JP2004138413A - Liquid chromatograph apparatus and its liquid supplying apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To achieve gradient easily and quickly by an inexpensive system while maintaining a mixture rate precision of an additive by an analysis method using the additive. <P>SOLUTION: A solvent selection valve 18 forms a channel for connecting either a solvent bottle 2a1 in a solvent A1 or a solvent bottle 2a2 in a solvent A2 to a pump 4a, and connecting either a solvent bottle 2b1 in a solvent B1 or a solvent bottle 2b2 in a solvent B2 to a pump 4b. The solvent selection valve 18 is periodically switched according to the concentration ratio of solvents A1: A2 set by a user and feeds the mixed liquid of the solvents A1 and A2 as a liquid A, and is similarly switched periodically according to a concentration ratio of the solvents B1: B2 set by the user and feeds the mixed liquid of the solvents B1 and B2 as a liquid B. Further, liquid feeding pumps 4a, 4b feeds liquid at each flow rate according to the total flow rate set by the user and the concentration ratio of the liquid A:B. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a liquid chromatograph device, particularly to a gradient liquid chromatograph device using a mobile phase while mixing a plurality of solvents, and a gradient liquid transfer device used in such a liquid chromatograph device. .
[0002]
[Prior art]
In liquid chromatography, there is a technique called a gradient in which a plurality of solvents are mixed and sent to a column as a mobile phase. There are two types of gradient technology, high-pressure gradient and low-pressure gradient technology.
[0003]
FIG. 2 shows an example of a conventional high-pressure gradient system. A liquid sending pump 4a and a liquid sending pump 4b are provided for each of the solution bottle 2a containing the solution A and the solvent bottle 2b containing the solution B, and the solvent from both the solution sending pumps 4a and 4b is provided. The mixture is mixed by the mixer 6 and sent to the column as a mobile phase.
[0004]
The liquid feed pump 4a and the liquid feed pump 4b may communicate with each other as two independent liquid feed pumps, or may be controlled by an external controller, or may have one liquid feed pump 4a and one liquid feed pump 4b. In some cases, a binary pump may be used as a unit. However, in any case, gradient liquid transfer is realized by replacing the set mixing ratio with the flow rate of each of the liquid transfer pumps 4a and 4b.
[0005]
In the case of performing the three-liquid gradient feeding, a solvent bottle 2c containing the C liquid is further provided, and a liquid-sending pump 4c is provided for the solvent bottle 2c. The three liquids are mixed by the mixer 6 and sent to the column as a mobile phase. A three-liquid high-pressure gradient system is constructed.
In such a system, the liquids are joined and mixed at a position where the liquid supply pressure is applied to the liquid on the discharge side of each liquid supply pump, and this is called a high-pressure gradient.
[0006]
FIG. 3 shows an example of a conventional low-pressure gradient system. The solvent bottles 2 a to 2 d containing the liquids A to D are connected to the liquid sending pump 4 through the solvent selection valve 8. The solvent selection valve 8 is configured so that only one of the solvent bottles 2a to 2d can be selected and connected to the pump 4. The solvent selection valve 8 is controlled by the liquid sending pump 4, and the liquid sending pump 4 sends the liquid to the column via the mixer 6 while periodically switching the solvent selection valve 8 so as to have a set mixing ratio. Realizes a gradient solution transfer.
In such a system, the liquids join and mix at the suction side of the liquid feed pump 4, that is, at a position where the liquids are depressurized, and this is called a low-pressure gradient.
[0007]
The high-pressure gradient system mixes the solvent under high pressure and generates few bubbles, and the accuracy of the gradient is almost determined by the performance of each liquid pump itself, so the concentration accuracy is excellent, but a liquid pump is required for each solvent. There is a disadvantage in terms of cost.
[0008]
On the other hand, in the low pressure gradient system, the concentration accuracy is inferior to the high pressure gradient system due to the generation of bubbles, the effect of the solvent compressibility, and the responsiveness of the solvent selection valve because the solvent is mixed under reduced pressure. There are merits such as the need for a stand and the number of solvents can be easily increased.
[0009]
In liquid chromatography, in the analysis of proteins and peptides, a method of performing a gradient with water and acetonitrile to which trifluoroacetic acid (TFA) is added is often used. However, when performing a high-pressure gradient system, a predetermined amount of TFA is used. Water and acetonitrile to which is added are prepared beforehand and used as solution A and solution B, respectively, but the amount of TFA added must be fixed at a predetermined amount.
[0010]
On the other hand, when using a low-pressure gradient system, water to which TFA has been added, pure water to which nothing has been added, solution A, solution B, acetonitrile to which TFA has been added, and acetonitrile to which nothing has been added, solution C, When used as solution D, the amount of TFA added can be easily changed, but the concentration accuracy cannot be expected very much.
