DE19907448A1 - Flowing system for carrying out e.g. analysis reactions comprises variable speed positive displacement pumps preceding a mixing point and set flow resistance, enabling variable and steady state conditions to be set up - Google Patents

Abstract

A flowing system that feeds separate sample, dilution, conditioning and reagent solutions, through one or more channels (6) of a small cross section following a transition and mixing point (3), is new. A new flowing system that feeds separate sample, dilution, conditioning and reagent solutions, through one or more channels (6) of a small cross section following a transition and mixing point (3) has pumping speeds that are precisely controlled over time. In this way, the fluids are precisely dosed as a given volume, over a set residence time. The volume and time are a function of pumping speed and time. Dispersion-free mixing, maintains precise, steady state concentration ratios and -gradients. The resultant concentration-time curve of the sample, indicator or reaction product, is continuously recorded by a flow meter (7).

Description

The invention relates to a method for flow-modulated concentration analysis in Pro volumes of dissolution. The field of application of the invention is the wet chemical concentration tion analysis in process media of chemical and biotechnological material conversion processes, flu food, surface, ground and drinking water as well as various solvents systems.

Previously known methods for flow analysis of defined sample solution volumes are based either on the principle of continuous flow analysis in air segmented and solvent-segmented reagent flows (L. T. Skeggs, Am. J. Pathol. 28 (1957) 311.) or on the principle of flow injection analysis (J. Ruzicka and E. H. Hansen, Flow Injection Analysis, John Wiley & Sons, New York, 1988) and flow injection analysis derived sequential injection analysis (J. Ruzicka, G. D. Graham and G. D. Christian, Anal. Chem. 62 (1990) 1861 .; J. Ruzicka and G.D. Marshall, Anal. Chim. Acta 237 (1990) 329.). In the continuous flow analysis, the sample solution to be analyzed sucked out of the sample reservoir by a peristaltic pump and by air or with a segmented immiscible liquid in the sample solution. This method needs relative large sample solution volumes and long rinsing times between two analyzes. At the häu Segmentation by air cushions, which is more commonly used, affects gas compressibility the reproducibility of the analyzes. Both the air and the solvent segments tation require special arrangements for segmentation in the flow and mostly special arrangements for phase separation, which complicate the analyzer arrangements unnecessarily adorns and makes them susceptible to faults.

The flow injection analysis defines discrete, more or less precisely Volumes of a sample or reagent solution using an injection valve or hydrodynamic Injection injected into a non-segmented carrier stream, whereby the principle of based on Ruzicka and Hansen (J. Ruzicka and E. H. Hansen, Flow Injection Analysis, John Wiley & Sons, 1988) defined controlled dispersion reproducible concentration-time Form profiles, so-called peak profiles and continuously through a flow detector  be recorded. The disadvantages of using injection valves are mechanical wear, the risk of blocking and in the more or less predetermined Injection volume. The hydrodynamic injection is based on the intermittent and switching on at least two pumps, one being between two branches channel section located is reproducibly filled with sample or reagent solution, for which an additional closing valve is necessary for practical applications. The dosing and injection accuracy is often much worse than when using injection veins til. The recorded peak signals are determined by the concentration of the samples substance, diffusion, convection and residence time due to the viscosity of the sample solution and affects the heat of reaction of the determination reactions used. Flow injection Analysis procedures must therefore be considered very frequently and very carefully the sample matrix in a defined, often narrow concentration range of the sample solution be calibrated. This results from the fact that in the vast majority of applications transient, d. H. very often kinetically controlled measurement signals are recorded and evaluated become.

