CN101169393A - Two-dimensional array liquid phase chromatogram-mass spectrometry combination method - Google Patents
Two-dimensional array liquid phase chromatogram-mass spectrometry combination method Download PDFInfo
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Abstract
The invention relates to a high-efficiency high-flux method of connecting two-dimension array liquid chromatogram and mass spectrum. After separated by the first-dimension chromatogram, the analyzed sample enters the second dimension for further high-efficiency separation; the first dimension enters the second-dimension chromatogram from the flow interface through a single chromatographic column; the mobile phase of the second dimension is hit by a chromatogram pump and then is divided by a dividing device through the interface and then hit into the second-dimension chromatographic column. The second-dimension chromatographic column adopts the array mode, and each chromatographic column in the array is synchronously separated with the same chromatographic conditions, the outlet of each chromatographic column of the second dimension is connected with one channel of the multi-channel detector to conduct synchronous high-sensitivity detection. The invention has the advantages of large system peak capacity, high separating efficiency, sensitive detection, simple online classification, large commercial prospect and extensive analyzing object.
Description
Technical field
The present invention relates to the multidimensional liquid-phase chromatographic analysis technology of complex sample, specifically a kind of two-dimensional array liquid phase chromatogram-mass spectrometry combination method.
Background technology
In recent years along with going deep into of studying, it has been recognized that a lot of difficult detected microcomponents may played the part of important role in the complex sample, so the full constituent analysis of complex sample (as biological sample, environmental sample, natural herbal etc.) just becomes the focus and the difficult point of analytical chemistry area research.The multi-dimensional chromatograph technology is acknowledged as powerful separating power.Than proven technique, comprehensive two dimensional gas chromatography provides powerful technical support (Tranchida, P.Q. for the full constituent analysis of volatility complex sample as wherein; Dugo, P.; Dugo, G.; Mondello, L.J.Chromatogr., A 2004,1054,3-16; Dall ü ge, J.; Beens, J.; Brinkman, UAT.J.Chromatogr., A2003,1000,68-108).By contrast,, imitate all far away from gas chromatography, make the research of LC * LC never obtain stem-winding progress (Kimura, H. because of the analysis speed and the post of liquid chromatography though two-dimensional liquid chromatography has bigger application; Tanigawa, T.; Morisaka, H.; Ikegami, T.; Hosoya, K.; Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Ueda, M.; Cabrera, K.; Tanaka, N.J.Sep.Sci. 2004,27,897-904).
In full two-dimensional liquid chromatography, in order to guarantee that current analysis can be finished before next flow point injects, the analysis speed of second dimension must be very fast.So under the guidance of this theory, the research focus of everybody common concern is the high-speed separation that how to realize second dimension on the basis of existing full two-dimensional model at present.Short column (Opiteck, G.J.; Jorgenson, J.W.; Anderegg, R.J.Anal.Chem.1997,69,2283-2291; Opiteck, G.J.; Ramirez, S.M.; Jorgenson, J.W.; Moseley, M.A.Anal.Biochem.1998,258,349-361; Wagner, K.; Miliotis, T.; Marko-Varga, G.; Bischoff, R.; Unger, K.K.Anal.Chem.2002,74,809-820), integral post (Kimura, H.; Tanigawa, T.; Morisaka, H.; Ikegami, T.; Hosoya, K.; Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Ueda, M.; Cabrera, K.; Tanaka, N.J.Sep.Sci.2004,27,897-904), high temperature HPLC (Stoll, D.R.; Carr, P.W.J.Am.Chem.Soc.2005,127,5034-5035) be used in succession, undeniable they injected new element for LC * LC, but very short disengaging time has limited their separating power.In order fundamentally to improve the separating power of two-dimensional liquid chromatography, just should suitably improve the analysis time of second dimension, increase the chromatogram column length of second dimension, but these disagree with the theory of separating fast of conventional two-dimensional second dimension all, also the full two-dimensional liquid chromatography of tradition is difficult to accomplish.
Summary of the invention
In order to overcome the subject matter that traditional system exists, how the present invention will realize that peak capacity combines as primary starting point with the effective of high analyte speed.System of the present invention peak capacity is big, the separation efficiency height; Detect sensitivity, simple at linear location; System design is simple, but the commercialization prospect is big; Analytic target is in extensive range.
