US3640809A - Chemically hardened gelatin-separating medium for chromatography and ion exchange and method of making the same - Google Patents

Chemically hardened gelatin-separating medium for chromatography and ion exchange and method of making the same Download PDF

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US3640809A
US3640809A US782652A US3640809DA US3640809A US 3640809 A US3640809 A US 3640809A US 782652 A US782652 A US 782652A US 3640809D A US3640809D A US 3640809DA US 3640809 A US3640809 A US 3640809A
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gelatin
separating medium
medium
separating
hardening
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Alfred Polson
Woolf Katz
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South African Inventions Development Corp
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    • 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
    • B01J20/291—Gel sorbents
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • 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
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography

Definitions

  • the present invention relates to a medium for separations, more particularly to a novel medium for separations, useful in particular for chromatography, in particular so-called exclusion chromatography or gel filtration by virtue of its ability to act as a molecular sieve, although other properties such as absorptive properties or ion exchange properties may be present as well to a greater or lesser degree which, in accordance with the present invention may be controlled and utilized when desired.
  • the invention also provides for the separating medium to be in the form of cast bodies adapted for electrophoresis.
  • the invention further provides for a process for the production of the separating medium, the use of the separating medium for carrying out separations, and includes in its scope the products of such separations.
  • a number of media suitable for exclusion chromatography are already known, some of the more important of which in the context of exclusion chromatography are cross-linked dextran of which different preparations may be used for retarding substances in the molecular weight range 300 to 300,000, agar-agar which in the same sense covers the molecular weight range of 10,000 to approximately 4,000,000 and polyacrylates which can retard substances up to a molecular weight of approximately 500,000.
  • separating media there exists a definite need for a larger variety of separating media to enable those skilled in the separating art to select separating media best suited to a particular separating problem.
  • the separating medium in accordance with the invention is of a macromolecular character and comprises, in a form suitable for a separating process a gelatin which has been chemically hardened and is indispersable at temperatures above 7 C., more particularly at least as high as room temperature, preferably at least up to 100 C., and is permeable to the flow therethrough of a chromatographic mobile phase. More par- .ticularly, the gelatin is selected with a high molecular weight in the sense of having an intrinsic viscosity of at least 0.9 dl./g., preferably at least 0.95 dl./g. more particular between 0.95 and 1.0 dl./g., say 0.95 dl./g. (deciliters per gram) measured in 4 molar urea, saline, neutral pH, prior to said chemical hardening.
  • sedimentation coefficient of the nonhardened gelatin which is preferably in excess of 2.4Xl cm./sec./dync (measured in 4M urea saline to avoid gelling or congealing), say at least 2.8 l0 cm./sec.dyne.
  • the medium should have a high ratio of surface area to volume of gelatin.
  • said ratio is at least l0 cm.', for smaller columns at least l00 cm. which in the case of beads of the separating medium corresponds to a bead diameter of 100 microns, the preferred bead diameter for chromatography being between 100 and 300 microns, preferably as nearly as possible to 200 microns for preparative columns and smaller, say -50 micron for micro-analytical columns, and intermediate between these values for intermediate sizes of columns.
  • the separating medium may also be applied as a coating on a substratum, e.g., as a coating of beads of inert material such as glass or any other surface suitable as a support, when the above preferred ratios of surface area to volume of active separating medium apply.
  • the method of preparation in accordance with the invention comprises transforming a selected gelatin into a form physically adapted as a separating medium for chromatography, permeable to the flow therethrough of a chromatographic mobile phase, including a chemical step of hardening the gelatin and insolubilizing it in the sense of rendering it indispersable in water above a temperature of 7 C. and preferably at least of room temperature or even higher.
