WO2014088498A1

WO2014088498A1 - Fluorescence and absorption spectrograph and method for analysing a fluid sample

Info

Publication number: WO2014088498A1
Application number: PCT/SE2013/051433
Authority: WO
Inventors: Thom LEIDING; Sindra PETERSSON ÅRSKÖLD
Original assignee: Probation Labs Sweden Ab
Priority date: 2012-12-04
Filing date: 2013-12-03
Publication date: 2014-06-12

Abstract

Invention regards a method for analysis of a fluid sample by absorption and fluorescence spectrometry, said method comprising the step of reconfiguring a spectrograph on the basis of an initial measurement. The parameters to be reconfigured are the integration time, the size of the detection range, the choice of the light source and the emitted light intensity. The invention further regards an apparatus for executing said method and an automated titration apparatus.

Description

FLUORESCENCE AND ABSORPTION SPECTROGRAPH AND METHOD FOR ANALYSING A FLUID SAMPLE

TECHNICAL FIELD

The disclosure relates to a system and a method for analysis of a fluid sample.

BACKGROUND

There exist several ways of studying properties of fluid samples in laboratory environment. One non-invasive, frequently used technique is the characterization of the sample by means of optical probing. More specifically, the sample is exposed to an incoming light signal and the resulting response signal is collected and subsequently analysed. By way of example, in an absorbance measurement, light with a certain intensity passes through the sample, whereupon its intensity decreases, i.e. a portion of the light has been absorbed by the sample. The attenuated light is then collected and analysed. Analogously, in a fluorescence measurement, a suitably prepared sample is energetically excited by light whereupon its molecules relax to their ground state while simultaneously emitting light, i.e. the sample fluoresces, said light subsequently being collected and analysed.

However, measurements of the above kind may be marred by errors. In this context, normally only one property of the sample (such as absorbance) may be studied at one time and it is common practice to prepare a fresh sample for each run. Consequently, and depending on the level of reproducibility of a certain sample, each parameter of the sample, e.g. pH, ionic strength and protein concentration, could exhibit a considerable change in value when studied in two subsequent runs. Obviously, these inadvertent variations could result in discrepancies between subsequent measurements of the specific sample property such as absorbance.

Similarly, these variations even increase the uncertainty when it comes to the reliability of the measurement regarding two different sample properties, when said measurement is performed during separate runs and a fresh sample is prepared for each run. Such a measurement could for instance be of interest when it is sought to establish a correlation between two sample properties.

Random noise and calibration errors (particularly present in thermal probes) in various instruments can also result in deviations between measurements.

On the above background, the analyst performing the experiment must decide whether observed differences between measurements are due to a new, previously unknown mechanism, or whether the differences are due to the experimental errors. In conjunction herewith, as is known in the particular technical field of optical sample probing, performing fast measurement sequences while maintaining full control of the experiment poses a great challenge. To at least partly address the above-mentioned problems, devices have been developed that are capable of measuring several sample properties in parallel. A further benefit of these devices is a significant saving of time and material, since all measurements are made during a single run. One such device is disclosed in the scientific article "A Multidimensional

Spectrophotometer for Monitoring Thermal Unfolding Transitions of

Macromolecules", authored by Glen Ramsay and Maurice R. Eftink and published on pages 516-523 of Biophysical Journal, Volume 31 , of February 1994. The device may be used in order to in a nearly simultaneous manner measure circular dichroism (CD) and fluorescence of a sample. In addition, dynode voltages obtained during the CD-measurement may be used to calculate changes in absorbance.

Structurally, the device, which is customized for studying processes involving biomacromolecules such as protein folding, comprises a lamp that emits polychromatic light, a plurality of optical elements such as slits and prisms for controlling light propagation, a monochromator for selecting light of certain wavelengths, several polarizers for converting the selected light into the polarized light necessary for CD-measurements and two light detectors, one used for fluorescence measurements and the other for CD-measurements. Photomultiplier tubes (PMT) are used as light detectors since these are particularly suitable for low intensity applications such as fluorescence measurements.

