KR101120944B1 - Automated dual online multi-functional liquid chromatography device - Google Patents

Abstract

The present invention relates to an automated dual on-line multifunction liquid chromatography apparatus, comprising: a sample inlet valve loaded with a sample to be analyzed; A first trap valve in fluid communication with the first solid phase extraction column and the first reversed phase liquid chromatography column; A second trap valve in fluid communication with the second solid phase extraction column and the second reversed phase liquid chromatography column; A first separation valve and a second separation valve for selectively discharging the fluid introduced into the first trap valve or the second trap valve, respectively; A multifunctional selection valve positioned in a flow path from the sample inlet valve to the first and second trap valves and connected to a sample separation column; And a connection valve for selectively supplying the fluid flowing out of the sample inlet valve to the multifunctional selection valve, the first trap valve, or the second trap valve. The automatic double online multifunction liquid chromatography according to the present invention. By using the device, one-dimensional separation function and sample separation column capable of desalination process and sample concentration can be performed, and two-dimensional separation function and online digestion function can be performed to increase the efficiency of desalination process and sample concentration. The experiment speed can be increased by minimizing the dead time to stop the experiment during the column equilibration time between each repeated experiment.

Description

AUTOMATIC DUAL ONLINE MULTI-FUNCTIONAL LIQUID CHROMATOGRAPHY DEVICE

The present invention relates to an automated dual on-line multifunctional liquid chromatography device, and more particularly, it is possible to perform online sample desalination, sample concentration, online digestion and phosphorylated peptide extraction, and column equilibration time between repeated experiments. Automated dual on-line multifunctional liquid chromatography that minimizes dead time to stop the experiment while increasing the experiment speed, enables rapid injection of samples, and highly repeatable with respect to the residence time of liquid chromatography Relates to a photography apparatus.

On-line solid phase extraction / capillary reverse-phase liquid chromatography has been recognized as an important technology for proteome research because of its excellent analytical efficiency. In particular, it is possible to effectively separate fine amounts of biomaterials and the wide range of analyte-solid reactions makes it possible to identify fine amounts of proteins with high efficiency.

Mass spectrometry-base methods have become a standard analytical platform for proteome research, and the shotgun method, or the bottom-up method, is a method for protein analysis prior to mass spectrometry. Is hydrolyzed to peptides, which increase the solubility of biological samples and produce peptide fragments that are readily ionizable and detectable in mass spectrometers.

However, this process inevitably leads to sample complexity, for example, in the case of the yeast proteome, one of the simplest proteome, more than 300,000 peptide fragments are produced from about 6,000 different proteins. Therefore, as a way to solve this sample complexity, various methods such as on- / off-line multidimensional protein identification technology have been developed (Link, AJ, Eng, J. , Schieltz, DM, Carmack, E., et al ., Nat . Biotechnol . 1999, 17 , 676-682; Chen, EI, Hewel, J., Felding-Habermann, B., Yates, JR III, Mol . Cell . Proteomics 2006, 5 , 53-56.), There is still a need to improve the efficiency and sensitivity of liquid chromatography columns. At this time, it has been known that the sensitivity of liquid chromatography / mass spectrometry experiments can be dramatically increased when the inner diameter is reduced while keeping the length of the separation column constant (Kim, M.-S., Choie, W. -S., Shin, YS, Yu, MH, Lee, S. -W., Bull. Korean Chem . Soc . 2004, 25 , 1833-1839.).

In addition, in the case of biological samples containing significant amounts of detergents and salts, the on-line desalting step is an essential process that must be performed prior to mass spectrometry. This interferes with the ionization process, reducing the detection sensitivity of the peptide sample. In this case, in consideration of time saving and sample loss, an on-line desalting process is more advantageous than an off-line desalting process.

On the other hand, the conventional reverse phase liquid chromatography device employs a solid phase extraction column to have only a one-dimensional separation function for desalting and concentrating a sample, or a two-dimensional separation function by connecting a strong cation exchange chromatography device and a reverse phase liquid chromatography online. Even in the case of a two-dimensional reverse phase liquid chromatography apparatus that performs the problem, it is difficult to perform accurate analysis due to mutual interference. In addition, such a two-dimensional on-line reverse phase liquid chromatography apparatus could not perform an on-line digestion function for digesting a protein in a peptide state or a function of selectively extracting only phosphorylated peptide. Furthermore, when filling capillary columns having long lengths and small inner diameters with a hydrophobic medium, a long time is required for column equilibration (or regeneration). For example, equilibration takes at least two hours to reuse a column having a length of 1 m and an internal diameter of 75 μm.

The present invention is to solve the above problems, the problem to be solved by the present invention is a two-dimensional separation function capable of desalting process and sample concentration, passing through the sample separation column to increase the efficiency of desalination process and sample concentration 2 In addition to dimensional separation function, online digestion function and extracting only phosphorylated peptides, the experiment speed is increased by minimizing dead time which should be stopped during column equilibration time between repeated experiments. The present invention provides an automated dual on-line liquid chromatography device capable of injecting a sample at a high speed and having a very high reproducibility with respect to the retention time of liquid chromatography.

The present invention to achieve the above object,

A sample inlet valve into which a sample to be analyzed is loaded;

A first trap valve in fluid communication with the first solid phase extraction column and the first reversed phase liquid chromatography column;

A second trap valve in fluid communication with the second solid phase extraction column and the second reversed phase liquid chromatography column;

A first separation valve and a second separation valve for selectively discharging the fluid introduced into the first trap valve or the second trap valve, respectively;

A multifunctional selection valve positioned in a flow path from the sample inlet valve to the first and second trap valves and connected to a sample separation column; And

It provides an automated dual on-line multi-function liquid chromatography device comprising a; connection valve for selectively supplying the fluid flowing out of the sample inlet valve to the multi-function selection valve, the first trap valve, or the second trap valve.

