JPS599860B2 - Method and apparatus for forming an eluent stream in liquid chromatography - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to liquid chromatography, and more particularly to a method for generating an eluent stream in a liquid chromatography system, and an apparatus for generating an eluent stream in a liquid chromatography system.

Several methods of forming eluent streams in liquid chromatography are currently known in the art. Uniform eluent flow in liquid chromatography is an essential condition for performing chromatography analysis. One method of forming an eluent stream in a liquid chromatography system involves displacing the eluent through the chromatography system under pressure.

As a pressure source, pumps of various designs are used (
J. J. Kirkland (ed.), “Modern Liquid Chromatography: The Best Practices” (MOdernPra)
activeOfLiquidChrOmatOgrap
hy), Wilmington, Delaware, E. I. IndustrialandBiOhemica of Dupont de Nouma & Co.
lDepartmentE. I. duPOntdeNe
mOursandCOMpaNy, WilmingtO
n, Delaware), WiIeyIntErscience, and ADivis, a division of John Wieley & Sons, Inc.
iOnOfJOhnWll-EyandSOns. In
c. ), (New York, London, Sydney and Toronto) 1971, see Chapter 2). This prior art method has certain drawbacks, the most important of which are the occurrence of pulsations and the high cost of the equipment necessary to carry out the method.

Additionally, modern liquid chromatography uses a wide range of pressures to force the eluent stream through the chromatography column at the desired rate. Changing the flow rate of the eluent involves delicate and labor-intensive pump adjustment operations for various pressures, which lengthens the analysis period and prevents the transition of the eluent from one flow rate to another. Providing a uniform flow of eluent in a liquid chromatograph by discharging the eluent from the container under the pressure of a piston mounted on the container with compressed gas or eluent applied to a flexible compartment. Methods of forming are known in the art.

This prior art method is realized by means of a device for forming the eluent stream in a liquid chromatography system, such as an air pump containing a rigid container in which a compressible container containing the eluent is placed. In this pump, compressed gas is supplied to a rigid container, so that said container is compressed and eluent is discharged therefrom. In various pump devices, plastic flasks, stainless steel or Teflon blowers are used as compressible containers. (J.J.
．． Edited by Kirk Rud, "The Practical Practice of Modern Liquid Chromatography" (Wilmington, Delaware, E.I. Dupont de Numas & Co., Industrial and Biochemistry Division), Uilleichi Interscience (J. Wille & Sons, Inc.) (New York, London,
(Sydney Toronto) 1971, see Chapter 2). However, said prior art methods and corresponding devices for forming eluent streams in liquid chromatography are characterized by a series of essential drawbacks.

The evolution of dissolved gases from the eluent impairs chromatographic parameters, such as resolution, and interferes with the operational stability of the zero-line variation sensor.

In order to implement the above prior art method using the above device,
A high pressure source, ie a container containing compressed gas, is required. As a result, the entire chromatography system is cumbersome, inconvenient to transport, and lacks self-reliability. The cylinder containing compressed gas operates at pressures below 150 atmospheres, so that the upper pressure limit of this pump system is limited.

The object of the present invention is to provide a method for forming an eluent stream in a liquid chromatograph, such that a uniform eluent stream can be formed without pulsation under any pressure used in the liquid chromatograph; The purpose is to provide such equipment.

J η This object is achieved by a method of forming an eluent stream in a liquid chromatography column by transferring the eluent under pressure to a chromatographic column, in which case, according to the invention, the transfer of the eluent is is ensured by the osmotic action of a solution of the substance whose concentration is kept constant.

If the same substance solution for generating the osmotic pressure is used as the eluent, the method of forming the eluent stream in liquid chromatography is substantially simplified.

A constant concentration of the substance solution is preferably maintained by contacting the substance solution with this substance in solid phase, the substance solution being isothermal at a temperature above the freezing point of the solvent.

The apparatus for carrying out the method according to the invention has a pressure source and a container containing an eluent in communication with this pressure source, according to the invention the pressure source is embodied in the form of two containers. an osmotic cell and a semipermeable membrane separating the two vessels, one of which is filled with a solvent and the other vessel is in communication with and contains a solid phase contained in the same vessel. It is filled with a saturated solution of said substance which is in contact with the substance.

