WO2013090972A1

WO2013090972A1 - Method for analysing a sample comprising a plurality of analytes

Info

Publication number: WO2013090972A1
Application number: PCT/AU2012/000142
Authority: WO
Inventors: Mark Jerome Eric TRUDGETT; Ross Andrew SHALLIKER
Original assignee: University Of Western Sydney
Priority date: 2011-12-23
Filing date: 2012-02-16
Publication date: 2013-06-27

Abstract

Disclosed herein is a method and system for analysing a sample comprising a plurality of analytes. The method comprises the steps of passing the sample through a first separating zone and a second separating zone, whereby each of the plurality of analytes have a first speed in the first separating zone and a second speed in the second separating zone; generating temporal information by sensing the plurality of analytes at a plurality of spaced apart positions at the second separating zone; and generating time dependent spectral information from the temporal information.

Description

METHOD FOR ANALYSING A SAMPLE COMPRISING A PLURALITY OF

ANALYTES

Field of the Invention

The invention generally relates to methods and systems for analysing a sample comprising a plurality of analytes, and specifically but not exclusively to

chromatographic and electrophoretic methods and systems for analysing a sample comprising a plurality of analytes.

Background of the Invention

A number of techniques that utilize time-based chemical separations can be used to analyse a sample. In such techniques, the separation of chemicals (analytes) within a sample is based on either the time required for each analyte to travel a given distance, or the distance travelled by any analyte in a given time. Such time-based separations include the analytical methods collectively known as chromatography and

electrophoresis.

Time-based chemical separations depend on the movement of analytes contained in a moving medium (the "mobile phase") through a zone containing non- moving material (the "stationary phase"). The time taken by the analytes to traverse the stationary phase (or the distance travelled by analytes relative to the stationary phase) depends on the chemical and physical affinities of each of the analytes with respect to both the mobile and stationary phases. Analytes in a sample can therefore be separated because of their differing relative affinities to the mobile and stationary phases and their susceptibility to any driving forces applied to or through the mobile phase.

Chromatography, electrophoresis and related techniques may therefore be used to characterize analytes in a sample.

In order to achieve separations of highly complex mixtures of analytes (where there is a risk that two or more analytes may traverse a stationary phase at the same rate), the use of multiple separation dimensions has been employed. These techniques are commonly referred to as "multidimensional" separations (a system having two separating dimensions is known as a two-dimensional separation, etc.).

Multidimensional separations usually employ two or more different stationary phases because it is unlikely that any two analytes in a sample will have the same chemical and physical affinities with both of the stationary phases. Multidimensional separations can generally be classified as either "offline" or "online" multidimensional separations.

In offline multidimensional separations, a comprehensive separation of analytes in a sample may be achieved. In practice, the eluate from the first separation dimension is fractionated, and each fraction is subsequently passed independently through the second separation dimension (the eluate of which may, in turn, be fractionated and passed similarly through further separation dimensions). The physically independent analysis of fractions of eluate through subsequent separation dimensions diminishes the possibility that analytes which passed through an earlier dimension at a different time will subsequently overlap at the end of the subsequent dimension (due to their differing speeds in the dimensions). Offline separations result in a more comprehensive separation of analytes in a sample, but at the cost of analysis times, which increases with the number of fractions and the number of separation dimensions.

In online multidimensional separations, the analytes are passed (in the eluate) from the first dimension to a second dimension via an interface that modulates the flow from the first dimension to the second dimension to match the relative speed differential between the two dimensions. Online separation systems are advantageous because the sample is never removed from the chromatographic system and they are typically much faster than offline systems. However, online separation systems often cannot provide a sample analysis as comprehensive as that provided by offline separation systems because of the so-called "wrap around effect", in which analytes exiting one dimension in a particular order become mixed with other analytes while passing through a subsequent dimension due to the analytes' differing relative speeds in each of the dimensions. Thus, the analytes can become randomly displaced across the

multidimensional separation domain, and it may not be possible to fully characterise the analytes in the sample.

Due to the different constraints imposed by analysis times (as described for both online and offline multidimensional separations), conventional multidimensional separations have been generally limited to separations in no more than three dimensions. Summary of the Invention

A first aspect of the invention provides a method for analysing a sample comprising a plurality of analytes, the method comprising the steps of:

passing the sample through a first separating zone and a second separating zone, whereby each of the plurality of analytes have a first speed in the first separating zone and a second speed in the second separating zone;

generating temporal information by sensing the plurality of analytes at a plurality of spaced apart positions at the second separating zone; and

generating time dependent spectral information from the temporal information.

Advantageously, the method of the invention may provide a sample analysis that is as comprehensive as an analysis undertaken using conventional offline

multidimensional separation techniques, but in a time similar to, or less than, that taken for conventional online multidimensional separations. Furthermore, because the method may not suffer the same analysis time considerations as conventional multidimensional separations, more separation dimensions may be employed, thus providing greater separating power.

