CN100542649C - The mobile phase of separating phospholipids in the high performance liquid chromatography - Google Patents
The mobile phase of separating phospholipids in the high performance liquid chromatography Download PDFInfo
- Publication number
- CN100542649C CN100542649C CNB2007100184931A CN200710018493A CN100542649C CN 100542649 C CN100542649 C CN 100542649C CN B2007100184931 A CNB2007100184931 A CN B2007100184931A CN 200710018493 A CN200710018493 A CN 200710018493A CN 100542649 C CN100542649 C CN 100542649C
- Authority
- CN
- China
- Prior art keywords
- mobile phase
- mutually
- phosphatide
- acid
- acetonitrile
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Treatment Of Liquids With Adsorbents In General (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The mobile phase of separating phospholipids in a kind of high performance liquid chromatography, the composition of the mobile phase of the present invention comprises: methyl alcohol 10%～50%, acetonitrile 50%～85%, fluoro organic acid 0.5%～3%, water 0.5～3%.The present invention is flowed and to be used through long-term mutually, does not produce any precipitation, can not cause damage to pumping system, and the pumping system of liquid chromatograph is not had any infringement; Can be used to detect separated phosphatide in EISD, compare with UV-detector, detect the baseline stability that obtains, be easy to quantitative analysis, highly sensitive, the application of its mobile phase is not subjected to any restriction; Compare with gradient method, use single pump can realize separating and detect; Flowing when adopting appropriate proportioning mutually,, can also isolate the unknown materials that other flow and can't separate mutually except can effectively separating to phosphatide.When the present invention is flowed when being applied to isocratic elution mutually, simple to operate, save time, favorable reproducibility, repeat easily.
Description
Technical field
The mobile phase of using when the present invention relates to a kind of high performance liquid chromatography separating phospholipids belongs to the organic chemistry analysis technical field.
Background technology
Method with the high performance liquid chromatography separating phospholipids comprises isocratic elution method and linear gradient elution method.Generally detect phosphatide with high performance liquid chromatograph and UV-detector coupling (HPLC-UV), high performance liquid chromatograph provides flow speed stability and pressure higher mutually mobile to chromatographic column, sample is separated in chromatographic column, detect separated phosphatide with UV-detector then.Each solvent ratios is constant mutually if flow when detecting, and then is the isocratic elution method, if instrument mixes different solvents automatically according to demand, then is linear gradient elution method.The proportioning time to time change of linear gradient elution method solvent, each separation all needs balance again, so analysis time, length, complicated operation, workload were big; And the isocratic elution method simple to operate, save time, favorable reproducibility, do not have again the problem of balance, so be widely used.But the condition of isocratic elution method is difficult to grope, and joining of each solvent of the phase that flows is difficult definite.
In addition, because the maximum absorption wavelength of phosphatide is about 205nm, and a lot of reagent also has strong absorption about wavelength 205nm, disturb the detection of phosphatide easily, causes UV-detector sensitivity to reduce.Therefore, the isocratic elution method is mainly used with the current downflow phase: the mobile phase of forming by acetonitrile-methyl alcohol-phosphoric acid/sulfuric acid, and with mutually mobile by what n-hexane (or chloroform/carrene)-isopropyl alcohol-ammoniacal liquor was formed, and other phase that similarly flows.Wherein, the phosphoric acid/sulfuric acid that acetonitrile-methyl alcohol-phosphoric acid/sulfuric acid is formed in flowing mutually can produce precipitation, makes the plunger of high performance liquid chromatograph mesohigh pump and sealing gasket friction produce cut and cause system's leakage.
Summary of the invention
The mobile phase that the purpose of this invention is to provide separating phospholipids in a kind of high performance liquid chromatography, the proportioning and the sedimentation problem of the phase that flows when high performance liquid chromatograph uses in the solution prior art isocratic elution method.
The mobile phase of separating phospholipids in the high performance liquid chromatography proposed by the invention, it forms by volume that percentage comprises: methyl alcohol 10%～50%, acetonitrile 50%～85%, fluoro organic acid 0.5%～3%, water 0.5～3%.The suitable volumes percentage of described mobile phase composition is: methyl alcohol 10%～25%, acetonitrile 75%～85%, fluoro organic acid 1%～2.5%, water 1%～3%.The preferred volume percentage of described mobile phase composition is: methyl alcohol 19～20%, acetonitrile 77～78%, fluoro organic acid 1～2%, water 1.5～2.5%.Described fluoro organic acid comprises any that trifluoroacetic acid, five fluorine propionic acid, hyptafluorobutyric acid, nine fluorine valeric acids, 11 fluorine caproic acids, ten trifluoro enanthic acid, 15 fluorine are sad.
