JP2003107063A - Deaeration apparatus and method of high performance liquid chromatography - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a deaeration apparatus that can be applied also to a normal phase system in a distribution chromatography where a deaeration operation need not be made repeatedly and the bubbling of a helium gas cannot be adopted in the deaeration of eluate in high performance liquid chromatography (HPLC), and a case for using a volatile eluate, and can achieve an analysis operation by the HPLC efficiently and accurately. SOLUTION: A helium gas is supplied to a pressure-resistance container 1 via a pressure reduction valve 4, and the inside of the pressure-resistance container 1 is retained at a set pressure. When the pressure in the pressure- resistance container 1 drops, the pressure reduction valve 4 is opened, the helium gas is supplied, and the eluate M does not come into contact with air for retaining in a deaeration state. Additionally, when the pressure in the pressure- resistance container 1 becomes higher than the set pressure, an exhaust valve 6 that is provided at exhaust piping 5 connected to the pressure-resistance container 1 is opened, the helium gas is exhausted, and the inside of the pressure-resistance container 1 is retained at the set pressure.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a degassing apparatus and a degassing method for high performance liquid chromatography, in which an eluent supplied to a column is supplied while maintaining a degassed state.

[0002]

2. Description of the Related Art Conventionally, liquid chromatography has been
The eluent obtained by adding the sample to be analyzed to the solvent is brought into contact with the packing material packed in the column to carry out the component analysis, and the packing material is analyzed by the difference in the adsorbing power of the eluent. Adsorption chromatography, ion exchange chromatography that performs analysis based on the difference in ionic bond strength, gel permeation chromatography that performs analysis based on the difference in the three-dimensional size of molecules, or the liquid is preliminarily permeated and adsorbed to the packing material. , A distribution chromatography in which an eluent (mobile phase) in which a sample is dissolved is passed through a solvent that is immiscible with the liquid (stationary phase) and the analysis is performed by partitioning between the liquids. In such liquid chromatography, if the particle size of the packing material is made smaller or the column is made longer in order to improve the analysis accuracy, the pressure inside the column becomes higher, and therefore the eluent is sequentially sent to the column using a pump. High performance liquid chromatography (High Performance Liquid Chromatography)
romatography Hereinafter referred to as HPLC. ) Was known. HPLC consists of a pump that supplies the eluent at a constant flow rate, a column that is filled with a packing material and that separates the components by the packing material, and the separated result is extracted as an electrical signal and compared with a solvent that does not contain a sample. It is composed of a detection device or the like for detecting components, and has excellent separation ability and high-sensitivity detection ability, and is widely used as a separation technique suitable for the analysis of a trace amount of components.

In such an HPLC, even if a slight amount of gas such as air is mixed in the eluent, it causes a malfunction of the pump and bubbles in the column cause column analysis efficiency. The degassing can be done by degassing the gas mixed in the eluent when supplying the eluent to the pump. A device is provided. For this deaerator, shortening the time required for deaerating operation,
Various proposals have been made so far such as simplification of work. For example, in Japanese Patent Laid-Open No. 11-137907, in order to measure a smaller amount of sample with high accuracy, the flow resistance when the eluent flows through the degassing device is reduced, and the time required for the degassing operation is shortened. It describes the device to do. Also disclosed is a method in which the degassing apparatus has a structure capable of heating and cooling, and optimal conditions are set irrespective of the external atmosphere, and thereby the degassing operation is efficiently performed, thereby realizing high-speed HPLC. Further, in JP-A-10-185892, in HPLC, for example, the first component of the solvent occupies most of the whole solvent and the second component is stirred so that sufficient mixing can be obtained even when the amount is extremely small. The tank is equipped with an ultrasonic vibration type mixer, and the stirring tank and the vacuum degassing device are connected by a hose, and stirring and degassing are performed simultaneously by the same device, so that the solvent can be adjusted quickly and accurately. A deaerator is disclosed.

