A solid state NMR and MS characterisation of the chemical composition of mimosa bark extract
| dc.contributor.advisor | Van der Westhuizen, J. H. | |
| dc.contributor.advisor | Bonnet, S. L. | |
| dc.contributor.advisor | Reid, D. | |
| dc.contributor.author | Senekal, Nadine D. | |
| dc.date.accessioned | 2015-11-13T08:33:54Z | |
| dc.date.available | 2015-11-13T08:33:54Z | |
| dc.date.issued | 2011-01 | |
| dc.description.abstract | English: Mimosa (Acacia mearnsii) also known as black wattle, and quebracho (Schinopsis balansae, Schinopsis lorentzii) are the major commercial sources of natural condensed tannins (proanthocyanidin oligomers) used today. Mimosa bark is harvested from commercial plantations in South Africa which, according to a survey done by the Department of Water Affairs and Forestry for 2001, cover an area of about 107 000 hectares in South Africa. Quebracho is extracted from the wood of natural forests in Brazil and Argentina. Mimosa bark is extracted with water (about 50% by weight). Tara (Cæsalpinia spinosa) and Italian chestnut (Castanea sativa) are the major commercial sources of hydrolysable tannins. The ability of water soluble hydrolysable and condensed tannins (polyphenols) to react with proteins, presumably via hydrogen bonds, lies at the heart of their ability to transform raw hide into leather and their commercial application as tannin agents. It explains their existence in nature as anti-feeding agents as it renders plants indigestible to insects and herbivores. It also explains the use of milk in tea where the complexation of milk proteins with tea tannins reduces astringency. The chemistry of this process however remains uncertain. The polyphenolic nature also renders tannin extracts very susceptible to oxidation and further polymerisation and rearrangements that render the extracts even more complex. This is evident in the transformation of green tea (high flavan-3-ol content and low condensed tannin content) into Indian or black tea (low flavan-3-ol content and high condensed tannin content). The quality of red wine is to a large extent determined by the amount and composition (which changes during ageing in a poorly understood way) of its condensed tannin. The tannins react with protein receptors on the tongue to impart “mouth feel” characteristics. Wood-aged wine not only contains condensed tannins from grape skin, but also hydrolysable tannins from the wooden barrels it is aged in. The polyphenolic nature of the aromatic rings allows reaction with electrophiles. This forms the basis of adhesive manufacturing, where formaldehyde is used to polymerise tannin extracts to form adhesives. Other commercial applications of tannin extracts include the use as anti-foaming agents in oil drilling and the manufacturing of amine containing resins (via the Mannich reaction) for water purification applications (removal of heavy metals). The production of mimosa condensed tannin is a sustainable process as trees are harvested every eight years. Tannins will become a more important source of feedstock nutrients, as crude oil, which is currently used, becomes depleted. It also creates employment in rural areas. Higher oligomers of condensed tannins are built up by successive addition of flavan-3-ol monomer extension units via C-4 to C-8 or C-4 to C-6 interflavanyl bonds. Higher oligomers are impossible to purify by chromatography and other methods of analysis are required. Acid catalysed fission of the interflavanyl bonds and trapping of the monomer intermediates with toluene-α-thiol or floroglucinol followed by analysis of the trapped products with HPLC is normally used to analyse condensed tannin composition. The analysis of mimosa and quebracho tannins is however compounded by the resorcinol type A-ring in these compounds. The absence of a 5-OH group imparts stability to the interflavanyl bond against acid hydrolysis. The high temperatures thus required to hydrolyse the interflavanyl bond in mimosa and quebracho tannins leads to decomposition. Mass spectrometry and 13C NMR (nuclear magnetic resonance) spectrometry in solution have also been used with varying degrees of success. The analysis of hydrolysable tannins is even more complex than that of condensed tannins. As a result, the composition of condensed and hydrolysable tannin extracts remains uncertain, after more than 50 years of research. Of particular