CA2009855A1 - Electrophoretic sieving in gel-free media with dissolved polymers - Google Patents
Electrophoretic sieving in gel-free media with dissolved polymersInfo
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Abstract
ELECTROPHORETIC SIEVING IN GEL-FREE MEDIA
WITH DISSOLVED POLYMERS
ABSTRACT OF THE DISCLOSURE
Separation of sample ions on the basis of molecular size is achieved by electrophoresis in an aqueous solution containing a dissolved non-crosslinked polymer.
The polymer has a molecular weight range which overlaps that of the sample ions being separated. Species which vary in molecular weight but not in charge/mass ratio are separated by this method, which is of particular interest in high performance electrophoresis in capillary columns where the use of gels would be awkward and inconvenient.
WITH DISSOLVED POLYMERS
ABSTRACT OF THE DISCLOSURE
Separation of sample ions on the basis of molecular size is achieved by electrophoresis in an aqueous solution containing a dissolved non-crosslinked polymer.
The polymer has a molecular weight range which overlaps that of the sample ions being separated. Species which vary in molecular weight but not in charge/mass ratio are separated by this method, which is of particular interest in high performance electrophoresis in capillary columns where the use of gels would be awkward and inconvenient.
Description
ELECTROPHORETIC SIEVING IN GEL-FREE MEDIA
WITH DISSOLVED POLYMERS
This invention relates to electrophoretic separa-tions, and to separations of species in a sample based on molecular size.
Molecular sieve electrophoresis is a powerful method for separating macromolecular solutes both among -themselves with high resolution on the basis of molecular size and from solutes of lesser molecular size. The gel media in which these separations take place however require careful preparation and special handling techniques, with problems in reproducibility and stability.
Capillary free zone electrophoresis, on the other hand, is also of interest for certain types of separations, since it permits the use of high voltages which provide the advantage of relatively high speed. The small size of the capillary further permits the separation of extremely small samples in a buffer solution without the use of complex media such as a gel or paper, and with essentially no band broadening. Capillary free zone electrophoresis is particularly useful in the separation of small peptides and proteins. Separation occurs on the basis of the charge/mass ratio, however, and for this reason certain separations are very difficult to achieve by this method, notably those involving high molecular weight polynucleotides and many SDS-treated proteins.
Gel media may be placed in capillaries for molec-ular sieve separations, but the preparation and use of such gels is particularly problematic, since they undergo phys-ical and chemical changes with each use and thus forpractical purposes can only be used once. This is detrimen-tal to the reproducibility of the separations and to the ~.
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WITH DISSOLVED POLYMERS
This invention relates to electrophoretic separa-tions, and to separations of species in a sample based on molecular size.
Molecular sieve electrophoresis is a powerful method for separating macromolecular solutes both among -themselves with high resolution on the basis of molecular size and from solutes of lesser molecular size. The gel media in which these separations take place however require careful preparation and special handling techniques, with problems in reproducibility and stability.
Capillary free zone electrophoresis, on the other hand, is also of interest for certain types of separations, since it permits the use of high voltages which provide the advantage of relatively high speed. The small size of the capillary further permits the separation of extremely small samples in a buffer solution without the use of complex media such as a gel or paper, and with essentially no band broadening. Capillary free zone electrophoresis is particularly useful in the separation of small peptides and proteins. Separation occurs on the basis of the charge/mass ratio, however, and for this reason certain separations are very difficult to achieve by this method, notably those involving high molecular weight polynucleotides and many SDS-treated proteins.
Gel media may be placed in capillaries for molec-ular sieve separations, but the preparation and use of such gels is particularly problematic, since they undergo phys-ical and chemical changes with each use and thus forpractical purposes can only be used once. This is detrimen-tal to the reproducibility of the separations and to the ~.
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efficiency of the technigue. In addition, it raises serious problems for those capillarieg which are incorporated into cartridges designed for automated instrumentation.
It has now been discovered that sample ions, and particularly biomolecules, may be separated from each other on the basis of molecular size by electrophoresis through an aqueous solution of a non-crosslinked polymer of a selected molecular weight (or molecular weight range) and concentration. The molecular weight of the polymer will be selected as described below to correspond to the molecular weight range of the sample ions in a manner which will inhibit the migration of the sample ions through the solution to ~arying degrees. Macromolecular sample ions and other biological species may thus be separated from each other and from sample ions of lesser size without the use of a gel. The terms "macromolecule" and "macromolecular" are used herein to refer to species having molecular weights of at least about 10,000.
The polymers used herein are generally non-crosslinked polymers. Branched or linear polymers may be used, linear polymers being preferred for many applications.
In addition, the polymers may be neutral or charged, neutral being preferred in applications where charge interaction between the sample ions and the polymer is sought to be avoided.
Cellulose derivatives have been used in capillary electrophoresis for suppressing electroendosmosis and other types of bulk flow by increasing the viscosity of the buffer solution, and for preserving the capillary as well. The quantities used for this purpose are small, however, with no substantial tendency to detain the sample ions during their migration or to affect their separation. The present j 35 invention resides in the discovery that dissolved. linear polymers in general produce a molecular sieving effect when used in certain amounts, these amounts being generally ., , :, , : -, : , . -, ,:: . :: : - :.
higher than the amounts of the cellulose derivatives used for suppressing bulk flow.
Aqueous media with dissolved polymers in accordance with this invention may be used for biomolecular separations in general, although they are of particular utility in separations performed in capillary columns with high voltage, i.e., high performance electrophoresis. The use of polymers in this manner permits the separation of species which vary in molecular weight with insufficient or no variation in charge/mass ratio, and lends itself to easy preparation of the separation media and high reproducibility.
As in the known use of cellulose derivatives referred to above, the dissolved polymers further serve to suppress bulk flow due to their inherent increase in viscosity. Examples of bulk flow occurring spontaneously are electroendosmosis, hydrokinetic flows (due to hydrostatic heads), and convection. At these polymer level~, however, the decrease in sample ion mobility caused by the presence of the polymers varies both with the size and concentration of the polymer and the size of the sample ion, a feature which does not occur at the low levels at which the cellulose derivatives have been used or suppressing bulk flow.