As a method of degassing the eluent of HPLC before sending it to the column, a method of degassing under reduced pressure mainly using an aspirator, a method of degassing using an aspirator and an ultrasonic oscillator, and helium gas elution The method includes deaeration by bubbling into the liquid at regular time intervals, depressurization using a hollow fiber membrane, and the like.

[0005]

However, the degassing method using an aspirator or an ultrasonic oscillator is preferable as a degassing method because the eluent is re-saturated with air in a short time after degassing. It wasn't something. For this reason, a degassing method by bubbling helium gas was also adopted, but this method replaces nitrogen and oxygen dissolved in the eluent with helium by dissolving helium in the eluent by bubbling. Since the dissolved helium volatilizes after the degassing and the air dissolves again, bubbling must be repeated at regular intervals to saturate the eluent with helium. It is important to keep the degassing time by bubbling helium gas constant, in order to keep the eluent composition constant.However, when the degassing operation is repeated many times, the bubbling time tends to fluctuate and the bubbling Was a problem in analysis accuracy.

Here, in partition chromatography, a hydrophobic solvent having a polarity lower than that of the stationary phase, which is preliminarily permeated and adsorbed by the packing material of the column, such as n.
-There are a normal phase system using hexane or the like as a mobile phase and a reverse phase system using a solvent such as water having a polarity higher than that of the stationary phase for the mobile phase. The degassing method by bubbling helium gas cannot be adopted because the composition of the mobile phase changes due to the evaporation of the solvent. Also, in adsorption chromatography and the like, when a volatile organic solvent is used as the eluent, the composition changes when bubbling of helium gas is carried out, so that it cannot be adopted.

The present invention has been made to solve the above problems, and an object of the present invention is to eliminate the need for repeated degassing operations in degassing the eluent of HPLC.
In addition, it can be applied to a normal phase system in partition chromatography in which bubbling of helium gas cannot be adopted and a case where a volatile eluent is used.
An object of the present invention is to provide a degassing device and a degassing method capable of efficiently performing LC analysis work and achieving high analysis accuracy.

[0008]

Means for Solving the Problems The present inventors have found that HPLC
The degassing method for simplifying the degassing operation of the mobile phase, further improving the analysis work, and further improving the analysis accuracy, as a result of diligent research on the degassing device, after degassing the eluent in the pressure vessel, It was found that by keeping the inside of this pressure vessel under a pressurized state of helium gas at all times, re-dissolution of air can be prevented. Therefore, the pressure vessel was connected to a helium cylinder by a pipe, and a decompression valve was installed in the pipe to keep the eluent after degassing at a constant pressure of helium gas. It was found that dissolution was prevented, and re-dissolution of air over time could be eliminated without performing degassing operation at regular intervals,
The present invention has been completed.

That is, the present invention is a degassing apparatus for high performance liquid chromatography, which supplies an eluent supplied to a column while maintaining a degassed state, in which a pressure resistant container for storing the eluent and a pressure resistant container The pressure-resistant container is connected by connecting a supply pipe for supplying an eluent to the column, the pressure-resistant container and a helium cylinder, and controlling the supply amount of helium gas supplied into the gas phase in the pressure-resistant container. A helium gas inlet pipe having a pressure reducing valve for maintaining the inside pressure at a predetermined pressure, and an exhaust pipe having an exhaust valve connected to the pressure resistant container for exhausting helium gas to maintain the pressure resistant container at a constant pressure. A degassing apparatus for high-performance liquid chromatography (claim 1) characterized in that, and a pressure-resistant volume for storing the degassed eluent when the eluent is supplied to a column for high-performance liquid chromatography. Gas phase within the supplying helium gas to be replaced by helium gas at a predetermined pressure, then,
By controlling the supply of helium gas to the gas phase in the pressure vessel so as to maintain a predetermined pressure in the pressure vessel,
A degassing method for high performance liquid chromatography, characterized in that the degassed state of the eluent is maintained, and helium gas is supplied into the eluent in the pressure resistant container to perform bubbling. The degassing method for high performance liquid chromatography according to claim 2, wherein helium gas is supplied to the gas phase of the pressure vessel after removing the dissolved gas therein and degassing.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The degassing apparatus for high performance liquid chromatography of the present invention is a degassing apparatus for high performance liquid chromatography, which supplies an eluent supplied to a column while maintaining a degassing state. Helium supplied to the gas phase in the pressure vessel, which is connected to the pressure vessel that stores the liquid, the supply pipe that is connected to the pressure vessel and that supplies the eluent to the column, the pressure vessel and the helium cylinder. Helium gas inlet pipe having a pressure reducing valve for maintaining a predetermined pressure in the pressure vessel by controlling the gas supply amount, and connected to the pressure vessel, the helium gas is exhausted to maintain a constant pressure inside the pressure vessel. There is no particular limitation as long as it is provided with an exhaust pipe having an exhaust valve that maintains the pressure.

