Betuliinijohdannaiset

Opinnäytetyön aiheena oli betuliinijohdannaiset. Tavoitteena oli tehdä synteesisarja, jossa betuliiniin ja betulonihappoon liitettiin esteröintireaktiolla erilaisia happoja, terpeenialkoholeja ja fenoleja. Lisäksi tavoitteena oli saada selvyys bioaktiivisista ominaisuuksista ja mahdollisista muista soveltuvuuksista. Työ tehtiin osana VTT:n Kuori- tutkimusprojektia, jossa betuliinille ja muille puiden kuori- ja uuteaineille pyritään löytämään kaupallisia käyttökohteita esimerkiksi lääkeaineina, pestisideinä ja kosmetiikka-alan raaka-aineina. Betuliini on pentasyklinen triterpeenialkoholi, jota saadaan eristettyä varsin tehokkaasti koivun kuoresta ja tuohesta uuttamalla. Betuliinilla ja sen johdannaisilla on todistettu olevan useita lääkinnällisiä vaikutuksia kuten esimerkiksi tulehduksia estäviä, antiviraalisia, antimalaarisia, antitumoraalisia sekä anti-HIV-aktiivisuutta. Betuliinissa, lup-20(29)-ene-3β,28-diolissa, on käytännössä kolme reaktiivista ryhmää, primäärinen hydroksyyliryhmä hiilessä 28, sekundäärinen hydroksyyliryhmä hiilessä 3 sekä alkeenirakenne hiilessä 20, joista jokaisen ryhmän reaktioilla voidaan helposti saada aikaan betuliinijohdannaisia. Betuliinin ja betulonihapon reaktioihin valittiin erilaisia esteröintimenetelmiä, joissa katalyytteinä käytettiin mm. p-tolueenisulfonihappoa, titaani(IV)isopropylaattia, N,N-dimetyyli-4-aminopyridiini ja N-(3-dimetyyliaminopropyyli)-N-etyylikarbodiimidihydroklori-dia. Synteesituotteiden puhdistusmenetelminä käytettiin uudelleen kiteyttämistä ja pylväskromatografiaa. Synteesituotteet analysoitiin NMR:llä, GC-MS:llä, IR-spektrometrillä ja alkuaineanalyysillä. Lisäksi reaktiotuotteista mitattiin sulamispisteet ja Rf-arvot. Synteesisarjaan suunnitelluista betuliini- ja betulonihappojohdannaisista onnistuivat betuliini-28-ferulihappoesteri, betulonihappo-28-vaniliiniesteri ja betulonihappo-28-karva-kroliesteri. Tuotteille on tehty ja tullaan tekemään erilaisia bioaktiivisuustestejä ja näin ollen kaikkia tuloksia ei ole vielä saatu. Osassa testeistä kyseiset tuotteet ovat osoittautuneet jo lievästi aktiivisiksi, joten kyseisten tuotteiden testaamista sekä betuliiniin ja sen johdannaisiin liittyvää syntetiikkaa ja tutkimustyötä kannattaa ehdottomasti jatkaa, koska betuliinijohdannaisilla voi olla ratkaiseva merkitys esimerkiksi sairauksien hoidossa.

Odor emission control and reduce in betaine manufacture

This thesis was made in Naantali plant of Finnfeeds Finland Oy. In this thesis the main study was in reducing, controlling, measuring and processing odour effluents in various methods. Also are considered legislation, marketing issues and environmental requirements of reducing of odour effluents. The literature review introduces odours complications, legislations and various methods of odour removal. There is also a review of volatile organic compounds detection and measuring methods. The experimental section consists TD-GC-MS-measurements and expansive measurements with electronic nose. Electronic nose is a new solution for recognition and measuring industrial odours. In this thesis the electronic nose was adapted into reliable recognition and measuring method. Measurements with electronic nose was made in betaine factory and main targets were odour removal process and other odours from factory. As a result of experimental work with TD-GC-MS-measurements becomes odour compound of 2-and 3- methylbutanal and dimethyldisulfide, which odour is sweet and fug. Extensive study with electronic nose found many developmental subjects. Odour balance measurements of factory and after calculation made adjustment of odour removal process, over all odour effluent to environment will reduce 25 %.

Fractionation of Wood Extracts

The aim of this thesis was to fractionate wood extracts into pure fractions using membrane technology, to observe membrane behaviour in solvents and to study the effect of conditioning on membrane performance. Four different wood extracts were used in the performed filtrations. In the literature part, the focus was on the effect of different solvent properties on polymeric membranes, especially on their retention and flux. Solute properties, such as shape, polarity and charge, were examined. Transport models, membrane stability and ways to improve the stability, when solvents were filtered, were also discussed. The experimental part consisted of a series of filtrations, where the effect of the wood extracts and solvent concentration on solute retention was observed. Polymeric and ceramic membranes were tested under different conditions and the solute analyses were performed with GC and GC-MS. It was discovered that it is possible to fractionate wood extracts using membrane technology to some extent, but more research must be done to understand the mechanisms behind the various interactions between the solvent and the membrane. Conditioning was also considered as an important part of the membrane pre-treatment.

