already present in the analytical device can be used without (essentially) any further effort. In addition to the function of the actual detector, such a detector may be configured as a flow cell having a detection volume (detection volume) in which the flow through the flow path can be controlled. Since air bubbles may be present in the flow path, the detector can be placed directly at the required position (without any further effort).

[0032] In one embodiment, the air bubble is located within the detection volume, especially air bubbles deep within the flow path, which may be very difficult to detect by other means and may have little or no effect on the pressure sensor signal (of the pump).

[0033] In one embodiment, the method includes generating a pressure signal by a pressure device (particularly a pressure device of an analytical device, more particularly an analytical pump or metering device). In a further example, the source for generating the pressure signal may be external (e.g., a pressurized air conduit connected to the instrument via an electromechanical valve) or attached to another device. This may offer the advantage that the pressure signal may be directly provided by existing established means, thereby saving costs and additional effort. In a preferred example, detectors and pumps already present in the analytical device may be used to detect bubbles without additional effort. Pressure modulation may allow for separation from static sources of unwanted or abnormally high intensity (e.g., surface contamination, attached particles, cracks, light leaks, etc.).

[0034] In one embodiment, the bubbles are gas bubbles, particularly air bubbles. Such bubbles may (often) occur in connection with analytical instruments such as HPLC. In one embodiment, the bubbles are located in a liquid solvent within the fluid path of the analytical instrument. Detecting the presence of bubbles in a liquid solvent (particularly a mobile phase, e.g., methanol) can significantly improve analytical results.

[0035] In one embodiment, the method further comprises processing the detected electromagnetic signal, inter alia, by at least one of the analytical device, a processing / computing device for controlling the analytical device, and an external processing / computing device. A variety of established signal processing techniques exist and can be applied depending on the desired results. This can further improve the detection of air bubbles and / or adapt it to a particular application. In a particular example, the electromagnetic signal can be transformed from the time domain to the frequency domain.

[0036] In one embodiment, the processing includes transforming the detected electromagnetic signal from the time domain to the frequency domain, particularly using a (fast) Fourier transform. This may provide the advantage of generating a different type of spectrum that highlights features that are potentially not prominent / visible / detectable in the time spectrum. In particular, frequency components of the signal (e.g., similar fundamental frequencies of the pressure signal and the electromagnetic signal) may be identified in the frequency spectrum. Such transforms may include, for example, DFT, FFT, Goertzel, correlation, etc. Modulation at a known frequency may isolate the effect from other uncorrelated noise sources (e.g., particles, random fluctuations, electrical noise, etc.). A major advantage may be found in that the modulation is independent of the DC component, thus making the measurement more stable. In one example, the DC component is not predictable and is not time-invariant.

[0037] In one embodiment, at least one peak in the frequency domain corresponds to an air bubble (see, for example, Figures 5 and 6), which may provide the advantage that this frequency component can be directly identified from the spectrum, allowing for unambiguous, fast, and reliable detection of air bubbles.

[0038] In one embodiment, the pressure signal varies with time. In one embodiment, the pressure signal is at least partially periodic. In one embodiment, the pressure signal is at least partially non-continuous. In a basic embodiment, the pressure signal may include a single pulse. In a more sophisticated embodiment, the pressure signal may be a continuous / periodic signal, e.g., a series of pulses. Depending on the complexity of the pressure signal, more reliable bubble detection may be provided.

[0039] In one embodiment, the method further includes removing the detected bubbles from the analytical device (see, for example, FIGS. 7A and 7B). Removing bubbles from the flow path can be done, for example, by at least one of flowing a fluid through the fluid path at a high flow rate, using a fluid to modify the surface tension of the fluid path, or adjusting the temperature of the fluid path. Based on the presence of detected bubbles, the bubbles may be removed in a subsequent step, thereby improving detection quality. Established and reliable methods may be applied (especially continuously) to remove the bubble(s) from the flow path (and analytical device).

[0040] In one embodiment, the method further includes characterizing at least one property (particularly size and / or location) of the detected bubble using the detected electromagnetic signal, particularly continuously, periodically, or sporadically. Primarily, this disclosure describes techniques for detecting (and confirming) the presence of a bubble. However, this disclosure can also be applied to receive more information about the bubble's properties (e.g., estimating its size).

