GB1591202A

GB1591202A - Absorption spectrometry

Info

Publication number: GB1591202A
Application number: GB35938/77A
Authority: GB
Original assignee: Philips Gloeilampenfabrieken NV
Current assignee: Koninklijke Philips NV
Priority date: 1976-08-30
Filing date: 1977-08-26
Publication date: 1981-06-17
Also published as: JPS5341286A; DE2736950A1; IT1065378B; FR2363102B1; FR2363102A1

Description

(54) ABSORPTION SPECTROMETRY (71) We, N.V. PHILIPS' GLOEILAMPENFABRIEKEN, a limited liability Company, organised and established under the laws of the Kingdom of the Netherlands, of Emmasingel 29, Eindhoven, the Netherlands do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention relates to a process and apparatus for measuring the concentration of a molecule having characteristic selective absorption and emission spectra, in a sample substance.

Many organic molecules (for example phenolic molecules) notably present very selective absorption and fluorescent or phosphorescent emission spectra, i.e. with relatively abrupt flanks.

A normal method of measuring the concentration of a molecule of this kind in a sample substance (frequently an aqueous solution of said molecules comprises irradiating the sample with a monochromatic excitation light beam having a wavelength lying within the absorption spectrum of the molecule, then filtering and sensing the corresponding light due to fluorescece or phosphorescece, which is emitted by the sample substance at a longer wavelength, and finally measuring the intensity of the collected light.

The measured intensity provides an indication of the concentration of the molecule under examination in the sample substance.

Although the said measurement method has various merits which make it preferable to other possible methods, it suffers from the disadvantage of providing a measurement which does not take account of inaccuracies due to the superimposition on the useful signal of spurious signals originating from diffused light and/or spurious fluorescece emitted by traces of other molecules contained in the sample substance.

It is an object of the present invention to provide an improved process and apparatus which can enable phenolic molecule concentration measurements to be made which are substantially free from inaccuracies due to spurious signals which are superimposed on the useful signal emitted by the molecule under examination when excited.

According to the invention there is provided a process for measuring the concentration of a molecule having characteristic selective absorption and emission spectra, in a sample substance, comprising subjecting the sample substance to a first monochromatic excitation light beam of wavelength lying within the absorption spectrum of the molecule under measurement, filtering and sensing a first light emission from the sample substance at a wavelength lying within the emission spectrum of the molecule, and determining the intensity of the sensed first monochromatic light emission, and further comprising subsequently subjecting the sample substance to a second monochromatic excitation light beam of a wavelength close to that of the first monochromatic excitation light beam but outside the absorption and emission spectra of the molecule, filter ing and sensing a corresponding second light emission from the sample substance at the same wavelength as the first, then determining the intensity of the sensed second light emission and subtracting this from that of the first so as to provide a signal representative of the concentration of said molecule in said sample substance and substantially free from spurious signals due to diffused light or to light resulting from spurious fluorescece or phosphorescece due to traces of other molecules in the sample substance.

According to the invention there is further provided apparatus for measuring the concentration of a molecule having characteristic selective absorption and emission spectra, in a sample substance, comprising an excita tion monochromator arranged to subject the sample substance to a first and subsequently to a second monochromatic excitation light beam of wavelengths which are close together but which lie respectively within the absorption spectrum of the molecule being measured, and outside the absorption and emission spectra of the same molecule, a collection monochromator arranged to filter and sense corresponding light emitted from the sample substance at a wavelength within the emission spectrum of the molecule, and electronic processing means arranged to determine the sensed light intensities corresponding to the respective emissions resulting from the first and second excitations and to subtract one from the other.

A process and apparatus in accordance with the invention is based on the use not of one but of two monochromatic excitation light beams of close wavelengths, one of which, falling within the absorption spectrum of the molecule under examination, causes the emission of a fluorescent or phosphorescent monochromatic light constituting the useful signal and on which are inevitably superimposed spurious signals due to diffused light and to possible fluorescence or phosphorescece emitted by traces of other substances, while the other, being of a wavelength lying outside the absorption and emission spectra of the molecule under examination, will only cause the emission of a corresponding spurious light signal due to diffusion of light and to the fluorescece or phosphorescece emitted by traces of other substances, when present.Since such spurious signals will be of substantially the same magnitude for the two excitation wavelengths, it will be apparent that subtraction of the second sensed light signal from the first will enable the effect of the spurious signal to be substantially cancelled, with the consequent isolation of the desired signal representing the effective concentration of the molecule under examination in the sample substance. The double monochromatic excitation provided by the present invention thus enables the inaccuracies accompanying the known single monochromatic excitation method to be reduced or removed.