A modification of the above device is proposed by the same authors in the subsequent article entitled "Modified Spectrophotometer for Multi-Dimensional Circular Dichroism/Fluorescence Data Acquisition in Titration Experiments:

Application to the pH and Guanidine-HCI Induced Unfolding of Apomyoglobin" and published on pages 701 -707 of Biophysical Journal, Volume 69, of August 1995. The modification consists of adding a syringe pump and a pH meter to the arrangement so as to allow titration experiments to be performed. However, as regards systems of this kind intended for use in research institutions, it is far more common to perform the titration part of the experiment manually. One

consequence thereof is rather poor precision of the experiment.

As regards the above-discussed devices, these are ridden with considerable drawbacks. More specifically, they, first of all, offer low temporal resolution.

Accordingly, a standard measurement of CD and fluorescence for a few different wavelengths lasts approximately three minutes. This is, as is widely known in the art, due to the fact that when using PMT-detection to characterize the spectrum, the desired sample property is measured individually for each wavelength of the spectrum whereafter the monochromator is adjusted so that the process of collecting the whole spectra is very time consuming.

Furthermore, none of the above devices permits direct measurement of the entire absorbance spectrum. This severely limits the applicability of these devices in time-resolved studies.

In addition, both types of devices are, as previously discussed, customized for studying processes involving biomacromolecules, in particular dynamics of protein folding. This severely reduces their field of application. Moreover, as is known in the art, protein samples are intrinsically unstable which severely limits the duration of the experiment. Fast detection would enable more data to be harvested from a delicate protein sample before it deteriorates.

Moreover, as shown above, each of the proposed arrangements has a large number of components, which makes the respective device bulky and relatively difficult to accommodate in a laboratory or relocate. One objective of the present invention is therefore to provide a system and a method for analysis of a fluid sample that eliminates at least some of the drawbacks associated with the current art.

SUMMARY

The above stated objective is achieved by means of a method and a system for analysis of a fluid sample according to the independent claims, and by the embodiments according to the dependent claims.

More specifically, a first aspect of the present invention provides a method that comprises the steps of configuring a spectrograph on the basis of a first set of measurement parameters, emitting a first light signal so that the sample is exposed to said first light signal, analysing, by means of the spectrograph, a second light signal coming from the sample in response to said first light signal, configuring said spectrograph on the basis of a second set of measurement parameters, emitting a third light signal so that the sample is exposed to said third signal, analysing, by means of the spectrograph, a fourth light signal coming from the sample in response to said third signal, wherein the reconfiguration of the spectrograph from the first to the second set of measurement parameters is instantaneous. It is to be noted that these method steps do not have to take place in the above order.

A second aspect of the present invention provides a system for analysis of a fluid sample, said system comprising a single spectrograph and a light source, wherein said fluid sample is, when the system is in use, so positioned that it is exposed to at least a portion of the light emitted by the light source, said system further comprising processing circuitry configured to: configure said spectrograph on the basis of a first set of measurement parameters, activate the light source so as to emit a first light signal so that the sample is exposed to said first light signal, analyse, by means of said spectrograph, a second light signal coming from the sample in response to said first light signal, configure said spectrograph on the basis of a second set of measurement parameters, activate the light source so as to emit a third light signal so that the sample is exposed to said third signal, analyze, by means of said spectrograph, a fourth light signal coming from the sample in response to said third signal, instantaneously reconfigure the

spectrograph from the first to the second set of measurement parameters. In this context, the term "measurement parameter" is to be construed to

encompass the adjustable parameters of the spectrograph, such as integration time and size of the detection range, as well as the adjustable parameters of the light source(s), such as the choice of light source and intensity of the emitted light. By enabling instantaneous reconfiguration of the spectrograph from the first to the second set of measurement parameters, a very fast measurement technique is achieved. Hence, a measurement of a sample property such as absorbance or fluorescence at any given measurement point is made in the low millisecond range. The instantaneous reconfiguration is made possible by, on one hand, integration of the spectrograph into the system and, on the other hand, by processing circuitry that leverages the full potential of the system components.

