AU733974B2

AU733974B2 - Immobilizate and process for the immobilization of a useful material

Info

Publication number: AU733974B2
Application number: AU40120/97A
Authority: AU
Inventors: Jurgen Engelhardt; Wolfgang Koch; Jorn-Bernd Pannek; Anant Vallabhbhai Patel; Klaus-Dieter Vorlop
Original assignee: Wolff Walsrode AG
Current assignee: Dow Produktions und Vertriebs GmbH and Co oHG
Priority date: 1996-08-08
Filing date: 1997-07-28
Publication date: 2001-05-31
Anticipated expiration: 2017-07-28
Also published as: ES2186918T3; EP0917492B1; AU4012097A; EP0917492A1; WO1998006489A1; JP2000516650A; TR199900256T2; DE59708675D1; DE19632032C1; PT917492E

Description

WO 98/06489 PCT/EP97/04081 -1- Immobilizate and process for the immobilization of a useful material The invention relates to an immobilizate which consists of a crosslinked outer shell including a useful material.

The invention further relates to a process for the immobilization of a useful material by enveloping it with an outer shell.

Numerous processes and materials are known with which a useful material can be included in a crosslinked outer shell and thereby immobilized. Many various applications have been proposed for ionotropic gels which crosslink with low molecular crosslinking reactants, one example being alginate. These gels can be used to produce solid or hollow spheres containing the useful material, said spheres surrounding e.g. a liquid core which can contain the crosslinking reactant. An outer shell can also be produced in the form of a membrane by the interfacial polymerization of an oil-in-water or water-in-oil emulsion, wherein a first polymerization reactant is incorporated in the aqueous phase and a second polymerization reactant is incorporated in the oily phase so as to form a membrane at the interfaces by copolymerization of the two polymerization reactants. Another process for the formation of an outer shell with predominantly purely synthetic materials is the microencapsulation process, wherein a useful material is surrounded with a liquid enveloping material and the whole is introduced into a crosslinking bath.

The known materials for the preparation of an immobilizate either have a low mechanical stability (ionotropic gels), or can only be prepared by elaborate means (copolymerization membrane), or present problems from the point of view of disposal (synthetic microcapsules) because they are non-degradable or difficult to degrade.

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D4 I t) I' WO 98/06489 PCT/EP97/04081 -2- The problem underlying the invention is therefore to provide a material for the preparation of an immobilizate, which material has a high mechanical and chemical stability, allows the simple preparation of generic immobilizates and is also extensively biodegradable.

Based on this problem, the outer shell of an immobilizate of the type mentioned at the outset consists according to the invention of a crosslinked cellulose ether containing sulfoalkyl groups, especially sulfoethyl groups, said cellulose ether preferably being sulfoethyl cellulose (SEC). Other suitable materials are especially sulfopropyl cellulose and mixed celluloses containing sulfoalkyl groups.

It has been found that the best results, particularly the highest mechanical stability, are achieved when the cellulose ether is crosslinked with polydimethyldiallylammonium chloride (PDMDAAC). Good results are also achieved by crosslinking the SEC with chitosan.

The immobilizate is prepared according to the invention by introducing the useful material into an anionic solution of a cellulose ether containing sulfoalkyl groups, preferably a solution of the sulfoethyl ether, and surrounding divided portions of the solution with a cationic crosslinking agent, for example by adding the cellulose ether solution dropwise to the crosslinking agent. When using chitosan or, preferably, PDMDAAC as the cationic crosslinking agent, it penetrates into the divided portions of the cellulose ether solution from outside and causes crosslinking. The crosslinking prevents further penetration of the crosslinking agent, so a hollow immobilizate, preferably a hollow sphere, is formed.

In an alternative process, the useful material can be introduced into a liquid which contains the cationic crosslinking agent and is then surrounded with the anionic cellulose ether solution. This procedure forms a hollow immobilizate with crosslinking from inside to outside. A secondary crosslinking can of course be [0o69 ON XH/XJL] 0:ZT U]M TO, VO/VO WO 98/06489 PCT/EP97104081 -3effected in all cases by storing the resulting hollow immobilizate in the crosslinking agent for a time.

The inmmobilizates according to the invention can be prepared with a high mechanical stability, are stable towards phosphates and acids and are biodegradable.

They arc therefore also particularly suitable for incorporating biological plant protection agents etc. into the soil. Particularly advantageously, an immobilizate according to the invention can enclose a seed in order to protect it, it being possible for the irunobilizate to contain protective chemicals or microorganisms and 10 optionally additives such as moisture stabilizers or nutrients for the microorganisms.

