JPS593258A - Gradient apparatus in liquid chromatography - Google Patents

Abstract

PURPOSE:To make it possible to prepare an elution liquid with a predetermined concn. gradient extremely simply, by providing a first flow line guiding a start solvent at the time of gradient start and a second flow line guiding a finish solvent at the time of gradient finish from the storage tank thereof. CONSTITUTION:A predetermined start solvent A is accommodated in a pump. cylinder 2 and sent and supplied through a start solvent flow line 4 being a first flow line by operating the piston of the pump cylinder 2. On the other hand, the finish solvent B accommodated in a storage tank 6 is sent and supplied through a finish solvent flow line (second flow line) 8 by a quantitative emission type pump 10 provided to said flow line 8. By this constitution, an elution liquid with a predetermined concn. gradient can be prepared extremely simply.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a gradient device for liquid chromatography, and particularly for semi-micro high performance liquid chromatography performed at a flow rate of approximately 1πl/min or less.
The present invention relates to an apparatus capable of effectively preparing an eluent in which the concentration of different solvents changes over time.

In recent years, liquid chromatography as a means of separation and analysis has been attracting attention in the fields of chemistry, biology, medicine, etc. In order to effectively elute the target substance from the separation column, it is necessary to Rather than using one type of solvent, methods have been adopted that use two or more different solvents, and one method in particular involves mixing different solvents and changing the mixing ratio over time to achieve separation. Gradient elution (gradient) methods for feeding into columns are attracting attention. On the other hand, such liquid chromatography requires 5Qkg/c due to its usage requirements.
High-performance liquid chromatography (I
(PLO).

By the way, there are two types of gradient devices conventionally used to perform gradient elution in liquid chromatography, especially high-performance liquid chromatography: high-pressure mixing type and low-pressure mixing type. This also applies to cases where the eluent flow rate is approximately 1πt/min or more, such as semi-micro high performance liquid chromatography, where the eluent flow rate is less than 1 m11 min, or even 300 m2/min.
If it is less than /1/1/min, problems will occur that are too large to be of practical use in any type of device.

In other words, in the case of a high-pressure mixing type, for example,
As shown in Publication No. 19959, etc., gradient elution is performed by changing the flow rate ratio of two pumps.
When adopting a mixing ratio of 99:1 in l/min, the flow rate ratio of the pump is 99 μl/min: 1 pl/min, which requires a pump that accurately delivers ultra-low flow rates.
This is technically extremely difficult, and also requires two
The system was not economical because several pumps were required.

On the other hand, in the other low-pressure mixing type device, as shown in Japanese Patent Publication No. 56-38664, etc., gradient elution is performed using one pump and a mixing proportional valve. Even if the dead volume from the proportional valve to the pump can be made as small as possible, the presence of about 2 to 8ynl5- is unavoidable, and if this continues, it will be difficult to program the separation column after starting the gradient flow diagram. This was completely impractical because it took 20 to 30 minutes for the mixed solvent to arrive.

Moreover, this type of dead volume is practical.”
200-300 corresponding to an acceptable 2-3 minute delay
It is extremely difficult to reduce the amount to less than μl due to the structure of the pump used, and considering that the suction amount per stroke of the pump is 50 to 150,711, it is difficult to draw a smooth gradient curve. It was impossible to mix two types of solvents.

The solvent is mixed gradiently with a proportional valve.
Since 50 to 150 μl of liquid is stored in the pump and gradually pumped out, it does not actually draw a smooth gradient curve, but a step-like gradient curve of 50 to 150 μβ. This is because it will happen.

In addition, Japanese Patent Publication No. 54-20157 describes a method for micro high performance liquid chromatography that is performed in an area with extremely few flowmeters, in which the discharge flow rate of the transfer liquid (eluent) is about 10 tt (about 1/min or less). As a gradient elution method used in A batch method has been disclosed in which an eluent with a For example, semi-micro high performance liquid chromatography, which requires a large amount of eluent, is technically very difficult, and also poses problems with the accuracy of the concentration gradient of the eluent. This results in the problem of long analysis time, and the operation is extremely troublesome because it requires frequent connection of gradient tubes and separation columns, etc., and separation operations. There are also inherent problems.

