Patent Description:
An optical spectrophotometric apparatus typically comprises a light emitter and a light detector which delimit opposite ends of a light-path into which a sample is positioned for analysis. A sample holder, such as comprising a sample cuvette for liquid or other flowable samples, is located in the light-path and used for positioning samples therein in a repeatable manner. A spectrometer, such as a monochromator or an interferometer, is also located in the lightpath to separate light from the light emitter into intensity dependent narrow spectral bands and to output the same for detection by the light detector and may be placed before or after the sample holder in the direction of travel of light from the light emitter to the light detector.

The sample holder has an internal sample receiving volume and is provided with surfaces, usually opposing surfaces, at least portions of which are transparent to the light traversing the light-path. The separation between these transparent portions determines the optical pathlength through the sample holder and thus through a sample which is being held in the sample receiving volume of the sample holder.

The usual manner of obtaining the necessary spectral data in any spectrophotometric apparatus is by generating a transmittance (or absorbance) spectrum of the sample. To do this a so-called single beam spectrum (SBS) is obtained which comprises spectral data relating to the sample and to the components of the apparatus employed to generate it. In order to isolate the spectral data related only to the sample, a similar single beam spectrum (SBZ) is typically measured on a so-called zero-material, such as water or a water based material if, for example, the sample to be measured is a liquid or air if, for example, the sample to be measured is a solid. Such single beam spectra (SBZ) include the same effects which are related to the components of the apparatus as do the sample spectra (SBS) but effects due to the sample are not present. The zero-material spectrum is then employed to provide a wavelength dependent zero level across the spectral region within which the spectral data is collected.

The single beam spectrum of the sample (SBS) is subsequently divided by the single beam spectrum of the zero-material (SBZ) at the same wavelengths throughout the respective spectra in order to obtain a so-called dual beam spectrum of the sample (DBS) which is essentially the transmittance spectrum of the sample relative to the zero-material and relates virtually only to the transmission properties of the sample. As is well known, taking the negative log<NUM> of this provides the absorbance spectrum for the sample. These operations are performed in an arithmetic unit of a computing device which is associated with the spectrophotometric apparatus and which is provided either integral with or separate but in operable connection to the apparatus, for example in the form of a suitably programmed personal computer.

Over time the output of the spectrophotometric apparatus tends to vary. An aspect of this variation may be described as an amplitude change as a result of which different amplitudes are measured at the same wavelengths for the same sample in two otherwise similar spectrophotometric apparatus or at two runs of the same spectrophotometric apparatus at different times. This is typically caused by the wear of the sample holder causing a change in the separation between the opposing transparent portions and hence to a change in the optical pathlength through the sample holder. As is known, according to the Beer-Lambert law, the absorbance of light by a sample at a given energy (wavelength or wavenumber) is proportional to the optical pathlength through the sample. Thus as the sample holder wears and the optical pathlength changes then the amplitude of the output of the spectrophotometric apparatus changes and needs to be compensated for at regular intervals.

In order to compensate for an amplitude change of the output of the spectrophotometric apparatus due to wear of the sample holder it is known, from for example <CIT> to employ dual beam spectrometric measurements on a so-called standardisation liquid (also often referred to as an "equalization liquid"). This standardisation liquid is a liquid that has a precisely controlled chemical composition resulting in an optical spectrum recorded by the spectrometer which shows a characteristic pattern with characteristic intensities in a predetermined frequency range. The standardisation liquid described in <CIT>, is propanol in water. Intensity information relating to the pattern is compared in the arithmetic unit to corresponding intensity information relating to the reference pattern that has previously been defined as the desired standard response from the standardisation liquid. Then, based on the comparison, the arithmetic unit generates a mathematical transform which describes the transformation of the intensities of the pattern of the optical spectrum recorded by spectrophotometric apparatus to those of the desired standard response the reference pattern. This mathematical transform is stored for access by the arithmetic unit for application to optical spectra of unknown samples that are subsequently recorded by the spectrophotometric apparatus in order to generate an optical spectrum in which amplitude changes due to sample holder wear are compensated for. A problem with this known compensation method is that it requires that the composition of the standardisation liquid is exactly controlled.