Also, in addition to being adapted to measure fluorescence, the system is highly suitable for effecting direct measurement of the entire absorbance spectrum of the sample. In this way, two key properties for characterisation of the sample, namely fluorescence and absorbance, may be measured in a single sample. More specifically, the light source may be arranged to emit light comprising wavelengths spanning from 200 nanometers to 1500 nanometers whereas the spectrograph is inherently capable of separating incoming light into a spectrum and subsequently recording this spectrum, i.e. simultaneously detecting the entire wavelength range. Thus, a very versatile system is obtained. In this context, the spectrograph being a powerful tool for analysis of fluid samples, its use in the system at hand opens for the possibility to collect an extensive amount of information about the sample. Obviously, the analyst performing the experiment may use this

information to characterize the studied sample in greater detail. In contrast to prior art that, as exemplified above, uses two separate light detectors, a single light-detecting unit of a spectrograph is employed in the invention at hand to, for a large number of applications, achieve comparable results. This, paired with the inherent structural simplicity of the system, i.e. a greatly reduced number of components compared to devices of the prior art, significantly reduces overall dimensions of the system, effectively converting the system at hand into a portable unit.

In an embodiment of the present invention, the emitted first light signal has a first spectral distribution and is suitable for measurement of absorbance in the sample, and said method further comprises the steps of: reconfiguring the spectrograph and the light source from a first to a second set of measurement parameters, emitting the third light signal that comprises a number of light pulses, each pulse having said first spectral distribution and being suitable for measurement of absorbance in the sample, wherein the number of emitted light pulses is determined by the maximum light intensity value present in the spectrum of the first light signal. Hereby, resolution of the measurement is vastly improved. More specifically, as is known in the art, the light-sensitive element of the spectrograph comprises an array with a plurality of identical light-sensitive array sections, each section corresponding to a certain wavelength. Typically, the spectrum of the incoming light signal is unevenly distributed and each photon of the signal incoming into the spectrograph is registered in a suitable array section. Hence, array sections corresponding to wavelengths of the light spectrum associated with high intensity could become saturated relatively quickly whereas array sections corresponding to wavelengths of the light spectrum associated with low intensity register rather few photons. In the present embodiment, during an absorbance measurement, the spectrograph is first configured so that the maximum light intensity value present in the spectrum of the first light signal doesn't lead to saturation of any one of the array sections. A second set of measurement parameters is thereafter generated, whereby the light source emits a plurality of light signals spectrally identical to the first signal and the spectrograph

simultaneously becomes modified to only register light in the chosen sub-range of wavelengths, said sub-range typically corresponding to wavelengths associated with rather few photons. Taking into account the maximum light intensity value present in the spectrum of the first light signal, the number of emitted signals is so designed that the superposition of all emitted signals in the chosen sub-range doesn't lead to saturation of the corresponding array section.

In a further embodiment, a synchronizing signal is generated, wherein the pulse width of the synchronizing signal is used to establish whether the third light signal that is suitable for measurement of fluorescence in the sample is to be emitted. Here, "pulse width" is to be construed as a time interval between the leading edge and trailing edge of a rectangular signal pulse. In this way, a command to perform a fluorescence measurement may be generated in a very simple manner, i.e. by comparing the pulse width of the actual pulse with a predetermined value.

In another embodiment, properties of the studied fluid sample are altered by means of a fully automated titration apparatus. Hereby, a system of

unprecedented precision is achieved. More specifically, resolution of the titration apparatus is in the low nanometer range (appr. 10 nl). As a comparison, the resolution of a state-of-the-art manual titration apparatus is, at best, 1 μΙ. In a further embodiment, no light is emitted while a titration is in progress. In this way, the exposure of samples to light is minimised. Often, the quality of samples deteriorates as a consequence of exposure to light. By avoiding light emission during the titration process, photo-bleaching is kept at a minimum and the useful life of the sample, and ultimately the duration of the experiment and the amount of useful data collected, is increased significantly. In yet another embodiment, the system comprises two light sources, the first source being adapted to emit a light signal suitable for the measurement of absorbance in the sample and the second source being adapted to emit a light signal suitable for exciting fluorescent molecules for the measurement of fluorescence in the sample while a single light-detecting unit of a spectrograph is sufficient to analyze both of these signals.

Further advantages and features of embodiments will become apparent when reading the following detailed description in conjunction with the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a highly schematical view of a system for analysis of a fluid sample according to an embodiment of the present invention where the system is suitable for performing absorbance measurements. Fig. 2 is a cross-sectional top view of a system for absorbance measurements according to an embodiment of the present invention.