[0041] In one embodiment, the fluid path is disposed between the analytical pump and the detector of the analytical device. In one embodiment, the fluid path extends at least partially through the (flow cell) detector (volume) of the analytical device. Such flow paths are common for analytical devices such as HPLC (e.g., compare FIG. 1).

[0042] In one embodiment, the bubbles respond to the pressure signal by contracting and expanding rhythmically, particularly at the same fundamental frequency. The inventors have discovered that bubbles in the flow channel do indeed respond in this way to an applied pressure signal. An imprint of the pressure signal on the bubbles is then detectable based on electromagnetic signal measurements.

[0043] In one embodiment, providing a pressure signal further comprises modulating piston movement, in particular piston velocity. In one embodiment, providing a pressure signal further comprises controlling the opening / closing of a valve connected to the pressure source. In one embodiment, providing a pressure signal further comprises controlling the connection / disconnection of a flow path to the pressure source. Thus, there can be many options for how to provide a pressure signal. In a preferred example, providing a pressure signal is already realized in the analytical device, so no additional effort is required. Furthermore, by controlling the opening / closing of a valve connected to a (constant) pressure source, the connection and disconnection (e.g., by a valve) of the flow cell to the pressure source can be controlled, respectively.

[0044] In one embodiment, the method further comprises detecting the pressure signal using a pressure sensor. This may provide the advantage that the pressure signal can be controlled / adjusted and / or compared to the detected electromagnetic signal. For such detection, a pressure sensor already installed in the analytical device may be used. For example, a pressure sensor associated with / associated with the (analytical) pump may be used to monitor the pressure signal.

[0045] In one embodiment, the analytical device is configured as a sample separation device, in particular a fluid chromatography device, more particularly an HPLC device.

[0046] In one embodiment, the sample separation device further comprises a mixing point where the sample is injected into the solvent, where the fluid compartment (analytical device) is located upstream or downstream of the mixing point.

[0047] In one embodiment, the sample separation device further comprises a solvent mixing point where at least two solvent portions can be mixed, where the fluid compartment (analytical device) is located upstream or downstream of the solvent mixing point.

[0048] In one embodiment, the sample separation device further comprises a solvent driver configured to drive a solvent as a mobile phase, where the fluid compartment (analytical device) is located upstream or downstream of the solvent driver.

[0049] From the embodiments just described, it can be seen that there is a great deal of design flexibility as to where the fluid compartments can be located within the analyzer / sample separator. Depending on the situation at hand and the measurement method being applied, different locations may be particularly preferred.

[0050] In one embodiment, the chromatography device includes a mobile phase (solvent) driver and a separation device, where the mobile phase driver is configured to drive a mobile phase through the separation device, and the separation device is configured to chromatographically separate compounds of a sample fluid in the mobile phase.

[0051] In one embodiment, the analytical device and / or sample separation device comprises a liquid chromatography system, where the sample fluid is a sample liquid, the mobile phase comprises one or more liquid solvents, and the separation device is a chromatography column configured to separate compounds of the sample dissolved in the mobile phase.

[0052] In one embodiment, the chromatography device is a fluid chromatography device, in particular an HPLC device.

[0053] Embodiments of the present disclosure may be implemented based on most conventionally available HPLC systems, such as the Agilent 1220, 1260, and 1290 Infinity LC series (provided by the applicant, Agilent Technologies).

[0054] The separation device preferably includes a chromatography column providing the stationary phase. The column can be a glass, metal, ceramic, or composite tube (e.g., 50 μm to 5 mm in diameter and 1 cm to 1 m in length) or a microfluidic column (e.g., as disclosed in European Patent Application No. 1577012 or the Agilent 1200 Series HPLC-Chip / MS system provided by the applicant, Agilent Technologies). Individual components are held separate by the stationary phase and separate from each other as they propagate through the column with the eluent at different velocities. At the end of the column, they elute at least partially separated from each other. Throughout the chromatographic process, the eluent may be collected in a series of fractions. The stationary phase, or adsorbent, in column chromatography is typically a solid material. The most common stationary phase in column chromatography is silica gel, followed by alumina.

