JPH01212356A - Diluted specimen preparation apparatus for liquid chromatography - Google Patents

Abstract

PURPOSE:To automatically prepare a specimen having high dilution accuracy and to simply alter a diluent, by a method wherein the diluent and an operating liquid are separated, and a diluent layer and an operating liquid layer are respectively sucked in a suction/emission pipe filled with the operating liquid in this order through an air layer to be held therein. CONSTITUTION:A downward suction/emission nozzle 3 is provided to the leading end of a liquid transfer suction/emission pipe 4 provided so as to extend from a suction/emission operating liquid storage tank 2 and a syringe type pump 5 is provided on the way of said pipe 4. The pipe 4 from the tank 2 to the nozzle 3 is filled with an operating liquid. This nozzle 3 is moved vertically and horizontally by a control part 16, and a small amount of air, a predetermined amount of a diluent, a small amount of air and a predetermined amount of a specimen are successively sucked and held between a diluent storage tank 13, a specimen container 14 and a diluted specimen preparing container 15 all of which are arranged at predetermined positions and emitted into the container 15. During this time, the rising speed of the nozzle is controlled to 1cm/sec or less by the control part 16 after the specimen is sucked.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial Application Field This invention relates to a diluted sample preparation device for liquid chromatography. More specifically, the present invention relates to an apparatus that can prepare and accurately prepare a diluted sample when introducing the diluted sample into liquid chromatography for separation analysis.

(b) Conventional technology Liquid chromatography has been widely used for the qualitative and quantitative determination of various analyte components, but due to the measurement range of the detector, column capacity, etc. It is often necessary to dilute the sample to a predetermined ratio with a diluent. Conventionally, such operations have been performed mainly manually, but they are far too cumbersome when a large number of samples are analyzed continuously.

Therefore, it has recently been considered to apply an automatic sample diluter or mechanism, such as those applied to general clinical biochemical analyzers. Such an automatic diluter generally has a syringe-type pump and a suction/discharge nozzle, and uses a suction/discharge pipe filled with diluent, and after aspirating a predetermined amount of sample through the nozzle, , a device is known that discharges a predetermined amount of diluent from a nozzle into an empty container.

However, in this device, diluent is used as a working fluid in the suction and discharge tubes, so it is difficult to change the diluent, and various diluents are required depending on the sample. It was not suitable for use as a pretreatment diluter for liquid chromatography.

Conventionally, the suction and discharge pipes are filled with a special working fluid (usually a solvent that can clean the pipes), and a predetermined amount of sample is aspirated from the sample container, discharged into the empty container, and then independently An apparatus for preparing a diluted sample by discharging a predetermined amount of diluent into the empty container from an installed diluent tank that can be easily replaced is used as a diluted sample preparation apparatus for liquid chromatography.

In order to improve dilution accuracy by preventing sample concentration and dilution preparation solution concentration due to evaporation during the dilution process,
The sample container and empty container used above are sealed with a diaphragm (septum), and correspondingly, the suction/discharge nozzle has a needle-like tip that can penetrate this diaphragm. Ta.

(c) Problems to be Solved by the Invention However, in the above-mentioned conventional diluted sample preparation apparatus, it was not possible to obtain sufficient dilution accuracy for accurate quantitative determination by liquid chromatography. The causes of this are not necessarily clear; for example, the sample may be spread into the hydraulic fluid due to contact between the working fluid in the suction/discharge pipe and the sample, or the nozzle may be pulled out of the sample container and placed into an empty container. When transferring, a part of the sample may fall out of the nozzle due to the instantaneous depressurization or shock inside the sample container.
This may be caused by a change in the amount of sample discharged.

For this reason, it is possible to interpose a small amount of air between the sample and the working fluid when aspirating the sample, but it has not been possible to achieve a sufficient improvement in dilution accuracy.

This invention was made under such circumstances, and in particular,
The present invention aims to provide a sample preparation device that can automatically prepare a diluted sample with high dilution accuracy suitable for liquid chromatography, and can also easily change the diluent.