For achieving the above object, the technical solution used in the present invention is:
A kind of two-dimensional array liquid phase chromatogram-mass spectrometry combination method, analyzed sample enter second dimension and carry out further high efficiency separation through the after separating of the first dimension chromatogram; First dimension enters the second dimension chromatogram through the flow point that single chromatographic column flows out through interface; The second dimension moving phase is shunted by part flow arrangement by interface after the chromatogram pump is got, and squeezes in the second dimension chromatographic column; The second dimension chromatographic column adopting array pattern, and every root chromatogram column carries out separated in synchronization with same chromatographic condition in the array; The outlet of the every root chromatogram column of second dimension links to each other with a passage of multichannel detector, carries out synchronous highly sensitive detection.
The described first dimension chromatogram can be the chromatogram of nearly all types such as reverse-phase chromatography, size exclusion chromatograph, ion-exchange chromatography or affinity chromatography; Interface can be sample storage ring, enriching column or T-valve etc.;
The second dimension chromatographic column array is made up of the identical or different chromatographic column more than two or two; The second dimension chromatographic column adopting and the different filler of the first dimension separating mechanism, it also can comprise the chromatogram of a lot of types such as reverse-phase chromatography, size exclusion chromatograph.Chromatographic column in the chromatographic column array wherein can be identical filler, also can be different fillers.But all adopt same elution requirement to carry out separated in synchronization; Preferably answer the damping of every root chromatogram column passage of living in the second dimension chromatographic column array identical; The present invention's second dimension chromatogram pump discharge adopts dividing technology, guarantees that each channel flow phase composition equates fully with flow velocity.
Described detecting device is a multichannel detector, can comprise hyperchannel mass detector, hyperchannel UV-detector, hyperchannel evaporative light-scattering detector etc.
Two-dimensional array liquid phase chromatogram-mass spectrometry combination of the present invention is the two-dimensional columns pattern of " 1+n " (n＞=1) with technology (2D-Array-HPLC/MS) employing, " 1 " refers to first dimension and adopts a conventional chromatogram post, " n " refers to same gradient elution, the separated in synchronization of common chromatographic column adopting that second dimension adopts the n root and the first dimension separating mechanism quadrature, after two dimensional separation efficiently, adopt the UV of a n passage and MS detecting device that n group effluent is detected synchronously respectively.
The invention has the beneficial effects as follows:
1. the second dimension chromatographic column can adopt various commercially available or homemade chromatographic columns, and the source is simple.
2. because second dimension adopts the chromatographic column array, therefore can suitably increase the analysis time of second dimension, improve system's peak capacity, increase separating power.
3. adopt array post and multichannel detector, can on the basis that increases system's separating power, reduce chromatogram pump number, the detecting device number of second dimension, make system construction simple.
4. adopt multiple detecting device synchronous detection, can guarantee to the full extent that the separate information of chromatogram do not lose, increase system's peak capacity.
5. applied widely, can be used for the high efficiency separation analysis of various complex samples.
Description of drawings
Fig. 1 is the two-dimensional array liquid phase chromatogram design frame chart.Among the figure: 1 is the chromatogram pump (pump 1) of first dimension, 2 is injector (injector), 3 is the chromatographic column (column 1) of first dimension, 4 is interface arrangement (interface), 5 is the chromatogram pump (pump 2) of second dimension, 6 is part flow arrangement (splitter), 7 is the chromatographic column array (columns array) of second dimension, 8 is multichannel mass detector (multi-channel MS detector), 9 is multichannel UV-detector (multi-channel UV detector), and 10 is workstation (computer).
Embodiment
Give further instruction below by example to the present invention, certainly, the present invention is not limited only to following embodiment.
Embodiment 1
Two-dimensional array liquid phase chromatogram-mass spectrometry combination is hidden the chloroform extraction position of oriental wormwood methanol extract liquid with the technical Analysis traditional Chinese medicine.
This test first dimension adopts the CN post, and (250 * 4.6mm), second dimension adopts the C18 post, and (150 * 2.1mm), bidimensional all adopts the gradient elution of acetonitrile (B) and water-0.5% formic acid (A).Because laboratory hardware is limited, that take at present is the 2D-Array-HPLC/MS of " 1+4 " pattern of off-line.Promptly first dimension adopts a cyano group post, and second dimension adopts four C18 posts.Wherein the chromatographic condition of first dimension is that 15%B rises to 75%B in the 72min internal linear, and flow velocity is 1mL/min, and detecting wavelength is 254nm.The chromatographic condition of second dimension is that 15%B rises to 39%B in the 20min internal linear, rises to 60%B in the 30min internal linear then, rises to 75%B in the 10min internal linear at last, and flow velocity is 0.2mL/min, adopts full wavelength scanner, and the monitoring wavelength is 254nm.The mass spectrum condition of second dimension is spray pressure power 35.0psi, dry gas flow velocity 8.0L/min, and 350 ℃ of baking temperatures, mass range 100 to 1000m/z, target mass-to-charge ratio 350m/z adopts the scanning of ESI positive ion mode.The moving phase of second dimension is got through binary pump, flows in four streams through a five-way valve average mark.In order to guarantee in order at utmost to guarantee the collimation of four passages, all strict equating of length of pipeline in the second dimension four-way in the test,, what four pillars adopted is the product of same batch of same company.