The pressure-resistant container is a pressure-resistant container for storing the eluent in which the sample is dissolved, and the stored eluent may be sent from a separate container. The container is preferably provided with a removable lid. A helium gas feed pipe and an exhaust pipe are connected to this lid.

The supply pipe connects the pressure vessel and the column,
The eluent in the pressure resistant container is supplied to the column, and a pump is provided so that a fixed amount of the eluent can be delivered to the column. Incidentally, the column is filled with a packing material that allows the eluent containing the sample to flow through and changes the output due to the contact of the eluent with the difference. Adsorption of the eluent, ion exchange, The analysis may be carried out by any method such as gel permeation and distribution, and a pump capable of uniformly supplying the eluent to the column without pulsation is preferable.

The helium gas feed pipe connects the helium gas cylinder and the pressure vessel to supply the helium gas to the pressure vessel, and the one that can be operated independently of the exhaust pipe is easy to handle. It is preferable that the pressure resistant container has a structure that is inserted and fixed in the lid. A pressure reducing valve is provided in the helium gas inlet pipe, the pressure inside the pressure vessel is lower than that inside the helium cylinder, and slightly pressurized, for example, 10 k.
The pressure is maintained at a constant pressure such as pa.

The exhaust pipe is connected to a pressure resistant container so that helium gas can be released from the pressure resistant container. An exhaust valve is provided in the exhaust pipe, and when the internal pressure of the pressure resistant container rises more than necessary, it is opened to maintain a constant pressure inside the pressure resistant container. It should be noted that the exhaust pipe provided with an exhaust valve may be connected to the pressure resistant container, but it may be installed in the helium gas feed pipe if it is on the downstream side of the pressure reducing valve.

Further, in the degassing method for high performance liquid chromatography of the present invention, when the eluent is supplied to the column for high performance liquid chromatography, the gas phase in the pressure resistant container in which the degassed eluent is stored is helium gas. Helium gas is supplied so that the pressure inside the pressure-resistant container is maintained at a predetermined pressure, and then the supply of helium gas to the gas phase inside the pressure-resistant container is controlled to maintain the pressure inside the pressure-resistant container. It is not particularly limited as long as it maintains a vapor state, but when the eluent can degas the dissolved gas by bubbling helium gas, helium gas in the eluent in the pressure vessel is used. Bubbling is performed to remove the dissolved gas in the eluent and degass it, and then to supply helium gas to the gas phase of the pressure vessel, degassing the eluent and degassing the eluent after degassing. Mood It can be carried out the original at the same device, preferably.

According to the degassing apparatus and the degassing method for high performance liquid chromatography of the present invention, helium gas is supplied into the gas phase in the pressure vessel for storing the eluent after the degassing operation, and a constant pressure is applied. By holding the eluent, it is possible to prevent the eluent from coming into contact with air and prevent the redissolution of air in the eluent. Therefore, in order to remove the air dissolved in the eluent again with the volatilization of the helium gas from the eluent with the passage of time, it is possible to avoid changing the composition due to repeated bubbling operation of the helium gas. Therefore, the analysis accuracy can be improved, and the analysis accuracy does not deteriorate due to the fluctuation of the bubbling time. Furthermore, even when the eluent is volatile, such as when the mobile phase in partition chromatography is a normal phase system, after degassing the eluent, by maintaining a constant pressure in the pressure vessel with helium gas, Since it is possible to prevent air from being redissolved in the eluent, it is possible to improve the analysis accuracy without changing the composition of the eluent.