As each of the plurality of analytes in the sample may generate a component of the temporal information that is localized in both time and frequency, these components may therefore be distinguishable in the time dependent spectral information. Thus, even if a number of analytes exit the first or second separation zone simultaneously, the temporal information generated by sensing the analytes at the second separating zone (from which the first and second speeds of the analytes can be determined) may enable the analytes to be distinguished from each other in the time dependent spectral information. The method of the invention may therefore be capable of distinguishing analytes that simultaneously exit the first and/or second separation zones. As discussed above, online multidimensional separation techniques can be compromised because it is sometimes not possible to precisely identify when certain analytes exit the first and/or second separation zone due to the "wrap around effect". Further, in the method of the invention it may not be necessary to collect aliquots from the first separating zone and separately pass each of those through the second separating zone because the temporal information necessary to provide an at least as comprehensive analysis as that of conventional offline multidimensional separation techniques may be generated whilst the analytes pass through the second separating zone. As will be appreciated, this can greatly reduce the time required to analyse a sample.

In an embodiment, the method comprises the step of correlating the time dependent spectral information with information associated with known substances. In an embodiment, the step of generating the time dependent spectral information comprises the step of determining the frequency components of only a portion of the temporal information.

In an embodiment, the step of generating the time dependent spectral information comprises the step of applying a short-time Fourier transform to the temporal information. Additionally or alternatively, the step of generating the time dependent spectral information comprises the step of applying a Wigner transform to the temporal information. Additionally or alternatively, the step of generating the time dependent spectral information comprises the step of applying a Gabor-Wigner transform to the temporal information. Additionally or alternatively, the step of generating the time dependent spectral information comprises the step of applying a wavelet transform to the temporal information. Additionally or alternatively, the step of generating the time dependent spectral information comprises the step of passing the temporal information through a plurality of band pass filters. The plurality of band pass filters may comprise analogue band pass filters. The band pass filters may comprise digital band pass filters. Generally, the time dependent spectral information may be generated using any suitable means.

In an embodiment, the step of generating the time dependent spectral information comprises the step of generating information indicative of a spectrogram of the temporal information. In an embodiment, at least some of the plurality of analytes are optically sensed. Additionally or alternatively, at least some of the plurality of analytes are sensed by one or more sensing techniques independently selected from the group consisting of: mass selective sensing; electrochemical sensing; electrical conductive sensing; thermal conductive sensing; light scattering sensing; thermal ionization sensing; electrical ionization sensing and electron capture sensing.

Substances which may be used in the first and second zones may comprise any substance through which a sample may be passed and which is capable of separating, at least to some extent, analytes in the sample. In an embodiment, the first separating zone comprises a first material and the second separating zone comprises a second material. The first material and the second material may, for example, be independently selected from the group consisting of: silica, fused silica, Squalane, dimethylsilicone, polyethyleneglycol, phenylmethylsilicone oils, diethyleneglycosuccinate, alumina, titania, zirconia, mixed oxide ceramics, HILIC, ion-exchange resins, ion exclusion resins, size exclusion resins, C8 and CI 8.

In an embodiment, the sample is a gas when passed through the first separating zone and the second separating zone. The first separating zone and the second separating zone may, for example, be columns in a gas chromatograph.

In an embodiment, the sample is a liquid when passed through the first separating zone and the second separating zone. The first separating zone and the second separating zone may, for example, be columns in a liquid chromatograph, columns in a high performance liquid chromatograph (HPLC), or capillaries in a capillary electrophoresis system.

In an embodiment, the sample is passed through the first separating zone and/or the second separating zone by pumping the sample through the first separating zone and/or the second separating zone, by sucking or blowing the sample through the first separating zone and/or the second separating zone or by applying a potential difference between the first separating zone and/or the second separating zone in order to induce an electro-driven flow. In an embodiment, the method comprises the further step of passing the sample through at least one further separating zone. In such an embodiment, the method may comprise the further steps of:

generating further temporal information by sensing the plurality of analytes at a plurality of spaced apart positions at the at least one further separating zone; and

generating further time dependent spectral information from the further temporal information.

A second aspect of the invention provides a system for analysing a sample comprising a plurality of analytes, the system comprising:

a first separating zone arranged for passing the sample therethrough;

a second separating zone adapted to receive the sample from the first separating zone and arranged for passing the sample therethrough, whereby the first separating zone and the second separating zone are arranged for the analytes to have a first speed in the first separating zone and a second speed in the second separating zone;

a temporal information generator arranged for generating temporal information, the temporal information generator comprising one or more sensors arranged to sense the plurality of analytes at a plurality of spaced apart positions at the second separating zone; and

a time dependent spectral information generator arranged to generate time dependent spectral information from the temporal information.

In an embodiment, the system comprises a correlator arranged to correlate the time dependent spectral information with information associated with known substances.

In an embodiment, the time dependent spectral information generator is arranged to apply a short-time Fourier transform to the temporal information.

Alternatively or additionally, the time dependent spectral information generator is arranged to apply a Wigner transform to the temporal information. Alternatively or additionally, the time dependent spectral information generator is arranged to apply a Gabor-Wigner transform to the temporal information. Alternatively or additionally, the time dependent spectral information generator is arranged to apply a wavelet transform to the temporal information. Alternatively or additionally, the time dependent spectral information generator has a plurality of band pass filters arranged to filter the temporal information. The plurality of band pass filters may comprise analogue band pass filters. The band pass filters may comprise digital band pass filters.

