JP3304394B2 - Gradient device for high-performance liquid chromatography - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a gradient device in a liquid chromatograph for analyzing components of a sample using a sample loop and a switching column.

[0002]

2. Description of the Related Art As shown in FIG. 8, a conventional high-performance liquid chromatograph gradient apparatus comprises two pumps P1 and P2 for sending mobile phases L1 and L2 contained in vessels F1 and F2, respectively. Is controlled by a control function such as a computer to change the flow rate of liquid sent to the lines 1 and 2, thereby changing the mixing ratio of the mobile phases L1 and L2 in the line 3 to obtain a gradient effect. .

[0003]

According to the above method,
For example, 100 μl (microliter) / m in line 3
The mobile phase of “in” is passed through the injector I to the separation column C,
When the liquid is sent to the detector MS, if the ratio of the mobile phase L1 to the mobile phase L2 is 99: 1, 99 μl / mi is added to the line 1.
n, pump P to send 1 μl / min to line 2
1 and the pump P2 will be controlled. However, the flow rate of a relatively inexpensive pump used for a liquid chromatograph or the like is normally guaranteed at about 50 μl / min or more. Therefore, if 1 μl / min is sent by such a pump, It means that stable liquid sending cannot be obtained. For this purpose, a high-precision pump capable of accurately feeding 1 μl / min is required, but such a pump is extremely expensive. Further, a mechanism for controlling the pumps P1 and P2 is also required, but since this control mechanism is also expensive, a gradient apparatus of a high-speed chromatograph that can obtain reproducibility is expensive according to the conventional method. Accordingly, an object of the present invention is to provide a gradient device in a high-speed chromatograph that can be configured at low cost and that can perform simultaneous analysis of multiple components with high reproducibility.

[0004]

Therefore SUMMARY OF THE INVENTION, Lou between line supplying line for supplying a first mobile phase and a second mobile phase
Sample loop, which is a tube-shaped conduit, and separate storage
A mixing mechanism capable of stirring is provided, the first mobile phase is configured to inject the sample into the sample loop after passing through the sample loop and the mixing mechanism in this order, and the second mobile phase is more than the first mobile phase. A sample having a strong solution output from the column was used to feed the sample to the column through the mixing mechanism and the sample loop in this order. Mixing mechanism
Has at least a storage tank and a stirring blade, and is provided in the storage tank.
Mixing by rotating the stirring blades
The structure may be used.

[0005]

The first mobile phase passes through the sampling loop and the mixing mechanism in this order to fill the inside of the sampling loop.
After being stored in the mixing mechanism, the sample is injected into the sampling loop. Then, the first mobile phase and the second mobile phase are mixed by the mixing mechanism while sending the two mobile phases, and the mixing ratio of the second mobile phase to the first mobile phase of the mobile phase stored in the mixing mechanism is gradually increased. increase. At this time, if a second mobile phase having a higher soluble output from the column than the first mobile phase is used, the mixed solution gradually increases the soluble output from the column. If a sample is sent out of the sample loop with such a mixture and sent to a separation column and a detector, a gradient effect is exhibited, and simultaneous analysis of multiple components can be performed with high accuracy and speed.

[0006]

Embodiments of the present invention will be described below. As shown in FIG. 1, lines 3 and 4 are connected between a line 1 for supplying a first mobile phase L1 and a line 2 for supplying a second mobile phase L2 via a switching valve V1. Line 1 is connected to a vessel F1 containing the first mobile phase L1. The first mobile phase L1 is a mobile phase for temporarily adsorbing the target component to the column, and a mobile phase having a low soluble output from the column is appropriately selected and used. Line 1
Is provided with a pump P1. Line 2 is connected to vessel F2 containing the second mobile phase L2. As the second mobile phase L2, a mobile phase having a stronger solution output from the column than the first mobile phase L1 is used. A pump P2 is provided in the middle of the line 2. The switching valve V1 is a six-way valve capable of switching lines, and has a state in which the entrance and exit are in communication with each other as shown by a solid line in the drawing, and a state shown by a dotted line, and the like. Can be appropriately switched to a state in which the entrances and exits communicate with each other. The line 1 is connected to the entrance and exit of the switching valve V1, and the line 2 is connected to the entrance and exit. The line 3 is connected to the entrance, and the line 4 is connected to the entrance. In addition, a separation line following the separation column C and the detector D is connected to the entrance and the exit, and a mass spectrometer MS is connected as needed on an extension line of the detector D. A drain line 6 for discharging the first mobile phase L1 is connected to the entrance.

