KR101078372B1 - liquid chromatography device - Google Patents
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- KR101078372B1 KR101078372B1 KR1020100004950A KR20100004950A KR101078372B1 KR 101078372 B1 KR101078372 B1 KR 101078372B1 KR 1020100004950 A KR1020100004950 A KR 1020100004950A KR 20100004950 A KR20100004950 A KR 20100004950A KR 101078372 B1 KR101078372 B1 KR 101078372B1
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Abstract
The present invention relates to a liquid chromatography apparatus, a sample to be analyzed is introduced, the first sample storage loop connection port and the second sample storage loop connection port connected to each other by a sample inflow port, a sample discharge port, a sample storage loop; A sample inlet valve including a solvent inlet port, and a solvent outlet port; In fluid communication with the solid phase extraction column and the reverse phase liquid chromatography column, the solid phase extraction column connection port, the reverse phase liquid chromatography column connection port, the first solvent inflow port, the second solvent inflow port, the solid phase extraction column connection port and the sample transfer loop. A trap valve including a sample transfer loop connection port connected by a solvent, and a solvent discharge port; And a solvent separation unit separating the flow of the solvent discharged from the sample inlet valve into the first solvent inlet port and the second solvent inlet port of the trap valve. The reverse direction can improve the separation resolution and provide the liquid chromatography apparatus which has high reproducibility.
Description
The present invention relates to a liquid chromatography apparatus, and more particularly, has the simplest form by changing the flow of solvent by using a specialized linkage of a trap valve formed with a T-type solvent separation section and a Z-type solvent flow path. The present invention relates to a liquid chromatography apparatus capable of improving separation resolution by reversely injecting a sample and dissolving a sample.
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. Hydrolyzes into peptides, which increase the solubility of biological samples and produce peptide fragments that are easily ionized and detectable in a mass spectrometer.
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. In addition, when a capillary column having a long length and a small inner diameter is filled 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.
On the other hand, the conventional liquid chromatography apparatus does not employ the solid phase extraction column, so that the injection time of the sample is long, or even if the solid phase extraction column is employed, the injection direction of the sample and the elution direction of the sample are the same, so that the separation resolution is deteriorated. there was.
In addition, in order to reverse the injection direction of the sample and the elution direction of the sample (so-called back flushing), the conventional liquid chromatography apparatus introduces a separate valve and / or pump, but this approach uses liquid chromatography. In addition to complicating the operation of the device, there was a problem of increasing the possibility of malfunction.
The problem to be solved by the present invention is to provide a liquid chromatography device that can improve the separation resolution by enabling the back flushing of the sample without the addition of a valve or pump by changing the flow of the solvent using the solvent separation unit It is.
In order to achieve the above object,
A sample to be analyzed is introduced, and includes 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. A sample inlet valve;
In fluid communication with the solid phase extraction column and the reverse phase liquid chromatography column, the solid phase extraction column connection port, the reverse phase liquid chromatography column connection port, the first solvent inflow port, the second solvent inflow port, the solid phase extraction column connection port and the sample transfer loop. A trap valve including a sample transfer loop connection port connected by a solvent and a solvent discharge port; And
It provides a liquid chromatography device comprising a; solvent separation unit for separating the flow of the solvent flowed out from the sample inlet valve to the first solvent inlet port and the second solvent inlet port of the trap valve.
Here, it is preferable that the solvent outlet port and the solvent separator of the sample inlet valve are in fluid communication with each other.
The apparatus may further include a solvent supply pump in fluid communication with the solvent inlet port of the sample inlet valve to supply a solvent to the sample inlet valve.
According to another embodiment of the present invention,
A sample to be analyzed is introduced, and includes 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. A sample inlet valve;
In fluid communication with the solid phase extraction column and the reverse phase liquid chromatography column, the solid phase extraction column connection port, the reverse phase liquid chromatography column connection port, the first solvent inflow port, the second solvent inflow port, the solid phase extraction column connection port and the sample transfer loop. A trap valve including a sample transfer loop connection port connected by a solvent, and a solvent discharge port; And
It provides a liquid chromatography apparatus comprising a; solvent separation unit for separating the flow of the solvent supplied to the sample inlet valve and the trap valve.
