DE1795235C3 - Separating agent for gel filtration - Google Patents

Description

CH ₂ = C - CO - NH - R ₂

in which R _{1 is} hydrogen or a methyl group and R _{2 is} hydrogen or a straight-chain or branched alkyl group optionally substituted by one or more hydroxyl and / or oxo groups. which may optionally be interrupted by one or more oxygen bridges mean, the copolymer having been obtained from an aqueous solution of the monomers. characterized in that the content of Ν, Ν'-methylene-bisacrylamide in the copolymer is 30 to 90 percent by weight of the copolymer.

2. Release agent according to claim 1, characterized in that the copolymer is in the form of spherical Particle is present.

3. Process: for the production of a release agent according to claim 1 and 2, by copolymerization of Ν, Ν'-methylene-bis-jcrylar..id and an acrylamide the formula

CH ₂ - C - CO - NH - R ₂

in which R _{1 is} hydrogen or a methyl group and R _{2 is} hydrogen or a straight-chain or branched alkyl group which is optionally substituted by one or more hydroxyl and / or oxo groups and which may optionally be interrupted by one or more oxygen bridges, or 1-vinyl-2 -pyrrolidinone. in aqueous solution, characterized in that the content of the aqueous solution -in Ν, Ν'-methylene-bisacrylamide makes up 30 to 90 percent by weight of the total weight of the two monomers.

4. The method according to claim 3, characterized in that that the total amount of Ν, Ν'-methylene-bis-acrylamide and optionally substituted Acrylamide or l-vinyl-2-pyrrolidinone in the aqueous solution is 5 to 20 percent by weight.

5. The method according to claim 3 or 4, characterized in that the monomer solution before Polymerisation emulsified into fine droplets in a liquid immiscible with water will.

6. The method according to claim 5, characterized in that that the droplets through a material insoluble in water and in the water-immiscible liquid, which through opposite the copolymer inert substances can be dissolved, and by a substantially hydrophobic surfactant are stabilized.

7. The method according to claim 6, characterized in that that the insoluble material consists of finely divided chalk coated with calcium stearate consists.

The present invention relates to a separating agent for gel filtration and a method for its production.

ίο The gel filtration represents a common method today to separate the components of a substance mixture in at least two fractions. The principle of gel filtration is that by means of, for example a layer or column of gel granules from a solution or suspension of a mixture of substances at least two fractions with different components of the mixture. depending on the size of the molecule and the resulting ability of the ingredients to penetrate into the gel granules.

The gel filtration is carried out in two stages. The first stage is the solution or suspension the substances to be separated are supplied to the gel granules, while in the second stage with a liquid is eluted.

The separating means according to the invention! for gel filtration consists of granules containing a copolymer an acrylamide of the formula

R,

CH ₂ = C - CO - NH - R ₂

in which R _{1 is} hydrogen or a methyl group and R _{2 is} hydrogen or a straight-chain or branched alkyl group optionally substituted by one or more hydroxyl and / or oxo groups, the chain of which may be interrupted by one or more oxygen bridges, or Vinyl-2-pyrrolidinone and Ν, Ν'-methylene-bisacrylamide, the copolymer being formed from an aqueous solution of the two components.

Release agents of this type are already known. Copolymers of the above type have been found to be spherical Granules neutralized by bead polymerisation. The resulting granules are not soluble in water, but swellable.

In this bead polymerization, the monomers are dissolved in water, -. before the resulting Solution with a phase which cannot be mixed with water emulsified and the polymerization is initiated (see bri-

5 "table patent specification 1004 669). The usability a copolymer as a separating agent in gel filtration depends on the pore size and the hardness of the swollen Copolymers. So far, the pore size was determined by changing the concentration of Ν, Ν'-Mcthylen-bis-aciyiamids in the aqueous solution varies. If the concentration of bin-acrylamide is increased, so the pore size will be yours while um = conversely, when the concentration is reduced, larger pores are achieved. The enlargement of the pores in a limit is set in this way that the gel granules gradually become too soft, whereby the flow through a layer or column of glass is obstructed.

