DE10348958B4 - Method for determining the temperature of aqueous liquids by optical means - Google Patents

Abstract

Method for determining the temperature of aqueous liquids in analysis vessels, in particular in multi-cuvettes and microplates, by optical means, in which the absorbances of the analysis vessel containing the solution to be examined at at least two wavelengths λ from the group of positive temperature-dependent absorbances of water (A ⁺ _λa ) , from the group of negative temperature-dependent absorbances of water (A ^- _λb ) and temperature-independent absorbances (A ⁰ _{λ c} ) are measured, from at least two of these Absorbanzen a signal size (S) is formed and from the thermal dependence of this signal size (S) by comparison with reference values as the calibration function, the temperature of the liquid to be examined is determined, characterized in that the signal size (S) as a quotient of at least one of arbitrarily selected spectral ranges positive temperature-dependent absorbance of water (A ⁺ _λa ) and at least one of arbitrary g selected spectral _regions negative temperature-dependent absorbance of water (A ^- _λb ) is formed.

Description

The The invention relates to a method and a device for determination the temperature of aqueous liquids in analytical vessels, preferably as multi cuvettes or are designed as microplates or microtiter plates. A Such detection for temperature control and for any eventual Control or regulation (tracking) of Temperature are in practice for the precision and Reproducibility of analytical investigations important.

Nearly All chemical and biological-chemical reactions are temperature-dependent. This dependence becomes known by the speed of molecular motion and the temperature-dependent changes the molecular conformation and / or the pH and / or the diffusion time.

Become chemical processes or reactions used for the analysis of substances, the type and / or the speed of product formation depends on which component is used and at which temperature the reaction takes place.

In In practice, the fluids to be examined are often in Reaction or sample vessels before. For analysis, either parts of the liquid mixtures are removed and in separate analysis vessels a fed offline analysis, or the investigations are advantageously also directly in the Reaction or sample vessels based suitable physical parameters performed online Reaction vessels can container of any size, cuvettes of any size Size or a plurality of containers or cuvettes be arranged in a specific grid. In the last Years are becoming increasingly Multiküvetten in the form of microplates or microtiter plates different Kavitätsdichte and configuration for Numerous analytical tasks in biology, in medicine and in environmental analysis to ultra-high throughput screening (UHTS) and used in combinatorial chemistry.

physical For example, parameters that are analyzed off-line or online are optical signals, such as absorbance, fluorescence intensity, luminescence, time-resolved Fluorescence, fluorescence polarization, light scattering, as well radioactivity and conductivity.

Various questions, such as kinetic enzyme analysis, kinetic analysis of cell function parameters (eg Kournikakis, J .: Bioluminescence Chemiluminesc., 10, 1995, 63-67, Ciapetti, J .: Biomed., Mater. Res. 41, 1998 , 455-460, Krause: Platelets 12, 2001, 423-430), the target search by temperature-dependent parameters, such as the de-and renaturation behavior of target ligand complexes, binding affinities of ligands, co-crystallization, etc. (z. B. WO 97/42500 ; US 6,036,920 ; Pantoliano, J .: Biomol. Screen. 6, 2001, 429-440) and Red-Ox systems (Pascual, C. Luminescence 14 (2) 1999, 83-89) presupposes the exact and rapid temperature adjustment in each reaction solution of all cavities shortly before and during the kinetic measurement process , For PCR techniques, a fast periodically alternating temperature setting is also required. The reaction solutions of a series examined in all cavities should have the same time and exactly the same temperature as possible.

For temperature measurement and control arise numerous problems, especially for multi cuvettes and Microplates.