interest are the average chain length of tannin extracts from different sources and the composition of the constituent monomers. In this thesis the potential of solid state NMR and electrospray mass spectroscopy to solve vexing problems in tannin chemistry was investigated. Solid state NMR is particularly useful to investigate insoluble samples, overcoming problems associated with selective extraction, chemical modifications during extraction and sample preparation and uncertainty regarding compounds that are not extracted. Electrospray mass spectrometry complements MALDITOF mass spectrometry in that molecules with masses below 500 Dalton are detected. We were able to assign all the resonances in solid state NMR of hydrolysable and condensed tannins by comparing liquid and solid state spectra of pure flavonoids and tannin extract. This allowed us to distinguish unequivocally between condensed tannins and hydrolysable tannins with a simple routine experiment, avoiding laborious chemical tests. A method was developed to identify and distinguish with confidence between quebracho and mimosa condensed tannins. This method is the only available method to identify quebracho, which is of interest to oenology (quebracho tannins are added to wine) and could hitherto only be identified chemically because it tests negatively for all the available tests for tannins. We established that no insoluble higher oligomeric condensed tannins or tannins covalently bonded to other insoluble bark components remain in spent mimosa bark (after extraction of tannins). It promises an easy way for the wattle industry to investigate lower extraction temperatures and extraction time and the associated energy savings. A fingerprinting method for mimosa was developed and is already used by the industry (Annex A). As the gum resonances do not overlap with the tannin resonances, the bark can be analysed directly without the requirement of manufacturing an extract. The only sample preparation required is to grind the bark (about 100mg) finely and pack the solid state NMR rotor. As carbon is magnetised via hydrogen, less than 30 minutes NMR time is required per sample. This provides an easy way to identify the bark of quebracho, mimosa and hydrolysable tannins. A solid state NMR spectrum of the spent bark not only indicated that no condensed tannins remain, but also supports the conclusion that spent bark consists of water insoluble gums (polymers of glucose and other sugars). We believe that this method will find application in identifying novel sources of tannins from indigenous plants. We expanded our investigation into tanned leather and developed an easy method to determine whether leather was tanned with mimosa, quebracho, Italian chestnut, tara, synthetic tanning material, chromium or aluminium. We believe this method can be used by the leather industry to determine tannin loading of tanned leathers. By combining our electrospray mass spectrometry data with published MALDI-TOF mass spectrometry data we could calculate the relative composition of monomers, dimers, trimers, tetramers etc. in condensed tannin sample. These calculations were used by the mimosa and quebracho tannin industry to comply with new European Union (EU) REACH (Registration, Evaluation, Authorisation and Restriction of Chemical substances) legislation. Without compliance mimosa extract cannot be exported to the EU. Sulfitation (treating mimosa and particularly quebracho extract with bisulfite) is routinely used in industry to enhance the extract’s properties (e.g. increase water solubility) and products with different levels of sulfitation are commercially available. The chemical changes associated with sulfitation remain speculation. The solid state NMR indicated that the C-ring is opened during the process. The electrospray MS conclusively demonstrated the existence of condensed tannin-sulfonate molecules for the first time. The m/e values correspond with ring opening and introduction of a sulfonate group on the C-2 position. | en_ZA |
