CN1724105A - Composition and method for high efficiency chromatography - Google Patents
Composition and method for high efficiency chromatography Download PDFInfo
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- CN1724105A CN1724105A CN 200510080509 CN200510080509A CN1724105A CN 1724105 A CN1724105 A CN 1724105A CN 200510080509 CN200510080509 CN 200510080509 CN 200510080509 A CN200510080509 A CN 200510080509A CN 1724105 A CN1724105 A CN 1724105A
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Abstract
The invention provides novel compositions, columns and methods for improved chromatographic separations. In particular, novel column packings are provided to improve overall chromatographic separation and efficiency. The invention provides a packing material having particles or mixtures of particles that are used for separation of small molecules, proteins or nucleic acids.
Description
Technical field
The present invention relates to be used to carry out high efficiency liquid chromatogram composition isolated and method, relate more specifically to be used for the new compositions and the method for chromatography column.
Background technology
Utilize the separation of high speed liquid chromatography (HPLC) to depend on many component solute molecules infiltrations in the fluid flow stream and pass particle-filled that is known as fixing phase.This allows efficiently that particle is separated from one another.Every kind of component has different affinity mutually to fixing, causes every kind of component different migration rate and time departure in post.The amount of its band expansion was determined when separative efficiency was passed through post by solute.
Plate theory is commonly used to describe solute and passes chromatographic column and band broadening.Plate theory is explained the band broadening according to many rate factors.For example, it is to carry out in the discrete hypothesis step of a plurality of continuous equalizations that separation can be considered as, and each step space all comprises fixing mutually with mutually mobile, and has all set up balance completely in each step.Each such stage is called " theory " column plate.In this case, by the theoretical cam curve in the separation degree calculating post.Column length is important for the calculating of this relevant theoretical tray.Column length/(theoretical cam curve that calculates) is called " height that is equivalent to a theoretical tray " or H, and is measuring of band broadening phenomenon.
The band broadening is important for separating, the quality that expression separates.For example, generally speaking, band is wide more in the separation, and separation or column efficiency are poor more.
Separate also based on the relation between the phase.In chromatography, one fixes mutually, and another flows mutually.Flowing flows through fixedly phase with fast relatively speed, so that does not in fact reach complete equipilibrium between this two-phase.Avoid the peak broadening and obtain clearly when separating efficiently when separating, must consider this point.
When chromatographic column was used plate theory, all solutes all were assumed that in the first column plate space that is present in post at first.Dispersion is based on distribution coefficient.In this case, for the solute concentration that is run into, distribution coefficient is constant, and solute distributes self fast between two-phase in each column plate space.
Provide the post of smallest peaks broadening to represent to separate clearly, and be that HPLC system and process are desired.It all is high-importance with separating efficiently that the filling mode that charges into filler attribute in the post and post obtains clear for sample component.Therefore, wish to make and variously determine relevant band broadenings and the process that column performance produces adverse effect is reached minimum.The influence of each coupled columns plate height H in these processes can through-rate theoretical and with the diameter d of for example flow phase velocity u, particle filled composite
pRelevant with the experimental variable of solute diffusion coefficient in flowing mutually and so on.Van Deemter equation is generally considered the master tape broadening process that parity price among the HPLC works in the height H of a theoretical tray.In addition, this equation can be reduced to and three important variablees: particle size, fluid velocity and diffusion coefficient are relevant.In this abbreviation equation, along with particle size increases, efficient should descend.Along with fluid velocity increases, prediction equation will determine efficient to the mass transfer item, and the product of deterioration and speed and particle diameter is proportional.Will be appreciated that: wish to make the band broadening to reach minimum and guarantee that the solute that obtains the best in analyzing chromatography, product purity and preparation chromatography separates.
But more the benefit brought aspect column efficiency of small-particle is necessarily offset by the shortcoming of higher back-pressure, and the shortcoming of higher back-pressure comprises by the higher instrument cost that system pressure caused to be increased and the reliability reduction.And, assert usually that in theory used particle should be monodispersed in the liquid chromatogram, and have narrow as far as possible particle size distribution.For example, Dewaele and the Verzele mixture that discloses the irregularly shaped material of (J.Chromatography, 260 (1983) 13-21) different particle sizes carries out the corresponding equal average particle size of packing ratio and produces bigger pressure.The work of Dewaele and Verzele is confined to the mixture of two kinds of components.Every kind of component in the mixture has the mean value of monodispersed distribution of particles and distribution.In addition, the mean value of particle size distribution differs by more than 40% each other.In other words, the mix particles body that differs greatly of average diameter does not have effect to the total back-pressure that reduces system.
Therefore, be desirable to provide one or more and allow new material efficient and that effectively separate, thereby avoid the back-pressure increase and separate and inefficient problem.