The apparatus may further include a first solvent supply pump and a second solvent supply pump for supplying a solvent to the automated dual on-line multifunction liquid chromatography apparatus.

In addition, it is preferable that the first solvent supply pump is in fluid communication with the sample inlet valve or the connection valve, and the second solvent supply pump is in fluid communication with the second separation valve.

In addition, it is preferable that the first solvent supply pump selectively supplies a solvent to the sample inlet valve or the connection valve through a T-type solvent separation tube.

In addition, it is preferable that at least one of the first solvent supply pump and the second solvent supply pump be provided with a solvent selection valve so as to supply a first solvent or a mixed solvent of the first solvent and the second solvent.

The sample inlet valve may include a sample inlet port, a sample discharge port, a first sample storage loop connection port and a second sample storage loop connection port, a solvent inlet port, and a solvent outlet port connected to each other by a sample storage loop. It is preferable.

The sample inlet valve may include: a first mode in which the sample inlet port and the first sample storage loop connection port are in fluid communication, and the second sample storage loop connection port and the sample discharge port are in fluid communication; And a second mode in which the first sample storage loop connection port and the solvent outlet port are in fluid communication, and the second sample storage loop connection port and the solvent inlet port are in fluid communication.

The connection valve may include a first inflow port, a second inflow port, a first connection port, a second connection port, a first outlet port, and a second outlet port.

The connecting valve may include a first mode in which the first inflow port, the first connection port, the second connection port, and the first outlet port are sequentially in fluid communication; And a second mode in which the second inflow port, the second connection port, the first connection port, and the second outlet port are sequentially in fluid communication.

In addition, the connection valve is preferably formed in the Z-type flow path between the ports in fluid communication with each other.

In addition, the multi-function selection valve, the fluid passage mode for introducing the fluid discharged from the sample inlet valve to discharge to the trap valve; A column passing mode through which the fluid discharged from the sample inlet valve passes through the sample separation column and is discharged to the trap valve; And a fluid closing mode for preventing the inflow of the fluid discharged from the sample inlet valve.

In addition, the multi-function selection valve, the inlet port, the outlet port, the first sample separation column connection port and the second sample separation column connection port respectively connected to both ends of the sample separation column, and other ports to implement the modes optional It may include a plurality of selection ports in fluid communication with.

In addition, the inflow port and the outlet port is in fluid communication in the fluid passage mode, the inlet port and the first sample separation column connection port, the outlet port and the second sample separation column connection port in the column pass mode Each in fluid communication with each other, it is preferable that the inlet port and the outlet port in the fluid closing mode is fluid disconnected.

In addition, the titanium dioxide column is further connected to the multi-function selection valve,

The multi-function selection valve, the fluid passage mode for introducing the fluid discharged from the sample inlet valve to discharge to the trap valve; A column passing mode through which the fluid discharged from the sample inlet valve passes through the sample separation column and is discharged to the trap valve; A fluid closing mode for preventing an inflow of fluid discharged from the sample inlet valve; And a titanium dioxide column passing mode through which the fluid discharged from the sample inlet valve passes through the titanium dioxide column and is discharged to the trap valve.

In addition, the multi-function selection valve, the inlet port, the outlet port, the first sample separation column connection port and the second sample separation column connection port respectively connected to both ends of the sample separation column, respectively connected to both ends of the titanium dioxide column A first titanium dioxide column connection port and a second titanium dioxide column connection port may include a plurality of selection ports selectively in fluid communication with other ports to implement the modes.

In addition, the inflow port and the outlet port is in fluid communication in the fluid passage mode, the inlet port and the first sample separation column connection port, the outlet port and the second sample separation column connection port in the column pass mode Each in fluid communication with each other, the inlet port and the outlet port are fluid-disconnected in the fluid closing mode, the inlet port and the first titanium dioxide column connecting port, the outlet port and the second port in the titanium dioxide column passing mode. Preferably, the titanium dioxide column connection ports are in fluid communication with each other.

The first trap valve may include a solid phase extraction column connection port in fluid communication with the first solid phase extraction column, a reverse phase liquid chromatography column connection port in fluid communication with the first reverse phase liquid chromatography column, a first inlet port, And a second inlet port, a sample moving loop connection port connected by the first solid phase extraction column connection port and a sample moving loop, and a discharge port, wherein the second trap valve is in fluid communication with the second solid phase extraction column. A solid phase extraction column connection port, a reverse phase liquid chromatography column connection port in fluid communication with the second reverse phase liquid chromatography column, a first inlet port, a second inlet port, and the second solid phase extraction column connection port and a sample moving loop It may include a connected sample transfer loop connection port, and the discharge port.

The first trap valve and the second trap valve may each include: a first mode in which the solid phase extraction column connection port and the first inlet port, the sample moving loop connection port, and the discharge port are in fluid communication with each other; And a second mode in which the reverse phase liquid chromatography column connection port, the solid phase extraction column connection port, and the second inflow port and the sample moving loop connection port are respectively in fluid communication with each other.

In addition, each of the first separation valve and the second separation valve may include a first inflow port, a second inflow port, a first outflow port, and a second outflow port.

The first separation valve and the second separation valve may each include: a first mode in which the first inflow port and the first outflow port, the second inflow port and the second outflow port are in fluid communication with each other; And a second mode in which the first inflow port and the second outflow port, the second inflow port and the first outflow port are in fluid communication with each other.

In addition, it is preferable to equilibrate in the second solid phase extraction column and the second reverse phase liquid chromatography column while the sample is analyzed in the first reverse phase liquid chromatography column.

