Patent Description:
Many substances absorb ultra-violet or visible light due to their chemical composition. The absorption of light by substances has been used as the basis for detecting the presence of, and measuring the concentration of, such substances for many years. The concentration of the substance can be determined by use of the Beer Lambert Law:
A=Ebc.

The UV region can be considered to consist of light of wavelength in the region of <NUM> to <NUM>, light of wavelength of <NUM> to <NUM> being known as 'deep UV'. Most analytical instruments for detecting substances which absorb in the deep ultra-violet (UV) region use a mercury- lamp, deuterium lamp or xenon flash lamp as a light source. One example of such an instrument is a flow cell in which a solution containing one or more UV absorbing substances is placed between a UV light source (e.g. a mercury-lamp) and a UV detector (e.g. a photomultiplier, a photodiode or a photo transistor) and changes in the intensity of UV light reaching the detector are related to the concentration of UV absorbing substances in the solution.

The detection of proteins, nucleic acids and peptides are of great importance in many sectors, including the environmental, biological and chemical sciences. Proteins have mainly two absorption peaks in the deep UV region, one very strong absorption band with a maximum at about <NUM>, where peptide bonds absorb, and another less intense peak at about <NUM> due to light absorption by aromatic amino acids (e.g. tyrosine, tryptophan and phenylalanine).

<CIT> and <CIT> describe the use of a UV LED as a source of light for analysis of the concentration of a substance in a liquid sample.

The incoming light to the measuring arrangement from the UV LED is divided into a reference light ray provided directly to a detector and a signal light ray provided through the sample and then to a detector. The detected signal light ray is greatly amplified and compared to an amplified version of the detected reference light ray. There may be problems in these kind of systems related to drift since the high detector amplification means that any tiny unequal performance change in the channels due to for example temperature fluctuations or humidity changes will cause a drift in the measured absorption signal. Systems measuring absorbance changes over time in a flowing sample are naturally much more sensitive to drift in the system compared to systems doing short measurements on a static sample.

Various other systems are also known for measuring absorbance or the like in both the medical field and also, for example, the petrochemical field. See, for example, <CIT>, <CIT> and <CIT>.

<CIT> describes a multiple photometer assembly for testing blood samples, which can be used in visible, infrared and ultraviolet region of the spectrum. The photometer module may include two flow cuvettes one of which is supplied with a reference fluid or the reference paths may be simply a fiber optical element. The light from each path is chopped at a different frequency by one of the two sets of holes and the two signals are thereafter optically combined so that they are detected at the same location on the surface of the photo-detector. After suitable amplification this combined signal may be electrically separated and appropriately processed.

An object of the present invention is to provide an improved method and a measuring device for measuring the absorbance of UV light by a protein in a solution in a measuring cell.

A further object of the present invention is to provide a method and a measuring device for measuring the absorbance of UV light by a protein in a solution in a measuring cell which are robust and reliable.

This is achieved in a method and a measuring device according to the independent claims.

Hereby only one detector is used to simultaneously measure both the reference light ray and the signal light ray which means that any drift caused by use of two separate detectors is eliminated.

Various aspects and embodiments of the present invention are thus defined by the appended claims.

<FIG> shows schematically a measuring device <NUM> of prior art for measuring the absorbance of a substance in a solution provided in a measuring cell <NUM>. The measuring device <NUM> comprises a light source <NUM> which is transmitting a first light ray <NUM>. Furthermore, the measuring device comprises a beam splitter <NUM> provided in the measuring device such that the first light ray <NUM> is divided by the beam splitter <NUM> into a signal light ray <NUM> and a reference light ray <NUM>. The measuring device <NUM> comprises further the measuring cell <NUM> which is positioned such that the signal light ray <NUM> will pass through the solution in the measuring cell <NUM>. Furthermore, the measuring device <NUM> comprises a first detector <NUM> arranged to detect the signal light ray <NUM> when it has passed the measuring cell <NUM> and a second detector <NUM> arranged to detect the reference light ray <NUM>. From the detected reference- and signal values the absorbance of the sample can be calculated by the formula A=log<NUM>(reference value/signal value). As discussed above drift is a problem in these kinds of systems, especially when doing long, continuous measurements as in liquid chromatography. Small fluctuations in for example temperature may affect the two different detector channels unequally and thereby causing drift and non-exact measurements.

