SE503661C2 - Methods for flow injection analysis and therefore adapted spectrophotometric flow cell - Google Patents

Abstract

A novel and highly efficient flow injection analysis method is disclosed, in which in order to attain an extremely high reaction rate of sample and reagent solutions at a reaction line, the solutions are injectedly supplied thereto alternately and respectively at a predetermined trace amount through independent two flow lines which are kept so as not to produce pulsation within the solutions. A thermostat bath accommodating the reaction line and a back pressure coil connected to the flow lines also make the aforementioned reaction rate stable and regular. Spectrophotometric flow cell succeeding the flow lines for the analytical determination of the sample solution has a specific range of optical path length and such optical path diameter which is widely diverged from its inlet.

Description

503 661 whose absorbance is measured for quantitative determination. The basic principle which is usually a precondition for a quantitative tative analysis, is thus that the determination must be made so close 100% as possible. On the other hand, the FIA method of the present invention the following basic features, namely, (1) reaction rate the FIA has the opportunity to get as close to 100% as possible (2) the reaction conditions are kept constant and thereby a constant reaction state is obtained, which is close to 100% although the reaction rate cannot be absolutely 100%, and (3) the amounts of reagent are sparse by using a fine pipes and a special pump.

However, this principle cannot be achieved with conventional FIA without difficulty, because a reagent solution and a sample solution only stays inside the tube for a short period of time, leading to them only for a short period of time exposed to reaction. This happens to just about everyone analysis operations, the reactions have not been completed to 100% but is about to end, with perfect determination hardly can be expected at conventional FIA.

The FIA method is usually performed using a reagent solution preferably in a fine non-corrosion-affected pipe manufactured by Teflon, a test solution of several dozen more hundreds of microliters are injected into the flow, a reaction compound is obtained by the reaction of the sample and the reagent inside the tube, and said compound is subjected to measurement inside a flow cell to determine a component of the pro ~ -t to analyzed using the physical or chemical properties of the reaction compound. In this respect, the apparatus is similar to In the FIA method, turn the one used in liquid chromatography.

However, the intention of the FIA method is to analyze only a single component in a homogeneous liquid phase containing many components, with liquid chromatography used to make a 503 661 separate analysis of each component in a homogeneous liquid phase holding many components.

Since the FIA method which is the subject of the present invention differs from the liquid chromatography method mainly in the case mentioned above, the following criteria must be taken into account upon successful execution of the method. (a) The sample and reagent solutions must be mixed thoroughly, and (b) only a single component shall be determined with high sensitivity and without interference from other components.

In order to meet these criteria, companies provide The present invention also discloses a specific flow cell.

-. In view of the above, it is the present invention task of providing a technical application regarding installation of flow lines, which will be described further below, and a method and apparatus which has improved with the following features in order to avoid the above disadvantages associated with a conventional FIA method and the to ensure more accurate determination by flow- the wires.

These features are: (I) The sample and reagent solutions are added alternately in holding to each other and in trace amounts, so that, inside one fine reaction tube, both solutions are subjected to liquid-addition liquid contact with wider reaction surfaces, mixing them carefully, resulting in improved reaction rate, and « (II) The flow rates of the reagent and sample solutions as well as the reaction temperature thereof is kept constant, the reaction tion rate is also kept constant.

First, the method according to the invention will be described according to following: 503 661 In the method, a sample solution and a reagent solution pass through flow lines, which are independent of each other, and thereafter they are sprayed alternately into a flow line for mixing in each a predetermined amount of trace, mixing and reacting geras carefully.

If necessary, at least a part of the flow line is arranged for mixing so that it passes through a thermostat bath, to keep the line at a constant temperature, they thereby achieved reaction rates can also be kept constant.

In addition, at the rear of a flow cell, a back pressure spiral connected so that bubbles can not form there and one even flow can be obtained with little irregular pulsation.

Since the spray feed rates of sample and reagent solution are preferably as small as possible, have these amounts preferably an accurate flow rate controlled by a supply pump, depending on the reagent and sample solutions viscosity, accuracy of the reaction tube diameter, etc.

Preferably, the size of this quantity and flow line internal diameter of the members as follows: If the inner pipe diameter of the flow lines is less than 0.25 mm it has been found that the frictional resistance between the pipes in walls and the solution flows become too large, the internal pressure in the pipes also become unfavorably high and efficient mixing of the sample and reagent solutions are difficult to obtain. On the other hand since the inside diameter exceeds 1.0 mm, the flow the resistance inside the pipes, and at the same time the inner pressure, consuming the sample and reagent solutions faster than required for reasonable analyzes, and thereby the costs of the analyzes will be high, but the production and the handling of the device including the pipes becomes easier.

Accordingly, preferred pipe diameters selected as above may limit a preferred range of flow rates through In practice, when two solutions are injected alternately in relative to each other to a mixture and reaction 503 661 line in the respective exact trace amounts, so that the reaction rate its properties can be kept high and constant, can be extreme accurate supply of the solutions is hardly obtained if it injected delivery rate per batch or each time is less than 1,25 Pl, and vice versa, if the amount exceeds 20 fl, the reagent and sample solutions are insufficiently mixed. Therefore is in the present invention the inner diameter of these flow lines for the sample solution and for the reagent solution, which are independently of each other, and the inner diameter of the flow line for mixing, preferably in the range 0.25-1.0 mn while the amount of solution sprayed alternately to the flow path the mixture for mixing is preferably 1.25-20 .mu.l at each injection kit.

Now comes the spectrophotometric flow cell provided with one detection elements according to the invention to be described.

If the inside diameter of the flow line for mixing, which is arranged according to the above-mentioned flow cell, is chosen between 0.25-1.00 mm, the diameter of the optical path of the flow cell must be expanded sharply compared to the diameter of the flow line for mixed ning, i.e. as much as in the order of 1.5-2.5 mm, while its optical path length is preferably 10-50 mm.

The invention will now be described in more detail below in connection with to the accompanying drawings, in which Fig. 1 is a view showing a flow line in the method according to present invention, Fig. 2 is an explanatory view showing the state of the liquid phases within a flow line for mixing, or a reaction tions spiral, Fig. 3 is a sectional view of an example of a flow cell operated in accordance with the present invention, and Fig. 4 is a side view of the flow cell shown in Fig. 3. 503 661 The present invention will now be described in more detail using the following examples: Example 1 Referring to Figures 1 and 2, in the present invention a sample solution C1 'and a reagent solution C2' are spray-fed. through a flow line C1 and another flow line C2, which are independent of each other, alternating into a flow line for mixing in trace amounts, as shown in Figure 2, so that the solutions have a larger contact area for efficient mixing hence.

To achieve such alternating spray feeding of the solutions, a pump P is used in the method of the present invention, which preferably of the non-pulsating double piston type, is a piston for the flow solution C1 of the sample solution and a flask for the reagent the solution flow line CZ, which are not synchronized with each other, the spray feed rate being controlled to approx. 5 Fl per stroke. This spray feed rate corresponds to the volume of a approx. 25 mm long Teflon hose with 0.5 mm inner diameter. For to obtain a constant flow rate without pulsations, It is recommended to use a double-piston type pump with displacement, which has a stroke of e.g. l mm, en slaguttömn ng of approx. 5 μl and a piston diameter of approx. 2-3 nn.

The letter B in Figure 1 refers to a hexagonal injection valve, which is located in the line C1 of the sample solution and by means of which a sample is fed under pressure to a carrier solution flowing in the wire Cl. RC represents a reaction coil in which they the two solutions are mixed and reacted. Preferably arranged the reaction coil RC inside a thermostat bath HB, as shown in the drawing, so that the reaction temperature of the solutions C1 ' and C2 'can be kept constant, the reaction rate thereof elevated and kept constant.

The solutions which have been carefully reacted in the reaction coil RC of a flow line mixing system, is carried to a flow cell FC where they are subjected to measurement by e.g. one spectrophotometer to obtain a measured value, which in turn 503 661 registered by a registration element R. The solutions that have measured is taken out of the pipes as a waste solution, using a back pressure coil BPC with an inner diameter of e.g. 0.20- 0.50 mm. This waste solution W is only emptied after it has been treated so that it does not give rise to any water soiling.The above-mentioned counter-pressure coil BPC also supervises that the solutions in the flow lines are prevented from forming bubbles, and that a stable, constant flow rate is obtained without pulsations. Sometimes pelister-type pumps are used to keep solutions inside flow lines in a pulsation-free manner condition, but these pumps are inferior to piston-type pumps with regard to their endurance. Consequently, preferably piston-type pumps, which have been described above.

It should be noted that features specific to the present invention can not only be applied to flow injection assays = but also on other flow line analyzes.

Example 2 This example describes an embodiment of a flow cell manufactured according to the present invention as advantageous could be used in a detection element of the present FIA apparatus, which is described in more detail below with reference to Figures 3 and 4.

In the FIA apparatus using, as shown in Figure 1, the flow cell made in accordance with the present invention, C1 'with the sample S and the reagent solution C2' of the non- pulsating pump P of double piston type to the reaction coil RC alternately via a mixed compound K-each in trace amounts from the flow lines C1 and C2, which are independent of each other. The solutions are mixed and reacted thoroughly. grant in the reaction coil RC, and is then sent to the flow the cell FC, which comprises a detection element and in which the component to be analyzed is detected and measured. Depot the values measured in this way are registered by the device R, while the solutions that have been subjected to the measurement pass through the back pressure coil BPC, after which they are discharged 503 661 outside the system as waste W. In this case, at least a part of the flow line for mixing, which is designated as as a reaction coil in Figure 1, through the thermostat bath HB so that the reaction rate in the mixing flow line can be increased and kept constant.

The flow cell FC which constitutes a detection or measuring element comprises, as can be seen most clearly in Figure 3, a tubular body 1 made of brass in which a cylindrical body 2 of Teflon is fitted and which at the central axis has one optical path channel 3. To the tubular brass body l both free ends are disc-shaped fittings 4 of brass arranged by means of screws 5, which fittings at the center have optical road hole] 4a, the diameter of which is as large as that of the optical one the road channel 3.

Reference numeral 6 shows transparent sheets of glass, which are used arranged at both ends of the optical path channel 3. The sheets of glass 6, which are closely attached to the cylindrical body 2 with by means of the fittings 4 through spacer elements 7, prevents solutions to be determined leak out of there.

The reacted solutions coming from the reaction signal RC enters the otitis road channel 3 through an inlet 8 of the flow cell FC and one end of the channel. When exposed to spectrophotometric measurement, they come out of an outlet 9 and is discharged as waste W after passing the back pressure coil BPC.

Below is an optical path diameter and an optical path length of the optical path channel 3, which is arranged in the flow cell operated according to the present invention, to be described.

Flow cells with different optical path diameters were manufactured, each at the optical path length was made constant, namely 10 mm, and the inside diameter of the mixing flow line was made 0.5 mm. 9,503,661 A 10.5 M picric acid solution was drained through line C1 The FIA apparatus of Figure 1 as a sample solution, while clean water was flowed through line C2. Absorbances (aAbs) was measured at 4000Å through the flow cell, which gave a result at approx. 0.05. The output signal of the spectrophotometer used at the measurement was 100 mV / Abs, while areas registered by the the control device R was 10, 5, 2 and 1 mV.

The above experimental results, i.e. relationships between different optical path diameters and S / N ratios, are given in Table 1 below.

TABLE 1 I * f Diameter (mm) Length (mm) of 5 Volume S / N for- Nr. of optical path optical path (Pl) holding 1 1.0 10 8 1 2 1.5 10 18 5 3 2.0 10 31 10 4, 10 50 20 5 3.0 10 71 20 6 4.0 10 130 20 7 5.0 10 200 20 From Table 1 above, it is clear that, the larger the the meter of the optical path, the better the S / N ratio resulting in improved electrical sensitivity of the flow cell. However, it should be noted that if the diameter of the optical path exceeds 2.5 mm, then the sensitivity increases when optical volumes increase sharply, while interference also increases as a result of this and S / N conditions thereby tend to saturated. If, on the other hand, the diameter of the optical path is smaller than 1.5 mm, the optical volume decreased significantly, which results in it being difficult to perform analyzes that are highly sensitive and interference-free. 503 661 10 Another series of experiments was done with respect to an optical path length of the flow cell in which the mixing flow path the inner diameter of the fluid was 0.5 mm, and the diameter of the flow cell their optical path was constantly 1.5 mm, while their optical road lengths were made different. The sample solution in line Cl, the solution in line C2 and other experimental conditions was the same as the above experiments according to Table 1.

Absorbances (AAbs) at 4000Å were measured as below Chart.

Table 2 i Å 2: ï Nr. Length (mm) I Cell in Diameter I § of optical in volume E (mm) of, AAbs path E (Pl) optical path AAbs holding 1 5 9 1.5 0.0250 0.50 2 10 18 1.5 0.0504 1.00 3 20 35 1.5 '0.102 2.02 4 50 88 1.5 0.255 5.06 5 100 | 155 1.5 0.520 10.30 As shown in Table 2 above, it appears that the longer it the optical path length is, the greater the absorbance ratio.

This means that the larger the optical wavelength is more precisely, a solution can be measured even if it has a low chromatic tet. In other words, when the optical path length is sufficiently long, even a very small amount of trace of a compound ing in a sample solution is determined with high sensitivity. However it should be noted that when the optical path length exceeds SO mm when determining a solution with a high concentration, saturate the absorbance thereof, whereby a difference in chromaticity cannot obtained. On the other hand, if it is less than 10 mm, the chromatic is not measured accurately. 503 661 ll Therefore, the optical path length of the flow cell should preferably be located within the range 10-50 mm.

In view of the above features of the invention, a number of beneficial effects are obtained, which are listed below.

(I) The present invention can provide an extremely high reaction rate which may be close to 100%. Furthermore, regular, ex- as the reaction rate can be kept constant Tremely accurate analyzes are done quickly and in large quantities reagents are not needed, which makes the analyzes very economical. nomiska.

(II) When the flow cell manufactured according to the present invention ing is used, the optical input value comes about the optical the road diameter is e.g. 2 mm, to be four times the value of one conventional liquid chromatography flow cell with an optical pathway whereby disturbances of other components such as and wherein S / N conditions diameter of 1 mm, not to be analyzed can be avoided, significantly improved and a component to be analyzed can actually be determined with high sensitivity.

(III) Compared to the inside diameters of the mixing pipe and reaction according to the invention, i.e. 0.25-1.00 mm, it is optical path diameter of the flow cell as large as 0.5-2.5 mm, which means that the solution to be analyzed is certainly forced to (IV) Furthermore, the invention has other beneficial effects, even dispersion when it reaches the flow rate. for example the improved detection sensitivity that flow the cell gives p.g.a. that its optical path length is chosen between 10- 50 mm.

Claims (10)

503 661 12 PATENT REQUIREMENTS Method for flow injection analysis where a sample solution and a reagent solution are flowed through flow lines, which are independent of each other, and then fed to a mixing flow line, characterized in that the sample and reagent solution are divided into a plurality of injection batches of predetermined amount, which are sprayed alternately to the mixing flow line. 2. A method according to claim 1, characterized in that each of the flow lines for the sample solution and the reagent solution has an inner diameter of 0.25-1.0 mm, and that the predetermined amount in which the sample solution and the reagent solution are sprayed to the mixing flow line, is 1.25-2.0 μl per injection batch. 3. A method according to claim 1 or 2, characterized in that at least a part of the mixing flow lines is housed in a thermostat bath. 4. A method according to claim 1, 2 or 3, characterized in that the mixing flow line at the end is connected to a spectrophotometric flow cell, which in turn is connected at the outlet to a back pressure coil for adjusting an internal pressure exerted inside the flow lines. 5. A method according to claim 1, 2, 3 or 4, characterized in that the sample and reagent solutions are fed by means of a pump of non-pulsating double-piston type. 6. A method according to claim 1, 2, 3, 4 or 5, characterized in that the diameter of the optical path of the spectrophotometric flow cell is designed so that it is greatly expanded compared to the diameter of the mixing flow path. ningen. 7. A method according to claim 6, characterized in that the diameter of the optical path of the spectrophotometric flow cell is 1.5-2.5 mm while the inner diameter of the mixing flow line is 0.25-1, 0 mm. 8. A method according to claim 7, characterized in that the optical path length of the spectrophotometric flow cell is 10-50 mm. Spectrophotometric flow cell to be used in a flow injection analysis apparatus, characterized in that it is arranged according to flow lines, each of which has an inner diameter of 0.25-1.0 mm, and has an optical path diameter of 1.5-2, 5 rum. Spectrophotometric flow cell according to claim 9, characterized in that the flow cell has an optical path length of 10-50 mm.