In order to ensure the mechanical strength of the semipermeable membrane, the permeation cell is provided with a substrate, on the surface of which the semipermeable membrane is arranged on the side facing the solvent flow.

If it is necessary to increase the operating pressure, a single eluent stream of a liquid chromatograph, consisting of at least two identical devices connected in series for forming a single eluent stream of a liquid chromatograph. It is advisable to use a mechanism for forming . The present invention is embodied as follows. A solvent is poured into one of the containers, which contacts the solution of the substance contained in the other container through the semipermeable membrane.

The container containing the solution of the substance is in turn communicated with a container 1, which contains an eluent. If the same substance solution is used as the eluent, the chromatography column can be connected directly to the container containing the substance solution. The eluent is transferred to the chromatographic column under the osmotic pressure of a substance solution whose concentration is kept constant. Osmotic pressure begins to develop from the moment a container containing a solution of a substance is brought into contact with a solvent. The solvent begins to permeate through the semipermeable membrane, as a result of which the total volume of the substance solution increases, thus causing a movement of the eluent under a certain pressure and with a constant flow rate. At present, the parameters determining the osmotic pressure value for concentrated solutions and the functional relationship between the parameters have not been sufficiently studied. Numerical determination of the osmotic pressure of a dilute solution can be performed using the Vantorhoff equation. The concentration of the substance is maintained constant, for example, by contacting the solution with the same substance in a solid phase. To provide a constant temperature solution, the container containing the substance solution is thermostatically controlled. Increased flow rates and increased pressure values of the eluent can be obtained by selecting the corresponding pairs of substances and solvents, the type and surface of the membrane, and the temperature which is isothermally adjusted.

Solutions of various compounds can be used in the method according to the invention.

As substances, both organic and inorganic compounds such as L
iCl, NaCl, NH4Cl, CaCl,, BaCl
2, MgCl2, KCI, MgC2O4, LiNO3,
Ca(NO3)2, Mg(to N)2, NaN0,,Na
2S04, MgS04, CUS04, MnS04, A1
C13, Na0H, KH2P04, (CH, COO)2
Mg1 cellulose nitrate, sugar, hemoglobin, gelatin,
and mixtures of compounds such as CsCl-KCIl sea salt, salt lake salt, etc. can be used. Although it is most convenient to use aqueous solutions of these materials, other solvents can also be used. The invention will become more fully apparent from the following detailed description of specific embodiments, taken in conjunction with the accompanying drawings.

Device 1 for forming an eluent stream for liquid chromatography (
FIG. 1) is a pressure source in the form of an osmotic cell, comprising an osmotic cell (pressure source) 2 communicating with a container 3 containing an eluent 4.

The eluent is contained in a variable volume 5 of the rigid container 3, the volume of which is limited by a movable impermeable septum 6.

The volume of said variable volume 5 is also limited by the interface between the eluent and a saturated solution 7 of a substance immiscible with the eluent and contained in the container 8 of the osmotic cell 2 which communicates with the rigid container 3. . In addition to the saturated solution 7, a solid phase substance 9 is present in the container 8, and the saturated solution and solid phase substance are separated from the solvent 11 by a semipermeable membrane 10. The solvent 11 is contained in a container 12 of the permeation cell 2, which is provided with an inlet pipe 13 for the supply of the solvent 11. When using the device according to this embodiment of the invention, the semipermeable membrane is placed on the substrate 14 (FIG. 2) in order to provide high pressure and high mechanical strength of the semipermeable membrane 10 (FIG. 1). Ru. As a semi-permeable membrane, it is porous and dynamic (D
yna-MlC) membranes, preformed membranes, sealing membranes, rigid structure membranes, composite membranes, isotropic membranes, anisotropic membranes, deposited membranes, impregnated membranes, sprayed membranes and deposited membranes. . Furthermore, non-porous (diffusible) membranes with sealing properties, rigid structures, isotropic or anisotropic membranes can be used. If it is necessary to increase the operating pressure, the present invention provides a system for forming an eluent stream in a column chromatography system consisting of at least two series-connected devices identical to the device 1 shown in FIG. provide.