  • the gelatin may be derived from any suitable nonnal source of gelatin e.g., bone, hides, or collagen, pigskin gelatin (acid processed) giving a very rigid gel even at low gel concentrations, particularly after chromiumtanning and being at present preferred as a source.
  • suitable nonnal source of gelatin e.g., bone, hides, or collagen, pigskin gelatin (acid processed) giving a very rigid gel even at low gel concentrations, particularly after chromiumtanning and being at present preferred as a source.
  • the preferred process includes a separating step by which gelatin fractions having an intrinsic viscosity outside the desired range are separated from a fraction within the desired range.
  • the preferred separating process is carried out by fractionation in aqueous medium with polyethylene glycol (PEG), preferably having a molecular weight above 300, more particularly above 1,000 say of approximately 6,000 to produce a heavy phase in which the high molecular weight gelatin is concentrated and the supernatant phase containing predominantly the lower molecular weight fraction.
  • PEG polyethylene glycol
  • the separation is for example carried out with PEG employed in concentrations between 5 and 8 percent, by weight based on the total, and the process is described in greater detail in our patent application Ser. No. 627,308 filing date Mar. 31, 1967 now US.
  • the desired high molecular weight fraction may be washed, for example with water to remove PEG and then with acetone or another suitable solvent to remove water and is then dried e.g., at room temperature.
  • the gelatin is dissolved in aqueous medium, more particularly water in a predetermined concentration, e.g., from l percent upwards, more particularly in the range of 4 percent to 45 percent depending on the molecular weight of the substances for the separation of which the medium is to be employed. From the aqueous dispersion thus produced it is now possible to produce beads of suitable particle size as a separating medium.
  • emulsifying with a substantially nonpolar medium, for example a mixture of toluene and carbontetrachloride, and in the presence of a suitable emulsifying agent, stirring, cooling and breaking the emulsion e.g., with methanol or ethanol.
  • a suitable emulsifying agent stirring, cooling and breaking the emulsion e.g., with methanol or ethanol.
  • concentration of the emulsifying agent and the rate of stirring influence the particle size of the beads.
  • a preferred emulsifying agent is a commercial epoxy vegetable oil product, e.g., that produced and marketed by Badische Anilin und Sodafabrik A.G. under the trade name Emulphor EL.
  • This emulsifying agent is employed in a preferred concentration of between 0.5 and 5 percent W/w more particularly 0.7 to 2 percent based on the aqueous gelatin solution.
  • the preferred ratio of water in the aqueous solution to said nonpolar medium is between 1:15 and 1:3 say l:2.
  • the preferred ratio of toluene to CCL, (when these are employed) is between Szl and 2:1.
  • the chemical hardening of the gelatin may be carried out either before or after the bead formation. In the latter case, the particles are filtered off and then treated, in the former case the chemical hardening agents are added prior to the step of cooling and allowing the beads to set.
  • the chemicals employed for the chemical hardening are substantially of the nature of tanning agents known as such in the leather-tanning art. They may be selected in accordance with the particular properties desired of the separating medium.
  • tannic acids or tannins e.g., spray-dried wattle tannin have been employed for the purpose to produce a separating medium having one particular set of separating characteristics, while hardening of the gelatin with formaldehyde (formalin) which reacts with the free NH groups of the gelatin or hardening by chromium tanning with a chromium composition known in the tanning art as Kromex or with a combination of the two produced totally different separating characteristics, both of which may be desirable under different circumstances.
  • formaldehyde formaldehyde
  • the gelatin In many cases it is advantageous to harden the gelatin with both formalin and Kromex, one after the other.
  • the preferred sequence may be Kromex first, followed by formaldehyde or vice versa.
  • tanning or hardening agent may even be selected to impart particular ion-exchange properties to the gelatin if such should be desired, or the gelatin may be subjected to chemical reactions prior to or after hardening with the exclusive object of introducing into the gelatin molecule specific ion-exchanging groups.