Fig. 3 is a highly schematical view of a system for analysis of a fluid sample according to another embodiment of the present invention, where the system is suitable for performing absorbance and fluorescence measurements nearly simultaneously.

Fig. 4 is a cross-sectional top view of a system for absorbance measurements additionally provided with a titration apparatus according to yet another

embodiment of the present invention.

Fig. 5 illustrates one inventive feature of the present invention. More specifically, it shows how a light source and a spectrograph are used in order to extract additional information from an absorbance measurement.

DETAILED DESCRIPTION

The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these

embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. In the drawings, like reference signs refer to like elements.

Fig. 1 is a highly schematical view of a system 2 for analysis of a fluid sample 14 according to an embodiment of the present invention where the system is suitable for performing absorbance measurements.

Said system, which is particularly suitable for non-invasive experiments in a laboratory environment, comprises a light source 6, in this case a Xenon flash lamp, emitting light across a broad range of wavelengths (200 - 1500 nm), said light also being synchronized with a light-detecting unit. The fluid sample 14, placed in a temperature-controlled sample cell 15 with stirring, typically includes a chromophore - such as an optical molecular probe or a naturally present chromophore - which changes its spectral properties in response to physical changes in the surrounding environment. A pinhole 10 guides the light into the cell 15 where it interacts with the sample 14. After having interacted with the sample 14, the light exits the sample cell 15, is focused by means of a lens 12 and enters the light-detecting unit, said unit being here part of a spectrograph 4, where the light is measured and registered. The thus collected data is subsequently presented to the user in a suitable way. The experiment is performed continuously and the experimental results are presented to the user in close to real time, i.e. with a delay in the millisecond range. With only minor modifications the system of the described kind, suitable for performing absorbance measurements, may be converted into a system for fluorescence measurements. Basically, owing to the versatility of the spectrograph serving inter alia for light detection, the only necessary modification is the exchange of the source of light to one suitable for fluorescence measurements, e.g. a light source positioned out of the optical line formed by the sample and the spectrograph. Systems of both types are

particularly suitable for dose-response measurements.

Fig. 2 is a cross-sectional top view of a system 2 for absorbance measurements according to an embodiment of the present invention. A sample holder 17, being positioned between a light source 6 and a light-detecting unit, and being enclosed in a casing 22, may be seen. The sample holder 17 is normally a standard cuvette with a quadratic cross-section (12,5 x 12,5 mm), having the internal sample volume of 1 or 3 ml. It is further equipped with magnetic stirring as well

temperature control in the span 4 - 80°C with 0,5°-accuracy. Temperature of the sample may be varied, e.g. by means of a Peltier-element. The chemical properties of the sample, for instance its pH-value, may be monitored using an electrode (not shown) that is insertable into a dedicated slot of the holder.

Moreover, properties of the fluid sample may be further altered using a fully automated titration apparatus (not shown in Fig. 2). This will be more fully described in conjunction with Fig. 4.

A light source 6, as described in conjunction with Fig. 1 , emits light that hits the fluid sample, where a portion of the light is absorbed, whereafter the attenuated light reaches the light-detecting unit of the spectrograph 4. The path of the light is shown by means of a dashed line. In analogy with the discussion in connection with Fig. 1 , with only minor modifications the system 2 shown in Fig. 2, suitable for performing absorbance measurements, may be converted into a system for fluorescence measurements. A control system, more thoroughly discussed further down, controls inter alia said titration process, the activation of light signals, the execution of measurements and the collection of measurement data and the regulation of the sample temperature.

The system 2 may be used, but is not limited to, to follow the response of the sample to changes in pH-value, changes of salt content and the addition of reagents. For this purpose, a number of different measurement-modes may be used, e.g. to make a controlled addition of a suitable substance and subsequently follow the sample in time, to repeatedly add a predetermined amount of the chosen substance and measure thereafter the effect on the sample, or to repeatedly add an amount of the chosen substance, wherein said amount is governed by the feedback originating from the sample.

The temporal resolution of the system at hand, i.e. how fast a whole measurement point is registered, depends on the chosen measurement parameters. It varies from the highest resolution obtainable from the light-detecting unit (currently appr. 1 ms) and upwards.

