WO1980000494A1

WO1980000494A1 - Method for measuring the concentration by total reflection spectroscopy

Info

Publication number: WO1980000494A1
Application number: PCT/DE1979/000078
Authority: WO
Inventors: G Mueller
Original assignee: G Mueller
Priority date: 1978-08-30
Filing date: 1979-08-02
Publication date: 1980-03-20
Also published as: EP0016166A1; JPS55500589A

Abstract

In a method for measuring the concentration by means of total reflection (ATR Spectroscopy), a transparent material with a high reflection index is used to regenerate a clean optical surface pertaining to the reflecting elements. This substance acts as an intermediate layer (12, 15) between the reflecting element (4) and the sample (6). Hence, a reproducible and faultless surface is obtained as desired between the reflecting element and the sample. The properties of this surface may be adapted to the needs. The surface of the intermediate layer may be liquid or solid. The method is particularly appropriate to determine the concentrations of biological substances.

Description

Concentration measurement method using total internal reflection spectroscopy

The invention relates to a method for measuring the concentration by means of the spectroscopy of the total internal reflection. Such a method is of great practical interest in particular for determining the concentration of biological substances, since it enables measurements with the smallest possible sample quantities directly in the biological environment.

Spectroscopy of internal total reflection has been known under the name ATR (attenuated total reflection) in the infrared spectral range since 1960. However, the strong absorption of water has hitherto prevented a general application of this test method also to natural biological and medical samples, which are generally strongly hydrated.

The use of intensive laser light sources has made it possible to reduce the difficulties which arise from water absorption. However, the difficulties resulting from the fact that many molecules, such as proteins, tend to adsorb on the surface of the reflection element used in the total internal reflection spectroscopy, so that there is a risk of contamination when changing the sample has not yet been overcome. In addition, the difficulties resulting from the fact that many of the reflection elements used in particular in the infrared spectral range are toxic and / or water-soluble have not been overcome.

It is the object of the present invention to create a method for concentration measurement by means of the spectroscopy of the total internal reflection, which enables reproducible measurements with high detection sensitivity in the infrared, visible and ultraviolet spectral range in a simple manner.

OMPI. - 2 -

This object is achieved according to the invention in that, for the regeneration of an optically clean surface of the reflection element, a transparent, highly refractive substance as an exchangeable intermediate layer between. Reflective element and sample is used.

The penetration depth of the transversely damped light wave penetrating into the adjacent sample substance during total reflection of the measuring beam path on the surface of the reflection element lies in the visible spectral range, e.g. in the range of fractions of a u and their field strength decreases exponentially with increasing depth of penetration. The method according to the present invention enables the production and restoration of clean and reproducible surfaces between the reflection element and sample. This measure is the only way to ensure that the concentration of substances can be measured correctly and at any time by evaluating the absorption spectrum in the ultraviolet (UV), visible ( VIS) and infrared (IR) spectral range.

The substance for the intermediate layer can be selected to match the measurement problem so that no disturbing adsorption of the sample to be measured occurs and that it forms a non-toxic layer between the sample to be examined and the reflection element.

The method according to the invention finds particularly advantageous application for determining the concentration of biological substances, since the intermediate layer can always be selected in such a way that no disruptive adsorption occurs and that toxic effects are prevented.

In order for the intermediate layer to be as thin as possible and yet to a large extent uniform in its thickness, it is advisable to use a liquid substance for its production. This liquid intermediate layer can also be easily removed after the measurement has been completed, for example by wiping or washing off. To produce the intermediate layer, a thin film with an opening is advantageously placed on the reflection element in such a way that the reflection points lie in the region of the opening. The liquid substance is then introduced into this opening and evenly distributed therein. However, a thin slide can also be used as the interchangeable intermediate layer, which consists of a material that refracts for the measuring radiation as strongly as or stronger than the reflection element and is arranged in optical contact with the reflection element by means of a suitable immersion liquid. Such an intermediate layer consisting of solid material has the advantage that it forms an even stronger barrier than a liquid between the reflection element and the sample, since diffusion processes in this intermediate layer practically do not take place at the temperatures in question.

Such a specimen slide can also be provided on its surface facing the sample with a, preferably vapor-deposited or chemically applied, coating made of material that is highly refractive for the measuring radiation. This makes it possible to obtain different interfaces between the sample and the intermediate layer on the one hand and the reflection element or the immersion liquid and the intermediate layer on the other hand, that is to say these interfaces with an overall one-piece intermediate layer with different requirements of the sample on the one hand and of the reflection element on the other hand.

Further features of the invention are specified in the subclaims.

The invention is explained below with reference to Figures 1 to 4 of the accompanying drawings; show it:

1 shows the representation of a measuring arrangement for carrying out a spectroscopy of the total internal reflection according to the prior art;

2 shows a first exemplary embodiment of an arrangement for carrying out the method according to the invention with a liquid substance as the intermediate layer;

3 shows another embodiment with a solid substance as an intermediate layer;

OMPI Fig. 4 shows another embodiment with a surface-coated solid substance as an intermediate layer.

