CA1213261A - Polyethylenimine bound chromatographic packing - Google Patents
Polyethylenimine bound chromatographic packingInfo
- Publication number
- CA1213261A CA1213261A CA000467924A CA467924A CA1213261A CA 1213261 A CA1213261 A CA 1213261A CA 000467924 A CA000467924 A CA 000467924A CA 467924 A CA467924 A CA 467924A CA 1213261 A CA1213261 A CA 1213261A
- Authority
- CA
- Canada
- Prior art keywords
- silica gel
- microns
- average
- pore size
- average particle
- 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.)
- Expired
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J39/00—Cation exchange; Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
- B01J39/26—Cation exchangers for chromatographic processes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3268—Macromolecular compounds
- B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J41/00—Anion exchange; Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
- B01J41/20—Anion exchangers for chromatographic processes
Abstract
POLYETHYLENIMINE BOUND CHROMATOGRAPHIC PACKING Abstract The reaction product of silica gel or controlled pore glass and polyethyleniminopropyl trimethoxy silane suitable for use as chromatographic column packing.
Description
I I
POLYETHYLENIMINE BOUND
C~ROMATOGRAPHIC PACKING
Brief Rescript_ n of Invention In accordance with the present invention, par-ticulate silica gel having an average particle die-meter of from about 3 to about 70 microns and an average pore size of from about 50 to about 1000 Angstrom units is reacted with po1yethylenimino-propel trimethoxy Solon (PEIPr--triMeO-silane~ having an average molecular weight of from about 400 to about 1800. The reaction product, non-crosslinked covalently bonded polyethy1eniminopropylsi1y1-silica gel ~PEI-PrSi-silica gel), is useful as column packing in liquid chromatography for the purification and separation of anions, serving as ion exchange media. For high-performance liquid chromatographic (HPLC) application, the PEI-PrSi-silica gel is useful in the separation and analysis of protein mixtures.
The subject PEI-PrSi-silica gel may also be converted into a ~arboxylated form suitable or the purification and Separation of cat ionic proteins.
Similarly useful products are also obtained from the initial interaction of particulate controlled pore glass having an average particle diameter of from about 37 to 177 microns and an average pore size of from about 40 to about 1000 Angstrom units with the PEIPr-triMeO-silane.
32~
Prior Art Alert and Regnier in J. ChromatogrO t85, 375-392 (1979) have shown that polyethylene mine (POW may be adsorbed to silica surfaces, thereby providing sufficient primary and secondary amino groups on I, adjacent adsorbed PHI molecules to be cross linked by multi functional oxirane~ into a polymeric layer.
Recently, the separation of synthetic oligonucleo-tides using high-performance liquid chromatography (HPLC) with columns of micro particulate silica coated with cross linked polyethylene mine has been reported in the literature by TUG. Lawson et at., Anal. Boo-3 chum. t33, 85-93 (1983). In contrast, the present invention provides a porous silica or glass support to which a non-crosslinked polyethyleniminopropyl Solon is covalently banded, rather than being , adsorbed thereon Detailed Descry Sheehan of Invention The non-crosslinked covalently bound PHI silica gel and glass products of the present invention are conveniently prepared in accordance with the follow-in steps:
A reacting either particulate silica gel having an average particle diameter of from about 3 to about 70 microns and an average pore size of from ! about 50 to about Tao Angstrom units, or paretic-slate controlled pore glass having an average Jo particle diameter of from about 37 to 177 microns and an average pore size of from about 40 to : -2-Jo about 1000 Angstroms, in an inert organic solvent slurry with a lower alkanolic solution of polyp ethyleniminopropyl trimethoxy Solon having an average molecular weight of from about 400 to about 1800, said reaction being conducted at am-, blent to refluxing temperature for about 2 to about 50 hours;
By recovering the resultant solid fraction from the reaction mixture; and C. heating said solid fraction at a temperature and for a time sufficient to dry and completely bond the Solon to the respective silica gel or con-trolled pore glass.
As used herein, the term "covalently bound" or "covalently bonded" mean that the PI moieties are covalently attached to the silica gel or controlled pore glass by way of chemical interaction resulting in a propyl~si~yl (Prows) linkage, and the term "non-cross linked" means that the amino and amino groups on adjacent covalently bound PHI moieties are not cross-inked, or reacted with a cross linking agent, to form a polymeric layer.
, Without being bound thereby it is believed that the reaction proceeds to completion in two steps as follows:
Step 1: Silica hydroxy1s and the methoxy groups on the Solon react to arm Swiss bonds and , _ lo ,, free methanol, with some residual methoxy groups remaining reacted OH Moo \ Prop i-O Prop ' I + I Jo Six f i- OH Moo Owe i-O Owe : Step I: Completion of the reaction with the residual methoxy groups is effected during heat cur-in by a) and b):
: a) O\ prop O\
¦ I Prop : it Owe -home 15 icky\ Rome I / \ I " ' -Prop it Prop it b) it prop it I -Mesh ; / \ heat i-O - Si-Pr-PEl i-O Owe ion i-O
Silica gel, consisting of amorphous silica, is commercially available in irregular and spherical ; - (preferred) particulate forms and in several common-`
coal grades with mesh sizes ranging from 3 through 325 (ASTM)~. Rather than relying upon a numerical ~2~3~
indication of mesh size however, more accurate India ala for purposes of this invention are the average diameter and average pore size of the silica gel par-tickles respectively, from about 3 to about 70 microns and from about 50 to about 1000, preferably 250-500, Angstrom units For end product use in packing PLUCK chromatographic columns, a silica gel starting material of from about 3 to about 10 microns is preferred, and, for packing low pressure cremate-graphic columns, from about 40 to about 70 microns is , preferred.
:-' Controlled pore glass (COG), which is a silicate containing support material chemically simian to silica for use in liquid chromatography, is common-isle available, for example, from the Pierce Comma-Jo eel Co., Rock ford, Illinois, with average particle diameter of 37-177 microns and average pore size of 40-1000 Angstroms, preferably 40-500 Angstroms . Among the inert organic solvents suitable for preparing the silica gel or COG slurry are aliphatic hydrocarbons such as, for example, hexane, Hutton and the like; aromatic hydrocarbons such as, for example, Bunsen, Tulane, zillion and the like; lower alkanols such as, for example, ethanol, isopropanol, buttonhole and the like; chlorinated methanes such as, for example, methane chloride, chloroform, carbon tetrachloride and the like (Caution: such sheller i solvents may react at higher temperatures!); and such Jo other inert solvents as tetrahydrofuran, glum, diglyme and the like. In general a 1:5 ratio of silica gel or COG in grams to solvent in milliliters I
affords a suitable slurry. Due to the fine, insole ruble nature of the particulate silica gel and COG, a slurry rather than a true solution is obtained.
Polyethyleniminopropyl trimethoxy Solon, also known as (N-trimethoxysi1y1propyl)-polyethylenimine~
is the reaction product of po1yethylenimine and aminopropyltrimethoxy Solon and is described by the following formula:
me MeO-~i-CH~CH2CE12-NH- CH2CH2NH)n -SHOESHINE 2 ( I ) Me wherein, for purposes of this invention, n is an integer from about 4 to about 37, or, if expressed in terms of average molecular weight, from about 400 to about 1B00.
The Solon I) is used in the reaction with the silica gel or COG in the form of a lower C1-C6 Balkan Olin solution using sufficient alXanol to syllables the Solon. A fifty percent w/w isopropanolic Scholl-lion is preferred. In general, about 25-100 grams of the Sweeney, or, alternatively, about 50-200 ml of a fifty percent w/w alkanolic solution of the Solon, is used to react with each 100 grams silica gel or COG. The reaction may be conducted at ambient them-portray although elevated temperatures up to the refluxing temperature of the reaction solvent system may be utilized to enhance the rate of reaction The reaction proceeds readily to substantial completion (Step 1) within 2-50 hours. Stirring during admix-3Q lure of the reactants is advantageously employed I
although the reaction thereafter may continue without further stirring. An hydrous conditions are not anti-eel, it having been found that the presence of a small amount of water, for example, about 0.1-1.0 ml per 50 ml of the slurry solvent, does not adversely affect the reaction The resultant solid fraction is recovered from the reaction mixture by conventional physical means, for example, filtration, centrif~gation, etc. In general, a filtering means sufficient to retain a particle size of 5 microns is suitable whereas eon--trifuging is suitable for a particle size of 3 j microns.
