Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/62—Detectors specially adapted therefor
  • G01N30/74—Optical detectors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
  • G01N27/44704—Details; Accessories
  • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
  • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
  • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
  • G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds

Definitions

• the present disclosure relates to an apparatus and method for obtaining information about a sample, in particular although not exclusively profiling a sample such as, for example, an oil sample.
• composition of petroleum products determines the product's upstream (production) and downstream (processing) behavior, product yields and quality, which all affect the economic value of the crude oil. Additionally, oil head engineers and scientists often need to know the details of oil as it appears from the ground, and are often required to cease production while oil samples are analysed. Oil head analytics therefore carry a very large added-value to the efficient operation of a production platform. Downstream analytics are also crucial, if usually less time-critical. A lack of knowledge of the chemical properties of petroleum species has to date limited the improvement of refining efficiency.
• compositions of saturated hydrocarbons are well characterized by a range of tools, including gas chromatography (GC); two dimensional gas chromatography (GCxGC); or by the coupling of GC or GCxGC with a mass spectrometer or other single or multichannel detector.
• ATEX process Appareils destines a etre utilises en ATmospheres Explosives
• gas chromatographs are already used on-line and in real-time (e.g. in refineries), however, GC is only useful in non- polar constituent analysis: comparatively little is known about the less abundant polar species or heavy biodegraded crude oils, whose compositional complexity far exceeds the peak capacity of typical analytical techniques. Indeed, the polar components (or combinations of polar and non- polar) may provide great insights into the oil composition, properties, origin, and transformations during ex situ or in situ upgrading processes.
• an apparatus for obtaining data about a sample comprising a separation conduit configured to separate a sample into constituents according to a separation characteristic as the sample travels through the conduit.
• the separation conduit comprises an injection port for introduction of the sample into the separation conduit.
• the apparatus also comprises a flow generator configured to generate flow of the sample in the conduit, a detector disposed relative to the separation conduit to generate a detector signal indicative of a constituent passing the detector, and a post-separation spectrometer disposed relative to the separation conduit to obtain a spectrum of the
• the apparatus enables improved profiling of a sample, for example, an oil sample.
• Profiling is improved by combining two profiling measurements (a separation characteristic and a post-separation spectrum) in a single apparatus, making profiling more rapid compared to sequential individual investigation and more comprehensive compared to individual investigations alone.
• the sample might be serum proteins; fatty acids, for example, from a marine environmental study; lipids, for example, from a biofuels investigation, or a very wide range of other materials from the biomedical, environmental, security, forensic, food and drink, renewable energy or other sectors.
• the apparatus further comprises a processor configured to determine a value of the separation characteristic of the constituent from the detector signal.
• the processor may be configured to determine, based on the detector signal, the time of arrival of the constituent at the post-separation spectrometer.
• the apparatus may further comprise a memory component to store the value associated with the spectrum.
• the memory component may be configured to store the value associated with the spectrum for at least some of the constituents as a profile of the sample.
• the processor and memory component enable the creation and population of a database. These memory and processor components may be physically separate from the apparatus. Communication between the memory and processor components and the apparatus may be by wireless or wired connection for the transmission of data.
• the processor and the memory component may be connected to a visualization tool, for example a computer terminal.
• the processor, memory component and/or visualization tool may be remote from the separation apparatus and may be connected to the apparatus or each other by a wired or wireless (including optical) connection for the
• data obtained may be recorded to a data storage device for use at a later date.
• a data storage device may be a USB device, base station, or other suitable storage device as will be apparent to those skilled in the art.
• the apparatus comprises a pre-separation spectrometer for obtaining a spectrum of the sample prior to separation.
• This pre-separation spectrometer enables a spectrum of the sample to be obtained before separation.
• This spectrum of the not yet separated sample may be used as a baseline against which the aggregated spectra of the separated constituents may be compared.
• the pre-separation spectrum of the sample obtained from the pre-separation spectrometer and the aggregated spectra of each constituent from the post-separation spectrometer may be compared to determine any differences.
• the processor may be configured to compare the sample obtained from the pre-separation spectrometer and the aggregated spectra of each constituent from the post-separation spectrometer to determine any differences for quality control purposes.
• a comparison between the pre-separation spectrum and the aggregated post-separation spectra may be used to determine the residue of oil remaining in the capillary.
• pre-preparation of the sample prior to the sample reaching the pre- separation spectrometer is done, for example, to facilitate flow of a sample through the separation conduit.