<FIG> shows schematically a measuring device <NUM> according to one embodiment of the invention. The measuring device <NUM> is arranged for measuring the absorbance of a substance in a solution provided in a measuring cell <NUM> of the measuring device. The measuring device <NUM> comprises a light source <NUM> which is transmitting a first light beam <NUM>. The light source could be for example a LED or any other type of suitable lamp. The measuring device comprises further a beam splitter <NUM> provided such that the first light beam <NUM> is divided by the beam splitter <NUM> into a signal light ray <NUM> and a reference light ray <NUM>. The beam splitter <NUM> can be for example a semitransparent mirror. The measuring device <NUM> comprises further the measuring cell <NUM> positioned such that the signal light ray <NUM> will pass through the solution in the measuring cell <NUM>. The measuring cell <NUM> can be a static measuring cell comprising a sample to be measured but it could also be a flow cell in which the sample is flowing through during continuous measurements.

The measuring device <NUM> furthermore comprises a first signal modulation device <NUM> arranged to modulate the signal light ray <NUM> and a second signal modulation device <NUM> arranged to modulate the reference light ray <NUM>. The first signal modulation device <NUM> is in this embodiment positioned between the beam splitter <NUM> and the measuring cell <NUM>. However, it could also be positioned after the measuring cell <NUM> but before a detector <NUM> also provided in the measuring device <NUM>. The detector <NUM> is according to the invention arranged to detect the signal light ray <NUM> when it has been modulated and passed the measuring cell <NUM> and also detect the reference light ray <NUM> when it has been modulated. In this shown embodiment the detector <NUM> is provided such that the signal light ray <NUM> is detected by the detector <NUM> without any further redirection. The reference light ray <NUM> is instead in this embodiment redirected by a light direction changing device <NUM> such that the reference light ray <NUM> after redirection by the light direction changing device <NUM> and modulation by the second signal modulation device <NUM> is detected by the detector <NUM>. The light direction changing device could be a mirror.

The second signal modulation device <NUM> is here shown to be provided after the light direction changing device <NUM> but it could also be provided between the beam splitter <NUM> and the light direction changing device <NUM>. Of course, the light direction changing device <NUM> could instead be provided in the signal light ray path and the detector <NUM> in the path way of the reference light ray, i.e. the direction of the reference light ray <NUM> directly after the beam splitter <NUM>. More than one light direction changing device could also be provided in the measuring device in order to direct the signal light ray and the reference light ray to the same detector in a suitable way.

With the measuring device according to the invention both the signal light ray <NUM> and the reference light ray <NUM> are detected by the same detector <NUM>. The detector <NUM> comprises or is connected to a processing device <NUM> which is configured for performing synchronous detection of the detected signal in order to reconstruct the intensities of the signal light ray <NUM> and the reference light ray <NUM> from the combined signal detected by the detector <NUM>. This is achieved by multiplying the detector signal by the modulation signals respectively and then low-pass filtering the results. In this way the two different signals are reconstructed and from them the absorption can be calculated. The processing device <NUM> can be completely made of hardware, completely made in software or a combination of the two. With this measuring device the signal light ray <NUM> and the reference light ray <NUM> can be detected simultaneously by the same detector <NUM>.

The detector <NUM> comprises also normally a photo diode, an amplifier and an AD converter (not shown in the Figure). Furthermore, an optical arrangement is usually provided between the light source <NUM> and the beam splitter <NUM>. However, this is not shown in the figures. This optical arrangement can comprise a collimating lens, an aperture and possibly also a filter.

The first signal modulation device <NUM> and the second signal modulation device <NUM> can for example be a chopper, a shutter, a movable mirror, a tuning fork or an adjustable grey filter. They could be separate devices or provided in the same device if the design of the measuring device and distances between the signal light ray and the reference light ray allows that. The first signal modulation device <NUM> and the second signal modulation device <NUM> should modulate the signals at different frequencies such that they can be differentiated in the detector. The modulation of the light rays creates an amplitude modulation at a set frequency. In one example the reference light ray can be modulated at a frequency somewhere in the range from <NUM> to a few kHz and the signal light ray is modulated at a frequency that differs with at least <NUM>% from the first frequency. The frequencies need to be chosen such that they do not interfere with other frequencies or their harmonics in the system.

The amplitude modulation can be sine modulation, square wave modulation or any other waveform that is suitable.