The recovered solid fraction is then heat cured at a temperature and for a time sufficient to dry and completely bond the Solon to the silica gel or COG
covalently. In general, from about 1-4 hours at about 40-120~C has keen found sufficient. The thus-obtained covalently bound, non-crosslinked final product pro-fireball contains from about 0.5 to about 3.8 percent nitrogen.
The thus-obtained weakly basic PEI-PrSi-silica gel or PEI-PrSi-CP~ products may be converted to a weakly acidic carboxylated form by conventional treat mint, for example, see So Gut et alp, Anal. Become.
128, 196-201 ~1983), with an appropriate dibasic acid android on an inert organic solvent Typical such androids include for example, succinic acid ashy-drive, glutaric cold android, dlglyco1ic acid ashy-drive and the like. Sufficient android is used to react with substantially all of the amino and amino :, functions on the PHI moiety. The number of carboxylic :, groups in the resultant succinoylated product, for example, may be determined by standard titration against suitable alkali. For purposes of this invent -I 5 lion, a carboxyl milliequivalent per gram of final product from about 0.3 to about 1.2 is preferred D
Accordingly, this invention provides a non-cross-linked polyethyleneimine (PHI) function cova1ently bound to silica gel or controlled pore glass by way of a propylsilyl (Prows) linkage. The subject Poppers-r silica gel or PEI-PrSi-CPG products constitute new and useful bonded phases for the purification and swooper-lion of anionic and, in the carboxylated form, cat ionic materials, e.g. proteins, oligonucleotides and other charged molecules by column chromatography I and are particularly suitable with modern HPLC incitory-mentation. The packing may be of various mesh sizes, for example, from about 50 to about 600 mesh. An j example of the methodology suitable for separations 1 20 operable herein is the same as previously reported in the literature for the adsorbed cross linked PEI-silica type of stationary phases, for example, the separation I-; of synthetic oligonucleotides as shown by TUG. Lawson et at., ibid.
, I EXAMPLE ?
A. To a slurry of 10 trams silica gel with aver-age particle diameter of 40 microns and average pore I size of 60 Angstroms, commercially available from J. T. Baker Chemical Co., Phillips burg, NJ, in irregu-far form as "Silica Gel #7024", in 50 ml Tulane is I
added with stirring 19.71 grams of a 50% w/w isopro-panolic solution of polyethy~eniminopropyl trimethoxy Sweeney having an average molecular weight of 400-600 (assume S00), commercially available from Tetrarch Systems Inc., Bristol, PA, as "(N-Trimethoxysilyl-propyl)-Po1yethylenimine PS076". The mixture is stirred at room temperature (about 25C~ for about 1 hr. 10 min. and then allowed to stand overnight (about 17 hours) without stirring. Stirring is again initiated for another S ho 40 min. at room tempera-lure and again the mixture is allowed to stand over-night. The mixture is next filtered over a medium frilled glass filter. The filtrate is washed with 50 m} Tulane twice and with 50 ml methanol twice to ensure removal of any excess Solon reactant and then oven dried at 80-85C for about 3 hr. 30 mint to yield about 12 gramslof the covalently bound PEI-silica gel product; about 3.9% N.
B. The procedure of Example I-A is repeated except that 1 ml water is added to the silica gel/
on Solon mixture. The yield of the PHI bonded silica gel product is about 13~3 trams; about 5.5% N.
A slurry of 20 grams silica gel with average particle diameter of 5.25 microns and average pore size of 330 Angstroms, commercially available from The Sop A Ray Tons Group, Hesperia, CA, as a spherical silica under the trademark "Vodka A", Catalog No 101T9B5, in 100 ml Tulane and 2 ml water is prepared and stirred for 10 minutes at room temperature. To this is added with stirring 39.4 grams of a 50% w/w isopropanolic solution of polyethyleniminopropyl in-methoxy Solon having an average molecular weight of 500 and the mixture is stirred for an additional 5 , 5 minutes. The mixture is then allowed to stand over-night at room temperature. The mixture is next film toned using a 1.0 micron filter funnel. The filtrate is washed with 50 ml Tulane twice and 50 ml methanol twice, then air dried on the funnel and finally oven dried at 80-85C for about 3 hr. 30 Min. to yield the PHI bonded silica gel product; about 2.85~ N.
A slurry of 20 grams of 230-400 mesh (ASTM) silica gel having an average particle diameter of 40-63 microns and an average pore size of 420 Angstroms, commercially available from E. Merck Reagents, Germ many, under the brand name "Fructose- 500", in 50 ml i methanol and 1 ml water is prepared and stirred for S
minutes at room temperature. A separate solution of 11.~ grams ox a 50~ w/w isopropanolic solution of polyethyleniminopropyl trimethoxy Solon having an average molecular weight of 1800 in JOG ml methanol is also prepared. The Solon solution is then added to the silica gel slurry over 5 minutes with stirring.
After addition is complete, stirring is discontinued and the mixture is allowed to stand at room tempera-lure for 50 hours. The mixture is next filtered over medium sized sistered glass. rho filtrate is washed with 3 x 50 ml methanol under vacuum and then oven I!
dried at 80-85C for about 4 hours to yield the PHI
bound silica gel product; about 1.1% N.
, . EXAMPLE 4 Thy following reaction mixtures are prepared in ' 5 accordance with the teachings of the preceding " examples:
Components A B C
Silica gel (5 microns, 330 Angstroms) 10 g 10 g 10 g Isopropanol 50 ml50 ml50 ml Water 0.5ml 0.25ml 0.1ml PEIPr-triMeO-silane (MOE) as 50~ w/w i-PrOH sown. 9.99 4.959 2 Each reaction mixture is stirred or 5 minutes at room temperature and then allowed to stand without ! stirring for 41 hr. on min. Each mixture is filtered, washed once with 50 ml isopropanol and twice with 50 I ml methanol. Each filtrate is oven dried at 80-85~C
'I for about 3 ho to min. to yield the respective PHI
j 20 bound silica gel products; A: 1~2% N; B: lo N; C:
0.9% N.
Example 5 . To a slurry of 10 grams silica gel with aver-! age particle diameter of 40 microns and average pore size of 50 Angstroms in 50 ml hexane is added 19.71 grams of a 50% w/w i Pro solution ox Popper trim Solon having an average molecular weight of 500~ The mixture is stirred or 5 minute at room temperature `
.
and then heated to reflex temperature for about 2 hours. The mixture is allowed to cool to room temper-azure, filtered and washed with 50 ml hexane twice and 50 ml methanol twice. The filtrate is then oven dried at 30-85 for about 3 hours to yield the PHI bound silica gel product.
B. The procedure of Example AYE is repeated ox-crept that an equal amount of controlled pore glass (125 microns, 240 Angstroms) is substituted for the silica gel used therein to yield the corresponding covalently bonded, non-crosslinked PEI-PrSi-CP~
product.
