CN103323544B - On-line pre-separating and separating analysis method for whole protein and liquid chromatogram system thereof - Google Patents
On-line pre-separating and separating analysis method for whole protein and liquid chromatogram system thereof Download PDFInfo
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Abstract
The invention provides an on-line pre-separating and separating analysis method for a whole protein and a liquid chromatogram system thereof. By utilizing orderly proportionately increased characteristic of hydrophobicity of Cn capture column, proteins in a protein mixture are orderly gradingly captured from large to small to implement pre-separation; and the proteins on the capture column are orderly matched by relative Cn analytical columns to implement secondary separation, and the analytical column is used together with the mass spectrum for analysis. The orderly proportionately increased capture columns Cn (n=1-18 or partial combination thereof, such as 1, 2, 4, 8 and 18) are used for on-line pre-separating samples, which greatly raises a pre-separation speed, sensitivity and resolution; and the separation columns and the capture column are pairwise matched (such as C4 capture and C4 separation column are matched), which have consistent hydrophobicity, thereby implementing maximization of separation resolution.
Description
Technical field
The present invention relates to the technology and method of the online classification pre-separation of a kind of whole protein and compartment analysis.
Background technology
At rear era gene now, the complete genome sequence of a lot of species all or just out determined, this makes comprehensive further investigation of protein group become possibility.Because most of biological function is finally all transmitted and realized by protein, the research of protein will promote more directly, more rapidly the development of biology and medical science on the basis of gene studies.Technology based on HPLC-MS can be carried out quick, sensitive Analysis and Identification to protein and composition thereof, is forward position and the first-selected research means of protein research.In more than ten years in the past, based on mass spectrographic protein analysis authenticate technology, be developed rapidly, can human body protein potpourri compared comprehensively and be characterized fast; But still can not the low-abundance disease marker albumen for clinical detection wherein effectively be monitored and be identified.Wherein chief reason is that the concentration of disease marker albumen is often all (every milliliter of pieck stage, 1 gram=1,000,000,000,000 pik), with other high-abundance proteins in human body (if albuminous concentration is at 40 milligrams about every milliliter, 1 gram=1000 milligrams) concentration differ 10 orders of magnitude, and the albumen that the method for current liquid chromatograph mass spectrography can only differ in 6 number order magnitude range concentration well detects.In order to allow liquid chromatography-mass spectrography low-abundance disease marker albumen can be detected, just must develop efficient Isolation method and first the protein mixture of large order of magnitude scope is divided into the section of some decimal order magnitude range before liquid chromatography-mass spectrography detects.
Traditional pre-separation technology is mainly ion-exchange chromatography, size exclusion chromatography etc.The eluent of ion-exchange chromatography generally includes inorganic salts, and these inorganic salts are incompatible with ensuing LC-MS analysis, must desalination.So, the necessary first collected offline of the cut of pre-separation, desalination.Because desalting process sample loss is very serious also more consuming time, while therefore doing pre-separation with ion-exchange chromatography, analysis speed is slow, and sensitivity is also lower.The eluent of size exclusion chromatography and LC-MS analysis are compatible, can on-line coupling, and analysis speed is fast, but its resolution is often very low.
In sum, the online pre-separation technology of development of new fast high-sensitive is that liquid chromatography-mass spectrography effectively detects the task of top priority of low-abundance disease marker albumen.
Summary of the invention
The trapping column C of hydrophobicity successively proportional increase for the present invention
n(n=1-18 or the combination of part wherein, as 1,2,4,8,18) sample is carried out to online pre-separation, greatly improved speed, sensitivity and the resolution of pre-separation; Separating column and trapping column match between two (as C4 trapping column and C4 separating column paired), hydrophobicity is consistent, has realized the maximization of isolation resolution.