The action principle of mobile phase proposed by the invention: along with the development of EISD, there are some reagent of strong absorption also can use at the 205nm place, the present invention finds that in conjunction with high performance liquid chromatography-EISD (HPLC-ELSD) multiple acid can replace additive such as phosphoric acid/sulfuric acid to be applied to the detection of phosphatide, and compatible very good with instrument, so high performance liquid chromatograph to the absorbing wavelength no requirement (NR) of the phase that flows, still has the mobile phase of the phosphoric acid/sulfuric acid of corrosion precipitation detrimental effect still to be widely used to high performance liquid chromatograph.By test and analysis, existing inorganic additives such as phosphoric acid/sulfuric acid in flowing mutually are limited in organic solubility in flowing mutually, undissolved phosphoric acid/sulfuric acid is present in mobile phase with precipitation form, finally is deposited in the pumping system, causes the friction infringement of plunger rod and sealing ring.And because fluoro organic acid structure such as trifluoroacetic acid, five fluorine propionic acid, hyptafluorobutyric acid, nine fluorine valeric acids, 11 fluorine caproic acids, ten trifluoro enanthic acid, 15 fluorine be sad is similar, character is close, replace inorganic acids such as phosphoric acid/sulfuric acid with it, can avoid the generation that precipitates fully, and can not damage the pump circulatory system, compatible very good with instrument can use for a long time.In order to reduce organic acid chromatographic column is caused damage, must in flowing mutually, add a certain proportion of water to improve the pH value of the phase that flows.
Proposed by the invention flowing has the following advantages mutually:
1, the present invention is flowed and to be used through long-term mutually, and the pumping system of high performance liquid chromatograph is not had any infringement.Described fluoro organic acid can dissolve each other fully with arbitrary proportion in the acetonitrile-methanol-water of arbitrary proportion, does not produce any precipitation, can not cause damage to pumping system, and operating position is good throughout the year.
2, of the present invention flowing can be used to detect separated phosphatide mutually in EISD, compares with UV-detector, and be highly sensitive; And from the reagent application point, the application of the mobile phase of the present invention is not subjected to any restriction substantially.
3, use the present invention to flow and detect the baseline stability that obtains mutually, be easy to quantitative analysis; And use the easy drift of UV-detector baseline, cause quantitative inaccuracy.
4, the present invention compares with gradient method, uses single pump can realize separating and detects; And gradient method needs many pumps or polynary blender to realize.
5, the present invention is flowed when adopting appropriate proportioning mutually, except can effectively separating phosphatide, can also isolate the unknown materials that other flow and can't separate mutually.
6, the present invention is added with a certain proportion of water in flowing mutually, can improve the pH value of the phase that flows, and reduces the infringement that organic acid causes chromatographic column.
7, flow when being applied to isocratic elution mutually as the present invention, simple to operate, save time, favorable reproducibility, repeat easily.
Accompanying drawing and explanation thereof
Accompanying drawing 1 is to adopt prior art methyl alcohol: acetonitrile: the phosphatide that extracts in the mobile relative erythrocyte membrane sample of 85% phosphoric acid (64.7%: 34.9%: 0.4%) separates the resulting HPLC-UV chromatogram of detection; It can isolate four kinds of main phosphatide (PS, PE, PC, SM) and a kind of unknown material (*), but long-term the use can be damaged the liquid phase systems high-pressure pump, causes the pump leakage.
Accompanying drawing 2 is for adopting methyl alcohol of the present invention: acetonitrile: trifluoroacetic acid: (29.5%: 68.8%: 0.7%: the phosphatide that extracts in the mobile relative erythrocyte membrane sample 1%) separated the resulting HPLC-UV chromatogram of detection to water; Main phosphatide (PS, PE, PC, SM) can be realized baseline separation substantially.
Accompanying drawing 3 is for adopting methyl alcohol of the present invention: acetonitrile: five fluorine propionic acid: (29.5%: 68.8%: 0.7%: the phosphatide that extracts in the mobile relative erythrocyte membrane sample 1%) separated the resulting HPLC-UV chromatogram of detection to water; Main phosphatide (PS, PE, PC, SM) can be realized baseline separation substantially.