Other features and advantages of the invention will be apparent from the description which follows.
The polyme~rs used in connection t~he present inven-tion must be water-soluble and, as stated above, are '~ preferably linear.
~¦ Selection of the polymer is optimally geared , toward the particular sample ions being separated. The j 35 molecular weight of the polymer is of primary in~erest in making this selection. In general, polymers varying widely in molecular weight may be used. Resolution of the sample .,.
' - ~:, ions will generally improve, however, as the polymer molecular weight approaches the range of the molecular weights of the sample ions. The best results are obtained with polymers having an average molecular weiyht which is between the lowest and highest molecular weights of the sample ions, and in particular witll polymers whose molecular weight range covers (i.e., is at least coextensive with) the molecular weight range of the sample ions. In preferred embodiments, the polymer has an average molecular weight lOwhich is from about 10,000 to about 2,000,000, and within about 0.1 to about 200 times, more preferably from about 0.2 to about 20 times, and most preferably from about 0.5 to about 2 times the average molecular weight of the sample ions.
15Within these parameters, the particular type of polymer may vary widely. For aqueous systems, examples of linear polymers which may be used are water-soluble cellulose derivatives and fully water-soluble polyalkylene glycols. Specific examples of such cellulose derivatives are sodium carboxymethyl cellulose, sodium carboxymethyl 2-hydroxyethyl cellulose, 2-hydroxyethyl cellulose, 2-hydroxypropyl cellulose, methyl cellulose, hydroxypropyl -methyl cellulose, hydroxyethyl methyl cellulose, hydroxybutyl methyl cellulose, and hydroxyethyl ethyl cellulose. Preferred cellulose derivatives are those with a highly hydrophilic character, and consequently high water solubility and minimal affinity to the sample ions. Methyl cellulose is particularly preferred. Celluloses are generally characterized in terms of the viscosity of aqueous solutions in which theyiare dissolved at specified concentrations and temperature. With this in mind, and depending on the size of the sample ions sought to be separated, the cellulose derivative may vary widely in terms of this viscosity characterization. For example, cellulose ¦ 35 derivatives may be used which are characterized as producing viscosities ranging from about 15 centipoise to about 17,000 centipoise when dissolved in water at 2 weight percent ....
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measured at 25C, although in the context of this invention they would be used at other concentrations. Polymers such as these are useful in separating polynucleotides with chains ranging from about 10 to about 10,000 base pairs.
Preferred cellulose derivatives for use in the present invention are those which would have viscosities of from about 1,000 to about 10,000 centipoise if prepared as 2%
aqueous solutions measured at 25C. It is to be understood that these viscosity characterizations are intended merely as an indication of the molecular weight of the polymer, and not of the actual viscosity when used in the context of the present invention.
Preferred polyalkylene glycols are polyethylene glycols having average molecular weights of at least about ' 15 10,000. Particularly preferred are polyethylene glycols ', with average molecular weights of at least about 20,000, most preferably at least about 30,000. As an example, mixtures of sample ions ranging in molecular weight from about 10,000 to about 100,000 may be separated with polyethylene glycols ranging in molecular weight from about 10,000 to about 100,000.
Examples of branched polymers which may be used in accordance with the invention are soluble starches and starch derivatives. A specific example is hydroxypropyl starch.
Mixtures of polymers in which varying molecular weights are purposely combined may also be used. This will be particularly useful in separating sample mixtures which have a wide range of molecular weights, thus providing separation over the entire range.
The use of charged polymers is an option which can provide a further separation parameter to the system. This will vary the interaction between the species and the polymer, and may thus be of use depending on the particular mixture Of species present in the sample, and the type Of separation sought among these species.
,.' The quantity of polymer to be dissolved in the resolving solution may vary widely, and will be any quantity which extends the retention time of the sample ions to such varying degrees that effective separation on the basis of molecular size is achieved. Clearly, this will vary with various parameters of the system, including for example the column configuration and length, the presence and effect of other factors influencing the separation such as charge and electrophoretic mobility, the molecular structure, intrinsic viscosity and interactive character of the polymer itself, and the range of and differences between the molecular weights of the sample ions. The degree to which the retention times for the sample ions should be extended for best results will vary with the sample composition and the polymer being used. For separations of macromolecular species, increases in retention time of at least about 25%, preferably at least about 35%, and most preferably at least about 50%, will provide the best results. For polyalkylene glycols, particularly polyethylene glycols, concentrations of at least about 2% by weight, preferably at least about 3%, and most preferably from about 3% to about 30% by weight will give the best results. For cellulose derivatives, preferred concentrations are at least about 0.1% by weight, with about 0.1% to about 30% by weight more preferred, and about 0.1% to about 10% by weight particularly preferred.
To conduct the separations in accordance with the present invention, operating conditions and procedures used in conventional electrophoretic separations, including appropriately selected buffer systems, may be used. The invention is of p~rticul!ar utility in high performance electrophoresis as performed in capillaries. Preferred capillaries are those having internal diameters of less than about 200 microns, more preferably less than about 100 microns, and most preferably about 25 microns to about 50 microns. Voltages of at least about 1000 volts.~re preferred, witll at least about 3000 volts particularly preferred.
The following examples are offered strictly for purposes of illustration, and are intended neither to define nor to limit the invention in any manner.
In each of these examples, electrophoresis was performed on an HPE-100 high performance electrophoresis instrument, a product of Bio-Rad Laboratories, Inc., Hercules, California. Capillary tubes of 20 cm length by 25 ~ inner diameter, and 50 cm length by 50 ~ inner diam-eter, coated with linear polyacrylamide as described in 10 Hjerten, U.S. Patent No. 4,680,201, issued July 14, 1987, were used. A conductivity bridge Model 31 from Yellow Springs Instrument Co., Yellow Springs, Ohio, was used, and detection was performed on-line in the capillary itself, by UV absorption. The hydroxypropyl methyl cellulose was obtained from Sigma Chemical Co., St. Louis, Missouri, in powder form, specified in terms of its viscosity when prepared as an aqueous solution at a concentration of 2% by weight and measured at 25C. The specified viscosities were 15, 50, 100 and 4000 centipoise, and the powdered polymer will be referred to herein for convenience as "15-centipoise," "50-centipoise," "100-centipoise," and "4000-centipoise hydroxypropyl methyl cellulose." The 4000-centipoise hydroxypropyl methyl cellulose was estimated to have an average molecular weight of approximately 900,000.