Here, the solvent separator is in fluid communication with the solvent inlet port of the sample inlet valve and the second solvent inlet port of the trap valve, the solvent outlet port of the sample inlet valve is in fluid communication with the first solvent inlet port of the trap valve Can be.
The apparatus may further include a solvent supply pump in fluid communication with the solvent separation unit to supply a solvent to at least one of the sample inlet valve and the trap valve.
In addition, the solvent separation unit may include a T-type solvent separation tube.
In addition, the trap valve may be formed in the Z-type solvent flow path between the ports in fluid communication with each other.
In addition, the sample inlet valve, the sample inlet port and the first sample storage loop connection port is in fluid communication, the second sample storage loop connection port and the sample discharge port is in fluid communication, the solvent inlet port and the A first mode in which the solvent outlet port is in fluid communication; And the sample inlet port and the sample outlet port are in fluid communication, the first sample storage loop connection port and the solvent inlet port are in fluid communication, and the second sample storage loop connection port and the solvent outlet port are in fluid communication. A second mode;
The trap valve may include a first mode in which the solid phase extraction column connection port and the first solvent inflow port are in fluid communication, and the sample transfer loop connection port and the solvent discharge port are in fluid communication; And a second mode in which the reverse phase liquid chromatography column connection port and the solid phase extraction column connection port are in fluid communication, and the second solvent inlet port and the sample transfer loop connection port are in fluid communication.
Further, when the sample inlet valve is in the first mode and the trap valve is in the first mode, the sample may be loaded into the sample storage loop.
Further, when the sample inlet valve is in the second mode and the trap valve is in the first mode, the sample may be injected into the solid phase extraction column through the solvent.
In addition, when the sample inlet valve is in the first mode and the trap valve is in the second mode, the sample injected into the solid phase extraction column through the solvent may be introduced into the reverse phase liquid chromatography column.
Further, when the sample inlet valve is in the second mode and the trap valve is in the first mode, the direction of the sample injected into the solid phase extraction column, and when the sample inlet valve is in the first mode and the trap valve is in the second mode. It is preferable that the directions of the samples eluted from the solid phase extraction column are opposite to each other.
In addition, the solvent supply pump is preferably supplying the solvent at a pressure of 5,000 psi to 20,000 psi.
In addition, the solvent supply pump may be provided with a solvent selection valve to supply a first solvent or a mixed solvent of the first solvent and the second solvent.
The reverse phase liquid chromatography column can also be connected to a mass spectrometer.
According to the present invention, the flow of fluid flowing into and out of the sample inlet valve and the trap valve is changed through a specialized linkage between the T-type solvent separator and the trap valve in which the Z-type solvent flow path is formed. High resolution and high reproducibility of liquid chromatographs can be achieved by reversing the directions of dissolution in the opposite directions, and desalination of the sample on-line and high repeatability with respect to the residence time of the liquid chromatography. It is possible to provide a graphics device.
1A schematically illustrates a valve configuration in a sample loading mode in a liquid chromatography apparatus according to an exemplary embodiment of the present invention.
1B schematically illustrates a valve configuration in a sample injection mode in a liquid chromatography apparatus according to an exemplary embodiment of the present invention.
1C schematically illustrates a valve configuration in a sample separation mode in a liquid chromatography apparatus according to an exemplary embodiment of the present invention.
FIG. 2A schematically illustrates a valve configuration in a sample loading mode in a liquid chromatography apparatus according to another exemplary embodiment of the present invention.
FIG. 2B schematically illustrates a valve configuration in a sample injection mode in a liquid chromatography apparatus according to another exemplary embodiment of the present invention.
FIG. 2C schematically illustrates a valve configuration in a sample separation mode in a liquid chromatography apparatus according to another exemplary embodiment of the present invention.
3 is a diagram showing a chromatogram of a sample separated from a liquid chromatography apparatus according to an embodiment of the present invention.
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.