A preferred embodiment of the degassing apparatus for high performance liquid chromatography of the present invention will be described below with reference to the drawings. 1. FIG. 1 is a block diagram of the HPLC degassing apparatus of the present invention. As shown in FIG. 1, the HPLC degassing apparatus of the present invention excludes air from being mixed in the eluent M supplied to the HPLC column (not shown) via the pump P, and the degassing state is achieved. A degassing apparatus for supplying the eluent M to the column while maintaining the temperature, and a pressure resistant container 1 in which the eluent M of HPLC is stored, and a removable lid 2 fitted in the pressure resistant container 1.
And a helium gas feed pipe 3 having one end inserted and fixed in the lid 2 and the other end connected to the helium cylinder B, and a depressurized pressure provided in the helium gas feed pipe 3 for keeping the pressure vessel 1 at a constant pressure. In consideration of the safety of the pressure vessel 1, the valve 4 includes an exhaust pipe 5 that is inserted and fixed in the lid 2, and an exhaust valve 6 installed in the exhaust pipe 5.

The pressure-resistant container 1 stores the eluent M, hermetically holds and holds the helium gas supplied from the helium cylinder B connected through the helium gas feed pipe 3, and keeps the inside at a predetermined pressure. Those having a mechanical strength that can be maintained are preferable. The pressure-resistant container 1 preferably has chemical resistance to the stored eluent M, and is preferably made of a material having excellent chemical properties such as acid / alkali resistance and resistance to an organic solvent, such as borosilicate glass. Those are preferably used. The volume of the pressure vessel 1 can be arbitrarily selected according to the volume of the eluent M required for the analysis, but generally, the volume of 1 to 2 L is often used. The shape is not particularly limited.

The lid 2 is fitted into the pressure-resistant container 1 and maintains high airtightness even in a pressurized state with helium gas. The lid 2 is provided with three holes 2a, 2b, 2c, and a helium gas inlet pipe 3, an exhaust pipe 5, and a helium gas inlet pipe 3, respectively.
A pump suction pipe S for delivering the eluent M is inserted and fixed.

The supply pipe S connects the pressure resistant container 1 and the column via a pump in order to supply the eluent to the column, and the suction end is immersed in the eluent M in the pressure resistant container 1. The filter 7 is mounted and the other end is connected to the pump P. The eluent M is circulated through the pump suction pipe S via the suction filter 7 by the operation of the pump P, and is delivered to the column. The pump P has no pulsation so that the eluent M can be uniformly supplied to the column.

The helium gas feed pipe 3 is inserted and fixed in the lid 2 so that one end thereof is arranged in the gas phase of the pressure-resistant container 1, and the other end is connected to the helium cylinder B.
Helium gas is appropriately supplied from the helium cylinder B into the gas phase of the pressure resistant container 1 via the pressure reducing valve 4. The material of the helium gas inlet pipe 3 is not particularly limited as long as it is a material having excellent pressure resistance and corrosion resistance, and a fluororesin material is preferably selected. The pipe diameter of the helium gas feed pipe 3 is determined from the conditions such as the flow rate and pressure of the helium gas. For example, when the holding pressure of the pressure resistant container is 10 Kpa, the pipe diameter is about 1.6 mm. preferable.