Summary of the invention
The invention provides the device, method and the filled compositions that are used for chromatogram.Filled compositions comprises that two or more have the component of particle size distribution (PSD) and particle size distribution mean value.Difference between the mean value of any two particle size distribution is less than 40% of the mean value of big component.
Particle can be to be fit to any inorganic of chromatogram purposes or to mix inorganic/organic material.
The present invention also provides filler and the chromatographic column that comprises the particle with PSD, and this PSD has been broadened with respect to the filler with higher back-pressure.
The present invention also provides and to have made and use particle or filler to separate the method for various organic and inorganic material.
At last, the invention provides useful particle dimer in total separation and increase efficient.
Description of drawings
Fig. 1 shows the plate height of HPLC system and the example of the van Deemter figure of mobile linear speed mutually.
Fig. 2 shows the particle size distribution figure that overlaps each other of single dispersion and two dispersed sample.
Fig. 3 shows for the back-pressure of the HPLC experiment of having compared the particle with size distribution shown in Fig. 2 and the figure of flow velocity.
Fig. 4 shows the van Deemter figure that tests shown in Fig. 3.
Fig. 5 shows the transmission electron microscopy figure of particle of the present invention.
It is the HPLC experiment of the mixture of macroparticle and small-particle that Fig. 6 shows for column packing wherein, the figure of efficient and macroparticle percentage.
Fig. 7 show for Fig. 6 in identical experiment, the figure of system pressure and macroparticle percentage.
The specific embodiment
Before describing the present invention in detail, must be noted that as used in this specification and the claims, singulative " a kind of " and " being somebody's turn to do " comprise plural object, unless context has clear regulation in addition.Thereby, for example mention that " a kind of material " then comprises the combination of multiple material, mention that " a kind of compound " then comprises the mixture of multiple compound, mention that " a kind of nitride " then comprises multiple nitride etc.
In description of the invention and claim, the term below will using according to the definition of following explanation.
Term " basically " when being used for representing rerum natura here, the meaning be described value 10% in.
Term " packing material " expression atresia, the packing material of porose, perhaps porose, atresia or surperficial porose mixing.This term is not to be used for restriction, and comprises the combination and the mixture of above-mentioned material.
Term " fixedly phase " expression comprises the phase of particle, and these particles comprise organic or inorganic material, and they randomly have the organic fragment with its bonding, thereby make particle surface be of value to some chromatographic isolation.In some cases, these particles or material are fixed in the post, do not move.The example of inorganic material is but is not limited to silica, zirconium and titanyl compound.The example of organic material is the SDVB copolymer.That inorganic material also can comprise is known in the art, comprise organic and inorganic fragment in same structure " mixing " particle, the United States Patent (USP) 4 that this particle is transferring Merck, 017,528 and transfer the United States Patent (USP) 6 of Waters, 528,167 and 6,686, description is all arranged in 035, and by reference the content of these three pieces of patents is incorporated into this.
Here with reference to figure the present invention is described.Figure does not draw in proportion, and particularly for clear statement, some size may have been exaggerated.
By well known by persons skilled in the art, the U.S. Patent No. 3,782 that is all transferring Du Pont for example, 075,3,855,172 and 4, (content with these three pieces of patents is incorporated into this by reference) standard method of describing in 010,242 prepares two samples of bonding silica particle.Utilize the bonding chemicals described in the U.S. Patent No. 4,705,725 (by reference its content being incorporated into this) also transfer Du Pont to come the bonding particle.Bonding is carbon 18 (octadecyl) material mutually.Should be noted that the particle size distribution (PSD) that these treatment technologies obtain limiting.This is common general knowledge in industrial standard.Though should be noted that under the situation of these methods and describe the present invention, the present invention should not be considered as being limited to the component of these particle size distribution.For example, can use or approximate identical or similar size, the particle in certain distributes not of Development and Production.The present invention also is applicable to the material and the situation of these types.
Fig. 2 represents by Coulter counter (Beckman Instruments, Hialeah, FL) particle size distribution of two samples of this that obtains.From be lower than 1 μ m to 6 μ m with 256 grid, calculate the number of particles in each grid.From Fig. 2 as seen, a sample (being labeled as A) is monodispersed, can see a peak at about 2 μ m places.Second sample provides two peaks, is that diameter is the mix particles body of 1.8 μ m and 2.2 μ m substantially, records as the Coulter counter.
Use standard technique that these two samples are inserted in the HPLC post of diameter as 4.6mm, long 30mm.(Agilent Technologies, Wilmington carry out HPLC on DE) and test and measure back-pressure and cross plate height that the function and measuring of the volume flow rate of post the converts function as linear flow rate as flow communication at Agilent 1100 instruments.Eluent is 85: 15 acetonitriles under 20 ℃ of temperature: water (volume ratio).Sample is the benzene octanone, carries out ultraviolet and detect under 245nm.