• Pre-preparation of the sample might include adding an amount of water, acetonitrile, methanol, or various proportions of each to the sample.
• the sample may flow from the pre-separation spectrometer past the detector and then past the post-separation spectrometer.
• the sample may flow from the pre-separation spectrometer past the post-separation spectrometer and then past the detector.
• the separation conduit is a linear conduit and, for example, extends from an inlet to a downstream outlet or connection to a waste chamber or other liquid handling structure.
• the separation conduit may be arranged in a loop by coiling the separation conduit back on itself such that the sample may flow around a coiled conduit.
• a separation conduit coiled to form a loop may extend from an injection port for introduction of a sample to the conduit to an outlet via which the sample may leave the conduit.
• the separation conduit may be coiled multiple times to form multiple loops.
• the post-separation spectrometer and the pre-separation spectrometer may be the same spectrometer.
• the resulting apparatus is of a simpler construction and can be manufactured at a lower component cost. This may be achieved in the case of a linear separation conduit, for example, by providing an input to the spectrometer at a first position along the separation conduit prior to separation of the sample, and an input to the spectrometer at a second position along the separation conduit after separation of the sample into
• the inputs may be provided, for example, using fibre optic cables to conduct light to the spectrometer from the respective positions.
• the separation conduit is arranged in a loop
• no such input means are required since the position at which the spectrometer measures spectra can be chosen at a point such that both the substantially unseparated sample and separated constituents will travel past this point. Therefore the spectrometer (or its input) may be disposed relative to the conduit at a single point, for example in the region between the injection port and the outlet.
• the loop itself can be of variable radius, in some embodiments, the radius may be selected to provide a circumference optimized for the separation.
• This loop could be in the same plane as the apparatus, or in a plane partially or wholly orthogonal to the plane of the apparatus.
• the sample may be separated into constituents by any suitable separation technique, for example, electrophoresis techniques, chromatography techniques, any other suitable technique, or a combination of two or more separation techniques, for example, capillary electrophoresis and chromatography.
• any suitable separation technique for example, electrophoresis techniques, chromatography techniques, any other suitable technique, or a combination of two or more separation techniques, for example, capillary electrophoresis and chromatography.
• CEC Capillary Electrophoresis Chromatography
• a stationary phase can be implemented (either in capillary
• the apparatus is configured to separate the sample by means of electrophoresis.
• the electrophoretic separation conduit can be implemented as a capillary (to implement capillary electrophoresis). In other embodiments, the electrophoretic separation conduit can be implemented as a conduit in a microfluidic chip.
• the flow generator comprises a power supply and a plurality of electrodes, for example two electrodes, positioned so to generate an electric field to cause flow of the sample and constituents.
• the apparatus is configured for capillary zone electrophoresis (CZE), in other embodiments the apparatus is configured for MicroChannel Zone Electrophoresis ( ⁇ ).
• CZE separation uses a buffer solution provided in the separation conduit, resulting in charge separation. The choice of buffer can be optimized for a given set of analytics which afford the greatest discrimination power (see An introduction High Performance Capillary Electrophoresis, by David Heiger. Publication Number 5968-9963E, 2000, Agilent Corporation).
• the velocity of each constituent is directly proportional to the effective charge of the constituent and the applied electric field, and inversely proportional to the viscosity of the buffer Therefore, in CZE, the value of the separation characteristic determined is the effective charge of the constituent.
• the term 'effective charge' is understood to mean the relative charge of the constituent compared with that of the surrounding environment, for example, the capillary wall and neighbouring liquids.
• the apparatus may be configured for gel electrophoresis, in particular capillary gel electrophoresis (CGE).
• CGE separation uses a gel matrix provided in the separation conduit, resulting in a mass-charge (m/q) separation.
• the gel can be tuned to pick out individual mass ranges, or functionalized in other ways to enhance its separation power.
• the velocity of each constituent is dependent on the ratio of the charge and mass of the constituent.
• the apparatus is configured to separate the sample by means of liquid chromatography (LC).
• LC liquid chromatography
• the constituents are separated based on the affinity of each constituent for a stationary phase and for a mobile phase.
• the velocity of each constituent is dependent on affinity of the constituent for the stationary phase and for the mobile phase.
• the value of the separation characteristic determined is largely a measure of the affinity of the constituent for the stationary phase, which may, for example, be proportional to the polarity of the constituent.