The Temperature must be in a variable variety of liquid samples be measured at the same time. Experience has shown that the temperature is despite uniform energy input not in all reaction solutions one plurality of reaction vessels, such as Microplates, same. This results from different disturbing influences the environment of the microplate, such. B. drift, evaporation, cooling or heating by sources of interference in measuring instruments. Would be advantageous it, eliminating these said disturbing influences to be able to or to compensate by different position-dependent readjustment.

in principle temperature can be controlled by thermocouples, thermistors, ohmmeters, infrared radiation detectors, Bimetallic devices, extensometers and State change measuring devices detected become. For measuring temperatures in the range of 20 ° C to 95 ° C in liquids with volumes of ≤ 250 μl are convenient however, only thermocouples applicable.

to Temperature measurement in a multiple number of vessels (cavities) divide however, also thermistors or other thermo-probes, since the measurement technically with increasing cavity density very expensive, practically not at the same time as the optical Measurement (www.inheco.com) and for the fluids contained in the cavities can not be carried out without influencing or entrainment can. Convenient stand for the ultramicroliter range (<10 μl) suitable thermocouples with negligible own heat capacity and heat dissipation not available. Furthermore is the temperature measured in individual cavities of the localization of the probe (edge, surface).

Method, which determine the temperature of the liquids by means of their Use infrared radiation, naturally only detect the surface temperature, the measurement is influenced by numerous parameters and the thus determined surface temperature therefore from the actual mean temperature in the liquid may differ significantly. These methods are in the IR spectral range Range of ≥ 3000 nm (thermo-IR) also consuming and not integrable in routine measuring instruments.

The enormous technical effort that must be made in order to be able to reliably determine calorimetric temperature statements using a plurality of sample solutions in the arrangement and in the volume range of microplates with high cavity density by means of thermo-IR measurements is known (for example, US Pat. US 20020098592 and US 20020098593 ).

In DE 41 30 584 and DE 199 28 056 describes methods and test kits, which succeed by the use of thermochromic indicator systems for absorbance and fluorescence measurement to characterize the temperature behavior of the temperature control unit of an optical measuring device for microplates. A disadvantage of these methods is that the indicator systems can not be used for online measurement during the actual determination of an analyte with the aid of the measuring device, since the basis of the temperature measurement is a pH change. A combination of indicator and analyte solution can therefore not be used for temperature measurement and control.

Due to the inadequate possibility for the correct simultaneous measurement of the temperatures in all individual cavities, the characterization of the temperature behavior in all sample liquids is also unsatisfactory for device manufacturers and scarcely sufficiently documented. In most cases, only the temperature parameters of the radiator or the heating plate, of the space surrounding the reaction vessels or of individual selected cavities are recorded and indicated (for example US 6,036,920 . US 5,459,300 . US 2002/0070208 ).

However, the actual temperature behavior of the sample liquids in the individual cavities is dependent on numerous parameters to varying degrees even with homogeneous energy input, as already described above. Examples include the current ambient temperature in the meter, type and volume of sample liquids, distance and contact surface of the respective cavity to the hot plate, material and material thickness of the microplate, called geometry and localization of the cavities on the microplate. Numerous solutions are known which are intended to minimize these interfering factors, such as the microplate recording in the measuring device, the thermal insulation of the microplates themselves (eg. US 5,459,300 ; US 2002/0070208 ) or their individual cavities and the evaporation protection.

The Most modern microplate readers today allow the versatile optical measurement of almost all commercially available microplate formats (6-1536 wells). However, there is still no satisfactory method that is suitable for multi-cuvettes or Microplates of any geometry and material a uniform temperature behavior of the liquids in the individual cavities within required limits, z. B. ± 0.1 K (IFCC, International Society for Clinical Chemistry, Eur. J. Clin. Chem. Clin. Biochem. 31, 1992, 901). It would be accordingly advantageous in analytical practice when the temperatures of the in the cavities samples individually and optionally individually could be corrected.

It There are numerous analytes that are temperature-dependent in aqueous solutions change optical properties. As examples of these are often used for optical test purposes Analytes, such as NADH and flavin nucleotides, called. The dependence However, the absorbances of the temperature is at usual Analyte concentrations and at usual wavelengths extremely low and therefore not usable for temperature measurement (eg Malcolm: Meth. Enzymol. 66, 1980, 8-11; Miller: Meth. Enzymol. 66, 1980, 350-360). In addition, the analyte concentration may of course change according to the analytical task, so that the signal height the temperature dependence by the concentration dependence falsified becomes.

One method developed for PCR techniques uses the temperature dependence of the fluorescence intensity of a primer nucleotide contained in the sample to feedback the heating of samples to specially designed microstructured supports in the range of 60 ° C to 95 ° C with this coupled fluorescent dye ( US 20020090641 ).