The present invention first openly method and technology scheme is: the method for the online classification pre-separation of a kind of whole protein and compartment analysis, it is characterized in that, and utilize C
n(combination of n=1-18 or part wherein, as 1,2,4,8,18) trapping column hydrophobicity is the characteristic of proportional increase successively, the albumen in protein mixture is carried out to classification trapping successively from big to small and realize pre-separation; Albumen in trapping column is used corresponding C more successively
n(as n=1,2,4,8,18) analytical column mates realizes the second-order separation, and analyzes with mass spectrometry.
Based on said method, the invention also discloses the system architecture technical scheme that realizes the method: the online classification pre-separation of a kind of whole protein and separated liquid chromatographic system, it is characterized in that, system comprises with C
n(n=1-18) some hydrophobicitys successively a series of trapping column being combined through series connections of proportional increase form pre-separation subsystem; System also comprises the second-order separation subsystem consisting of a series of analytical columns matching with described pre-separation subsystem, described analytical column and trapping column are matched between two, between analytical column, walk abreast independent operating is successively set separately, each analytical column output terminal and mass spectrometry carry out subsequent analysis.
The concrete example of the present invention's protection: the online classification pre-separation of a kind of whole protein and compartment analysis liquid chromatographic system, it is characterized in that, comprise N root trapping column, N root separating column, sampling valve, three post selector valves and a binary efficient liquid-phase chromatographic pump (referred to as binary pump), three post selector valves are respectively the second post selector valve, the 3rd post selector valve, the 4th post selector valve; Pairing between two between trapping column and separating column; Described sampling valve is for sampling; Described binary efficient liquid-phase chromatographic pump is installed on sampling valve, and after having sampled, the binary efficient liquid-phase chromatographic pump while is as the voltage supply source of pre-separation subsystem and the second-order separation subsystem; Described trapping column is connected in series the periphery that is arranged in the second post selector valve successively, N root trapping column is successively and increases progressively and being combined through series connection formation pre-separation subsystem according to hydrophobicity, after pre-separation completes, in trapping column, according to hydrophobicity, increases progressively and traps respectively the albumen that hydrophobicity is successively decreased with it; Described separating column is parallel being arranged between the 3rd post selector valve and the 4th post selector valve in turn, form second level isolated subsystem, according to hydrophobicity, increase progressively as sequencing and step by step the pre-separation albumen in trapping column is carried out to the second-order separation successively, finally by the 4th post selector valve, export to analytic system and detect.
Described binary pump is exported buffer A (water+1% formic acid as 99%) when sample introduction; When separated, export gradient buffering liquid B(as 99% acetonitrile+1% formic acid), the ratio of buffer B (with respect to buffer A) is more and more higher in time.For example, in 60 minutes, the ratio of buffer B and buffer A rises to 100% by 0 linearity.
The microfluidic valve adopting in technical scheme, for existing parts, is not the contribution part of the present invention to prior art, therefore the structure of microfluidic valve, principle of work, manner of execution is not introduced in this manual in detail.
The trapping column adopting in technical scheme, separating column parts itself, and before analytical column separation, by trapping column, sample is carried out to pre-trapping and all belong to prior art.
Compared with prior art, the inevitable obtainable beneficial effect of technical solution of the present invention:
1. the present invention has simplified protein mixture pre-separating system, simple in structure.The pre-separation part of this chromatographic system, only with a microfluidic valve, has replaced traditional one dimension chromatographic system completely, completes pre-separation step, than conventional ion exchange or size exclusion chromatography, has saved an expensive binary pump.
2, system flexibility is high.Be suitable for the pre-separation of all capillary chromatographic columns with separated.Capillary chromatography column internal diameter is less, and sensitivity is higher.Little internal diameter (as 15 microns) capillary column can be realized the separated evaluation of hypersensitivity to the precious biology of denier or clinical sample (as tumor tissue cell).
3. the trapping column in chromatographic system of the present invention and separating column (such as C4 trapping column must be matched C4 separating column) in pairs, so by successively pre-separation, and separated step by step, the isolation resolution of whole system is maximized.The pre-separation of coupling and separatedly realized to greatest extent full coverage, high-resolution, the high-effective classifying separation from big to small of holoprotein group.