The effluent of first dimension is collected in the sample introduction bottle on a time period, then on the injector of four ports sample introduction to four a C18 post successively, sample size is 400 μ L, in order to reduce the peak broadening that large volume sample injection brings, adopt " column cap enrichment " (interface can adopt T-valve) technology that solute band is retained in first section very narrow zone of chromatographic column herein, adopt gradient elution then, carry out sample separation.The outlet of four root chromatogram columns connects one zero dead volume threeway separately, and two outlets of threeway link to each other with multichannel UV-detector with multichannel mass detector respectively.Can obtain ultraviolet, the mass spectrum information of institute's separating compound so simultaneously.
The gained result gets rid of same compound contained between adjacent component through data analysis, and it is individual that this method detects compound 118 altogether, wherein can make structure by online ultraviolet-mass spectrum information for 9.And under common one dimension chromatographic separation condition, one has 20 good discretely chromatographic peaks in ultraviolet figure and mass spectrogram.The compounds that obtain that separate mostly are microcomponent greatly more, under one-dimensional condition, they be overshadowed in detect under the big peak less than.This shows the great ability that two-dimensional array liquid phase chromatogram-mass spectrometry combination is analyzed complex sample with technology.
System's peak capacity of this method gained is 24300, and the full two-dimensional liquid chromatography of tradition has than before had great advance.
Two-dimensional array liquid phase chromatogram-mass spectrometry combination is hidden the extracting n-butyl alcohol position of oriental wormwood methanol extract liquid with the technical Analysis traditional Chinese medicine.
Adopt with embodiment 1 and adopt same chromatographic system, be used for analyzing the n-butanol portion that Chinese medicine is hidden oriental wormwood.Wherein the chromatographic condition of the first dimension cyano group post is that 10%B rises to 18%B in the 16min linearity, then rises to 45%B in the 5min internal linear, rises to 100%B in the 4min internal linear then, and flow velocity is 1mL/min, and detecting wavelength is 254nm.The chromatographic condition of the second dimension C18 post is that 10%B rises to 25%B in the 18min internal linear, then rises to 32%B in the 14min internal linear, rises to 65%B in the 33min internal linear then, and flow velocity is 0.2mL/min, adopts full wavelength scanner, and the monitoring wavelength is 254nm.The mass spectrum condition of second dimension is spray pressure power 35.0psi, dry gas flow velocity 8.0L/min, and 350 ℃ of baking temperatures, mass range 100 to 1000m/z, target mass-to-charge ratio 600m/z adopts the scanning of ESI positive ion mode.Hide the oriental wormwood n-butanol portion and under common one-dimensional condition, can examine 95 chromatographic peaks altogether, and can detect 575 components altogether with technical Analysis, wherein can make structure by online ultraviolet-mass spectrum information for 11 through two-dimensional array liquid phase chromatogram-mass spectrometry combination.
Show by above-mentioned example, adopt two-dimensional array liquid phase chromatogram-mass spectrometry combination technology provided by the present invention, can be widely used in efficient, the high flux compartment analysis of complex sample.This method has the separation efficiency height, system's peak capacity is big, and operating equipment is simple, in characteristics such as the linear location ability are strong.
Claims (6)
1. two-dimensional array liquid phase chromatogram-mass spectrometry combination method is characterized in that: analyzed sample enters second dimension and carries out further high efficiency separation through the after separating of the first dimension chromatogram; First dimension enters the second dimension chromatogram through the flow point that single chromatographic column flows out through interface; The second dimension moving phase is shunted by part flow arrangement by interface after the chromatogram pump is got, and squeezes in the second dimension chromatographic column; The second dimension chromatographic column adopting array pattern, and every root chromatogram column carries out separated in synchronization with same chromatographic condition in the array; The outlet of the every root chromatogram column of second dimension links to each other with a passage of multichannel detector, carries out synchronous highly sensitive detection.
2. according to the described two-dimensional array liquid phase chromatogram-mass spectrometry combination method of claim 1, it is characterized in that: the described first dimension chromatogram can be reverse-phase chromatography, size exclusion chromatograph, ion-exchange chromatography or affinity chromatography.
3. according to the described two-dimensional array liquid phase chromatogram-mass spectrometry combination method of claim 1, it is characterized in that: described interface can be sample storage ring, enriching column or T-valve.