The pressure reducing valve 4 has a fixed set pressure lower than the upstream side, that is, the helium cylinder B side in the downstream side pipe of the helium gas inlet pipe 3 and the pressure resistant container 1, for example, 10
It is held at kpa. Therefore, when the pressure inside the pressure resistant container 1 becomes lower than the set pressure of the pressure reducing valve 4, the pressure reducing valve 4 is automatically opened and helium gas flows into the pressure resistant container 1.
When the internal pressure of the pressure resistant container 1 is increased and the pressure inside the pressure resistant container 1 rises to a set pressure, the pressure reducing valve 4 is automatically closed and the inflow of helium gas is stopped. Pressure reducing valve 4
The type is not particularly limited, but a diaphragm valve can be suitably selected as a self-powered pressure reducing valve. The diaphragm valve is preferable because it has good pressure responsiveness, does not require instrumentation, etc., and can simplify the equipment. Further, in order to improve the accuracy of pressure responsiveness, a sensor type control valve can be appropriately employed as necessary.

One end of the exhaust pipe 5 is inserted into and fixed to the lid 2 of the pressure-resistant container 1, and the helium gas in the pressure-resistant container 1 is discharged through the exhaust valve 6. The material of the exhaust pipe 5 is
Similar to the helium gas feed pipe 3, the material is not particularly limited as long as it has excellent pressure resistance and corrosion resistance, and the same material as the helium gas feed pipe 3 is preferably used. The pipe diameter of the exhaust pipe 5 is determined from the pressure and flow rate of the helium gas.

The exhaust valve 6 is, together with the pressure reducing valve 4, the pressure-resistant container 1.
Gas is exhausted from the inside to maintain the inside of the pressure resistant container 1 at a constant set pressure, and is provided in the exhaust pipe 5. When the pressure inside the pressure vessel 1 rises above a set pressure, for example, 10 kpa, helium gas is automatically opened to the outside, and when the pressure inside the pressure vessel 1 returns to a predetermined pressure, the exhaust valve 6 automatically The internal pressure of the pressure resistant container 1 does not exceed the set pressure and rise. The type of exhaust valve 6 is
A diaphragm type regulating valve or the like can be used.

The HPLC degassing method of the present invention using such an HPLC degassing apparatus will be described. Eluent M
Is a method capable of adopting a degassing method by bubbling helium gas, for example, in the case of an eluent using a non-volatile solvent such as water as in a reverse phase system of partition chromatography, that is, bubbling with helium gas. When the eluent M does not change its composition when
By applying the degassing apparatus for HPLC of the present invention, bubbling with helium gas can be performed to remove gas dissolved in the eluent M. To perform bubbling of helium gas, a helium gas feed pipe 3 is inserted through a hole 2a provided in the lid 2 of the pressure-resistant container 1 and fixed so that its tip is placed in the eluent M. The exhaust valve 6 is opened, the valve of the helium cylinder B is opened, and the helium gas whose pressure is adjusted by the pressure reducing valve 4 is supplied into the eluent M in the pressure resistant container 1 to perform bubbling and dissolve in the eluent M. The gas is replaced with helium gas and removed to degas.

After that, the degassed state of the degassed eluent M is maintained. In order to maintain the degassed state of the eluent M, the helium gas feeding pipe 3 is inserted into the hole 2a provided in the lid 2 of the pressure resistant container 1, and the tip thereof is arranged in the gas phase inside the pressure resistant container 1. So that it is fixed. The valve of the helium cylinder B is opened, and helium gas is supplied to the gas phase in the pressure vessel 1 through the pressure reducing valve 4 whose pressure is adjusted to a predetermined pressure, for example, 10 kpa. At this time, the exhaust valve 6 is opened, and the gas in the pressure resistant container 1 is exhausted from the exhaust valve 6 and replaced with helium gas. Then, the pressure vessel 1 filled with helium gas is maintained at the set pressure, and the exhaust valve 6 is closed. When the pressure in the pressure vessel 1 is reduced, the pressure reducing valve 4 is opened, helium gas is supplied from the helium cylinder B to the pressure vessel 1, and the eluent M does not come into contact with air. The air is not dissolved and the degassing state is maintained. Further, when the pressure inside the pressure vessel 1 becomes higher than the set pressure, the exhaust valve 6 is opened, the set pressure is maintained inside the pressure vessel 1, and safety is ensured.