The related application that the present invention is possible:
1. the online classification pre-separation of whole protein of the present invention being built and separation chromatography system and high-resolution, high-performance mass spectrometer (as track trap mass spectrometer) coupling, identify for the separation of any whole protein group;
2. with clinical hospitals cooperation, qualitative, quantitative look-matter combined instrument is identified for the differential liberation of a clinical disease large-scale protein quality sample, be devoted to discovery and the checking of disease marker;
3. give full play to the high feature of self-assembly systems dirigibility, attempt less internal diameter (as 50 microns, 30 microns, even 15 microns), the more application of high sensitivity capillary column, the precious biology of denier or clinical sample (as tumor tissue cell) are realized to hypersensitivity is separated to be identified.
4. the whole online classification pre-separation of protein of the present invention and method for separating and analyzing, promoting the use of all kinds of groups learns in research, the research and development of all kinds of groups of, systems biology and the personalized medicines of Jiang Wei China are made contributions, simultaneously also for the exploitation of commercialization instrument accordingly provides template.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, technical solution of the present invention is described further.
The whole online classification pre-separation of protein of Fig. 1 and compartment analysis system, wherein protein sample is injected device and squeezes into sample loop.
Sample in Fig. 2 binary pump pumping mobile phase A (100% water+0.1% formic acid) carrier band sample loop passes through T1, T2, T3, T4 and T5 trapping column successively; In trapping column, load successively fixedly phase of C1, C2, C4, C8 and C18;
The sample that Fig. 3 is captured in T1 trapping column passes through the separation of A1 analytical column and by Mass Spectrometer Method, loads fixedly phase of C1 in A1 analytical column;
The sample that Fig. 4 is captured in T2 trapping column passes through the separation of A2 analytical column and by Mass Spectrometer Method, loads fixedly phase of C2 in A2 analytical column;
The sample that Fig. 5 is captured in T3 trapping column passes through the separation of A3 analytical column and by Mass Spectrometer Method, loads fixedly phase of C4 in A3 analytical column;
The sample that Fig. 6 is captured in T4 trapping column passes through the separation of A4 analytical column and by Mass Spectrometer Method, loads fixedly phase of C8 in A4 analytical column;
The sample that Fig. 7 is captured in T5 trapping column passes through the separation of A5 analytical column and by Mass Spectrometer Method, loads fixedly phase of C18 in A5 analytical column;
Explain: T1, T2, T3, T4, loads respectively C1 in T5 trapping column, C2, C4, C8, C18 is phase fixedly; A1, A2, A3, A4, loads respectively C1 in A5 trapping column, C2, C4, C8, C18 is phase fixedly.
Fig. 8 is captured in T1, T2, T3, T4 and albumen in the T5 trapping column base peak chromatogram after, Mass Spectrometer Method separated with A5 analytical column through A1, A2, A3, A4 respectively.
Fig. 9 is captured in that albumen in T1 trapping column is separated through A1 analytical column, the two-dimentional exploded view after Mass Spectrometer Method.
Figure 10 is captured in that albumen in T2 trapping column is separated through A2 analytical column, the two-dimentional exploded view after Mass Spectrometer Method.
Figure 11 is captured in that albumen in T3 trapping column is separated through A3 analytical column, the two-dimentional exploded view after Mass Spectrometer Method.
Figure 12 is captured in that albumen in T4 trapping column is separated through A4 analytical column, the two-dimentional exploded view after Mass Spectrometer Method.
Figure 13 is captured in that albumen in T5 trapping column is separated through A5 analytical column, the two-dimentional exploded view after Mass Spectrometer Method.
Embodiment
As shown in Figure 1, in sampling valve: its first interface a1 is connected with sampling injector (this syringe can change automatic sampler into realize robotization); Its second interface b1 is connected by sample loop with the 5th interface e1; Its 3rd interface c1 is connected with binary pump outlet; Its 4th interface d1 is connected with the entrance of the T1 trapping column of the first selection post; Its 6th interface f1 is for effluent discharge, and the 6th interface f1 uses with collection container (meaning not shown in the figures) and be connected.Sampling valve is connected with the first post selector valve by interface d1.