The methyl cellulose was also obtained from Sigma Chemical Co., similarly specified as producing a viscosity of 4000 centipoise in a 2% aqueous solution at 25C, and will be referred to herein in a manner similar to the hydroxypropyl methyl cellulose. The myoglobin was Type III from horse 30 heart, the albumin was Fraction V bovine serum albumin. The -myoglobin, albumin and the substance P were also obtained from Sigma Chemical Co. The DNA fragments used in Example 4 were a mixture used as Low Range Size Standards supplied by Bio-Rad Laboratories, Hercules, California, and included fragments containing 88, 222, 249, 279, 303, 634~ 800, 1434 and 1746 base pairs. -~
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This example demonstrates the effect of polyethyl-ene glycol (PEG) as a solute in an electrophoretic medium used for separating myoglobin and substance P. The first part of this example serves as a control test performed in the absence of the PEG, while the second shows the effect which the PEG has on the component separation.
A. Electrophoresis without sieve-Pr motina polvmer.
A sample solution was prepared by dissolving substance P and myoglobin in 10 mM pH 2.5 phosphate buffer to achieve concentrations of 100 ~g/mL of substance P and 50 ~g/mL of myoglobin. A 3-~L sample of the solution was loaded electrophoretically on a coated 20 cm x 25 ~
capillary cartridge filled with the buffer solution.
Electrophoresis was performed on the loaded sample by applying a potential of 8000 V across the capillary, with detection at 200 nm with a sensitivity range of 0.02 AUF.
The myoglobin eluted at a retention time of 2.8 minutes, and the substance P at 3.3 minutes. Note that the myoglobin migrated through the capillary faster than the substance P.
B. Electrophoresis in presence of PEG.
The experiment of Part A was repeated, the only difference being that the buffer solution in the capillary further contained PEG with an average molecular weight of approximately 35,000 at a concentration of 5 weight percent.
The sample components eluted in a reversed elution order and with lengtheneid retention times. The substance P
eluted first at 4.9 minutes, followed by the myoglobin at 5.9 minutes.
This example illustrates the use of PEG in the electrophoretic separation of the monomer, dimer and trimer 9 20~9855 of albumin. The first part of this example i5 a control test, while the second includes the use of PEG.
A. Electrophoresis without sieve-promotinq polymer.
A sample was prepared by dissolving albumin as a mixture of the monomer, dimer and trimer in 10 mM pH 2.5 phosphate buffer to a total albumin concentration of 100 ~g/mL. The sample was loaded, run and detected on the same column using the same conditions as in Example lA. The result was a single sharp peak at a retention time of 3.5 minutes.
B. Electrophoresis in presence of_PEG.
The experiment of Part A was repeated with the lS inclusion of PEG with an average molecular weight of approximately 35,000 at a concentration of 5 weight percent in the column solution. Monomer, dimer and trimer formed separate peaks at retention times of 5.6 minutes, 6.8 minutes and 7.7 minutes, respectively.
This example illustrates several conditions where water-soluble polymers were in the buffer solution but incomplete or no separation occurred due to insufficient amvunts of polymer or due to polymer chains of insufficient length.
A. Albumin with 2~ PEG of molecular weiqht 35.000.
The experiment of Example 2B was repeated, using 2% of the PEG rather than 5%. The monomer was detected at a retention time of 3.7 minutes, and the dimer at 4.0 minutes, with the trimer peak not distinguishable. The monomer and ;; dimer peaks overlapped.
Z0~9855 B. Albumin with 5~ PEG of molecular we~ght 6C00.
The experiment of Example 2B was repeated, using PEG of an average molecular weight of 6000 rather than 35,000. All of the albumin components passed the detector as a single peak at a retention time of 4.3 minutes, with no separation a~ong the three.
C. Albumin and myoalobin/ _bstance P with 0.2~ 4000-centi~oise hvdroxyDropy~ yl_cell_lose.
A solution was prepared by dissolving 4000-centipoise hydroxypropyl methyl cellulose in 0.1 M pH 2.5 phosphate buffer at 0.25 weight percent. The experiment of Example 2B (albumin sample) was then repeated, followed by the experiment of Example lB (sample containing myoglobin and substance P), using this hydroxypropyl methyl cellulose-containing buffer solution in place of the PEG-containing buffer solution in the capillary in each case.
The monomer, dimer and trimer components of albumin passed the detector together as a single peak at a retention time of 3.1 minutes. In the myoglobin/substance P
run, the myoglobin displayed a retention time of 3.1 minutes ~ and the substance P a retention time of 3.7 minutes, which 3 are essentially the same and in the same order as when no polymer was present in the buffer solution (EY~ample lA).
This result suggests that the sieve passages around the 0.25% hydroxypropyl methyl cellulose were too --large to have any effect on the protein, although they might well create a sieving effect with DNA.
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D- 10~ Ethylene alycol and 10/_glycerin.
As the monomer of PEG, ethylene glycol has similar hydrophilicity characteristics. This experiment demonstrates that, like PEG, ethylene glycol and its analog glycerin (1,2,3-propanetriol) both increase the solution viscosity, but neither produce the sieving effec~
attributable to PEG. Viscosity increases in themselves are therefore not responsible for the sieving effect.
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Separate solutions were prepared, one containing ethylene glycol and the other glycerin, both at 10% by weight in 0.1 M pH 2.5 aqueous phosphate buffer. A third solution, containing 0.05% of the 4000-centipoise hydroxy-propyl methyl cellulose solution in the same buffer, wasprepared for comparison. The quantity of the latter is the amount used in the prior art for suppressing bulk flow.