Surprisingly, it has now been shown that with a considerable increase in the content of Ν, Ν'-methylene-bis-acrylamide Gel granules that show large pore size and at the same time high hardness are obtained previously unattained combination of properties.

The lower limit, called the effect mentioned, begins, is about 30 parts by weight of N, N'-methylenebis-acrylamide, based on 100 parts by weight of the both monomers, the upper limit at about 90 parts by weight. Due to the increased hardness of the accordingly produced gel granules one achieves improved flow rates in the filtration layer or -s.iule. The novel gel granules according to the invention are particularly suitable for separating mixtures which contain substances with molecular weights above 50,000, especially proteins.

In working out the present invention, numerous polymerization experiments were carried out, in which the starting material consists of a mixture of N, N'-Meihylen-bis-acrylamide and consisted of acrylamide, and the content of the first-mentioned component between 20 and 90 percent by weight, based on the total weight of the monomers was varied. It was found that in the different proportions of copolymers which differ considerably in pore size and hardness. With a Ge-ί.alt of 25% N.N'-methylene-bis-acrylamide the Exclusion limit at a molecular weight of about 500,000 (protein). With increasing content of Ν, Ν'-methylene-bis-acrylamide increases the pore size then to.

The present invention is therefore a copolymer of Ν, Ν'-methylene-bis-acrylamide and the Acrylamide mentioned above with a content of 30 to 90 percent by weight, based on the total Copolymer of Ν, Ν'-methylenebis-aufylamid. preferably the content of N, N'-methylene - '^ - acrylamide 35 to 50 percent by weight.

According to one embodiment of the invention, the polymer particles are spherical.

As radicals R ₂ according to the above formula are, for example, ^r eii surface radicals such as the methyl, hydroxymethyl. Ethyl and isopropyl up to the complicated radicals, / B. called the l, l-dimethyl-3-oxobutyl radical.

In the process outlined above, spherical balls are produced. Instead of bead polymerization, the copolymer may also be formed by Hiockpolymerisaüon however, in which case the block polymer is crushed subsequently, / .. example by milling to give granules having an irregular shape is obtained. However, the spherical granules are preferred.

The invention also relates to a method of manufacture of the separating agent described above, which consists in the fact that an aqueous solution of N.N'-methylene-bis-acrylamide and an acrylamide of the formula

R ₁

= C CO NH

R ₂

in which R ₁ denotes hydrogen or a methyl group and R ₂ denotes hydrogen or a straight-chain or branched alkyl group optionally substituted by one or more hydroxyl and / or oxo groups, the chain of which may optionally be interrupted by one or more oxygen bridges, or 1-vinyl -2-pyrrolidinone, in which the proportion of N, N'-methylene-bif-acrylamide makes up 30 to 90 percent by weight of the two monomers, is subjected to polymerization conditions. The process is carried out in such a way that the solution of the monomers is emulsified into droplets in a liquid which is immiscible with water and the polymerization is then initiated.

According to a preferred embodiment of the invention, the total amount is from N.N'-methylene-bis-acrylamide and optionally substituted acrylamide or 1-vinyl-2-pyrrolidone in the aqueous Solution 5 to 20, especially 7 to 15 percent by weight.

According to another embodiment, the droplets obtained in the emulsification become partial by a very finely divided liquid which is not soluble in water and in the water-immiscible liquid Material produced by substances that make up the copolymer

does not harm, can be dissolved, and partly by a mainly hydrophobic, surface-active Substance stabilized. In the polymerization processes employed in devising the present invention became one in water and the

- ~ organic liquid insoluble granulated material used to stabilize the sub-suspension of droplets in the water-immiscible liquid and to prevent coagulation. For this purpose, granulated chalk coated with calcium carbonate was found to be particularly suitable.