[0007] A switching valve V2 is provided between the line 3 and the line 4, and the line 3 is connected to the entrance of the switching valve V2, and the line 4 is connected to the entrance. Further, a mixing mechanism MC is provided in the middle of the line 4. The mixing mechanism MC is a means having a function of storing and stirring the mobile phase, and includes, for example, means as shown in FIG. 2 and FIG. FIG. 2 shows line 4
Is a means in which a storage tank 10 is formed in the middle of the process, and a stirrer 12 of a magnetic stirrer 11 is charged therein. FIG. 3 shows a means in which a stirring blade 13 is disposed inside a storage tank 10 formed in the middle of the line 4 and is rotated and stirred by a motor 14. Note that the entrance and exit of the line 4 may be provided at the same height above the storage tank 10 as shown in FIG. 2, or may be provided at different heights as shown in FIG.

[0008] The switching valve V2 is also a six-way valve capable of switching lines similarly to the switching valve V1 described above. As shown by a solid line in FIG.
It is possible to appropriately switch between a state in which the entrances and exits communicate with each other, and a state in which the entrances and exits communicate with each other as shown by the dotted line. In the switching valve V2 as described above, a line 7 is connected so as to connect the entrance and the exit, and a sample loop SL is provided in the middle of the line 7. In addition, a line 8 provided with an injector I for injecting a sample is connected to the entrance and exit,
A line 9 for discharging excess liquid is connected to the entrance. The switching valve V2 as described above
It is also possible to omit the line 8 by directly incorporating the injector at the entrance. That is, as shown in FIG. 4, a commercially available product injector (manufactured by Leodyne) has a sample loop SL in the middle of the line 7 connecting the entrance and exit, and the injector I is incorporated in the entrance and exit. Goods may be used. Each of the lines 1 to 9 is made of a stainless steel tube.

In FIG. 1, the first mobile phase sent by the pump P1 of the line 1 is a switching valve V1.
Of the switching valve V2 → the line 7 and flows through the sample loop SL. Thus, the first mobile phase that has filled the inside of the sample loop SL further flows into the inlet / outlet → line 4 of the switching valve V2, enters the mixing mechanism MC, and is stored. The surplus first mobile phase that can no longer be stored in the mixing mechanism MC is the switching valve V1
Is discharged from the line 6 through the entrance of the port. On the other hand, in the line 2, the mobile phase L2 is sent by the pump P2, and the mobile phase L2 flows in the order of the inlet / outlet of the switching valve V1, the line 5, the separation column C, the detector D, and, if necessary, the mass spectrometer MS. deep. Next, the sample is injected from the injector I by connecting the inlet and outlet of the switching valve V2 with the switching of the flow path of the switching valve V2. The injected sample passes through the line 8 → the inlet / outlet of the switching valve V2, flows into the line 7, and enters the sample loop SL. Excess sample and first mobile phase L
1 is discharged from the line 9 through the entrance and exit of the switching valve V2. In the mechanism in which the injector I is incorporated at the entrance and exit of the switching valve V2 as shown in FIG. 4, the injected sample flows into the line 7 through the entrance and exit of the switching valve V2 and enters the sample loop SL. Next, the switching valves V1 and V2 are simultaneously switched while the sample is in the sampling loop SL as described above, so that the switching valve V1 is in a state where the entrance and exit of the switching valve V1 are communicated with the switching valve V2. ,When,
And the entrance and exit are in communication. Thus, the mobile phase L2 sent by the pump P2 passes through the line 2 → the inlet / outlet of the switching valve V1, flows into the line 4, and enters the mixing mechanism MC in the middle of the line 4.
In the mixing mechanism MC, the first mobile phase L1 and the second mobile phase L2 are gradually mixed. However, since the mixing is performed while the second mobile phase L2 is being supplied, the second mobile phase L1 and the second mobile phase L2 are stored in the mixing mechanism MC. The 2 mobile phase L2 gradually increases the mixing ratio with respect to the 1st mobile phase L1 and thus flows into the line 7 through the inlet / outlet of the switching valve V2 while exhibiting a gradient effect, and is provided in the middle of the line 7. The sample in the sample loop SL is pushed out. The extruded sample passes through the inlet / outlet of the switching valve V2 → the line 3 → the inlet / outlet of the switching valve V1 together with the liquid mixture of the first mobile phase L1 and the second mobile phase L2, flows into the line 5 and is separated by the separation column C. And is detected by the detector D. In addition, it is measured by the mass analyzer MS as needed.
The first mobile phase L1 sent by the pump P1 passes through the entrance and exit of the switching valve V1 and is discharged from the line 5. As described above, a favorable gradient effect can be obtained, and simultaneous analysis of multiple components can be performed with high accuracy and speed.