In an embodiment, the time dependent spectral information generator is arranged to generate information indicative of a spectrogram of the temporal information.

In an embodiment, the one or more sensors comprise at least one optical sensor. Alternatively or additionally, the one or more sensors may comprise at least one of: a mass selective sensor; an electrochemical sensor; an electrical conductive sensor; a thermal conductive sensor; a light scattering sensor; a thermal ionization sensor; an electrical ionization sensor and an electron capture sensor.

In an embodiment, the first separating zone comprises a first material and the second separating zone comprises a second material. The first material and the second material may be independently selected from the group consisting of: silica, fused silica, Squalane, dimethylsilicone, polyethyleneglycol, phenylmethylsilicone oils, diethyleneglycosuccinate, alumina, titania, zirconia, mixed oxide ceramics, HILIC, ion- exchange resins, ion exclusion resins, size exclusion resins, C8 and CI 8.

An embodiment of the system is arranged for the sample to be a gas. The system may, for example, comprise a gas chromatograph.

An embodiment of the system is arranged for the sample to be a liquid. The system may for example, comprise a liquid chromatograph, a high performance liquid chromatograph (HPLC) or a capillary electrophoresis system.

An embodiment of the system comprises a pump arranged to pump the sample through the first separating zone and/or the second separating zone, a suction device arranged to suck the sample through the first separating zone and/or the second separating zone, a source of static pressure (e.g. a gas cylinder) arranged to blow or push the sample through the first separating zone and/or the second separating zone, or a potential difference source arranged to apply a potential difference between the first separating zone and/or the second separating zone in order to induce an electro-driven flow.

An embodiment of the system may comprise at least one further separating zone. A third aspect of the invention provides a method comprising the steps of:

(a) receiving temporal information, the temporal information having been generated by:

(i) passing a sample comprising a plurality of analytes through a first separating zone and a second separating zone, whereby each of the plurality of analytes have a first speed in the first separating zone and a second speed in the second separating zone; and

(ii) sensing the plurality of analytes at a plurality of spaced apart positions at the second separating zone; and

(b) generating time dependent spectral information from the temporal information.

A fourth aspect of the invention provides a system, the system comprising: (a) a temporal information receiver arranged to receive temporal information, the temporal information having been generated by:

(b) a time dependent spectral information generator arranged to generate time dependent spectral information from the temporal information.

Where possible, features of any one of the aspects of the invention may be combined. Brief description of the figures

In order to achieve a better understanding of the nature of the present invention, embodiments of the invention will now be described, by way of example only, with reference to the accompanying figures in which:

Figure 1 shows a flow diagram of the steps in the method of the first aspect;

Figure 2 shows a block diagram of a system in accordance with an embodiment of the system of the second aspect;

Figure 3 depicts the combination of a number of signals from multiple sensors to produce a combined signal for analysis;

Figure 4 depicts an embodiment of the method of the first aspect being used to process multiple sensor signals within a system employing multiple separation dimensions;

Figure 5 depicts an embodiment of the system of the second aspect; and Figure 6 depicts another embodiment of the system of the second aspect having a further separation zone than the system of Figure 5.

Detailed description of embodiments of the invention

Figure 1 shows a flow diagram of the steps in a method for analysing a sample comprising a plurality of analytes, the flow diagram being generally indicated by the numeral 10. Figure 2 shows a block diagram of an embodiment of a system of the second aspect, generally indicated by the numeral 20, the system being arranged for analysing a sample in accordance with the method of figure 1.

Referring to Figures 1 and 2, in a step 12 of method 10, a sample 22 comprising a plurality of analytes is passed through a first separating zone 24 and a second separating zone 26. Each of the plurality of analytes have a first speed in the first separating zone 24 and a second speed in the second separating zone 26.

In another step 14 of the method 10, temporal information is generated by sensing the plurality of analytes at a plurality of spaced apart positions 28, 30 at the second separating zone 26. In system embodiment 20, the temporal information is generated by a temporal information generator 42 having sensors 32, 34 and a sensor signal combiner 40. The sensors 32, 34 are at each of the spaced apart positions 28, 30. In another embodiment (not shown), the sensors may be remote from the spaced apart positions and sensed remotely, as may be arranged for a sensor comprising a laser beam and light detector, for example. The sensors 32, 34 are arranged to sense the plurality of analytes at their respective spaced apart positions 28, 30 and generate respective sensor information. The sensor information may (but not necessarily) be indicative of analyte concentration at the respective point at the second separating zone.

In system embodiment 20, the sensors 32, 34 are in communication with the signal combiner 40, which receives and combines the sensor information. In other system embodiments (not shown), there may be more than two spaced apart positions and sensors and, in such embodiments, the combiner may be in communication with the more than two spaced apart positions and sensors. In system embodiment 20 the combiner 40 combines the information by summing the sensor information. In other embodiments (not shown), the sensor information may be combined in any suitable way and/or conditioned before and/or after combination as suitable. For example, a DC offset or noise may be removed by conditioning means such as a digital or analogue signal conditioner. The signal conditioner may be incorporated in a combiner, for example, or be arranged to receive the temporal signal from a combiner before the signal is passed to other components and/or units.