  • gel concentrations not less than 25 percent, say from 30 percent upwards is preferred for reasons of adequate ion-exchange capacities.
  • the ion-exchange properties may in accordance with a preferred feature of the invention be calibrated by electro-osmosis through a membrane of the medium, and the invention includes in its scope the media thus calibrated.
  • a fractionating process which comprises introducing a mixture to be fractionated into a separating medium in accordance with the invention, bringing about movement of a component of the mixture through the separating medium and relative to at least one other component of the mixture at least in part by percolation of a liquid through said medium, and recovering at least one of said components separately from the other said component.
  • Said movement may be brought about by electrophoresis where the substances to be fractionated lend themselves thereto, i.e., to movement under the influence of an applied potential difference.
  • Important embodiments of the invention provide for said movement to be brought about by percolation of a liquid through a bed of the separating medium.
  • the separating process in accordance with the invention may be carried out substantially in a manner known per se but employing a separating medium as defined above or as prepared in accordance with the above process.
  • the ratio of water to gelatine in the gel affects the molecular sieve properties.
  • FIG. 5 represents a set of calibration diagrams obtained by electro-osmosis on four different samples of chemically hardened gelatin for use as a separating medium in accordance with the invention
  • FIGS. 6 and 7 represent chromatograms obtained with ion exchangers in accordance with Examples 10 and I l.
  • Example 2 a Preparation of gelatin of high-gel strength Gelatin (I60 g.) was dissolved in 3,800 ml. of distilled water over a boiling water bath with gentle stirring. When all the material had dissolved a solution of polyethylene glycol (240 g. PEG dissolved in 200 ml. of water) was added to the warm solution of gelatin and well mixed. The solution was kept at room temperature for 2-3 hours when it was found that the high gel strength fraction separated out as a viscous oily layer. The upper fraction was completely removed and discarded, and the remaining portion was slowly poured into 4 litres of distilled water at 4 C. which was slowly agitated with a mechanical stirrer.
  • polyethylene glycol 240 g. PEG dissolved in 200 ml. of water
  • the insoluble gelatin collected round the stirrer and was removed prior to storing it with the water at 4 C. overnight.
  • the following daythe ropey product was collected in a large precooled filter funnel containing a cotton wool plug to prevent the gelatin from passing through with the filtrate.
  • the gelatin was washed repeatedly with large volumes of cold distilled water until free of PEG.
  • the swollen strands were squeezed to express water and then dried by immersion in two changes of acetone. Excess acetone was removed by extraction with light petroleum (boiling range 4060 C.) and the material was finally dried by passing a warm current of air over it. The temperature of the air should not exceed 50 C. as at higher temperatures of drying a large proportion of the gelatin was found to become insoluble.
  • the same gelatin fractionated with PEG 6,000 had an intrinsic viscosity in 4 M Urea, saline, neutral pH, 1p 0.95 dl./g.
  • a known amount of the high molecular weight gelatin was dissolved in 300 ml. of distilled water over a boiling water bath with gentle stirring.
  • the organic liquid phase toluene carbon-tetrachloride) containing the dissolved stabilizer Emulphor EL (Badische Anilin und Soda-Fabrik AG, Ludwigshafen am Rhein, Germany) was preheated to 50 C. and added to the dissolved gelatin.
  • the mechanical stirrer was switched on and after approximately 1 minute the flask containing the suspension was lowered with the stirrer into a beaker containing crushed ice.
  • the beads of the different formulations differed in their ability to retain substances of different maximum molecular weights.
  • Untanned beads prepared as described above were trans ferred to a beaker containing 1 liter of a solution of Kromex 4 percent (w/v) and 2 percent (w/v) sodium chloride at pH 2.8. The suspension was well stirred and tanning was allowed to continue for 4 hours. A harder preparation is produced by tanning for 24 or preferably 48 hours. The beads were collected on a Buchner filter and well washed with 0.85 percent (w/v) saline until quite free of Kromex and kept in phosphate buffered saline pH 7.0 with sodium azide (l/l0,000) as a preservative.