Sequential Injection Analysis (J. Ruzicka, G. D. Marshall and G. D. Christian, Anal. Chem. 62 (1990) 1861-1866, J. Ruzicka, G.D. Marshall, Anal. Chim. Acta 237 (1990) 329-343) or sequential flow analysis after sample injection are the sample that Reagent solution and possibly further solutions sequentially, by switching one Multi-way valve or by selecting different solution reservoirs using a hose pinch valves and a branch in a channel section upstream of a pump sequentially sucked in, arranged and by later, often multiple switching over Flow direction mixed reproducibly. In addition to the improved mixing results increased dispersion that reduces sensitivity. Sequential Injection Analysis Like flow injection analysis, it is based on the principle of controlled dispersion on the setting of a more or less high dispersion to achieve a sufficient appropriate mixture between analyte and reagent and can by Do not avoid dilution caused by dispersion.

A disadvantage of the previously known methods for concentration analysis is that the The upstream pump section can only be cleaned incompletely, i. H. can be rinsed out or the pump has to be contacted with the sample solution, as regards its applicability severely limits the selection of the analyzable sample solutions. Although the Vermi  conditions, d. H. e.g. B. the sample solution dilution is adjustable in wide ranges this is always due to the intensity of the mixing at the sometimes extreme volumes conditions that in turn can make efficient mixing very difficult or even exclude. The mixing ratios are also reproducibly adjustable however, it is very difficult to predict, which requires a high calibration effort. At all FIA and SIA methods and derived methods depend on the dispersion, the dilution tion, the mixing and thus the analysis results of the viscosity of sample, Carrier and reagent solution. Even when using low-calibration and particularly sig nal stable flow detectors must often and taking into account the pro benmatrix can be calibrated. The sample solution injection is the same as for liquid chroma topography a displacement injection, the subsequent mixing with the reagent solution solution to avoid annoying blind peaks or other artifacts.

It is the case with reaction kinetic examinations and often required higher accuracy often necessary to enter stationary concentrations with high precision and sufficiently quickly put. From the concentrations of standard or reagent solutions used and the flow rates set in their dosage can be the output concentrations can be calculated in a simple manner. With slow determination reactions, it is often necessary, in the case of precisely defined concentration ratios Reaction or dwell times can be set precisely over a wide range. With convention stopped-flow arrangements (Q. H. Gibson, in: K. Kustin (Ed.), S. P. Colowick, N. O. Kaplan (Series Eds.), Methods in Enzymology, Vol. 16, 1969, 187-228) it is necessary nimble to flush the pistons of the precision pumps used before each experiment clean and refill, which is a lot of manual work and often too high Consumption of reagents and sample solutions means.

So far, no measuring arrangements are known in which discrete and small sample volumes, preferably less than 200 ul regardless of the dispersion, d. H. dispersion-free, of dif fusive mass transfer, of viscosity gradients between the sample solutions and others Solutions as well as possibly independent of the temperature can be analyzed. Especially for the automatic calibration of e.g. B. running in flow channels Ana lysing and measurements would be beneficial by simply programming one computer-controlled timing standard solutions with constant concentration in to be generated in situ from a stock solution, calibrated and immediately afterwards for analysis of the sample solutions.  

The invention is therefore based on the object of a method for precise concentration tion analysis in discrete sample solution volumes in flow channels, the un depending on the amount caused by diffusion, convection and laminar flow profiles gentle dispersion, of viscosity and temperature differences and the precise setting of dwell times, concentrations and concentration gradients and optionally of sta tional and transient concentration-time functions.

This object is achieved according to the invention by a method with the features of the claim 1 solved.

With this method for flow-controlled concentration analysis in sample solution volume mina become in one or more flow channels with at least one transition a smaller flow area only through precise timing of Pumping speeds of previously separate sample, dilution, conditioning and reagent solutions in a wide, by the pump speeds and the pump dosed volume range with high accuracy as well as residence times and through dispersion-free mixing concentrations, concentration ratios and concentration served precisely and stationary and the concentration-time courses of the samples substance, an indicator substance or a reaction product continuously, preferred recorded by a flow detector.