It has already been stated that light for the second monochromatic excitation has a wavelength close to the first but outside the absorption and emission spectra of the molecule under examination. In reality, the wavelength of the light for said second monochromatic excitation should preferably lie within the range between the two spectra if both the spurious signals due to diffused light and those due to the interfering light emitted by traces of other substances contained in the sample are to be effectively cancelled, while it should be just greater than the maximum wavelength of the emission spectrum of the molecule under examination if it is required only to remove spurious signals due to diffused light or if it is technically not possible to provide the second monochromatic light excitation at a wavelength between the two spectra.As these latter are usually spaced apart by approximately 30 nm the distance between the two excitation wavelengths may therefore vary from about 15 to about 60 nm. In general, it will not be a case of individual wavelengths but of very narrow wave-bands, which could vary from 0.1 to 10 nm according to the type of molecule under examination and various other constructional and operational factors.

Likewise the isolated and collective wavelengths for the fluorescece or phosphorescece emitted by the sample substance, will not have a single value, but will in reality consist of a narrow band of wavelengths variable from 1 to 100 nm according to the actual circumstances.

Preferably the two monochromatic excitation light beams are emitted in very rapid time succession, so as to enable the measurement to be made on flow samples, i.e.

samples in movement relative to the measurement apparatus. In particular the time lag should preferably lie in the range from 0.1 to 10 ,u sec.

This can cause difficulties with regard to the choice of a suitable form of monochromator, because most normal commercially available monochromators which have various moving parts are unable to provide a sufficiently rapid change-over from one wavelength to another. In accordance with the invention, this problem is preferably solved by using a stationary form of monochromator (based only on a rapid alternation of the illumination of two lamps) as described in the UK Patent Specification No.

1,482,475, to which reference should be made for further details.

An embodiment of the present invention will now be described by way of example, with reference to the accompanying drawings, of which: Figure 1 is a diagrammatic overall view of measurement apparatus in accordance with the present invention, Figure 2 shows the same apparatus in further detail, Figures 3 and 4 are graphs which show the location of the excitation wavelength and fluorescent or phosphorescent emission wavelength relative to the absorption and emission spectra of the molecule under examination when cancelling both forms of unwanted signal, and when cancelling only the unwanted signals due to diffused light respectively, Figure 5 shows on a common time scale various wave-forms present during operation of the apparatus.

The measurement apparatus shown in the drawings, and in particular in Figures 1 and 2, generally comprises an excitation monochromator 1, a sample cell 2 containing the sample substance 3 for which the concentration of the pre-determined molecule having characteristic of selective absorption and emission spectra, is to be measured (for example a phenol in aquous solution), a measuring monochromator 4 and finally an analogue, digital or mixed electronic processor 5.

The excitation monochrometer 1 is of the static type described in UK Patent Specification No. 1,482,475 and comprises two lamps 6 and 7 which are alternately illuminated in rapid succession (repeated several times) to produce corresponding monochromatic excitation light beams 8 and 9 (Fig. 2) very close together in time (0.1 to 10 IL sec.) and of wavelengths A1 and A2 which are also very close (15 to 60 nm apart).

It will be assumed that the molecule under examination has a characteristic absorption spectrum Sa and a characteristic emission spectrum Se shown in Figures 3 and 4, accompanied by a constant diffused light Ld and possible secondary spectra Sa and Se (Fig. 3) due to traces of other molecules in the same substance.

If the wavelength hl of a first monochromatic excitation light is made substantially to coincide with the centre of the absorption spectrum Sa, as is in fact done, the molecule thus excited emits a corresponding fluorescent (or phosphorescent) light at a corresponding wavelength x3 substantially coinciding with the centre of the emission spectrum Se. Said main emission of light has superimposed on it at wavelength A3 a certain diffused light Ld and, in certain cases, an emission of secondary light due to the excitation effect determined by the light of X1 fal- ling on possible traces of molecules having spectra Sa and Se also present in the sample substance.