This system is illustrated in FIG. 3, where, for convenience in explaining the operation of the system, one structural member of the device has a reference letter that differs from that of the same member in another device. The liquid chromatography system for forming one eluent stream is connected to a second similar device 1 (FIG. 3) through an inlet pipe 13 (FIG. 2) for supplying the solvent.
The device 1 (FIG. 2) communicates with the outlet of a rigid container 15 (FIG. 3). A second similar device is a rigid container 15 containing a solvent 11 (the same solvent present in container 12 of FIG. 1) in a flexible container 16.

The solvent 11 is separated by a movable partition 17 from a saturated solution 18 of a substance stored in a container 15 which communicates with the vessel 19 of the osmosis cell 2, the saturated solution 18 of substance 18 and the same substance 20 in solid phase being separated from the solvent supply. From the solvent 23 contained in the other container 24 of the infiltration cell 2, which is provided with an inlet pipe 25 for the substrate 2
2 is separated by a semipermeable membrane 21 on top. The operation of an apparatus for carrying out the method of forming an eluent stream in liquid chromatography is illustrated in FIG.

At the initial stage, the eluent 4 is poured into the variable vessel 5 of the container-3, and the outlet of the container-3 is connected to the chromatography column. The container 12 is supplied with the same solvent 11 (used for dissolving the saturated solution 7 of the substance) via a tube 13 . A saturated solution 7 of the substance is then poured into the container 8 and the same substance 9 in the solid phase is applied at a selected flow rate of the eluent (ω for a predetermined time (γ)) to ensure a constant concentration of the saturated solution of the substance in the container 8. (
Cs). The amount of solid material is determined by the following relationship: Q=K-Cs.
αγ [where K is a proportionality coefficient].

When the solvent 11 comes into contact with the semipermeable membrane 10, the osmotic pressure begins to increase in the container 8 due to the penetration of the solvent 11 into the saturated solution 7 of the substance, and under this osmotic pressure the saturation of the substance increases. The sum solution 7 is in the container 8.
from container 1 containing eluent 4.

Under the action of the osmotic pressure of the saturated solution 7 of the substance, the flexible impermeable septum 6 or the interface between the eluent and the saturated solution 7 of the eluent-immiscible substance begins to move, so that the container - From 3, the eluent 4 moves towards the chromatography column (the direction of movement of the moved eluent towards the chromatography column is indicated by arrow A). To increase the working pressure, apart from selecting a suitable pair of solvents, a liquid chromatography mechanism for forming an eluent stream as shown in FIG. 3 can be used. .

The operation of this mechanism is as follows.

At an initial stage, the eluent 4 is poured into the flexible container 5 (FIG. 3) of the container 3, and the outlet of the container 3 is connected to the chromatography column. Inlet pipe 13 to container 12
The same solvent (same as the solvent 11 that is poured to fill the flexible vessel of container-15) is introduced through, and the outlet of container-15 is connected to tube 13. A solvent 23, which may be the same as the solvent 11 or different, is poured into the container 24 via an inlet pipe 25. Containers 8 and 1
At 9, solid phase substances 9 and 20 are introduced (the substances may be the same or different). The containers are filled with saturated solutions 7 and 18 of the corresponding substances. When the solvent 23 comes into contact with the saturated solution of substance 18 through the semipermeable membrane 21 on the substrate 22, the solvent 23 begins to permeate into the container 19 where the osmotic pressure begins to increase. Under the action of this pressure, a saturated solution 18 of the substance is transferred from the variable volume 16 of the container 15 to the solvent 2.
3 in communication with the container 15 via a pipe 13
2, thereby increasing the initial pressure of the solvent 11 in subsequent osmotic cells. The solvent 11 now passes through the semipermeable membrane 10 into the container 8 containing the saturated solution 7 of the substance, creating there an osmotic pressure equal to the sum of the osmotic pressure values developed in the two osmotic cells. Under this pressure, the eluent 4 is transferred from the variable volume 5 of the container 3 to the chromatography column.