Regardless of the sample property being examined, the system for analysis of a fluid sample comprises a single spectrograph and a light source, wherein said fluid sample is, when the system is in use, so positioned that it is exposed to at least a portion of the light emitted by the light source (this may be clearly seen in Fig. 2).

Said system further comprises processing circuitry configured to:

configure said spectrograph 4 on the basis of a first set of measurement parameters,

emit a first light signal so that the sample is exposed to said first light signal, analyse, by means of said spectrograph 4, a second light signal coming from the sample in response to said first light signal,

configure said spectrograph 4 on the basis of a second set of measurement parameters,

emit a third light signal so that the sample is exposed to said third signal, analyze, by means of said spectrograph 4, a fourth light signal coming from the sample in response to said third signal,

instantaneously reconfigure the spectrograph 4 from the first to the second set of measurement parameters. In this context, the term "measurement parameter" is to be construed to

encompass the adjustable parameters of the spectrograph, such as integration time and size of the detection range, as well as the adjustable parameters of the light source(s), such as choice of light source and intensity of the emitted light. By enabling instantaneous reconfiguration of the spectrograph 4 from the first to the second set of measurement parameters, a very fast measurement technique is achieved. Hence, a measurement of a sample property such as absorbance or fluorescence at any given measurement point is made in the low millisecond range. The instantaneous reconfiguration is made possible by, on one hand, integration of the spectrograph 4 into the system and, on the other hand, by processing circuitry that leverages the full potential of the system components.

Also, in addition to being adapted to measure fluorescence, the system is highly suitable for effecting the direct measurement of the entire absorbance spectrum of the sample. In this way, two key properties for characterisation of the sample, namely fluorescence and absorbance, may be measured in a single sample. More specifically, a light source may be arranged to emit light comprising wavelengths spanning from 200 nanometers to 1500 nanometers whereas the spectrograph is inherently capable of separating incoming light into a spectrum and subsequently recording this spectrum, detecting the entire wavelength range simultaneously. Thus, a very versatile system is obtained. In this context, the spectrograph being a powerful tool for analysis of fluid samples, its use in the system at hand opens up the possibility of collecting an extensive amount of information about the sample. Obviously, the analyst performing the experiment may use this

information to characterize the studied sample in greater detail.

In one embodiment, the processing circuitry may be integrated in the system itself and in other embodiments it is at the exterior of the system, e.g. being part of a computer (not shown). When the system 2 comprises dedicated processing circuitry, said processing circuitry is typically controlled by the computer that also controls sample properties such as temperature and pH-value. This computer normally controls the spectrograph 4 as well. In addition, all measurement parameters are set by the user using a program running on the computer.

A measurement (absorbance and/or fluorescence) is performed for a given set of measurement parameters, as defined above, and given sample properties (temperature, pH-value ... ) in each measurement point. Accordingly, in each measurement point, and for a given sample temperature and pH-value, the sample may be characterized in a number of different ways, depending on the chosen set of measurement parameters. This procedure is repeated for all measurement points, whereafter the experiment is completed.

In an embodiment, a typical measurement sequence (absorbance) comprises following steps:

- the spectrograph 4 receives a command from the computer to adjust the temperature of the sample and the stirring frequency,

- the spectrograph 4 receives a command from the computer regarding choice of the light source and intensity of the light emitted by the flash lamp,

- the light detecting unit of the spectrograph 4 receives a command from the computer to perform a measurement for a given set of measurement parameters,

- the light source 6 is activated,

- the detecting unit detects the received light and forwards the measured data to the computer program,

- the computer program retrieves data from the system 2 regarding the sample properties such as temperature and pH-value,

- data for the particular measurement point and the set of measurement parameters is saved,

- the computer program moves for the same measurement point to the next set of measurement parameters until it reaches the last one. In case of a fluorescence measurement, the above sequence is slightly modified. More specifically, if a synchronizing signal that normally is generated by the detecting unit has a pulse width within a predetermined range, then the light signal that is suitable for measurement of fluorescence in the sample is emitted. Here, "pulse width" is to be construed as a time interval between the leading edge and trailing edge of a rectangular signal pulse. In this way, a command to perform a fluorescence measurement may be generated in a very simple manner, i.e. by comparing the pulse width of the actual pulse with a predetermined value.

Fig. 3 is a highly schematical view of a system 2 for analysis of a fluid sample according to another embodiment of the present invention where the system is suitable for simultaneously performing absorbance and fluorescence

measurements.