The present invention has been made in view of the above circumstances, and its purpose is to provide a gradient device for liquid chromatography, particularly a gradient device for liquid chromatography in the semi-micro region. It is about providing.

Another object of the present invention is to provide an economical and inexpensive gradient device with a simple structure that can change the mixing ratio of different solvents over time and continuously supply the mixture to a separation column of a predetermined liquid chromatography device. The aim is to provide

In order to achieve these objectives, the gradient device according to the present invention has (a) a 10th channel for guiding the starting solvent at the beginning of the gradient, and (1) a terminal solvent at the end of the gradient. (C) a pump means provided on the second flow path for feeding the finished solvent; (d) the second and second flow paths; 〇 A third flow path provided on the downstream side of the flow path and connected to any of them, and (0) for the starting solvent provided in the third flow path -L and containing 1 volume. (f) an eluent reservoir equipped with a stirring mechanism and a variable capacity mechanism that mixes the final solvent to form an eluent whose concentration changes over time, and (f) is provided on the downstream side of the flow path No. (g) a 40th channel connected to either the 30th channel or the 20th channel to guide the liquid guided through the channels to the separation column of the liquid chromatography apparatus; connecting the second flow path and the fortieth flow path at the same time; connecting the twenty flow path and the thirtieth flow path; At the same time, the present invention is characterized in that it includes a switching valve mechanism that can selectively perform a connection operation for connecting the third flow path and the fourth flow path.

Therefore, according to the present invention, the eluent reservoir contains the starting solvent in advance, and the starting solvent is sequentially diluted by the continuous introduction of the finishing solvent supplied by the pump means, so that the concentration changes over time. This eluent is used as an eluent, and this eluent is continuously fed through a flow path to a separation column of a liquid chromatography device.
It has become possible to prepare an eluent having a predetermined concentration gradient extremely easily, and it has also become possible to prepare such an eluent continuously. Moreover, since the eluent can be formed in the eluent reservoir simply by switching the flow path using the switching valve means, it is possible to significantly simplify the structure as well as the operation. Even in semi-micro high performance liquid chromatography where the flow rate of eluent supplied to the separation column is approximately 1 ml/min or less, preferably 300 μl/min or less, the gradient elution operation can now be performed continuously. . Furthermore,
According to the present invention, the starting solvent and the ending solvent can be arbitrarily introduced into the eluent reservoir by switching the flow path using the switching valve means, so that the eluent preparation cycle having a concentration gradient is automatically repeated over time. This made it possible to perform continuous gradient elution operations.

EMBODIMENT OF THE INVENTION Hereinafter, in order to clarify the present invention more specifically, examples of the present invention will be described in detail based on the drawings.

10- First, in FIG. 1, 2 is a pump syringe, in which a predetermined starting solvent A is accommodated. and,
The piston action of the pump syringe 2 causes the starting solvent A to be fed through the starting solvent flow path (No. 10 flow path) 4. Further, the finished solvent B stored in the storage tank 6 is passed through the finished solvent channel (second channel) 8 to a constant discharge type pump 10 provided on the channel 8.
It is designed to be delivered by. These starting solvent channel 4 and ending solvent channel 8 are connected to each boat of a six-way valve 12, which is a switching valve mechanism, and are connected to the other two boats of this six-way valve, at the upstream end and the downstream end. An eluent forming channel (channel No. 30) 14 whose ends are connected to form a loop is provided.

An eluent reservoir 16 is provided in the eluent forming channel 14, which does not form a loop with respect to the six-way valve 12. This eluent reservoir 16 has a magnetic stirrer 18 at its lower part. As is well known, the magnetic stirrer 18 is a stirrer housed in the eluent reservoir 16. The magnetic stirrer 18 is used to stir the liquid in the eluent reservoir 16. Further, a piston 24 as a capacity variable mechanism is provided at the upper part of the eluent reservoir 16 so as to be able to slide in liquid-tight contact with the eluent reservoir 16. The volume within the liquid reservoir 16 can be changed arbitrarily.