In addition, there is an almost unmanageable number of pH indicators that, as dyes and fluorescers, change their optical properties in a pH-dependent manner (eg Practical Handbook of Biochemistry and Molecular Biology, Fasman, GD, Ed., CRC Press, Boca Raton , 1990, 561-563). In combination with temperature-dependent changes in the pH, such indicators can in principle be used for temperature measurement. Disadvantage of these solutions (see also DE 4 130 584 and DE 199 28 056 ) is that such indicator buffer systems are not without problems compatible with all analytical issues.

One Analyte present in virtually all biological reactions and analytical Tests by nature as a solvent in highest and practically constant concentration (≥ 50 mol / L) is water. water absorbs only slightly in the visible spectral range. It has different strengths in the near and middle infrared range pronounced and with the wavelength growing absorbance maxima. Many of these maxima are for a variety of analytical Issues used.

The Absorbance maxima ≥ 1600 nm become known used for moisture determination of various commercial products (e.g., Rader, J .: Assoc., Off., Anal. Chem., 50, 1967, 701-703; Stokvold, J .: Pharmaceutical Biomed. Anal. 28, 2002, 867-873). Typically the absorbances at λ> 1000 nm for detection of water and its quantification in the meteriology and interstellar research (for example, www.eos.ubc.ca/research/moc2/workshop_2002).

Water is known to have multiple, smaller near infrared (NIR) maxima. There are absorbance maxima around 960 nm, 1150 nm, 1390 nm and 1900 nm. The dependence of the absorbance on the temperature and the state of matter of the water at selected wavelengths or wavenumbers in the entire range from 600 nm to ≥ 1900 nm is well known ( US 6,320,662 ; Angell, J .: Chem. Phys. 80, 1984, 6245-6252; www.dartmouth.edu/~etransfer/water.htm). Nevertheless, for example, the wavelength range around 980 nm is used for layer thickness correction in vertical photometers ( US 6,320,662 ). This is not measured at the wavelength of the highest absorption (962 nm), which is temperature-dependent, but in the isosbestischen range between 988 nm and 1008 nm at the Absorbanzflanke.

In the spectral ranges z. B. around 1020 nm, 1250 nm, and 1650 nm Absorbance minima, but also a weak temperature dependence show the absorbances.

The absolute absorbances or absorbance constants of all maxima and Minima grow strongly with the wavelength up to λ = 3 μm (Hale, G.H. et al .: Applied Optics 12, 1973, 555-563). That is why a reliable one Measurement of NIR absorbances (0.50-2.0 OD) in each of the temperature-dependent spectral regions only with respectively defined irradiated water layers, d. H. Layer thickness range possible.

However, the determination of the layer thickness is not always exactly possible, such. B. in microtiter plates in which is analyzed by vertical photometry. Among other things, it depends on the precision of the dosing device, microplate intolerances and meniscus formations. However, the layer thickness determination can be carried out with the aid of the absorbance of water ( US 6,320,662 ). It should also be noted that polymers used for the preparation of the analysis vessels, for example microplates or microtiter plates, such as currently used polystyrene, polycarbonate, polyethylene and polypropylene, also absorb light in the NIR and IR range from about 1600 nm ,

To the Water-proofing and reliable layer thickness determination are more obvious Only isosbestic wavelength ranges usable. Temperature determination by absorbance measurement in one of the temperature-dependent Wavelength ranges is only possible with known and constant layer thickness, but due to the small changes Insensible per degree.

By forming quotients of absorbances in a wavelength range in which the absorbance increases in temperature (A ⁺ ), and absorbances in a wavelength range in which the absorbance temperature dependent (A ^- ), however, the temperature-dependent signal is significantly amplified and independent of the layer thickness. In particular for microtiter plates, a correction of the blank value (A ⁰ ), which is caused by the absorbance of the microtiter plate itself, is often necessary for the precision improvement of the analytical signal. For this purpose, the absorbance is measured in a wavelength range that does not affect the analytical wavelengths. By subtracting the blank value A ⁰ of, for example, two temperature-dependent optical signals (A ⁺ and A ^- ), the correction is performed.