Example 6 The procedure of Example 2 is repeated except that 25 grams silica gel (5.25 microns; 330 Angstroms) in 125 ml Tulane and 2.5 ml water is reacted with 50 grams of the 50% wow i-PrOH solution of PEIPr-triMeO-Solon (Moe 500) to yield about 29.4 grams ox the PHI
bonded silica gel product. This product is then mixed I with 125 ml Tulane and 10 grams su~cinic android and the mixture rotated in an B0~C water bath for 2 hours. At the end of this time, 20 ml methanol is added and the mixture is filtered, The recovered succinoylated PHI bound silica vet product is success lively washed with 1 x 50 Tulane, 2 x SO ml methanol and 1 x 50 ml ethyl ether. The product is then dried at about 80C for about 48 minutes. Titration of the product against ON sodium hydroxide indicates a carboxyl milliequivalent of about owe per gram of product.
~32~.
f Example 7 A methanolic (25 ml) slurry of the product of ' Example 2 (3.6 grams) is pressure-packed into stain-.:~ less steel columns 250 x 4.6 mm. and the packed , 5 columns are equilibrated by pumping through 0.025M
j potassium phosphate buffer, pi I a: a flow rate of 1 ml/min. After equilibration these singular) columns are used for chromatographic analysis ox a protein solution containing commercial grade swept-I chrome C9 alpha1-acid glycopr~tein, ovalbumin, and beta-lactoglobulin. A 4-mg sample of thy protein solution containing 1 my of each protein it applied to the equilibrated column, and separation is achieved with a 20 min. linear gradient from 0.25 potassium phosphate, pi 6.8, to 0.50M potassium phosphate, pi 6.8l at a flow rate of 1 ml/min. The proteins chrome-to graph as sharp symmetrical peaks with retention -.
times of about 2.8 min. for cytochrome c, ZOO min. for alpha acid glycoprotein, 9.7 min. for oval`bumin, and a doublet at 10.5 and 11~0 min. for beta~lactoglobu-fin. All of the 280 no absorbing material of each protein analyzed separately are collected which per-mitt ~uantitation by comparing its absorbency to known standards of that protein Recovery is ~95% for each US protein.
It it Jo be understood that anionic protein soul-lions can be retained by the weakly basic type of columns herein described and can be separated by grad-tent elusion by varying either the ionic strength or pi of the mobile phase.
:
I
. `
, A methanolic (25 ml) slurry of the succinoylated product of Example 6 (3.6 grams) is pressure-packed into stainless steel columns 250 x 4.6 mm and the packed columns are equilibrated by pumping through 0.010M potassium phosphate buffer, pi 6.0, at a flow ; rate of 1 mL/min. after equilibration, these (sing-far) columns are used for chromatographic analysis of a protein solution containing commercial grade oval-cumin, cytochrome c, hemoglobin and lysozyme. A 4-mg sample of the protein solution containing 1 my of each protein is applied to the equilibrated column, and separation is achieved with a 20 min. linear gradient from O.01OM potassium phosphate, pi 6.0, to O.750M
lo 15 potassium phosphate, pi 6.0 at a foe rate of 1 my/
'I min. The proteit1s chromatography as sharp symmetrical peaks with retention times of about 3.0 min. for oval-cumin, l1.4 min. for hemoglobin, 13.5 min. for swept-I chrome c, and 18 min. for lysozyme. All of the 280 no absorbing material of each protein analyzed separately are collected which permits quantitation by comparing its absorbency to known standards of that protein.
Recovery is greater than 95% for each protein It is to be understood that cat ionic protein solutions can be retched by the weakly cat ionic type of columns herein described and can be separated by ¦ gradient elusion by varying either the ionic strength or pi of the mobile phase.
, '
POLYETHYLENIMINE BOUND
C~ROMATOGRAPHIC PACKING
Brief Rescript_ n of Invention In accordance with the present invention, par-ticulate silica gel having an average particle die-meter of from about 3 to about 70 microns and an average pore size of from about 50 to about 1000 Angstrom units is reacted with po1yethylenimino-propel trimethoxy Solon (PEIPr--triMeO-silane~ having an average molecular weight of from about 400 to about 1800. The reaction product, non-crosslinked covalently bonded polyethy1eniminopropylsi1y1-silica gel ~PEI-PrSi-silica gel), is useful as column packing in liquid chromatography for the purification and separation of anions, serving as ion exchange media. For high-performance liquid chromatographic (HPLC) application, the PEI-PrSi-silica gel is useful in the separation and analysis of protein mixtures.
The subject PEI-PrSi-silica gel may also be converted into a ~arboxylated form suitable or the purification and Separation of cat ionic proteins.
Similarly useful products are also obtained from the initial interaction of particulate controlled pore glass having an average particle diameter of from about 37 to 177 microns and an average pore size of from about 40 to about 1000 Angstrom units with the PEIPr-triMeO-silane.
32~
Prior Art Alert and Regnier in J. ChromatogrO t85, 375-392 (1979) have shown that polyethylene mine (POW may be adsorbed to silica surfaces, thereby providing sufficient primary and secondary amino groups on I, adjacent adsorbed PHI molecules to be cross linked by multi functional oxirane~ into a polymeric layer.
Recently, the separation of synthetic oligonucleo-tides using high-performance liquid chromatography (HPLC) with columns of micro particulate silica coated with cross linked polyethylene mine has been reported in the literature by TUG. Lawson et at., Anal. Boo-3 chum. t33, 85-93 (1983). In contrast, the present invention provides a porous silica or glass support to which a non-crosslinked polyethyleniminopropyl Solon is covalently banded, rather than being , adsorbed thereon Detailed Descry Sheehan of Invention The non-crosslinked covalently bound PHI silica gel and glass products of the present invention are conveniently prepared in accordance with the follow-in steps:
A reacting either particulate silica gel having an average particle diameter of from about 3 to about 70 microns and an average pore size of from ! about 50 to about Tao Angstrom units, or paretic-slate controlled pore glass having an average Jo particle diameter of from about 37 to 177 microns and an average pore size of from about 40 to : -2-Jo about 1000 Angstroms, in an inert organic solvent slurry with a lower alkanolic solution of polyp ethyleniminopropyl trimethoxy Solon having an average molecular weight of from about 400 to about 1800, said reaction being conducted at am-, blent to refluxing temperature for about 2 to about 50 hours;
By recovering the resultant solid fraction from the reaction mixture; and C. heating said solid fraction at a temperature and for a time sufficient to dry and completely bond the Solon to the respective silica gel or con-trolled pore glass.
As used herein, the term "covalently bound" or "covalently bonded" mean that the PI moieties are covalently attached to the silica gel or controlled pore glass by way of chemical interaction resulting in a propyl~si~yl (Prows) linkage, and the term "non-cross linked" means that the amino and amino groups on adjacent covalently bound PHI moieties are not cross-inked, or reacted with a cross linking agent, to form a polymeric layer.
, Without being bound thereby it is believed that the reaction proceeds to completion in two steps as follows:
Step 1: Silica hydroxy1s and the methoxy groups on the Solon react to arm Swiss bonds and , _ lo ,, free methanol, with some residual methoxy groups remaining reacted OH Moo \ Prop i-O Prop ' I + I Jo Six f i- OH Moo Owe i-O Owe : Step I: Completion of the reaction with the residual methoxy groups is effected during heat cur-in by a) and b):
: a) O\ prop O\
¦ I Prop : it Owe -home 15 icky\ Rome I / \ I " ' -Prop it Prop it b) it prop it I -Mesh ; / \ heat i-O - Si-Pr-PEl i-O Owe ion i-O
Silica gel, consisting of amorphous silica, is commercially available in irregular and spherical ; - (preferred) particulate forms and in several common-`
coal grades with mesh sizes ranging from 3 through 325 (ASTM)~. Rather than relying upon a numerical ~2~3~
indication of mesh size however, more accurate India ala for purposes of this invention are the average diameter and average pore size of the silica gel par-tickles respectively, from about 3 to about 70 microns and from about 50 to about 1000, preferably 250-500, Angstrom units For end product use in packing PLUCK chromatographic columns, a silica gel starting material of from about 3 to about 10 microns is preferred, and, for packing low pressure cremate-graphic columns, from about 40 to about 70 microns is , preferred.