Furthermore, an eluent supply path (40th flow path) 28 is connected to one boat of the six-way valve 12, and a liquid chroma i - The sample injector 30, separation column 32, and further detector 34 of the imaging device are connected in sequence.

Note that the six boats 36, 38, 40, 42, 44, and 46 arranged in an annular manner of the six-way valve 12 have an end solvent flow path 8. Eluent supply path 28. The downstream end of the eluent forming channel 14, the drain channel 48. Starting solvent flow path4. The upstream ends of the eluent forming channels 14 are connected in a circular manner, and by rotating the handle 50 between the solid line position and the broken line position, each boat can be connected to either of the adjacent boats on both sides. It is designed to be connected to. That is, as shown in the figure, the handle 5
When 0 is at the solid line position, connections are made between ports (36, 46, 38, 40, 42, and 44) as shown by the solid line, and when 0 is at the broken line position, connections are made as shown by the broken line. Between the boats (36 and 38.40 and 42.44 and 46)
connections are made.

Therefore, in the gradient apparatus having such a configuration, the starting solvent A in the pump syringe 2 is first supplied and contained in the eluent reservoir 16 prior to gradient elution. For this operation, the handle 50 of the six-way valve 12 is
is moved to the dashed line position, thereby connecting the boat 44 connected to the starting solvent flow path 4 and the boat 46 connected to the upstream end of the eluent forming flow path 14, while Boat 40 connected to the downstream end of 14
is connected to the boat 4213- connected to the drain channel 48, whereby the starting solvent A in the pump syringe 2 is transferred to the starting solvent channel 4213- by the actuation of the piston of the pump syringe 2. The solvent is supplied into the eluent reservoir 16 through the six-way valve 12 and the eluent forming channel 14, and the solvent in the eluent reservoir 16 is thereby forced out and discharged through the drain channel 48. . In addition, by switching the six-way valve 12, the boat 3 to which the end solvent flow path 8 is connected
6 and the boat 38 to which the eluent supply path 28 is connected, the finished solvent B in the storage tank 6 is sent to the separation column 32 by driving the pump 10, and The filler and the like filled therein will be washed by the finishing solvent B.

When the eluent reservoir 16 is filled with the starting solvent, the handle 50 of the six-way valve 12 is rotated from the dashed line position to the solid line position, thereby causing the boat 3
6 and the boat 46 are communicated, and the solvent B in the storage tank 6 is
is terminated by the suction 14-discharge operation of the pump 10. From the solvent flow path 8 to the eluent forming flow path 1
4 and flow into the eluent reservoir 16. Then, the starting solvent A in the eluent reservoir 16 is gradually diluted by the finishing solvent B flowing in, and the stirring action by the magnetic stirrer 18 causes a uniform concentration that changes over time. The eluent is used as an eluent, and is led from the connected boat 40 through the boat 38 to the eluent supply path 28, and then sent to the separation column 32.

Note that the gradient curve of the eluent formed in the eluent reservoir 16 is given by the following equation.

t ■OC (also) = C (-(Cc -C; ) ・ e
However, C(1.): Concentration of the eluent formed after time t C1: Initial concentration in the eluent reservoir 16, in other words, concentration of the starting solvent A C': End of the eluent flowing into the eluent reservoir 16 Concentration of solvent B ■0: Volume of eluent reservoir 16 Q: Discharge flow rate of pump 10 Therefore, concentration of eluent formed after 1 hour CCt'
Therefore, l is proportional to Q/V o .

In other words, the inclination of the eluent gradient can be controlled by Q/Vo. Therefore, in the above example, a variable capacity mechanism (24 , 26).However, the position of the piston 24 of the variable capacity mechanism is fixed, and the pump 10 for feeding the finished solvent B is changed from a constant discharge type pump to a variable discharge rate pump. By this,
It is also possible to control the slope of the eluent gradient that is formed.