The method can be carried out in different ways. So it is possible that in a simple flow channel system with a transition to smaller flow cross-sections with a continuous flow of reagent, conditioning or dilution solution, a pump transporting the sample solution is switched on for a defined time and the pump speed V _{R, or} one of the other solutions transporting pump is reduced by the amount of the flow rate of the sample solution V _S to V _R , so that a constantly flowing volume defined by the switch-on time of the sample solution pump and the selected flow rate V _S causes a zone of stationary concentration ratios which is sufficiently long for the stable detection. A subsequent flow channel section with a sufficiently reduced flow cross section ensures that even with small sample solution volumes metered in, a flow zone which is unaffected by the dispersion, ie by convection, laminar flow profile formation and diffusion, is formed with preferably constant or stationary concentration ratios. In the absence of a material turnover, there is a constant concentration ratio. This enables the adjustment of the measuring range of a subsequent flow detector to the sample substance concentration to be determined.

With a mass conversion, for example between the sample substance S and the reagent R in accordance with the reaction (1), this determines the steady-state concentration ratios depending on the residence time, ie the longitudinal coordinate of the flow channel.

aS + bR → reaction products (1)

If a reaction product P is detected, the sample substance concentration is calculated [S] from the measured concentration [P], the stoichiometry of reaction (1) and the reac tion conversion U, which in turn depends on the reaction rate. Are the samples- [S] and the reagent concentration [R] known can be obtained from the at different dwellings times measured product concentrations derived speed laws and Ge velocity constants are calculated, none due to diffusion and / or convection tion caused or influenced secondarily by temperature and viscosity differences Dispersion must be taken into account.

With complete conversion of the sample substance, constant concentration ratios again result, from which, based on the stoichiometry of the determination reaction with the stoichiometric factors a and b and the reagent consumption [R] _o U, which is the result of multiplication of the starting reagent concentration [R] _o with the degree of conversion U des Reagent results, the analyte concentration to be determined can be calculated according to equation (2).

[S] = a / bU [R] _o .V _R / V _S (2)

The flow detector measures the resulting concentration [R] _{t of} the reagent or the concentration of the reaction product [P] via a calibration function of the form (3) with the increase m and the blank value h _o , where h is the respective measured variable.

h = mf ([P]) + h _o (3)

It is also possible to stop the pumps if after the together menfluss Y has set a steady concentration-time course in the flow channel, what  a practically unlimited extension of the dwell time without diffusion, Convection and thermal convection on the one at a spatial coordinate of the flow channel imposed concentration.

In order to ensure that the standing sample solution is mixed with the measuring errors caused by flowing reagent solution, it is possible that on Confluence between two solutions, one on the receiving end and one on the dosing side free changeover valve the solution to be mixed from the receiving solution separates and for admixing with the receiving solution for a defined time connects.

In addition, it is possible that two pumps are controlled so that they are right Form corner-shaped pumping speed-time profiles, which when mixing through the pumps transported solutions of two or more non-reacting with each other Substances in a section of the developing concentration profile to constant Concentration ratios between the substances lead.

It is also possible to use more than two pumps to save the sample solution several other solutions containing reagents or buffers in one on one mix the following confluence flow channel so that stationary Form concentration-time courses, which subsequently result from chemical conversion change tongues.

With several mixing points connected in series and immediately following connected flow channels, it is possible to move the sample solution in succession dene reagent, indicator, conditioning and / or standard solutions to mix z. B. to implement several sequential determination and derivatization reactions.

The method will be explained in more detail below with the help of the drawings. Show it:

Fig. 1a-d a process for durchflußgesteuerten Analysis of concentrations of ethanol, based on

• - a - a flow analysis arrangement with inclusion of discrete Sample solution volumes,
• - b - a timing diagram,
• - c - a pump speed-time profile and of
• - d - concentration-time courses for one to complete Analyte turnover ongoing determination reaction.

Fig. 2a, a process for amperometric analysis of thioglycolate b based on

• - a - a flow analysis arrangement and
• - b - a timing diagram

Fig. 3a, a method for analysis of sucrose concentrations durchflußgesteuerten b based on

• - a - a flow analysis arrangement and
• - b - a timing diagram.

Fig. 4 based on a method for flow-controlled enzymatic determination of glutamine

• - a - a flow analysis arrangement and
• - b - a timing diagram.

In Fig. 1a a Durchflußanalysenanordnung is shown, the two computer-controlled precision pumps 1 and 2, a confluence 3, a change-over valve 4, a flow channel designed as a knotted flow pipe 6 with a reduced inner cross-section and adapted to this cross section, the photometric flow detector 7 has. The transition 5 to the smaller, through-flow cross section is located immediately after the changeover valve 4 . First, a sample solution volume V _S defined by the product of the pump speed V _{P1 of} the pump 1 and the suction time t _{DOS is} transported into the sample feed channel 8 via the changeover valve 4 . After switching from pump 1 to the other pumping direction and valve 4 and switching on pump 2 , the flow rates V _P1 and V _{P2 are} set. In this way, the ethanol-containing sample solution and serving as a reagent solution sulfuric dichromate solution are pressed simultaneously in the ratio of their flow rates V _P1 / V _P2 into the flow channel 6 and pumped to the flow detector 7 . Distilled water is used as the displacement solution. The flow detector records the entire concentration profile of the Cr (III) ions formed in accordance with the determination reaction (4) at an absorption wavelength of 605 nm

C ₂ H ₅ OH + 2 C ₂ O ₇ ^2- + 16 H ⁺ → 2 CO ₂ + 4 Cr ³⁺ + 11 H ₂ O (4)

on. The evaluation computer only determines the zone that is not influenced by the dispersion the concentration-time curve in which a constant or stationary concentration Develops time history, recorded and used to calculate the ethanol concentration pulled.

Fig. 1b shows the corresponding timing diagram of this method for photometric determination of ethanol.

It is also possible to pump the dichromate solution continuously through the flow channel 6 and the detector 7 . According to the diagram shown in FIG. 1c, the flow rate V _P2 of pump 2 is reduced by the amount 0.1 ml / min ^-1 of V _P1 simultaneously with the activation of pump 1 . This ensures a uniform distribution of the metered sample solution over the relevant reagent flow zone over time t. Through the transition into the tapered, tightly knotted flow channel 6 , the mixing zone of injected sample solution with the reagent zone is pulled apart and mixed so far that a zone of constant concentration ratios is formed in the absence of any reaction.

In both process variants, the determination reaction ( 4 ) creates a concentration zone of analyte and reagent substance that is determined almost exclusively by the reaction kinetics. Fig. 1d shows the resulting concentration-time profiles of dichromate, chromium (III) ions and ethanol compared to the dichromate concentration profile in the absence of ethanol.

Exceeding a minimum dwell time results in full turnover of the Ethanol to carbon dioxide and dichromate to Cr (III) ions, which are measured photometrically at a Wavelength of 605 nm can be detected. The flow detector flows through, among other things a zone of constant concentrations of the Cr (III) ion. From which to a constant Value-adjusting detector signal is measured via the photometrically concentrated product tration and the stoichiometry of the determination reaction (4) the ethanol concentration calculated.

In Fig. 2a a working on the same principle flow arrangement for ampe rometric determination of thioglycolate is shown, with the low-volume order switching valve 9 , in which the upper valve part is switched between positions P1 and P2, and with low-pulsation or -damped hose pumps 1 and 2 works. First, the triiodide solution serving as a reagent flows through the valve 9 and the flow channel 6 to the flow detector 7 and the sample solution flows through the valve channel 10 into the drain. After switching the valve 9 into the metering position P2, a volume V _S defined by the product of the pump speed V _P1 and the metering time t _{DOS is} transported into the flow channel 6 . At the moment of valve switching, the pump speed V _{P2 of} the pump 2 is reduced to V _P2 -V _P1 to V _P2 *, so that the flow-dependent amperometric flow detector 7 is always flowed through at the same speed. FIG. 2b shows the timing chart of the determination method. The corresponding pump speed-time profiles are shown, which can be changed to set the volume ratios V _S / V _R for the purpose of setting the determination range.

In flow channel 6 , the thioglycolate to be determined reacts after reaction (5) with triiodide to the corresponding disulphide. The remaining triiodide is on a glassy carbon electrode

2 HS-CH ₂ -COO ^- + I ₃ ^- → ^- OOC-CH ₂ -SS-CH ₂ -COO ^- + 3I ^- (5)

cathodically detected at a potential of 0 mV against the Ag / AgCl / 0.1 M KCl reference electrode. The thioglycolate concentration to be determined is determined from the difference between the steady-state measured triadid concentration before and after metering in the sample solution, the stoichiometric factors of the determination reaction taking place after (5), the set ratio V _S / V _{R of} that for the sample solution V _S to that for the reagent solution set pump speed V _{R is} calculated.

Fig. 3a shows the flow measuring arrangement for carrying out the enzymatic determination of sucrose, which differs from that shown in Fig. 1a only in that three liquid flows meet at the confluence. A defined volume of the sucrose-containing sample solution PL is transported via the changeover valve 4 into the sample solution channel 8 . After the flow reversal of the pump 1 and switching of the valve 4 , the sample solution is transported to the confluence 3 via the tapered connecting capillary 11 . Shortly before the arrival of the sample solution, the pumps 2 , 12 and 13 are started, so that an invertase solution R1, a conditioning buffer R2 and optionally a glucose oxidase / mutarotase solution R3 are pressed into the flow channel 6 simultaneously with the sample solution. The pump speeds V _P2 , V _P12 and V _P13 are expediently set such that the optimum reaction conditions, pH 6.3, 1.5 U invertase per ml, 5 U glucose oxidase per ml and 20 U mutarotase per ml are set in the flow channel. At the moment when the sample solution is added, the pump speeds V _P2 , V _P12 and V _{P13 are} reduced proportionally by the total amount V _P1 , so that the detector 7, designed as an amperometric flow measuring cell, is flowed through at a constant speed. After calibration of the detector 7 and complete conversion, the hydrogen peroxide concentration present can be calculated from the stationary amperometric signal stream and the saccha rose concentration can be calculated from this and the stoichiometry of the determination reactions ( 6-8 ). Fig. 3b shows the timing diagram of this determination method.

Sucrose + H ₂ O → fructose + α-D-glucose (6)

α-D-glucose ⇆ β-D-glucose (7)

β-D-glucose + O ₂ → D-gluconate + H ₂ O ₂ + H ⁺ (8)

FIG. 4a shows the Durchflußanalysenanordnung for the enzymatic determination of glutamine by means of two-stage enzymatic reaction and fluorescence detection. The cofactor solution C adjusted to pH 4.9: 1 mM NAD ⁺ , 0.1 M sodium acetate / acetic acid is continuously transported by pump 1 through valves 9 and 14 , flow channels 6 and 15 and the fluorescence detector. By time-controlled switching of the valve 9 , a sample solution volume defined by the metering time t _DOS and the pump speed V _P2 is pressed into the flow channel 6 , which contains immobilized glutaminase in this application example. At the moment of valve switching, the pumping speed V _P1 is reduced by the amount of V _P2 , so that the resulting Durchflußge speed is equal to V _P1 so remains constant in the flow analysis channel. In the flow channel 6 , the hydrolysis of glutamine to glutamate catalyzed by glutaminase takes place