In system embodiment 20, the sensors 32, 34 are electrically connected to the combiner 40 by electrical connections 36, 38 in the form of conductive tracks on a printed circuit board that hosts the sensors and the combiner. However, any suitable electrical or optical connection may be used. For example, in another embodiment (not shown), the electrical connections are coaxial cables terminated with BNC connectors. In other embodiments, the connections may be an optical connection such as a digital fibre optic connection. In other embodiments, the connections may be a wireless connection, for example a WiFi™ link, or any suitable type of connection. Other connections within system embodiment 20, such as connection 44, may also be any suitable type of connection such as an electrical, optical or wireless connection.

The combined signal information from the combiner 40 is indicative of the presence of analytes at any one of the spaced apart positions 28, 30, over the sensing period. Generally, the sensing period commences when the sample 22 is introduced to - li the first separating zone 24 and ends when the sample leaves the second separating zone 26. However, other sensing periods may be used as is suitable.

The sensing of the passage of the analytes past the spaced apart positions 28, 30 generates a plurality of temporal pulses. The pulses have a regular period and thus a corresponding frequency. For example, figure 3(a) shows example pulses in the sensor information produced by each of the sensors in a system embodiment having 5 spaced apart positions and 5 sensors as an analyte travels through the second separating zone. Figure 3(b) shows an example combination of the pulses of figure 3(a). The passage of each analyte past the sensors contributes a note of characteristic period and

consequently frequency to the combined signal at an output of a combiner. The frequency is dependent on the speed at which the analyte passes the plurality of spaced apart points. The frequency of the note increases with the speed of the analyte through the second separating zone. The note also has a characteristic position in the sensing period. The position of the note within the sensing period is dependent on the speed of the analyte through the first and second separating zones. The strength of the note is dependent on the interaction of the sensors and the analytes and may be indicative of the analyte concentration at the spaced apart positions.

While system embodiment 20 has two spaced apart positions 28, 30 and associated sensors 32, 34, other embodiments may have more spaced apart positions. There may, for example, be 3, 4, 5, 6 or generally any number of such spaced apart positions, in which case the spacing between adjacent spaced apart positions may be constant. Furthermore, it is not necessary to provide a sensor for each of the spaced apart positions. For example, a single sensor (referred to herein as a multiplexed sensor) may be arranged to record signals at one or more of the spaced apart positions. The performance of the system may increase with the number of spaced apart positions because the frequency bandwidth of the generated notes decreases with the number of spaced apart positions at which the analytes are sensed, improving note definition. The characteristic (centre) frequencies of the notes are not dependent on the number of spaced apart positions.

In another step 16 of method 10, time dependent spectral information is generated from the temporal information. The system embodiment 20 has a time dependent spectral information generator 46 that receives the temporal information from the temporal information generator 42 via connection 44. Conceptually, the time dependent spectral information generator, at least in this embodiment, maps the notes in the temporal information onto a 2 dimensional frequency-time map that is indicative of the timing and characteristic frequency of the notes. The frequency-time map is conceptually similar to a musical score that positions notes in frequency (vertical position on a musical stave) and time (horizontal position on the musical stave). A frequency-time map, when represented visually, is referred to as a spectrogram.

The time dependent spectral information generator 46 is arranged to apply a short-time Fourier transform to the temporal information, which generates the time dependent spectral information. The short-time Fourier transform may be implemented using a known Fast Fourier Transform algorithm with a Gaussian function (or any suitable windowing function such as triangular) centred at the time within the sensing period of interest, applied to the temporal signal data. In other embodiments, however, the time dependent spectral information generator may be alternatively or additionally arranged to apply at least one of a Wigner transform to the temporal information, a Gabor-Wigner transform to the temporal information and a wavelet transform to the temporal information. Generally, any suitable algorithm or transformation that maps the notes may be used. Definitions of these transformations and suitable code for their implementation may be found at the Numerical Recipes™ website www.nr.com, for example. Other references include Numerical Recipes in C (1992).

In system embodiment 20, the time dependent spectral information generator 46 comprises a computational unit 48 which computes the transformation. In another system embodiment (not shown), the computational unit applies digital band pass filters to the temporal signal, each filter being configured to pass a note having a particular characteristic frequency. In system embodiment 20, the computational unit 48 is an industrial PC running suitable transformation or band pass software. In other embodiments, however, the computational unit may comprise application specific hardware having a logic device such as a field programmable array or ARM processor, for example, an embedded system, a remote server connected to the temporal information generator over the internet, or any suitable unit having suitable computational capability.

The signal combiner 40 and time dependent spectral information generator 46, and other units, may comprise software units. For example, the sensors may be connected to an analogue-to-digital card in communication with a processor running Labview™ or equivalent software. The information generated by the sensors, which in one embodiment is an analogue electrical signal, is digitised by the card, and the sensor information may be combined by a fist Labview™ software unit, and then further processed in a second Labview™ software unit that generates the time dependent spectral information digitally. The time dependent spectral information may be rendered by a graphics unit on a display such as a LED flat panel display in communication with the processor. The time dependent spectral information may be saved to a file on a storage device such as a hard drive in communication with the processor, for example, and accessed by a correlator software unit. In this embodiment, the processor may comprise a suitable microprocessor such as, or similar to, the INTEL PENTIUM™, connected over a bus 148 to a random access memory of around 100 Mb and a non-volatile memory such as a hard disk drive or solid state nonvolatile memory having a capacity of around 1 Gb. The processor may have input/output interfaces such as a universal serial bus and a possible human machine interface e.g. mouse, keyboard, display etc.