The chalk can be added to the phase consisting of the water-immiscible liquid in which it can be easily dispersed due to the presence of calcium stearate. The coating with calcium stearate at the same time ensures that the chalk does not pass into the aqueous phase. Appropriate together with the chalk, a smaller amount of a surface-active agent that is used for Formation of a water-in-oil emulsion is capable of added. The calcium carbonate particles form a protective layer around the droplets from the aqueous Monomer solution and prevents the droplets from running together during polymerization. The Calcium carbonate can be dissolved out later.

z. B. with acetic acid. The stearic acid can be mixed with a suitable solvent such as toluene or trichlorethylene be washed out.

example 1

Copolymer of acrylamide and N, N'-methylenebisacrylamide

33 g of cellulose acetate butyrate were in 1000 ml Ethylene dichloride dissolved and placed in a reaction flask. The reaction flask was then placed in a Place a water bath at 50 ° C. In 500 ml of water at 50 ° C., 50 g of a mixture of 43 parts were added N, N'-Meihylen-bis-acrylamide and 57 parts of acrylamide were dissolved, and the solution was passed through a hot Filtered glass filter. Then 6 g of ammonium penilfate were added as an initiator to the monomer solution Whole was poured into the reaction flask. The phase mixture was at 200 r.p.m. touched. To Nitrogen was passed in for 15 minutes in order to displace the oxygen from the reaction vessel. The reaction was terminated after 4 hours. The beads formed were washed with acetone and water

«5 washed and sieved while still moist. The diameter the pearl was between 53 and 200 μm. After Shrinking with acetone and drying gave about 44 g yield.

Example 2

Zopolymer from acrylamide and N, N'-methylenebis acrylamide

The following materials were used:

25 g acrylamide,

25 g of Ν, Ν'-methylene-bis-acrylamide.

6 g ammonium sulfate.
500 g distilled water,
25 g emulsifier (dodecylphenoxypoly- (ethylcnoxy) -ethanol),

100 g granulated chalk, coated with calcium stcarate,
1000 m! Toluene.

Chalk and emulsifier were slurried in toluene in a reaction vessel, which is in a Water bath at 50 ° C. The monomers were dissolved in water at 50 ° C. After adding the Ainmonpersulfats for the monomer solution was this poured into the reaction vessel. The two-phase mixture was stirred vigorously. After 4 hours it was the reaction ended. The beads formed were washed with dilute acetic acid, toluene, water and acetone washed.

The particle diameters were between 50 and 250 am. Yield after drying: 47 g.

Example 3

Copolymer of acrylamide and N.N'-methylcn-bisacrylamide

The following materials were used:

27.4 g of acrylamide.

22.5 g Ν, Ν'-methylene-bis-acrjlamid, 6 g ammonium sulfate.

5 (X) g of distilled water.
25 g emulsifier (dodecylphenoxypoly- (ethylcnoxy) -ethanol).

100 g granulated chalk, coated with calcium stearate,
1000 ml of toluene.

The procedure of Example 2 was followed.

The particle diameter was between 75 and 275 | j.m, the yield at 46 g.

Example 4

Copolymer of acrylamide and N.N'-methylene-bisacrylamide

The following materials were used:

30.5 g of acrylamide.

19.5 g of N.N'-methylene-bis-acrylamide. 6 g ammonium persulfate,
500 ml of distilled water,
33 g of cellulose acetate butyrate, 1000 ml of ethylene dichloride.

The cellulose acetate butyrate was dissolved in the reaction flask placed in a water bath at 50 ° C. Ammon persulfate was added as an initiator to the monomer solution and the solution was immediately poured into the reaction vessel. The two-phase mixture was stirred at 200 rpm. After 15 minutes, oxygen was removed from the reaction vessel by introducing nitrogen. The reaction was terminated after 4 hours. The beads formed were washed with acetone and water, the diameter was 75 to 225 μm.
; After shrinking with acetone and drying, about 47 g of product were obtained.

