PROCESS FOR INJECTING A MINUTE VOLUME OF A SOLUTION AND AN APPARATUS THEREFOR

Abstract: Process for injecting a minute volume of a solution from a nozzle device by positive pressure applied from a source of compressed air with or without positive pressure applied in parallel from a cylinder device, comprising applying the posi­ tive pressure from the compressed air source at least in a final period of injection. The process enables in injecting minute volumes of solution to remarkably decrease the injection error due to a small fraction of solution remaining at the lower end of nozzle and can be applied to advantage to those appar­ atus for analyses and measurements which strictly require precision in injection.

Description: The present invention relates to a process and an automated apparatus for injecting a minute volume of a solution from a nozzle device in accordance with the generic clauses of Claims 1 and 2. Such a process and apparatus are known from US-A-3 735 902. From US-A-4 088 446, such an apparatus is also known. It corresponds principally to the embodiment of the invention shown in Figure 1. The control valve and inert gas source therein are used to purge any air from the probe before the intake position of the nozzle is assumed and to expel the sample residual from the nozzle device while the discharge position is assumed by the probe. An injection apparatus for delivering minute volumes each ranging from several µℓ to several hundred µℓ of a sample solution is generally used in biochemical immunoassay (IA) and liquid chromatography. Previous injection apparatus is usually provided with a cylinder device connected to the cavity of a nozzle device into which an object solution is sucked. The solution is sucked by the backward motion of a piston of the cylinder device and injected by the forward motion of the piston. However, when this kind of an injection apparatus is used to inject minute volumes of a solution as mentioned above, significant errors of injection have often been met. Provided that the lower end of the nozzle in an inverted conical shape has a diameter of about 0.3 - 0.5 mm and the tube wall is about 0.3 - 0.5 mm thick, an injection error of several µℓ is inevitable when about 200 µℓ of a solution is sucked and injected by the forward and backward motion of a piston in the cylinder device. The error is not negligibly small when a high precision is required in injection. The error of course becomes more remarkable when a minute volume such as several µℓ to several ten µℓ is concerned. It is the object of the present invention to provide a process and an apparatus for injecting a minute volume of solution with a far improved precision in injection and being suitably employed in bioassay systems to pipet and deliver sample solutions. This object is solved by a process and an apparatus having the features of Claims 1 and 2. A preferred embodiment is disclosed in Claim 3. Detailed Description of the Invention: According to the inventors' investigation, the mentioned injection error comes about by the loss of the object solution to be delivered. Particularly a part of the solution flows around to the outer wall of the nozzle or remains on the inner wall of the nozzle and is left there undelivered. This probably has occurred mainly because the speed of injection is lower in the final and in the initial period of injection than in the midway of injection, if the injection is made by the positive pressure applied from the cylinder device. This phenomenon becomes remarkable when a sample solution wets the nozzle material as solutions of biological samples do and also when the piston of the cylinder device is driven by the screw-driven mechanism using a pulse motor for delivering minute volumes. This is explained, for example, by referring to Figs. 5(a), (b) and 6(a), (b). Fig. 5(a) shows the speed of piston when the cylinder device is driven in the rectangular mode with a pulse motor, while Fig. 6(a) in the trapezoid mode with a pulse motor. The speed of injection from the nozzle, as shown in Fig. 5(b) and Fig. 6(b), forms gradual slope in the final (also in the initial) period of injection. By applying the air pressure operation in injecting a solution from the nozzle the volume of the solution which may remain undelivered at the lower end of the nozzle is minimised. The air pressure applying operation can be employed at least in the final period of injection, particularly to improve the completeness in adding drops of solution. Fig. 3(a) - (c) illustrate the case known from the prior art when the air pressure applying operation is adopted in the final period of injection. In Fig. 4(a) - (c), the air pressure applying operation is adopted in both final and initial periods of injection, in accordance with the invention. The cylinder device is employed to suck a solution in the nozzle and is also used in parallel for injecting the solution. Very high precision in injection can be obtained by employing the cylinder operation throughout the whole period of injection and the air pressure applying operation in parallel only in the initial and final periods of injection. According to the invention, the valve device is opened only for a short time after the initiation of the cylinder operation in the initial period of injection, to assure rapid growth of the speed with which the solution in the nozzle is delivered. Then the injection of the solution in the nozzle is continued by the cylinder operation and, in the final period, the valve device is opened again to assure the air pressure for rapid delivery of solution and the complete removal of the last drop from the lower tip of the nozzle. The air pressure applying operation in the final period, as described above, should, preferably, continue in ordinary cases for 0.2 - 1 sec, more preferably, 0.2 - 0.5 sec, and the same in the initial period preferably for 0.1 - 0.2 sec. The nozzle devices to which the present invention can be applied include vertically movable nozzles as well as stationary nozzles. There is no restriction on