There is known a device for analyzing a medical sample ( DE 42 03 202 A1 ), in which the optical absorbances of a test liquid in a cuvette are measured at at least two wavelengths (1405 nm and 1534 nm) in the NIR range and the temperature of the test liquid is determined by comparison with reference values (calibration data set) determined in a calibration step. Already in the DE 42 03 202 A1 was pointed to the problems with unavoidable temperature gradients between the environment of the reaction vessels and the reaction vessels themselves, which lead to significant deviations of the actual temperature in the analysis vessels of setpoints or determined values. These problems become all the greater, the more complicated the vessel shapes (different from plane-parallel vessel walls of the optical cuvette) are. Microplates and multi-cuvettes are used to hold and treat a large number of small sample volumes and by no means have plane-parallel, optically delimiting vessel walls. Differences in the thickness of the optically effective and in Their temperature to be determined layer, which are irradiated vertically unlike in optical cuvettes affect due to the small layer thickness itself, due to tolerances in shape and dimensioning of the analysis vessel and by metering inaccuracies, surface tension and randomness of the also temperature-dependent meniscus formation immensely on the accuracy the optical measurement, even if the measurement results are not absolute, but also used in a known manner under difference for the evaluation. That in the DE 42 03 202 A1 described method is therefore not suitable for universal applicability, especially in small vessels, such as microplates and multi-cuvettes.

Of the The invention is therefore based on the object, the temperature of aqueous liquids universally applicable and as possible aufwandgering, each fast, accurate and sensitive, especially for a thermal Control and / or correction. The temperature detection should also be for a variety of sample liquids, which are in the cavities, for example, multi-cuvettes and microplates are located, with reasonable effort for each cavity immediately before or during be possible for the analytical sample examination. Neither should the liquids or parts thereof or their analytical investigations by the Temperature determination impaired or disabled.

To solve this problem, in a method for determining the temperature of aqueous liquids in analysis vessels, especially in multi-cuvettes and microplates, with optical means, wherein the absorbances of the analysis vessel containing the solution to be examined at at least two wavelengths λ from the group of positively temperature-dependent absorbances from water (A ⁺ _λa ), from the group of negative temperature ^- dependent absorbances of water (A ^- _λb ) and temperature ^- independent absorbances (A ⁰ _λc ) are measured, from at least two of these absorbances a signal size (S) is formed and from the thermal dependence of these Signal size (S) is determined by comparison with reference values as a calibration function, the temperature of the liquid to be examined, according to the invention the signal size (S) as a quotient of at least one arbitrarily selected spectral ranges positive temperature-dependent absorbance of water (A ⁺ _λa ) _sowi e at least one of arbitrarily selected spectral ranges negative temperature-dependent absorbance of water (A ^- _λb ) is formed.

By the Qutientenbildung from absorbances in a wavelength range in which the absorbance temperature-dependent _growth (A ⁺ _λa ) and absorbances in a wavelength range in which the absorbance temperature dependent falls (A ^- _{λ b} ), the temperature-dependent signal is on the one hand significantly amplified and regardless of the often unknown or incorrectly determined layer thickness. In this case, the accuracy of the temperature determination can be further increased if, of the said absorbances of the at least two monochromatic light beams of different wavelengths before the quotient formation, the absorbances of non-water portions of the vessel containing the liquid, such as. B. from the vessel wall forming polymers and / or dissolved in the liquid analyte (A ⁰ _λc ) are subtracted (difference quotient).

to execution of the method is preferably a photometric measurement and Evaluation device, consisting of a radiation source for generating the at least two monochromatic light beams different Wavelength, optical Elements for guiding the light rays through the vessels with the fluids to be examined, at least one optical receiver for measuring the light intensities and a preferably computer-aided evaluation, in particular for the Determination of absorbances and signal sizes, for comparison with the reference values and for the Data output or display.