In addition, it is preferable that equilibration is performed in the first solid phase extraction column and the first reverse phase liquid chromatography column while the sample is analyzed in the second reverse phase liquid chromatography column.

In addition, in the first solid phase extraction column, the flow direction of the incoming sample and the flow direction of the sample eluted toward the first reverse phase liquid chromatography column are opposite to each other, and in the second solid phase extraction column, Preferably, the flow direction and the flow direction of the sample eluted toward the second reversed phase liquid chromatography column are opposite to each other.

Using an automated dual on-line liquid chromatography apparatus according to an embodiment of the present invention, the separation efficiency by connecting the one-dimensional separation and reverse phase two-dimensional separation using the one-dimensional separation function and the sample separation column capable of desalting and sample concentration The two-dimensional separation function and high pressure in the solvent can be produced to perform on-line digestion function to digest protein into peptide state. In addition, it is possible to selectively extract only the phosphorylated peptide using a titanium dioxide column. In addition, by using a plurality of columns and a plurality of pumps, the experiment speed can be increased by minimizing a dead time to stop the experiment during the column equilibration time required between repeated experiments each time. That is, when the solvent of the first solvent supply pump flows to the first reverse phase liquid chromatography column, the solvent of the second solvent supply pump flows to the second reverse phase liquid chromatography column, while the other column is As the solvent flows, it washes the path that the sample moved and equilibrates the column state, thereby minimizing downtime and increasing sample analysis efficiency.

1 is a block diagram of each valve in the sample loading step in the automated dual on-line reverse phase liquid chromatography apparatus according to an embodiment of the present invention.
2 is a block diagram of each valve in the process of performing an on-line fire extinguishing function in an automated dual on-line reverse phase liquid chromatography apparatus according to an embodiment of the present invention.
Figure 3 is a block diagram of each valve in the process of performing the one-dimensional separation function in the automated dual on-line reverse phase liquid chromatography apparatus according to an embodiment of the present invention.
Figure 4 is a block diagram of each valve in the process of performing the two-dimensional separation function in the automated dual on-line reverse phase liquid chromatography apparatus according to an embodiment of the present invention.
5 is a block diagram of each valve in the process of performing the phosphorylated peptide extraction function in the automated dual on-line reverse phase liquid chromatography apparatus according to an embodiment of the present invention.
6 shows an equilibrium in a second solid phase extraction column and a second reverse phase liquid chromatography column during sample analysis in a first reverse phase liquid chromatography column in an automated dual on-line reverse phase liquid chromatography apparatus according to an embodiment of the present invention. It is a block diagram of each valve in the process made.

Hereinafter, the present invention will be described in more detail with reference to preferred examples. However, these examples are intended to illustrate the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited thereby.

Automated dual on-line multifunction liquid chromatography apparatus according to an embodiment of the present invention, as shown in Figure 1, the sample inlet valve 100 is loaded with the sample to be analyzed; A first trap valve 400a in fluid communication with the first solid phase extraction column 410a and the first reversed phase liquid chromatography column 420a; A second trap valve 400b in fluid communication with the second solid phase extraction column 410b and the second reversed phase liquid chromatography column 420b; A first separation valve 600a and a second separation valve 600b for selectively discharging the fluid introduced into the first trap valve or the second trap valve, respectively; A multi-function selection valve 300 positioned in a flow path from the sample inlet valve to the first and second trap valves and connected to a sample separation column 310; And a connection valve 200 for selectively supplying the fluid flowing out of the sample inlet valve to the multifunctional selection valve, the first trap valve, or the second trap valve.

Automated dual on-line multifunction liquid chromatography apparatus according to an embodiment of the present invention by changing the mode (or position) of each of the valves described above, that is, the sample is analyzed in one column while performing a variety of functions with only a simple operation of the valve The other column can be used to wash down the path that the sample has traveled as solvent flows and to equilibrate the column to minimize downtime. Here, the sample inlet valve 100, the connection valve 200, the first and second separation valve (600a, 600b), the first and second trap valve (400a, 400b) may include two modes, The multifunctional selector valve 300 used to perform various functions of the liquid chromatography apparatus may include three or four modes. Hereinafter, a process of analyzing a sample in a first reversed phase liquid chromatography column and a process of equilibrating a column at the same time in a sample analysis process will be described as an example.

1 is a view illustrating a valve configuration of an automated dual on-line liquid chromatography device in a sample loading step.

The sample inlet valve 100 is a valve for loading a sample by introducing a sample to be analyzed, and includes a first sample storage loop connected to each other by a sample inlet port 101, a sample discharge port 102, and a sample storage loop 107. The connection port 103 and the second sample storage loop connection port 104, the solvent inlet port 105, and the solvent outlet port 106 may be included.

Automated dual on-line liquid chromatography apparatus according to an embodiment of the present invention by using two solvent supply pump (500a, 500b) as shown in Figure 1 of the sample in one column of two reverse phase liquid chromatography column Allows column equilibration in the other column during analysis. In addition, such an automated dual on-line liquid chromatography device by changing the flow path of the fluid by using the multi-function selection valve 300, the one-dimensional separation function and the one-dimensional separation using the sample separation column and desalination process and sample concentration In addition to the two-dimensional separation function to increase the separation efficiency by connecting reverse phase two-dimensional separation, and the on-line digestion function that digests the protein into peptide state by generating high pressure in solvent, it selectively extracts only the phosphorylated peptide using titanium dioxide column. Function can be performed.