The present invention allows a uniform flow of eluent to be produced in a liquid chromatograph without pulsation within a wide range of pressures used in the liquid chromatograph.

Furthermore, the method according to the invention is very simple with respect to the equipment used.

In the device for carrying out the method according to the invention there are no moving parts. Unlike prior art mechanisms where operation of the pump system causes contamination of the eluent with impurities, such contamination does not occur in the method according to the invention. In order to better understand the invention, some specific examples are illustrated below.

Example 1 A solvent, ie water, is introduced into a steel container 12 having an internal volume of 200 m1 through an inlet pipe 13.

A variable volume 5 with a capacity of 500 ml is filled with heptane.
Enough 2009 Mg to replace the total volume of eluent
The SO4 solid salt is placed in a container 8 with an internal volume of 500 m1. Next, a saturated aqueous solution of MgSO4 is poured into container 8. Container 12 is separated from container 8 by a semipermeable cellulose acetate film membrane 10 supported by a cermet substrate 14. The surface area of the membrane is equal to 25c111. Water penetrates into the container 8 from the side of the cermet substrate and creates an osmotic pressure inside it. The pressure at the inlet of the chromatography column and the eluent flow rate through the column are determined by the solid salt MgSO
4 is maintained constant until it is completely dissolved in the water supplied to container 8. The constant flow rate of heptane is 4 m1/h and the pressure at the column inlet is 120 atmospheres absolute. Example 2 The procedure of Example 1 is repeated, except that the semipermeable diaphragm is a plasma-deposited 4-vinylpyridine membrane supported on a "Millibore" substrate manufactured by Millibore, Inc.

The flow rate of the eluent is 60 ml/h at 78 atmospheres absolute. Example 3 The method of Example 1 is repeated, but the resistance of the chromatography column is varied by decreasing the length of the column.

At a constant heptane flow rate of 12 ml/h, the pressure at the column inlet is equal to 40 atmospheres absolute. Example 4 Repeat the method of Example 1, but
However, a saturated aqueous solution of sutucarose is poured into the container 8.

At a constant flow rate of the eluent of 10 TfL1/h, the pressure at the inlet of the chromatography column is equal to 110 atmospheres absolute.

Example 5 The procedure of Example 1 is repeated, but while the cyclohexanone is poured into container 12, the saturated solution of cellulose nitrate in cyclohexanone is poured into container 8, and a polyimide film is used as the membrane.

At a constant eluent flow rate of 6 ml/h, the inlet pressure of the chromatography column is 90 atm absolute. Example 6 The procedure of Example 1 is repeated, except that while the ethanol is being poured into container 12, the saturated ethanol solution of sutucarose is poured into container 8.

At a constant flow rate of the eluent of 9 mj/h, the inlet pressure of the chromatography column is 70 atm absolute.

EXAMPLE 7 The process is carried out in a similar manner to Example 1, but the saturated solution of cellulose nitrate in ethyl acetate is poured into container 8 while the ethyl acetate is poured into container 12, and acrylonitrile film is used as membrane material.

At a constant eluent flow rate of 6 d/h, the inlet pressure of the chromatography column is equal to 64 atmospheres absolute.

EXAMPLE 8 A liquid chromatography system for forming a single eluent stream as shown in FIG.

At the initial stage, the variable volume 5 (FIG. 3) of the container 3 is poured with heptane 4 and the outlet of the container 3 is connected to the chromatography column. Water is introduced into the container 12 through the inlet pipe 13. The variable volume 16 of the container 15 is also filled with water, while the outlet of the container 15 is connected to the pipe 13. Solvent, ie water, is introduced into container 23 through inlet tube 25. The solid salt MgSO49,2O is introduced into vessels 8 and 19 in an amount of 2009 per vessel. Thereafter, saturated aqueous solutions of MgSO4 7 and 18 are poured into the same containers 8 and 19. When the water and the saturated MgSO4 solution 18 come into contact through the semi-permeable membrane 21, the water begins to permeate into the container 19 where an osmotic pressure begins to develop.