Alternatively, the processor may comprise a programmed Field Programmable Array having an analogue-to-digital converter and suitable algorithms as described above written therein. This may facilitate particularly fast analysis. In another embodiment, custom software may be written for improved performance together with custom printed circuit boards and/or circuits. The processor may be an embedded system.

Alternatively, the spectral information generator (e.g. spectral information generator 46 in system embodiment 20) may not be a computational device. The spectral information generator may be an analogue device. The spectral information generator may comprise a plurality of analogue band pass filters each arranged to filter the temporal information and pass a note having a particular characteristic frequency. The output from each band pass filter may then be plotted in real time on a piece of paper, for example. Generally, the spectral information generator may comprise any combination of digital and analogue components as suitable.

Each of the analytes typically generates a component of the temporal signal that is localized in time and frequency. Consequently, these components may be distinguished in the time dependent spectral information. Even if two analytes share their location in one of time and frequency, then they can still be distinguished. The simultaneous differentiation of analytes in time and frequency may therefore provide superior differentiation between analytes than provided for to date.

The method of the first aspect may comprise the optional step of correlating the time dependent spectral information with information associated with known substances (e.g. analytes that are suspected to be present in the sample).

Correspondingly, the system embodiment 20 has a correlator 50 arranged to correlate the time dependent spectral information with information associated with known substances. For example, prior to performing the method of the first aspect on a sample, the method may be performed separately for known substances. The resulting features in the time dependent spectral information, now attributable to known substances, may be saved to a database 52. The correlator 50 may then seek to identify these features in the time dependent spectral information for a later analysed sample. Alternatively, the information in the database 52 may be calculated using information about substances and the system embodiment 20, rather than being empirically generated.

In the methods and systems of the invention, the analytes may be sensed using any technique that will sense the analytes. In some embodiments, at least some of the analytes are optically sensed (e.g. using UV, Vis, I , RI, fluorescence or

chemiluminescence sensors or detectors). For example, at least some of the analytes may be sensed by sensing a light that has been partially absorbed or scattered by at least one of the analytes. In other embodiments, at least some of the analytes may be sensed by sensing luminescence from the analyte. For example, at least some of the analytes may be sensed by sensing chemiluminescence or fluorescence from the analyte. In other embodiments, at least some of the analytes may be sensed by light scattering sensing. In other embodiments, the sensors may not be optical, but may be configured for mass selective sensing, electrochemical sensing, electrical conductive sensing, thermal conductive sensing, thermal ionization sensing, electrical ionization sensing and/or electron capture sensing.

In some embodiments, a single sensor can be used to sense the plurality of analytes at a plurality of the spaced apart positions at the second separating zone. Such sensors are commonly referred to as multiplexed sensors or multiplexed detectors. Multiplexed sensors may comprise a single sensor that alternately receives signals from two or more of the spaced apart positions. A multiplexed sensor may, for example, consist of a single potentiometer (voltage sensor) that is alternately electrically switched between electrodes located at one or more spaced apart positions to alternately measure the potential difference at each of those spaced apart positions. Additionally or alternatively, multiplexed sensors may comprise a single sensor that is physically moved from one spaced apart position to another. For example, a single optical spectrograph may be mechanically moved to sense the presence or absence of light at one or more of the spaced apart positions.

In some embodiments, two or more sensors can be used to sense the plurality of analytes at the plurality of spaced apart positions at the second separating zone.

Substances which may be used in the first and second zones in the methods and systems of the invention may comprise any substance through which a sample may be passed and which is capable of separating, at least to some extent, analytes in the sample. In some embodiments, the first separating zone has a first material and the second separating zone has a second material. For example, the first separating zone may comprise a first stationary phase and the second separating zone may comprise a second stationary phase. The first material and the second material may, for example, be independently selected from the following substances: silica; fused silica; Squalane; dimethylsilicone; polyethyleneglycol; phenylmethylsilicone oils;

diethyleneglycosuccinate; alumina; titania; zirconia; mixed oxide ceramics, or any ceramic; phenyl or phenyl-type phases, for example, diphenyl, or pentafluoro phenyl; HILIC; amide; ion-exchange, ion exclusion or size exclusion resins; C8, CI 8, other alkyl chains, including substituted alkyl chains, or polar embedded stationary phases, or polar end-capped stationary phases or stationary phases made from polymers, peptides or proteins.

Alternatively, the first separating zone and the second separating zone may be at different temperatures. In such embodiments, provided the zones having different temperatures are capable of separating, at least to some extent, analytes in the sample, the first separating zone and the second separating zone could comprise the same substance.

In some embodiments, the first separating zone and the second separating zone may be contained within separate physical units, such as, for example, separate HPLC columns or separate gas chromatograph capillaries. In other embodiments, the first separating zone and the second separating zone may be contained within a single physical unit.