With The invention is an accurate determination of the average temperature of watery Liquids, For example, analyze, optically possible, which also in one multiple numbers (microplates, multi cuvettes, etc.) even smaller volume wells with their layer thickness related tolerances regarding meniscus, vessel shape and size as well Sample loading may be present. The liquids (Analyzer) do not need to be taken from the vessels for temperature determination too are not mechanically in contact with measuring equipment. The Danger of falsification and carryover of the liquids in vascular cavities not given.

The optical methods not only allow an accurate, sensitive and rapid temperature determination of the liquids in a variety of cavities with reasonable effort, but also allows the temperature determination or control thereof immediately before or during the analytical measurement of the material to be analyzed.

To For this purpose, the said photometric measuring and evaluation device advantageous in known per se photometer or microplate reader with the aim of simultaneous or simultaneous temperature detection for one Integrated precision analysis become.

Furthermore, it is possible to use the photometric measuring and evaluation device as part of a rule to form device for tracking or keeping the temperature constant. The subclaims contain expedient embodiments of the method according to the invention and the device for its implementation.

The Invention will be described below with reference to the drawing embodiments be explained in more detail.

It demonstrate:

1 : Temperature dependence of the absorbance spectra ( 1a ) and the absorbance difference spectra ( 1b ) of pure water;
Difference spectra: Difference in absorbance values at a given temperature from the absorbance values at 20.6 ° C

2 Tabulated list of parameters of linear calibration functions y = aT + b and polynomial calibration functions y = cT ² + dT + e corresponding to different signal quantities (S), like the absorbance values 1 as well as to selected absorbance quotients, cumulative quotients, product quotients and quotient quotients have been adjusted;
with the temperature increasing absorbance values at λa1: 962 nm, λa2: 1152 nm and λa3: 1385 nm;
temperature decreasing absorbance values at λb4: 1025 nm, λb5: 1236 nm and λb6: 1600 nm;
isosbestic absorbance values at λc7: 996 nm, λc8: 1099 nm and λc9: 1188 nm;
a _r = 100 * a / z, a _r : relative increase in the calibration line in the range from 20.5 ° C. to 78 ° C., z: value of the absorbance, the absorbance quotient, the cumulative quotient, the product quotient or the difference quotient at 20, 5 ° C;
R ² : correlation coefficient for the adjusted calibration function;
Sensitivity of temperature measurement = E = mean standard deviation of the differences in the temperatures and measured temperatures obtained from the calibration function recorded on the same day

3 : Deviation of the parameters of the linear calibration function y = aT + b of aqueous solutions of buffer substances, salts, solvents, sucrose, glycerol and Triton X-100 from the parameters of pure water

4a : Principle design for recording the temperature-dependent absorbances of the water content of sample liquids located in cavities of a microplate

4b : Dependence of the difference quotient on the measured temperature for two cavities of the microplate filled with the same sample volume

4c : Deviation of the temperature measured by thermosensor from the temperature that can be calculated from the linear calibration curve of all measuring points

4d : Temperature dependence of the simple quotient as well as different difference quotients for cavities of the microplate filled with different volumes

embodiment 1:

Temperature dependence of the absorbance of pure water:

In 1 is the dependence of the absorbance spectra ( 1a ) and the absorbance difference spectra ( 1b ) from the temperature in the range of about 20 ° C to 80 ° C. The spectra were recorded in glass cuvettes with a Cary 500 with thermostated cuvette holder at a layer thickness (d) of 3 mm.

The wavelength ranges of 950-990 nm, 1110-1170 nm and 1310-1410 nm show, as indicated by the arrows, a growing absorbance (A ⁺ _λa ) with temperature increase, the wavelength ranges of 1000-1090 nm, 1190-1300 nm and 1430-1700 nm show an absorbance _decrease (A ^- _λb ) with increasing temperature. The absorbances in the range of 996 ± 1 nm, 1099 ± 1 nm, 1188 ± 1 nm and 1304 ± 1 nm are temperature-independent (A ⁰ _{λ c} ) and represent so-called isosbestic points.

embodiment 2:

Signal quantities (S) as absorbances, absorbance quotients, Sum quotients, product quotients and difference quotients as Function of the temperature in glass cuvettes:

From the in 1a The absorbance _values A ⁺ _λa1 at 962 nm, A ⁺ _λa2 at 1152 nm, A ⁺ _{λa 3} at 1385 nm (increasing with temperature), A ^- _λb4 at 1025 nm, A ^- _λb5 at 1236 nm and A ^- _{λb6 were measured} at 1600 nm (falling with the temperature) and A ⁰ _λc7 at 996 nm, A ⁰ _λc8 at 1099 nm and A ⁰ _λc9 at 1188 nm (temperature-independent) extracted and the temperature dependence (as a mean increase a _r of each parameter based on the measuring point at 20.5 ° C) and the sensitivity E of the temperature measurement (as the standard deviation of the difference of the temperature calculated on the basis of the respective calibration function from the actually measured temperature). For comparison of the increases a _r , a linear function was in each case adapted to the signal variables in a good approximation (correlation coefficient R ² > 0.990), although significant systematic deviations from most straight lines are evident. To determine the sensitivity, the adaptation (linear or polynomial) which yields R ² > 0.999 was used.

2 shows the relative increases a _r , the correlation coefficients for the respective linear or polynomial calibration function and the found sensitivities for the temperature determination by means of absorbance, absorbance quotient, absorbance sum quotient, absorbance product quotient or absorbance difference quotient. Columns four and six in the table ( 2 ) Listen the found sensitivities E as standard deviations of the deviations of all Dm measured temperatures _T, (of the calculable based on the respective calibration function temperatures T _soll). The following applies: Δm = T is - T should ,

It can be seen that, compared to the simple absorbance values by the formation of quotients and sum quotients, the temperature dependence of the signal (a _r , column 3) increases by a factor of 2 to 6, and by the formation of product and difference quotients by a factor of 10 to reach 30. The sensitivity (E) of the temperature determination, recognizable by the smaller deviations of the calculated from the measured temperatures (columns 4 and 6) becomes depending on the adjustment function for quotients, sum and difference quotients against the calibration functions determined from the simple Absorbanzwerten up to 0.1 K improved. Although the product quotients show a higher relative increase a _r , no equally improved temperature sensitivity.

embodiment 3:

modification the calibration function parameter of the temperature dependence of the absorbance of Water in the presence of dissolved materials:

It the spectra of aqueous solutions recorded analogously to application example 1. In the water were defined Amounts of various buffer substances, salts, solvents, Sucrose, glycerol and Triton X-100 dissolved.

The absorbance _values A ⁺ _λa3 at 1385 nm and A ^- _λb6 at 1600 nm were extracted from the spectra and the temperature dependence of the absorbance _quotients (A ⁺ _λa3 / A ^- _λb6 ) was _adjusted to a linear function y = aT + b. The parameters of the calibration functions obtained are compared with those of pure water. As a comparison, the mean values of the parameters a and b for pure water, which had been determined in three independent measurements on three different days, were used.

In 3a It can be seen that all increases a of the calibration functions decrease in the presence of solutes compared to pure water (0). The extent of the decrease in the increase in commonly used physiological concentrations of 0.1 M buffers and low concentrations of ethanol, DMSO, glycerol, salts and the detergent Triton X-100 is only marginal. At 1 M buffer concentration, a significant deviation is observed.

In the 3b The change in the ordinate intersection shown in the table is different. There are aqueous solutions which show no significant deviations from pure water, such as 0.1 M buffer, 1% (0.171 M) ethanol and 1% (0.141 M) DMSO, but also solutions which cause a sharp reduction in the point of intersection, such as 1 M Buffer, 10% (1.71 M) ethanol and 10% (1.41 M) DMSO, but also increases in b are possible, as with 1 M NaCl and NaJ and in detergent-containing solutions.