  • Example 2 Gelatin beads prepared with a spray gun A shallow precooled glass trough was partially filled with a cold solution of Kromex-saline pH 2.8 into which was poured twice to three times the volume of ether at 0 C. The trough was surrounded with crushed ice to prevent the temperature from rising above about 40 C. A spray gun having a nozzle with a pore diameter of 0.1 mm. or less was used for spraying the warm solution of gelatin (of various concentrations) previously fractionated as described in Example la, directly onto the cold surface of the ether, from a height of approximately 30 cm. The gelatin beads passed through the ether and came into contact with the Kromex solution. The ether was removed, and the Kromex bead suspension was kept at 4 C. until the tanning had reached completion. The remaining ether was removed under negative pressure and the beads were collected and preserved as previously described. To obtain spheres of different porosities the gelatin concentration was varied between 4 and 30 percent.
  • Example 3 Preparation of tanned gelatin granules
  • Gelatin of known concentration, previously fractionated as described in Example la was dissolved in water over a boiling water bath with gentle stirring. The hot solution was allowed to cool to approximately 50 C. and was immediately poured into a precooled enamel dish which was surrounded with crushed ice and water. Once the solution had gelled it was cut into small pieces (about l-cm. cubes) with a knife and transferred to a beaker containing l liter of a solution of Kromexsaline pH 2.8 at 4 C. The tanning was continued at 4 C. for 24 hours after which the entire contents of the beaker were homogenized in a Waring blender until the particles were small enough for use in chromatography.
  • pearls (beads) made by the emulsifying technique in which organic solvents are used are less porous than the granules which are made by the disintegration of the tanned gelatin gel of the same initial concentration.
  • a possible explanation is that the surface of the beads is covered with a shell which has a higher concentration due to loss of water to the organic solvents.
  • Example 4 The procedure in accordance with Example 1 is modified as follows: e.g.,
  • the fractionated gelatin is dissolved in water and treated with nonpolar solvent and stabilizer as before. immediately after starting the stirring the desired tanning agent, e.g., 1.5 grams Kromex and/or vegetable wattle tannin was added and stirring was continued for l0-l 5 minutes prior to cooling.
  • desired tanning agent e.g. 1.5 grams Kromex and/or vegetable wattle tannin
  • FIGS. la and 1b illustrate the chromatographic partial separation of two haemocyanins, J asus lalandii (Mol. Wt. 500,000) and Burnupena cincta (Mol. Wt. 6,600,000), confirmed by analytical ultracentrifugation.
  • the separations were carried out with columns of 4 percent gelatin beads prepared in accordance with Examples 2e and 111 respectively.
  • the elluting fluid was phosphate buffered saline, pH 6.9 and the experiments were conducted at 21, the effluent being monitored with an optical recording unit.
  • FIGS. 2a and 2b illustrate the chromatographic separation of human serum employing 4 percent gelatin beads prepared in accordance with examples ld and le respectively.
  • the elluting fluid was phosphate bufiered saline, pH 6.9 and the experiments were conducted at 21, the effluent being monitored with an optical recording unit.
  • Example 7 It was possible to resolve chromatographically cobra venom to a considerably greater degree using beads prepared from a 30 percent aqueous gelatin solution in accordance with Example le (FIG. 3b) than with the commercial preparation Sephadex G-50" (FIG. 3a).
  • the elluting fluid was phosphate buffered saline, pH 6.9 and the experiments were conducted at 21, the effluent being monitored with an optical recording unit.
  • FIGS. 4a4f illustrate the separation of Jasus lalandii haemocyanin (J), Myoglobin (M) and tryptophan (T) using various beads produced in accordance with Example l
  • the elluting fluid was phosphate buffered saline, pH 6.9 and the experiments were conducted at 21, the effluent being monitored with an optical recording unit.
  • Example 9 Calibration of separating medium Samples of the same gelatin as used for the production of beads are gelled from aqueous dispersion (in this example 20 percent strong) in the form of membranes having a thickness of between about 0.05 and 0.06 cm. The membranes are then tanned as described in the various examples above.
  • the membranes are subjected to electro-osmosis with0.05 M glycine as a reference substance, a potential difference of 100 volt, the current being in the range of between 3 and 5.5 milliamperes.