The state of the sample inlet valve shown in FIG. 1 is a step in which a sample is loaded, and the sample inlet port 101 and the first sample storage loop connection port 103, the sample discharge port 102 and the second sample storage loop are illustrated in FIG. Since the connection ports 104 are in fluid communication with each other, the sample may be introduced into the sample storage loop 107 through the sample inlet port 101. When the user determines that the concentration of the sample is too low through the sample storage loop 107, a sufficient sample concentration can be obtained by repeating the sample injection a plurality of times.

The sample storage loop 107 preferably has a volume of 1 μl to 10 μl, and when the volume of the sample storage loop 107 is less than 1 μl, there is a problem in that the handling of the sample is difficult. It is not preferable because the sample injection time is long.

In addition, the sample inlet valve 100 includes a sample discharge port 102, which discharges the extra sample so that the sample can be accommodated within the above-described volume range in the sample storage loop 107. Play a role.

As described above, when the inflow of the sample is completed, the connection state of the sample inlet valve is changed by using a mode switching switch (not shown) of the sample inlet valve 100 in the first mode shown in FIG. Switch to 2 mode.

In the second mode of the sample inlet valve 100, the first sample storage loop connection port 103 and the solvent outlet port 106 are in fluid communication, and the second sample storage loop connection port 104 and the solvent inlet port 105 are in fluid communication. Is configured to be in fluid communication.

On the other hand, the automated dual on-line multifunction liquid chromatography apparatus according to an embodiment of the present invention may further include a first solvent supply pump 500a and a second solvent supply pump 500b for supplying a solvent. As shown in FIG. 1, the first solvent supply pump 500a may be in fluid communication with the sample inlet valve 100 or the connection valve 200, and the second solvent supply pump 500b may include a second separation valve ( 600b) may be in fluid communication.

Each of these solvent supply pumps 500a and 500b preferably supplies solvent at a pressure of 5,000 psi to 20,000 psi. When the solvent supply pressure is less than 5,000 psi, the length of the usable column is shortened to reduce separation resolution. There is a problem, and if it exceeds 20,000 psi, there is a possibility of solvent leakage from the valve, which is undesirable.

The solvent supplied from the solvent supply pumps 500a and 500b may be a first solvent or a mixture of the first solvent and the second solvent. To this end, a solvent selection valve (not shown) may be installed in the solvent supply pumps 500a and 500b to supply a first solvent or a mixed solvent in which a first solvent and a second solvent are mixed at a predetermined ratio.

In addition, the T-type solvent separation tube 510 for selectively supplying a solvent to the sample inlet valve 100 or the connection valve 200 may be connected to the first solvent supply pump 500a.

2 is a block diagram of each valve in the process of performing an on-line fire extinguishing function in an automated dual on-line reverse phase liquid chromatography apparatus according to an embodiment of the present invention.

The first solvent is introduced into the sample inlet valve 100 from the solvent inlet port through the first solvent supply pump 500a, and the sample remaining in the sample storage loop 107 by the hydraulic pressure of the supplied first solvent is the solvent outlet port. It is discharged through the 106, and reaches the connecting valve 200.

The connection valve 200 includes a first inlet port 201, a second inlet port 202, a first connection port 203, a second connection port 204, a first outlet port 205, and a second outlet port. 206, the first inlet port 201 of the connecting valve 200 is in fluid communication with the solvent outlet port 106 of the sample inlet valve 100.

In the process of performing the on-line fire extinguishing function, the connection valve 200 sequentially fluidly communicates the first inlet port 201, the first connection port 203, the second connection port 204, and the first outlet port 205. Has a modified mode. Therefore, the first solvent introduced from the solvent supply pump together with the sample includes the first inlet port 201, the first connection port 203, the second connection port 204, and the first outlet port of the connection valve 200. It passes through the connecting valve while passing through 205). At this time, as shown in FIG. 2, the connection valve is formed with a Z-type flow path between the ports in fluid communication with each other.

The first solvent passing through the connection valve 200 reaches the multifunction selection valve through the inlet port 301 of the multifunction selection valve 300 in fluid communication with the first outlet port 205 of the connection valve 200.

As shown in FIG. 2, the multi-function selection valve 300 includes a first sample separation column connection port 302 connected to both ends of the inlet port 301, the outlet port 307, and the sample separation column 310, respectively. And a second sample separation column connection port 303, a first selection port 304, a second selection port 305, and a third selection port 306 configured to be selectively connected to these ports. have. In addition, the titanium dioxide column 311 may be further connected for the phosphorylated peptide extraction function, wherein the multi-function selection valve 300 'is connected to the first titanium dioxide column connection port 308 connected to both ends of the titanium dioxide column 311. ) And a second titanium dioxide column connection port 309 may be further included.

The multi-function selection valve 300 may be located in a flow path facing a trap valve to which a reversed phase liquid chromatography column in which a sample loaded from a sample inlet valve into which a sample is loaded in the liquid chromatography device is analyzed is connected.

The multi-function selection valve 300 according to the present invention can implement three modes by changing the connection state of each pod, and if the titanium dioxide column 311 is connected can implement four modes.

The multi-function selector valve 300 shown in FIG. 2 represents a fluid closing mode that performs an on-line fire extinguishing function. That is, in the fluid closing mode, the fluid discharged from the sample inlet valve is prevented from entering the multifunction selection valve 300. As shown in Fig. 2, in this mode, the inlet port 301 and the outlet port 307 are fluidly disconnected from each other. To this end, the second selection port 305 and the first sample separation column connection port 302, the outlet port 307 and the first selection port 304, the second sample separation column connection port 303 and the third selection port 306 may each be in fluid communication with each other. Therefore, the incoming first solvent does not pass through the multifunctional selector valve 300, and thus the pressure of the supplied first solvent gradually increases. As such, the on-line digestion function of digesting the protein into the peptide state is performed by generating a high pressure by blocking the flow of the solvent during the transport of the solvent. In this case, since the efficiency of the on-line fire extinguishing function is improved as the pressure of the solvent is increased, it is preferable to increase the pressure of the first solvent to the maximum allowable pressure of the valve.