(d) Means for Solving the Problems Thus, according to the present invention, (a) a storage tank for suction/discharge hydraulic fluid, a liquid transfer pipe extending from the storage tank, and a tip of the liquid transfer pipe. The suction/discharge device is composed of a downwardly connected needle-shaped suction/discharge nozzle and a syringe-type pump attached in the middle of this liquid transfer pipe, and is filled with the working fluid from the storage tank to the suction/discharge nozzle. (b) a nozzle drive unit that moves the suction/discharge nozzle vertically and horizontally; (c) diluent storage tanks each disposed at a predetermined position below the horizontal movement path of the suction/discharge nozzle; It is equipped with a diaphragm-sealed sample container and a diluted sample preparation container, and (d) a control unit that controls the operation of the syringe type pump and the nozzle drive unit, and the control unit controls the horizontal and vertical movement of the discharge/suction nozzle. A small amount of air, a predetermined amount of diluent in the diluent storage tank, a small amount of air, and a predetermined amount of sample in the sample container are suctioned and held in the suction/discharge pipe in this order, and then the retained diluent and sample are There is provided a diluted sample preparation device for liquid chromatography, comprising a control means for discharging the sample into a diluted sample preparation container and for limiting the rising speed of the suction/discharge nozzle after suctioning the sample to 1 cm/sec or less. Ru.

This invention separates the diluted liquid and the working liquid, and suctions and holds the diluted liquid layer and the sample liquid layer in this order through an air layer in the suction/discharge pipe filled with the working liquid, and in this state. The structure is such that a diluted sample can be prepared by discharging both of these layers into a diluted sample R container (empty container). Furthermore, the configuration is such that the speed of the suction/discharge nozzle that is pulled out from the sample container is significantly reduced.

In this invention, the diluent and the sample are always sucked into the suction/discharge tube first.

If it is reversed, it is difficult to obtain high dilution accuracy.

Further, the diluent and the sample are each sucked and held through a small amount of air. If the sample is held by suction without using air, high dilution accuracy may not be obtained, for example, when multiple samples are sampled from the same sample container, which is not suitable in terms of reliability of the apparatus.

Note that the diluent is appropriately selected depending on the solubility of the sample to be used. On the other hand, the suction/discharge hydraulic fluid is not particularly limited as long as it is chemically stable, and usually water or an alcohol-based solvent may be used.

On the other hand, the rising speed of the suction/discharge nozzle is 1 cm/sec or less. If the control is performed faster than this, it is not suitable because a part of the aspirated sample will fall from the nozzle tip due to the sudden pressure reduction and impact within the sample caused by lifting the nozzle through the diaphragm, causing fluctuations in the sample amount.

(e) Function The diluent sucked first in the suction/discharge pipe acts to reliably prevent contact and mixing between the sample to be sucked later and the suction/discharge hydraulic fluid in the pipe. At the time of dispensing, this diluted liquid is transferred to the suction where the sample was held.
Since the sample is discharged through the discharge tube, particularly through the suction/discharge nozzle, it serves to prevent volume errors that may occur due to the sample adhering to the tube wall. Furthermore, since the rising speed of the suction/discharge nozzle that is withdrawn from the sample container is set to 1 cm/sec or less, partial dropout of the sample from the nozzle tip that may occur during this operation is reliably prevented.

These effects work together to perform a dilution operation with high dilution accuracy.

(f) Example FIG. 1 is a perspective view showing an example of a diluted sample preparation device for liquid chromatography according to the present invention, and FIG. 2 is a diagram illustrating the configuration thereof.

In the figure, diluted sample preparation device for liquid chromatograph 1
is the storage p1 of suction/discharge hydraulic fluid (e.g. methanol)
It has a built-in suction/discharge pipe 4 made of polytetrafluoroethylene which is connected from the needle-shaped suction/discharge nozzle 3 to a needle-shaped suction/discharge nozzle 3. A syringe type pump 5 is provided. Here, the inside of the suction/discharge pipe 4 is filled with the above-mentioned working fluid.