In another embodiment of the invention one of the first or the second signal modulation device <NUM>, <NUM> is instead a device which is controlling the light source <NUM>. Controlling the light source <NUM>, such as for example turn it on and off by a specific frequency will of course affect both the signal light ray and the reference light ray but in combination with one signal modulation device in either the signal light ray or the reference light ray the two signals can still be differentiated from each other in the detector in a correct way if the measured signal is adjusted by adding the difference between the "light on"/"modulator on" and the "light on"/"modulator off" value to each "light off"/"modulator on" portion of the signal.

According to the invention the beam splitter <NUM> is an asymmetrical beam splitter where a larger portion of the first light beam <NUM> is directed into the signal light ray <NUM> than the reference light ray <NUM>. This could be useful since a larger part of the dynamic range of the detector then is used for the signal light ray.

<FIG> is a flow chart of a method for measuring the absorbance of light of a substance in a solution in a measuring cell <NUM> according to one embodiment of the invention. The method comprises the steps as described below, also with reference to <FIG>:.

However, if the construction and distances between the signal light ray and the reference light ray allows it the first and second signal modulation devices <NUM>, <NUM> could be built into one and the same device.

In one embodiment of the invention the step of modulating the signal light ray comprises modulating the signal light ray at a first frequency and the step of modulating the reference light ray comprises modulating the reference light ray at a second frequency which is different from the first frequency. Furthermore, one possibility to modulate the signal light ray and the reference light ray is to create an amplitude modulation to both the signal light ray and the reference light ray. The amplitude modulation waveform could be a sine, square wave or any suitable waveform.

S9: Providing the measuring cell <NUM> such that the signal light ray <NUM> passes through the measuring cell.

S11: Detecting a signal in a detector <NUM>, which signal is the combined signal intensity of the signal light ray <NUM> and the reference light ray <NUM> detected by the detector <NUM>.

S15: Performing synchronous detection of the detected signal in order to reconstruct the intensities of the signal light ray <NUM> and the reference light ray <NUM> from the combined signal detected by the detector <NUM> based on the modulation performed to the signal light ray <NUM> and the reference light ray <NUM>.

The detecting of the signal light ray <NUM> in the detector <NUM> and the detecting of the reference light ray <NUM> in the same detector <NUM> can with this method be performed simultaneously.

In one embodiment the method further comprises the step of changing direction of one or both of the signal light ray 31and the reference light ray <NUM> with at least one light direction changing device <NUM> such that both the signal light ray and the reference light ray can be detected by the same detector <NUM>.

Claim 1:
A method for measuring the absorbance of UV light by a protein in a solution in a flow cell (<NUM>), said method comprising the steps of:
- transmitting (S1) a first light beam (<NUM>) from an ultraviolet, UV, light source (<NUM>) of a wavelength of <NUM> to <NUM> towards a beam splitter (<NUM>);
- dividing (S3) the first light beam (<NUM>) into a signal light ray (<NUM>) and a reference light ray (<NUM>) by the beam splitter (<NUM>);
- modulating (S5) the signal light ray (<NUM>);
- modulating (S7) the reference light ray (<NUM>);
- providing (S9) the measuring flow cell (<NUM>) such that the signal light ray (<NUM>) passes through the measuring flow cell (<NUM>);
- detecting (S11) a signal in a detector (<NUM>), which signal is the combined signal intensity of the signal light ray (<NUM>) and the reference light ray (<NUM>) detected by the detector (<NUM>);
- performing synchronous detection (S15) of the detected signal in order to reconstruct the intensities of the signal light ray (<NUM>) and the reference light ray (<NUM>) from the combined signal detected by the detector (<NUM>), said synchronous detection being based on the modulation performed to the signal light ray and the reference light ray, wherein said dividing of the first light beam into signal and light rays is performed by an asymmetrical beam splitter (<NUM>) such that a larger portion of the first light beam is directed to the signal light ray than to the reference light ray; and
- wherein the step of modulating (S5) the signal light ray (<NUM>) comprises modulating the signal light ray at a first frequency and the step of modulating (S7) the reference light ray (<NUM>) comprises modulating the reference light ray at a second frequency which is different from the first frequency, and wherein the reference light ray (<NUM>) and the signal light ray (<NUM>) are detected in the same detector (<NUM>) simultaneously.