The document <CIT> discloses determining an optical pathlength based on a known concentration of the analyte in an interrogation region (measurement cell). This document also discloses a cleaning solution for removing hydrophobic materials (fat, oils) from the measurement cell, the cleaning solution having <NUM>% transmission and thus allowing a measurement of the incident light for calibration purposes.

A method to compensate for amplitude changes in the output of the spectrophotometric apparatus without using a specially composed standardisation liquid is known from <CIT>. Here, the liquid zero-material, which is typically water and which requires no difficult fabrication, is used instead of the separate standardization liquid. The single beam spectrum of the zero-material (SBZ), typically water, is employed in determining a mathematical transform which, again, describes a transformation of a spectrum recorded by the spectrophotometric apparatus to one which is unaffected by sample holder wear. Unfortunately, the so recorded zero beam absorption spectrum (SBZ) includes information not only on the zero-material in the cuvette but also background information on elements, including those in atmospheric air and those associated with optical components, within the light-path between the light emitter and the light detector which is unrelated to the zero material but which influences the light intensity. In order to remove this background information, one solution proposed by <CIT> is to determine a single beam absorption spectrum for air (SBA) which will then contain the same background information as that of the zero beam absorption spectrum (SBZ) of the water but, of course, without any contribution from water. Thus a dual beam spectrum of the zero-material (DBZ), being essentially the transmittance spectrum of the zero-material relative to air, will relate virtually only to the transmission properties of the zero-material. However, the separation between the opposing transparent windows of the typical cuvette is around <NUM>. This makes it difficult to ensure that all sample is removed and only air is present in the cuvette during such background measurements. Dismantling and thoroughly drying the cuvette for each compensation measurement is impractical, as is replacing the sample cuvette with a dry one for each compensation measurement. Moreover, interference fringes in the recorded spectra which arise upon the introduction of air into the cuvette due to multiple reflections from the cuvette windows further complicate the analysis.

Another solution proposed in <CIT> is to make a mathematical estimation of the background information. However, such an estimation has shown to be insufficiently accurate in certain circumstances and for certain applications.

It is an aim of the present invention to mitigate one or more of the problems associated with at least one of the known methods.

According to a first aspect of the present invention there is provided a method for determining an optical pathlength of a cuvette of a spectrophotometric apparatus, the spectrophotometric apparatus including a spectrometer and having associated therewith a computing device, the method comprising: obtaining into the computing device by means of the spectrometer a single beam spectrum of a liquid zero-material held in the cuvette at least in a first energy region in which the liquid zero-material absorbs; obtaining into the computing device by means of the spectrometer a single beam spectrum of a second liquid at least in the first energy region, the second liquid replacing the liquid zero material in the cuvette and having a composition excluding the liquid zero-material and having no absorption in the first energy region; determining in the computing device a dual beam spectrum of the liquid zero-material relative to the second liquid at least in the first energy region from the two single beam spectra; and calculating in the computer device an optical pathlength through the cuvette in dependence of spectral information obtained from the first energy region of the determined dual beam spectrum. Since the energy region employed is that in which the second liquid shows no appreciable absorption then it is not critical that the amounts of components of the second liquid which give rise to characteristic absorptions is controlled.

In the invention the liquid zero material is water. This has an advantage that no special preparation, such as precise mixing of chemical components, is necessary.

In the invention the second liquid and the liquid zero-material are immiscible (at least to the extent that the presence of one liquid in the other is in amounts that does not detectably alter the single beam spectrum recorded for that other liquid), i.e. the second liquid is a hydrophobic liquid, such as a vegetable oil, a siloxane based oil (for example a silicone oil) or a mineral oil, and the liquid zero-material is water. This helps to ensure that the second liquid and the liquid zero-material can be completely exchanged for measurement of their respective single beam spectra.