Accompanying drawing 4 is for adopting methyl alcohol of the present invention: acetonitrile: hyptafluorobutyric acid: water (49.1%: 49.1%: 1.1%: the HPLC-ELSD chromatogram that the phosphatide that extracts in the mobile relative erythrocyte membrane sample 0.7%) separates to detect gained; Resulting baseline is more stable, main phosphatide (PS, PE, PC, SM) can be realized baseline separation.
Accompanying drawing 6 is for adopting methyl alcohol of the present invention: acetonitrile: five fluorine propionic acid: water (32.7%: 65.5%: 1.1%: the HPLC-ELSD chromatogram that the phosphatide that extracts in the mobile relative erythrocyte membrane sample 0.7%) separates to detect gained; Can be with the complete baseline separation of main phosphatide (PS, PE, PC, SM).
Accompanying drawing 7 is for adopting methyl alcohol of the present invention: acetonitrile: trifluoroacetic acid: water (19.4%: 77.4%: 1.9%: the HPLC-ELSD chromatogram that the phosphatide that extracts in the mobile relative erythrocyte membrane sample 1.3%) separates to detect gained; Can be with main phosphatide (PS, PE, PC, SM) and the complete baseline separation of a kind of unknown material (*).
Accompanying drawing 8 is for adopting methyl alcohol of the present invention: acetonitrile: five fluorine propionic acid: water (19.4%: 77.4%: 1.9%: the HPLC-ELSD chromatogram that the phosphatide that extracts in the mobile relative erythrocyte membrane sample 1.3%) separates to detect gained; Can be fully with main phosphatide (PS, PE, PC, SM) and the complete baseline separation of a kind of unknown material (*).
Accompanying drawing 9 is for adopting methyl alcohol of the present invention: acetonitrile: trifluoroacetic acid: water (16.2%: 80.9%: 0.6%: the HPLC-ELSD chromatogram that the phosphatide that extracts in the mobile relative erythrocyte membrane sample 2.3%) separates to detect gained; More main phosphatide (PI, PS, PE, PC, SM) can be realized separating with unknown material (*), but the hangover of SM peak shape is serious.
Among the figure abscissa be retention time (minute); Ordinate is response voltage (volt);
SF among the figure is solvent front (solvent front); * be without the material of identifying, can only know it is a kind of fat at present; PI is phosphatidylinositols (phosphatidylinositol); PS is a phosphatidyl silk amino acid (phosphatidylserine); PE is phosphatidyl-ethanolamine (phosphatidyl-ethanolamine); PC is phosphatid ylcholine (phosphatidylcholin); SM is sphingomyelins (Sphingomyelin), and above chromatogram all is to extract and adopt the present invention to flow from erythrocyte membrane or tissue to reach the result that chromatographic column separation detection obtains mutually, and explanation can effectively separate the phosphatide in these cell membranes or the tissue.
The specific embodiment
Flow mutually below in conjunction with accompanying drawing to the present invention that the concrete enforcement and the effect thereof of each set of dispense ratio describe, and to adopting the present invention to flow to be separated chromatographic condition explanation concrete when detecting.
High performance liquid chromatograph comprises system controller, UV-detector, efficient liquid phase pump system, degas system and column oven.Chromatographic column is anti-phase analytical column C18 (250mm * 4.6mm I.D.), 80 ℃ of EISD drift pipe temperature, nitrogen flow rate 2L/ minute; Flow rate of mobile phase 1.0ml/ minute.
Separate test experience and separate detection phosphatide with HPLC-UV at first, the phosphatide of sample for from the swine erythrocyte film, extracting, the volume ratio of the phase that flows is a methyl alcohol: acetonitrile: 85% phosphoric acid (64.7%: 34.9%: 0.4%), testing result is seen accompanying drawing 1, but finds the pump leakage of high performance liquid chromatograph.
For fear of the pump leakage, will flow changes methyl alcohol mutually into: acetonitrile: trifluoroacetic acid: water (29.5%: 68.8%: 0.7%: 1%), testing result was seen accompanying drawing 2, though can be with the basic baseline separation of each phosphatide, baseline has certain drift.Owing to use the UV detector, so the adjustment of trifluoroacetic acid is restricted, and trifluoroacetic acid is high more, and the baseline noise is big more, and sensitivity is low more.
And adopt five fluorine propionic acid also can produce same problem, flowing changes methyl alcohol mutually into: acetonitrile: five fluorine propionic acid: (29.5%: 68.8%: 0.7%: 1%), testing result was seen accompanying drawing 3 to water.