Fig. 3 shows the back-pressure figure of HPLC experiment.Wherein show sample A and B, and shown in sample B as the optimum flow rate judged by van Deemter figure among Fig. 4 under back-pressure than low 20% of sample A.
Fig. 4 shows the van Deemter curve of sample column.Utilize identical average particle size, calculate the conversion plate height of these two posts.Optimum flow rate with regard to maximum column efficiency is judged by the position of the minimum of a value among the figure, and as seen from the figure, around minimum plate height, plate height differs and is no more than several percentages (0.00385mm of sample B is to the 0.00372mm of sample A); Minimum of a value is enough wide in the curve, thereby basic identical in the performance of the whole operating rate scope center pillar of post.
Therefore, the broad particle size distribution of sample B proof has obviously reduced back-pressure effectively in the HPLC experiment, do not have sacrificial posts efficient simultaneously again.
Fig. 5 shows the slide glass that above-mentioned wide PSD particle is housed and amplifies 500 times transmission electron microscopy figure (TEM).As if though generally the Coulter counter data can not be associated with the TEM data, the many particles in the micrograph link the formation dimer.Therefore, though do not wish limit by mechanism, may cause by dimeric structure as measured some or all the bimodal characteristics of PSD of Coulter counter.Here the present invention for required protection scope comprises the PSD that wherein has the dimer particle structure.
Preparation have with embodiment 1 in identical bonding phase, internal diameter be 4.6mm, longly be the HPLC post of 30mm.These posts are mounted with the mix particles body of the unimodal particle size distribution of 1.8 μ m average diameters and 3.5 μ m average diameters.The HPLC experiment condition is as follows:
Instrument: Agilent 1100 (Agilent Technologies, Wilmington, DE)
Volume injected: 2 μ l
Detect: 254nm ultraviolet with 1.8ul pond
Phase flows: 60/40 methanol
Flow velocity: 1.5ml/min
Temperature: room temperature
Sample: uracil, phenol, 4-chloronitrobenzene and toluene
Table 1
| 3.5 the percentage by weight of μ m particle in mixture | Efficient (column plate) | Pressure (bar) |
| 0 | 7500 | 377 |
| 10 | 7200 | 357 |
| 20 | 7200 | 333 |
| 25 | 7200 | 330 |
| 30 | 7100 | 320 |
| 40 | 6900 | 316 |
| 50 | 5900 | 299 |
As seen, more the odds for effectiveness of small-particle 1.8 μ m is more kept under the situation of macroparticle substantially adding up to 30%, and system pressure is saved 57bar from table 1.Fig. 6 and 7 clearly show that this influence, and wherein the percentage curve of efficient and 3.5 μ m particles shows that adding still is platform-like up to 40% particle, and the percentage curve of pressure and 3.5 μ m particles is linear substantially.
Can not know directly that from discussion of " background technology " part the mixture of this multiple particle size why should be able to show this high efficiency of the back-pressure required with respect to operation post.Under the situation of not wishing to be subjected to mechanism to limit, to column efficiency may contribute the filling that may come from post easier, thereby chromatographic efficiency more approaches the more theoretical maximum of small-particle.Another possible contribution may come to fix with this border of fixing between the wall of having inserted post wherein mutually fills effect.
Though the embodiment that more than provides has limited concrete particle size distribution and chromatographic condition setting, it is to be subject to these conditions that the present invention should not be considered as.For example, with clear, filler can comprise that other is not the material of bonding silica to those skilled in the art in the post from present disclosure, and still keeps identical with the silica post benefit of being brought by PSD.
In addition, it is bimodal that PSD needs not be, and has the narrow PSD of identical average particle size as long as compare, its wide required reduction that is enough to provide to back-pressure.Those skilled in the art does not need too much experiment just can develop the PSD particle of the iptimum relationship with the back-pressure of showing and efficient.
Fixing particle in mutually needn't be all the hole that all has same degree.For example, have the mix particles body in hole in various degree fixing can comprising mutually, for example the porose particle of the particle of atresia or surface.The example of the porose particle in surface provides in the U.S. Patent No. 4,477,492 that transfers Du Pont, and by reference its content is incorporated into this.In a word, the present invention cover any mixture with respect to the essentially identical fixedly back pressure of efficient multiple sized particles that reduce, that porous is different.
The average particle size of particle of the present invention should not be considered as being subject to the foregoing description yet.Average particle size only is subjected to the availability limitations of particle as the chromatographic stationary phase, and usually in the scope of 0.5 μ m～100 μ m and 1 μ m～10 μ m.Though the embodiment that more than provides relates to the use of particle in analytical applications, obviously as seen the advantage that the present invention is used in the preparative chromatography will wherein can obtain higher efficient for given back-pressure.