In the liquid chromatography apparatus according to the exemplary embodiment of the present invention, as illustrated in FIG. 1A, a first sample storage connected to each other by a
The liquid chromatography apparatus according to the present invention operates in three modes: sample loading mode, sample injection mode and sample separation mode. And, the connection relationship between the
As shown in FIG. 1A, the liquid chromatography apparatus according to the present invention includes two valves, that is, a
The
In the sample loading mode, the sample to be analyzed is introduced through the
The
In addition, the
When the inflow of the sample is completed as described above, the mode switching switch (not shown) of the
Liquid chromatography according to an embodiment of the present invention is further in fluid communication with the
It is preferable that the
The solvent supplied from the
In the sample injection mode, the first solvent is introduced from the
In particular, the present invention is characterized in that the sample and the solvent passing through the
Here, the
The
As shown in FIG. 1B, the connection relationship between the trap valve in the sample injection mode is that the solid phase extraction
Therefore, the sample transferred with the first solvent from the
In the present invention, as illustrated in FIGS. 1A to 1C, the
The solid
In addition, in the present invention, a stainless steel liner of an internal inducer is used as the solid
On the other hand, the flow rate of the sample flowing into the solid
By this process, the flow rate in the solid phase extraction column during the desalination of the sample can be adjusted from 1.8 μl / min to 2.0 μl / min.
The reason for limiting the flow rate in the solid phase extraction column to 1.8 to 2.0 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.
Until the above process, the sample is filled in a predetermined amount in the
Next, the process of separating the sample injected into the solid
1C illustrates a state in which the
As the solvent in the sample separation mode, 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.
In addition, referring to FIG. 1C, the connection relationship between the
Therefore, in the present invention, the injection direction of the sample and the dissolution direction of the sample are reversed by using the
Sample separation in the solid
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 reverse phase
In the liquid chromatography apparatus according to another embodiment of the present invention, as shown in FIG. 2A, a sample to be analyzed is introduced, a
At this time, the
In this case, the liquid chromatography apparatus is connected to the inlet side of the
FIG. 2A illustrates a connection relationship between the
That is, in the
2b and 2c illustrate a connection relationship between the
That is, as illustrated in FIGS. 2A to 2C, the liquid chromatography apparatus according to another embodiment of the present invention also includes a trap valve including two solvent inlet ports even though it is further provided with a valve or additionally installed with a solvent supply pump. By using the 200 and the
Example
Sample pretreatment
As analytical sample, inolase (purchased from Sigma-Aldrich, St. Louis, MO, USA) isolated from bakers yeast was used, and the pretreatment of the sample was sequence-modified pork trypsin (Promega, Purchased from Madison, WI, USA). Pretreatment of the sample was carried out according to the following procedure.
The inolase was dissolved in 100 mM NH 4 HCO 3 buffer, then thermally denatured at 90 ° C. for 10 minutes, cooled to room temperature, methanol was added and trypsin was added so that the weight ratio of substrate to enzyme was 50: 1. Was added. Hydrolysis by trypsin was performed at 37 ° C. for 12 hours.
On the other hand, in order to perform analysis on more complex proteome samples, trypsin digest peptide of whole lysate was used. Yeast proteome is Y2805 ( MATa pep :: his3 prb1-D1.6R can1 his1-200 ura3-52 ) and AF-2 ( HMLa or HMRa ho ade2-1 trp1-1 can1-, S. cerevisiae haploid strains) 100 leu2-3,112 his3-11,15 ura3-1 ssd1 ) (Kim, M.-S., Choie, W.-S., Shin, YS, Yu, MH, Lee, S.-W., Bull.Korean Chem. Soc. 2004, 25 , 1833-1839.). In this case, the protein was dissolved in 100 mM NH 4 HCO 3 buffer, then trypsin was added and hydrolyzed at 37 ° C. for 20 hours. The result was completely dried using a SpeedVac system (SPD1010; ThermoSavant, Holbrook, NY, USA) and then stored at -20 ° C until the next experiment was performed.
device
A first solvent of 0.1% formic acid (purchased from Merck (Darmstadt, Germany)) was used, and a second solvent of 100% acetonitrile containing 0.1% formic acid (purchased from JT Baker (Phillipsburg, NJ, USA). )) Was used.