4. according to the described two-dimensional array liquid phase chromatogram-mass spectrometry combination method of claim 1, it is characterized in that: the described second dimension chromatographic column array is made up of the identical or different chromatographic column more than two or two.
5. according to the described two-dimensional array liquid phase chromatogram-mass spectrometry combination method of claim 1, it is characterized in that: described multichannel detector comprises hyperchannel UV-detector and/or hyperchannel mass detector.
6. according to the described two-dimensional array liquid phase chromatogram-mass spectrometry combination method of claim 1, it is characterized in that: the damping of every root chromatogram column passage of living in is identical in the described second dimension chromatographic column array.
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| CN101747407A (en) * | 2008-12-02 | 2010-06-23 | 杭州中肽生化有限公司 | High-efficient integrated system for separation and purification |
| CN103212217A (en) * | 2013-04-20 | 2013-07-24 | 复旦大学 | Two-dimensional conventional column array type chromatographic separation system and method for removing high-abundance proteins |
| CN103293258A (en) * | 2012-02-24 | 2013-09-11 | 青岛海洋地质研究所 | Device and method for analyzing composition of natural gas hydrate gas |
| CN103930778A (en) * | 2011-03-23 | 2014-07-16 | 明尼苏达大学评议会 | Valve and splitting system for multi-dimensional liquid analysis |
| CN104569223A (en) * | 2015-01-31 | 2015-04-29 | 苏州汇通色谱分离纯化有限公司 | Method for preparing impurity standard substance of pesticide through two-dimensional multi-time central cutting preparative chromatography |
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| CN101747407A (en) * | 2008-12-02 | 2010-06-23 | 杭州中肽生化有限公司 | High-efficient integrated system for separation and purification |
| CN103930778A (en) * | 2011-03-23 | 2014-07-16 | 明尼苏达大学评议会 | Valve and splitting system for multi-dimensional liquid analysis |
| US11692980B2 (en) | 2011-03-23 | 2023-07-04 | Regents Of The University Of Minnesota | Valve and splitting system for multi-dimensional liquid analysis |
| US10429361B2 (en) | 2011-03-23 | 2019-10-01 | Regents Of The University Of Minnesota | Valve and splitting system for multi-dimensional liquid analysis |
| CN103293258A (en) * | 2012-02-24 | 2013-09-11 | 青岛海洋地质研究所 | Device and method for analyzing composition of natural gas hydrate gas |
| CN103293258B (en) * | 2012-02-24 | 2015-04-15 | 青岛海洋地质研究所 | Method for analyzing composition of natural gas hydrate gas |
| CN103212217A (en) * | 2013-04-20 | 2013-07-24 | 复旦大学 | Two-dimensional conventional column array type chromatographic separation system and method for removing high-abundance proteins |
| CN104569223A (en) * | 2015-01-31 | 2015-04-29 | 苏州汇通色谱分离纯化有限公司 | Method for preparing impurity standard substance of pesticide through two-dimensional multi-time central cutting preparative chromatography |
| CN105241984A (en) * | 2015-10-23 | 2016-01-13 | 厦门色谱分析仪器有限公司 | Array multi-dimensional liquid-phase chromatographic column system |
| CN106248832A (en) * | 2016-09-07 | 2016-12-21 | 中国科学院微生物研究所 | A kind of new metabolite edge analysis full Two-dimensional Liquid matter method for combined use and detection kit thereof |
| CN109844521A (en) * | 2016-10-04 | 2019-06-04 | 密执安州立大学董事会 | Multichannel Multidimensional Comprehensive gas-chromatography |
| CN106501429A (en) * | 2016-11-01 | 2017-03-15 | 聊城大学 | A kind of full two-dimensional highly effective liquid phase chromatographic instrument of the multichannel with Multifunctional switching valve and purposes |
| CN107037169B (en) * | 2016-11-01 | 2018-12-04 | 聊城大学 | A kind of full two-dimensional highly effective liquid phase chromatographic instrument and chromatography separating method with Multifunctional switching valve |
| CN106501429B (en) * | 2016-11-01 | 2018-03-09 | 聊城大学 | A kind of full two-dimensional highly effective liquid phase chromatographic instrument of the multichannel with Multifunctional switching valve and purposes |
| CN107037169A (en) * | 2016-11-01 | 2017-08-11 | 聊城大学 | A kind of full two-dimensional highly effective liquid phase chromatographic instrument and chromatography separating method with Multifunctional switching valve |
| CN109100459A (en) * | 2018-10-10 | 2018-12-28 | 兰州东立龙信息技术有限公司 | A kind of chromatography block |
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