Under the action of this pressure, the saturated aqueous solution 18 of MgSO4 moves water from the variable volume 16 of the container-15 to the vessel 12, which is connected to the container-15 by a tube 13, so that the water contained in the vessel 12 Initial pressure increases. This water in turn permeates through the semipermeable membrane 10 made of cellulose acetate film placed on the cermet substrate 14 to form MgS.
It enters the container 8 containing a saturated aqueous solution of O4, creating an osmotic pressure equal to the sum of the values of the osmotic pressures occurring in the two osmotic systems. Osmotic pressure is equal to 235 atmospheres absolute. Under this pressure, heptane 4 is transferred from variable volume 5 of container-3 to the chromatography column. 4 at a pressure of 235 atmospheres absolute with a column length twice that of Example 1.
A heptane flow rate equal to m1/h is obtained.

Example 9 Ca.
4509 solid salts of (NO3)2 are charged in advance.

Into the same container 26 is poured a saturated aqueous solution of Ca(NO,)2.

A 500 mt variable volume 31 of a rigid container 32 is filled with heptane 33 and connected to a chromatography column. The filled container 26 is placed in a tank 34 containing a solvent 35, ie water. As soon as the container 26 is placed in the tank 34, water begins to percolate through the semipermeable membrane 27 into the container 26, where it creates an osmotic pressure. Under the influence of this pressure, heptane moves into the chromatographic column.

The range of operating pressure values occurring in this osmotic system is from 5 to 150 atmospheres absolute.

The heptane flow rate used as eluent is 18 ml/h. Example 10 A 1,000 ml titanium container 26 (FIG. 4) with a cylindrical surface area defined by a polyvinyl chloride semipermeable membrane partition 27 supported on a reinforced cermet substrate 28 is pre-charged with the solid salt NaCl 29. do.

The same container 26 is charged with a saturated aqueous solution 30 of NaCl.
In contrast to Example 4, in this example the role of eluent is performed by a saturated aqueous solution 30 of NaCl. The outlet of container 26 is connected to a chromatography column (not shown). The container 26 is placed in a tank 34 filled with a solvent 35, which is water. As soon as the container 26 is placed in the tank 34, water passes through the semi-permeable septum 27 into the container 26, where it develops an osmotic pressure. Under this pressure, a saturated aqueous solution of NaCl 30 is discharged into a chromatography column. A predetermined flow rate of NaCl saturated aqueous solution equal to 60 ml/h under a pressure of 48 atmospheres absolute is obtained.

Example 11 The method of Example 9 is repeated, but using a saturated aqueous solution of CH3COONa.

A constant flow rate of eluent equal to 60 ml/h under a pressure of 57 atmospheres absolute is ensured.

Example 12 120 ml container 36 with walls having a polyvinyl chloride semipermeable membrane 37 supported on a reinforced cermet substrate 38
(FIG. 5), a heater 39 is provided.

250g of Mg (NO3) in 600Tn1 steel container 40
241 and its saturated aqueous solution 42 is also charged thereto at 25°C.

Pour hexane 45 into the 500 d variable volume 43 of the rigid container 44 and connect the outlet of the container 44 to the chromatography column. Vessel 40 is in communication with container 44.

Thereafter, a solvent 46, which is water, is charged into the container 36, and the temperature of the solvent 46 is adjusted to 50° C. by a heater 39. As soon as water is poured into container 36, it passes through semipermeable membrane 37 into container 40, where it develops an osmotic pressure. Under the above pressure, hexane is transferred into the chromatography column. Depending on the isothermally regulated temperature, the osmotic pressure value will vary and therefore the flow rate of the eluent through the chromatographic column will vary. 5 at osmotic pressure of 190 absolute atmospheres
A predetermined flow rate of a saturated aqueous Mg(NO3)2 solution equal to 30 ml/h at a temperature of 0° C. is ensured.