• Electrophoresis High Pressure Liquid Chromatography, Gas Chromatographic Separation or other Separation modalities.
• the post-separation spectrometer and/or pre-separation spectrometer are configured to measure absorption spectra, in some embodiments UV absorption spectra. This has the advantage that tagging of the sample is not required.
• the post- separation spectrometer and/or pre-separation spectrometer may be an emission, radiation or fluorescence spectrometer.
• the post-separation spectrometer and pre-separation spectrometer obtain different or the same type of spectra. For example, it might be
• the UV source for the pre-separation spectrometer could be the same or different source as the source which induces the induced or intrinsic fluorescence or Raman spectrum for the post-separation spectrometer to measure.
• a spectrometer as used in the present disclosure is an instrument for measuring received light (or other radiation) intensity as a function of light wavelength to produce a signal or data indicative of: a) the absorption of light or other radiation received when a sample or constituent is in a measurement light path of the spectrometer in the case of absorption spectrometry, or b) light emission as a function of wavelength whether induced (by for example, laser
• an absorption spectrum is detected when the sample or constituent absorbs light that would otherwise reach an intensity detector of the spectrometer.
• a fluorescence, Raman or emission spectrum is detected when the sample or component emits light that is then detected, either spontaneously or when stimulated.
• the value of the separation characteristic is associated with the corresponding spectrum based on the order in which the constituents pass the detector, which corresponds to the order they pass the post-separation spectrometer in a linear arrangement and in an arrangement comprising a single loop.
• the order in which the constituents pass the detector corresponds to the order in which they pass the post-separation spectrometer for the first time.
• the order in which the constituents pass the detector will be the same as the order in which they pass the post-separation spectrometer (for the first time in the case of a multiple loop arrangement) irrespective of which instrument the constituents pass first.
• the time of arrival of a constituent at a particular point may be determined based on the detector signal.
• the passage of the constituent, or 'analyte' causes a change in photocurrent, for example, passage of the constituent may cause a drop in photocurrent associated with the wavelength-specific absorption in that constituent.
• the detector may comprise a number of pixels provided along the direction of travel of the constituents. As a constituent passes each pixel it is detected by the detector. The time at which the constituent passes each pixel is recorded. From this position and time information the speed of travel of the constituent can be calculated, typically using a linear fit. This fit can be achieved using data acquired by the detector itself by producing a trajectory, plotting distance against time elapsed, of the changes in photocurrent in the detector, associated with the passage of the constituents.
• This fit may be refined by also including a point corresponding to the time and location at which the sample is introduced into the separation conduit.
• This additional constraint has the merit of allowing the association of a given constituent with a specific injection time and the association of a given constituent with any particular injection. This improves the reliability and accuracy in any given spectral measurement and allows a correlation in information gained through the separation stage and the spectral analysis stage.
• the linear fit may then be extrapolated or interpolated to determine the time at which a given constituent will be at a particular point along the separation conduit, for example, the post-separation spectrometer.
• the processor is configured to determine the time of arrival of each constituent at the post-separation spectrometer.
• the time of arrival of a constituent at any point along the separation conduit may be determined. This may be achieved by evaluating a fit function to determine the time of arrival of a constituent at a given point, for example the post-separation spectrometer. For example, in the case where the constituents flow past the detector and then past the post-separation spectrometer, a linear fit may be extrapolated to predict the time at which a constituent will arrive at the post-separation spectrometer.
• a linear fit may be extrapolated back or interpolated (taking account of the injection point and time) to determine the time at which a constituent was at the post-separation spectrometer.
• Linear extrapolations may be desirable for simplicity, alternatively, any other extrapolation technique may be used.
• some or all of the constituents travel at a constant velocity and hence a linear fit may provide an accurate model of the actual trajectories. In some embodiments, some or all of the constituents travel at a non-constant velocity.
• a linear fit may be used as an approximation or, alternatively, a non-linear fit may be used, using an analytical or otherwise parameterized (e.g. piecewise linear, spline, etc) function may be used.
• Statistical techniques such as Gaussian Processes may be used in some embodiments.
• one or more detectors may be placed along the trajectory to obtain additional data points for fitting the trajectories.
• a functional form may be modeled using information concerning a driving force and separation environment. Other information may also be used as will be apparent to those skilled in the art.
• more than one sample may be profiled/measured simultaneously for example, as described in WO02/13122.