:-' Controlled pore glass (COG), which is a silicate containing support material chemically simian to silica for use in liquid chromatography, is common-isle available, for example, from the Pierce Comma-Jo eel Co., Rock ford, Illinois, with average particle diameter of 37-177 microns and average pore size of 40-1000 Angstroms, preferably 40-500 Angstroms . Among the inert organic solvents suitable for preparing the silica gel or COG slurry are aliphatic hydrocarbons such as, for example, hexane, Hutton and the like; aromatic hydrocarbons such as, for example, Bunsen, Tulane, zillion and the like; lower alkanols such as, for example, ethanol, isopropanol, buttonhole and the like; chlorinated methanes such as, for example, methane chloride, chloroform, carbon tetrachloride and the like (Caution: such sheller i solvents may react at higher temperatures!); and such Jo other inert solvents as tetrahydrofuran, glum, diglyme and the like. In general a 1:5 ratio of silica gel or COG in grams to solvent in milliliters I
affords a suitable slurry. Due to the fine, insole ruble nature of the particulate silica gel and COG, a slurry rather than a true solution is obtained.
Polyethyleniminopropyl trimethoxy Solon, also known as (N-trimethoxysi1y1propyl)-polyethylenimine~
is the reaction product of po1yethylenimine and aminopropyltrimethoxy Solon and is described by the following formula:
me MeO-~i-CH~CH2CE12-NH- CH2CH2NH)n -SHOESHINE 2 ( I ) Me wherein, for purposes of this invention, n is an integer from about 4 to about 37, or, if expressed in terms of average molecular weight, from about 400 to about 1B00.
The Solon I) is used in the reaction with the silica gel or COG in the form of a lower C1-C6 Balkan Olin solution using sufficient alXanol to syllables the Solon. A fifty percent w/w isopropanolic Scholl-lion is preferred. In general, about 25-100 grams of the Sweeney, or, alternatively, about 50-200 ml of a fifty percent w/w alkanolic solution of the Solon, is used to react with each 100 grams silica gel or COG. The reaction may be conducted at ambient them-portray although elevated temperatures up to the refluxing temperature of the reaction solvent system may be utilized to enhance the rate of reaction The reaction proceeds readily to substantial completion (Step 1) within 2-50 hours. Stirring during admix-3Q lure of the reactants is advantageously employed I
although the reaction thereafter may continue without further stirring. An hydrous conditions are not anti-eel, it having been found that the presence of a small amount of water, for example, about 0.1-1.0 ml per 50 ml of the slurry solvent, does not adversely affect the reaction The resultant solid fraction is recovered from the reaction mixture by conventional physical means, for example, filtration, centrif~gation, etc. In general, a filtering means sufficient to retain a particle size of 5 microns is suitable whereas eon--trifuging is suitable for a particle size of 3 j microns.
The recovered solid fraction is then heat cured at a temperature and for a time sufficient to dry and completely bond the Solon to the silica gel or COG
covalently. In general, from about 1-4 hours at about 40-120~C has keen found sufficient. The thus-obtained covalently bound, non-crosslinked final product pro-fireball contains from about 0.5 to about 3.8 percent nitrogen.
The thus-obtained weakly basic PEI-PrSi-silica gel or PEI-PrSi-CP~ products may be converted to a weakly acidic carboxylated form by conventional treat mint, for example, see So Gut et alp, Anal. Become.
128, 196-201 ~1983), with an appropriate dibasic acid android on an inert organic solvent Typical such androids include for example, succinic acid ashy-drive, glutaric cold android, dlglyco1ic acid ashy-drive and the like. Sufficient android is used to react with substantially all of the amino and amino :, functions on the PHI moiety. The number of carboxylic :, groups in the resultant succinoylated product, for example, may be determined by standard titration against suitable alkali. For purposes of this invent -I 5 lion, a carboxyl milliequivalent per gram of final product from about 0.3 to about 1.2 is preferred D
Accordingly, this invention provides a non-cross-linked polyethyleneimine (PHI) function cova1ently bound to silica gel or controlled pore glass by way of a propylsilyl (Prows) linkage. The subject Poppers-r silica gel or PEI-PrSi-CPG products constitute new and useful bonded phases for the purification and swooper-lion of anionic and, in the carboxylated form, cat ionic materials, e.g. proteins, oligonucleotides and other charged molecules by column chromatography I and are particularly suitable with modern HPLC incitory-mentation. The packing may be of various mesh sizes, for example, from about 50 to about 600 mesh. An j example of the methodology suitable for separations 1 20 operable herein is the same as previously reported in the literature for the adsorbed cross linked PEI-silica type of stationary phases, for example, the separation I-; of synthetic oligonucleotides as shown by TUG. Lawson et at., ibid.
, I EXAMPLE ?
A. To a slurry of 10 trams silica gel with aver-age particle diameter of 40 microns and average pore I size of 60 Angstroms, commercially available from J. T. Baker Chemical Co., Phillips burg, NJ, in irregu-far form as "Silica Gel #7024", in 50 ml Tulane is I
added with stirring 19.71 grams of a 50% w/w isopro-panolic solution of polyethy~eniminopropyl trimethoxy Sweeney having an average molecular weight of 400-600 (assume S00), commercially available from Tetrarch Systems Inc., Bristol, PA, as "(N-Trimethoxysilyl-propyl)-Po1yethylenimine PS076". The mixture is stirred at room temperature (about 25C~ for about 1 hr. 10 min. and then allowed to stand overnight (about 17 hours) without stirring. Stirring is again initiated for another S ho 40 min. at room tempera-lure and again the mixture is allowed to stand over-night. The mixture is next filtered over a medium frilled glass filter. The filtrate is washed with 50 m} Tulane twice and with 50 ml methanol twice to ensure removal of any excess Solon reactant and then oven dried at 80-85C for about 3 hr. 30 mint to yield about 12 gramslof the covalently bound PEI-silica gel product; about 3.9% N.
B. The procedure of Example I-A is repeated except that 1 ml water is added to the silica gel/
on Solon mixture. The yield of the PHI bonded silica gel product is about 13~3 trams; about 5.5% N.
A slurry of 20 grams silica gel with average particle diameter of 5.25 microns and average pore size of 330 Angstroms, commercially available from The Sop A Ray Tons Group, Hesperia, CA, as a spherical silica under the trademark "Vodka A", Catalog No 101T9B5, in 100 ml Tulane and 2 ml water is prepared and stirred for 10 minutes at room temperature. To this is added with stirring 39.4 grams of a 50% w/w isopropanolic solution of polyethyleniminopropyl in-methoxy Solon having an average molecular weight of 500 and the mixture is stirred for an additional 5 , 5 minutes. The mixture is then allowed to stand over-night at room temperature. The mixture is next film toned using a 1.0 micron filter funnel. The filtrate is washed with 50 ml Tulane twice and 50 ml methanol twice, then air dried on the funnel and finally oven dried at 80-85C for about 3 hr. 30 Min. to yield the PHI bonded silica gel product; about 2.85~ N.
A slurry of 20 grams of 230-400 mesh (ASTM) silica gel having an average particle diameter of 40-63 microns and an average pore size of 420 Angstroms, commercially available from E. Merck Reagents, Germ many, under the brand name "Fructose- 500", in 50 ml i methanol and 1 ml water is prepared and stirred for S
minutes at room temperature. A separate solution of 11.~ grams ox a 50~ w/w isopropanolic solution of polyethyleniminopropyl trimethoxy Solon having an average molecular weight of 1800 in JOG ml methanol is also prepared. The Solon solution is then added to the silica gel slurry over 5 minutes with stirring.
After addition is complete, stirring is discontinued and the mixture is allowed to stand at room tempera-lure for 50 hours. The mixture is next filtered over medium sized sistered glass. rho filtrate is washed with 3 x 50 ml methanol under vacuum and then oven I!
dried at 80-85C for about 4 hours to yield the PHI
bound silica gel product; about 1.1% N.