The sample may be a gas when passed through the first separating zone and the second separating zone. The first separating zone and the second separating zone may be columns in a gas chromatograph, for example. In this embodiment, the system is arranged for the sample to be a gas and may be broadly described as a gas

chromatograph, for example.

Alternatively, the sample may be a liquid when passed through the first separating zone and the second separating zone. The first separating zone and the second separating zone may, for example, be columns in a liquid chromatograph, columns in a high performance liquid chromatograph (HPLC), or capillaries in a capillary electrophoresis system. In this embodiment, the system is arranged for the sample to be a liquid. The system may comprise a liquid chromatograph, a high performance liquid chromatograph (HPLC) or a capillary electrophoresis system, for example.

Alternatively, in some embodiments, the sample may be a supercritical fluid when passed through the first separating zone and the second separating zone.

In some embodiments, the sample may be a liquid when passed through the first separating zone and a gas when passed through the second separating zone, or vice versa. Further, the methods and systems of the invention may utilize combinations of time-based separation techniques (e.g. a HPLC coupled with a liquid chromatograph or vice versa, or a liquid chromatograph coupled with a capillary electrophoresis system or vice versa).

In some embodiments, the sample is passed through the first separating zone and the second separating zone by pumping the sample through the first separating zone and the second separating zone. Embodiments of such systems may thus comprise a suitable pump, which may be arranged proximal to the entry of the first separating zone.

In some embodiments, the sample is passed through the first separating zone and the second separating zone by sucking the sample through the first separating zone and the second separating zone. Embodiments of such systems may thus comprise a suitable suction device, which may be arranged proximal to the exit of the second separating zone.

In some embodiments, the sample is passed through the first separating zone and the second separating zone by blowing the sample through the first separating zone and the second separating zone. Embodiments of such systems may thus comprise a suitable blowing device (e.g. a source of pressure such as a pressurized gas cylinder), which may be arranged proximal to the entry of the first separating zone. For example, in gas chromatograph systems, a sample may be carried into the separating zone in a flow of gas flowing from a pressurized gas cylinder.

In other embodiments, the sample is passed through the first separating zone and the second separating zone using a high voltage (e.g. in capillary electrophoresis systems). Embodiments of such systems may thus comprise a suitable high voltage source.

In some embodiments, different techniques may be used to cause the sample to pass through the separation zones. For example, in one embodiment, a sample may be pumped through the first separating zone and then sucked through the second separating zone. In an embodiment, the method comprises a further step of passing the sample through at least one further separating zone. The corresponding embodiment of the system may comprise at least one further separating zone. The method may also comprise the further steps of: generating further temporal information by sensing the plurality of analytes at a plurality of spaced apart positions at the at least one further separating zone; and generating further time dependent spectral information from the further temporal information. The further temporal information may be used to provide an even further improved separation of the analytes in the sample.

The methods and systems of the invention are applicable to time-based separation techniques that depend upon the movement of analytes imparted by a moving medium (mobile phase) in contact with a non-moving reference (stationary phase). Such time -based separations include, but are not limited to analytical methods collectively known as chromatography and electrophoresis.

As will be appreciated, in the invention, the separation steps discussed above could be carried out independently of the analysis steps discussed above. For example, a portable system adapted to perform the separation steps discussed above could send the data obtained over the internet (or via other means), to a separate system arranged to perform the analysis steps discussed above.

Thus, in a third aspect, the invention provides a method comprising the steps of:

(b) generating time dependent spectral information from the temporal information. In a fourth aspect, the invention provides a system comprising:

(a) a temporal information receiver arranged to receive temporal information, the temporal information having been generated by:

Specific embodiments of the invention will now be described.

Figures 4(a) to 4(c) show example representations of information generated by an embodiment of a method and a corresponding embodiment of a system of the invention having 5 spaced apart positions and 5 detectors in a second separating zone. Figure 4(a) shows a representation of the temporal information generated by the 5 detectors from a sample having analytes A to E. The analytes B, D and E have different characteristic frequencies, but overlap in time and are therefore not physically separated. The analyte pair A and D and the analyte pair C and E have overlapping frequencies, but do not overlap in time. Figure 4(b) shows the combined sensor signal information. Figure 4(c) shows a representation of time dependent spectral information, in the form of a spectrogram, generated from the temporal information of figure 4(b). Even though the analytes B, D and E and not distinguishable in figure 4(b), they are in the spectrogram of figure 4(c). The analyte pair A and D and the analyte pair C and E are distinguishable as shown in Figure 4(c) when time dependent spectral information is obtained from the time-dependent signal encompassing analytes A to E.

Figure 5 shows another embodiment of a system of the second aspect, generally depicted as system embodiment 70.

System embodiment 70 has mobile phase introduction means 72, which may be via static or dynamic pressure, by use of electrical potential difference, mobile phase pumps, or any other suitable means. Flow of the mobile phase may, for example, be achieved by using one or more syringe pumps or piston pumps, by using the static pressure provided by a cylinder of compressed gas, or by using a pressure differential whereby the outlet of either dimension has a lower pressure induced by a means to generate a vacuum.