The light signal of wavelength A3 thus emitted, indicated in Fig. 2 by the reference numeral 10, and consisting in practice of a desired signal component of monochromatic light emitted by the molecule under examination and, superimposed thereon, a spurious signal component due to the presence of diffused light and, possible, monochromatic light emitted by traces of other molecules, is filtered and collected by the measuring monochromator 4, which for this purpose comprises an inlet filter 11 of very narrow band centred on A3, a positionable mirror 12 and a photomultiplier 13 the purpose of which is to convert the weak light signal 10 into an amplified electrical signal 14 (fig. 2).

While the wavelength A of the first mono- chromatic excitation light is substantially centred on the absorption spectrum Sa of the molecule under examination, the wavelength A2 of the second monochromatic excitation light is located outside said absorption spectrum, namely between the two spectra Sa and Se (Fig. 3) or at a value greater than Se (fig.

4). It is therefore not able to excite the molecule under examination to make it emit, as in the case of light of wavelength At, , a fluorescent or phosphorescent light of wavelength A3, but is however able to provide the measuring monochromator 4 with a spurious light signal 15 of wavelength A3 (Fig. 2) which coincides with the spurious part of the signal 10 determined by the previous excitation at A2, and represents in the case of Figure 3 both the diffused light and the monochromatic light emitted by the traces of molecules with spectra Sa and Se, and in the case of Figure 4 only the diffused light.

Said spurious light signal 15 is filtered by the filter 11 and converted into a corresponding electrical signal 16 by the photomultiplier 13, from which two signals are emitted repeated several times in rapid succession, namely a signal 14 due to the excitation at wavelength At and comprising the useful signal with a spurious signal superimposed thereon, and a signal 16 due to the excitation at wavelength X2 and comprising only a spurious signal of value equal to that of the spurious signal contained in the signal 14.

Said signal 14 and 16, of obviously different amplitude, are shown graphically by the wave form C of Fig. 5, the wave form A representing the pattern of the monochromatic excitation light signals 8 and 9, clearly of equal amplitude, and the wave form B represents the pattern of electric signals 17 and 18 generated by a photodiode 19 (Figs. 1 and 2) to represent predetermined fractions 20 and 21 of the monochromatic excitation light signals 8 and 9.

Both the succession of electrical emitted light signals 14 and 16 and the succession of electrical reference signals 17 and 18 are transferred by the photomultiplier 13 and photodiode 19 respectively to the electronic processor 5 where two memory amplifiers 22 and 23, also known as "buffer memories", amplify and store them. Said successions of signals are then compared in a signal normaliser 24 which, comparing the emitted light signals 14 and 16 with the signals representing excitation light 17 and 18, produces output signals 25 and 26 which correspond to the signals 14 and 16 but also take account of, and are compesated for variations in the intensity of the excitation light emitted by the monochromator 1.

The normalised signals 25 and 26 are then integrated in an integrator divider 27 which takes the mean of all signals 25 and all signals 26 so as to give rise to respective mean signals 28 and 29 which are free from small variations due to slight variations in operation of the parts of the apparatus which have generated them, in particular of the photomultiplier 13, which notably usually operates by counting photons.

Finally said mean signals 28 and 29, which may also be indicated separately by respective output indicators 30 and 31, are compared and the second is subtracted from the first in a subtraction device 32, which provides a single output signal 33 indicated visually by an output indicator (of analogue or digital type) 34.

As the signal 28 represents the desired monochromatic emission signal component representative of the molecule under examination with a superimposed spurious signal component determined by the diffused light and by any spurious monochromatic light emitted by traces of other molecules contained in the sample substance, and the signal 29 only represents said spurious signal component (with the same magnitude), it will be evident that the result of the subtraction carried out in the subtraction device 32 will be the desired signal, substantially free of superimposed spurious signals. Said desired signal is shown in the wave-form D in Fig. 5.

WHAT WE CLAIM IS: 1. A process for measuring the cocnentration of a molecule having characteristic selective absorption and emission spectra, in a sample substance, comprising subjecting the sample substance to a first monochromatic excitation light beam of wavelength lying within the absorption spectrum of the molecule under measurement, filtering and sensing a first light emission from the sample substance at a wavelength lying within the emission spectrum of the molecule, and determining the intensity of the sensed first -monochromatic light emission and further comprising subsequently subjecting the sample substance to a second monochromatic excitation light beam of a wavelength close to that of the first monochromatic excitation light beam but outside the absorption and emission spectra of the molecule, filtering and sensing a corresponding second light emission from the sample substance at the same wavelength as the first, then determining the intensity of the sensed second light emission and subtracting this from that of the first so as to provide a signal representative of the concentration of said molecule in said sample substance and substantially free from spurious signals due to diffused light or to light resulting from spurious florescece or photphorescece due to traces of other molecules in the sample substance.