Using each of the three solutions, a sample con-taining the albumin and fragments of substance P ranging from 4 to 11 amino acids in size was subjected to electro-phoresis using the same operating column and conditions as in Example 2B. In the comparison run, the substance P
fragments separated into individual peaks, but the albumin components passed the detector as a sinyle peak at a reten-tion time of 2.5 minutes, showing no separation betweenmonomer, dimer and trimer. In the ethylene glycol run, all peaks passed the detector in the same order as in the con-trol run, again with no separation of the albumin components I into separate peaks. The albumin retention time was 3.4 i 20 minutes. In the glycerin run as well, all peaks passed the detector in the same order as in the control run, again with no separation of the albumin components into separate peaks.
The albumin retention time was 3.5 minutes.
This example illustrates the separation of DNA
fragments of differing lengths, using the Low Range DNA Size Standards of Bio-Rad Laboratories. The first part of this example is a control test, while the second includes the use 30 of hydroxypropyl methyl cellulose at a concentration high enough to cause a sieving effect.
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A. Electroph_r sis without sie~ romo in polymer.
A 3-~L sample of the mixture was electropho-3S retically loaded onto a 50 cm x 50 ~ capillary filled with a buffer solution made up of 0.089 M Tris-boric acid, 0.002 M
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20C~9855 ethylenediamine tetraacetic acid, and 0.1% sodium dodecyl sulfate, at a pH of 8Ø
Detection was done at 260 nm with a sensitivity range of 0.005 AUF. The DNA fragments in the sample passed 5 the detector in three overlapping peaks at retention time~
of about 5-7 minutes, indicating poor if any separation of the fragments.
8. Electrophoresis with hvdro~syprQpvl methyl cellulose in 10sieve-promotina amount.
The experiment of Part A of this example was repeated, the sole difference being that 0.5% of the 4000-centipoise hydroxypropyl methyl cellulose was additionally included in the buffer solution. The result this time was 15 that the nine sizes of DNA fragments were well separated, with the retention times listed in Table I:
TABLE I
ELECTROPHORESIS OF DNA FRAGMENTS
Retention Fraament size Time 88 base pairs 16.2 minutes 222 18.0 249 18.4 279 18.7 303 19.4 634 22.1 800 22.8 30 1434 24.0 1746 24.4 This example illustrates the separation of a 35 mixture of at least fifteen lengths of DNA fragments, the lengths differing by 123 base pairs, beginning with 246 base :: :
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pairs. Methyl cellulose was used to obtain the sieving effect.
A 3-~L sample of the mixture was electropho-retically loaded onto a 50 cm x 50 ~ capillary filled with a buffer solution made up of 0.089 M Tris-boric acid, 0.002 M
ethylenediamine tetraacetic acid, and 0.5% of the 4000-centipoise methyl cellulose solution at a pH of 8Ø
Detection was done at 260 nm with a sensitivity range of 0.005 AUF. The fragments passed the detector as separate peaks, with the retention times listed in Table II:
TABLE II
ELECTROPHORESIS OF DNA FRAGMENTS
Retention Fraament size Time 246 base pairs 20.3 minutes 369 22.2 492 23.8 20615 25.3 738 26.~ -861 27.2 984 27.7 1107 28.2 251230 28.5 1353 28.7 1476 28.9 1599 29.0 1722 29.2 301845 29.3 ,, :.
This example demonstrates the lack of effect of 1%
hydroxypropyl methyl cellulose on ths mobility of small ions, using a range of amounts of the polymer including amounts which are sieve-promoting in the preceding examples.
The solute in this case is sodium chloride.
'., , '.' The polymers used in this group of tests were 15-centipoise, 50-centipoise, 100-centipoise, and 4000 centipoise hydroxypropyl methyl celluloses (HMC's). Each type of HMC was dissolved at 1% by weight in both water and a 20 mM aqueous sodium chloride solution. The conductivities of the resulting solutions were then measured and compared as indications of the effect of the polymer on the mobility of the sodium and chloride ions. The results were as listed in Table III below.
TABLE III
CONDUCTIVITY OF NaCl SOLUTIONS
conductivity conductivity 15Type of HMCin waterin 20mM ~laCl used at 1% at 1%
~entipolse)(~ hos) _ ~Imhos~
---* 2.85 3900 ~ Control: no polymer present.
3 The lack of variability of the numbers in the right column indicates that the mobility of the sodium and chloride ions ~, is unchanged by the presence of the polymer.
~ 35 The foregoing is offered primarily for purposes of `. illustration. It will be readily apparent to those skilled in the art that modifications and variations in the pro-cedures, materials, quantities and operating conditions described above may be made without departing from the spirit and scope of the invention.
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It has now been discovered that sample ions, and particularly biomolecules, may be separated from each other on the basis of molecular size by electrophoresis through an aqueous solution of a non-crosslinked polymer of a selected molecular weight (or molecular weight range) and concentration. The molecular weight of the polymer will be selected as described below to correspond to the molecular weight range of the sample ions in a manner which will inhibit the migration of the sample ions through the solution to ~arying degrees. Macromolecular sample ions and other biological species may thus be separated from each other and from sample ions of lesser size without the use of a gel. The terms "macromolecule" and "macromolecular" are used herein to refer to species having molecular weights of at least about 10,000.
The polymers used herein are generally non-crosslinked polymers. Branched or linear polymers may be used, linear polymers being preferred for many applications.
In addition, the polymers may be neutral or charged, neutral being preferred in applications where charge interaction between the sample ions and the polymer is sought to be avoided.
Cellulose derivatives have been used in capillary electrophoresis for suppressing electroendosmosis and other types of bulk flow by increasing the viscosity of the buffer solution, and for preserving the capillary as well. The quantities used for this purpose are small, however, with no substantial tendency to detain the sample ions during their migration or to affect their separation. The present j 35 invention resides in the discovery that dissolved. linear polymers in general produce a molecular sieving effect when used in certain amounts, these amounts being generally ., , :, , : -, : , . -, ,:: . :: : - :.
higher than the amounts of the cellulose derivatives used for suppressing bulk flow.