, . EXAMPLE 4 Thy following reaction mixtures are prepared in ' 5 accordance with the teachings of the preceding " examples:
Components A B C
Silica gel (5 microns, 330 Angstroms) 10 g 10 g 10 g Isopropanol 50 ml50 ml50 ml Water 0.5ml 0.25ml 0.1ml PEIPr-triMeO-silane (MOE) as 50~ w/w i-PrOH sown. 9.99 4.959 2 Each reaction mixture is stirred or 5 minutes at room temperature and then allowed to stand without ! stirring for 41 hr. on min. Each mixture is filtered, washed once with 50 ml isopropanol and twice with 50 I ml methanol. Each filtrate is oven dried at 80-85~C
'I for about 3 ho to min. to yield the respective PHI
j 20 bound silica gel products; A: 1~2% N; B: lo N; C:
0.9% N.
Example 5 . To a slurry of 10 grams silica gel with aver-! age particle diameter of 40 microns and average pore size of 50 Angstroms in 50 ml hexane is added 19.71 grams of a 50% w/w i Pro solution ox Popper trim Solon having an average molecular weight of 500~ The mixture is stirred or 5 minute at room temperature `
.
and then heated to reflex temperature for about 2 hours. The mixture is allowed to cool to room temper-azure, filtered and washed with 50 ml hexane twice and 50 ml methanol twice. The filtrate is then oven dried at 30-85 for about 3 hours to yield the PHI bound silica gel product.
B. The procedure of Example AYE is repeated ox-crept that an equal amount of controlled pore glass (125 microns, 240 Angstroms) is substituted for the silica gel used therein to yield the corresponding covalently bonded, non-crosslinked PEI-PrSi-CP~
product.
Example 6 The procedure of Example 2 is repeated except that 25 grams silica gel (5.25 microns; 330 Angstroms) in 125 ml Tulane and 2.5 ml water is reacted with 50 grams of the 50% wow i-PrOH solution of PEIPr-triMeO-Solon (Moe 500) to yield about 29.4 grams ox the PHI
bonded silica gel product. This product is then mixed I with 125 ml Tulane and 10 grams su~cinic android and the mixture rotated in an B0~C water bath for 2 hours. At the end of this time, 20 ml methanol is added and the mixture is filtered, The recovered succinoylated PHI bound silica vet product is success lively washed with 1 x 50 Tulane, 2 x SO ml methanol and 1 x 50 ml ethyl ether. The product is then dried at about 80C for about 48 minutes. Titration of the product against ON sodium hydroxide indicates a carboxyl milliequivalent of about owe per gram of product.
~32~.
f Example 7 A methanolic (25 ml) slurry of the product of ' Example 2 (3.6 grams) is pressure-packed into stain-.:~ less steel columns 250 x 4.6 mm. and the packed , 5 columns are equilibrated by pumping through 0.025M
j potassium phosphate buffer, pi I a: a flow rate of 1 ml/min. After equilibration these singular) columns are used for chromatographic analysis ox a protein solution containing commercial grade swept-I chrome C9 alpha1-acid glycopr~tein, ovalbumin, and beta-lactoglobulin. A 4-mg sample of thy protein solution containing 1 my of each protein it applied to the equilibrated column, and separation is achieved with a 20 min. linear gradient from 0.25 potassium phosphate, pi 6.8, to 0.50M potassium phosphate, pi 6.8l at a flow rate of 1 ml/min. The proteins chrome-to graph as sharp symmetrical peaks with retention -.
times of about 2.8 min. for cytochrome c, ZOO min. for alpha acid glycoprotein, 9.7 min. for oval`bumin, and a doublet at 10.5 and 11~0 min. for beta~lactoglobu-fin. All of the 280 no absorbing material of each protein analyzed separately are collected which per-mitt ~uantitation by comparing its absorbency to known standards of that protein Recovery is ~95% for each US protein.
It it Jo be understood that anionic protein soul-lions can be retained by the weakly basic type of columns herein described and can be separated by grad-tent elusion by varying either the ionic strength or pi of the mobile phase.
:
I
. `
, A methanolic (25 ml) slurry of the succinoylated product of Example 6 (3.6 grams) is pressure-packed into stainless steel columns 250 x 4.6 mm and the packed columns are equilibrated by pumping through 0.010M potassium phosphate buffer, pi 6.0, at a flow ; rate of 1 mL/min. after equilibration, these (sing-far) columns are used for chromatographic analysis of a protein solution containing commercial grade oval-cumin, cytochrome c, hemoglobin and lysozyme. A 4-mg sample of the protein solution containing 1 my of each protein is applied to the equilibrated column, and separation is achieved with a 20 min. linear gradient from O.01OM potassium phosphate, pi 6.0, to O.750M
lo 15 potassium phosphate, pi 6.0 at a foe rate of 1 my/
'I min. The proteit1s chromatography as sharp symmetrical peaks with retention times of about 3.0 min. for oval-cumin, l1.4 min. for hemoglobin, 13.5 min. for swept-I chrome c, and 18 min. for lysozyme. All of the 280 no absorbing material of each protein analyzed separately are collected which permits quantitation by comparing its absorbency to known standards of that protein.
Recovery is greater than 95% for each protein It is to be understood that cat ionic protein solutions can be retched by the weakly cat ionic type of columns herein described and can be separated by ¦ gradient elusion by varying either the ionic strength or pi of the mobile phase.
, '
Claims (5)
1. The covalently bound, non-crosslinked poly-ethylenimine reaction product of particulate silica gel having an average particle diameter of from about 3 to about 70 microns and an average pore size of from about 50 to about 1000 Angstrom units, or particulate controlled pore glass having an average particle diam-eter of from about 37 to about 177 microns and an average pore size of from about 40 to about 1000 Angstrom units, with polyethyleniminopropyl trimethoxy silane having an average molecular weight of from about 400 to about 1800.
2. A covalently bound, non-crosslinked poly-ethylenimine, chromatographic column packing consisting essentially of the reaction product of particulate silica gel having an average particle diameter of from about 3 to about 70 microns and an average pore size of from about 50 to about 1000 Angstrom units with polyethyleniminopropyl trimethoxy silane having an average molecular weight of from about 400 to about 1800.
3. The packing of Claim 2 wherein the particu-late silica gel has an average particle diameter of from about 5 to about 40 microns and an average pore size of from about 50 to about 330 Angstrom units and the polyethyleniminopropyl trimethoxy silane has an average molecular weight of from about 400 to about 600.
4. The packing of Claim 2 wherein the particu-late silica gel has an average particle diameter of about 40-62 microns and an average pore size of about 420 Angstrom units and the polyethyleniminopropyl tri-methoxy silane has an average molecular weight of about 1000.
5. The reaction product of Claim 1 which is converted to a weakly acidic carboxylated product containing from about 0.3 to to about 1.2 carboxyl milliequivalent per gram by reaction with a dibasic acid anhydride in an inert organic solvent.
6. The reaction product of Claim 1 which is converted to a weakly acidic carboxylated product containinging about 0.8 carboxyl milliequivalent per gram by reaction with succinic acid anhydride in an inert organic solvent.
7. A method for preparing a covalently bound, non-crosslinked polyethylenimine chromatographic col-umn packing which comprises:
a. reacting particulate silica gel having an average particle diameter of from about 3 to about 70 microns and an average pore size of about 50 to about 1000 Angstrom units, or particulate controlled pore glass having an average particle diameter of from about 37 to about 177 microns and an average pore size of from about 40 to about 1000 Angstrom units, in an inert organic solvent slurry with a lower alkanolic solution of poly-ethyleniminopropyl trimethoxy silane having an average molecular weight of from about 400 to about 1800, said reaction being con-ducted at ambient to refluxing temperature for about 2 to about 50 hours;
b. recovering the resultant solid fraction from the reaction mixture; and c. heating said solid fraction at a temperature and for a time sufficient to dry and complet-ly bond the silane to the respective silica gel or controlled pore glass.