the structure of nozzle device, but the device is generally used with a replaceable nozzle tip attached at the lowest end of the nozzle. The cylinder device which is connected to cavity in the nozzle device and is mostly employed for sucking a solution may include those of the mechanical type in which piston is moved by a screw driven mechanism operated by a pulse motor and those operable by the air pressure. A certain amount of a solution is sucked by the backward movement of the piston from the initial position to generate negative pressure in the cylinder connected to the nozzle. The piston is moved to the initial position during or after the injection of solution. The compressed air source which is connected for the air pressure applying operation to the injection from the nozzle device and the valve device installed en route on the connecting line are usually assembled of an accumulator for the air pressure, a pump or a compressed air tank, and an electromagnetic valve of the ordinarily closed type. Preferably a flow rate adjusting valve is inserted as part of the valve device on the connecting line. The operation of on-off of the valves including the electromagnetic valve on an automated apparatus can be performed with the aid of a sequential control circuit which works in good time with other devices for the injection. The air pressure applying operation for injection is conducted for the purpose of applying an air pressure large enough to allow as little solution as possible to remain on the inner and outer surface of lower end of the nozzle. The pressure should amount generally to 0.1 - 5, preferably 0.1 - 0.5 atmosphere by gauge. Too small an air pressure is not sufficient to eliminate the remaining solution, but on the other hand too large a pressure may cause the solution from the lower end of the nozzle to be spattered. The air flow rate is adjusted by an air flow adjusting value to a range 20 to 100 mℓ/min, preferably 40 to 60 mℓ/min. The time duration in which the valve device is momentarily opened to apply an air pressure may be, at the initial period, selected so as to apply an amount of air big enough to increase the solution ejection speed up to a prescribed rate in a short time, but not more than the amount of the solution contained in the nozzle. At the final period, the air pressure necessary for the last period of ejection of solution is assured, and thus the amount of remaining solution can be minimized, and an almost constant amount of the solution injection can be assured from the initial period through the final period. In the case that the compressed air source employed is of such a kind as to generate a pressure only when required, the valve device may be composed of a check valve (a single direction valve). The present invention will be described in detail with reference to the accompanying drawings. Brief Explanation of the Drawings: Fig. 1 and Fig. 2 show constitutional examples of embodiments of this invention. Figs. 3 and 4 are for illustrating the speed of injection of solution according to the prior art and the invention respectively, while Figs. 5 and 6 show how the speed of injection varies when the process is carried out with the sole cylinder operation. Detailed Description of the Preferred Embodiments: In Fig. 1, 1 is a nozzle tip which is replaceable and fitted to a nozzle holder 2, and the replacement is made by a replacing device not shown in the figure. A flow route is provided through the nozzle holder 2 connecting the axial hole in the nozzle tip 1 with an air tube 3. The air tube 3, at the other end than that connected to the nozzle holder 2, is branched and one branch is connected to a cylinder room 5 in a cylinder device 4 which is screw driven by a pulse motor 6, while the other branch is connected to an aerial pressure accumulator not shown in the figure via a valve device 9 which is composed of an ordinarily closed electromagnetic valve 7 and a flow rate adjusting valve 8. The electromagnetic valve 7 is connected to a control circuit not shown in the figure and is motivated, when required during the injection, temporarily to open the route in time with the operation of other devices. Operations take place as follows. At first, the nozzle tip 1 is fitted to the nozzle holder 2 and the lower end is dipped to a certain depth in a sample solution placed in the sample vessel. Under the situation mentioned, the piston in the cylinder device 4 is moved to the pull direction (downwards in the figure) to suck the sample solution in the nozzle tip 1. The amount of sample taken can be controlled with a sufficiently high precision by controlling the distance of movement of the piston. It is also possible in the present embodiment to perform injection of a diluted solution. For this purpose, a quantity of dilution water is first sucked in the nozzle tip, followed by suction of a sample solution. Subsequently, the nozzle tip is pulled out of the sample vessel with the piston in the cylinder device being fixed and is brought just above a sample injection vessel. Then the solution in the nozzle tip 1 is transferred by injection into the vessel. This is performed, for example, by moving the piston in the cylinder device to the "insert" direction or to the home position with the electromagnetic valve 7 momentarily switched to the "open" state at the same time in order to momentarily apply a pressure of compressed air determined by the flow rate adjusting valve 8. When the injection has been finished, the electromagnetic valve 7 is momentarily switched to the "open" state to minimize the amount of solution remaining on the surface of the tip. Thus, a course of injection is completed. The operations should be repeated for further procedure. Using the apparatus shown in Fig. 1, injections of a solution from the nozzle tip 1 were performed according to the conditions and procedures described below. Precisions of injection are shown in CV-values in Table 1. Comparison example: Injection of solution according to the prior art by the cylinder operation alone. Example 