Below HPLC-ELSD is used in experiment, the phosphatide that employing extracts from the swine erythrocyte membrane sample, the volume ratio of the phase that flows this moment is a methyl alcohol: acetonitrile: hyptafluorobutyric acid: water (49.1%: 49.1%: 1.1%: 0.7%), testing result is seen accompanying drawing 4, each main phosphatide is substantially by baseline separation, and baseline stability.
Change the phase component ratio that flows, flowing is methyl alcohol mutually: acetonitrile: trifluoroacetic acid: (32.7%: 65.5%: 1.1%: 0.7%), testing result was seen accompanying drawing 5 to water, and each main phosphatide is by complete baseline separation, and baseline stability, finding simultaneously has a unknown peak that certain reservation is arranged.
Trifluoroacetic acid changes five fluorine propionic acid in will flowing mutually, and flowing is methyl alcohol mutually: acetonitrile: five fluorine propionic acid: (32.7%: 65.5%: 1.1%: 0.7%), testing result was seen accompanying drawing 6 to water, and each main phosphatide is still by baseline separation, and there is better reservation at unknown peak.
Further adjust each component ratio mutually that flows, mobile phase volume is than being methyl alcohol: acetonitrile: trifluoroacetic acid: water (19.4%: 77.4%: 1.9%: 1.3%), testing result is seen accompanying drawing 7, and each main phosphatide and unknown peak are all by baseline separation, and the sensitivity of each phosphatide is all high.
Trifluoroacetic acid changes five fluorine propionic acid in will flowing mutually, flowing is methyl alcohol mutually: acetonitrile: five fluorine propionic acid: water (19.4%: 77.4%: 1.9%: 1.3%), testing result is seen accompanying drawing 8, and each main phosphatide and unknown peak are equally all by baseline separation, and the sensitivity of each phosphatide is all high.
Continue to reduce the middle mutually methyl alcohol ratio that flows, flowing is methyl alcohol mutually: acetonitrile: trifluoroacetic acid: water (16.2%: 80.9%: 0.6%: 2.3%), testing result is seen accompanying drawing 9, by finding out among the figure, having in above experiment does not severally have material retained to be retained, and basic baseline separation, but the hangover at SM peak is comparatively serious.
Trifluoroacetic acid changes nine fluorine valeric acids in will flowing mutually, flowing is methyl alcohol mutually: acetonitrile: nine fluorine valeric acids: water (16.2%: 80.9%: 0.6%: 2.3%), testing result is seen accompanying drawing 10, having does not equally severally have material retained to be retained, and basic baseline separation, but the hangover at SM peak is comparatively serious.
Claims (3)
1, the mobile phase of separating phospholipids in a kind of high performance liquid chromatography is characterized in that, the composition of the phase that flows by volume percentage by methyl alcohol 10%～50%, acetonitrile 50%～85%, fluoro organic acid 0.5%～3%, water 0.5%～3% constitutes; Described fluoro organic acid is selected from sad any of trifluoroacetic acid, five fluorine propionic acid, hyptafluorobutyric acid, nine fluorine valeric acids, 11 fluorine caproic acids, ten trifluoro enanthic acid, 15 fluorine.
2, mobile phase according to claim 1 is characterized in that, the percent by volume of described mobile phase composition is: methyl alcohol 10%～25%, acetonitrile 75%～85%, fluoro organic acid 1%～2.5%, water 1%～3%.