  • the flow rate is measured in terms of l/TXIOO mm. at different pH values, the pH being changed with either 0.05 M HCl or 0.05 M NaOI-I.
  • curve 1 represents that obtained after tanning with formalin only
  • curve 2 is that obtained with a membrane tanned with Kromex followed by formaldehyde
  • curve 3 is that obtained with a membrane first tanned with formaldehyde and then with Kromex
  • curve 4 is that obtained with a membrane tanned with Kromex only.
  • the suspension of granules was collected on a sintered glass filter (pore No. 3) and the filter cake washed with 0.1 N NaOH.
  • the filter cake was suspended in 1 liter of 0.l N HCl, excess acid removed on the filter and then resuspended in 0.l N NaOH and left overnight at room temperature.
  • gelatin granules were collected on the filter, washed with water and then with 0.006 M-PO, buffer pH 6.9. Once equilibrated against the buffer the granules were packed into a column 2.5X40 cm.
  • a sample of horse antidog or antibaboon lymphocyte serum was dialysed against the buffer, centrifuged at 10,000 r.m.p. for 10 min. and l ml. of the clear SNF applied to the column.
  • the elution diagram is shown in FIG. 6.
  • the first peak (which did not adsorb to the column) was 'y-globulin which was identified in the ultracentrifuge (78).
  • the second peak was that due to albumin and related fractions, and had been adsorbed to the column.
  • the fraction was removed with a NaCl gradient starting with l M NaCl in the reservoir vessel.
  • the suspension was collected on a filter and repeatedly washed with l0 percent CH COOH and then with distilled water.
  • the filter cake was washed with water until neutral and then equilibrated against 0.006 M KH PO pH 5.6.
  • the first peak (FIG. 7) was that due to albumin and related fractions and was not adsorbed.
  • the second peak was that due to y-globulin and had been adsorbed and was eluted with a NaCl gradient starting with 0.4 M-NaCl in the reservoir.
  • a separating medium of a macromolecular character for chemical separations by chromatography comprising a chemically hardened gelatin in a form suitable for a chromatographic separating process and being indispersable in water at temperatures at least'above 7 C., and being provided with flow passage means or interleading pores is permeable to the flow therethrough of a chromatographic mobile phase.
  • a separating medium as claimed in claim 1 which is indispersable in water at temperatures at least up to C.
  • a separating medium as claimed in claim 1 in a form suitable for chromatography and having a ratio of surface area to volume of at least l cm.
  • a separating medium as claimed in claim 14 wherein the bead diameter is from 300 microns downwards.
  • a separating medium as claimed in claim 1 having ionexchange properties calibrated by electro osmosis.
  • a separating medium as claimed in claim 1 when incorporated in a chromatographic apparatus when incorporated in a chromatographic apparatus.
  • a calibrated separating medium of a macromolecular character for chemical separations by chromatography comprising a chemically hardened gelatin medium in a form suitable for a chromatographic separating process, said medium being indispersable in water at temperatures at least above 7 C. and permeable to the flow therethrough of a chromatographic mobile phase and having ion-exchange characteristics which are calibrated by forming a membrane and carrying out electro-osmosis through said membrane at a plurality of pH values.
  • a method as claimed in claim 19 including the step of fractionating the gelatin on a molecular weight basis and recovering a fraction of increased average molecular weight.
  • a fractionating process which comprises introducing a mixture to be fractionated into a separating medium of a macromolecular character for chemical separations by chromatography, said medium being chemically hardened gelatin in a form suitable for a chromatographic-separating process and being indispersable in water at temperatures at least above 7 C., and permeable to the flow therethrough of a chromatographic mobile phase,
  • a process as claimed in claim 29 which comprises bringing about said movement by percolation of a liquid through a bed of the separating medium.
  • a process as claimed in claim 30 carried out by way of exclusion chromatography.
  • a process as claimed in claim 29 which comprises adjusting conditions of separating at least in part for ion exchange to take place as at least one factor determining the fractionation.
  • a process as claimed in claim 29 further comprising bringing about said movement by electrophoresis.