[0055] The mobile phase (or eluent) can be either a pure solvent or a mixture of different solvents. It can also contain additives, i.e., a solution of additives in a solvent or solvent mixture. It can be selected, for example, to adjust the retention of compounds of interest and / or the amount of mobile phase required for chromatography. The mobile phase can also be selected to effectively separate different compounds. The mobile phase may contain an organic solvent, such as methanol or acetonitrile, often diluted with water. For gradient operation, water and organic solvent are provided in separate containers, from which a gradient pump delivers the planned mixture to the system. Other commonly used solvents include isopropanol, THF, hexane, ethanol, and / or any combination thereof, or any combination of these with the aforementioned solvents.

[0056] The sample fluid may include any type of process liquid, a natural sample such as juice, a bodily fluid such as plasma, or may be the result of a reaction such as from a fermentation broth, a bioreactor, a digestion, or other type of sample preparation.

[0057] The fluid is preferably a liquid, but may also be or include a gas and / or a supercritical fluid (e.g., as used in Supercritical Fluid Chromatography (SFC) as disclosed in U.S. Pat. No. 4,982,597).

[0058] The pressure of the mobile phase may range from 2 to 200 MPa (20 to 2000 bar), in particular from 10 to 150 MPa (100 to 1500 bar), more particularly from 50 to 130 MPa (500 to 1300 bar).

[0059] The HPLC system may further include a detector for detecting separated compounds of the sample fluid, a fractionation unit for outputting separated compounds of the sample fluid, or any combination thereof. Further details of the HPLC system are disclosed in relation to the aforementioned Agilent HPLC series offered by the applicant, Agilent Technologies.

[0060] In an exemplary embodiment, one aspect of the present disclosure can be found in detecting the presence or absence of (stationary) air bubbles (in the mobile phase) at the location of optical detection, i.e., the flow cell. A specific pressure pattern (e.g., a square wave) is applied by a (chromatographic) pump, and the air bubbles respond with rhythmic contractions and expansions at the same fundamental frequency of the pressure pattern, which can then be optically detected. The signal from such air bubbles is significantly stronger than signals from any other source, thus enabling the unambiguous identification of the presence of such air bubbles at the location of optical detection.

[0061] In an exemplary embodiment, the method includes generating a frequency pattern (by a chromatography pump), performing optical detection with a detector (flow cell), and performing frequency analysis of the optical detection signal to detect air bubbles in the liquid.

[0062] In an exemplary embodiment, a method for detecting gas bubbles in the detection volume of an LC flow cell (particularly, but not exclusively, a fluorescence detector) is described. A pressure square wave is applied to the system using a pump. Air bubbles respond by rhythmically contracting and expanding at the same fundamental frequency as the pressure square wave. The aforementioned effects may modulate the detected intensity. The signal contains components at the fundamental frequency of the pressure square wave and multiples thereof. Evaluation of the signal by frequency and amplitude (DFT, FFT, Goertzel, correlation, etc.) and discrimination by threshold or pattern may further be achieved.

[0063] In one embodiment, the method may be used as a system diagnostic procedure, for example, the method may be performed automatically as part of a start-up procedure or system health check or the like.

[0064] In one embodiment, the signals may be processed automatically and the detection of conditions (bubbles present / absent, large / small bubbles, etc.) may also be performed automatically, for example by pattern recognition, especially in conjunction with artificial intelligence / machine learning models. Automatic condition detection may also be applied to the process of removing detected bubbles.

[0065] Once the detected air bubble, or a sufficient portion thereof, is determined to have been removed, the system may automatically initiate, continue, or repeat an operation, such as analyzing the sample.

[0066] In one embodiment, the user / operator may be automatically notified of detected bubbles (e.g., alert on software / system display, push message, email, call, etc.). Through the same channel, the user may be notified of the success or failure of the bubble removal measurement.

[0067] In one embodiment, it may be envisaged that the system will independently determine measures to remove bubbles, for example, or try several possibilities (flow rate, solvent composition, temperature, etc.) one after the other or in combination. Feedback from signal analysis may be used to trigger further measurements. Additionally or alternatively, various possible solutions may be presented to the user, who may choose none, one or more.

[0068] Other objects and many of the attendant advantages of the presently disclosed embodiments will be readily appreciated and better understood by reference to the following more detailed description of the embodiments when taken in conjunction with the accompanying drawings, in which features that are substantially or functionally the same or similar are indicated by the same reference numerals. [Brief explanation of the drawings]

[0069] [Figure 1] FIG. 1 illustrates an analytical device implemented as a liquid chromatography device, according to an exemplary embodiment.