These changes, which can not be explained solely by the decrease of the pure water concentration, but from a change the water structure can result suggest that for each used aqueous solution a separate temperature calibration must be made, as the calibration functions of water and water solutions in both parameters, a and b, can differ, causing significant deviations in the temperature correctness lead can.

embodiment 4:

modification the calibration function parameter of the temperature dependence of the absorbance of Water in the presence of dissolved analyte:

The Experiments as described in Application Example 3 were carried out with important analytes in biochemical analysis, such as NADH (0.5 mM) and fluorescein (40 μM) repeated. Both analytes do not show their own NIR absorbance ranges. It However, in both analytes, there is not one due to the low concentration change explainable Reduction of the increase in the absorbance quotient by approx. 4.5%, but no change in the additive member b.

embodiment 5:

precision and sensitivity of the temperature measurement by means of absorbance of Water in microplates (layer thickness d 3-4 mm):

In a simple photometer principle construction accordingly 4a the light absorbances of a thermostated microplate filled with water are recorded as a function of the temperature. The cavities of the microplate are through 96 in the microplate grid in a radiator 1 passively pressed single corrugations 2 realized. The temperature control of all individual wells 2 done by the radiator 1 and an upper heating 3 , Both the radiator 1 as well as the upper heating 3 be through a calculator 4 temperature-controlled. In the individual wells 2 there is an aqueous liquid 5 whose temperature in selected single wells 6 each through a thermistor 7 (∅ 0.04 mm) is recorded synchronously with the absorbance measurement. Light in the range of 800 nm (A ⁰ _λc10 ), 1350 nm (A ⁺ _λa3 ) and 1550 nm (A ^- _λb6 ) is _detected by optical filters 8th , which are preferably arranged in a filter wheel, not shown, from a continuous spectrum of a controlled tungsten lamp 9 generated and by means of fiber optic cable 10 over the selected single wells 6 guided. Several single waves 6 can be successively brought into the beam path and thus measured successively. Under the single waves 6 is a photodetector (photodiode) 11 arranged. The principle of the photometer is provided by an opaque housing 12 completed. For temperature evaluation in the selected individual wells 6 located liquid 5 are the thermistors with a computer 13 connected. To this computer 13 are also the photodetectors 11 connected to the evaluation of the radiation intensities detected by the same.

In 4b is the difference quotient (A ⁺ _λa3 - A ⁰ _λc10 ) / (A ^- _λb6 - A ⁰ _λc10 ) of two selected _ones each with 100 μl of the liquid 4 (Water) filled single wells 6 applied as a function of the measured temperature. The straight lines for both wells almost overlap.

4c shows the deviation of the measured from the determined from the associated linear equation temperatures using the example of Well a with the values 4b , On average, a deviation of ± 0.183 K was found in the temperature range of about 25-36 ° C and in the entire temperature range of ± 0.439 K investigated.

In 4d shows the temperature dependence of the simple quotient (A ⁺ _λa3 / A ^- _λb6 ) and the difference quotient for two wells filled with different volumes of a microplate. The differences in absorbance resulting from the unequal filling lead to different characteristics of the quotient for both wells, so that the characteristic obtained from one well can not be used to determine the temperature of any other well with a different filling volume due to the imprecision of the doser used. By the difference quotient formation with (A ⁰ _λc10 ), with λc10 = 800 nm as temperature-independent absorbance (corresponding 4b However, these differences are eliminated and obtained for both wells almost identical characteristics. Thus, when using the difference quotient using a characteristic curve determined for one well, the temperature in other wells can be determined with a deviation from the accuracy of only 0.323K. The mean deviation (analogous to 4c ) of the calculated temperature in the well with which the characteristic was determined, from this calibration curve is 0.249 K and from the other well from the same calibration curve at 0.251 K.

1

radiator

2, 6

Single Well

3

top heater

4, 13

computer

5

liquid

7

thermistor

8th

optical filter

9

tungsten lamp

10

optical cable

11

photodetector (Photodiode)

12

casing

Claims (17)