On the other hand, in the nine-port multi-function selection valve 300 'connected to the titanium dioxide column can be implemented in the fluid closing mode by closing the first titanium dioxide column connection port 308.

After the on-line digestion is performed, a one-dimensional separation process for desalination and concentration of the sample may be performed. Figure 3 is a block diagram of each valve in the process of performing the one-dimensional separation function in the automated dual on-line reverse phase liquid chromatography apparatus according to an embodiment of the present invention.

As shown in FIG. 3, in order to perform the one-dimensional separation function, the multi-function selection valve 300 is changed to a fluid passage mode, that is, to directly in fluid communication with the inlet port 301 and the outlet port 307. . At this time, in the seven-port multifunction selection valve 300, the second selection port 305, the third selection port 306, the second sample separation column connection port 303 and the first selection port 304 are each other It may be in fluid communication. In addition, in the nine-port multifunction selection valve 300 ', the first selection port 304, the second titanium dioxide column connection port 309, the second selection port 305, and the second selection port 306 are each other. It may be in fluid communication. It is apparent to those skilled in the art that the connection of such ports is only one embodiment, and may have a connection of various ports for a desired mode.

The solvent introduced from the inlet port may be discharged toward the first trap valve 400 through the outlet port without passing through the sample separation column 310 according to the connection relationship between the ports.

The first solvent passing through the multifunction selector valve reaches the first separation valve. The first separation valve is a valve that selectively discharges the incoming fluid to the first trap valve 400a or the second trap valve 400b. To this end, the first separation valve 600a may include a first inflow port 601, a second inflow port 602, a first outflow port 603, and a second outflow port 604.

The first separation valve 600a may include a first mode in which the first inflow port 601 and the first outflow port 603, the second inflow port 602 and the second outflow port 604 are in fluid communication with each other, The first inlet port 601 and the second outlet port 604, the second inlet port 602 and the first outlet port 603 may include a second mode in fluid communication with each other. The first separation valve 600a is in the above-described first mode state so that the first solvent passing through the multifunction separation valve 300 reaches the first trap valve 400a.

In a state where the first separation valve 600a is in the first mode, the first solvent is transferred to the first trap valve 400a. The first trap valve 400a is a solid phase extraction column connecting port 402 in communication with the first solid phase extraction column 410a, and a reverse phase liquid chromatography column connecting port 406 in communication with the first reverse phase liquid chromatography column 420a. ), A first inlet port 401, a second inlet port 404, a sample moving loop connection port 403 connected by a solid phase extraction column connection port and a sample moving loop 407, and an emission port 405. can do.

In order to introduce the first solvent containing the sample into the first solid phase extraction column, the first trap valve 400a may include a first inlet port 401 and a solid phase extraction column connection port 402, as shown in FIG. 3. ), The sample transfer loop connection port 403 and the discharge port 405, the reverse phase liquid chromatography column connection port 406 and the second inlet port 404 may be set in fluid communication with each other.

Accordingly, the sample discharged from the multifunctional selection valve 300 and transferred together with the first solvent is injected into the first solid phase extraction column 410a through the solid phase extraction column connection port 402 via the first inlet port 401. do. At this time, the flow direction of the sample to be injected (or the flow direction of the first solvent) is the same as the arrow direction shown.

Solid phase extraction column (410a. 410b) is a column directly bonded to the solid phase extraction column connecting port 402, in the present invention, the solid phase extraction column has an inner diameter of 50㎛ 500㎛ and length of 1cm to 4cm, which It has a very short length compared to the conventional solid-phase extraction column, the length of the solid-phase extraction column is very small as described above in spite of being operated at a very high pressure of about 20,000 psi in the present invention, to maximize the separation resolution of the sample It becomes possible. In addition, as will be described later, since the flow direction of the sample flowing in and the flow direction of the sample eluted toward the reversed phase liquid chromatography column are opposite to each other, further improved sample separation resolution can be achieved.

In addition, in the present invention, a stainless steel liner of an internal inducer may be used as the solid phase extraction columns 410a and 410b. By blocking the outflow of the packing by blocking with a stainless steel screen having a degree of pore size, a solid solid phase extraction column capable of withstanding high pressures can be used.

In addition, the discharge port 405 also releases the salt component contained in the sample, thereby enabling an efficient desalting process.

By this process, the flow rate in the solid phase extraction column during the desalination of the sample can be adjusted to 1.8 μl / min to 3.0 μl / min.

The reason for limiting the flow rate in the solid phase extraction column to 1.8 to 3.0 μl / min is to ensure the increase in desalination efficiency, the prevention of the resulting internal pressure increase and the minimization of sample loss in the solid phase extraction column.

Next, the two-dimensional separation function that allows the first solvent to pass through the sample separation column to fractionate the sample and increase the desalination and concentration efficiency of the sample.

Figure 4 is a block diagram of each valve in the process of performing the two-dimensional separation function in the automated dual on-line reverse phase liquid chromatography apparatus according to an embodiment of the present invention.

In order to perform the two-dimensional separation function in the first solid phase extraction column 410a of the first trap valve 400a, the sample inlet valve 100, the connection valve 200, and the first trap valve 400b in the one-dimensional separation function are performed. The valve configuration of the) is maintained as it is, and the mode of the multi-function selection valve 300 may be switched from the fluid passage mode to the column passage mode. That is, as shown in FIG. 4, the column passing mode of the multi-function selection valve is such that the incoming fluid passes through the sample separation column 310 to be discharged toward the trap valve. In this column passing mode, the inflow port 301 and The first sample separation column connection port 302, the outlet port 307, and the second sample separation column connection port 303 are in fluid communication with each other, and at this time, the second selection port 305 and the third selection port 306. ) May be in fluid communication with each other.