*1 The mechanical stability was verified by the following comparative experiments: To dissolve the cellulose derivatives, SEC as the polymer was added in successive o. 15 portions to deionized water at room temperature with stirring (700 rpm). After the polymer had dissolved, stirring was normally stopped after 5 hours. The pH of the solutions used was in the range 5.6 to 7.8.

9 9 Due to the preparative process, the solutions of SEC samples contained a small 20 proportion of insoluble fibre. The samples make up a series with increasing degree of substitution (DS) in the range 0.25 to 1.26.

Table 1 below collates the cellulose derivatives used by the Applicant. Blanose Cellulose Gum® Type 7 MFD (CMC) was also tested.

tz /VH I19Z BtrZG z 19+! aAR3 UoS I I|l( |0 1S3 I A dL0;Z[I![ -V -V tLY~ 8~8 L9±~ aA~UOSiILOD seIA2g~d~D:Zt~tD-~-v [I'OV6 ON XH/YlI OO:ZT a134 TO, 10/170 WO 98/064S9 PCT/EP97/04081 -4- I~b Ue n

S

(1) (2) (3) (4) (6) (7) (8) Brookfield no. 2 spindle, I rpm. 60 s, 2% solution Brookfield no. 4 spindle, I rpmn, 60 s, 2% solution Brookfield no. 2 spindle, 2 rpm, 30 s, 2% solution rotational viscometer, 2.52 rps, 2% solution commercial produ~ct Roto. 2.55 rps, 2% solution viscosity methyl substitution z 9 L LgYz 8 v z6 E L 94 9 A 93 UO S I I 103 SO I A 20 V4d 0 Z L Q-V -t [OV69 ON YH/YI] OO:ZT UM TO, 1O/17 WO 98/06489 PCT/EP97/04081 Abbreviations used:

CM/CMC

DCM/DCMC

SE/SEC

HE

M

carboxymnethyl/carboxymethyl cellulose dicarboxymethyl/dicarboxymethyl cellulose sulfoethyl/sulfoethyl cellulose hydroxyethyl methyl 0

OSO*

OOSS

S

Four different PDMDAAC products were used: Age floco (Ag) I-IV. These are listed in the Table below.

PDMDAAC polymers used Abbreviation Product Molecular weight Ag I Age floc WT 50 SLY 10,000 20,000 A&II Age flec WT 35 VLV 20,000 30.000 Ag II Age floc WT 40 50,000 100,000 A IV i Age floc WT 40 HV 100,000- 200,000 ~osi~yJSo1utioa 1%] Viscosity Soludon 200 cps 50 191 cps 35 1860 cps 40 10,000 cps 40 pH 5.7 5.9 5.3 13 In the experiments, the polymer solutions Ag I IV were used at a concentration of 2%.

The solutions of the starting polymers were used in the non-sterile form for screening and for the subsequent systematic studies. By means of a syringe and cannula (0.90 x 40 mm 20 G x 1.5 Gr. 1; Braun Melsungen), the aqueous solutions of the cellulose derivatives and polyacrylates were added dropwise to a stirred PDMAAC solution (in small glass vessels with snap-on covers) at room temperature. The drop height was ca 8 cm, it being necessary to ensure that the chosen drop height is not too large because otherwise the spheres burst. UIf the drop height is too small, the hollow spheres (HS) do not sink into the crosslinking agent The experimental set-up is illustrated schematically in Figure 1.

H1 8VI' z L9+! 9 A D U0 SI I 0 D S 81 A 0 N dL 0 Z 1 0 WO 98/06489 PCT/EP97/04081 -6- In the screening experiments, the HS obtained were separated from the crosslinking agent by sieving after a crosslinking time of 15 minutes and, if sufficiently stable, were stored in deionized water and 0.45% and 0.9% NaCl solution, and their stability was determined at times to, t24h and t48h.

The most stable of the previously screened HS were systematically studied by being tested as a function of the crosslinking times in PDMDAAC. This was done by leaving the HS in the stirred crosslinking agent for 1, 15, 30, 60, 120 or 200 min or until they began to shrink, and then storing them in deionized water and 0.45% and 0.9% NaCI solution. The HS obtained were characterized by measurement of the stability, the diameter and the membrane structure or thickness.