8. A method for preparing a covalently bound, non-crosslinked polyethylenimine chromatographic col-umn packing which comprises:
a. reacting particulate silica gel having an average particle diameter of from about 3 to about 70 microns and an average pore size of about 50 to about 1000 Angstrom units in an inert organic solvent slurry with a lower alkanolic solution of polyethyleniminopropyl trimethoxy silane having an average molecu-lar weight of from about 400 to about 1800, said reaction being conducted at ambient to refluxing temperature for about 2 to about 50 hours;
b. recovering the resultant solid fraction from the reaction mixture; and c. heating said solid fraction at a temperature and for a time sufficient to dry and complet-ly bond the silane to the silica gel.
9. The method of Claim 8 wherein heating step c is conducted at about 40-120°C for about 1-4 hours.
10. A method for preparing a covalently bound, non-crosslinked polyethylenimine chromatographic col-umn packing which comprises:
a. reacting particulate silica gel having an average particle diameter of from about 3 to about 70 microns and an average pore size of about 50 to about 1000 Angstrom units, or particulate controlled pore glass having an average particle diameter of from about 37 to about 177 microns and an average pore size of from about 40 to about 1000 Angstrom units, in an inert organic solvent slurry with a lower alkanolic solution of poly-ethyleniminopropyl trimethoxy silane having an average molecular weight of from about 400 to about 1800, said reaction being con-ducted at ambient to refluxing temperature for about 2 to about 50 hours;
b. recovering the resultant solid fraction from the reaction mixture;
c. heating said solid fraction at a temperature and for a time sufficient to dry and complet-ly bond the silane to the respective silica gel or controlled pure glass, and d. converting the resultant covalently bound, non-crosslinked product to a weakly acidic carbox-ylated product containing from about 0.3 to about 1.2 carboxyl milliequivalent per gram by reaction with a dibasic acid anhydride in an inert organic solvent.
11. A chromatographic column suitable for liquid chromatography packed with covalently bound, non-crosslinked polyethylenimine reaction product of par-ticulate silica gel having an average particle diam-eter of from about 3 to about 70 microns and an aver-age pore size of from about 50 to about 1000 Angstrom units, or particulate controlled pore glass having an average particle diameter of from about 37 to about 177 microns and an average pore size of from about 40 to about 1000 Angstrom units, with polyethylenimino-propyl trimethoxy silane having an average molecular weight of from about 400 to about 1800.
12. A chromatographic column suitable for liquid chromatography packed with covalently bound, non-crosslinked polyethylenimine reaction product of par-ticulate silica gel having an average particle diam-eter of from about 3 to about 70 microns and an aver-age pore size of from about 50 to about 1000 Angstrom units with polyethyleniminopropyl trimethoxy silane having an average molecular weight of from about 400 to about 1800.
13, A chromatographic column suitable for liquid chromatography packed with covalently bound, non-crosslinked polyethylenimine reaction product of par-ticulate silica gel having an average particle diam-eter of from about 5 to about 40 microns and an aver-age pore size of from about 50 to about 300 Angstrom units with polyethyleniminopropyl trimethoxy silane having an average molecular weight of from about 400 to about 600.
14. A chromatographic column suitable for liquid chromatography packed with covalently bound, non-crosslinked polyethylenimine reaction product of par-ticulate silica gel having an average particle diam-eter of about 40-62 microns and an average pore size of about 420 Angstrom units with polyethylenimino-propyl trimethoxy silane having an average molecular weight of about 1000.
15. A chromatographic column suitable for liquid chromatography packed with the carboxylated product of
5. The reaction product of Claim 1 which is converted to a weakly acidic carboxylated product containing from about 0.3 to to about 1.2 carboxyl milliequivalent per gram by reaction with a dibasic acid anhydride in an inert organic solvent.
6. The reaction product of Claim 1 which is converted to a weakly acidic carboxylated product containinging about 0.8 carboxyl milliequivalent per gram by reaction with succinic acid anhydride in an inert organic solvent.
7. A method for preparing a covalently bound, non-crosslinked polyethylenimine chromatographic col-umn packing which comprises:
a. reacting particulate silica gel having an average particle diameter of from about 3 to about 70 microns and an average pore size of about 50 to about 1000 Angstrom units, or particulate controlled pore glass having an average particle diameter of from about 37 to about 177 microns and an average pore size of from about 40 to about 1000 Angstrom units, in an inert organic solvent slurry with a lower alkanolic solution of poly-ethyleniminopropyl trimethoxy silane having an average molecular weight of from about 400 to about 1800, said reaction being con-ducted at ambient to refluxing temperature for about 2 to about 50 hours;
b. recovering the resultant solid fraction from the reaction mixture; and c. heating said solid fraction at a temperature and for a time sufficient to dry and complet-ly bond the silane to the respective silica gel or controlled pore glass.
8. A method for preparing a covalently bound, non-crosslinked polyethylenimine chromatographic col-umn packing which comprises:
a. reacting particulate silica gel having an average particle diameter of from about 3 to about 70 microns and an average pore size of about 50 to about 1000 Angstrom units in an inert organic solvent slurry with a lower alkanolic solution of polyethyleniminopropyl trimethoxy silane having an average molecu-lar weight of from about 400 to about 1800, said reaction being conducted at ambient to refluxing temperature for about 2 to about 50 hours;
b. recovering the resultant solid fraction from the reaction mixture; and c. heating said solid fraction at a temperature and for a time sufficient to dry and complet-ly bond the silane to the silica gel.
9. The method of Claim 8 wherein heating step c is conducted at about 40-120°C for about 1-4 hours.
10. A method for preparing a covalently bound, non-crosslinked polyethylenimine chromatographic col-umn packing which comprises:
a. reacting particulate silica gel having an average particle diameter of from about 3 to about 70 microns and an average pore size of about 50 to about 1000 Angstrom units, or particulate controlled pore glass having an average particle diameter of from about 37 to about 177 microns and an average pore size of from about 40 to about 1000 Angstrom units, in an inert organic solvent slurry with a lower alkanolic solution of poly-ethyleniminopropyl trimethoxy silane having an average molecular weight of from about 400 to about 1800, said reaction being con-ducted at ambient to refluxing temperature for about 2 to about 50 hours;
b. recovering the resultant solid fraction from the reaction mixture;
c. heating said solid fraction at a temperature and for a time sufficient to dry and complet-ly bond the silane to the respective silica gel or controlled pure glass, and d. converting the resultant covalently bound, non-crosslinked product to a weakly acidic carbox-ylated product containing from about 0.3 to about 1.2 carboxyl milliequivalent per gram by reaction with a dibasic acid anhydride in an inert organic solvent.
11. A chromatographic column suitable for liquid chromatography packed with covalently bound, non-crosslinked polyethylenimine reaction product of par-ticulate silica gel having an average particle diam-eter of from about 3 to about 70 microns and an aver-age pore size of from about 50 to about 1000 Angstrom units, or particulate controlled pore glass having an average particle diameter of from about 37 to about 177 microns and an average pore size of from about 40 to about 1000 Angstrom units, with polyethylenimino-propyl trimethoxy silane having an average molecular weight of from about 400 to about 1800.
12. A chromatographic column suitable for liquid chromatography packed with covalently bound, non-crosslinked polyethylenimine reaction product of par-ticulate silica gel having an average particle diam-eter of from about 3 to about 70 microns and an aver-age pore size of from about 50 to about 1000 Angstrom units with polyethyleniminopropyl trimethoxy silane having an average molecular weight of from about 400 to about 1800.