[0011]
[Problems to be solved by the invention]
In liquid chromatography, in the analysis of proteins, peptides, and the like, a method of performing a gradient with water and acetonitrile to which TFA has been added is often used.However, when performing a high-pressure gradient system, water and a predetermined amount of TFA are added to water. Acetonitrile is prepared and used as solution A and solution B, however, the amount of TFA added must be fixed at a predetermined amount.
[0012]
On the other hand, when using a low-pressure gradient system, water to which TFA has been added, pure water to which nothing has been added, solution A, solution B, acetonitrile to which TFA has been added, and acetonitrile to which nothing has been added, solution C, When used as solution D, the amount of TFA added can be easily changed, but the concentration accuracy cannot be expected very much.
[0013]
In the analysis method using such additives, the amount of additive added is an important parameter of the analysis, but it is easy to maintain the analysis accuracy using the conventional high-pressure gradient system or low-pressure gradient system while maintaining the analysis accuracy. Gradients could not be realized with a fast, inexpensive system.
[0014]
Therefore, the present invention solves such a problem, and uses an additive-based analysis method, while maintaining the mixing ratio accuracy of the additive, a simple, quick, and inexpensive gradient liquid sending device that realizes a gradient with an inexpensive system. , And a liquid chromatograph provided with such a gradient liquid sending device.
[0015]
[Means for Solving the Problems]
The liquid-feeding device of the present invention is provided on a suction side of at least one liquid-feeding pump of a high-pressure gradient mechanism for changing the flow rate of a plurality of liquid-feeding pumps to an arbitrary mixed concentration, and at least one liquid-feeding pump of the high-pressure gradient mechanism. And a low-pressure gradient mechanism for providing a desired mixed concentration by periodically switching the solvent selection valve that can switch a plurality of solvents.
[0016]
By placing a low-pressure gradient mechanism in front of at least one liquid-feeding pump of the high-pressure gradient mechanism, a solvent obtained by mixing two or more solvents at a low pressure gradient at a set concentration ratio by the low-pressure gradient mechanism is further added to a plurality of liquid-feeding pumps. And high-pressure mixing at a set mixing ratio by a high-pressure gradient mechanism according to the above.
[0017]
In the liquid sending pump provided with a low-pressure gradient mechanism in the preceding stage, the solvent is mixed in the first stage by the low-pressure gradient mechanism provided by the solvent selection valve provided in the preceding stage, and the solvent is mixed by the high-pressure gradient mechanism provided by the respective liquid sending pumps in the second stage. Further, a plurality of solvents are mixed to form a mobile phase, which is then sent.
[0018]
The liquid chromatograph device of the present invention is a column for separating a sample introduced together with a mobile phase into components, a gradient liquid sending device for sending a mobile phase to the column while preparing a mobile phase by mixing a plurality of solvents. An injector provided in a flow path between the liquid sending device and the column for injecting a sample, and a detector for detecting a sample component eluted from the column, and the liquid sending device of the present invention as a gradient liquid sending device. Is used.
[0019]
BEST MODE FOR CARRYING OUT THE INVENTION
FIG. 1 shows a liquid chromatograph apparatus according to one embodiment. Reference numeral 12 denotes a column for separating a sample introduced together with the mobile phase into components. The left side of the figure indicated by reference numeral 20 is a gradient liquid sending device of one embodiment. The gradient liquid sending device 20 mixes a plurality of solvents A1, A2, B1 and B2 with the mixer 6 to prepare a mobile phase. Then, the mobile phase is sent to the column 12. An injector 10 for injecting a sample is provided in a flow path between the gradient liquid sending device 20 and the column 12. Downstream of the column 12, a detector 14 for detecting a sample component eluted from the column 12 is provided.
[0020]
In the gradient liquid sending device 20, the solvent bottles 2a ₁ and 2a ₂ containing the solvents A1 and A2 are connected to the suction side of the liquid sending pump 4a via the solvent selection valve. Similarly, solvent bottles 2b ₁ and 2b ₂ containing the solvent B1 solution and the solution B2 are connected to the suction side of the liquid sending pump 4b via the solvent selection valve 18.
[0021]
The solvent selection valve 18, either the solvent bottle 2a ₂ of the solvent bottle 2a ₁ or solvents A2 solvents Al was connected to the pump 4a, either solvent Bl solvent bottle 2b ₁ or solvents B2 solvent bottle 2b ₂ One of them constitutes a flow path which can be connected to the pump 4b. The solvent selection valve 18 sends the mixed solution of the solvent Al and A2 as the liquid A while being periodically switched according to the concentration ratio of the solvent A1: A2 set by the user, and the solvent B1: B2 similarly set by the user. The mixture of the solvents B1 and B2 is sent as the liquid B while being periodically switched according to the concentration ratio of the liquid. Further, the liquid sending pumps 4a and 4b send liquid at each flow rate according to the total flow rate set by the user and the concentration ratio of the liquid A: liquid B.