According to a second aspect of the present invention there is provided a method of correcting for an amplitude change in an output of a spectrophotometric apparatus, the spectrophotometric apparatus including a cuvette for holding a liquid sample and a spectrometer and having associated therewith a computing device, the method comprises exposing an unknown liquid sample in the cuvette to electromagnetic radiation at a plurality of energies; obtaining into the computing device using the spectrometer a single beam spectrum of the unknown liquid sample; determining in the computing device a dual beam spectrum of the unknown liquid sample relative to a liquid zero-material; and applying by means of the computer device a mathematical transform to the dual beam spectrum to correct for an amplitude change in the output of the spectrophotometric apparatus, which mathematical transform describes a transformation of the amplitude values of the determined dual beam spectrum to a desired amplitude values; wherein the mathematical transform is dependent on an optical pathlength of the cuvette, the method including calculating the optical pathlength by the method according to the first aspect of the present invention.

As will be appreciated by the skilled artisan, the energy of electromagnetic radiation employed in and/or detected by the spectrophotometric apparatus may be expressed using a number of inter-related units such as wavenumber, wavelength, frequency or channel number whilst remaining within the scope of the invention as described and claimed.

The aforementioned and other advantages associated with the present invention will become apparent from a consideration of the following description of aspects of non-limiting exemplary embodiments of the present invention which is made with reference to the accompanying figures, of which:.

In the following, an embodiment of the spectrophotometric apparatus <NUM> will be described with reference to <FIG> and <FIG> in the context of absorption spectroscopy. The apparatus <NUM> comprises a radiation device <NUM>, a spectrometer <NUM>, which in the present embodiment is an interferometric arrangement, a detector <NUM>, a measuring device <NUM> and a sample holder <NUM> for holding a sample to be analysed.

The radiation device <NUM> comprises a radiation source <NUM> which is arranged to emit polychromatic radiation from within some or all of the ultraviolet to infrared energy range of the electromagnetic spectrum in the direction as indicated by the letter R in <FIG> and <FIG>. In the present embodiment, and by way of example only, the radiation source <NUM> is configured to emit only from within the infrared energy range. It will be appreciated that the energy range is to be selected in dependence of the expected absorption characteristics of the types of liquid sample to be measured by the apparatus <NUM> and may typically extend from within an energy region (or regions) between the ultraviolet and the infrared energy ranges of the electromagnetic spectrum.

The spectrometer <NUM> of the present embodiment comprises necessary equipment for implementing Fourier transform spectroscopy, as is well-known to a person skilled in the art. For example, the spectrometer <NUM> comprises a collimator which collimates the infrared radiation and additional equipment comprised in an interferometer, for example optical components such as moveable and static mirrors, beam splitters and lenses. Other equipment for implementing other types of optical spectrometer known in the art may be employed in other embodiments.

The detector <NUM> is arranged to detect incoming infrared radiation which has been transmitted through the sample holder <NUM>, see further below.

The measuring device <NUM> is connected to the detector <NUM> for collecting unprocessed data about the detected infrared radiation and transmits it to an associated computing device <NUM>. The computing device <NUM> is connected to, and in some embodiments is integral with, the measuring device <NUM> by means of a connection, which may be wired or wireless. The measuring device <NUM> is, by means of this computing device <NUM>, configured to determine a transmittance in a discrete number of channels positioned equidistantly along a wavenumber axis. The computing device <NUM> comprises a processor for processing the collected data, suitable computing software, as well as additional equipment well-known to a person skilled in the art. Moreover, the computing device <NUM> is arranged to store the collected data and the processed data in an associated memory. According to the present embodiment, a routine using Fourier transform algorithms is used in order to transform the unprocessed data from the detector <NUM> into data about the intensity as a function of the wavenumber. Additionally, the computing device <NUM> may be configured to operate to present the data graphically in terms of two-dimensional plots, see <FIG> and <FIG> referred to below.