13, A chromatographic column suitable for liquid chromatography packed with covalently bound, non-crosslinked polyethylenimine reaction product of par-ticulate silica gel having an average particle diam-eter of from about 5 to about 40 microns and an aver-age pore size of from about 50 to about 300 Angstrom units with polyethyleniminopropyl trimethoxy silane having an average molecular weight of from about 400 to about 600.
14. A chromatographic column suitable for liquid chromatography packed with covalently bound, non-crosslinked polyethylenimine reaction product of par-ticulate silica gel having an average particle diam-eter of about 40-62 microns and an average pore size of about 420 Angstrom units with polyethylenimino-propyl trimethoxy silane having an average molecular weight of about 1000.
15. A chromatographic column suitable for liquid chromatography packed with the carboxylated product of
Claim 5.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US06/555,368 US4540486A (en) | 1983-11-25 | 1983-11-25 | Polyethylenimine bound chromatographic packing |
| US555,368 | 1983-11-25 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1213261A true CA1213261A (en) | 1986-10-28 |
Family
ID=24217004
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000467924A Expired CA1213261A (en) | 1983-11-25 | 1984-11-15 | Polyethylenimine bound chromatographic packing |
Country Status (12)
| Country | Link |
|---|---|
| US (1) | US4540486A (en) |
| EP (1) | EP0143423B1 (en) |
| JP (1) | JPS60135761A (en) |
| KR (1) | KR890000825B1 (en) |
| AT (1) | ATE53307T1 (en) |
| AU (1) | AU565046B2 (en) |
| CA (1) | CA1213261A (en) |
| DE (1) | DE3482414D1 (en) |
| IE (1) | IE57720B1 (en) |
| IL (1) | IL73496A (en) |
| NZ (1) | NZ210126A (en) |
| ZA (1) | ZA848886B (en) |
Families Citing this family (56)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5110784A (en) * | 1984-04-09 | 1992-05-05 | Dow Corning Corporation | Dual surface porous material |
| US4606825A (en) * | 1985-04-22 | 1986-08-19 | J. T. Baker Chemical Company | Purification of immunoglobulin G |
| US4680120A (en) * | 1985-05-07 | 1987-07-14 | J. T. Baker Chemical Company | Bonded phase of silica and carboalkoxyalkyl silanes for solid phase extraction |
| US4640909A (en) * | 1985-05-07 | 1987-02-03 | J. T. Baker Chemical Company | Bonded phase of silica and carboalkoxyalkyl silanes for solid phase extraction |
| ATE78499T1 (en) * | 1985-05-28 | 1992-08-15 | Pcr Group Inc | POLYMERS MODIFIED BY SILANE. |
| US4680121A (en) * | 1986-02-20 | 1987-07-14 | J. T. Baker Chemical Company | Bonded phase of silica for solid phase extraction |
| US4650784A (en) * | 1986-02-20 | 1987-03-17 | J. T. Baker Chemical Company | Bonded phase of silica for solid phase extraction |
| US4661248A (en) * | 1986-03-06 | 1987-04-28 | J. T. Baker Chemical Company | Sulfonic derivatives of acylated polyethyleneimine bonded phase silica products |
| US4721573A (en) * | 1986-03-06 | 1988-01-26 | J. T. Baker Chemical Company | Use of sulfonic derivatives of acylated polyethyleneimine bonded phase silica products |
| US5066395A (en) * | 1986-03-06 | 1991-11-19 | J. T. Baker Inc. | N-acylated derivatives of polyethyleneimine bonded phase silica products |
| JPS62225244A (en) * | 1986-03-27 | 1987-10-03 | Nippon Shokubai Kagaku Kogyo Co Ltd | Adsorbent |
| US4804686A (en) * | 1986-05-13 | 1989-02-14 | Purdue Research Foundation | Cation-exchange support materials and method |
| US6251278B1 (en) * | 1987-06-08 | 2001-06-26 | Chromatochem, Inc. | Process for separating a substance from a mixture |
| US5240602A (en) * | 1987-06-08 | 1993-08-31 | Chromatochem, Inc. | Chromatographic material |
| US4855054A (en) * | 1987-06-17 | 1989-08-08 | Dow Corning Corporation | Using liquid chromatography dual zone packing materials |
| US4941974A (en) * | 1987-06-17 | 1990-07-17 | Dow Corning Corporation | Method of making liquid chromatography dual zone packing materials |
| US4897197A (en) * | 1987-06-17 | 1990-01-30 | Dow Corning Corporation | Using liquid chromatography packing materials |
| US4778600A (en) * | 1987-06-17 | 1988-10-18 | Dow Corning Corporation | Liquid chromatography dual zone packing materials |
| US4959340A (en) * | 1987-06-17 | 1990-09-25 | Dow Corning Corporation | Method of making liquid chromatography packing materials |
| US4773994A (en) * | 1987-06-17 | 1988-09-27 | Dow Corning Corporation | Liquid chromatography packing materials |
| US4929560A (en) * | 1988-02-03 | 1990-05-29 | Damon Biotech, Inc. | Recovery of tissue plasminogen activator |
| US4920051A (en) * | 1988-02-03 | 1990-04-24 | Damon Biotech, Inc. | Recovery of urokinase compounds |
| US4950634A (en) * | 1988-02-11 | 1990-08-21 | Dow Corning Corporation | Method for producing dual zone materials by use of an organosilane mixture |
| US4950635A (en) * | 1988-02-11 | 1990-08-21 | Dow Corning Corporation | Method for producing dual zone materials by catalyzed halosilylation |
| EP0386926A3 (en) * | 1989-03-02 | 1991-12-18 | Supelco, Inc. | Silica gel supports suitable for chromatographic separations |
| US5085779A (en) * | 1989-06-07 | 1992-02-04 | J. T. Baker, Inc. | Polyethyleneimine matrixes for affinity chromatography |
| US5092992A (en) * | 1989-06-07 | 1992-03-03 | J. T. Baker Inc. | Polyethyleneimine matrixes for affinity chromatography |
| CA1332598C (en) * | 1989-06-20 | 1994-10-18 | Laura J. Crane | Polyethyleneimine matrixes for affinity chromatography |
| US5087359A (en) * | 1989-08-07 | 1992-02-11 | J. T. Baker Inc. | Quaternized PEI silica solid supports for chromatography |
| EP0416748B1 (en) * | 1989-08-07 | 1994-03-30 | J.T. Baker Inc. | Quaternised pei silica solid supports for chromatography |
| US5164427A (en) * | 1989-10-12 | 1992-11-17 | Macherey, Nagel & Co. | Column-packing material for gel-permation chromatography method for preparation, and applications |
| DE3934068A1 (en) * | 1989-10-12 | 1991-04-18 | Macherey Nagel & Co Chem | POROESE PARTICLES FILLED WITH POLYMER GELS FOR GELPERMEATION CHROMATOGRAPHY AND METHOD FOR THE PRODUCTION THEREOF |
| WO1992011933A1 (en) * | 1991-01-04 | 1992-07-23 | Perseptive Biosystems, Inc. | Sulfonamide bonded hydrophilic coatings |
| EP0534316A1 (en) * | 1991-09-21 | 1993-03-31 | Hoechst Aktiengesellschaft | Use of alkylated polyethylenimines to concentrate bile acids |
| US5190660A (en) * | 1992-06-04 | 1993-03-02 | Lindoy Leonard F | Ion complexation by silica-immobilized polyethyleneimines |
| US5695882A (en) * | 1995-08-17 | 1997-12-09 | The University Of Montana | System for extracting soluble heavy metals from liquid solutions |
| US5869152A (en) * | 1996-03-01 | 1999-02-09 | The Research Foundation Of State University Of New York | Silica materials |
| US6987079B2 (en) * | 2001-08-14 | 2006-01-17 | W.R. Grace & Co.-Conn. | Supported catalyst systems |