The eluent thus formed in the eluent reservoir 16 is guided into the eluent supply path 28 through the connected boats 40 to 38, and is sent to the separation column 32. Become. Note that the bow 1 44 of the six-way valve 12 connected to the starting solvent flow path 4 is connected to the boat 42 connected to the drain flow path 48, but the pump syringe 2 is not operated. Therefore, the starting solvent A in the syringe 2 will not be discharged into the drain channel 48. In addition, such eluent supply path 28
As is well known, the eluent whose concentration changes over time is supplied through the sample injector 30, develops the sample in the separation column 32, separates it into its respective components, and then The light is guided to a detector 34, where each component is detected.

In this way, according to the gradient device having such a configuration,
The eluent that is formed in the eluent reservoir 16 and has a concentration gradient over time can be formed extremely easily by simply switching the six-way valve 12, and as a result, the eluent that has a concentration gradient over time can be formed extremely easily. minutes, especially 300
It has become possible to effectively perform gradient elution operations in high performance liquid chromatography in the 7-micron range of microliters per minute or less, and it has become possible to put it into practical use1.
7- It is.

Moreover, the gradient eluent can be prepared simply by flowing the finished solvent B into the eluent reservoir 16, unlike the conventional method of using two pumps or a proportional valve. Therefore, in addition to the effect that only one pump 10 needs to be used, it is also possible to achieve a simple structure in terms of the device.

Further, according to the embodiment with such a configuration, the eluent preparation operation is sufficient simply by flowing the finished solvent B into the eluent reservoir 16, so the presence of dead volume in the pump 10 etc. is almost eliminated. It is possible to ignore it.

Furthermore, according to the above-mentioned apparatus, the pump syringe 2 is placed in the eluent reservoir 16 by simply switching the six-way valve 12.
Since the starting solvent A in the storage tank 6 and the ending solvent B in the storage tank 6 can be introduced at will, it also has the great feature that the gradient elution operation can be performed automatically and continuously.

Incidentally, the effectiveness of this device can be seen from the following experimental results. It is easily recognized.

That is, in Figure 2, 0.05% of acetone is used as the starting solvent A.
This is a gradient curve showing the concentration change when a gradient eluate was prepared using methanol containing % of % and methanol as the final solvent B. Afterwards, a clear continuous change in concentration was observed, and the line was stable, so it was determined that the two solvents were sufficiently mixed. It is also understood that it is desirable to inject the sample (from the sample injector 30) after an appropriate amount of time has elapsed from the start of the gradient.

In addition, Fig. 3 shows a chromatography obtained by performing gradient elution of aromatic compounds using the apparatus shown in Fig. 1.
), as well as (a) when an isocratic eluent is used,
(0) is shown. The measurement conditions are as follows.

Flow rate i I 00 μl / ”10 Separation column; /lS r 'N EP AK
+S' L0181 Made by JASCO Corporation @): Filled with "silica gel chemically bonded with octadecyl groups" with an inner diameter of 1°5 mm and a length of 250 mm.

Ultraviolet spectroscopic detector, JA8COUVIDEC-1oo-1
11 [manufactured by JASCO Corporation].

Detection wavelength: 254 nm As is clear from the results in Figure 3, the device according to the present invention (
Good separation performance has been obtained by gradient elution using the method (Fig. 1).

Also shown in FIG. 4 is another embodiment of the invention which is different from the gradient apparatus as shown in FIG. There, instead of the six-way valve 12 in the device of FIG. 1, there are two valves, an inlet valve 52 and an outlet valve 54.
This differs greatly from the previous example in that the eluent reservoir (6) is provided before and after the eluent reservoir (6). That is, the inlet valve 52 and the outlet valve 54 are operated simultaneously, and the 6
Similarly to the direction valve 12, the connection operation of connecting the starting solvent channel 4 and the eluent forming channel 14 and simultaneously connecting the ending solvent channel 8 and the eluent supply channel 28, and It is possible to selectively perform a connection operation of connecting the eluent forming channel 14 and the eluent supplying channel 28 at the same time as connecting the channel 8 and the eluent forming channel 14, The drawing shows the state in which the former connection operation has been performed. In addition,
This is slightly different from the previous example in that the termination solvent flow path 8 and the eluent supply path 28 are not directly connected, but a short-circuit flow path 56 is provided between them. There is. Further, 58 is a drain channel, which is used to drain the eluent by means of the outlet valve 14 in order to drain other liquids from the eluent reservoir when the starting solvent A is being supplied to the eluent reservoir 16. It is provided so as to communicate with the reservoir 16.