In the figures of the drawing, a representation of the boundary area between the reflection element and the sample at the point at which the total reflection takes place is shown at the top right in each case a greatly enlarged representation compared to the rest of the drawing scale.

Furthermore, the optical refractive indices n relating to the spectral range of the measuring beam for the various media through which the measuring light beam used for the spectroscopy passes from the entry into the reflection element to the exit from it are indicated in the drawing. The indices correspond to the reference numbers with which the respective element to which the refractive index provided with this index is assigned.

For a better understanding of the invention, a conventional measuring arrangement for carrying out a spectroscopy of the total internal reflection according to the prior art will first be briefly explained with reference to FIG. 1. This measuring arrangement has a light source 1, which is preferably designed as a laser and which generates a light bundle 2 that is as well collimated as possible. This light beam 2 passes through the entrance surface 3 of a reflection element 4 and is then totally reflected with a suitable choice of the angle of incidence θ inside the reflection element 4 at the interface 5 between this reflection element and a sample 6 provided thereon, the number of reflections depending on the geometry of the Reflection element depends and should be adapted to the particular measurement problem. The selection of a suitable form of the reflection element 4 is carried out according to criteria known per se, as described, for example, in the book "Internal Reflection Spectros copy" by NJ Harrick, published by the publisher John Wiey & Son Inc., New York 1967 is. The transverse attenuated light wave which occurs during the total reflection of the light bundle 2 in the optically thinner medium, ie the sample 6, is influenced by the sample substance. The spectral composition of the incident light beam 2 thus changes speaking of the absorption properties of the sample 6, so that a light bundle 7 is obtained when exiting the reflection element 4, the spectral composition of which is characteristic of the sample 6 in comparison to the light bundle 2. This change is determined as the absorption spectrum by means of the spectral apparatus 8 and the detector 9, an amplifier 10 is fed from there and is recorded, for example, by means of a recorder 11.

In the exemplary embodiment in FIG. 2, just as in FIGS. 3 and 4, the light source 1, the spectral apparatus 8, the detector 9, the amplifier 10 and the recording device 11 have been omitted for the sake of a simplified illustration.

In the exemplary embodiment in FIG. 2, a liquid intermediate layer 12 is provided between the reflection element 4 and the sample 6. In order to obtain a uniform and thin liquid intermediate layer, a mask 13, which consists of a thin film and a central opening 13 'provided therein, is first placed on the reflection element 4 as an aid. A small amount of liquid substance which is highly refractive for the measuring radiation is introduced into the opening 13 *, for example by means of a burette, with a glass rod, by dripping or the like, and the like

Excess liquid substance, which protrudes to a certain extent beyond the upper edge of the opening 13 ', or the like with a knife, a spatula. wiped off, so that a liquid intermediate layer 12 is obtained which has the same thickness as the film 13. In this way, by using very thin foils 13, it is possible to achieve extremely thin layer thicknesses of the liquid intermediate layer 12 with a uniform thickness of this intermediate layer, as is desirable. Finally, the sample 6 is layered over the liquid intermediate layer 12 (in the lower left part of FIG. 2 the sample is not shown but only a top view of the reflection element 4 and the film 13 with the opening 13 'is shown), specifically so that the sample 6 does not go beyond the outer edge 14 of the film 13. This outer edge 14 can be designed to be higher than the other surface of the film 13 in order to reduce the risk that part of the sample runs beyond this edge and can reach the surface of the reflection element 4 directly.

O PI The liquid 12 must be such that it does not mix with the sample substance 6 and, of course, does not enter into an interfering connection with it. For the measurement in the UV and VIS spectral range, 12 immersion oils such as Br, (CH) or C, H ₇ Br or polymers such as polyvinyl carbazole and also resins are suitable as the high-refractive liquid.

In the infrared spectral range up to 7.5 u, polytrifluoroethylene oil, in particular, can be used as the liquid substance for the intermediate layer 12

Permeability in the range of 2.5 to 7.5 u lies between 90 and 98%

20 and that has a refractive index n of 1.94. A further liquid substance for the intermediate layer 12, which is suitable for use in the infrared spectral range from 1 to 3.3 JU and 3.9 to 6.5 µ and 7.5 to 40 JU, is paraffin oil, the optical permeability of which is in the wavelength ranges mentioned is essentially above 90% and its refraction

The number n _{β is} between 1.480 to 1.484.

After the spectroscopic measurement has been carried out, the film 13 is removed and the liquid intermediate layer 12 is wiped and / or washed off by the reflection element 4. Then in the Wei described above, a new liquid intermediate layer 12 is brought up by means of a new film 13 and in this way the next sample 6 is prepared for a spectroscopic examination.

3 shows an exemplary embodiment in which a slide 15 made of solid material is arranged as an intermediate layer between the sample 6 and the reflection element 4. An immersion liquid 16 is provided to establish good optical contact between the reflection element 4 and the object carrier 15.