Aqueous media with dissolved polymers in accordance with this invention may be used for biomolecular separations in general, although they are of particular utility in separations performed in capillary columns with high voltage, i.e., high performance electrophoresis. The use of polymers in this manner permits the separation of species which vary in molecular weight with insufficient or no variation in charge/mass ratio, and lends itself to easy preparation of the separation media and high reproducibility.
As in the known use of cellulose derivatives referred to above, the dissolved polymers further serve to suppress bulk flow due to their inherent increase in viscosity. Examples of bulk flow occurring spontaneously are electroendosmosis, hydrokinetic flows (due to hydrostatic heads), and convection. At these polymer level~, however, the decrease in sample ion mobility caused by the presence of the polymers varies both with the size and concentration of the polymer and the size of the sample ion, a feature which does not occur at the low levels at which the cellulose derivatives have been used or suppressing bulk flow.
Other features and advantages of the invention will be apparent from the description which follows.
The polyme~rs used in connection t~he present inven-tion must be water-soluble and, as stated above, are '~ preferably linear.
~¦ Selection of the polymer is optimally geared , toward the particular sample ions being separated. The j 35 molecular weight of the polymer is of primary in~erest in making this selection. In general, polymers varying widely in molecular weight may be used. Resolution of the sample .,.
' - ~:, ions will generally improve, however, as the polymer molecular weight approaches the range of the molecular weights of the sample ions. The best results are obtained with polymers having an average molecular weiyht which is between the lowest and highest molecular weights of the sample ions, and in particular witll polymers whose molecular weight range covers (i.e., is at least coextensive with) the molecular weight range of the sample ions. In preferred embodiments, the polymer has an average molecular weight lOwhich is from about 10,000 to about 2,000,000, and within about 0.1 to about 200 times, more preferably from about 0.2 to about 20 times, and most preferably from about 0.5 to about 2 times the average molecular weight of the sample ions.
15Within these parameters, the particular type of polymer may vary widely. For aqueous systems, examples of linear polymers which may be used are water-soluble cellulose derivatives and fully water-soluble polyalkylene glycols. Specific examples of such cellulose derivatives are sodium carboxymethyl cellulose, sodium carboxymethyl 2-hydroxyethyl cellulose, 2-hydroxyethyl cellulose, 2-hydroxypropyl cellulose, methyl cellulose, hydroxypropyl -methyl cellulose, hydroxyethyl methyl cellulose, hydroxybutyl methyl cellulose, and hydroxyethyl ethyl cellulose. Preferred cellulose derivatives are those with a highly hydrophilic character, and consequently high water solubility and minimal affinity to the sample ions. Methyl cellulose is particularly preferred. Celluloses are generally characterized in terms of the viscosity of aqueous solutions in which theyiare dissolved at specified concentrations and temperature. With this in mind, and depending on the size of the sample ions sought to be separated, the cellulose derivative may vary widely in terms of this viscosity characterization. For example, cellulose ¦ 35 derivatives may be used which are characterized as producing viscosities ranging from about 15 centipoise to about 17,000 centipoise when dissolved in water at 2 weight percent ....
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measured at 25C, although in the context of this invention they would be used at other concentrations. Polymers such as these are useful in separating polynucleotides with chains ranging from about 10 to about 10,000 base pairs.
Preferred cellulose derivatives for use in the present invention are those which would have viscosities of from about 1,000 to about 10,000 centipoise if prepared as 2%
aqueous solutions measured at 25C. It is to be understood that these viscosity characterizations are intended merely as an indication of the molecular weight of the polymer, and not of the actual viscosity when used in the context of the present invention.
Preferred polyalkylene glycols are polyethylene glycols having average molecular weights of at least about ' 15 10,000. Particularly preferred are polyethylene glycols ', with average molecular weights of at least about 20,000, most preferably at least about 30,000. As an example, mixtures of sample ions ranging in molecular weight from about 10,000 to about 100,000 may be separated with polyethylene glycols ranging in molecular weight from about 10,000 to about 100,000.
Examples of branched polymers which may be used in accordance with the invention are soluble starches and starch derivatives. A specific example is hydroxypropyl starch.
Mixtures of polymers in which varying molecular weights are purposely combined may also be used. This will be particularly useful in separating sample mixtures which have a wide range of molecular weights, thus providing separation over the entire range.
The use of charged polymers is an option which can provide a further separation parameter to the system. This will vary the interaction between the species and the polymer, and may thus be of use depending on the particular mixture Of species present in the sample, and the type Of separation sought among these species.
,.' The quantity of polymer to be dissolved in the resolving solution may vary widely, and will be any quantity which extends the retention time of the sample ions to such varying degrees that effective separation on the basis of molecular size is achieved. Clearly, this will vary with various parameters of the system, including for example the column configuration and length, the presence and effect of other factors influencing the separation such as charge and electrophoretic mobility, the molecular structure, intrinsic viscosity and interactive character of the polymer itself, and the range of and differences between the molecular weights of the sample ions. The degree to which the retention times for the sample ions should be extended for best results will vary with the sample composition and the polymer being used. For separations of macromolecular species, increases in retention time of at least about 25%, preferably at least about 35%, and most preferably at least about 50%, will provide the best results. For polyalkylene glycols, particularly polyethylene glycols, concentrations of at least about 2% by weight, preferably at least about 3%, and most preferably from about 3% to about 30% by weight will give the best results. For cellulose derivatives, preferred concentrations are at least about 0.1% by weight, with about 0.1% to about 30% by weight more preferred, and about 0.1% to about 10% by weight particularly preferred.
To conduct the separations in accordance with the present invention, operating conditions and procedures used in conventional electrophoretic separations, including appropriately selected buffer systems, may be used. The invention is of p~rticul!ar utility in high performance electrophoresis as performed in capillaries. Preferred capillaries are those having internal diameters of less than about 200 microns, more preferably less than about 100 microns, and most preferably about 25 microns to about 50 microns. Voltages of at least about 1000 volts.~re preferred, witll at least about 3000 volts particularly preferred.
The following examples are offered strictly for purposes of illustration, and are intended neither to define nor to limit the invention in any manner.
