Vpliv funkcionalizacij in struktur metakrilatnih monolitov na pretočnosti in kapacitete za različno velike molekule

Frankovič, Vida

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Title:	Vpliv funkcionalizacij in struktur metakrilatnih monolitov na pretočnosti in kapacitete za različno velike molekule
Authors:	ID Frankovič, Vida (Author) ID Krajnc, Peter (Mentor) More about this mentor... ID Podgornik, Aleš (Comentor)
Files:	DR_Frankovic_Vida_2009.pdf (1,37 MB) MD5: 545B8683D3A13A5D5DE0BEE7A8E5FC1A PID: 20.500.12556/dkum/d6c0665d-0f78-42f4-bb4c-2967a711dbe0
Language:	Slovenian
Work type:	Dissertation
Organization:	FKKT - Faculty of Chemistry and Chemical Engineering
Abstract:	V zadnjih desetletjih je bilo veliko narejeno na področju priprave in izboljšave kromatografskih nosilcev za doseganje čim boljše ločbe biomolekul. Convection Interaction Media (CIM) ionsko-izmenjevalni metakrilatni monolitni kromatografski nosilci vedno bolj pridobivajo na pomenu pri hitrih ločbah in čiščenju velikih biomolekul, kot so večji proteini, DNA, virusi, bakteriofagi, itd. Zaradi njihove posebne odprto-porozne strukture, ki omogoča hitro izmenjavo molekul med mobilno in stacionarno fazo na osnovi konvektivnega prenosa, izkazujejo pretočno neodvisno ločbo in dinamično vezno kapaciteto. Velikost por monolitnih nosilcev igra zelo pomembno vlogo pri ločevanju različno velikih molekul. Pore morajo biti dovolj velike, da omogočajo neovirano vezavo in ločbo biomolekul določene velikosti. Velike molekule se lahko v premajhnih porah ujamejo in zamašijo pretočne kanale, kar se lahko odrazi v manjših izkoristkih pri ločevanju in čiščenju. Poleg tega lahko zaradi zamašitve tlak na nosilcu močno naraste in pride do fizičnih poškodb le tega. Pri izbiri nosilca z določeno velikostjo por je potrebno biti pazljiv, saj se lahko vezna kapaciteta zmanjša na račun večjih por, zaradi zmanjšanja specifične površine. Predmet mojega doktorskega dela je bil najti ustrezen način funkcionalizacije površine por monolita, ki bo zagotavljala zvišanje kapacitete biomolekul ob čim nižjem padcu tlaka. Najpogostje se v ta namen uporablja uvajanje polimernih verig (lovk) s tehniko pripajanja (graftiranje). Rezultati doktorske disertacije so pokazali najmanj dva do tri kratno povišanje dinamične vezne kapacitete za številne različno velike molekule, vključno s plazmidno DNA na graftiranih monolitnih nosilcih. Čeprav graftiranje na površino metakrilatnih monolitov povzroča nižjo prepustnost, je njihova kapaciteta mnogo višja od ne-graftiranih monolitov, ki imajo isto prepustnost pri enakih pogojih. Zaradi dokazanih pretočno neodvisnih lastnosti, lahko graftirani metakrilatni monoliti postanejo primerna izbira za čiščenje različno velikih molekul. Izboljšanje procesov čiščenja biomolekul je mogoče na področju produktivnosti, selektivnosti in enostavnejše uporabe. Idealno so vse tri karakteristike zajete v enem samem kromatografskem nosilcu, kar omogoča čiščenje v enem samem kromatografskem koraku. Z graftiranjem hidrofobnih linearnih verig in naknadno pretvorbo preostalih epoksi v anionske skupine nam je uspelo pripraviti monolit z mešanimi adsorpcijskimi lastnostmi, ki se je izkazal kot učinkovit v procesu čiščenja pDNA.
Keywords:	graftiranje (pripajanje), funkcionalizacija, CIM monoliti, anionsko-izmenjevalni kromatografski nosilci, DEAE, padec tlaka, prepustnost, dinamična vezna kapaciteta, proteini, pDNA, število veznih mest (z-faktor), kombinirani monoliti
Place of publishing:	[Maribor
Publisher:	V. Frankovič]
Year of publishing:	2009
PID:	20.500.12556/DKUM-12536
UDC:	678.744.32:544.725(043.3)
COBISS.SI-ID:	13657878
NUK URN:	URN:SI:UM:DK:STIWDDUR
Publication date in DKUM:	01.12.2009
Views:	4950
Downloads:	385
Metadata:
Categories:	KTFMB - FKKT
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Secondary language

Language:	English
Title:	The effect of functionalization and structure of methacrylate monoliths on permeability and capacity for differently sized molecules
Abstract:	In recent decades much work has been done on the development and optimisation of chromatographic supports in order to achieve efficient purification of biomolecules. Convection Interaction Media (CIM) methacrylate ion-exchange monolithic chromatographic supports are becoming the material of choice for fast separations and purifications of large biomolecules such as large proteins, DNA, viruses, bacteriophages, etc. Due to the specific open-porous structure of monoliths that allows rapid exchange of molecules between the mobile and stationary phase based on the convective transport, the flow independent separation and dynamic binding capacity is exhibited. Pore size of the monolithic media plays an important role in the separation of different sized molecules. Pores should be large enough to allow an access of the target macromolecules. Large molecules can be trapped in the small pores which become clogged. This may result in less efficient separation and lower purification yield. In addition, pressure on chromatographic support might increase significantly and this can cause a physical damage of the support. The choice of the support with a particular pore size should be made carefully since the binding capacity can be reduced too, due to the reduction of surface area. The subject of my doctoral thesis was to find an appropriate way to functionalize the monolith in order to obtain high binding capacity for biomolecules at the lowest possible pressure drop. One of the frequently used techniques is the introduction of polymer chains (tentacles) by grafting. The results of this study demonstrate that the grafted monoliths exhibit at least two to three fold higher dynamic binding capacity over a broad range of molecular sizes (including pDNA) than the non-grafted analogues. Although the introduction of polymer chains onto the surface of methacrylate monoliths led to a decrease of permeability, their capacity is still significantly higher then those of the non-grafted monoliths having the same permeability. Since the flow-unaffected properties were preserved, grafted methacrylate monoliths may become a resin of choice for downstream processing of various macromolecules. Further improvement of the purification process of biomolecules can be made for productivity, selectivity and simpler handling. Ideally, single chromatographic medium would have all three characteristics, which would allow purification in a single chromatographic step. By the grafting of linear hydrophobic chains and the subsequent functionalization of the remaining epoxy groups to the anion groups, we managed to prepare a monolith with mixed adsorption properties, which has proved effective in the pDNA purification process.
Keywords:	grafting, functionalization, CIM monoliths, anion-exchange chromatographic supports, DEAE, pressure drop, permeability, dynamic binding capacity, proteins, pDNA, number of binding sites (z-factor), mixed-mode monoliths

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