Example 5

Copolymer of methacrylamide and Ν, Ν'-methylene bis-acrylamide

30 g methacrylamide.

20 g of N.N'-methylcn-bis-acrylamide.

5 g ammonium persulfate,
5 (K) g of distilled water.

25 g emulsifier (Dodecylphene \ ypoly- (ethylcn-

oxy) -alhanol),

100 g fine-grain chalk, coated with stcarat. 100 ml of toluene.

Chalk and emulsifier were placed in a reaction vessel which was open in a water bath at 50 C. Slurried toluene. The monomers were dissolved in water at 50 C, the solution: was with the Initiator is added and then immediately into the reaction vessel ecgossen. The second mixture was stirred vigorously. After 15 minutes nitrogen was introduced, to force the oxygen out of the reaction vessel. The reaction was ended after 4 hours. The beads formed were washed with dilute F.ssL: - acid. Toluene. Washed with water and acetone.

The particle diameter of the wet pearls lage.i between 50 and 200 μm.

The yield after drying was about 4d g.

B e i s ρ i e I 6

Copolymer of 1-vinyl-2-pyrrolidinone and N.N'-methylcn-bis-acrylamicl

Starting materials:
20 ml of 1-vinyl-2-pyrrolidinone.

30 g of N.N'-methylene-bis-acrylamide,

5 g ammonium sulfate,
480 ml of distilled water.

100 ρ fine-grained chalk, coated with calcium carbonate.

1.5 g emulsifier (dodecylphenoxypolyethylcn-

oxy (ethanol).
1000 ml of n-heptane.

The procedure of Example 5 was followed, however, n-heptane is used instead of toluene.

The diameter of the pearls was; at 50 to 300 μm the yield at 44 g.

Example 7
Copolymer of N-hydroxymethyl-acrylamide and

N.N'-methylene-bis-acrylamide
Starting materials:

46 ml of N- (hydroxymethyl) acrylamide (60%
aqueous solution),

22.5 g Ν, Ν'-methylene-bis-acrylamide,

5 g ammonium persulfate,
454 ml of distilled water,

100 g fine-grained chalk, with calcium stearate over it pulled,

25 g emulsifier (dodecylphenoxypolyethylene

oxy) ethanol).
1000 ml of toluene.

The procedure of Example 5 was followed.

The diameter of the moist beads were between 100 and 300 μm. The yield of dry pearls was 47 g.

B e i s ρ i e 1 8

Copolymer of acrylamide and N, N'-methylenebisacrylamide

27.5 g acrylamide,

22.5 g of N.N'-methylene-bis-acrylamide,

5 g ammonium persulfate,
500 ml of distilled water,
100 g fine-grain chalk coated with stearate, 25 g emulsifier (dodecylphenoxypoly- (ethylene-

oxy) ethanol),
1000 ml of toluene.

The procedure of Example 5 was followed.

The diameter of the moist beads were between 100 and 300 μm. The yield of dry pearls was 47 g.

Example 9

Copolymer of N- (l, l-dimethyl-3-oxobutyl) acrylamide and N, N'-methylene-bis-acrylamide

Starting materials:

27.5 g of NU.l-dimethyl-S-oxobutyO-acrylamide,
22.5 g of N.N'-methylene-bis-acrylamide,

5 g ammonium persulfate,
500 ml of distilled water,

100 g fine-grain chalk, coated with calcium stearate,
3 g emulsifier (dodecylphenoxypoly- (ethylene-

oxy) ethanol,
1000 ml of n-heptane.

The procedure of Example 6 was followed.

The diameter of the particles was between 60 and 250 μm. The yield of dry beads was 42 g.