Here, the suction/discharge nozzle 4 has a built-in pulse motor with variable speed and reverse capability, and this motor and linear gear 10
It is attached to a nozzle support 9 that can be moved up and down in combination with the nozzle support 9. On the other hand, 18 in the figure indicates a nozzle support 9.
This is a supporting moving device for moving the nozzle supporting device in the horizontal X direction together with the storage box 24, and a moving means consisting of a pulley 7 and a motor 8 for moving the nozzle supporting device is built inside.

Further, this support moving tool 18 itself is also configured to be movable in the horizontal Y direction, and a pulley 1 that performs this movement is provided.
1 and a motor 12 are built into the main body 22 of the device. That is, the nozzle 3h (X-Y (7)) is configured to be movable horizontally or vertically by driving each of these motors 8,9,1277).

Further, the syringe type pump 5 is provided with a pump driving means 6 consisting of a cam 61 and a pulse motor 62.
The motor 62 is controlled to perform a suction operation or an extrusion operation of the pump 5.

On the other hand, below the horizontal movement path of the nozzle 3, there is a storage tank 13 for a diluent (for example, methanol), a plurality of sample containers 14 containing samples, and a plurality of diluted sample preparations consisting of empty containers. Container 15... is arranged,
Each of these is covered with a rubber diaphragm 25.degree. 26.27 on the upper side to be in a sealed state. In addition, in the figure, 19 shows a sample container mounting stand, and 20 shows a diluted sample preparation container mounting stand, respectively.

Further, in the figure, reference numeral 16 denotes a control unit for controlling the drive of each motor and magnetic valve 1 as will be described later, and is controlled by a program control method using a microprocessor, and 17 is a keyboard. Note that 21 indicates waste liquids for temporarily storing cleaning waste liquid from the nozzle 3.

A series of control steps of the control section 16 in the apparatus 1 and a dilution step thereof will be described below.

First, when the dilution operation start key on the keyboard 17 is pressed, the control unit 16 horizontally moves the nozzle 3 onto the diluent storage tank 13, and there drives the pump 5 to release a small amount (usually 3 to 531 (2)). After suctioning the air, the nozzle 3 is lowered to penetrate the diaphragm 25, and the nozzle lowering is stopped with the tip of the nozzle inserted into the diluent.In this state, the pump 5 is subsequently driven to dispense a predetermined amount. The diluted liquid is sucked into the nozzle 3 (first step). Here, the suction time is determined in accordance with the amount input in advance from the keyboard 17.

After this suction step is completed, the nozzle 3 is raised (ascending speed 5 CIIIl/sec; high speed mode) and pulled out from the diluent storage tank 13, and then placed horizontally on the sample container t4 containing the sample intended for dilution preparation. move it. Here, the movement position is the sample container I entered in advance from the keyboard 17.
It is determined in accordance with the D number, etc.

In this state, after driving the pump 5 to suck in a small amount of air similar to the above, the nozzle 3 is lowered to penetrate the diaphragm 26, and the nozzle lowering is stopped with the nozzle tip inserted into the sample. . Subsequently, the pump 5 is driven to suck a predetermined amount of sample into the nozzle 3 (second step). The suction time here is determined in accordance with the amount input in advance from the keyboard 17 in the same manner as described above.

Thereafter, the nozzle 3 is raised and pulled out from the sample container 14, and then horizontally moved onto a predetermined diluted sample preparation container 15. However, the nozzle rising speed at this time is different from the above-mentioned rising and falling speeds, and is adjusted to an extremely warm speed of lc'+/second (low speed mode). As a result, a predetermined amount of the aspirated sample can be reliably collected without being affected by the reduced pressure inside the container or vibrations during ascent.

FIG. 3 shows the state of the diluted liquid and sample sucked and held in the nozzle through the above steps. In the diagram, A is the hydraulic fluid, B
C indicates the diluted solution, and C indicates the sample. In this way, sample C is isolated from hydraulic fluid A by the layer of diluent B, so sample C is not brought into hydraulic fluid A. The slight diffusion of the sample liquid C into the diluted liquid via the air layer does not affect the dilution accuracy since they are the objects to be mixed in the end. Note that the slight diffusion of the diluent B into the working fluid A does not substantially adversely affect the dilution accuracy.