[0022]
In the present embodiment, the solution A and the solution B are mixed by a high-pressure gradient, so that the concentration accuracy is superior to that of the four-liquid low-pressure gradient system. Further, since the mixing section having the mixing ratio A1: A2 of the liquid A and the mixing ratio B1 = B2 of the liquid B is mixed by a low-pressure gradient, it is more cost-effective than a high-pressure gradient system using four liquid feed pumps.
[0023]
In the present embodiment, two independent liquid feed pumps are used, but a form of a binary pump in which two liquid feed pumps are integrated is also included.
In this embodiment, there are only two liquid feed pumps, but the present invention is not limited to two pumps, and includes a system having three or more liquid feed pumps.
[0024]
In the present embodiment, a selection valve of two flow paths is connected in front of each liquid sending pump, but the present invention is not limited to two flow paths, and a selection valve of three or more flow paths may be connected. included.
In the present embodiment, the flow path selection valve is connected to both of the two liquid feed pumps. However, the present invention is not limited to this, and includes a mode in which the flow path selection valve is connected to only one side.
[0025]
【The invention's effect】
In the present invention, the low-pressure gradient system is added before the high-pressure gradient system, so that the analysis method using the additive can be performed easily, quickly, and at a low cost while maintaining the mixing ratio accuracy of the additive. Can be realized.
To explain this more specifically, in the conventional apparatus, considering a four-liquid gradient of A, B, C, and D, whether a high-pressure gradient or a low-pressure gradient, the set value needs to be A + B + C + D = 100%. On the other hand, in the present invention, in the case of the embodiment, since the setting is made so that A + B = 100%, A1 + A2 = 100%, and B1 + B2 = 100%, the concentration setting of the additive can be intuitively grasped. .
For example,
A1: Pure water A2: 0.1% TFA water B1: Pure acetonitrile B2: As acetonitrile containing 0.1% TFA, 40% 0.08% TFA water and 60% acetonitrile containing 0.08% TFA Suppose you do. Here, the reason why the TFA concentrations of the two solutions are made equal is that it is a general analysis method to keep the concentrations of the additives of the two solutions finally mixed the same.
When such a mobile phase is prepared, the liquid is sent as follows in the conventional method and the present invention.
(1) In the case of a conventional two-liquid gradient (regardless of whether it is a high-pressure gradient or a low-pressure gradient):
From the beginning, 0.08% TFA water is prepared as a liquid A, and acetonitrile containing 0.08% TFA is prepared as a liquid B, and the liquid A is sent as 40% and the liquid B is sent as 60%.
(2) In the case of a conventional four-liquid gradient (regardless of whether it is a high-pressure gradient or a low-pressure gradient):
A1: 8%, A2: 32%, B1: 12%, B2: 48% are sent. In this case, the concentration of the additive and the ratio of water / acetonitrile are not intuitively understood.
(3) In the case of the present invention:
A1: 20%, A2: 80%, B1: 20%, B2: 80%, A (= A1 + A2): 40%, and B (= B1 + B2): 60%.
In the case of the present invention, there are six parameters, but actually A1 = 100% −A2
B1 = 100% −B2
A = 100% -B
Therefore, the number of setting parameters may be three.
As described above, the concentration of the additive and the ratio of water / acetonitrile can be used as set values as they are.
In addition, while it is common to perform a gradient in which the mixing ratio of water / acetonitrile is gradually changed while keeping the concentration of the additive constant, in the case of a conventional four-liquid gradient as in (2), The result is a very complex gradient, which is not practical. Therefore, the present invention is the most excellent.
[Brief description of the drawings]
FIG. 1 is a flow chart showing one embodiment.
FIG. 2 is a flow chart showing an example of a conventional high-pressure gradient liquid sending device.
FIG. 3 is a flow chart showing an example of a conventional low-pressure gradient liquid sending device.
[Explanation of symbols]
2a ₁ , 2a ₂ , 2b ₁ , 2b ₂ Solvent bottles 4a, 4b Liquid feed pump 6 Mixer 10 Injector 12 Column 14 Detector 18 Solvent selection valve 20 Gradient liquid feeder

Claims (2)

A high-pressure gradient mechanism for changing the flow rate of a plurality of liquid sending pumps to an arbitrary mixed concentration,
A solvent selection valve is provided at least on the suction side of one of the liquid sending pumps of the high-pressure gradient mechanism, and is capable of switching a plurality of solvents. The solvent selection valve is periodically switched to obtain an arbitrary mixed concentration. A liquid sending device comprising a low-pressure gradient mechanism. A column for separating a sample introduced together with a mobile phase into components, a gradient liquid sending device for sending the mobile phase to the column while preparing a mobile phase by mixing a plurality of solvents, and between the liquid sending device and the column. Injector provided in the flow path of the injector for injecting a sample, and a liquid chromatograph device having a detector for detecting a sample component eluted from the column,
A liquid chromatograph device using the liquid feeding device according to claim 1 as the gradient liquid sending device.

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