1: Injection of solution according to the prior art by the cylinder operation, successively followed by the air pressure applying operation (operation in accordance with Fig. 3). Example 2: Injection of solution according to the invention by the successive operations of the temporary air pressure applying operation, the cylinder operation and the air pressure applying operation (operation in accordance with Fig. 4). Diameter of nozzle tip (at the lower end): 0.3 mm Thickness of wall of nozzle tip: 0.3 - 0.5 mm Sample: 8 % BSA in Saline Sample amount of injection (sucked and injected): See Table 1. Total amount of injection (Sample + Dilution): 200 µℓ Operations of injection Cylinder operation: 200 µℓ/2 sec Pressure of air : 0.1 atmosphere by gauge Duration of pressure application Example 1 (final period) : 0.5 sec Example 2 (initial period) : 0.1 sec (final period) : 0.5 sec Flow rate adjustment: 10 mℓ/10 sec Amount of injection (µℓ) Comparison example CV (value) Example 1 CV (value) Example 2 CV (value) 100 1.88 1.60 0.35 50 1.53 1.85 0.22 25 5.29 0.85 0.97 10 3.13 1.05 0.96 5 6.05 0.86 0.78 In the table, CV (value) where n: frequency (10 for each example) x : mean of x i 's The result manifests the superiority of the precision in injection as obtained by the present invention (Example 2) to those in the other examples. This invention can be expected to contribute those analytical systems in which precisions in injection seriously influence analytical results. In Example 2 where the pressure application in the initial period of injection accelerates the injection up to a certain speed from the start of the process, the solution to be injected does not flow around to the outer wall of the nozzle. When a viscous solution is involved, the solution flows more slowly in the nozzle. This solution is slowly injected by the pressure of the cylinder and the acceleration by the aerial pressure in the final period can effect complete injection of solution without leaving significant trace at the lower end of the nozzle. Of course, the present invention can be realized in various modified forms not restricted to the embodiment indicated in Fig. 1. The example shown in Fig. 2 employs a compressor (not shown in the figure) and a pressure regulator 12 as compressed air source. Other components in the assembly are the same as in Fig. 1 except the electromagnetic valve 7' which is usually placed in the open air. This example is evaluated to be advantageous because the compressed air from the compressed air source is usually released via the electromagnetic valve 7' into atmosphere instead of to the cylinder device. When the injection apparatus of this example is employed in an analytical measurement system, the compressor can be also used for energy source for other instruments. This permits increased freedom in designing the system and the common use of an apparatus. As has been described above, the present invention enables in injecting minute volumes of solution to remarkably decrease the injection error due to a small fraction of solution remaining at the lower end of nozzle and therefore this invention can be applied to the advantage to those apparatus for analyses and measurements which strictly require precision in injection. In the case that a cylinder device prepared for suction of a solution is used for injection purpose with the intention to realize complete delivery of the last drop of the solution, the stroke of the piston on injection is usually longer than the stroke on suction and therefore there is a void stroke on suction. Nowadays air cylinder devices employ smaller diameters and longer stems for the sake of more precision. The trend may probably give rise to troubles in controlling the amount of suction and adjustment of the injection speed. On the contrary the apparatus of this invention is free from such a trouble and permits a small and precise injection to be realized with a large effect.

Claims: Process for injecting a minute volume of a solution from a nozzle device by applying a positive pressure by a movement of a piston in a cylinder device (4) and applying a positive pressure from a source of compressed air (10) in a final period of the injection when an end position of said piston in said cylinder device (4) is reached, characterized in that said pressure applied from said source of compressed air (10) is applied additionally in an initial period of injection starting with the movement of said piston and ending before the beginning of said final period, such that no pressure is applied from said source of compressed air (10) in an intermediate period which endures from the end of said initial period to the beginning of said final period. An automated apparatus for injecting a minute volume of a solution comprising a nozzle to suck and inject said solution, a cylinder device (4) with a movable piston, said cylinder device (4) being connected to said nozzle (3) and a compressed air source (10) connected to said nozzle (3) to apply a positive pressure for ejecting said solution from said nozzle and an electromagnetic valve device (9) which is placed on a line connecting said compressed air source (10) to said nozzle and which is operated with the aid of a sequential control circuit characterized in that said sequential control circuit is such that said valve device (9) is open in a final period of injection which starts when an end position of said piston in said cylinder device (4) is reached and that said valve device (9) is also open in an initial period of injection which starts with the movement of said piston and ends before the beginning of said final period, such that said valve device (9) is closed in an intermediate period which endures from the end of said initial period to the beginning of said final period. Apparatus of Claim 2 wherein the nozzle device is attachable with a replaceable disposable tip.

Inventors: HAYASHI, HIDECHIKA, HIGO, YUJI, IWASAKI, SHUJI
Applicants: TOSOH CORPORATION
Application year: 1986
Main classification: G01N1
IPC codes: G01N30, G01N1