• the technique described above may be applied to extrapolate or interpolate the linear fit to determine the time at which each constituent was introduced into the separation conduit and hence which sample it forms part of. This enables an increase in the throughput of samples through the apparatus.
• the value of the separation characteristic can be calculated from the velocity of the constituent.
• the apparatus may be calibrated to determine the value from the velocity of the constituent. For example, in the case of CZE separations, the effective charge of the constituent is acquired.
• a specific and well-characterised marker for example anthracine or chrysene, can be added to the mix and its subsequent identification in the separation spectra can be used to calibrate the remaining objects.
• the use of such a marker is advantageous in calibrating the time domain of the separation.
• the separation conduit comprises a bifurcation connected to a branch conduit for removal of a selected constituent.
• the apparatus may comprise a switching arrangement configured to direct a selected constituent from the separation conduit into the branch conduit.
• Devices for separating the constituents of a sample are described in WO 2004/005910 and WO 2010/004236, the disclosures of both of these applications are herein incorporated by reference in their entirety.
• the processor may be configured to determine the time of arrival of each constituent at the bifurcation, thereby enabling removal of selected constituents. Methods for determining the time of arrival as described above may be used.
• the post-separation spectrometer is associated with the branch conduit, rather than the conduit in which separation occurs.
• multiple loops may be provided in series, for example by coiling a conduit back on itself several times, such that the separated constituents can travel around one loop followed by another loop thereby enabling multiple measurements to be taken by the detector and/or spectrometers.
• This enables a better signal to noise ratio to be achieved. For example, better signal to noise ratio may be achieved where the constituent band suffers broadening.
• the first time past the detector can be used to predict the time of arrival at the post-separation spectrometer for each loop.
• Subsequent detections can be used to refine this prediction.
• Prediction of the time of arrival of a constituent at a point along the conduit is facilitated since the relationship between distance travelled and time elapsed is well-understood and in any case may be independently measured.
• different conditions may be provided in each loop.
• the varying conditions could advantageously allow extra information to be acquired in the same separation, resulting in a higher dimensionality of the information extracted from the separation.
• These varying conditions could include, but not be limited to, the use of a different buffer or the use of a different electric field gradient.
• the conduit may be divided into a number of segments such that each segment may be provided with different conditions.
• further spectrometer and/or detector inputs, or further spectrometers and/or detectors may be required to measure a sample and/or its constituents at each segment.
• a method for profiling a sample comprising separating the sample into a plurality of constituents according to a respective separation characteristic using a non-destructive separation process and obtaining a value of the separation characteristic for at least one of the plurality of constituents.
• the method also comprises carrying out a spectral analysis of the at least one of the plurality of constituents to obtain a spectrum for each of the at least one of the plurality of constituents and storing the value associated with the spectrum.
• the value of the separation characteristic of the at least one constituent is obtained from a detector signal indicative of each of the at least one of the plurality of constituents passing a detector.
• the method may comprise the use of the apparatus as previously described.
• the detector may be configured to detect the passage of a constituent by absorption of light that would otherwise reach the detector by the constituent passing the detector.
• the light may be ultra violet light, generally in the range 10-400nm, for example, in the range 200-400nm, alternatively in the UV-Vis range of 200- 500nm, for example, in the case of oil constituents.
• Other wavelength ranges or even single wavelengths (as in the use of a laser) may also be used.
• the UV range is Near Infra-Red (NIR) 750nm - 1400nm and indeed other ranges are possible as will be apparent to those skilled in the art.
• the detector may be configured to detect fluorescence or emission radiation from a constituent as the constituent passes the detector.
• the apparatus and method described herein are particularly advantageous in the rapid profiling of oil-head crude oil, in particular in off-line near-real-time and in-line real-time analysis.
• Other advantageous applications are point of care analysis of a blood sample, analysis of biological samples including proteins in general or, for instance, DNA, environmental sampling, analysis of chemicals, including but not limited to Toxic Industrial Chemicals (TICs) or Chemical Warfare Agents (CWAs).
• TICs Toxic Industrial Chemicals
• CWAs Chemical Warfare Agents
• the apparatus and method described herein have applications in the oil and gas analytics sector, but also in biomedicine, environmental analysis, security analytics, the food and drink sector and in the renewable, hydrocarbon and nuclear power sectors. Samples will generally be liquid in nature, but with a suitable liquefaction preliminary step, could be gaseous.