2. A process as claimed in Claim 1, wherein said second monochromatic excitation light has a wavelength lying between said absorption and emission spectral regions relating to the molecule.

3. A process as claimed in Claim 1, wherein said second monochromatic excitation light has a wavelength greater than those within the emission spectrum of the molecule.

4. A process as claimed in Claim 1, 2 or 3, including the insertion of a time delay of approximately 0.1 - 10 Er. sec between the applications of the first and second monochromatic excitation light beams.

5. An apparatus for measuring the concentration of a molecule having characteristic selective absorption and emission spectra, in a sample substance, comprising an excitation monochromator arranged to subject the sample substance to a first and subsequently to a second monochromatic excitation light beam of wavelengths which are close together but which lie respectively within the absorption spectrum of the molecule being measured, and outside the absorption and emission spectra of the same molecule, a collection monochromator arranged to filter and sense corresponding light emitted from the sample substance at a wavelength within the emission spectrum of the molecule, and electronic processing means arranged to determine the sensed light intensities corresponding to the respective emissions resulting from the first and second excitations and to subtract one from the other.

6. An apparatus as claimed in Claim 5, wherein said collection monochromator comprises a narrow band input filter and a photomultiplier arranged to convert the light intensities emitted by the sample substance and filtered through said inlet filter into corresponding electrical emitted-light signals and to amplify said signals.

7. An apparatus as claimed in Claim 5 or 6, wherein said electronic processing means comprise storage means for storing said electrical emitted-light signals and for storing further electrical signals representing the excitation light intensities, signal normalising means arranged to compare said electrical emitted-light signals and said further electrical signals to give normalised signals insensitive to variations in excitation light intensity, integrator divider means arranged to take the mean of the normalised signals produced from the first monochromatic excitation light and of the normalised signals produced subsequently by the second monochromatic excitation light and subtraction, means arranged to subtract the mean signal produced by the second monochromatic excitation light from the mean signal produced by the first monochromatic excitation light to enable a signal to be obtained representing the magnitude of the monochromatic light signal emitted by the molecule under examination.

8. An apparatus for measuring the concentration of a molecule having characteristic selective absorption and emission spectra,

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. the parts of the apparatus which have generated them, in particular of the photomultiplier 13, which notably usually operates by counting photons. Finally said mean signals 28 and 29, which may also be indicated separately by respective output indicators 30 and 31, are compared and the second is subtracted from the first in a subtraction device 32, which provides a single output signal 33 indicated visually by an output indicator (of analogue or digital type) 34. As the signal 28 represents the desired monochromatic emission signal component representative of the molecule under examination with a superimposed spurious signal component determined by the diffused light and by any spurious monochromatic light emitted by traces of other molecules contained in the sample substance, and the signal 29 only represents said spurious signal component (with the same magnitude), it will be evident that the result of the subtraction carried out in the subtraction device 32 will be the desired signal, substantially free of superimposed spurious signals. Said desired signal is shown in the wave-form D in Fig. 5. WHAT WE CLAIM IS:

1. A process for measuring the cocnentration of a molecule having characteristic selective absorption and emission spectra, in a sample substance, comprising subjecting the sample substance to a first monochromatic excitation light beam of wavelength lying within the absorption spectrum of the molecule under measurement, filtering and sensing a first light emission from the sample substance at a wavelength lying within the emission spectrum of the molecule, and determining the intensity of the sensed first -monochromatic light emission and further comprising subsequently subjecting the sample substance to a second monochromatic excitation light beam of a wavelength close to that of the first monochromatic excitation light beam but outside the absorption and emission spectra of the molecule, filtering and sensing a corresponding second light emission from the sample substance at the same wavelength as the first, then determining the intensity of the sensed second light emission and subtracting this from that of the first so as to provide a signal representative of the concentration of said molecule in said sample substance and substantially free from spurious signals due to diffused light or to light resulting from spurious florescece or photphorescece due to traces of other molecules in the sample substance.

in a sample, substantially as herein described with reference to the accompanying drawings.