Process steps (cf. Figure 1): Mixing of the material to be included with aqueous polyanion solution (capsule core); introduction into a suitable crosslinking agent containing polycations

(PDMDAAC);

removal of excess crosslinking agent; transfer of the capsules to the medium intended for storage or use.

The spherical transparent simplex membrane forms during the process of immersion of the drop at the interface between polyanion and polycation and is based on electrostatic bonding.

The autoclaved 2.6% polymer solution SEC II (stored for 10 min at 121 0 C, 2 weeks at 4 0 C) was added dropwise to 2% PDMDAAC solution Ag III (stored for 3 days at 4°C) by means of a cannula (internal diameter 0.9 mm) and crosslinked for 30 min at room temperature at a stirrer speed of 300 rpm.

WO 98/06489 PCT/EP97/04081 -7- The spheres were stored in deionized water and 0.9% NaC1 solution. One day later the resulting spheres were subjected to secondary crosslinking. The secondary crosslinking times were 0, 5, 15, 30, 60 and 120 min. 0.25% PDMDAAC solution Ag III in water, pH 6.8, was chosen as the crosslinking agent for the spheres stored in deionized water; the spheres stored in NaC1 solution were subjected to secondary crosslinking in 0.25% PDMDAAC solution Ag III in 0.9% NaCl solution, pH 6.8.

All the spheres subjected to secondary crosslinking were stored in 0.9% NaCI solution. The stability of the HS was measured after storage for 4 days at 4 0

C.

Determination of the membrane structure and the membrane density For exact determination of the membrane thickness, the SEC HS polymer solution SEC II in 2% PCMDAAC solution Ag III, cannula of internal diameter 0.9 mm for large HS, cannula of internal diameter 0.4 mm for small HS, crosslinking time 50 min) were fixed by the method of Karnovsky (1965) and the HS were carefully torn apart in order to be able to determine the strength of the membrane.

After the surface had been sputtered with gold, the membrane was photographed in a scanning electron microscope (SEM). The photographs provide evidence of the structure of the membrane and its thickness.

Determination of the diameter of the spheres The diameter do of the HS at time to was determined by means of a stereoscopic magnifying glass. After storage of the HS in deionized water and 0.45% and 0.9% NaC1 solution, the diameters d2 4 h and d48h were measured again after 24 and 48 h respectively, thereby enabling the swelling behaviour of the HS to be determined.

The swelling was calculated according to the following formula: diameter of the swollen sphere A0 swelling 100%] 100 6 Ll4A diameter of the sphere at time To cok 0 7 [068 ON XH/XL] 00:ZT a3Ml TO, VO/1O WO 98/06489 PCT/EP97/04081 -8- The diameter of 10 of each of the SEC HS was determined in order to ascertain whether the inmmobilizates swell or shrink during continued growth in maize flour medium.

Measurement of the stability of the spheres (cf. Figure 2) The stability of the HS was measured immediately. The HS were kept in deionized water while being conveyed to the measuring apparatus. After storage of the HS for 24 and 48 h in multititre plates at 4°C in deionized water and 0.45% and 0.9% NaCI S10 solution, their stability was measured again.

The measuring apparatus used, according to Washausen (1980), is illustrated schematically in Figure 2.

The load cell was moved up and down by means of a threaded rod driven by a motor with control gear The pressure piston (PP) is fixed to the load cell.

For determination of the rupture point, an HS was placed on the support plate The pressure piston was lowered by hand until it just made contact with the HS. The diameter of the bead could be read off directly on an integrated calliper gauge. The apparatus was then started and the load cell moved down at a speed of 1.5 umm/min.

The rate of advance was determined experimentally (distance/time). The measuring signal corresponding to the particular compressive force was transferred from the load cell to the recorder via a measuring amplifier The HS was slowly compressed as the pressure piston was lowered. The force exerted increased continuously as the piston advanced. The maximum force capable of being exerted on an HS could be detected at the rupture point (HS burst).

If the HS did not rupture, a limit switch changed the direction of movement of the load cell when the distance between the pressure piston and the support plate was S/L 6 uos LI /LL L191 8t Z 2 9 U03 I I IosilIo S 1A 2 0 A d LZL 0 WO 98/06489 PCT/EP97/04081 -9- 0.15 mm. (There would be little point in compressing the HS further because the spheres had a membrane thickness of ca. 40 [jm.) The measuring signal on the recorder resulted from the constant vertical movement of the load cell and the corresponding force transferred to the HS. After prior calibration by means of a weight, the applied force could be read off immediately from the deflection of the recorder during the measurements.

The results obtained are collated in Table 2 below.