In each of these examples, electrophoresis was performed on an HPE-100 high performance electrophoresis instrument, a product of Bio-Rad Laboratories, Inc., Hercules, California. Capillary tubes of 20 cm length by 25 ~ inner diameter, and 50 cm length by 50 ~ inner diam-eter, coated with linear polyacrylamide as described in 10 Hjerten, U.S. Patent No. 4,680,201, issued July 14, 1987, were used. A conductivity bridge Model 31 from Yellow Springs Instrument Co., Yellow Springs, Ohio, was used, and detection was performed on-line in the capillary itself, by UV absorption. The hydroxypropyl methyl cellulose was obtained from Sigma Chemical Co., St. Louis, Missouri, in powder form, specified in terms of its viscosity when prepared as an aqueous solution at a concentration of 2% by weight and measured at 25C. The specified viscosities were 15, 50, 100 and 4000 centipoise, and the powdered polymer will be referred to herein for convenience as "15-centipoise," "50-centipoise," "100-centipoise," and "4000-centipoise hydroxypropyl methyl cellulose." The 4000-centipoise hydroxypropyl methyl cellulose was estimated to have an average molecular weight of approximately 900,000.
The methyl cellulose was also obtained from Sigma Chemical Co., similarly specified as producing a viscosity of 4000 centipoise in a 2% aqueous solution at 25C, and will be referred to herein in a manner similar to the hydroxypropyl methyl cellulose. The myoglobin was Type III from horse 30 heart, the albumin was Fraction V bovine serum albumin. The -myoglobin, albumin and the substance P were also obtained from Sigma Chemical Co. The DNA fragments used in Example 4 were a mixture used as Low Range Size Standards supplied by Bio-Rad Laboratories, Hercules, California, and included fragments containing 88, 222, 249, 279, 303, 634~ 800, 1434 and 1746 base pairs. -~
~: :
This example demonstrates the effect of polyethyl-ene glycol (PEG) as a solute in an electrophoretic medium used for separating myoglobin and substance P. The first part of this example serves as a control test performed in the absence of the PEG, while the second shows the effect which the PEG has on the component separation.
A. Electrophoresis without sieve-Pr motina polvmer.
A sample solution was prepared by dissolving substance P and myoglobin in 10 mM pH 2.5 phosphate buffer to achieve concentrations of 100 ~g/mL of substance P and 50 ~g/mL of myoglobin. A 3-~L sample of the solution was loaded electrophoretically on a coated 20 cm x 25 ~
capillary cartridge filled with the buffer solution.
Electrophoresis was performed on the loaded sample by applying a potential of 8000 V across the capillary, with detection at 200 nm with a sensitivity range of 0.02 AUF.
The myoglobin eluted at a retention time of 2.8 minutes, and the substance P at 3.3 minutes. Note that the myoglobin migrated through the capillary faster than the substance P.
B. Electrophoresis in presence of PEG.
The experiment of Part A was repeated, the only difference being that the buffer solution in the capillary further contained PEG with an average molecular weight of approximately 35,000 at a concentration of 5 weight percent.
The sample components eluted in a reversed elution order and with lengtheneid retention times. The substance P
eluted first at 4.9 minutes, followed by the myoglobin at 5.9 minutes.
This example illustrates the use of PEG in the electrophoretic separation of the monomer, dimer and trimer 9 20~9855 of albumin. The first part of this example i5 a control test, while the second includes the use of PEG.
A. Electrophoresis without sieve-promotinq polymer.
A sample was prepared by dissolving albumin as a mixture of the monomer, dimer and trimer in 10 mM pH 2.5 phosphate buffer to a total albumin concentration of 100 ~g/mL. The sample was loaded, run and detected on the same column using the same conditions as in Example lA. The result was a single sharp peak at a retention time of 3.5 minutes.
B. Electrophoresis in presence of_PEG.
The experiment of Part A was repeated with the lS inclusion of PEG with an average molecular weight of approximately 35,000 at a concentration of 5 weight percent in the column solution. Monomer, dimer and trimer formed separate peaks at retention times of 5.6 minutes, 6.8 minutes and 7.7 minutes, respectively.
This example illustrates several conditions where water-soluble polymers were in the buffer solution but incomplete or no separation occurred due to insufficient amvunts of polymer or due to polymer chains of insufficient length.
A. Albumin with 2~ PEG of molecular weiqht 35.000.
The experiment of Example 2B was repeated, using 2% of the PEG rather than 5%. The monomer was detected at a retention time of 3.7 minutes, and the dimer at 4.0 minutes, with the trimer peak not distinguishable. The monomer and ;; dimer peaks overlapped.
Z0~9855 B. Albumin with 5~ PEG of molecular we~ght 6C00.
The experiment of Example 2B was repeated, using PEG of an average molecular weight of 6000 rather than 35,000. All of the albumin components passed the detector as a single peak at a retention time of 4.3 minutes, with no separation a~ong the three.
C. Albumin and myoalobin/ _bstance P with 0.2~ 4000-centi~oise hvdroxyDropy~ yl_cell_lose.
A solution was prepared by dissolving 4000-centipoise hydroxypropyl methyl cellulose in 0.1 M pH 2.5 phosphate buffer at 0.25 weight percent. The experiment of Example 2B (albumin sample) was then repeated, followed by the experiment of Example lB (sample containing myoglobin and substance P), using this hydroxypropyl methyl cellulose-containing buffer solution in place of the PEG-containing buffer solution in the capillary in each case.
The monomer, dimer and trimer components of albumin passed the detector together as a single peak at a retention time of 3.1 minutes. In the myoglobin/substance P
run, the myoglobin displayed a retention time of 3.1 minutes ~ and the substance P a retention time of 3.7 minutes, which 3 are essentially the same and in the same order as when no polymer was present in the buffer solution (EY~ample lA).
This result suggests that the sieve passages around the 0.25% hydroxypropyl methyl cellulose were too --large to have any effect on the protein, although they might well create a sieving effect with DNA.
.j .
D- 10~ Ethylene alycol and 10/_glycerin.