In the sample introduction stage, as shown in Figure 1, the rotor of sampling valve location is communicated with syringe, a1, b1, sample loop, e1, f1, and syringe pressurization makes to pour in sample loop liquid to be measured, more than collection liquid to be measured by f1, discharge sampling valve.
As shown in Figure 2, in the first post selector valve: the first post selector valve is connected with sampling valve by trapping column T1.The outlet of trapping column T1 is connected with the entrance of trapping column T2 with interface b2 respectively by a three-way pipe; The outlet of trapping column T2 is connected with the entrance of trapping column T3 with interface a2 respectively by a three-way pipe; The outlet of trapping column T3 is connected with the entrance of trapping column T4 with interface e2 respectively by a three-way pipe; The outlet of trapping column T4 is connected with the entrance of trapping column T5 with interface d2 respectively by a three-way pipe; The outlet of trapping column T5 is connected with interface c2, and interface c2 is connected with the f2 of valve central authorities again, and the first post selector valve is circulated to the second post selector valve by the f2 of valve central authorities.
In preliminary separation stage, first adjust the rotor of sampling valve, the rotor of sampling valve is reorientated binary pump, c1, b1, sample loop, e1, d1 is communicated with, d1 interface is connected with the entrance of the trapping column T1 of first post selector valve simultaneously, and secondly first post selector valve rotor original location is connected in series T1, T2, T3, T4, T5 successively.After binary pump work pressurization, in first post selector valve, flow through trapping column T1, T2, T3, T4, T5 of liquid to be measured is collected in trapping column T1, T2, T3, T4, T5 successively according to protein is descending, realizes thus pre-separation.
As shown in Figure 3, in the second post selector valve: its central interface g3 is connected with first post selector valve central interface f2; Its first interface f3 is waste discharge mouth, and interface f3 is connected with a waste collection container; The 5th interface b4 of its second interface e3 and the 3rd post selector valve is connected, for the treatment of analytical column A5; The 4th interface c4 of its 3rd interface d3 and the 3rd post selector valve is connected, for the treatment of analytical column A4; The 3rd interface d4 of its 4th interface c3 and the 3rd post selector valve is connected, for the treatment of analytical column A3; Second interface e4 of its 5th interface b3 and the 3rd post selector valve is connected, for the treatment of analytical column A2; Second interface f4 of its 5th interface a3 and the 3rd post selector valve is connected, for analytical column A1.
At separation phase, Treatment Analysis post A1, as shown in Figure 3:
First adjust the rotor of the first post selector valve, the rotor of the first post selector valve is reorientated trapping column T1, b2, f2 is communicated with, the rotor of the second selector valve is reorientated central interface g3 is communicated with a3, thereby under forcing pump effect, the C1 protein in trapping column T1 is carried out to second level separation, in T1, the C1 protein of trapping is emptying, meanwhile, under the rotor location of the 3rd post selector valve, interface f4 exports the 3rd post selector valve by central interface g4 by C1 protein the second-order separation thing.
At separation phase, Treatment Analysis post A2, as shown in Figure 4:
First adjust the rotor of the first post selector valve, the rotor of the first post selector valve is reorientated trapping column T2, a2, f2 is communicated with, the rotor of the second selector valve is reorientated central interface g3 is communicated with d3, thereby under forcing pump effect, the C2 protein in trapping column T2 is carried out to second level separation, in T2, the C2 protein of trapping is emptying, meanwhile, under the rotor location of the 3rd post selector valve, interface e4 exports the 3rd post selector valve by central interface g4 by C2 protein the second-order separation thing.
At separation phase, Treatment Analysis post A3, as shown in Figure 5:
First adjust the rotor of the first post selector valve, the rotor of the first post selector valve is reorientated trapping column T3, e2, f2 is communicated with, the rotor of the second selector valve is reorientated central interface g3 is communicated with c3, thereby under forcing pump effect, the C4 protein in trapping column T3 is carried out to second level separation, in T3, the C4 protein of trapping is emptying, meanwhile, under the rotor location of the 3rd post selector valve, interface d4 exports the 3rd post selector valve by central interface g4 by C4 protein the second-order separation thing.