Further below, a method for correcting intensity deviations (also referred to as 'amplitude changes') of this spectrophotometric apparatus <NUM> will be described.

The sample holder <NUM> is, in the present embodiment, placed between the interferometric arrangement which forms the spectrometer <NUM> and the detector <NUM>. Furthermore, the sample holder <NUM> is arranged to hold a liquid sample <NUM> which is to be spectrally analysed, here by monitoring infrared radiation transmitted through it and generating a single beam spectrum SBs therefrom. In the invention, a water sample <NUM> is employed as a reference or so-called "zero" fluid and is used in order to perform a determination of a cuvette pathlength and corrections to the amplitude of signals recorded by the spectrometer dependent thereon in a manner according to the present invention, see further below. The water sample <NUM> is placed in a cuvette <NUM> of the sample holder <NUM>, which cuvette <NUM> is in part made out of calcium flouride. The outer surface of the cuvette <NUM> is shaped as a rectangular parallelepiped. The cuvette <NUM> comprises inner walls <NUM>, window elements <NUM>, spacers <NUM>, cavities <NUM> and a sample space <NUM> for holding the liquid sample <NUM>, see the cross-sectional top view in <FIG>. The inner walls <NUM> and the window elements <NUM> are transparent to the infrared radiation which is emitted by the radiation source <NUM> and sent through the liquid sample <NUM>. It is noted that the spacers <NUM> do not need to be transparent. For example, the spacers <NUM> may be comprised out of a plastic. The volume of the sample space <NUM> may be varied by varying the extension of the spacers <NUM>. Furthermore, there is an inlet <NUM> for introducing the liquid sample <NUM> into the sample space <NUM> and an outlet <NUM> for removing the liquid sample <NUM> from the space <NUM>. According some embodiments, the liquid sample <NUM> is kept in motion during the measurement, flowing from the inlet <NUM> to the outlet <NUM> via the sample space <NUM>, as indicated by the arrows in <FIG>. In other embodiments, however, the liquid sample <NUM> is kept stationary in the sample space <NUM> during the measurement, in which embodiments the inlet <NUM> and the outlet <NUM> may be omitted.

The distance covered by the infrared radiation in the sample space <NUM> is referred to as an optical pathlength L. Since, in the present embodiment the radiation is transmitted through the liquid sample <NUM> at right angles with respect to a side edge of the cuvette <NUM>, in the direction R in <FIG> and <FIG>, the optical pathlength L coincides with an inner length extension of the cuvette <NUM>, between the window elements <NUM>. If the cuvette <NUM> wears down, the optical pathlength L will change (increase).

In other embodiments the optical radiation which is measured in the spectrometer may be radiation which has traversed the liquid sample <NUM> in the sample space <NUM> a plurality of times (for example after reflection from a one of the window elements <NUM>). In such embodiments the optical pathlength L will not coincide with the inner length extension of the cuvette <NUM> but will be some multiple of this depending, in a known manner, on the detection geometry of the system. However, it will be appreciated that the optical pathlength L will still depend on the inner length extension such that any changes to this inner length extension will manifest as changes in amplitude of optical radiation detected by the detector <NUM>.

In fact, since the window elements <NUM> making contact with the water sample <NUM> are made from calcium flouride, they will be dissolved over time. During its lifetime, the cuvette <NUM> may also have been deteriorated by other chemicals. For example, the thickness T (see <FIG>) of the window elements <NUM> will become smaller over time. Consequently, the separation between the windows <NUM> will increase over time and give rise to changes in the optical pathlength L. In addition, cuvettes <NUM> placed in different apparatuses of the same type <NUM> may have different optical pathlengths L. For instance, differing optical pathlengths L may have resulted from having dissolved the window elements <NUM> of the cuvettes <NUM> to differing degrees, even if the cuvettes <NUM> have been substantially similar at some point in time. Moreover, the extension of the spacers <NUM> may vary between different cuvettes <NUM>, thereby giving rise to varying optical pathlengths L. Therefore, in order to make the characteristics of different apparatuses of the same type <NUM> more similar and the characteristics of a same apparatus <NUM> more stable over time, the optical pathlength L need to be determined and any changes compensated for.