[0070] [Figure 2] 10A-10C illustrate electromagnetic signals with and without an air bubble present, according to an illustrative embodiment;

[0071] [Figure 3] FIG. 10 illustrates pressure signals detected by a pressure detector and a flow detector according to an exemplary embodiment.

[0072] [Figure 4A] FIG. 1 illustrates a comparison of electromagnetic and pressure signals according to an exemplary embodiment. [Figure 4B] FIG. 1 illustrates a comparison of electromagnetic and pressure signals according to an exemplary embodiment.

[0073] [Figure 5] 1A-1C illustrate electromagnetic and pressure signals in the frequency domain in the presence of an air bubble according to an exemplary embodiment;

[0074] [Figure 6] 10A-10C illustrate electromagnetic signals in the frequency domain in the presence of air bubbles in different ways, according to an exemplary embodiment;

[0075] [Figure 7A] 10A-10C illustrate electromagnetic signals as air bubbles are removed from a flow path according to an exemplary embodiment. [Figure 7B] 10A-10C illustrate electromagnetic signals as air bubbles are removed from a flow path according to an exemplary embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0076] Detailed Description of the Drawings Referring now more particularly to the drawings, FIG. 1 shows a schematic diagram of an analytical apparatus 10, here embodied as a high-performance liquid chromatography (HPLC) apparatus. A solvent driver 20 (which may be used as a pressurizing device, such as a pump) receives a solvent as a mobile phase from a solvent source 25. The solvent driver 20 drives the mobile phase through a separation apparatus 30 (such as a chromatography column), which may be viewed here as the analytical region of the apparatus. A sample injector 40 (also called a sampler, sampling space, sample injection device, sample dispatcher, etc.) is provided between the solvent driver 20 and the separation apparatus 30 to expose or add a portion of one or more sample fluids to the mobile phase flow at a mixing point 45 (often referred to as sample introduction). The separation apparatus 30 is adapted to separate compounds of a sample fluid (e.g., a liquid). A detector 50 is provided for detecting the separated compounds of the sample fluid. A fractionation unit 60 may be provided to output the separated compounds of the sample fluid. In one embodiment, at least a portion of the sample injector 40 and the fractionation unit 60 may be combined, for example, in the sense that the same common hardware is used when applied by both the sample injector 40 and the fractionation unit 60.

[0077] The separator 30 may include a stationary phase configured to separate compounds of the sample fluid. Alternatively, the separator 30 may be based on a different separation principle (e.g., field-flow fractionation).

[0078] The mobile phase can contain only one solvent, but may also be a mixture of solvents (solvent source 25). Such mixing may be low-pressure mixing and occur upstream of solvent driver 20, so that solvent driver 20 already receives and pumps the mixed solvent as the mobile phase. Alternatively, solvent driver 20 may include multiple individual pumping units, each receiving and pumping a different solvent or mixture, so that mixing of the mobile phase (as it is received by separator 30) occurs downstream (or as part of) mobile phase driver 20 at high pressure. The composition (mixture) of the mobile phase may remain constant over time (so-called isocratic mode) or may change over time (so-called gradient mode).

[0079] A data processing device (controller) 70, which can be a conventional PC or workstation, can be coupled (as indicated by the dotted arrows) to one or more devices within the analytical device 10 for receiving information and / or controlling operations.

[0080] The flow path can extend, for example, from the solvent driver 20 through the sample injection device 40 and the separation device 30 (analysis region) to the detector 50. However, the flow path can also extend only through the detector 50. In this example, the detector is configured as a flow cell including a detection volume through which a fluid sample may be passed for detection by electromagnetic (optical) radiation. In a particular example, the flow cell may include an at least partially transparent body having a hollow interior space through which the fluid sample may flow, in which case the fluid sample may be electromagnetically / optically detected while passing through the hollow interior space (flow path). In a particular example, the detector 50 is configured as a fluorescence detector that measures the fluorescence of the sample fluid passing through the flow path in the flow cell. The presence of air bubbles in the flow path may interfere with reliable detection results, and therefore, the presence of such bubbles may need to be efficiently detected.