Here, the sample separation column 310 may be any column capable of performing a two-dimensional separation function by combining with a reversed phase liquid chromatography column. For example, Strong Cation eXchange (SCX) columns, Weak Anion eXchange (WAX) columns, Hydrophilic Interaction Chromatography (HILIC) columns, and SCX-WAX MIXED columns. It can be used as a sample separation column.

In the nine-port multi-function selector valve 300 ', the inflow fluid must be discharged through the sample separation column 310 without passing through the titanium dioxide column 311, so that the first selection port 304 and the second titanium dioxide are discharged. The column connection port 309, the second selection port 305, and the third selection port 306 may be in fluid communication with each other.

The first solvent introduced into the multifunctional selection valve 300 through the configuration of the valve may be injected into the first solid phase extraction column 410a of the first trap valve 400a by passing through the sample separation column 310. Therefore, after the fractionation of the sample is made it is possible to further increase the desalination of the sample and the concentration of the sample.

5 is a block diagram of each valve in the process of performing the phosphorylated peptide extraction function in the automated dual on-line reverse phase liquid chromatography apparatus according to an embodiment of the present invention.

In order to perform the phosphorylated peptide extraction function in the first solid phase extraction column 410a of the first trap valve 400a, the sample inlet valve 100, the connection valve 200, and the first trap valve 400b in the two-dimensional separation function are performed. The valve configuration of the) is maintained as it is, the titanium dioxide column (311) is to be connected to the multi-function selection valve (300 '), and change the mode of the multi-function selection valve (300') to the titanium dioxide column passing mode. That is, as shown in FIG. 5, the titanium dioxide column passing mode of the multi-function selection valve is such that the incoming fluid passes through the titanium dioxide column 311 and is discharged toward the trap valve. In the titanium dioxide column passing mode, the inlet port ( 301, the first titanium dioxide column connection port 308, the outlet port 307 and the second titanium dioxide column connection port 309 are in fluid communication with each other, wherein the first selection port 304 and the first sample The separation column connection port 302, the second selection port 305, and the third selection port 306 may be in fluid communication with each other.

Through the configuration of the valve, the first solvent introduced into the multifunctional selection valve 300 ′ may be injected into the first solid phase extraction column 410 a of the first trap valve 400 a by passing through the titanium dioxide column 311. This allows extraction of phosphorylated peptides.

Until now, the sample is filled in a predetermined amount in the sample storage loop 107 through the sample inlet port 101, and then the on-line extinguishing function of the sample is performed using the first solvent, and the first fluid is in fluid communication with the first trap valve 400a. The process of performing the 1-dimensional separation function, the 2-dimensional separation function and the phosphorylated peptide extraction function in the solid phase extraction column 410a has been described.

Next, the process of analyzing the sample by separating the sample injected into the first solid phase extraction column 410a by using the second solvent should be performed, which will be described below.

6 shows an equilibrium in a second solid phase extraction column and a second reverse phase liquid chromatography column during sample analysis in a first reverse phase liquid chromatography column in an automated dual on-line reverse phase liquid chromatography apparatus according to an embodiment of the present invention. It is a block diagram of each valve in the process made.

As a solvent in the sample analysis process, a mixed solvent of the first solvent and the second solvent is used, and the sample is separated through the solvent gradient by varying the ratio of the mixed solvent.

Referring to FIG. 6, a mixed solvent of the first solvent and the second solvent is supplied to the first trap valve 400a through the second solvent supply pump 500b. At this time, the second separation valve 600b serves to guide the mixed solvent supplied from the second solvent supply pump 500b to the first trap valve 400a.

The second separation valve 600b is a valve that selectively discharges the fluid flowing in the same manner as the first separation valve 600a described above to the first trap valve 400a or the second trap valve 400b. To this end, the second separation valve 600b may include a first inflow port 601, a second inflow port 602, a first outflow port 603, and a second outflow port 604.

The second separation valve 600b may include a first mode in which the first inflow port 601 and the first outflow port 603, the second inflow port 602 and the second outflow port 604 are in fluid communication with each other, The first inlet port 601 and the second outlet port 604, the second inlet port 602 and the first outlet port 603 may include a second mode in fluid communication with each other. In order to transfer the mixed solvent supplied from the second solvent supply pump 500b to the first trap valve 400a, the mode of the second separation valve is changed to the above-described second mode as shown in FIG.

The mixed solvent passing through the second separation valve 600b flows into the first trap valve 400a, and the reverse phase liquid chromatography is performed to analyze the sample in the first reverse phase liquid chromatography column in fluid communication with the first trap valve. The mode of the first trap valve 400a is changed such that the graph column connecting port 406, the solid phase extraction column connecting port 402, the second inflow port 404 and the sample moving loop connecting port 403 are in fluid communication with each other. do. At this time, the first inlet port 401 and the discharge port 404 may be in fluid communication with each other. Therefore, the mixed solvent introduced through the second inflow port 404 reaches the first solid phase extraction column 410a via the sample moving loop 407, and connects the solid phase extraction column connection port 402 to the reverse phase liquid chromatography column. The first reversed phase liquid chromatography column 420a is passed through the port 406. In this case, the flow direction of the sample eluted toward the first reverse phase liquid chromatography column 420a (or the flow direction of the mixed solvent) is the same as an arrow, which indicates the flow of the sample flowing into the first solid phase extraction column 410a. Since the opposite direction (or the flow direction of the first solvent), the separation resolution of the sample can be further improved.