Table 2 Abbreviation pH DS Ag I Ag II Ag III Ag IV CMC I 6.9/9.0 0.8 CMC II 6.6/8.0 0.8 6.8/11.0 CMC III 7.1/4.0 0.9 CMC IV 6.9/3.5 0.7 CMCV 6.4/4.0 1.8 CMC VI 6.9/6.0 0.6 CM/DCMI 7.2 3.0 0.41 0.29 DCM I 7.4/3.0 0.21 CMC/HE I 6.1/3.0 0.67/0.86 CMC/HE II 6.3 3.0 0.67 0.86 CMC/HE/iM I 7.2 2.0 0.72 0.25 1.59 CMC/M I 7.5 2.0 0.94 0.64 CMC/SE I 6.7 6.0 0.49 0.37 CMC/SE II 6.0 10.0 0.92 0.23 CMC/SE III 6.2 5.0 1.27 0.47 SEC I 6.6 1.0 0.25 SEC II 7.0 2.0 0.39 SEC III 5.6/4.0 0.45 SEC IV 6.8 2.0 0.52 SEC V 7.8/4.0 0.54 SEC VI 7.2/4.0 0.57 SEC VII 6.1/3.0 0.57 SEC VIII 7.4 2.0 0.67 +-ii SEC IX 6.7/4.0 1.26 already shrunk [0168 ON XH/XL] 00:ZT aHAM TO, V0/V0 WO 98/06499 PCT/EP97/04081 Table 2 shows that the PDMDAAC should preferably have a molecular weight of between 20,000 and 100,000 for stable SEC spheres. In isolated cases, it is also possible to obtain highly stable spheres with molecular weights of between 10,000 and 20,000 and of 100,000 to 200,000 for suitable SEC solutions.

The comparative results show that spheres of carboxymethyl cellulose (CMC), in particular, do not give comparable mechanical stability results.

The reference to any prior art in this specification is not, and should not be taken as, o an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge in Australia.

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Claims

1. Immobilizate consisting of a crosslinked outer shell including a useful material, characterized in that the outer shall is formed of a crosslinked cellulose ether containing sulfoalkyl groups.

2. Immobilizate according to claim 1, characterized in that the outer shell is formed of a crosslinked sulfoalkyl cellulose. 10 3. Immobilizate according to claim 2, characterized in that the outer shell is formed of sulfoethyl cellulose (SEC). Immobilizate according to any one of claims 1 to 3, characterized in that the cellulose ether is crosslinked with polydimethyldiallylammonium chloride 15 (PDMDAAC).

5. Immobilizate according to any one of claims 1 to 4, characterized in that the cellulose ether is crosslinked with chitosan.

6. Process for the immobilization of a useful material by enveloping it with an outer shell, characterized in that the useful material is introduced into an anionic solution of a cellulose ether containing sulfoalkyl groups, and in that divided portions of the solution are surrounded with a cationic crosslinking agent.

7. Process according to claim 6, characterized in that the cellulose ether solution is added dropwise to the crosslinking agent.

8. Process for the immobilization of a useful material by enveloping it with an outer shell, characterized in. that the useful material, in a liquid containing a cationic crosslinking agent, is surrounded with an anionic solution of a cellulose -ether containing sulfoalkyl groups. 2 6 9 8 t Z S L 9 aA9 UOSI Io0 s@eIAP0!IJd LO :LO-t -V [R969 ON XH/Xl] T:GT flHl TO0, -12-

9. Process according to any one of claims 6 to 8, characterized in that a sulfoalkyl cellulose solution is used as the anionic solution. Process according to claim 9, characterized in that a sulfoethylene cellulose solution is used as the anionic solution. 1. Process according to any one of claims 6 to 10, characterized in that chitosan is used as the cationic crosslinking agent. 10 12. Process according to any one of claims 6 to 9, characterized in that polydimethyldiallylammonium chloride (PDMDAAC) is used as the cationic crosslinking agent.

13. Immobilizates or processes for their preparation, substantially as hereinbefore 15 described with reference to the examples and accompanying drawings. C *9 0* eq C C C E C C C S.C. C.. C C .RC, C C C C 9 C C DATED this 30th day of March, 2001 WOLF WALSRODE AKTIENGESELLSCHAFT 20 By its Patent Attorneys DAVIES COLLISON CAVE 9 /S LL9Z 8vrZ6 Z 19+! aAe uos i 3 s aII I OLD S 8 A a d Z 0-t7 -S