The basic setup shown in Fig. 3 resembles that of Fig. 1 . However, the system 2 of Fig. 3 comprises two light sources 16, 18, the first source 16 being adapted to emit a (first) light signal suitable for measurement of absorbance in the sample, i.e. emitting light across a broad range of wavelengths, and the second source 18 being adapted to emit a (third) light signal suitable for measurement of

fluorescence in the sample, i.e. at least one, but preferably a plurality of narrow wavelength bands. A single light-detecting unit of the spectrograph 4 is sufficient to collect both of these signals for subsequent analysis. As indicated in the schematic drawing, the two light sources are so arranged that the two light beams are perpendicular to each other and intersect in a sample 14. It is clear that the ability to nearly simultaneously and directly analyze two sample properties, absorbance and fluorescence, adds to the usefulness of the system 2.

This setup also allows for the detection of absorbance changes in the sample that result from photo-excitation by a second light source in photosynthetic- and/or photovoltaic applications. In such an embodiment, the exciting light may be chosen at a much higher intensity than the light source contributing the light input for the absorbance measurement. The light that excites the sample may be delivered in flashes or as continuous illumination.

Fig. 4 is a cross-sectional top view of a system 2 for absorbance measurements additionally provided with a fully automated titration apparatus according to yet another embodiment of the present invention. By means of titration, properties of the fluid sample may be altered where the step of the incremental change may be adjusted. The basic setup shown in Fig. 4 resembles that of Fig. 2. A titration apparatus 20 has been added. Said titration apparatus 20 comprises one or a pair of syringe pumps connected with a cuvette by means of capillaries. Hereby, a system of unprecedented precision is achieved. More specifically, resolution of the titration apparatus 20 is in the low nanoliter range (appr. 10 nl). As a comparison, the resolution of a state-of-the-art manual titration apparatus is, at best, 1 μΙ.

In a variant, as the titration apparatus 20 is fully automated, the only thing a user needs to do is to define a desired state of the sample, i.e. a particular

concentration and/or temperature, and the system automatically adjusts the sample so that it matches the specified state whereupon a predefined

measurement cycle is performed (on the basis of measurement parameters), the purpose of which is to suitably characterize the sample.

In addition to all the process steps described in conjunction with Fig. 2, and after, for a given measurement point, measurements have been performed for all sets of measurement parameters, the program gives a command to the instrument to move on to the next measurement point. Here, measurement points may be defined as points where the sample has a particular concentration and/or temperature. This could mean that a new temperature is set, pH-value or ion concentration is changed, a predetermined amount of liquid is added to the sample so as to incrementally change its composition, or to wait for a certain period of time. Thereafter, the procedure with different sets of measurement parameters, as described above, is repeated.

As an advantage, the system arranges for no light emission while the titration is in progress. In this way, the exposure of the samples to light is minimised. Often, the quality of samples deteriorates as a consequence of exposure to light. By avoiding light emission during the titration process, photo-bleaching is kept at a minimum and the useful life of the sample, and ultimately duration of the experiment and the amount of useful data that can be collected, is increased significantly.

Fig. 5 illustrates one inventive feature of the present invention. More specifically, it shows how a light source and a spectrograph are used in order to extract additional information from an absorbance measurement. This is in Fig. 5 visualised by means of a graph that shows the relationship between the

wavelength (λ) and the relative intensity across the continuous light spectrum. More specifically, as is known in the art, the light-sensitive element of the spectrograph comprises an array with a plurality of identical light-sensitive array sections, each section corresponding to a certain wavelength. Typically, the spectrum of the incoming light signal is unevenly distributed (exemplified by a dashed line in Fig. 5) and each photon of the signal incoming into the

spectrograph is registered in a suitable array section. Hence, array sections corresponding to wavelengths of the light spectrum associated with high intensity could become saturated relatively quickly, whereas array sections corresponding to wavelengths of the light spectrum associated with low intensity register rather few photons.