A method <NUM> for determining an optical pathlength L of a cuvette <NUM> is described with reference to the flow diagram of <FIG>. The description is exemplified with respect to the spectrometer <NUM> depicted in <FIG> and <FIG>, the detector <NUM> of which is arranged to detect incoming infrared radiation which is transmitted through the sample holder <NUM> along an optical lightpath of pathlength L at right angles with respect to a side edge of the cuvette <NUM>, in the direction R in <FIG> and <FIG>, which then coincides with an inner length extension of the cuvette <NUM>, between the window elements <NUM>.

According to the present exemplary embodiment the method utilizes the single beam spectrum SBZ of the liquid zero-material sample, which is here a nominally pure water sample (the water sample may contain small amounts of other components, such as around <NUM>% by volume detergent, which do not impact the measured single beam spectrum SBZ), for detecting the optical pathlength L through the cuvette <NUM>. After the spectrophotometric apparatus <NUM> has been corrected using measurements on the water sample, it may be used for measurements on other liquid samples, such as milk or wine in order to make quantitative determinations of components of interest in these samples in a manner well known in the art.

At a step <NUM> the liquid zero-material is introduced into sample space <NUM> and a single beam spectrum of the zero liquid sample SBZ is obtained into the computing device <NUM> using the spectrometer <NUM>. Such a spectrum A is illustrated in <FIG> which illustrates a plot of the intensity of detected radiation (here in the infrared portion of the electromagnetic spectrum) indexed against wavenumber and is collected at least in a first energy region (identified as "Relevant region" in the figure) at which the zero liquid material absorbs at least a portion of the incident optical radiation emitted by the radiation source <NUM>.

At a step <NUM> the liquid zero-material sample <NUM> in the cuvette <NUM> is replaced with a second liquid sample which is characterised by having a composition excluding the liquid zero-material and by having no absorption in the first energy region. A single beam spectrum of the second liquid sample SB<NUM> is obtained into the computing device <NUM>, again using the spectrometer <NUM>. An example of such a spectrum B is also illustrated in <FIG> for a vegetable oil which is here, for example, corn oil and is collected at least in the first energy region.

In the invention the second liquid is immiscible with the zero liquid. This helps ensure that the liquid <NUM> already in the cuvette <NUM>, which here by way of example is the zero liquid, is completely replaced by the other liquid, which here by way of example is the second liquid. It will be appreciated that the order of performing the steps <NUM> and <NUM> may be reversed so that the zero liquid replaces the second liquid in the cuvette <NUM> for measurement. In the invention, wherein the liquid zero-material is water, the hydrophobic second liquid may be, for example, a liquid selected from a vegetable oil, such as sunflower, olive, corn or grape oil; a siloxane based oil, such as a silicone oil; and a mineral oil.

At a step <NUM> the computing device <NUM> operates to determine a dual beam spectrum of the zero material relative to the second material DBZ and an absorbance spectrum therefrom, at least in the first energy region. Such a dual beam absorbance spectrum is illustrated in <FIG>.

At a step <NUM> the optical pathlength L through the cuvette <NUM> is calculated in the computing device <NUM>. In some embodiments this is done from the application of the Beer-lambert law to the absorbance spectrum in the first energy region which is determined at the step <NUM>, using a knowledge of the molar absorptivity of water. In some embodiments this optical pathlength L is determined from the application of a chemometric model, such as a PLS model, which links features in the absorbance spectrum to the optical pathlength L through the cuvette <NUM>. This model is generated in a manner well known in the art of chemometrics from a multivariate data analysis, such as a Partial Least Squares (PLS) analysis, of a plurality of dual beam absorbance spectra of water (or generally a "liquid zero material") relative to the oil (or generally a "second liquid") obtained using cuvettes <NUM> having different known reference optical pathlengths Lref.