If the only important thing is to avoid surface contamination, but in the special case there is no other incompatibility between the material of the reflection element 4 and the substance of the sample 6, the slide 15 can be made of the same material as the reflection element. However, the slide 15 can also consist of another suitable material which has a high refractive index in the spectral range of the measurement radiation. For the measurement in the UV and VIS spectral range, the slide 15 can be made of glass or a polymer, for example. For the measurement in the IR spectral range, the slide 15 can be made of germanium, silicon or selenium, or also particularly expediently from the material commercially available under the name "chalcogenide glass", which is characterized by a high transmission and a refractive index nft, 5 in the range 4 to 13 JJ and which is resistant to water and weak acids.

In the embodiment shown in FIG. 4, the fixed slide 15 is provided on its surface facing the sample 6 with a coating 17, which is applied to the slide 15, for example, by vapor deposition or by chemical means. This coating makes it possible, inter alia, to achieve an even higher refractive index at the interface between the sample 6 and the specimen slide 15 or, if the material of the specimen slide 15 is relatively free to choose, a material which is particularly suitable for the particular type of sample substance 6 currently used to provide the interface between the sample 6 and the slide 15.

In addition to those already mentioned, the following substances can also be provided in the IR spectral range as object carrier 15 or coating 17: aN0 ₃ , calcite, quartz, LiF, MgF ^, CaF, SrF, PbF ₂ , T-12, NaF, BaF ₂ , KF, CsF, NaCl, NaBr, KCl, NaJ, KBr, CsCL, AgCl, TICl, KRS-6, AgBr, KJ, TIBr, CsBr, KRS-5, CsJ. It is also possible to use a polymer which is transparent and highly refractive to infrared radiation as the object carrier 15 or as the coating 17, a copolymer of polyethylene and polypropylene being suitable as a polymer, in particular preferably one with a ratio of 97, for the fingerprint area : 3 between polyethylene and polypropylene.

It is important that in all cases of the exemplary embodiments described above, between the optical refractive indices n, the reflection element 4, n, the sample 6, r .. ~, the liquid intermediate layer 12, n .., - of the slide 15, -., the immersion liquid 16 and n → - the coating 17 of the slide 15, the relationships are observed, which are shown in FIGS. 2 to 4.

OMPI IPO - 8th -

In addition to achieving a regenerable surface, the described method enables the prevention of undesired adsorption on the surface of the reflection element and, in addition to the prevention of an undesirable influence of the reflection element on the sample owing to its toxicity and / or water solubility, with a suitable selection of the intermediate layer, the achievement of an increased detection sensitivity by using the intermediate layer material in this way selects that certain substances from the sample are adsorbed on this intermediate layer.

Claims

1. A method for measuring the concentration by means of spectroscopy of the total internal reflection, characterized in that a transparent, highly refractive substance is used as a replaceable intermediate layer between the reflection element and the sample for regenerating an optically clean surface of the reflection element.

10

2. The method according to claim 1, characterized in that a liquid, high refractive index for the measuring radiation is used as an intermediate layer (12) for regenerating the optically clean surface.

15 3. The method according to claim 2, characterized in that to obtain a uniform, thin, liquid intermediate layer (12), a thin film (13) with an opening (13 *) is used, so on the reflection element (4) it is imposed that the refl ection points lie in the area of the opening (13 ') and that the substance for the formation of the

20 liquid intermediate layer (12) is introduced into this opening (13 ') and is evenly distributed therein.

4. The method according to claim 1, characterized in that a thin slide (15) is used as interchangeable intermediate layer, the _-5 from a for the measuring radiation as strong as or stronger than that

Reflective element (4) consists of refractive material and is arranged in optical contact with the reflective element (4) via an immersion liquid (16).

5. The method according to claim 4, characterized in that the surface of the specimen slide (15) facing the sample (6) is provided with a coating (17) made of a material which is highly refractive for the measuring radiation.

_OMPI

6. The method according to any one of claims 1 to 5, characterized in that the interchangeable intermediate layer (12, 15) or the coating (17) is renewed for each measurement.

7. The method according to any one of claims 1 to 5, characterized in that the measurement is carried out in the ultraviolet spectral range and the intermediate layer is formed from a substance which is highly refractive in this range.

8. The method according to any one of claims 1 to 5, characterized in that the measurement is carried out in the visible spectral range and the intermediate layer is formed from a substance which is highly refractive in this range.

9. The method according to any one of claims 1 to 5, characterized in that the measurement is carried out in the infrared spectral range and the intermediate layer is formed from a substance which is highly refractive in this range.

10. The method according to any one of claims 7 to 9, characterized in that the intermediate layer is formed from a plastic.

11. The method according to any one of claims 1-10, characterized by its application for determining the concentration of biological substances.

12. The method according to any one of claims 1-10, characterized by its application to measurement tasks in the context of clinical chemistry.