In the present embodiment, during absorbance measurements, the spectrograph is first configured so that the maximum light intensity value present in the spectrum of the first light signal doesn't lead to saturation of any one of the array sections. This is visualised by the dashed line in Fig. 5 (ground signal) and the long, continuous line in Fig. 5 that represents a subsequent signal consisting of the sum of two identical pulses, i.e. the ground signal times two. The number of pulses in the subsequent signal is chosen so as not to saturate any array section of the light-sensitive element of the spectrograph. A further set of measurement parameters is thereafter generated, whereby the light source emits a plurality of light signals spectrally identical to the previous signals and, the spectrograph simultaneously becomes modified only to register light in the chosen sub-range of wavelengths, said sub-range typically corresponding to wavelengths associated with rather few photons. This situation is visualised by means of a short, continuous line in Fig. 5. Taking into account the maximum light intensity value present in the spectrum of the subsequent light signal, the number of emitted signals is so designed that superposition of all emitted signals in the chosen subrange doesn't lead to saturation of the corresponding array section. In this particular case, the short, continuous line in Fig. 5 represents a signal consisting of eight identical pulses, i.e. the ground signal times eight or, rather, signal times four.

It is clear that this vastly improves the sensitivity of the measurement for wavelengths of the light spectrum associated with low intensity, resulting in more qualitative data from the sample. In the drawings and specification, there have been disclosed typical preferred embodiments of the invention and, although specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation, the scope of the invention being set forth in the following claims.

Claims

1 . A method for analysis of a fluid sample (14), said method comprising the steps of:

- configuring a spectrograph (4) on the basis of a first set of

measurement parameters,

emitting a first light signal so that the sample (14) is exposed to said first light signal,

analysing, by means of the spectrograph (4), a second light signal coming from the sample (14) in response to said first light signal,

configuring said spectrograph (4) on the basis of a second set of measurement parameters,

emitting a third light signal so that the sample (14) is exposed to said third signal,

- analysing, by means of the spectrograph (4), a fourth light signal coming from the sample (14) in response to said third signal,

wherein the reconfiguration of the spectrograph (4) from the first to the second set of measurement parameters is instantaneous.

2. A method according to any of the preceding claims, said method further comprising the step of configuring a light source (6) on the basis of at least one of the first and the second set of measurement parameters.

3. A method according to claim 2, wherein the emitted first light signal has a first spectral distribution and is suitable for measurement of absorbance in the sample (14), said method further comprising the steps of:

reconfiguring the spectrograph (4) and the light source (6) from the first to the second set of measurement parameters,

emitting the third light signal that comprises a number of light pulses, each pulse having said first spectral distribution and being suitable for measurement of absorbance in the sample (14),

wherein the number of emitted light pulses is determined by the maximum light intensity value present in the spectrum of the first light signal.

4. A method according to claim 1 or 2, said method further comprising the step of generating a synchronizing signal, wherein the pulse width of the synchronizing signal is used to establish whether the third light signal that is suitable for measurement of fluorescence in the sample (14) is to be emitted.

5. A method according to any of the preceding claims, said method further comprising the step of altering of the properties of the fluid sample (14) by means of titration.

6. A method according to claim 5, wherein no light is emitted while titration is in progress.

7. A system (2) for analysis of a fluid sample, said system comprising a single spectrograph (4) and a light source (6), wherein said fluid sample (14) is, when the system is in use, so positioned that it is exposed to at least a portion of the light emitted by the light source (6), said system (2) further comprising processing circuitry configured to:

configure said spectrograph (4) on the basis of a first set of measurement parameters,

activate the light source (6) so as to emit a first light signal so that the sample (14) is exposed to said first light signal,

analyse, by means of said spectrograph (4), a second light signal coming from the sample (14) in response to said first light signal,

configure said spectrograph (4) on the basis of a second set of

measurement parameters,

activate the light source (6) so as to emit a third light signal so that the sample (14) is exposed to said third signal,

analyze, by means of said spectrograph (4), a fourth light signal coming from the sample (14) in response to said third signal,

instantaneously reconfigure the spectrograph (4) from the first to the second set of measurement parameters.

8. A system according to claim 7, wherein the system (2) comprises two light sources (6), the first source (16) being adapted to emit a light signal suitable for measurement of absorbance in the sample (14) and the second source (18) being adapted to emit a light signal suitable for measurement of fluorescence in the sample (14).

9. A system according to claim 7 or 8, wherein the system (2) further comprises a fully automated titration apparatus (20).