• the information from the separation stage and post-separation spectrometer can be stored and analysed.
• the individual separated constituents may be correlated - or connected - to the individual spectra of the respective separated constituents.
• Several approaches are proposed in certain embodiments. For example, the storing of individual constituent spectra (whether absorption, fluorescent or Raman in whatever wavelength range is required for the particular embodiment) associated with a given separation band, which will correspond to a single molecular type or a combination of such molecular types which are too close in the separation space to separate. Correlation can be made in an ad hoc way which allows a variety of analytical methods. Automated
• correlation of separation data relating to a constituent may be made with existing bodies of profiling data, for example mass spectrometry (MS), gas chromatography (GC) and GC-MS libraries, via the corresponding spectral data obtained.
• a separation technique e.g. high performance capillary electrophoresis (HPCE)
• HPCE high performance capillary electrophoresis
• Fig 1 schematically illustrates an apparatus according to a first embodiment
• Fig 2 schematically illustrates a processor unit coupled to the detector and post-separation spectrometer of the apparatus of Fig 1
• Fig 3 illustrates a flow diagram of a method of profiling a sample
• Fig 4 illustrates prediction of trajectories/constituent positions with the apparatus of Fig 1 .
• Fig 5 schematically illustrates an apparatus according to a second embodiment.
• an apparatus 2 comprises a linear separation conduit 4 through which a sample may flow in the direction shown by arrow 'A' in response to a driving force.
• the separation conduit 4 is a capillary.
• the separation conduit is a microfluidic channel or other separation conduit
• An injection port 6 enables introduction of a sample into the conduit 4, enabling the sample to travel along the separation conduit 4, to exit the separation conduit 4 at a (optional) bifurcation 8 along a branch conduit 10, or to continue to travel past the bifurcation 8 along the separation conduit 12.
• the apparatus 2 is arranged to separate the sample into a plurality of constituents 14, as the sample and its constituents 14 travel in the direction shown by arrow A. Separation of the sample is by capillary electrophoresis, for example, capillary zone electrophoresis and will be described in more detail below.
• a post-separation spectrometer 16 is positioned adjacent the separation conduit 4, upstream of the bifurcation 8. The post-separation spectrometer 16 is configured to obtain a spectrum 18 of each constituent 14 as it passes the post-separation spectrometer 16. The post-separation spectrometer 16 is a UV spectrometer and so obtains UV absorption spectra 18 of the constituents 14.
• a pre-separation spectrometer 20 is positioned adjacent the separation conduit 4, downstream of the injection port 6 and upstream of the post-separation spectrometer 16. The pre-separation spectrometer 20 is configured to obtain a spectrum 22 of the sample prior to any substantial separation occurring. The pre-separation spectrometer 20 is a UV spectrometer and is configured to obtain a UV absorption spectrum 22 of the sample.
• a detector 24 is provided adjacent the separation conduit 4 between the post-separation spectrometer 16 and the pre-separation spectrometer 20.
• the detector 24 comprises a number of pixels provided along the direction of travel A of the constituents 14. As a constituent 14 passes each pixel it is detected by the detector 24 and generates a detector signal. Each constituent 14 that passes the detector 24 creates a distinct signal in the detector readout 26.
• the detector 24 is a photonic detector, for example, a UV detector.
• this detector may be capable of matching the wavelength(s) of the UV light source (for example a Xenon or Deuterium lamp, or a diode laser, among other possibilities) and also matching the absorption properties of the constituents.
• the detector may use wavelengths in the 200-400nm range, for example, 200-300nm, for example, around 254nm.
• the apparatus 2 comprises a light source 27, for example a UV light source, on one side of the separation conduit 4 and the detector 24 on the other. As a constituent 14 passes between the light source 27 and the detector 24, there is a reduction in the intensity of light reaching the detector 24 providing the detected signal.
• a light source 27 for example a UV light source
• the apparatus 2 comprises two electrodes 28, 30.
• the first electrode 28 coupled to a first end 32 of the separation conduit 4, and the second electrode 30 is coupled to a second end 34 of the separation conduit 4.
• the electrodes 28, 30 are connected to a power supply 36 such that an electric field can be generated across the separation conduit 4, between the first and second ends 32, 34 to drive separation.
• separation is driven by a pressure differential across the length of the separation medium 4.
• electrodes are inserted into an electrically contiguous part of the separation medium.
• a pressure gradient is imposed across the separation conduit 4.