The drawing illustrates the properties of the release agent according to the invention.
In the drawing shows

F i g. 1 shows a diagram in which the flow rate in ml / cm ² / h is plotted on the vertical as a function of the pressure drop along a column of 30 × 15 cm on the horizontal (in cm of water column per cm of layer height). The curves 1 to 7 correspond to gels with different contents of Ν, Ν'-methylene-bis-acrylamide in the monomer mixture. Curves 1 to 7 correspond to gels made from monomer mixtures with the following N, N'-methylene-bis-acrylamide content: 45, 39, 37, 33, 29, 25 and 15%. It can be seen that the flow rate improves fundamentally when the bis-acrylamide content exceeds 25%. During the polymerization, the concentration of monomers in the aqueous solution was 10 percent by weight. The particle size distribution was practically the same in all cases.

F i g. 2 shows a fig. 1 analog diagram in which the flow velocity is dependent on is shown by the pressure drop. Curve 1 relates to a copolymer of 60% methacrylamide and 40% Ν, Ν'-methylcn-bis-acrylamide, curve 2 on a copolymer from 60% N- (hydroxymethyl) acrylamide and 40% N.N'-methylene-bis-acrylamide; the copolymer curve I was obtained from a 10% strength monomer solution, the copolymer of curve 2 from a 15% strength monomer solution manufactured. The particle diameter of the The polymerization product was in both cases between 50 and 200 μm.

In order to demonstrate the large pore size of the gels _{according to the invention, the value Λ "η} ι · for gamma globulin for the gels prepared according to Examples 2 to 4 is given below.

The value A ' _nr appears in the following equation

V _t = V ₀ I Καν (Vt - V ₀ )

in which V _{t denotes} the total volume of the layer, V ₀ denotes the ^J o volume of the spaces between the pearls and V denotes the volume of the eluting liquid used to denote a substance. Kar is thus the part of the pearl that is accessible to the substance in question. If the volume of the interstices V ₀ ^a 5 is unknown, the parameter VJVt is often used instead of AO ₁ to indicate the penetration of the substance into the gel beads.
The A ^ I ₁ values are as follows:
Copolymer of Example 4 (61% acrylamide, 39% N.N'-methylene-bis-acrylamide): 0.61;

Copolymer of Example 3 (55% acrylamide, 45% Ν, Ν'-methylene-bis-acrylamide): 0.75;

Copolymer of Example 2 (50% acrylamide, 50% Ν, Ν'-methylene-bis-acrylamide): 0.76.
For comparison, it should be mentioned that the AO, value for gamma globulin in the most porous, e.g. Currently commercially available dextran and polyacrylamide gels are around 0.20.

F i g. 3 and 4 illustrate the separating effect of a separating medium according to the invention. Substances with different molecular weights were percolated through a layer (1.5 x 30 cm) of the release agent.

In Fig. 3, the Ä * _at values of some dextrans of different molecular weights (in the vertical) are plotted against the logarithm of the molecular weight of the dextrans (in the horizontal). The synthesis of the gel used here is described in Example 3.

In Fig. 4 are the values V _e / Vt for some proteins δ «" of different molecular sizes, namely cytochrome C (molecular weight = 13,000), / f-lactoglobulin (molecular weight = 38,000), albumin (molecular weight = 69,000), gamma globulin ( Molecular weight = 156,000) and α-crystalline (molecular weight = 1,000,000) plotted as a function of the logarithm of the molecular weight.

The gel used was prepared according to the procedure of Example 7, with the exception that the The proportion of Ν, Ν'-methylene-bis-acrylamide was 40% (instead of 45%).

The diagram shows that the gel is suitable for separating mixtures of high molecular weight compounds. Satisfactory separation is achieved even with molecular weights above 10 ^7.

1 sheet of drawings

Claims (2)

Claims: J. Separating agent for gel filtration, consisting of a copolymer of 1. Ν, Ν'-methylene-bis-acrylamide and 2. 1-vinyl-2-pyrrolidinone or an acrylamide of the formula R,