FIG. 5 shows a means in which the mixing mechanism MC provided on the line 4 in FIG. 1 is omitted, and a mixing mechanism MC is provided on the line 7 instead. Other configurations are the same as those in FIG. The line 7 is provided with both a mixing mechanism MC and a sample loop SL. Also, as shown in FIG. 4, the injector may be directly installed at the entrance and exit of the switching valve V2 as shown in FIG. In the above, the first mobile phase L1 sent by the pump P1 in the line 1 passes through the inlet / outlet of the switching valve V1, the line 3, and the inlet / outlet of the switching valve V2, flows into the line 7, and flows into the line 7. The sample loop SL provided in the middle of 7 is filled and stored in the mixing mechanism MC. The surplus first mobile phase L1 that can no longer be stored in the mixing mechanism MC is the switching valve V
It is discharged from the line 6 through the entrance and exit of the line → line 4 → the entrance and exit of the switching valve V1. on the other hand,
In the line 2, the mobile phase L2 is sent by the pump P2, and the mobile phase L2 flows in the order of the inlet / outlet of the switching valve V1, the line 5, the separation column C, the detector D, and, if necessary, the mass spectrometer MS. Next, switching valve V2
The sample is injected into the injector I by switching the flow path of (1) and (2) to communicate with the entrance and exit of (2). The injected sample passes through the line 8 → the inlet / outlet of the switching valve V2, flows into the line 7, and enters the sample loop SL. The first moving layer L1 in the sample loop SL passes through the mixing mechanism MC, the entrance and exit of the switching valve, and is discharged from the line 9. Next, while the sample is in the sample loop SL as described above, the switching valves V1 and V2 are simultaneously switched, so that the switching valve V1 is in a state where the entrance and exit of the switching valve V1 are communicated with the switching valve V2.
,, And, the entrance and exit are in communication. As a result, the mobile phase L2 sent by the pump P2 passes through line 2 → the entrance and exit of the switching valve V1 → line 4 → the entrance and exit of the switching valve V2,
It flows into the line 7, enters the mixing mechanism MC in the middle of the line 7, and is gradually mixed in the mixing mechanism MC. Since the mixing is performed while sending the second mobile phase L2, the mobile phase L2 stored in the mixing mechanism MC gradually increases the mixing ratio with respect to the first mobile phase L1, thus exerting a gradient effect, and thus, the sample loop. Extrude sample into SL. The extruded sample is taken from the inlet / outlet of the switching valve V2 → line 3 → switching valve V1.
, Flows into the line 5 through the inlet / outlet, is separated by the separation column C, and is detected by the detector D. Also, it is measured by the mass spectrometer MS as needed. The first mobile phase L1 sent by the pump P1 passes through the inlet and outlet of the switching valve V1 and is discharged from the line 6.
Thus, even with the system as described above, a good gradient effect can be obtained as before, and simultaneous analysis of multiple components can be performed with high accuracy and speed.

Next, FIG. 6 shows, via a switching valve V, a line 21 for sending the second mobile phase L 2 and a line 22 for sending the sample and the mobile phase to the analytical column C and the detector D. , A line 23 is provided. The switching valve V is similar to the switching valve V1 shown in FIG. The line 21 is connected to the container F containing the second mobile phase L2, and sends the liquid by operating the pump P. The line 21 is connected to the entrance and exit of the switching valve V. The line 23 is connected so as to connect the entrance and exit of the switching valve V, and a mixing mechanism MC and a sample loop SL are provided in the middle of the line 23. The mixing mechanism MC is a means for storing and agitating the mobile phase as shown in FIGS. 2 and 3, for example. The line 22 connects the inlet / outlet of the switching valve V and the detector D, and has a separation column C on the way. If necessary, the line 22 can be extended, and the mass spectrometer MS can be connected to the end of the detector D. In addition, a line 24 provided with a sample inlet I for injecting a sample is connected to the inlet / outlet of the switching valve V, and a line 25 for discharging excess liquid is connected to the inlet / outlet. First of all, the injector I was used to
The mobile phase L1 is injected, and the first mobile phase L thus injected is injected.
1 passes through the line 24 → the entrance and exit of the switching valve V, and flows into the line 23. Further, the first mobile phase L1
Pass through the sample loop SL, fill the inside of the sample loop SL, and are stored in the mixing mechanism MC. The first mobile phase L1, which can no longer be stored in the mixing mechanism MC,
Line 2 passing through the entrance and exit of switching valve V
Exhausted from 5 Next, a sample is injected from the injector I. The injected sample passes through the line 24, the entrance and exit of the switching valve V, flows into the line 23, and enters the sample loop SL. The first mobile phase L 1 in the sample loop SL passes through the mixing mechanism MC, passes through the entrance and exit of the switching valve V, and is discharged from the line 25. Next, after switching the flow path of the switching valve V, the second mobile phase L2 is moved by the pump P to the line 21.
Solution. The second mobile phase L2 passes through the inlet / outlet of the switching valve V, flows into the line 23, enters the mixing mechanism MC, and the first mobile phase L1 is gradually mixed.
At this time, as the second mobile phase L2, a solution having a stronger solution output from the separation column C than the first mobile phase L1 is used. Second mobile phase L
Since the mixing is carried out while the liquid 2 is supplied, the mobile phase L2 stored in the mixing mechanism MC gradually becomes the first mobile phase L1.
, Through the inlet / outlet of the switching valve V, enters the line 22, is separated by the separation column C, and is detected by the detector D, while exhibiting a gradient effect. Also, if necessary, mass spectrometer MS
Is measured. A good gradient effect can be obtained with the system configured as described above, and simultaneous analysis of multiple components can be performed with high accuracy and speed.