In some situations it may be appropriate to incorporate one or more additional variables in the multivariate analysis. An example of such other variable which may affect the absorbance spectra collected by the spectrophotometric apparatus <NUM> is temperature. In this case the spectra that are collected for use in generating the model are also collected at different known temperatures, preferably across a temperature range spanning temperatures expected to be experienced by the apparatus <NUM> during normal operation. Thereby, variations in the one or more other variables may be compensated for in the finally calculated of optical pathlength L.

In some embodiments the so-calculated optical pathlength L may be compared in the computing device <NUM> with a preset value and a difference ΔL determined. The computing device <NUM> may then be programmed to generate a warning, indicating that the cuvette <NUM> has become excessively worn and requires replacing, when the difference ΔL exceeds or equals a preset value.

Knowledge of the actual optical pathlength L as determined at step <NUM> may be employed in some embodiments in the calculation in the computing device <NUM> of a correction factor Icorr for use in correcting for the effects of changes in the optical pathlength on the intensities of radiation measured by the spectrophotometric apparatus <NUM>.

According to a second aspect of the present invention an additional step <NUM> is provided at which the correction factor Icorr is determined in the computing device <NUM> as dependent on a ratio of a nominal optical pathlength L<NUM> to the determined optical pathlength L.

In some embodiments a step <NUM> is provided at which subsequently obtained spectra are corrected using this correction factor Icorr.

At this step <NUM> Icorr may be employed in the computing device <NUM> in order to correct measured intensities Am to those (Anom) expected at the nominal pathlength L<NUM> by applying, for example, the relationship <MAT>.

In some embodiments, at this step <NUM>, the correction factor Icorr may be employed in the computing device <NUM> to generate a control signal dependent on this correction factor Icorr by which a gain stage <NUM> of the detector <NUM> may be set in order to correct the amplitudes of measured intensities of incident radiation to those expected at the nominal optical pathlength L<NUM>.

In some embodiments the x axis (or wavenumber scale) of the single beam spectra collected using the spectrometer <NUM> of the spectrophotometric apparatus <NUM> is standardized before the y axis (amplitude) correction is performed (i. e before the correction factor Icorr is applied). This may be achieved in a manner that is well known in the art by applying a mathematical transform to the single beam spectrum by which transform measured data is standardized along the x axis. In some embodiments, the x axis standardisation is based on the CO<NUM> peak of air in the infrared range, as described below.

Claim 1:
A method for determining an optical pathlength (L) of a cuvette (<NUM>) of a spectrophotometric apparatus (<NUM>), the spectrophotometric apparatus (<NUM>) including a spectrometer (<NUM>) and having associated therewith a computing device (<NUM>) , the method comprising: obtaining (<NUM>) into the computing device (<NUM>) by means of the spectrometer (<NUM>) a single beam spectrum (SBZ) of a liquid zero-material held in the cuvette (<NUM>) at least in a first energy region in which the liquid zero-material absorbs; obtaining (<NUM>) into the computing device (<NUM>) by means of the spectrometer (<NUM>) a single beam spectrum (SB<NUM>) of a second liquid at least in the first energy region, the second liquid replacing the liquid zero material in the cuvette (<NUM>) and having a composition excluding the liquid zero-material and having no absorption in the first energy region; determining (<NUM>) in the computing device (<NUM>) a dual beam spectrum (DBZ) of the liquid zero-material relative to the second liquid at least in the first energy region from the two obtained single beam spectra (SBZ; SB<NUM>); and calculating (<NUM>) in the computer device (<NUM>) an optical pathlength (L) through the cuvette (<NUM>) in dependence of spectral information obtained from the first energy region of the determined dual beam spectrum (DBZ),
characterized in that:
the second liquid is immiscible with the liquid zero-material,
wherein the liquid zero-material is water and the second liquid is a hydrophobic liquid.