| US6802966B2 (en) | 2001-08-14 | 2004-10-12 | W. R. Grace & Co. Conn. | Solid compositions for selective adsorption from complex mixtures |
| AU2008201441B2 (en) * | 2002-07-22 | 2010-08-12 | Cytec Technology Corp. | Method of preventing or reducing aluminosilicate scale in a Bayer process |
| CA2492450A1 (en) * | 2002-07-26 | 2004-02-05 | Applera Corporation | Microfluidic device including purification column with excess diluent, and method |
| US6923895B2 (en) * | 2002-09-09 | 2005-08-02 | Beckman Coulter, Inc. | Coated capillary electrophoresis tubes and system |
| KR100528959B1 (en) * | 2003-02-11 | 2005-11-16 | 학교법인 포항공과대학교 | Silica gel bonded with cucurbiturils |
| US7964380B2 (en) * | 2005-01-21 | 2011-06-21 | Argylia Technologies | Nanoparticles for manipulation of biopolymers and methods of thereof |
| US20090208919A1 (en) * | 2005-01-21 | 2009-08-20 | Argylla Technologies, Llp | Particle matrix for storage of biomolecules |
| US8288169B2 (en) * | 2005-01-21 | 2012-10-16 | Argylla Technologies | Surface mediated self-assembly of nanoparticles |
| MY144940A (en) * | 2005-01-25 | 2011-11-30 | Avantor Performance Mat Inc | Chromatographic media |
| DE102005023107A1 (en) * | 2005-05-13 | 2006-11-16 | Basf Ag | Modified polyamines |
| US7999065B2 (en) | 2006-10-13 | 2011-08-16 | Cytec Technology Corp. | Hydrophobically modified polyamine scale inhibitors |
| JP5643642B2 (en) * | 2007-07-25 | 2014-12-17 | ジーイー・ヘルスケア・バイオ−サイエンシズ・アーベー | Separation matrix |
| JP5284893B2 (en) * | 2009-06-29 | 2013-09-11 | 富士シリシア化学株式会社 | Separation method of hydrophilic organic compound and filler for hydrophilic interaction chromatography |
| RU2015114330A (en) | 2012-09-17 | 2016-11-10 | У.Р. Грейс Энд Ко.-Конн. | CHROMATOGRAPHIC MEDIA AND DEVICES |
| AU2014245532B2 (en) * | 2013-03-25 | 2016-11-10 | National Institute Of Advanced Industrial Science And Technology | Adsorbent for rare earth element and method for recovering rare earth element |
| ES2929099T3 (en) | 2014-05-02 | 2022-11-24 | Grace W R & Co | Functionalized support material and methods of manufacturing and use of functionalized support material |
| JP2016006410A (en) * | 2014-05-27 | 2016-01-14 | Jnc株式会社 | Chromatography carrier and protein purification method using the same |
| JP2018517559A (en) | 2015-06-05 | 2018-07-05 | ダブリュー・アール・グレース・アンド・カンパニー−コーンＷ Ｒ Ｇｒａｃｅ ＆ Ｃｏ−Ｃｏｎｎ | Adsorbing bioprocess clarifier and method for producing and using the same |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3722181A (en) * | 1970-05-22 | 1973-03-27 | Du Pont | Chromatographic packing with chemically bonded organic stationary phases |
| US4212905A (en) * | 1976-06-30 | 1980-07-15 | Board of Reagents, for and on behalf of the University of Florida | Method of coating supports using addition copolymers of aminimides |
| US4290892A (en) * | 1978-10-23 | 1981-09-22 | Varian Associates, Inc. | Anion exchange chromatographic separation of polyfunctional compounds |
| US4245005A (en) * | 1979-02-28 | 1981-01-13 | Purdue Research Foundation | Pellicular coated support and method |
| US4340496A (en) * | 1979-03-02 | 1982-07-20 | Varian Associates, Inc. | Anion exchange chromatographic composition for separation of polyfunctional compounds |
| ZA801718B (en) * | 1979-03-27 | 1981-10-28 | British Petroleum Co | Functionalised inorganic oxide products and their use in the removal of heavy metals,transistion metals and actinidemetals from solution |
| US4384954A (en) * | 1980-04-16 | 1983-05-24 | Kuraray Co., Ltd. | Column for adsorption of blood proteins |
| GB2097280B (en) * | 1981-04-27 | 1984-11-07 | Public Helath Lab Service Boar | High pressure liquid affinity chromatography |
-
1983
- 1983-11-25 US US06/555,368 patent/US4540486A/en not_active Expired - Lifetime
-
1984
- 1984-11-06 NZ NZ210126A patent/NZ210126A/en unknown
- 1984-11-12 IL IL8473496A patent/IL73496A/en not_active IP Right Cessation
- 1984-11-13 AU AU35369/84A patent/AU565046B2/en not_active Expired
- 1984-11-14 ZA ZA848886A patent/ZA848886B/en unknown
- 1984-11-15 CA CA000467924A patent/CA1213261A/en not_active Expired
- 1984-11-19 KR KR1019840007240A patent/KR890000825B1/en not_active IP Right Cessation
- 1984-11-19 JP JP59242585A patent/JPS60135761A/en active Granted
- 1984-11-20 AT AT84114034T patent/ATE53307T1/en not_active IP Right Cessation
- 1984-11-20 DE DE8484114034T patent/DE3482414D1/en not_active Expired - Lifetime
- 1984-11-20 EP EP84114034A patent/EP0143423B1/en not_active Expired - Lifetime
- 1984-11-23 IE IE3005/84A patent/IE57720B1/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0370181B2 (en) | 1991-11-06 |
| IL73496A0 (en) | 1985-02-28 |
| NZ210126A (en) | 1987-04-30 |
| EP0143423B1 (en) | 1990-06-06 |
| AU3536984A (en) | 1985-05-30 |
| DE3482414D1 (en) | 1990-07-12 |
| EP0143423A3 (en) | 1986-03-12 |
| AU565046B2 (en) | 1987-09-03 |
| KR850004109A (en) | 1985-07-01 |
| IE57720B1 (en) | 1993-03-24 |
| ATE53307T1 (en) | 1990-06-15 |
| EP0143423A2 (en) | 1985-06-05 |
| KR890000825B1 (en) | 1989-04-10 |
| US4540486A (en) | 1985-09-10 |
| IE843005L (en) | 1985-05-25 |
| IL73496A (en) | 1987-12-20 |
| JPS60135761A (en) | 1985-07-19 |
| ZA848886B (en) | 1985-07-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA1213261A (en) | Polyethylenimine bound chromatographic packing | |
| KR890002847B1 (en) | Acylated polyethylenimine bound chromatographic packing | |
| CA1283856C (en) | Purification of immunoglobulin g | |
| US4847159A (en) | Substrates coated with organo-silanes that are sterically-protected | |
| US4469630A (en) | Purification of monoclonal antibodies | |
| JPS5839576B2 (en) | A novel substance that can reversibly immobilize biological macromolecules and its production method | |
| IL229774A (en) | Chromatography matrices including novel staphylococcus aureus protein a based ligands | |
| KR910005189B1 (en) | Sulfonic derivatives of acylated polyethyleneimine bonded phase silica products | |
| WO1995000463A1 (en) | Optical isomer separating agent and process for producing the same | |
| EP0162462B1 (en) | Purification of type igm monoclonal antibodies | |
| EP0270095B1 (en) | Packing material for liquid chromatography | |
| US4721573A (en) | Use of sulfonic derivatives of acylated polyethyleneimine bonded phase silica products | |
| EP3762121B1 (en) | Composite material for bioseparations | |
| US5066395A (en) | N-acylated derivatives of polyethyleneimine bonded phase silica products | |
| CN107652352B (en) | Affinity chromatography medium for purifying histidine-tagged protein | |
| CN114618451A (en) | Weak cation exchange chromatography stationary phase and preparation and application thereof | |
| JPH02169590A (en) | Isolation and purification of lysergic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry | ||
| MKEX | Expiry |
Effective date: 20041115 |