The system may also have a means for varying the composition of the mobile phase 74. This may be achieved by inserting a mixing chamber into the mobile phase flow path and or by varying the pumping rate of one or more pumps.

The system may also have a means of removing contaminants or undesirous components from the mobile phase 76. Dissolved gases for example, may be removed from a liquid mobile phase using a mobile phase degasser or water vapour removed from a gaseous mobile phase by passing the gaseous mobile phase through a water- absorbing material. The system has a means for sample introduction into the mobile phase flow 78, by electrically or manually operated valves or switches, sample injection valves, electrical potential difference, capillary action or other means. Sample may be introduced into the mobile phase flow by, for example, manual injection with a syringe directly through a septum into the mobile phase flow. Alternatively, an automated means may be used, whereby a microprocessor controlled syringe loads sample from a sample reservoir and injects the sample into a switching valve that then delivers the sample into the mobile phase.

The system may also have flow moderation means (not shown), by regulation devices, valves or other means (e.g. servo-motor or stepper-motor controlled pistons, electronically controlled needle valves or variable electrical potential).

The system may also have switching valves (not shown) within the flow path of the mobile phase. Switching valves may be configured, for example, to include or to exclude chosen separation dimensions or to periodically remove portions of the mobile phase or to vary the composition of the mobile phase. The system may also have heating or cooling means (not shown). These may be for heating or cooling the system or parts of the system or to maintain those parts at specific temperatures or to control the temperature of those parts.

System embodiment 70 also has a first separating zone 80 and second separating zone 82. Four detectors 84 are located at spaced apart positions 85 of second separating zone 82. One of the detectors 84 is a multiplexed detector 88, whilst the other three detectors 89 are not. Multiplexed detector 88 comprises a single sensor that alternately receives signals from three of the spaced apart positions 85. The multiplexed detector may, for example, consist of a single potentiometer (voltage sensor) that is alternately electrically switched between electrodes located at one or more spaced apart positions at the second separating zone to alternately measure the potential difference at each of those spaced apart positions. Additionally or alternatively, multiplexed detectors may comprise a single sensor that is moved from one spaced apart position to another. In an embodiment, a single optical sensor, for example, may be mechanically moved to sense the presence or absence of light at one or more of the spaced apart positions.

The detector 88, 89 signals may be combined and processed using processor 86 in the manner described above.

Figure 6 shows another embodiment of a system of the second aspect, generally depicted as system embodiment 90. In addition to the features of system embodiment 70, system embodiment 90 has 3 (or more) separating zones. Signal information is generated by the detectors located on the second and subsequent zones. Alternatively, signal information may only be generated by detectors located only on a subset of separating zones. The extra information may be used to improve performance.

Now that embodiments of methods and systems for analysing a sample comprising a plurality of analytes have been described, it will be appreciated that some embodiments have some of the following advantages:

• Analytes may be better differentiated;

• A greater number of analytes may be differentiated;

• Analysis time may be reduced; • The ability to distinguish between a plurality of analytes may not be diminished or lost by the passage of the analytes through a second separating zone;

• The analysis may be performed with a degree of analyte differentiation similar to an offline analysis but in a shorter time.

It will be appreciated that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. For example, the separating zones and the temporal information generator may be connected over a network, examples of which include but are not limited to a WAN, the internet and a LAN, to the time dependent spectral information generator. The time dependent spectral information generator may be any suitable combination of hardware and/or software as appropriate, and may comprise a server, for example. Consequently, the processing of the temporal information may be a considerable distance from the separating zones, perhaps in different nations. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

It is to be understood that prior art publication referred to herein does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

Claims

1. A method for analysing a sample comprising a plurality of analytes, the method comprising the steps of:

passing the sample through a first separating zone and a second separating zone, whereby each of the plurality of analytes have a first speed in the first separating zone and a second speed in the second separating zone; generating temporal information by sensing the plurality of analytes at a plurality of spaced apart positions at the second separating zone; and

generating time dependent spectral information from the temporal information.

2. A method defined by claim 1 comprising the step of correlating the time

dependent spectral information with information associated with known substances.

3. A method defined by claim 1 or 2 wherein the step of generating the time dependent spectral information comprises the step of determining the frequency components of only a portion of the temporal information.

4. A method defined by any one of the preceding claims wherein the step of generating the time dependent spectral information comprises the step of applying a short-time Fourier transform to the temporal information.

5. A method defined by any one of the preceding claims wherein the step of generating the time dependent spectral information comprises the step of applying a Wigner transform to the temporal information.

6. A method defined by any one of the preceding claims wherein the step of generating the time dependent spectral information comprises the step of applying a Gabor-Wigner transform to the temporal information.

7. A method defined by any one of the preceding claims wherein the step of generating the time dependent spectral information comprises the step of applying a wavelet transform to the temporal information.

A method defined by any one of the preceding claims wherein the step of generating the time dependent spectral information comprises the step of passing the temporal information through a plurality of band pass filters.

A method defined by any one of the preceding claims wherein the step of generating the time dependent spectral information comprises the step of generating information indicative of a spectrogram of the temporal information.

A method defined by any one of the preceding claims, wherein at least some of the plurality of analytes are optically sensed.