[Brief explanation of the drawing]

FIG. 1 is a longitudinal sectional view of an embodiment of an apparatus for carrying out the method according to the invention, and FIG. A longitudinal section in elevation of another embodiment of the device according to the invention, FIG. 3 shows a system consisting of two similar devices (according to FIG. 2) connected in series for carrying out the method of the invention. FIG. 4 is an elevational view in longitudinal section, FIG. 4 is a front view in longitudinal section, FIG.
The figure shows a longitudinal section in elevation of another embodiment of the device for carrying out the method according to the invention. 1... Device for forming an eluent flow in liquid chromatography, 2... Pressure source (osmotic system),
3... Container 1, 4... Eluent, 7
... Saturated solution of substance, 8 ... Container containing saturated solution of substance, 9 ... Solid phase substance, 10
... Semipermeable membrane, 11 ... Solvent, 12 ...
. . . Container containing solvent, 14 . . . Substrate.

Claims (1)

[Claims] 1. A saturated solution consisting of a solvent and a substance dissolved therein;
the eluent by contacting the substance with the solvent through a semipermeable membrane and pushing a variable volume of the eluent with the liquid chromatography eluent by the resulting pressure increase of the saturated solution. A method of forming an eluent stream comprising transferring an eluent to a liquid chromatography column. 2 As substances, Mg(NO_3)_2, MgSO_4
, NaCl, Ca(NO_3)_2, CH_3COON
a. The method according to item 1 above, wherein cellulose nitrate, cellulose, and sucrose are used. 3. The method according to item 1 above, wherein water, ethanol, ethyl acetate, cyclohexanone is used as the solvent. 4. The method according to item 1 above, wherein as eluent the same substance solution as for the creation of osmotic pressure is used. 5. Process according to paragraph 1 above, wherein a constant concentration of the substance solution is maintained by contacting said solution with the same substance in a solid phase, and wherein the substance solution is thermostatically regulated at a temperature above the freezing point of the solvent. 6 In a device for producing a flow of eluent in liquid chromatography, a rigid container 3 having an outlet leading to a chromatographic column and containing a variable volume 5 containing eluent 4; two vessels 8 and 12; , an osmotic cell (pressure source) 2 consisting of a semipermeable membrane 10 that separates these two containers, and a base 14 that supports the semipermeable membrane;
However, the container 12 is filled with a solvent 11 and the container 8 is in communication with the container 3 and is filled with a saturated solution of a substance soluble in the solvent, which saturated solution is in the same container 8. device for generating a flow of eluent, which is in contact with the solid substance 9 contained in the container, and the saturated solution is in contact with the variable volume 5 via a movable impermeable partition 6. 7. Apparatus according to clause 6, in which the osmotic system is provided with a substrate and the semipermeable membrane is placed on the surface of the substrate from the side of the substrate facing the solvent flow. 8 As a semipermeable membrane, cellulose acetate, polysulfonic acid,
7. The device of item 6 above, having a polyimide and acrylonitrile membrane. 9. In an apparatus for producing a flow of eluent in a liquid chromatograph consisting of at least two apparatuses 1 (FIG. 2), one of the apparatuses 1 has an outlet leading to the chromatographic column and has an eluent flow. a rigid container 3 containing a variable volume 5; an osmotic cell (pressure source) consisting of two containers 8 and 12, a semipermeable membrane separating the two containers, and a substrate 14 supporting the semipermeable membrane; 2; wherein said container 12 is filled with a solvent 11, said container 8 communicates with said container 3 and filled with a saturated solution of a substance 9 dissolved in said solvent, the saturated solution is in contact with the substance 9 in solid form contained in the same container 8, and the saturated solution is in contact with the variable volume 5 via a movable impermeable partition 6; Another of the devices 1 has: a rigid container 15 with an outlet leading to the container 12 of the previous device 1 and containing a variable volume 16 containing a solvent identical to that in that container 12; two containers 19; and 24, and a semipermeable membrane 21 that separates these two containers,
an osmotic cell (pressure source) 2 consisting of a substrate 22 supporting the semipermeable membrane; the container 24 is filled with a solvent 23; the container 19 communicates with the container 15; is filled with a saturated solution of a substance 20 soluble in the solvent, the saturated solution is in contact with the solid substance 20 contained in the container 19, and the saturated solution is mobile in contact with the variable volume 16 via an impermeable partition 17 and, if required, any number of devices 1
a device for producing a flow of eluent, connected in succession to the previous one in the same way.