Though embodiment shows the present invention and is used for little organic molecule (uracil, phenol, toluene and 4-nitrobenzene) that the present invention also is applicable to and separates big polymer, protein, nucleic acid and peptide.Utilize also separable other biomolecule known in the art of the present invention.
Claims (22)
1. post that is used in the chromatographic isolation comprises:
(a) housing; With
(b) be arranged on the interior fixedly phase of described housing, should fixing comprise packing material mutually, described packing material comprises two or more components, and each component has a particle size distribution and a mean particle diameter, and the difference of wherein any two average diameters is less than or equal to 40% of big mean value.
2. post as claimed in claim 1, the wherein said fixing material that is selected from organic material, inorganic material, hybrid materials or its combination that comprises mutually.
3. post as claimed in claim 1, the difference between the diameter at the place, two peaks of wherein said distribution be less than or equal to these two peaks everywhere diameter mean value 35%.
4. post as claimed in claim 1, wherein said post can be used in the liquid chromatogram separation.
5. post as claimed in claim 4, wherein said post can be used in the preparation separation.
6. post as claimed in claim 1, wherein said post can be used in the gas-chromatography separation.
7. post as claimed in claim 1, wherein said packing material comprises dimer.
8. post that is used in the chromatographic isolation comprises:
(a) housing; With
(b) be arranged on fixedly phase in the described housing, this is fixing comprise mutually population between 500 and 3000, average particle diameter first component between 1 and 4 micron and population between 500 and 3000, second component of average particle diameter between 1 and 4 micron.
9. be used in the chromatogram packing material as the fixing composition of matter of phase, described packing material comprises the particle with bimodal number average particle size distribution or has the mixture of the different porous particles of bimodal number average particle size distribution, in described particle size distribution, the difference between the diameter at the place, two peaks of described distribution be less than or equal to this place, two peaks diameter mean value 40%.
10. composition as claimed in claim 9, the wherein said fixing material that is selected from organic material, inorganic material, hybrid materials or its combination that comprises mutually.
11. composition as claimed in claim 9 wherein saidly fixing comprises the particle with bimodal distribution mutually, in described distribution, the difference between the diameter at the place, two peaks of described distribution be less than or equal to this place, two peaks diameter mean value 35%.
12. composition as claimed in claim 9, wherein said composition can be used in the liquid chromatogram separation.
13. composition as claimed in claim 12, wherein said composition can be used in the preparation separation.
14. composition as claimed in claim 9, wherein said composition can be used in the gas-chromatography separation.
15. composition as claimed in claim 9, wherein said packing material comprises dimer.
16. composition as claimed in claim 2, wherein said packing material comprise the particle of spheroidal particle and the dimeric compoiste adhering structure of described spheroidal particle.
17. a chromatography separating method comprises:
(a) make the sample contact have the fixedly phase of packing material, this packing material comprises the particle with particle size distribution, in described distribution of particles, the difference between the particle diameter at the place, two peaks of every kind of distribution be less than or equal to this place, two peaks diameter mean value 40%; With
(b) sample separation component.
18. chromatography separating method as claimed in claim 17, the wherein said fixing material that is selected from organic material, inorganic material, hybrid materials or its combination that comprises mutually.
19. chromatography separating method as claimed in claim 17, wherein said post can be used in the liquid chromatogram separation.
20. chromatography separating method as claimed in claim 17, wherein said post can be used in the gas-chromatography separation.
21. post as claimed in claim 1, wherein said packing material comprise the particle of spheroidal particle and the dimeric compoiste adhering structure of described spheroidal particle.
22. a chromatography separating method comprises:
(a) make fixedly phase of sample contact, the described fixing packing material that comprises first and second components that has mutually, the number average particle size distribution of described first component is wideer than the number average particle size distribution of described second component, and the maximum particle size diameter of wherein said first component is similar to the maximum particle size diameter of described second component.
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| US10/884,344 US7309426B2 (en) | 2004-04-23 | 2004-07-01 | Composition and method for high efficiency chromatography |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102844111A (en) * | 2010-04-26 | 2012-12-26 | 通用电气健康护理生物科学股份公司 | Method for production of chromatography media |
| CN111615415A (en) * | 2017-10-12 | 2020-09-01 | 弗朗索瓦·帕门蒂尔 | Chromatography method |
-
2005
- 2005-06-30 CN CN 200510080509 patent/CN1724105A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102844111A (en) * | 2010-04-26 | 2012-12-26 | 通用电气健康护理生物科学股份公司 | Method for production of chromatography media |
| CN111615415A (en) * | 2017-10-12 | 2020-09-01 | 弗朗索瓦·帕门蒂尔 | Chromatography method |
| CN111615415B (en) * | 2017-10-12 | 2023-03-10 | 弗朗索瓦·帕门蒂尔 | Material exchange method |
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Open date: 20060125 |