| dc.description.abstract | Afrikaans: Mimosa (Acacia mearnsii) ook bekend as swart wattle, en quebracho (Schinopsis balansae, Schinopsis lorentzii) is die hoof kommersiële bronne van natuurlik gekondenseerde tanniene (proantosianidien oligomere) wat vandag gebruik word. Mimosa bas word geoes in kommersiële plantasies in Suid Afrika wat, volgens’n opname deur die Departement van Waterwese en bosbou in 2001, ‘n area van ongeveer 107 000 hektaar beslaan. Quebracho word geëkstraeer van die hout van natuurlike woude in Brazilië en Argentinië. Mimosa bas word in water geëkstraeer (ongeveer 50% per gewig). Tara (Cæsalpinia spinosa) en italiaanse kastaiing (Castanea sativa) is die hoof kommersiële bronne van hidroliseerbare tanniene. The vermoeë van wateroplosbare hidroliseerbare en gekondenseerde tanniene (polifenole) om met proteïene, waarskynlik via waterstof bindings, te reageer, lê naas hul vermoë om ongebreide vel in leer te omskep, en in hul kommersiële toepassing as tannien agente. Dit verklaar ook hul bestaan in die natuur as teen-voedings agente wat plante onverteerbaar maak vir insekte en herbivore. Verder verklaar dit die gebruik van melk in tee, waar die kompleksering van melk proteïene met tee tanniene die bitterheid van tee verminder, alhoewel die chemie van hierdie proses steeds onduidelik is. Die polifenoliese aard maak tannien ekstrakte baie vatbaar vir oksidasie, asook verdere polimerisasie en herrangskikking, wat die ekstrak meer komplekseer. Dit is duidelik in die transformasie van groen tee (hoë flavan-3-ol inhoud en lae gekondenseerde tannien inhoud) na indiese of swart tee (lae flavan- 3-ol inhoud en hoë tanien inhoud). Die kwaliteit van rooiwyn word tot ‘n groot mate bepaal deur die hoeveelheid en samestelling (wat verander gedurende veroudering tydens ‘n proses wat tot hede nog moeilik verklaarbaar is) van die gekondenseerde tanniene. Die tanniene reageer met proteïen reseptore op die tong om die kenmerkende mondgevoel te gee. Houtverouderde wyn bevat nie net gekondenseerde tanniene van die druiweskil nie, maar ook hidroliseerbare tanniene van die houtvate waarin dit verouder word. Die polifenoliese aard van die aromatiese ringe laat reaksies met elektrofiele toe. Dit vorm die basis van kleefmiddel vervaardiging, waar formaldehied gebruik word om tannien ekstrakte te polimeriseer om kleefmiddels te vorm. Ander kommersiële gebruike van tanniene sluit in anti-skuim agente in olie ontginning en die vervaardiging van amiene wat hars bevat (via die Mannich reaksie) vir water suiwerings toepassing (verwydering van swaar metale). Die produksie van mimosa gekondenseerde tanniene is ‘n volhoubare proses aangesien die bome elke agt jaar geoes word. Dis sal ‘n belangriker bron van chemiese roumateriaal vir dierevoeding word, soos wat die huidig gebruikte ru-olie bronne uitgeput raak. Dit skep ook werksgeleenthede in landelike areas. Hoër oligomerise gekondenseerde tanniene bestaan uit opeenvolgende eenhede van flavan-3- ol monomere via C-4 tot C-8 of C-4 tot C-6 inter-flavaniel bindings. Hoër oligomere kan nie met chromatografie gesuiwer word nie, en ander metodes van analise word benodig. Suur gekataliseerde splyting van inter-flavaniel bindings en die opvang van monomeriese intermediêre produkte met tolueen-α-thiol of floroglusinol, gevolg deur analise van die geïsoleerde produkte met HPLC, word normaalweg gebruik om gekondenseerde tannien samestellings te bepaal. Die analise van mimosa en quebracho tanniene word egter bemoeilik deur die resorsinol-tipe A-ring in hierdie verbindings. Die afwesighed van ‘n 5- OH groep maak die inter-flavaniel binding stabiel teen suur hidrolise. Die hoë temperature wat dus benodig word vir die hidrolise van die inter-flavaniel binding in mimosa en quebracho, lei tot ontbinding. Massa spektrometrie en 13C KMR (kern magnetise resonans) spektrometrie in oplossing is ook al met wisselende grade van sukses gebruik. Die analise van hidroliseerbare tanniene is selfs meer kompleks as die van gekondenseerde tanniene. As gevolg hiervan, bly die samestelling van gekondenseerde en hidroliseerbare tanien ekstakte onseker, selfs na 50 jaar se navorsing. Van besondere belang is die gemiddelde ketting lengte van tannien ekstrakte van verskillende bronne en die samestelling van die monomere wat dit vorm. In dié skripsie word die potensiaal ondersoek van vaste toestand KMR en elektrosproei massa spektrometrie om die ingewikkelde probleme van die tannien industrie op te los. Vaste toestand KMR is veral geskik om onoplosbare monsters te ondersoek, probleme met selektiewe ekstraksie, asook chemiese verandering gedurende ekstraksie en monster voorbereiding en onsekerheid oor verbindings wat nie geëkstraeer is nie, te oorkom. Elektrosproei spektrometrie