(Experimental Example) The results of an experiment conducted by the present inventors on the line described with reference to FIG. 1 will be described below. Using a sample containing 1.0 mg / ml each of methyl benzoate, ethyl benzoate, propyl benzoate, butyl benzoate, pentyl benzoate, hexyl benzoate, heptyl benzoate, and octyl benzoate, the first mobile phase L1 30:70 acetonitrile and water
And a condition of a liquid sending flow rate of 1.0 ml / min. The mobile phase L2 used was a mixture of acetonitrile and ethanol in a ratio of 50:50, the flow rate was 20 μl / min, and the separation column C was Inertsilicon.
ODS-2 (φ0.7mm × 150mm), detection UV
254 mm was used. FIG. 7 shows the experimental results under the above conditions. In the figure, the horizontal axis indicates the time required to separate the sample into each component in the separation column C, and the vertical axis indicates the detected amount of each component thus separated. as a result,
As shown in FIG. 7, very good separation was obtained. The same mobile phase was used, and the liquid sending flow rate was 100 μl /
min, and the separation column C was Inertsil ODS-
When the elution time of each component was repeated 10 times when using 2 (φ2.1 mm × 250 mm), the variation coefficient of the elution time of each component was 0.1% or less, and a very accurate gradient was obtained. Was done.

[0013]

According to the gradient device for high performance liquid chromatography of the present invention, the following effects can be obtained. (1) A gradient can be obtained with an inexpensive device. (2) The gradient at a low flow rate has reproducibility and high accuracy, so that reliable experimental data can be obtained. (3) Since compounds of high polarity to low polarity can be simultaneously separated and analyzed, simultaneous analysis of multiple components becomes possible. (4) Separation of each component is improved. (5) Since the diffusion is reduced, the peak of each component becomes sharp and the sensitivity becomes high. (6) Each setting condition of the high-performance liquid chromatograph is easily set, and in particular, a solvent having a strong dissolving power can be uniformly used as the second mobile phase. (7) As described above, the cost, accuracy, simplicity, separability and sensitivity are remarkably improved, so that it has extremely high practical value.

[Brief description of the drawings]

FIG. 1 is a block diagram of a gradient device.

FIG. 2 is a sectional view of a mixing mechanism.

FIG. 3 is a sectional view of a mixing mechanism.

FIG. 4 is an enlarged view of a switching valve V2.

FIG. 5 is a block diagram of a gradient device according to another embodiment.

FIG. 6 is a block diagram of a gradient device according to another embodiment.

FIG. 7 is a graph showing measurement results obtained by the apparatus of the present invention.

FIG. 8 is a block diagram of a chromatograph showing a conventional technique.

[Explanation of symbols]

 L1 First mobile phase L2 Second mobile phase 1 Line for supplying first mobile phase 2 Line for supplying second mobile phase SL Sample loop MC Mixing mechanism

────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Satada 7-20-10 Tsukushino, Abiko City, Chiba Prefecture (56) References JP-A-6-266954 (JP, A) (58) Fields investigated (Int. . ^7, DB name) G01N 30/34

Claims (2)

(57) [Claims] 1. A sample which is a loop-shaped conduit between a line supplying a first mobile phase and a line supplying a second mobile phase.
Loop and separate mixin that can be stored and stirred
A first mobile phase passes through the sample loop and the mixing mechanism in order to inject the sample into the sample loop, and the second mobile phase has a higher output of the melt output from the column than the first mobile phase. A gradient device in a high-performance liquid chromatograph configured to send a sample to a column through a mixing mechanism and a sample loop in the order of using a strong one. 2. A mixing mechanism comprising: a storage tank and a stirring blade;
And the stirring blades rotate in the storage tank.
2. The group according to claim 1, wherein the mixing is performed by
Radiant device.