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US782652A 1967-12-18 1968-12-10 Chemically hardened gelatin-separating medium for chromatography and ion exchange and method of making the same Expired - Lifetime US3640809A (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4130470A (en) * 1975-12-12 1978-12-19 Aminkemi Ab Method for generating a pH-function for use in electrophoresis
US4148943A (en) * 1975-05-22 1979-04-10 Agfa-Gevaert Ag Process for the preparation of matted photographic layers containing gelatine
US4275196A (en) * 1979-03-23 1981-06-23 The Cleveland Clinic Foundation Glyoxal agarose
US4971833A (en) * 1986-11-03 1990-11-20 Excorim Kb Method of coating solid particles with a hydrophilic gel
US5427729A (en) * 1989-10-03 1995-06-27 The Perkin-Elmer Corporation Method of making gel packed columns suitable for use in chromatography

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111760469A (zh) * 2020-07-03 2020-10-13 华东理工大学 一种单宁酸与明胶亲水涂层的膜的制备方法

Citations (9)

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US611814A (en) * 1898-10-04 Adam millar
US1201133A (en) * 1915-10-19 1916-10-10 Chemische Producte Vormals H Scheidemandel Ag F Solid gelatinous product.
US2101574A (en) * 1935-04-12 1937-12-07 Du Pont Hardening of proteins
US2196128A (en) * 1936-03-27 1940-04-02 Acheson Colloids Corp Coating and impregnating composition and method of coating surfaces
US2227982A (en) * 1936-10-23 1941-01-07 Eastman Kodak Co Method of hardening photographic gelatin
US2381752A (en) * 1943-12-31 1945-08-07 Du Pont Process of insolubilizing portein and product
US2868773A (en) * 1956-05-15 1959-01-13 Quaker Chemical Products Corp Process of insolubilizing proteins
US3369007A (en) * 1963-09-24 1968-02-13 Pharmacia Ab Molecular sieving agent consisting of mainly ball shaped gel grains and a method for their manufacture
US3411972A (en) * 1966-06-30 1968-11-19 Monsanto Res Corp Method for molding gelatin products

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US611814A (en) * 1898-10-04 Adam millar
US1201133A (en) * 1915-10-19 1916-10-10 Chemische Producte Vormals H Scheidemandel Ag F Solid gelatinous product.
US2101574A (en) * 1935-04-12 1937-12-07 Du Pont Hardening of proteins
US2196128A (en) * 1936-03-27 1940-04-02 Acheson Colloids Corp Coating and impregnating composition and method of coating surfaces
US2227982A (en) * 1936-10-23 1941-01-07 Eastman Kodak Co Method of hardening photographic gelatin
US2381752A (en) * 1943-12-31 1945-08-07 Du Pont Process of insolubilizing portein and product
US2868773A (en) * 1956-05-15 1959-01-13 Quaker Chemical Products Corp Process of insolubilizing proteins
US3369007A (en) * 1963-09-24 1968-02-13 Pharmacia Ab Molecular sieving agent consisting of mainly ball shaped gel grains and a method for their manufacture
US3411972A (en) * 1966-06-30 1968-11-19 Monsanto Res Corp Method for molding gelatin products

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Helfferich, Ion Exchange, McGraw Hill Co., (1962). *
McDonald, Ionography, The Year Book Publishers, Inc. (1955). *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4148943A (en) * 1975-05-22 1979-04-10 Agfa-Gevaert Ag Process for the preparation of matted photographic layers containing gelatine
US4130470A (en) * 1975-12-12 1978-12-19 Aminkemi Ab Method for generating a pH-function for use in electrophoresis
US4275196A (en) * 1979-03-23 1981-06-23 The Cleveland Clinic Foundation Glyoxal agarose
US4971833A (en) * 1986-11-03 1990-11-20 Excorim Kb Method of coating solid particles with a hydrophilic gel
US5427729A (en) * 1989-10-03 1995-06-27 The Perkin-Elmer Corporation Method of making gel packed columns suitable for use in chromatography

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