At separation phase, Treatment Analysis post A4, as shown in Figure 6:
First adjust the rotor of the first post selector valve, the rotor of the first post selector valve is reorientated trapping column T4, d2, f2 is communicated with, the rotor of the second selector valve is reorientated central interface g3 is communicated with d3, thereby under forcing pump effect, the C8 protein in trapping column T4 is carried out to second level separation, in T4, the C8 protein of trapping is emptying, meanwhile, under the rotor location of the 3rd post selector valve, interface c4 exports the 3rd post selector valve by central interface g4 by C8 protein the second-order separation thing.
At separation phase, Treatment Analysis post A5, as shown in Figure 7:
First adjust the rotor of the first post selector valve, the rotor of the first post selector valve is reorientated trapping column T5, c2, f2 is communicated with, the rotor of the second selector valve is reorientated central interface g3 is communicated with e3, thereby under forcing pump effect, the C18 protein in trapping column T5 is carried out to second level separation, in T5, the C18 protein of trapping is emptying, meanwhile, under the rotor location of the 3rd post selector valve, interface b4 exports the 3rd post selector valve by central interface g4 by C18 protein the second-order separation thing.
The 3rd post selector valve, its 6th interface a4 emptying; Its central interface g4 is connected with the nozzle needle of electron spray by a metal two-way; The required high-tension electricity of electron spray is added in metal two-way by crocodile clip, and nozzle needle spraying is received by mass spectrographic entrance.
Embodiment
The compartment analysis of the whole protein group of the Escherichia coli of take below illustrates the course of work of this liquid chromatographic system as example.
Step 1, sample introduction: in sampling valve, protein sample is injected device and squeezes into sample loop, as shown in Figure 1;
Step 2, pre-separation: the sample in mobile phase A (100% water+0.1% formic acid) carrier band sample loop passes through T1, T2, T3, T4 and T5 trapping column successively, as shown in Figure 2;
Step 3, C1 is separated: be captured in sample in T1 trapping column (filling C1 is phase fixedly) by (filling C1 the is phase fixedly) separation of A1 analytical column and by Mass Spectrometer Method, the link position of each chromatogram valve is as shown in Figure 3; Through the chromatogram of A1 analytical column (filling C1 is phase fixedly) LC-MS analysis gained as shown in Figure 8, the albumen determinand in C1 interval range obtains good separation to the albumen of imprison in T1 trapping column (filling C1 fixedly phase); Corresponding single isotopic mass with the two-dimentional exploded view of scanning of the mass spectrum spectrum number as shown in Figure 9.
Step 4, C2 is separated: be captured in sample in T2 trapping column (filling C2 is phase fixedly) by (filling C2 the is phase fixedly) separation of A2 analytical column and by Mass Spectrometer Method, the link position of each chromatogram valve is as shown in Figure 4; Through the chromatogram of A2 analytical column (filling C2 is phase fixedly) LC-MS analysis gained as shown in Figure 8, the albumen determinand in C2 interval range obtains good separation to the albumen of imprison in T2 trapping column (filling C2 fixedly phase); Corresponding single isotopic mass with the two-dimentional exploded view of scanning of the mass spectrum spectrum number as shown in figure 10;
Step 5, C4 is separated: be captured in sample in T3 trapping column (filling C4 is phase fixedly) by (filling C4 the is phase fixedly) separation of A3 analytical column and by Mass Spectrometer Method, the link position of each chromatogram valve is as shown in Figure 5; Through the chromatogram of A3 analytical column (filling C4 is phase fixedly) LC-MS analysis gained as shown in Figure 8, the albumen determinand in C4 interval range obtains good separation to the albumen of imprison in T3 trapping column (filling C4 fixedly phase); Corresponding single isotopic mass with the two-dimentional exploded view of scanning of the mass spectrum spectrum number as shown in figure 11;
Step 6, C8 is separated: be captured in sample in T4 trapping column (filling C8 is phase fixedly) by (filling C8 the is phase fixedly) separation of A4 analytical column and by Mass Spectrometer Method, the link position of each chromatogram valve is as shown in Figure 6; Through the chromatogram of A4 analytical column (filling C8 is phase fixedly) LC-MS analysis gained as shown in Figure 8, the albumen determinand in C8 interval range obtains good separation to the albumen of imprison in T4 trapping column (filling C8 fixedly phase); Corresponding single isotopic mass with the two-dimentional exploded view of scanning of the mass spectrum spectrum number as shown in figure 12;