• the apparatus 2 comprises a processor unit 37 having a processor 38 and a memory component 39.
• the processor unit 37 receives input from the detector 24 and the post-separation spectrometer 16 (the remaining features of Fig. 1 being omitted to aid clarity of presentation).
• the processor 38 is configured to determine a value of the separation characteristic of the constituent 14 from the detector signal.
• the memory component 39 is configured to store the value of the separation characteristic associated with the spectrum 18.
• the separation conduit 4 is filled with suitable buffer solution comprising, for example an aqueous mixture of salts or other electrolytes, for example, brine, TBE, Tris, or any other suitable buffer as will be known in the art.
• suitable buffer solution comprising, for example an aqueous mixture of salts or other electrolytes, for example, brine, TBE, Tris, or any other suitable buffer as will be known in the art.
• the precise buffer solution will depend on the mixture to be separated.
• a sample for example, a crude oil sample, is then introduced into the separation conduit 4 by injection into injection port 6.
• an absorption spectrum 22 of the sample is obtained by the pre-separation spectrometer 20.
• An electric field is generated across the separation conduit 4 by means of the power supply 36 and electrodes 28, 30, causing the sample to separate into a plurality of constituents 14, indicated by step 40.
• Each constituent 14 travels through the separation conduit 4 at a different velocity based on the electrophoretic mobility of the constituent 14 and the magnitude of the electric field applied across the conduit 4. As described previously, other separation techniques may also be used. Since each constituent 14 travels through the conduit 4 at a different speed (that speed may be moderated by diffusion effects), the constituents 14 of the sample separate along the length of the conduit 4, and therefore travel past the detector 24 at different times. Similarly the constituents 14 travel past the post-separation spectrometer 16 at different times.
• each constituent 14 travels along the separation conduit 4 from the pre-separation spectrometer 20 in the direction of travel indicated by arrow TV, they pass detector 24. As a constituent 14 passes each pixel of the detector 24 its passage of each pixel is detected and generates a detector signal, creating a distinct signal in the detector readout 26.
• the value of the separation characteristic, in the case of CZE the effective charge, for each constituent 14 is determined by the processor 38 from the velocity of the constituent 14 as determined from the detector signal. This is illustrated by step 42 in Fig 3. Determination of constituent charge from constituent velocity is known in the art, see for example An introduction High Performance Capillary Electrophoresis, by David Heiger, as referenced above.
• each constituent 14 continues to travel along the separation conduit 4 from the detector 24 to the post-separation spectrometer 16. As each constituent 14 passes the post-separation spectrometer 16, the post-separation spectrometer 16 obtains an absorption spectrum 18 of the constituent 14. This is illustrated by step 44 in Fig 3.
• the constituents 14 continue to travel along the separation conduit 4 from the post-separation spectrometer 16, either leaving the separation conduit 4 at the bifurcation 8 along the branch conduit 10, or continuing along the separation conduit 12.
• the value of the separation characteristic and spectrum for each constituent 14 is then compared by the processor 38 with a database of separation characteristics associated with respective spectra to identify the sample, as shown by step 46 in Fig 3, or in some
• embodiments is simply stored as a sample profile for later use.
• comparison by the processor 38 between the spectrum 22 of the sample prior to separation obtained by the pre-separation spectrometer 20 with that of the aggregated spectra 18 of each constituent 14 obtained by the post-separation spectrometer 16 is used in some embodiments to identify any differences which may exist for the purposes of quality control to determine whether all constituents 14 of the sample have reached the post-separation spectrometer 16.
• the processor 38 is in some embodiments configured to raise a quality control alert if the constituent spectra do not add up, to a set tolerance level, to the spectrum obtained for the sample.
• the information obtained from the detector 24 may be used to predict when the constituent 14 will arrive at the post-separation spectrometer 16, thereby aiding the association between the separation information from the detector 24 and the spectral information from the post-separation spectrometer 16 for each constituent 14.
• the individual constituents may not fully separate, for example, where more than one constituent travels along the separation conduit 4 at the same or a similar speed. In this case, the detector will measure the aggregate of the more than one constituent.
• the resulting spectrum will have contributions from the more than one constituent.
• the separation stage and the spectral stages provide complementary information, and therefore increase the likelihood of discriminating between the components of interest, and quantifying them accurately.