Capillary columns (75 μm ID × 360 μm OD × 80 cm length), ie reverse phase liquid chromatography columns, were prepared by slurry packing fused-silica capillaries with C18-bound particles (Shen, Y., Moore, RJ, Zhao , R., Blonder, J., et al. , Anal. Chem. 2003, 75 , 3596-3605; Shen, Y., Tolic N., Masselon, C., Pasa-Tolic L. et al. , Anal. Chem. 2004, 76 , 144-154; Shen, Y., Smith, RD, Unger KK, Kumar, D., Lubda, D., Anal.Chem . 2005, 77 , 6692-6701).
Solid phase extraction columns were constructed using internal reducers purchased from VICI (Houston, TX, USA). It was fabricated by filling a 1 cm liner (250 μm ID) located in an internal reducer (1/16 "to 1/32") with C18 material at a pressure of 10,000 psi. After the filling step was completed, the column was sonicated for 5 minutes while maintaining a pressure of 10,000 psi against the column, and the pressure was lowered overnight before the column was used to prevent the charged C18 materials from dispersing. In addition, a stainless steel screen (2 μm pores) was attached to both ends of the liner prior to use in the actual experiment.
On the other hand, a mass spectrometer connected to a reverse phase liquid chromatography column includes a 7-tesla Fourier-transform ion cyclotron equipped with a nanoelectrospray ionization interface. resonance mass spectrometer) (FTICR, LTQ-FT, ThermoFinnigan) was used.
Evaluation of the analysis result
The inolase trypsin digest produced by the pretreatment was analyzed using a liquid chromatography apparatus according to an embodiment of the present invention.
In order to demonstrate the reproducibility of the liquid chromatography apparatus according to the present invention, a total of six analyzes were carried out under the same conditions, and the peptide samples were sent to a solid phase extraction column, which was then separated by reverse phase chromatography, and then subjected to mass spectrometry. The chromatogram of the isolated sample obtained is shown in FIG. 3.
It was confirmed that the standard deviation of each experiment was within 0.1% for a total dissolution time of 120 minutes, through which it can be seen that the liquid chromatography apparatus according to the present invention has excellent experimental reproducibility.
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 (16)
In fluid communication with the solid phase extraction column and the reverse phase liquid chromatography column, the solid phase extraction column connection port, the reverse phase liquid chromatography column connection port, the first solvent inflow port, the second solvent inflow port, the solid phase extraction column connection port and the sample transfer loop. A trap valve including a sample transfer loop connection port connected by a solvent, and a solvent discharge port; And
And a solvent separator separating the flow of the solvent flowing out of the sample inlet valve into a first solvent inlet port and a second solvent inlet port of the trap valve.
And the solvent outlet port and the solvent separation part of the sample inlet valve are in fluid communication with each other.
And a solvent supply pump in fluid communication with the solvent inlet port of the sample inlet valve and supplying a solvent to the sample inlet valve.
In fluid communication with the solid phase extraction column and the reverse phase liquid chromatography column, the solid phase extraction column connection port, the reverse phase liquid chromatography column connection port, the first solvent inflow port, the second solvent inflow port, the solid phase extraction column connection port and the sample transfer loop. A trap valve including a sample transfer loop connection port connected by a solvent, and a solvent discharge port; And
And a solvent separator separating the flow of the solvent supplied into the sample inlet valve and the trap valve.
The solvent separator is in fluid communication with the solvent inlet port of the sample inlet valve and the second solvent inlet port of the trap valve,
And a solvent outlet port of the sample inlet valve is in fluid communication with a first solvent inlet port of the trap valve.
And a solvent supply pump in fluid communication with the solvent separation unit for supplying a solvent to at least one of the sample inlet valve and the trap valve.
The solvent separation unit liquid chromatography apparatus, characterized in that it comprises a T-type solvent separation tube.
And a Z-type solvent flow path formed between the ports in fluid communication with the trap valve.