komplimenteer MALFI-TOF massa spektrometrie deurdat molekules met ‘n massa laer as 500 Dalton gesien kan word. Ons kon al die resonansies in vaste toestand KMR van hidroliseerbare en gekonsentreede tanniene toeken deur die vergelyking tussen vloeibare en vaste toestand spektra van suiwer flavonoiëde en tannien ekstrakte. Dit het ons toegelaat om onomwonde te onderskei tussen gekondenseerde tanniene en hiroliseerbare tanniene deur eenvoudige roetine eksperimente, sonder langdradige chemiese toetse. ‘n Metode is ontwikkel om met sekerheid tussen mimosa en quebracho gekondenseerde tanniene te onderskei en hulle te identifiseer. Dié metode is die enigste beskikbare metode om quebracho te identifiseer, wat van belang is in wynkunde (quebracho tanniene word by wyn gevoeg). Quebracho kon tot nou toe slegs chemies geidentifiseer word, aangesien dit negatief toets in alle beskikbare toetse vir tanniene. Ons het vasgestel dat geen onoplosbare hoër oligomeriese gekondenseerde tanniene of tanniene kovalent gebind aan ander onoplosbare bas komponente, oorbly in die gebruikte mimosa bas nie (na ekstraksie van tanniene). Dit verskaf ‘n eenvoudige metode vir die wattel industrie om die gebruik van laer ekstraksie temperature en -tye na te vors, met geassosieerde energie besparing. ‘n Vingerafdruk-metode vir mimosa is ontwikkel en word reeds in die industrie gebruik (Aanhangsel A). Aangesien hars resonansies nie met tannien resonansies oorvleuel nie, kan die bas geanaliseer word sonder om ‘n ekstak te vervaardig. Die enigste voorbereiding behels om die bas fyn te maal (ongeveer 100 mg) en in die vaste toestand KMR rotor te pak. Aangesien koolstof via waterstof gemagnetiseer word, is minder as 30 minute KMR tyd per monster voldoende. Dit versaf ‘n maklike metode om die bas van quebracho, mimosa en hidroliseerbare tanniene te identifiseer. ‘n Vaste toestand KMR spektrum bewys dat die gebruikte bas nie net geen gekondenseerde tanniene bevat nie, maar staaf ook die afleiding dat gebruikte bas uit water-onoplosbare harse (polimere van glukose en ander suikers) bestaan. Ons glo dat hierdie metode aangewend kan word om nuwe bronne van tanniene van inheemse plante te identifiseer. Ons het die navorsing uitgebrei na gebreide leer en ‘n eenvoudige metode ontwikkel om te bepaal of die leer met mimosa, quebraco, italiaanse kastaiing, tara, kunsmatige brei materiaal, chroom of aluminium gebrei is. Ons glo dat hierdie metode deur die leer industrie gebruik kan word vir die bepaling van the tannien lading in gebreide leer. Deur elektrosproei massa spektrometrie te kombineer met gepubliseerde MALDI-TOF massa spektrometrie data, kan ons die relatiewe samestelling van die monomere, dimere en trimere bereken. Hierdie berekeninge word gebruik in die mimosa en quebracho tannien industrie om te voldoen aan die nuwe Europese Unie (EU) REACH (Registration, Evaluation, Authorisation and Restriction of Chemical substances) wetgewing. Sonder nakoming hiervan kan mimosa ekstrak nie na die EU uitgevoer word nie. Sulfitering (behandeling van mimosa en veral quebracho ekstrak met bisulfiet) word gereeld industrieël gebruik om die ekstrak se eienskappe te verbeter (bv. verhoogde water oplosbaarheid) en produkte met verskillende vlakke van sulfitering is kommersieël beskikbaar. Die chemiese veranderinge geassosieër met sulfitering bly spekulatief. Die vaste toestand KMR dui daarop dat die C-ring oopmaak tydens dié proses. Die elektrosproei MS bewys vir die eerste keer onomwonde die bestaan van gekondenseerde tannien-sulfonaat molekules. Die m/e waarde stem ooreen met die ring wat oopgaan en die invoeging van ‘n sulfonaat groep op die C-2 posisie. | af |
| dc.description.sponsorship | University of the Free State | en_ZA |
| dc.description.sponsorship | THRIP | en_ZA |
| dc.description.sponsorship | Mimosa Extract Company (Pty) Ltd. | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/11660/1638 | |
| dc.language.iso | en | en_ZA |
| dc.publisher | University of the Free State | en_ZA |
| dc.rights.holder | University of the Free State | en_ZA |
| dc.subject | Tannins | en_ZA |
| dc.subject | Mimosa | en_ZA |
| dc.subject | Bark | en_ZA |
| dc.subject | Dissertation (M.Sc. (Chemistry))--University of the Free State, 2011 | en_ZA |
| dc.title | A solid state NMR and MS characterisation of the chemical composition of mimosa bark extract | en_ZA |
| dc.type | Dissertation | en_ZA |