Next, the control unit 16 lowers the nozzle 3, inserts its tip into the preparation container 15, and discharges the sample C and diluent B into the container 15 (third step). At this time, sample C is discharged first and then diluted liquid B is discharged, so the diluted liquid exerts a kind of cleaning effect on the tube wall, and the sample remains attached to the inner wall of the nozzle tip, which has a negative effect on dilution accuracy. It also prevents the effects of Therefore, a diluted sample with the intended high dilution accuracy is prepared in the container 15.

After the series of dilution steps described above is completed, the nozzle 3 is transferred onto the waste liquid 21 shown in Fig. 1, where the inside of the pipe is cleaned by discharging a predetermined amount of working fluid, and subsequent dilution is performed. It is considered to be in the preparation state for the process.

The sequentially prepared diluted samples are then transferred to the sample introduction port of the liquid chromatograph by another syringe type pump (not shown) and subjected to separation analysis.

In addition, using the above-mentioned apparatus 1, methanol is used as a diluent,
When a 5-fold dilution was prepared using diphenyl as a sample, it was confirmed that the dilution error was significantly improved to within 0.5%.

(G) Effects of the Invention According to the diluted sample preparation device of the present invention, it is possible to automatically prepare a diluted sample with high dilution accuracy suitable for separation and quantification by liquid chromatography. Since the working fluid is also separated from the diluent, the diluent can be changed freely, making it extremely suitable for liquid chromatography.

[Brief explanation of the drawing]

FIG. 1 is a perspective view showing an embodiment of the diluted sample preparation device of the present invention, FIG. 2 is an explanatory view of the configuration, and FIG. 3 is an explanatory view showing the state of the liquid in the nozzle during the dilution process. . ! ......Diluted sample preparation device for liquid chromatography, 2...Storage tank, 3...Suction/
Discharge nozzle, 4...Suction/discharge pipe, 5...
...Syringe type pump, 6...Pump driving means, 61...Cam, 62...Pulse motor, 7.11...Pulley, 8.12・
...Motor, 9...Nozzle support,
lO... Linear gear, 13... Diluent storage tank, 14... Sample container, 15...
- Diluted sample preparation container, 16...Control unit, 1
7... Keyboard, 18... Support moving tool, 19... Sample container mounting stand, 20...
・Diluted sample preparation container mounting stand, 21...waste liquid bottle,
22... Body, 23... Solenoid valve, 24... Storage box, 25.26.27.
...Diaphragm, A...Hydraulic fluid, B...
- Diluent, C...sample solution. Figure 1

Claims (1)

[Claims] 1. (a) A storage tank for suction/discharge hydraulic fluid, a liquid transfer pipe extending from the storage tank, and a needle-shaped suction/discharge pipe connected downward to the tip of the liquid transfer pipe. (b) a suction/discharge pipe consisting of a nozzle and a syringe-type pump attached midway through the liquid transfer pipe, and filled with the working fluid from the storage tank to the suction/discharge nozzle; and (b) the suction/discharge nozzle. (c) a diluent storage tank, a diaphragm-sealed sample container, and a diluted sample preparation container, each disposed at a predetermined position below the horizontal movement path of the suction/discharge nozzle; and (d) a control unit that controls the operation of the syringe-type pump and the nozzle drive unit, and the control unit moves a small amount of air into the diluent storage tank by horizontally and vertically moving the discharge/suction nozzle. a predetermined amount of diluent,
A small amount of air and a predetermined amount of the sample in the sample container are suctioned and held in the suction/discharge tube in this order, then the retained diluent and sample are discharged into the diluted sample preparation container, and the suction/discharge after the sample suction is performed. A diluted sample preparation device for liquid chromatography, comprising a control means for limiting the rising speed of a discharge nozzle to 1 cm/sec or less.