• the detector 24 comprises a number of pixels 48 provided along the direction of travel, A, of the constituents 14. As a constituent 14 passes each pixel 48 it is detected by the detector 24. The time at which the constituent 14 passes each pixel is recorded and plotted 50 as shown in Fig 3. From this position and time information the speed of travel of the constituent can be calculated, typically using a linear fit 52, the gradient of this line equating to the speed of travel of the constituent 14. This fit 52 may optionally be refined by also including the time 54, 56 at which the sample is introduced into the separation conduit 4.
• the linear fit 52 may then be extrapolated to determine the time at which a given constituent 14 will be at a particular point along the separation conduit 4.
• the fit itself may be a linear fit, calculated using a least-squares or maximum likelihood approach. However, a linear fit (constant velocity trajectory) is not presupposed and other trajectories are possible and useful in this regard. Statistical techniques other then linear regression may be used to determine the trajectory. As shown in Fig 4, the time of arrival at the post-separation
• spectrometer 16 for each constituent 14 is calculated at points along the line indicated by the number 58.
• the time of arrival of a desired constituent 14 at the bifurcation 8 may also be calculated so that selected constituents 14 may be extracted. This feature could be used for subsequent off-line analysis of any given constituent, if required, using, for example, a Mass Spectrometer.
• the separation conduit 4 is configured as a loop by coiling a conduit back on itself.
• the sample is injected into the conduit 4 via injection port 6.
• the sample and its constituents 14 travel around the loop in the direction of travel indicated by arrow A.
• the sample leaves the loop along a branch conduit 10, or travels past a bifurcation 8 along the separation conduit 12.
• a single spectrometer 60 is provided adjacent the conduit 4, such that the sample travels from the injection port 6 past the spectrometer 60.
• the spectrometer 60 performs the function of both the post-separation and pre-separation spectrometer described in relation to the first embodiment.
• the sample is introduced to the separation conduit 4 and passes the spectrometer 60 prior to any significant separation of the sample. At this point a spectrum of the sample prior to separation is obtained.
• the detector 24 which is configured to operate as described above in relation to the first embodiment.
• the constituents then continue past the spectrometer 60 for a second time. As each constituent 14 passes the spectrometer 60, the spectrometer 60 obtains a spectrum for each constituent. The sample then leaves the loop via the branch conduit 10 or separation conduit 12.
• the detector 24 information and spectral data obtained for the sample and the constituents 14 may then be processed in the same way as described in relation to the first embodiment.
• the separation conduit 4 may be coiled to form multiple loops such that in the first loop, both the sample prior to separation and the constituents 14 post separation pass the spectrometer 60 in the first loop. In subsequent loops, the constituents 14 pass the spectrometer 60 once per loop.
• separation techniques other than CZE, such as CGE, may be used as well as separation techniques other than electrophoresis, such as liquid chromatography, could be used.
• spectrometers and/or detectors other than UV absorption spectrometers and/or detectors may be used, for example, spectrometers or detectors configured to obtain emission, radiation (including but not limited to Raman) or fluorescence spectra.
• the apparatus is not limited to having a separation conduit comprising a linear or looped capillary, but, the separation conduit may be provided in any other suitable configuration.
• pixel is employed for components of the detector 24 configured to detect constituents/light intensity along the direction of the conduit 4, this term and the corresponding components, as used herein, are not limited to a specific geometric arrangement but intended to cover any such arrangement, for example a 2-D or 1-D array of detector units such as a Photodiode Array (PDA), Charge Coupled Device (CCD) array, or a miniaturized array of Photo Multiplier Tubes (PMT), which is capable of defecting signals as described above.
• PDA Photodiode Array
• CCD Charge Coupled Device
• PMT Photo Multiplier Tubes
• processor 38 is depicted as a single functional unit in Figure 2, it will be understood that this also covers a distributed arrangement of several physical processor, some or all of which may be integrated with the apparatus described above, or provided in a separate device, for example a desktop computer and indeed covers any physical arrangements of processing circuitry capable of performing the necessary processing.
• the processing circuitry may be of a general purpose type specifically programmed to carry out the functions described above or may be provided by an application specific integrated circuit (ASIC). Any computations may be performed, for example, in software, hardware, firmware, middleware or any suitable combination thereof.

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• 2014
  • 2014-03-17 GB GBGB1404756.7A patent/GB201404756D0/en not_active Ceased
• 2015
  • 2015-03-17 WO PCT/GB2015/050784 patent/WO2015140543A1/en active Application Filing

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