When the eluent M is volatile, such as a normal phase system in partition chromatography, the mobile phase of the eluent M is degassed by bubbling with helium gas. Therefore, after degassing using an ultrasonic oscillator or an aspirator, the mobile phase is supplied into the pressure resistant container 1 and the degassed state of the mobile phase stored in the pressure resistant container 1 is the same as above. And keep it. That is, helium gas is supplied to the gas phase in the pressure-resistant container 1 that stores the mobile phase, the gas in the pressure-resistant container 1 is replaced with helium gas, and the supply of the helium gas is controlled so that the inside of the pressure-resistant container 1 is controlled to a predetermined value. By maintaining the pressure, the contact of the mobile phase with air can be eliminated, and the degassed state can be maintained without changing the composition of the mobile phase.

The eluent M thus maintained in the degassed state
Is sent from the pressure resistant container 1 to the supply pipe S through the suction filter 7 by the operation of the pump P, injected into the HPLC column, and the analysis work is started. The pressure in the container 1 changes,
The pressure reducing valve 4 is opened, helium gas flows in, and a predetermined pressure is maintained in the pressure resistant container 1. For this reason, the contact of the eluent M with air is eliminated, the air is not redissolved in the eluent M, and the eluent M maintained in the degassed state is injected into the HPLC column, which is highly accurate. Analysis is performed.

[0029]

As is clear from the above description, according to the degassing apparatus and degassing method for high performance liquid chromatography of the present invention, helium gas is stored in the pressure resistant container for storing the eluent in the HPLC analysis work. Is supplied and kept at a predetermined pressure, whereby redissolution of air in the eluent can be prevented. Therefore, it is possible to avoid repeated bubbling operation of helium gas and improve the analysis accuracy, and the analysis accuracy does not decrease due to the fluctuation of the bubbling time. Further, even when the mobile phase in partition chromatography is volatile such as normal phase system, by keeping the gas phase in the pressure vessel at a constant pressure with helium gas, it is possible to prevent re-dissolution of air into the eluent. Therefore, it is possible to improve the analysis accuracy without changing the composition of the eluent.

When the eluent is non-volatile, bubbling with helium gas is performed using the same device,
Further, since the degassed state can be maintained, efficient and highly accurate analysis work by HPLC can be performed.

[Brief description of drawings]

FIG. 1 is a configuration diagram of a degassing apparatus for high performance liquid chromatography according to the present invention.

[Explanation of symbols] 1 ... Pressure resistant container 3 …… Helium gas inlet pipe 4 ... Pressure reducing valve 5 ... Exhaust piping 6 ... Exhaust valve M: Elution phase B: Helium cylinder S: Supply piping

Claims (3)

[Claims] 1. A degassing apparatus for high performance liquid chromatography, which supplies an eluent supplied to a column while maintaining a degassed state, comprising: a pressure container for storing the eluent; and a pressure container connected to the pressure container. A supply pipe for supplying an eluent to a column is connected to the pressure-resistant container and a helium cylinder, and the pressure inside the pressure-resistant container is controlled by controlling the amount of helium gas supplied into the gas phase inside the pressure-resistant container. Helium gas inlet pipe having a pressure reducing valve for maintaining the pressure of, and exhaust pipe connected to the pressure vessel and having an exhaust valve for exhausting helium gas to maintain the pressure vessel at a constant pressure. Characteristic degassing device for high performance liquid chromatography. 2. When supplying an eluent to a column for high performance liquid chromatography, the gas phase in the pressure vessel, in which the degassed eluent is stored, is replaced with helium gas, and helium gas is supplied so as to have a predetermined pressure. Then, the supply of helium gas to the gas phase in the pressure resistant container is controlled so as to maintain the pressure inside the pressure resistant container at a predetermined pressure, and the degassed state of the eluent is maintained. Degassing method for high performance liquid chromatography. 3. A helium gas is supplied to the eluent in the pressure vessel to perform bubbling, the dissolved gas in the eluent is removed and deaerated, and then the helium gas is supplied to the gas phase of the pressure vessel. The degassing method for high performance liquid chromatography according to claim 2.