[0081] 2 shows the electromagnetic signal 110 with and without the presence of an air bubble 150, according to an exemplary embodiment. The Y-axis represents the signal intensity at the detector (e.g., a fluorescence detector signal) in arbitrary units, and the X-axis represents time (also in arbitrary units). A pressure device (here, an analytical pump) provides a pressure signal (in this case, a periodic pressure signal) by controlling the pump piston in a specific manner. When no air bubbles are present in the flow path, only a flat line (no air bubbles) is detected as the electromagnetic signal, even though a pressure signal is provided by the pressure device.

[0082] However, if an air bubble is present (bubble present), a periodic electromagnetic signal 110 can be detected by the detector 50. The electromagnetic signal 110 varies over time between an upper position (high intensity) and a lower position (low intensity) (here a sawtooth square wave). In other words, the two exponential functions never reach an equilibrium state; for example, the system behaves like a first-order low pass driven by a fast square wave. It is shown diagrammatically that small air bubbles may generate only a small electromagnetic signal 110, while large air bubbles may generate a large electromagnetic signal 110.

[0083] The presence of bubbles in the flow path can be detected based on the electromagnetic signal 110 detected in response to the pressure signal provided by the pressurizing device. Specifically, this is because bubbles respond to pressure signals by rhythmically contracting and expanding, particularly at the same fundamental frequency. In other words, the cyclic pressure signal provided by the pump is transmitted to the bubbles, and the resulting cyclic movement of the bubbles is detected by the electromagnetic signal detector of the analyzer detector 50.

[0084] 3 shows a pressure signal 120 detected by both a pressure detector and a flow detector (of the analyzer 10), according to an exemplary embodiment. The pressure signal 120 is provided by moving a piston of the pump 20 of the analyzer 10, which creates a periodic pattern 121 (measured in bar by the pressure detector). The flow detector measures the flow rate in mL / min, and the pressure signal 120 is predicted in a digital manner (such as 1s and 0s) based on the displacement of the piston.

[0085] 4A and 4B show a comparison of an electromagnetic signal 110 and a pressure signal 120, according to an example embodiment.

[0086] FIG. 4A shows the superposition of a pressure signal 121 (e.g., as detected by a pressure sensor in bar, based on the flow rate detected by the pump; see the left side of the Y-axis) and an electromagnetic signal 110 (see the right side of the Y-axis) as detected by an electromagnetic radiation detector in the analyzer 10. The X-axis shows the time (in seconds) for both signals 110, 121. Two situations can be observed: i) the periodic pattern of the electromagnetic signal 110 corresponds to the periodic pattern of the pressure signal 121; and ii) the electromagnetic signal 110 caused by the bubble weakens (decreases in amplitude) over time, thereby indicating that the bubble is gradually (rather than suddenly) removed from the flow path. In other words, the pump cycle is imprinted on the detector signal, and the signal amplitude (presumably) corresponds to the bubble size (volume of gas remaining in the cell).

[0087] FIG. 4B shows the corresponding pressure signal 120 measured by the flow detector in mL / min (compare with FIG. 3).

[0088] FIG. 5 illustrates the electromagnetic signal 110 (as detected by the electromagnetic signal detector) and the pressure signal 120 (as detected by the flow detector) when transferred from the time domain (see FIGS. 3 and 4) to the frequency domain, according to an exemplary embodiment. In this case, a Fast Fourier Transform (FFT) is used, so the X-axis currently represents frequency (Hz), while the Y-axis represents signal intensity. It can be seen that the signals are fairly consistent with each other. Therefore, even small features / variations in the pressure signal 120 may be observable in the detector signal 110. A large peak corresponding to the presence of an air bubble 150 can be observed in the frequency domain. While the peak would be observable in the pressure signal 120 anyway, it is only observable in the electromagnetic signal 110 if an air bubble 150 is present. In the absence of an air bubble 150, the electromagnetic signal 110 in the frequency domain is a rather smooth line.

[0089] FIG. 6 shows the electromagnetic signal 110 in the frequency domain when a bubble 150 is present in a different way, according to an exemplary embodiment. Compared to FIG. 5, only the electromagnetic signal 110 is shown. The presence of the bubble 150 can be confirmed by a large peak (the presence of the peak does not convey any clear information). In other words, FIG. 6 shows the conversion of the detector signal 110 into the frequency domain, confirming that the pump cycle is indeed imprinted on the detector signal 110, with the first prominent peak in the trace corresponding to the pump cycle (1 / 12 sec ≈ 0.083 Hz).