In this way, the same effects as in the previous example can be achieved by switching the flow paths using the two inlet valves 52 and outlet valves 54.

The present invention can be advantageously employed when the flow rate of the solution to the separation column as described above is set to 10 μ4 to 1 m11 minutes, but it can also be applied in areas where the flow rate is extremely low, e.g. It is possible to apply it even at a rate of about 0.01 μl/min.

Furthermore, the composition of the starting solvent A and the ending solvent B may be composed of a single component or a plurality of components.

Furthermore, it goes without saying that various other changes and modifications can be made to the present invention based on the knowledge of those skilled in the art, without departing from the spirit of the present invention as defined in the claims.

[Brief explanation of drawings]

FIG. 1 is a system diagram schematically showing an example of a liquid chromatography device equipped with a gradient device according to the present invention, and FIG. 2 is a graph showing a gradient curve obtained using the device shown in FIG. , Fig. 3(a), (b), (
C) shows the chroma22-togellum obtained by gradient elution using an isocratic eluent, an eluent formed by the apparatus of FIG. 1, and another isocratic eluent, respectively; FIG. FIG. 3 is a system diagram showing a chromatography apparatus equipped with a device showing another embodiment of the invention. 2: Pump syringe 4: Starting solvent flow path 6: Storage tank
8; End solvent flow path 10: Pump 1
2: 6-way valve 14: Eluent forming channel 16: Eluent reservoir 18: Magnetic stirrer 20: Stirrer 22: Electromagnetic drive section 24: Piston 26: Nut 28: Eluent supply channel 30: Sample injector 32-Separation column 34: Detector 36.88, 40, 42, 44, 46: Boat 48:
Drain channel 50: Handle 52: Inlet valve 54: Outlet valve 56: Short-circuit channel 58 Nidrain channel Applicant JASCO Corporation 23- Figure 1 Figure 2 OIo 20 30 m1n0
10 (min)

Claims (5)

[Claims] (1) A 10th flow path for guiding the starting solvent at the start of the gradient, a 20th flow path for guiding the ending solvent from the storage tank at the end of the gradient, and the above-mentioned flow path provided on the second flow path. a pump means for feeding the finished solvent; a 30th flow path provided on the downstream side of the 1st and 20th flow paths and connected to any of them; and a 30th flow path on the third flow path. an eluent reservoir provided with a stirring mechanism and a volume variable mechanism for mixing the termination solvent into the contained starting solvent to form an eluent whose concentration changes over time; and the 30th stream. a 40th channel that is provided on the downstream side of the channel, is connected to either the 30th channel or the 20th channel, and guides the liquid guided through the channels to the separation column of the liquid chromatography device; and a connecting operation of connecting the 10th flow path and the 30th flow path and simultaneously connecting the 20th flow path and the 4th flow path;
A switching pulp mechanism capable of selectively performing a connecting operation of connecting the 20th flow path and the 3rd flow path and simultaneously connecting the 30th flow path and the 40th flow path; , a gradient apparatus in liquid chromatography, characterized in that it comprises: (2) The switching valve mechanism is a 6-type valve, and the six boats arranged in an annular manner are connected to the downstream end of the 20th flow path, the 40th flow path, the downstream end of the third flow path, and the drain flow path. 11. The channels, the 10th channel, and the 3rd channel are connected in an annular manner in the order of the second end of the channel; 11. 2. The device according to claim 1, wherein each boat is connected to either of the adjacent boats on both sides by a switching operation. (3) The device according to claim 1, wherein the pump means is a constant discharge pump that performs discharge at a constant flow rate. (4) The apparatus according to claim 1, wherein the starting solvent is entirely introduced into the first flow path by a pump syringe containing and holding the starting solvent. (5) Approximately 10 μl to 1.0 μl is discharged through the 40th flow path by the discharge operation of the pump means. 2. The apparatus according to claim 1, wherein the liquid is fed to said separation column at a rate of ml/min.