Step 7, C18 is separated: be captured in sample in T5 trapping column (filling C18 is phase fixedly) by A5 (filling C18 is phase fixedly) analytical column separation and by Mass Spectrometer Method, the link position of each chromatogram valve is as shown in Figure 7; Through the chromatogram of A5 (filling C18 is phase fixedly) analytical column LC-MS analysis gained as shown in Figure 8, the albumen determinand in C18 interval range obtains good separation to the albumen of imprison in T5 trapping column (filling C18 fixedly phase); Corresponding single isotopic mass with the two-dimentional exploded view of scanning of the mass spectrum spectrum number as shown in figure 13.
Chromatogram from Fig. 8 can find out, it is separated that the albumen of trapping column pre-separation is obtaining high-resolution on corresponding separating column.
From the variation of Fig. 9 to Figure 13, can find out, whole protein sample is by successfully realized from big to small pre-separation in T1 to T5 trapping column.As T1 has trapped the above albumen of more 20KDa, T2 has trapped the above albumen of more 15KDa, and T3 has trapped the above albumen of more 10KDa.
Claims (3)
1. a method for the online classification pre-separation of whole protein and compartment analysis, is characterized in that, utilizes C
ntrapping column hydrophobicity is the characteristic of proportional increase successively, the albumen in protein mixture is carried out to classification trapping successively from big to small and realize pre-separation; Albumen in trapping column is used corresponding C more successively
nanalytical column mates realizes the second-order separation, and analyzes with mass spectrometry.
2. the online classification pre-separation of whole protein and a separated liquid chromatographic system, is characterized in that, system comprises with C
nsome hydrophobicitys successively a series of trapping column being combined through series connections of proportional increase form pre-separation subsystem; System also comprises the second-order separation subsystem consisting of a series of analytical columns matching with described pre-separation subsystem, described analytical column and trapping column are matched between two, between analytical column, walk abreast independent operating is successively set separately, each analytical column output terminal and mass spectrometry carry out subsequent analysis.
3. the online classification pre-separation of a kind of whole protein as claimed in claim 2 and separated liquid chromatographic system, it is characterized in that, comprise N root trapping column, N root separating column, a sampling valve, three post selector valves and a binary efficient liquid-phase chromatographic pump, three post selector valves are respectively the second post selector valve, the 3rd post selector valve, the 4th post selector valve; Pairing between two between trapping column and separating column; Described sampling valve is for sampling; Described binary efficient liquid-phase chromatographic pump is installed on sampling valve, and after having sampled, the binary efficient liquid-phase chromatographic pump while is as the voltage supply source of pre-separation subsystem and the second-order separation subsystem; Described trapping column is connected in series the periphery that is arranged in the second post selector valve successively, N root trapping column is successively and increases progressively and being combined through series connection formation pre-separation subsystem according to hydrophobicity, after pre-separation completes, in trapping column, according to hydrophobicity, increases progressively and traps respectively the albumen that hydrophobicity is successively decreased with it; Described separating column is parallel being arranged between the 3rd post selector valve and the 4th post selector valve in turn, form second level isolated subsystem, according to hydrophobicity, increase progressively as sequencing and step by step the pre-separation albumen in trapping column is carried out to the second-order separation successively, finally by the 4th post selector valve, export to analytic system and detect.
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