Sample separation in the first solid phase extraction column 410a is performed by changing the ratio of the first solvent and the second solvent in the mixed solvent supplied from the second solvent supply pump 500b over time. That is, as the ratio of the second solvent in the mixed solvent increases, the degree of dissociation of the sample bound to the first solid phase extraction column 410a increases to flow into the first reverse phase liquid chromatography column 420a, and then through separation of the sample. The analysis is made.

As the first solvent and the second solvent, various combinations of solvents may be selected to achieve the object as described above, but is not limited thereto, and 0.1% formic acid aqueous solution may be used as the first solvent, 90% of acetonitrile aqueous solution may be used as the second solvent. That is, it can be said that the system adopts a system in which the degree of dissociation of the sample bound to the solid phase extraction column increases as the content of acetonitrile in the total solvent increases.

The first reverse phase liquid chromatography column 420a, in which the separation of the sample is performed, preferably has an inner diameter of 10 μm to 150 μm and a length of 10 cm to 150 cm, and the first reverse phase liquid chromatography column 420a is a mass spectrometer. By connecting to a mass spectrometer, subsequent analytical processes can be performed.

As described above, while the sample is analyzed in the first reverse phase liquid chromatography column 420a, the second solid phase extraction column 410b and the second reverse phase liquid chromatography column using the first solvent supplied from the first solvent supply pump. 420b may be equilibrated.

To this end, as shown in FIG. 6, the mode of the connection valve 200 is changed such that the first solvent from the first solvent supply pump 500a passes through the second trap valve 400b.

That is, the second inlet port 202, the first solvent of the connecting valve 200 such that the first solvent supplied from the first solvent supply pump 500a reaches the second separating valve 600b via the connecting valve 200. The mode of the connection valve 200 is changed such that the second connection port 204, the first connection port 203, and the second outlet port 206 are sequentially in fluid communication. Even in this case, the Z shape of the flow path in the connection valve may be maintained.

At this time, since the first inflow port 601 and the second outflow port 604 are in fluid communication with each other in the second separation valve 600b, the first solvent passes through the second separation valve and the second trap valve 600b. You can head to).

Meanwhile, in order to equilibrate the second solid phase extraction column 410b and the second reverse phase liquid chromatography column 420b, the reverse phase liquid chromatography column connection port 406 and the solid phase extraction column connection port 402 and the second inflow port The mode of the second trap valve 400b is changed such that 404 and the sample moving loop connection port 403 are in fluid communication with each other.

Therefore, the first solvent introduced into the second trap valve 400b passes through the second solid phase extraction column 410b and the second reverse phase liquid chromatography column 420b, thereby enabling equilibration thereof.

Up to now, the process of equilibrating the second solid phase extraction column and the second reverse phase liquid chromatography column while analyzing the sample in the first reverse phase chromatography column has been described, but the reverse process, for example, the second reverse phase chromatography column Of course, it is possible to equilibrate the first solid phase extraction column and the first reverse phase liquid chromatography column during the analysis of the sample.

In addition, in the present embodiment, the on-line digestion, 1-dimensional separation, 2-dimensional separation, and phosphorylated peptide extraction function are all performed using the first solvent, and then the process of separating and analyzing the sample using a mixed solvent is performed. Although described as an example, various combinations are possible as needed. For example, before separating and analyzing a sample using a mixed solvent, perform only one-dimensional separation function, or perform a combination of one-dimensional separation function and two-dimensional separation function, or use an online digestion function first. After performing, one-dimensional separation function or two-dimensional separation function can be selected and performed. Furthermore, the phosphorylated peptide extraction function may be additionally performed in each of the above processes.

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (23)