3, mobile phase according to claim 1 is characterized in that, the percent by volume of described mobile phase composition is: methyl alcohol 19%～20%, acetonitrile 77%～78%, fluoro organic acid 1%～2%, water 1.5%～2.5%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2007100184931A CN100542649C (en) | 2007-08-16 | 2007-08-16 | The mobile phase of separating phospholipids in the high performance liquid chromatography |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2007100184931A CN100542649C (en) | 2007-08-16 | 2007-08-16 | The mobile phase of separating phospholipids in the high performance liquid chromatography |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101156995A CN101156995A (en) | 2008-04-09 |
| CN100542649C true CN100542649C (en) | 2009-09-23 |
Family
ID=39305304
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2007100184931A Active CN100542649C (en) | 2007-08-16 | 2007-08-16 | The mobile phase of separating phospholipids in the high performance liquid chromatography |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100542649C (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102156174A (en) * | 2011-05-06 | 2011-08-17 | 吉林大学 | Detection of phosphatidylcholine in yolk lecithin by using high performance liquid chromatography |
| CN102680630A (en) * | 2012-05-21 | 2012-09-19 | 天津大学 | Method for analyzing micro-algae phospholipid group |
| CN110514776A (en) * | 2019-09-03 | 2019-11-29 | 中国水产科学研究院黄海水产研究所 | The detection method of phosphatide in a kind of antarctic krill oil |
-
2007
- 2007-08-16 CN CNB2007100184931A patent/CN100542649C/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN101156995A (en) | 2008-04-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Walter et al. | Recent innovations in UHPLC columns and instrumentation | |
| Audunsson | Aqueous/aqueous extraction by means of a liquid membrane for sample cleanup and preconcentration of amines in a flow system | |
| Nováková et al. | Modern analytical supercritical fluid chromatography using columns packed with sub-2 μm particles: a tutorial | |
| Rohrschneider | Solvent characterization by gas-liquid partition coefficients of selected solutes | |
| Noga et al. | A practical guide to nano‐LC troubleshooting | |
| CN106442752B (en) | A kind of liquid chromatogram-ion chromatography combined system and method | |
| US8992778B2 (en) | Methods and apparatus for generating solvent gradients in liquid chromatography | |
| Liu et al. | Continuous flow liquid membrane extraction: a novel automatic trace-enrichment technique based on continuous flow liquid–liquid extraction combined with supported liquid membrane | |
| Hu et al. | Determination of inorganic anions in ethyl acetate by ion chromatography with an electromembrane extraction method | |
| CN100542649C (en) | The mobile phase of separating phospholipids in the high performance liquid chromatography | |
| CN105612424B (en) | Liquid chromatography device for quickly measuring | |
| JP2003014718A (en) | Mobile-phase gradient device and high-performance liquid chromatograph using the same | |
| Lin et al. | Development of in‐tube solid‐phase microextraction coupled to pressure‐assisted cec and its application to the analysis of propranolol enantiomers in human urine | |
| CN107167535B (en) | Method for detecting raltitrexed enantiomer by reversed phase liquid chromatography | |
| CN104678041A (en) | Method for simultaneously testing inorganic anions and aniline compounds by two-dimensional system | |
| El Ouahabi et al. | On-line solid-phase extraction to enhance sensitivity in peptide biomarker analysis by microseparation techniques coupled to mass spectrometry: Capillary liquid chromatography versus capillary electrophoresis | |
| CN202614733U (en) | Device for detecting content of toluene formyl benzotriazole passivators in transformer oil | |
| Trocewicz | Sample preparation of amphetamine and methamphetamine by means of supported liquid membrane technique for high‐performance liquid chromatography analysis | |
| Fountain et al. | Practical applications of charged surface hybrid (CSH) technology | |
| Kolobova et al. | Application of ionic liquids based on imidazole to the electrophoretic determination of amino acids in urine | |
| Liu et al. | Evaluation of an on-line coupled continuous flow liquid membrane extraction and precolumn system as trace enrichment technique by liquid chromatographic determination of bisphenol A | |
| Gimeno et al. | Determination of aromatic sulfonates in coastal water by on-line ion-pair solid-phase extraction/ion-pair liquid chromatography with UV detection | |
| CN104897838B (en) | Method for separating and analyzing is extracted while a kind of highly polar organic acids and base mixture | |
| Zhang et al. | Extraction and separation of triazine herbicides in soybean by ionic liquid foam-based solvent flotation and high performance liquid chromatography determination | |
| CN106979985B (en) | Liquid chromatogram atomic spectrum combined system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C56 | Change in the name or address of the patentee |
Owner name: SHAANXI BAIMEI GENE CO., LTD. Free format text: FORMER NAME: SHAANXI LIFEGEN CO., LTD. |
|
| CP01 | Change in the name or title of a patent holder |
Address after: 386 box 710069, Northwestern University, 229 Taibai North Road, Shaanxi, Xi'an Patentee after: Shaanxi Baimei Gene Co., Ltd. Address before: 386 box 710069, Northwestern University, 229 Taibai North Road, Shaanxi, Xi'an Patentee before: Shaanxi Beimei Gene Co., Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200509 Address after: 710086 4-2-4620, floor 2, building 4, Free Trade Industrial Park, No. 2168, Zhenghe Fourth Road, Fengdong new town, Xi'an City, Shaanxi Province Patentee after: Xi'an blood oxygen Biotechnology Co., Ltd Address before: 386 box 710069, Northwestern University, 229 Taibai North Road, Shaanxi, Xi'an Patentee before: Shaan Xi Lifegen Co.,Ltd. |