As the monomer of PEG, ethylene glycol has similar hydrophilicity characteristics. This experiment demonstrates that, like PEG, ethylene glycol and its analog glycerin (1,2,3-propanetriol) both increase the solution viscosity, but neither produce the sieving effec~
attributable to PEG. Viscosity increases in themselves are therefore not responsible for the sieving effect.
.
;~
.q . ~ " ~ .~: .. ~ . , ~ .. . ~ i .... . .. ... .... ..
Separate solutions were prepared, one containing ethylene glycol and the other glycerin, both at 10% by weight in 0.1 M pH 2.5 aqueous phosphate buffer. A third solution, containing 0.05% of the 4000-centipoise hydroxy-propyl methyl cellulose solution in the same buffer, wasprepared for comparison. The quantity of the latter is the amount used in the prior art for suppressing bulk flow.
Using each of the three solutions, a sample con-taining the albumin and fragments of substance P ranging from 4 to 11 amino acids in size was subjected to electro-phoresis using the same operating column and conditions as in Example 2B. In the comparison run, the substance P
fragments separated into individual peaks, but the albumin components passed the detector as a sinyle peak at a reten-tion time of 2.5 minutes, showing no separation betweenmonomer, dimer and trimer. In the ethylene glycol run, all peaks passed the detector in the same order as in the con-trol run, again with no separation of the albumin components I into separate peaks. The albumin retention time was 3.4 i 20 minutes. In the glycerin run as well, all peaks passed the detector in the same order as in the control run, again with no separation of the albumin components into separate peaks.
The albumin retention time was 3.5 minutes.
This example illustrates the separation of DNA
fragments of differing lengths, using the Low Range DNA Size Standards of Bio-Rad Laboratories. The first part of this example is a control test, while the second includes the use 30 of hydroxypropyl methyl cellulose at a concentration high enough to cause a sieving effect.
: .
A. Electroph_r sis without sie~ romo in polymer.
A 3-~L sample of the mixture was electropho-3S retically loaded onto a 50 cm x 50 ~ capillary filled with a buffer solution made up of 0.089 M Tris-boric acid, 0.002 M
. '' , ~ ''i'; ' ': : ': ' ~ : :' ': ' ~ ~
20C~9855 ethylenediamine tetraacetic acid, and 0.1% sodium dodecyl sulfate, at a pH of 8Ø
Detection was done at 260 nm with a sensitivity range of 0.005 AUF. The DNA fragments in the sample passed 5 the detector in three overlapping peaks at retention time~
of about 5-7 minutes, indicating poor if any separation of the fragments.
8. Electrophoresis with hvdro~syprQpvl methyl cellulose in 10sieve-promotina amount.
The experiment of Part A of this example was repeated, the sole difference being that 0.5% of the 4000-centipoise hydroxypropyl methyl cellulose was additionally included in the buffer solution. The result this time was 15 that the nine sizes of DNA fragments were well separated, with the retention times listed in Table I:
TABLE I
ELECTROPHORESIS OF DNA FRAGMENTS
Retention Fraament size Time 88 base pairs 16.2 minutes 222 18.0 249 18.4 279 18.7 303 19.4 634 22.1 800 22.8 30 1434 24.0 1746 24.4 This example illustrates the separation of a 35 mixture of at least fifteen lengths of DNA fragments, the lengths differing by 123 base pairs, beginning with 246 base :: :
" . , ,, , . ,.. , ,,.. j . ~ , , ~, ... , :. - .
pairs. Methyl cellulose was used to obtain the sieving effect.
A 3-~L sample of the mixture was electropho-retically loaded onto a 50 cm x 50 ~ capillary filled with a buffer solution made up of 0.089 M Tris-boric acid, 0.002 M
ethylenediamine tetraacetic acid, and 0.5% of the 4000-centipoise methyl cellulose solution at a pH of 8Ø
Detection was done at 260 nm with a sensitivity range of 0.005 AUF. The fragments passed the detector as separate peaks, with the retention times listed in Table II:
TABLE II
ELECTROPHORESIS OF DNA FRAGMENTS
Retention Fraament size Time 246 base pairs 20.3 minutes 369 22.2 492 23.8 20615 25.3 738 26.~ -861 27.2 984 27.7 1107 28.2 251230 28.5 1353 28.7 1476 28.9 1599 29.0 1722 29.2 301845 29.3 ,, :.
This example demonstrates the lack of effect of 1%
hydroxypropyl methyl cellulose on ths mobility of small ions, using a range of amounts of the polymer including amounts which are sieve-promoting in the preceding examples.
The solute in this case is sodium chloride.
'., , '.' The polymers used in this group of tests were 15-centipoise, 50-centipoise, 100-centipoise, and 4000 centipoise hydroxypropyl methyl celluloses (HMC's). Each type of HMC was dissolved at 1% by weight in both water and a 20 mM aqueous sodium chloride solution. The conductivities of the resulting solutions were then measured and compared as indications of the effect of the polymer on the mobility of the sodium and chloride ions. The results were as listed in Table III below.
TABLE III
CONDUCTIVITY OF NaCl SOLUTIONS
conductivity conductivity 15Type of HMCin waterin 20mM ~laCl used at 1% at 1%
~entipolse)(~ hos) _ ~Imhos~
---* 2.85 3900 ~ Control: no polymer present.
3 The lack of variability of the numbers in the right column indicates that the mobility of the sodium and chloride ions ~, is unchanged by the presence of the polymer.
~ 35 The foregoing is offered primarily for purposes of `. illustration. It will be readily apparent to those skilled in the art that modifications and variations in the pro-cedures, materials, quantities and operating conditions described above may be made without departing from the spirit and scope of the invention.
, ~".,,~ ", . ~ , -" ',
Claims (27)
1. A method of separating a mixture of sample ions of varying molecular weights in a sample into components, said method comprising electrophoretically passing said sample through a separation column containing a gel-free aqueous solution of a substantially linear polymer having a molecular weight of about 10,000 to about
2,000,000, said molecular weight being within a range of about 0.1 to about 200 times the average molecular weight of said macromolecular species in said mixture, the concentration of said polymer in said solution being sufficient to retard the flow of said species through said separation column to degrees which vary with the molecular weights of said species.