The sample inlet valve,
The sample inlet port and the first sample storage loop connection port are in fluid communication, the second sample storage loop connection port and the sample discharge port are in fluid communication, and the solvent inlet port and the solvent outlet port are in fluid communication. 1 mode; And
The sample inlet port and the sample outlet port are in fluid communication, the first sample storage loop connection port and the solvent inlet port are in fluid communication, and the second sample storage loop connection port and the solvent outlet port are in fluid communication. 2 modes; and
The trap valve is,
A first mode in which the solid phase extraction column connection port and the first solvent inlet port are in fluid communication, and the sample transfer loop connection port and the solvent release port are in fluid communication; And
And a second mode in which the reverse phase liquid chromatography column connection port and the solid phase extraction column connection port are in fluid communication, and the second solvent inlet port and the sample transfer loop connection port are in fluid communication. Device.
And the sample is loaded into the sample storage loop when the sample inlet valve is in the first mode and the trap valve is in the first mode.
And when the sample inlet valve is in the second mode and the trap valve is in the first mode, the sample is injected into the solid phase extraction column through the solvent.
When the sample inlet valve is in the first mode and the trap valve is in the second mode, a sample injected into the solid phase extraction column through the solvent is introduced into the reversed phase liquid chromatography column .
The direction of the sample injected into the solid phase extraction column when the sample inlet valve is in the second mode and the trap valve is in the first mode, and the solid phase when the sample inlet valve is in the first mode and the trap valve is in the second mode. Liquid chromatography apparatus, characterized in that the direction of the samples eluted from the extraction column are mutually reverse.
The solvent supply pump is a liquid chromatography apparatus, characterized in that for supplying the solvent at a pressure of 5,000 psi to 20,000 psi.
And a solvent selection valve is installed in the solvent supply pump to supply a first solvent or a mixed solvent of a first solvent and a second solvent.
And said reverse phase liquid chromatography column is connected to a mass spectrometer.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101574272B1 (en) | 2014-08-20 | 2015-12-07 | 한국표준과학연구원 | Two dimensional liquid chromatography system for Heart-cut method |
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| CN109030647B (en) * | 2018-07-30 | 2024-03-22 | 天津海关动植物与食品检测中心 | Online immunoaffinity purification detection device for terbutaline, salbutamol, ractopamine and clenbuterol |
| CN115144510A (en) * | 2021-03-30 | 2022-10-04 | 上海润达榕嘉生物科技有限公司 | Automatic pretreatment device for clinical mass spectrum |
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|---|---|---|---|---|
| US5462660A (en) | 1994-04-22 | 1995-10-31 | The United States Of America As Represented By The Secretary Of Agriculture | High performance liquid chromatography injection system for the simultaneous concentration and analysis of trace components |
| JPH1137985A (en) | 1997-07-15 | 1999-02-12 | Tosoh Corp | Liquid chromatograph using changeover valve |
| KR100757512B1 (en) | 2006-11-16 | 2007-09-11 | 고려대학교 산학협력단 | Ultrahigh-pressure dual on-line solid phase extraction/capillary reverse-phase liquid chromatography system |
| KR100924369B1 (en) | 2007-12-04 | 2009-10-30 | 고려대학교 산학협력단 | Ultrahigh-pressure cation exchanger dual on-line solid phase extraction/capillary reverse-phase liquid chromatography system |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5462660A (en) | 1994-04-22 | 1995-10-31 | The United States Of America As Represented By The Secretary Of Agriculture | High performance liquid chromatography injection system for the simultaneous concentration and analysis of trace components |
| JPH1137985A (en) | 1997-07-15 | 1999-02-12 | Tosoh Corp | Liquid chromatograph using changeover valve |
| KR100757512B1 (en) | 2006-11-16 | 2007-09-11 | 고려대학교 산학협력단 | Ultrahigh-pressure dual on-line solid phase extraction/capillary reverse-phase liquid chromatography system |
| KR100924369B1 (en) | 2007-12-04 | 2009-10-30 | 고려대학교 산학협력단 | Ultrahigh-pressure cation exchanger dual on-line solid phase extraction/capillary reverse-phase liquid chromatography system |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101574272B1 (en) | 2014-08-20 | 2015-12-07 | 한국표준과학연구원 | Two dimensional liquid chromatography system for Heart-cut method |
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