[0090] 7A and 7B show the electromagnetic signal 110 as measured by the electromagnetic signal detector 50 when an air bubble 150 is removed from the flow path, according to an exemplary embodiment. In both figures, the X-axis represents time (in minutes) and the Y-axis represents detector signal strength. Furthermore, in both examples, the air bubble 150 is continuously removed (e.g., by flushing at high speed). It can be clearly seen that the electromagnetic signal 110 (in response to the pressure signal 120) continuously weakens over time (the signal is periodic and does not decay). [Explanation of symbols]

[0091] 10: Analyzer 20: Solvent drive unit 25: Solvent source 30: Separation device 40: Sample injector 50: Detector 60: Fractionation unit 70: Data processing device, control device 110: Electromagnetic Signal 120: Pressure signal measured by the detector 121: Pressure signal in bar 150: Bubbles

Claims

1. A method for detecting an air bubble (150) in an analytical device (10), comprising: detecting an electromagnetic signal (110) in the fluid path responsive to the provided flow / pressure signal (120); determining the presence of an air bubble (150) in the fluid path based on the detected electromagnetic signal (110).

2. 2. The method of claim 1, wherein the electromagnetic signal (110) is detected in a detector (50) of the analytical device (10), in particular a fluorescence detector.

3. 3. The method according to claim 1 or 2, further comprising generating the pressure signal (120) by a pressure device (20), in particular a pressure device (20) of the analytical device (10), more particularly an analytical pump or a metering device.

4. the bubbles (150) are gas bubbles, in particular air bubbles, and / or The method of any one of claims 1 to 3, wherein the gas bubble (150) is located in a liquid medium in the fluid path of the analytical device (10).

5. 5. The method according to claim 1, further comprising processing the detected electromagnetic signal (110), in particular by at least one of the analytical device (10), a processing device for controlling the analytical device (10), and an external processing device.

6. The method of claim 5, wherein said processing comprises transforming said detected electromagnetic signal (110) from the time domain to the frequency domain, in particular using FT.

7. The method of claim 6, wherein at least one peak in the frequency domain corresponds to the gas bubble (150).

8. The following characteristics: The pressure signal (120) varies with time. the pressure signal (120) is at least partially periodic; The method of any one of claims 1 to 7, wherein the pressure signal (120) is at least partially discontinuous.

9. 9. The method of claim 1, further comprising removing detected bubbles (150) from the analysis device (10) by at least one of: flowing a fluid through the fluid path at a high flow rate, using a fluid with specific properties, in particular to modify the surface tension of the fluid path; adjusting the temperature of the fluid path.

10. 10. The method of claim 1, further comprising using the detected electromagnetic signal (110) to characterize at least one property of the detected gas bubble (150), in particular its size and / or position, in particular continuously, periodically, temporarily or sporadically.

11. The following characteristics: The fluid path is disposed between an analytical pump (20) and a detector (50) of the analytical device (10); The method of any one of claims 1 to 10, wherein the fluid path extends at least partially through a detector (50) of the analytical device (10).

12. The method according to any one of the preceding claims, wherein the bubbles (150) respond to the pressure signal (120) by contracting and expanding, particularly rhythmically, particularly at the same fundamental frequency.

13. Providing the pressure signal (120) comprises: Modulating piston movement, in particular piston velocity, and / or Controlling the opening / closing of a valve connected to a pressure source, and / or The method of any one of claims 1 to 12, further comprising controlling connection / disconnection of a fluid path to the pressure source.

14. Detecting the pressure signal (120) using a pressure sensor; and / or The method of any one of claims 1 to 13, further comprising detecting the flow rate using a flow sensor.

15. a pressure device (20), in particular a pump or metering device, configured to provide a pressure signal (120) to the fluid path; an electromagnetic signal detector (50) configured to detect an electromagnetic signal (110) in the fluid path responsive to the provided flow / pressure signal (120); a controller (70) configured to determine the presence of an air bubble (150) in the fluid path based on the detected electromagnetic signal (110).

16. An analytical device (10), comprising:

16. An apparatus according to claim 15; An analytical device (10) coupled to said device (100) and comprising an analytical area, in particular a chromatographic area, configured to analyze a fluid sample.

17. 17. The analytical device (10) of claim 16, configured as a sample separation device (10), in particular a fluid chromatography device, more particularly a high performance liquid chromatography (HPLC) device.

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