A sample inlet valve into which a sample to be analyzed is loaded;
A first trap valve in fluid communication with the first solid phase extraction column and the first reversed phase liquid chromatography column;
A second trap valve in fluid communication with the second solid phase extraction column and the second reversed phase liquid chromatography column;
A first separation valve and a second separation valve for selectively discharging the fluid introduced into the first trap valve or the second trap valve, respectively;
A multifunctional selection valve positioned in a flow path from the sample inlet valve to the first and second trap valves and connected to a sample separation column; And
And a connection valve for selectively supplying the fluid flowing out of the sample inlet valve to the multifunctional selection valve, the first trap valve, or the second trap valve. The method of claim 1,
And a first solvent supply pump and a second solvent supply pump for supplying a solvent to the automated dual on-line multifunction liquid chromatography apparatus. The method of claim 2,
And the first solvent supply pump is in fluid communication with the sample inlet valve or the connecting valve, and the second solvent supply pump is in fluid communication with the second separation valve. The method of claim 3,
The first solvent supply pump is an automated dual on-line multifunction liquid chromatography device, characterized in that for supplying the solvent selectively to the sample inlet valve or the connecting valve through the T-type solvent separation tube. The method of claim 2,
At least one of the first solvent supply pump and the second solvent supply pump is an automated dual on-line multi-functional liquid chromatography, characterized in that the solvent selection valve is installed to supply the first solvent or a mixed solvent of the first solvent and the second solvent Photography device. The method of claim 1,
The sample inlet valve may include a sample inlet port, a sample discharge port, a first sample storage loop connection port and a second sample storage loop connection port, a solvent inlet port, and a solvent outlet port connected to each other by a sample storage loop. Dual online multifunction liquid chromatography device. The method of claim 1,
The sample inlet valve,
A first mode in which the sample inlet port and the first sample storage loop connection port are in fluid communication, and the second sample storage loop connection port and the sample discharge port are in fluid communication; And
And a second mode in which the first sample storage loop connection port and the solvent outlet port are in fluid communication, and the second sample storage loop connection port and the solvent inlet port are in fluid communication. Liquid chromatography apparatus. The method of claim 1,
The connecting valve includes a first inlet port, a second inlet port, a first connection port, a second connection port, the first and second outlet port, characterized in that the automated dual on-line multifunction liquid chromatography device. The method of claim 8,
The connection valve,
A first mode in which the first inflow port, the first connection port, the second connection port, and the first outlet port are sequentially in fluid communication; And
And a second mode in which the second inlet port, the second connection port, the first connection port, and the second outlet port are in fluid communication with each other in sequence. 2. 10. The method of claim 9,
The connection valve is an automated dual on-line multifunction liquid chromatography device, characterized in that the Z-shaped flow path is formed between the fluid communication ports. The method of claim 1,
The multifunction selection valve,
A fluid passage mode for introducing the fluid discharged from the sample inlet valve and discharging the fluid to the trap valve;
A column passing mode through which the fluid discharged from the sample inlet valve passes through the sample separation column and is discharged to the trap valve; And
Automated dual on-line multifunction liquid chromatography device comprising a; fluid closing mode for preventing the inflow of the fluid discharged from the sample inlet valve. The method of claim 11,
The multi-function selection valve may include an inlet port, an outlet port, a first sample separation column connection port and a second sample separation column connection port respectively connected to both ends of the sample separation column, and fluids selectively connected to other ports to implement the modes. An automated dual on-line multifunction liquid chromatography device comprising a plurality of selection ports communicated with each other. The method of claim 12,
In the fluid passage mode, the inlet port and the outlet port is in fluid communication,
In the column passing mode, the inlet port, the first sample separation column connection port, the outlet port and the second sample separation column connection port are in fluid communication with each other,
And the inlet port and the outlet port are fluidly disconnected in the fluid closing mode. The method of claim 1,
Titanium dioxide column is further connected to the multi-function selection valve,
The multifunction selection valve,
A fluid passage mode for introducing the fluid discharged from the sample inlet valve and discharging the fluid to the trap valve;
A column passing mode through which the fluid discharged from the sample inlet valve passes through the sample separation column and is discharged to the trap valve;
A fluid closing mode for preventing an inflow of fluid discharged from the sample inlet valve; And
And a titanium dioxide column passing mode through which the fluid discharged from the sample inlet valve passes through the titanium dioxide column and is discharged to the trap valve. 2. The method of claim 14,
The multifunctional selection valve may include an inlet port, an outlet port, and a first sample separation column connection port and a second sample separation column connection port respectively connected to both ends of the sample separation column, and first ends connected to both ends of the titanium dioxide column. And an optional titanium dioxide column connection port and a second titanium dioxide column connection port, and a plurality of selection ports in fluid communication with other ports to implement the modes. 16. The method of claim 15,
In the fluid passage mode, the inlet port and the outlet port is in fluid communication,
In the column passing mode, the inlet port, the first sample separation column connection port, the outlet port and the second sample separation column connection port are in fluid communication with each other,
In the fluid closing mode, the inlet port and the outlet port is fluid disconnected,
In the titanium dioxide column passing mode, the inlet port, the first titanium dioxide column connection port, the outlet port and the second titanium dioxide column connection port are in fluid communication with each other, characterized in that each of the automated dual online multifunction liquid chromatography device . The method of claim 1,
The first trap valve,
Solid phase extraction column connection port in fluid communication with the first solid phase extraction column, reverse phase liquid chromatography column connection port in fluid communication with the first reverse phase liquid chromatography column, a first inlet port, a second inlet port, the first solid phase A sample moving loop connecting port connected to the extraction column connecting port and the sample moving loop, and a discharge port,
The second trap valve,
Solid phase extraction column connection port in fluid communication with the second solid phase extraction column, reverse phase liquid chromatography column connection port in fluid communication with the second reverse phase liquid chromatography column, a first inlet port, a second inlet port, and the second solid phase An automated dual on-line multifunction liquid chromatography apparatus comprising a sample transfer loop connection port connected by an extraction column connection port and a sample transfer loop, and an emission port. The method of claim 17,
Each of the first trap valve and the second trap valve,
A first mode in which the solid phase extraction column connection port and the first inlet port, the sample moving loop connection port, and the discharge port are in fluid communication with each other; And
And a second mode in which the reverse phase liquid chromatography column connection port, the solid phase extraction column connection port, and the second inflow port and the sample moving loop connection port are in fluid communication with each other. Chromatography Apparatus. The method of claim 1,
Each of the first separation valve and the second separation valve,
An automated dual on-line multifunction liquid chromatography apparatus comprising a first inlet port, a second inlet port, a first outlet port and a second outlet port. 20. The method of claim 19,
Each of the first separation valve and the second separation valve,
A first mode in which the first inflow port and the first outflow port, the second inflow port and the second outflow port are in fluid communication with each other; And a second mode in which the first inflow port and the second outflow port, the second inflow port and the first outflow port are in fluid communication with each other, respectively. The method of claim 1,
Equilibrium is performed in the second solid phase extraction column and the second reverse phase liquid chromatography column while the sample is analyzed in the first reversed phase liquid chromatography column. The method of claim 1,
Equilibrium is performed in the first solid phase extraction column and the first reverse phase liquid chromatography column while the sample is analyzed in the second reversed phase liquid chromatography column. The method of claim 1,
In the first solid phase extraction column, the flow direction of the sample flowing in and the flow direction of the sample eluted toward the first reverse phase liquid chromatography column are opposite to each other,
In the second solid-phase extraction column, the flow direction of the sample flowing in and the flow direction of the sample eluted toward the second reverse phase liquid chromatography column is opposite to each other, characterized in that the automated dual on-line multifunction liquid chromatography device.

Images