Method for determining the temperature of aqueous liquids in analysis vessels, in particular in multi-cuvettes and microplates, by optical means, in which the absorbances of the analysis vessel containing the solution to be examined at at least two wavelengths λ from the group of positive temperature-dependent absorbances of water (A ⁺ _λa ) , from the group of negative temperature-dependent absorbances of water (A ^- _λb ) and temperature-independent absorbances (A ⁰ _{λ c} ) are measured, from at least two of these Absorbanzen a signal size (S) is formed and from the thermal dependence of this signal size (S) by comparison With reference values as a calibration function, the temperature of the liquid to be examined is determined, characterized in that the signal size (S) as a quotient of at least one arbitrarily selected spectral ranges positive temperature-dependent absorbance of water (A ⁺ _λa ) and at least one of any ig selected spectral _regions negative temperature-dependent absorbance of water (A ^- _λb ) is formed. A method according to claim 1, characterized in that the signal size (S) is formed according to the formula S = A ⁺ _λa / A ^- _λb . A method according to claim 1, characterized in that the signal size (S) according to the formula S = (A ⁺ _λa - A ⁰ _λc ) / (A ^- _λb - A ⁰ _λc ) is formed. Method according to Claim 1, characterized in that the signal magnitude (S) is formed from the summation quotient (ΣA ⁺ _{λa (1-n)} ) / (ΣA ^- _{λb (1-n)} ). A method according to claim 1, characterized in that the signal magnitude (S) from the product _quotient (A ⁺ _{λa (1)} * A ⁺ _{λa (2)} * .... * A ⁺ _{λa (n)} ) / (A ^- _{λb ( 1)} * A ^- _{λb (2)} * .... * A ^- _{λb (n)} ). Method according to claim 1, characterized in that the reference values each before the temperature determination under the same physical conditions for the solution to be tested as Calibration function can be determined. A method according to claim 1, characterized in that the absorbances A ⁺ _λa in the wavelength range of 930 nm to 990 nm, 1130 nm to 1170 nm, 1340 nm to 1430 nm and 1800 nm to 1900 nm, the absorbances A ^- _λb in the wavelength range of 1010 nm to 1100 nm, 1190 nm to 1290 nm, 1445 nm to 1700 nm and 1950 nm to 2100 nm and the absorbances A ⁰ _λc in the wavelength range from 200 nm to 900 nm and at 996 ± 10 nm, 1099 ± 10 nm, 1188 ± 10 nm and 1304 ± 10 nm, respectively. A method according to claim 1, characterized in that the signal size (S) of liquids in vessels with layer thicknesses ≥ 10 mm, for example, cuvettes and containers, with absorbances (A ⁺ _λa ) in the wavelength range of 930 nm to 990 nm and with absorbances (A ^- _{λb ) is determined} in the range of 1010 nm to 1100 nm. A method according to claim 1, characterized in that the signal size (S) of liquids in vessels with layer thicknesses ≤ 1 mm, for example, cuvettes and microplates, with absorbances (A ⁺ _λa ) in the range of 1800 nm to 1900 nm and with absorbances (A ^- _{λ b} ) is determined in the range from 1950 nm to 2100 nm. A method according to claim 1, characterized in that the signal size (S) of liquids in vessels with layer thicknesses of 4.5 mm to 10 mm, for example, cuvettes and microplates, with absorbances (A ⁺ _{λ a} ) in the range of 1130 nm to 1170 nm and with absorbances (A ^- _λb ) ranging from 1190 nm to 1290 nm. A method according to claim 1, characterized in that the signal size (S) of liquids in vessels with layer thicknesses of 1.5 mm to 4.5 mm, for example microplates, with absorbances (A ⁺ _λa ) in the range of 1340 nm to 1400 nm and with absorbances (A ^- _λb ) in the range of 1445 nm to 1700 nm. Method according to claim 11, characterized in that the signal size (S) of liquids in vessels with layer thicknesses between 3 mm and 4 mm, for example microplates with 96 or 384 cavities, from absorbances (A ⁺ _λa ) in the range from 1350 nm to 1390 nm and from absorbances (A ^- _λb ) in the range of 1540 nm to 1600 nm. Method according to one or more of the preceding claims 1 to 12, characterized that the temperature determination coincides with an analytical investigation of liquids or performed immediately before becomes. Method according to one or more of the preceding claims 1 to 12, characterized that the temperature determination for the documentation of the analytical Examination of the fluids is used. Method according to one or more of the preceding claims 1 to 12, characterized that the temperature determination for temperature control or correction, for example, temperature control, for the analytical study of liquids is used. Method according to claim 14, characterized in that the temperature determination for determination of correction values for the analytical examination of the liquids is carried out. Method according to claim 14, characterized in that the results of the analytical examination of liquids be precisely specified by the temperature-dependent correction values.