A method defined by any one of the preceding claims, wherein at least some of the plurality of analytes are sensed by sensing a light that has been partially absorbed by at least one of the analytes, or sensing luminescence from the analyte.

A method defined by any one of the preceding claims wherein at least some of the plurality of analytes are sensed by one or more sensing techniques independently selected from the group consisting of: mass selective sensing; electrochemical sensing; electrical conductive sensing; thermal conductive sensing; light scattering sensing; thermal ionization sensing; electrical ionization sensing and electron capture sensing.

A method defined by any one of the preceding claims, wherein the first separating zone comprises a first material and the second separating zone comprises a second material.

A method defined by claim 13, wherein the first material and the second material are independently selected from the group consisting of: silica, fused silica, Squalane, dimethylsilicone, polyethyleneglycol, phenylmethylsilicone oils, diethyleneglycosuccinate, alumina, titania, zirconia, mixed oxide ceramics, HILIC, ion-exchange resins, ion exclusion resins, size exclusion resins, C8 and C18.

A method defined by any one of the preceding claims, wherein the sample is a gas when passed through the first separating zone and the second separating zone.

A method defined by claim 15, wherein the first separating zone and the second separating zone are columns in a gas chromatograph.

A method defined by any one of claims 1 to 14, wherein the sample is a liquid when passed through the first separating zone and the second separating zone.

A method defined by claim 17, wherein the first separating zone and the second separating zone are columns in a liquid chromatograph, columns in a high performance liquid chromatograph (HPLC), or capillaries in a capillary electrophoresis system.

A method defined by any one of the preceding claims, wherein the sample is passed through the first separating zone and the second separating zone by pumping the sample through the first separating zone and the second separating zone, by sucking or blowing the sample through the first separating zone and the second separating zone, or by applying a potential difference between the first separating zone and the second separating zone in order to induce an electro- driven flow.

A method defined by any one of the preceding claims, comprising the further step of passing the sample through at least one further separating zone.

A method defined by claim 20, comprising the further steps of:

- generating further temporal information by sensing the plurality of analytes at a plurality of spaced apart positions at the at least one further separating zone; and

- generating further time dependent spectral information from the further temporal information.

A system for analysing a sample comprising a plurality of analytes, the system comprising:

a first separating zone arranged for passing the sample therethrough; a second separating zone adapted to receive the sample from the first separating zone and arranged for passing the sample therethrough, whereby the first separating zone and the second separating zone are arranged for the analytes to have a first speed in the first separating zone and a second speed in the second separating zone;

A system defined by claim 22 comprising a correlator arranged to correlate the time dependent spectral information with information associated with known substances.

A system defined by claim 22 or 23 wherein the time dependent spectral information generator is arranged to apply a short-time Fourier transform to the temporal information.

A system defined by any one of claims 22 to 24 wherein the time dependent spectral information generator is arranged to apply a Wigner transform to the temporal information.

A system defined by any one of claims 22 to 25 wherein the time dependent spectral information generator is arranged to apply a Gabor-Wigner transform to the temporal information.

A system defined by any one of claims 22 to 26 wherein the time dependent spectral information generator is arranged to apply a wavelet transform to the temporal information.

A system defined by any one of claims 22 to 27 wherein time dependent spectral information generator has a plurality of band pass filters arranged to filter the temporal information.

A system defined by any one of claims 22 to 28 wherein the time dependent spectral information generator is arranged to generate information indicative of a spectrogram of the temporal information.

A system defined by any one of claims 22 to 29, wherein the one or more sensors comprise at least one optical sensor.

A system defined by any one of claims 22 to 30 wherein the one or more sensors comprise at least one of: a mass selective sensor; an electrochemical sensor; an electrical conductive sensor; a thermal conductive sensor; a light scattering sensor; a thermal ionization sensor; an electrical ionization sensor and an electron capture sensor.

A system defined by any one of claims 22 to 31, wherein the first separating zone comprises a first material and the second separating zone comprises a second material.

A system defined by claim 32, wherein the first material and the second material are independently selected from the group consisting of: silica, fused silica, Squalane, dimethylsilicone, polyethyleneglycol, phenylmethylsilicone oils, diethyleneglycosuccinate, alumina, titania, zirconia, mixed oxide ceramics, HILIC, ion-exchange resins, ion exclusion resins, size exclusion resins, C8 and C18.

A system defined by any one of claims 22 to 33, wherein the system is arranged for the sample to be a gas.

A system defined by claim 34, wherein the system comprises a gas

chromatograph.

36. A system defined by any one of claims 22 to 33, wherein the system is arranged for the sample to be a liquid. A system defined by claim 36, wherein the system comprises a liquid chromatograph, a high performance liquid chromatograph (HPLC) or a capillary electrophoresis system.

A system defined by any one of claims 22 to 37, comprising a pump arranged to pump the sample through the first separating zone and the second separating zone, a suction device arranged to suck the sample through the first separating zone and the second separating zone, a source of static pressure arranged to blow the sample through the first separating zone and the second separating zone, or a potential difference source arranged to apply a potential difference between the first separating zone and the second separating zone in order to induce an electro-driven flow.

A system defined by any one of claims 22 to 38 comprising at least one further separating zone.

A method comprising the steps of:

A system comprising: