CA1321080C - Flow injection analysis method and apparatus thereof - Google Patents

Abstract

Abstract of the Disclosure A novel and highly efficient flow injection analysis method is disclosed, in which in order to attain an extreme-ly 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 aforemen-tioned 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

-` 1 32 1 080 Title of the Invention Flow Injection Analysis Method and Apparatus thereof Backqround of the Invention This invention relates to a method of quantitative analysis by which a chemical component of a substance is quantitatively determined and also to an apparatus thereof, and more particularly it relates to an improved Slow injec-tion analysis (hereinafter, simply called as FIA) and impro-ved detection members in an apparatus employed for said FIA 10 method.
Further more particularly, this invention relates to a technical method of analysis in which a sample solution (in other words, a carrier solution containing a sample) and a reagent solution are flown respectively at a trace amount in an anticorrosive tube such as a tube made from Teflon and - ~

are reacted therein, their reaction product is measured , within a flow cell by means of its physical or chemical characteristics such as done generally in a spectrophotomet-ric method, and chemical component or components to be 20 analyzed in the sample are thus determined, and the inven-tion also relates to improvements of an apparatus which can advantageously be employed in such analysis method.
In general, as wet chemical analysis method, there are gravimetric analysis, volumetric analysis, spectrophotomet-ric analysis and so on.
In the gravimetric analysis, chemical components to be '~

* trade mark ,.-~r- . . - , :, , ,:: : : ~ -- :

.
.

analyzed shall be compl~tely of their 100% separated by filtration to the insoluble precipitates, which are in turn weighed for their quantitative determlnation. And, also in the volumetric analysis and the spectrophotometric analysis, a solution containing sample components to be analyzed shall be reacted completely with a reagent solution by 100%.
Hence, it is prerequisite in the spectrophotometric method that the reaction proceeds quantitatively without being accompanied by any side reaction. It shall be noted also that similarly to other wet chemical analyses in a 10 spectrophotometric method too, a component to be analyzed has to be changed completely of its 100% to a colored com-pound, absorbance of which is measured for a quantitative determination. Thus, the prerequisite basic principle common to a quantitative analysis is that the determination has to be made completely as nearly as possible to its 100%.
On the other hand, this invention of FIA method has been made with the following fundamental design conceptions, that is, (1) to ailow a reaction rate of FIA to come as nearly as possible to 100%, (2) to keep the reaction condi- 20 tions constant, and thereby to obtain a constant reaction state which is close to 100% even if the reaction rate can not be absolutely 100%, and (3) to save the amount of~
reagents by using a fine tube and a special pump.
However, this principle can not easily be attained in the conventional FIA. Because, a reagent solution and a sample solution stay within the tube only for a short period of , .

-- ' time and consequently they are subjected to a reactlon only for a short period of time. This results that in almost all analysis operations, their reactions have not been completed by 1oo% but are on the way to completion, whereby perfect determination can hardly be expected by a conventional FIA.
The FIA method is generally practiced by flowing a reagent solution in a fine anticorrosive tube preferably made from Teflon, injecting into the flow a sample solution of several tens to several hundreds ~ , obtaining a reaction compound by the reaction of said sample and reagent within 10 the tube, and subjecting said compound to the measurement within a flow cell for the determination of a component in the sample to be analyzed by means of physical or chemical characteristics of the reaction compound. In this respect, an apparatus used in the FIA method resembles to the one used in liquid chromatography.
However, the FIA method intends to analyze only a single component in a multicomponent homogeneous liquid phase, while a liquid chromatography is to make a separate analysis of each component in a multicomponent homogeneous 20 liquid phase.
Since the FIA method which is the subject of this invention differs from a liquid chromatography method basi-cally and noticeably as mentioned above, following remarks have to be taken into consideration in carrying out said method successfully.
(a) Sample and reagent solutions have to be mixed up ' ' ' ~

thoroughly arld (b) A single component is to be determined exclusively with high sensitivity and without interference of other components.
In order to observe the remarks, this invention is also provide a specific flow cell.

Brief SummarY of the Invention In view of the above, this invention is to provide an application engineering about assembly of flow lines, embo-died herein as a method and an apparatus which have been 15improved with the following features for solving the afore-mentioned drawbacks accompanied to a conventional FIA method and consequently for ascertaining more precise determination through the flow lines.
Such features are:
(I) Sample and reagent solutions are supplied alternately to each other and respectively at a trace amount so that within a fine reaction tube, both solutions can make liquid-to-liquid contacts with wider reaction surface areas, whereby they are thoroughly mixed up, resulting in the improvement of a reaction rate, and (II) Flow rates of the reagent and sample solu-tions as well as a reaction temperature thereof are made constant, whereby a reaction rate is also made constant.

~,, , More particularly, the flow injection analysis method according to the invention as claimed hereinafter is characterized in that a sample solution and a reagent solution are fed through flow lines which are independent to each other and each of which has an inner diameter of 0.25-1.0 mm, and subsequently they are injectedly supplied to a mixing flow line alternately and respectively at a prede-termined amount of 1.25-2.0 ~e per injection batch, at least some part of the mixing flow line being accommodated in a thermostat bath, an end of said mixing flow line being connected to an spectrophotometric flow cell which is in turn connected at its outlet to a back pressure coil for the adjustment of an inner pressure exerting within the flow lines.

4a ' " 1 32 1 080 In said method, a sample solution and a reagent solu-tion pass throuqh flow lines which are independent to each other, and then, they are injectedly supplied to a mixing flow line alternately to each other and respectively at a predetermined trace amount, whereby they are mixed and reacted thoroughly.
And, when required, at least a part of said mixing flow line is passed through a thermostat bath so that the line is kept at a constant temperature, whereby reaction rate achieved thereby can also be made constant. And, in addi- 10 tion, there is connected at a back portion of a flow cell a back pressure coil so that bubbles are prevented to form thereat and a smooth flow can be obtained with few irregular pulsation.
While it is preferable that an injection supply amount of the sample and reagent solutions are as small as possib-le, said amount is apparently subject to a flow rate accura-cy controlled by a supply pump, viscosities of the reagent and sample solutions, diametric accuracy of a reaction tube, ..
and others. Nevertheless, the most appropriate range of 20 said amount and an inner diameter of flow lines can be selected as follows:
To wit, it has been found that when an inner tubular diameter of flow lines is less than 0.25mm, frictional resistance between inner walls of the tubes and solution flows becomes too high, inner pressure in the tubes becomes also disadvantageously high, and efficient mixing of sample , .
:

1 32 1 08~
and reagent solutions can hardly be obtained. On the contrary, when the inner diameter exceeds 1.0mm, flow resis-tance within the tubes lowers, and concurrently inner pressure lowers also, whereby sample and reagent solutions are consumed at an amount more than those required for reasonable analyses and whereby costs for analyses become high, although the manufacture and handling of an apparatus including the tubes become easier.
Preferable tubular diameters thus selected above can accordingly confine a preferable range of flow rates through 10 the tubes. In practice, when two solutions are injected into a mixing and reaction line alternately to each other and respectively at a precisely trace amount so that reac-tion rates thereof can be kept high and constant, extremely accurate supply of the solutions can hardly be attained if an injection supply amount per a batch or at each time is less than 1.25 ,ue, and on the contrary, if said amount exceeds 20,u~, the reagent and sample solutions would insuf-ficiently be mixed.
Therefore, in this invention, inner diameters of these 20 flow lines for the sample solution and for the reagent solution which are independent to each other, and an inner diameter O'L the mixing flow line shall preferably be within a ~ange of 0.25-1.0mm, while an amount of the solutions which are injectedly supplied into the mixing flow line alternately shall preferably be 1.25-20~ at each injection batch.

1 32 1 0~0 Secondly, the spectrophotometric flow cell provided to a detecting member in this invention is briefed below.
When an inner diameter of the mixing flow line which is followed by the above-mentioned flow cell is selected as 0.25-1.Omm, an optical path diameter of the flow cell has to be expanded largely than that of the mixing flow line, that is, as much as a range of 1.5-2.5mm, while its o~tical path length shall preferably be 10-50mm.

Brief DescriPtion of the Drawinq 10 In the accompanying drawing which illustrates preferred embodiments of this invention;
Fig. 1 is a view showing a flow linè in the method made in accordance with this invention, Fig. 2 is an explanatory view showing states of liquid phases within a mixing flow line or a reaction coil, Fig. 3 is a sectional view of an example of flow cell made in accordance with this invention, and Fig. 4 is a side view of said flow cell shown in Fig.
3. 20 Detaild Description of the Invention Now, this invention is described more in detail by way of the following examples:
Example 1.
With reference to Figs. 1 and 2, in this invention, a sample solution C1' and a reagent solution C2' are injected-, 1 32 1 0~0 ly supplied, through a flow line C1 and another flow line C2which are independent to each other, into a mixing flo~ line alternately to each other and respectively at a trace amount as shown in Fig. 2 so that the solutions may have larger contact sur~aces for efficient mixing thereof.
In order to achieve such alternate injection supplies of the solutions, a pump P used in this invention method shall preferably be those of a non-pulsation double plunger type, one of which plungers for the sample solution flow line C1 and another of which plungers for the reagent solu- 10 tion flow line C2 are not synchronized to each other, and injection supply amount made by which is controlled to be about s,uQ per a stroke. This injection amount is correspon-dent to a volume of about 25mm length of a Teflon tube of 0.5mm in inner diameter. And, in order to obtain a constant rate of flow without pulsation, it is recommendable to employ a phase different double plunger type pump having, for example a stroke length of 1mm, a stroke delivery of about 5,uQ, and a plunger diameter of about 2-3 mm. The letter B in Fig. 1 represents a hexagonal injection valve 20 which is located in thesample solution line C1 and by which a sample is supplied under pressure into a carrier solution flown in the line C1. RC represents a reaction coil in which the two solutions are mixed and reacted. It is prefe-rable, as shown in the drawing, to accommodate the reaction coil RC within a thermostat bath HB so that a reaction temperature of the solutions C1' and C2' ~ay be kept 1 32 1 08~) constant wnere~v reaction r~te thereo~ is raised high and kept constant.
The solutions which have been reacted thoroughly in the reaction coil RC of a mixing rlow line system, are sent to a flow cell FC where they are subjected to a measurement by an spectrophotometer and others to obtain a measured value which is in turn recorded by a recorder member R. And, the solutions which have been measured are brought outside of the lines as a waste solution, by means of a back pressure coil BPC of an inner diameter of 0.20-0.50mm for example. 10 This waste solution W is discharged only after it has been treated so as not to bring about any water pollution. The above-mentioned back pressure coil BPC works also to prevent the solutions throughout the flow lines from producing bubbles, and as well to obtain a stable constant rate of flow without pulsation. While it is observed sometimes to employ a pelister type pump in order to keep solutions within the flow lines under a condition without pulsations, this type of pumps are inferior to plunger type pumps with respect to their endurance. Hence, it is preferable to 20 employ plunger type pumps as explained above.
It shall be noted that features specific to this inven-tion could be applicable not only to flow injection analyses but also to other flow line analyses.
Example 2.
In this example, an example of flow cell which is made in accordance with this invention and could advantageously be employed in a detectLng member of the present FIA appara-tus, is described in more detail with reference to Figs. 3 and ~.
In the FIA apparatus which uses, as shown by Fig. 1, the flow cell made in accordance with this invention, the solution C1' with the sample S and the reagent solution C2' are supplied by the non-pulsation double plunger type pump P
to the reaction coil RC via a mixing joint M alternately and respectively at a trace amount from the flow lines C1 and C2 which are independent to each other. The solutions are 10 mixed and reacted thoroughly in the reaction coil RC, and then sent to the flow cell FC which constitutes a detecting member and in which the component to be analyzed is detected and measured. The values measured thus are recorded by the recorder R, while the solutions which have been subjected to the measurement pass through the back pressure coil BPC, and then discharged outside of the system as the waste W. In this instance, at least a part of the mixing flow line which is designated as the reaction coil in Fig. 1 is passed through the thermostat bath HB so that a reaction rate in 20 the mixing flow line can be raised high and also constant.
The flow cell FC which constitutes a detecting or measuring member is consisted of, as best shown in Fig. 3, a tubular body 1 made from brass to which there is insertedly fitted a cylindrical body 2 made from Teflon and having at its central axis an optical path channel 3. To the both free ends of the brass tubular body 1, there are fitted by screws 5 discal frames 4 made from brass and having at their centers optical path holes 4a, ~iameter of which is equal to the optical path channel 3.
Numeral 6 indicates transparent sheets of glass which are fitted to the both ends of the optical path channel 3.
The glass sheets 6 which are closely fitted to the cylindri-cal body 2 by means of the frames 4 through spacers 7, prevent solutions to be determined from leaking therefrom.
The reacted solutions sent from the reacti~on coil RC
enter into the optical path channel 3 through an inlet 8 of 10 the flow cell FC and one end of said channel. When they are subjected to an spectrophotometric measurement, they come out from an outlet 9 and are discharged as the waste W after having passed the back pressure coil BPC.
In the following, an optical path diameter and an optical path length of the optical path channel 3 which is provided in the flow cell made in accordance with this invention, is explained.
The flow cells of different optical path diameters were made, while their optical path length was made constant, 20 viz., 1Omm and the inner diameter of the mixing flow line was made 0.5mm.
A 10-5M picric acid solution was flown through the line C1 of the FIA apparatus of Fig. 1 as a sample solution thereof, while a pure water through the line C2. They were measured of their absorbance (aAbs) at 4,000A by the flow cell, showing the result of about 0.05. An output of the :

. .,. ~-:

1 32 1 0~0 spectrophotometer emploved ln t.ie ~easurement was 100mV/Abs, r~hlle ranges recorded bv the recorder R were 10, 5, 2, and lmv.
The above experimental results, that is, relations of different optical path diameters and S/N ratios are given in the following Table 1.

Table 1.
No. Optical Path o.P. length (mm) Volume (,uQ) S/N ratio Diameter(mm) 10 1 1.0 10 8 1 2 1.5 10 18 5 3 2.0 10 31 10 4 2.5 10 50 20 5 3.0 10 71 20 6 4.0 10 130 2G

7 5.0 10 20~ 20 As readily be understood from the above Table 1, larger the optical path diameter is, better the S/N ratio becomes, 20 resulting in improving electrical sensitivity of the flow cell. However, it is noticed that when the optical path diameter exceeds 2.5mm, sensitivities were raised up as optical volumes increased sharply, while strays also increase accordingly and S/N ratios thereby tend to saturate.
On the contrary, when the optical path diameter was less than 1.5 mm, an optical volume remarkably decreased, resul-..

ting in making it difficult to conduct such analyses whichare of high sensitivity and free from other interferences.
Another series of e.Yperiments were made with respect to an optical path length of the flow cell, in which an inner diameter of the mixing flow line was made 0.5mm, and an optical path diameter of flow cells were made constant as 1.5mm, while their optical path lengths were made different.
The sample solution in the line C1, the solution in the line C2, and other experimental conditions were same to the above experiments of the Table 1. 10 Absorbances (~Abs) at 4,000A were measured as shown in the following Table.

Table 2.
_ No. Optical Path Cell O.P. ~Abs aAbs Length (mm) Volume (JuQ) Diameter (mm) ratio _ 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 20 5 100 _150 1.5 0.520 10.30 As shown in the above Table 2, it becomes known that longer the optical path length is, greater the absorbance ratio becomes. This means that the more the optical path length is, the more precisely a solution even of a low chromaticity can be measured. In other words, when the o?~ical ~ath length is sufficientl~ long, even a ~ery trace amount of a component in a sampLe solution can be determined with a high sensitivity. However, it is noticed that "hen the optical path length exceeds 50mm in the determination of a solution of a high concentration, absorbance thereof satu-rates whereby a difference of chromaticity can hardly be obtained. On the contrary, when it is less than 1Omm, chromaticity can not precisely be measured.
Therefore, an optical path length of the flow cell shall preferably be within a range of 10-50mm. 10 In view of the explanation made above with respect to this invention, various remarkable effects obtained by it are enumerated below.
(I) This invention can provide an extremely high reaction rate which can indefinitely be close to 100%~ And in addition, since the reaction rate can regularly be kept constant, extremely high precise analyses can be made prom-ptly and can avoid the use of excessive amount of reagents, whereby the analyses become very economic.
(II) When the flow cell made in accordance with this 20 invention is employed, an entrance optical value in case of its optical path diameter being 2mm for example, will be four times of that of a conventional liquid chromatographic flow cell of an optical path diameter of 1mm, whereby inter-ferences by other components which are not to be analyzed can be well avoided, whereby S/N ratios are greatly improved and a component to be analyzed can accordingly be determined determined ~ith a high sensitivity.
(III) Compared to inner diameters of the mixing and reaction line in accordance with this invention, viz., 0.25-1.0mm, an optical path diameter of the flow cell is as large as 0.5-2.5mm, whereby the solution to be analyzed is positi-vely urged to disperse evenly when it reaches the flow cell.
(IV) In addition, the invention has other advantageous effects such as the improved detecting sensitivity the flow cell has attained on account of its optical path length being selected as 10-50mm. 10 -' : . .

Claims (7)

1. A flow injection analysis method according to the invention as claimed hereinafter is characterized in that a sample solution and a reagent solution are fed through flow lines which are independent to each other and each of which has an inner diameter of 0.25-1.0 mm, and subsequently they are injectedly supplied to a mixing flow line alternately and respectively at a predetermined amount of 1.25-2.0 µ? per injection batch, at least some part of the mixing flow line being accommodated in a thermostat bath, an end of said mixing flow line being connected to an spectrophotometric flow cell which is in turn connected at its outlet to a back pressure coil for the adjustment of an inner pressure exerting within the flow lines.

2. The method as claimed in claim 1, in which the sample and reagent solutions are supplied by a pump of non-pulsation double plunger type.

3. The method as claimed in claim 2, in which an optical path diameter of the spectrophotometric flow cell is made so as to be sharply widened compared to that of the mixing flow line.

4. The method as claimed in claim 3, in which the optical path diameter of the spectrophotometric flow cell is 1.5-2.5 mm, while an inner diameter of the mixing flow line is 0.25-1.0 mm.

5. The method as claimed in claim 4, in which an optical path length of the spectrophotometric flow cell is 10-50 mm.

6. A spectrophotometric flow cell to be used in an apparatus for flow injection analysis method, which is characterized by that it succeeds to flow lines each of an inner diameter of 0.25-1.0 mm and has an optical path diameter of 1.5-2.5 mm.

7. The spectrophotometric flow cell as claimed in claim 6, in which the flow cell has an optical path length of 10-50 mm.