Glutamine + H ₂ O + H ⁺ → Glutamate + NH ₄ ⁺ (9)

according to reaction (9). According to the schedule shown in FIG. 4b, in the second analysis step, 0.3 M sodium phosphate buffer, pH 8.1 is metered by time-controlled switching of valve 14 . The reaction mixture conditioned in this way now passes through the flow channel 15 , which contains immobilized glutamate dehydrogenase. According to reaction (10), NADH is formed, which is detected fluorimetrically at an excitation wavelength of 365 nm and an emission wavelength of 440 nm. The advantage of this special embodiment of the method is that the inevitable high dilution of the sample solution to the ratio of the metered solutions is minimized in conventional flow-through methods. If the reagent R is a redox dye, e.g. B. iodonitrotetrazolium chloride INT and diaphorase added, the reaction (11) also takes place, in which a formazan is formed, which is photo metrized at 492 nm. Starting from the molar formazan concentration, which is accessible via the Lambert-Beer law, the glutamine concentration to be determined can be calculated via the stoichiometry of reactions 9-11.

Glutamate + NAD ⁺ → α-Ketoglutarate + NH ₄ ⁺ + NADH (10)

NADH + INT + H ⁺ → NAD ⁺ + Formazan (11)

List of reference numbers

1

,

2nd

,

12th

,

13

Precision pumps

3rd

Confluence

4th

Diverter valve

5

Transition to a smaller flow-through cross-section

6

Flow channel

7

Flow detector

8th

Sample delivery channel

9

Low dead volume changeover valve

10th

Drain

11

tapered connecting capillary

14

second low-volume changeover valve

15

second flow channel

Claims (8)

1. Method for flow-modulated concentration analysis, in which in one or more flow channels ( 6 , 14 ) with at least one transition ( 3 , 5 , 11 ) to a smaller flow cross-section by precise timing of pump speeds, previously separated sample, dilution, Conditioning and reagent solutions are metered with a high degree of accuracy in a wide volume range determined by the pumping speeds and pumping times, and dwell times and by dispersion-free mixing concentrations, concentration ratios and gradients are set precisely and steadily, and the concentration-time courses of the sample substance, one Indicator substance or a reaction product can be recorded continuously, preferably by a flow detector ( 7 ). 2. The method of claim 1, wherein with a continuous flow of a reagent, conditioning or dilution solution, the pump ( 1 ) transporting the sample solution is switched on and the pump speed of the other pump is reduced by the amount of the flow rate of the sample solution, so that at resulting, constant flow rate forms a sufficiently long zone of steady-state concentrations or concentration ratios. 3. The method according to claims 1 and 2, wherein after the mixing of the metered Sample solution and the reagent solution a chemical reaction setting statio nary concentration-time courses of the sample substance, the reagent and the reac tion products. 4. The method according to claims 1 and 2, wherein after setting Kon stationary concentration-time profiles, the sample and reagent solution transporting pumps ( 1 , 2 ) are stopped by a certain dwell time to achieve a desired degree of conversion of the determination reaction adjust. 5. The method according to claims 1 to 4, wherein in order to avoid the concentration analysis previous mixtures between the sample and the reagent, conditioning and the dilution solution a receiving and donor side dead volume-free switching valve ( 9 , 14 ) each to separates the solution to be mixed from the received solution and connects it to the receiving solution for admixing for a certain time. 6. The method according to claim 1 and at least one of claims 2 to 5, wherein the the sample solution and a pump transporting the other solutions controls that with a constant sum of the flow rates Ver Ratio of both flow rates is changed so that the desired Volume mixing ratio, e.g. B. dilution over a sufficiently long time is set stationary. 7. The method according to claims 1 to 6, wherein more than two pumps are used to transport in addition to the sample and the reagent solution further reagent solutions to a common mixing point ( 3 ) and to mix in the subsequent flow channel ( 6 ). 8. The method according to claims 1 to 7, wherein to a continuously flowing carrier or the sample solution itself via series-connected dead volume-free or low-switching valves ( 9 , 14 ) successively several reagent solutions or other solutions in each case in an assigned flow channel ( 6 , 15 ), preferably at a constant resulting flow rate.