2. A method in accordance with claim 1 in which said sample ions are macromolecular species, and said concentration of said polymer is sufficient to increase the retention time of said macromolecular species in said column by at least about 25%.
2. A method in accordance with claim 1 in which said sample ions are macromolecular species, and said concentration of said polymer is sufficient to increase the retention time of said macromolecular species in said column by at least about 25%.
3. A method in accordance with claim 1 in which said sample ions are macromolecular species, and said concentration of said polymer is sufficient to increase the retention time of said macromolecular species in said column by at least about 35%.
4. A method in accordance with claim 1 in which said sample ions are macromolecular species, and said concentration of said polymer is sufficient to increase the retention time of said macromolecular species in said column by at least about 50%.
5. A method in accordance with claim 1 in which said polymer has an average molecular weight which is between the lowest and highest molecular weights of said sample ions in said mixture.
6. A method in accordance with claim 1 in which said polymer has a molecular weight range which is at least coextensive with that of said sample ions.
7. A method in accordance with claim 1 in which said polymer has an average molecular weight which is within a range of about 0.2 to about 20 times the average of the lowest and highest molecular weights of said sample ions.
8. A method in accordance with claim 1 in which said polymer has an average molecular weight which is within a range of about 0.5 to about 2 times the average of the lowest and highest molecular weights of said sample ions.
9. A method in accordance with claim 1 in which said separation column is a capillary tube with an internal diameter of less than about 200 microns.
10. A method in accordance with claim 1 in which said separation column is a capillary tube with an internal diameter of less than about 100 microns.
11. A method in accordance with claim 1 in which said separation column is a capillary tube with an internal diameter of from about 25 microns to about 50 microns.
12. A method in accordance with claim 1 in which said separation column is a capillary tube with an internal diameter of less than about 100 microns, and the passing of said sample therethrough is achieved by applying a voltage of at least about 1000 volts across said capillary tube.
13. A method in accordance with claim 1 in which said separation column is a capillary tube with an internal diameter of less than about 100 microns, and the passing of said sample therethrough is achieved by applying a voltage of at least about 3000 volts across said capillary tube.
14. A method in accordance with claim 1 in which said polymer is a water-soluble polyalkylene glycol.
15. A method in accordance with claim 1 in which said polymer is a polyethylene glycol having an average molecular weight of at least about 10,000.
16. A method in accordance with claim 1 in which said polymer is a polyethylene glycol having an average molecular weight of at least about 20,000.
17. A method in accordance with claim 1 in which said polymer is a polyethylene glycol having an average molecular weight of at least about 30,000.
18. A method in accordance with claim 1 in which said polymer is a polyethylene glycol having an average molecular weight of at least about 20,000, and the concentration of said polymer in said solution is at least about 2% by weight.
19. A method in accordance with claim 1 in which said polymer is a polyethylene glycol having an average molecular weight of at least about 20,000, and the concentration of said polymer in said solution is at least about 3% by weight.
20. A method in accordance with claim 1 in which said polymer is a polyethylene glycol having an average molecular weight of at least about 30,000, and the concentration of said polymer in said solution is from about 3% to about 30% by weight.
21. A method of separating a mixture of macro-molecular species having molecular weights ranging from about 10,000 to about 100,000 in a sample, said method comprising electrophoretically passing said sample through a capillary column containing a gel-free aqueous solution of polyethylene glycol having a molecular weight ranging from about 10,000 to about 100,000, the concentration of said polyethylene glycol in said solution being from about 3% to about 10% by weight.
22. A method in accordance with claim l in which said polymer is a water-soluble cellulose derivative.
23. A method in accordance with claim 1 in which said polymer is a water-soluble cellulose derivative characterized in terms of the viscosity of a 2% aqueous solution thereof being within a range of about 15 centipoise to about 17,000 centipoise at 25°C.
24. A method in accordance with claim 1 in which said polymer is a water-soluble cellulose derivative characterized in terms of the viscosity of a 2% aqueous solution thereof being within a range of about 1,000 centipoise to about 10,000 centipoise at 25°C.
25. A method in accordance with claim 1 in which said polymer is a water-soluble cellulose derivative characterized in terms of the viscosity of a 2% aqueous solution thereof being within a range of about 1,000 centipoise to about 10,000 centipoise at 25°C, and the concentration of said polymer in said solution is at least about 0.1% by weight.
26. A method in accordance with claim 1 in which said polymer is a water-soluble cellulose derivative characterized in terms of the viscosity of a 2% aqueous solution thereof being within a range of about 1,000 centipoise to about 10,000 centipoise at 25°C, and the concentration of said polymer in said solution is from about 0.1% to about 10% by weight.
27. A method of separating a mixture of poly-nucleotide chains in a sample, said polynucleotide chains each containing from about 10 to about 10,000 base pairs, said method comprising electrophoretically passing said sample through a capillary column containing a gel-free aqueous solution of a substantially linear polymer selected from the group consisting of methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose, and hydroxybutyl methyl cellulose, said polymer characterized in terms of the viscosity of a 2% aqueous solution thereof being within a range of about 1,000 centipoise to about 10,000 centipoise at 25°C, and the concentration of said polymer in said solution is from about 0.1% to about 0.5% by weight.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002009855A CA2009855A1 (en) | 1990-02-12 | 1990-02-12 | Electrophoretic sieving in gel-free media with dissolved polymers |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002009855A CA2009855A1 (en) | 1990-02-12 | 1990-02-12 | Electrophoretic sieving in gel-free media with dissolved polymers |
| EP19900301508 EP0442177B1 (en) | 1990-02-13 | 1990-02-13 | Electrophoretic sieving in gel-free media with dissolved polymers |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2009855A1 true CA2009855A1 (en) | 1991-08-12 |
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ID=25673945
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002009855A Abandoned CA2009855A1 (en) | 1990-02-12 | 1990-02-12 | Electrophoretic sieving in gel-free media with dissolved polymers |
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| Country | Link |
|---|---|
| CA (1) | CA2009855A1 (en) |
-
1990
- 1990-02-12 CA CA002009855A patent/CA2009855A1/en not_active Abandoned
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