Cuticular and Suberin Polymers of Edible Plants. Analysis by Gas Chromatographic-Mass Spectrometric and Solid State Spectroscopic Methods

Cutin and suberin are structural and protective polymers of plant surfaces. The epidermal cells of the aerial parts of plants are covered with an extracellular cuticular layer, which consists of polyester cutin, highly resistant cutan, cuticular waxes and polysaccharides which link the layer to the epidermal cells. A similar protective layer is formed by a polyaromatic-polyaliphatic biopolymer suberin, which is present particularly in the cell walls of the phellem layer of periderm of the underground parts of plants (e.g. roots and tubers) and the bark of trees. In addition, suberization is also a major factor in wound healing and wound periderm formation regardless of the plants’ tissue. Knowledge of the composition and functions of cuticular and suberin polymers is important for understanding the physiological properties for the plants and for nutritional quality when these plants are consumed as foods. The aims of the practical work were to assess the chemical composition of cuticular polymers of several northern berries and seeds and suberin of two varieties of potatoes. Cutin and suberin were studied as isolated polymers and further after depolymerization as soluble monomers and solid residues. Chemical and enzymatic depolymerization techniques were compared and a new chemical depolymerization method was developed. Gas chromatographic analysis with mass spectrometric detection (GC-MS) was used to assess the monomer compositions. Polymer investigations were conducted with solid state carbon-13 cross polarization magic angle spinning nuclear magnetic resonance spectroscopy (13C CP-MAS NMR), Fourier transform infrared spectroscopy (FTIR) and microscopic analysis. Furthermore, the development of suberin over one year of post-harvest storage was investigated and the cuticular layers from berries grown in the North and South of Finland were compared. The results show that the amounts of isolated cuticular layers and cutin monomers, as well as monomeric compositions vary greatly between the berries. The monomer composition of seeds was found to differ from the corresponding berry peel monomers. The berry cutin monomers were composed mostly of long-chain aliphatic ω-hydroxy acids, with various mid-chain functionalities (double-bonds, epoxy, hydroxy and keto groups). Substituted α,ω-diacids predominated over ω-hydroxy acids in potato suberin monomers and slight differences were found between the varieties. The newly-developed closed tube chemical method was found to be suitable for cutin and suberin analysis and preferred over the solvent-consuming and laborious reflux method. Enzymatic hydrolysis with cutinase was less effective than chemical methanolysis and showed specificity towards α,ω-diacid bonds. According to ₁₃C CP-MAS NMR and FTIR, the depolymerization residues contained significant amounts of aromatic structures, polysaccharides and possible cutan-type aliphatic moieties. Cultivation location seems to have effect on cuticular composition. The materials studied contained significant amounts of different types of biopolymers that could be utilized for several purposes with or without further processing. The importance of the so-called waste material from industrial processes of berries and potatoes as a source of either dietary fiber or specialty chemicals should be further investigated in detail. The evident impact of cuticular and suberin polymers, among other fiber components, on human health should be investigated in clinical trials. These by-product materials may be used as value-added fiber fractions in the food industry and as raw materials for specialty chemicals such as lubricants and emulsifiers, or as building blocks for novel polymers.

Analytical methods for analyzing organic peroxides

Työn tarkoituksena oli kehittää analyyttinen erotusmenetelmä eräässä valmistusprosessissa käytettävän hapettavan aineen ja liuottimen välillä syntyvien reaktiotuotteiden tutkimiseen ja analysoimiseen. Lisäksi tarkoituksena oli tutkia prosessiolosuhteiden turvallisuutta. Kirjallisuusosassa käsitellään erilaisia orgaanisia peroksideja, niiden käyttötarkoituksia ja niiden käyttöön liittyviä huomioitavia asioita. Lisäksi tarkastellaan yleisimpiä analyysimenetelmiä, joita on käytetty erilaisten peroksidien analysoinnissa. Näitä analyysimenetelmiä on useimmiten käytetty nestemäisten näytteiden tutkimuksissa. Harvemmin on analysoitu kaasu- ja kiintoainenäytteitä. Kokeellisessa osassa kehitettiin kirjallisuuden perusteella peroksidiyhdisteille identifiointimenetelmä ja tutkittiin prosessin näytteet. Analyysimenetelmiksi valittiin iodometrinen titraus ja HPLC-UV-MS-menetelmä. Lisäksi käytettiin peroksidimittaukseen soveltuvia testiliuskoja. Tutkimus osoitti, että iodometrisen titrauksen ja testiliuskojen perusteella näytteissä oli vähäisiä määriä peroksideja viikon jälkeen peroksidilisäyksestä. HPLC-UV-MS-analyysien perusteella näytteiden analysointia häiritsi selluloosa, jota löytyi jokaisesta näytteestä.

Ionic liquid mediated biomass deconstruction: from analysis challenges to fermentable sugars

Ionic liquids, ILs, have recently been studied with accelerating interest to be used for a deconstruction/fractionation, dissolution or pretreatment processing method of lignocellulosic biomass. ILs are usually utilized combined with heat. Regarding lignocellulosic recalcitrance toward fractionation and IL utilization, most of the studies concern IL utilization in the biomass fermentation process prior to the enzymatic hydrolysis step. It has been demonstrated that IL-pretreatment gives more efficient hydrolysis of the biomass polysaccharides than enzymatic hydrolysis alone. Both cellulose (especially cellulose) and lignin are very resistant towards fractionation and even dissolution methods. As an example, it can be mentioned that softwood, hardwood and grass-type plant species have different types of lignin structures leading to the fact that softwood lignin (guaiacyl lignin dominates) is the most difficult to solubilize or chemically disrupt. In addition to the known conventional biomass processing methods, several ILs have also been found to efficiently dissolve either cellulose and/or wood samples – different ILs are suitable for different purposes. An IL treatment of wood usually results in non-fibrous pulp, where lignin is not efficiently separated and wood components are selectively precipitated, as cellulose is not soluble or degradable in ionic liquids under mild conditions. Nevertheless, new ILs capable of rather good fractionation performance have recently emerged. The capability of the IL to dissolve or deconstruct wood or cellulose depends on several factors, (e.g. sample origin, the particle size of the biomass, mechanical treatments as pulverization, initial biomassto-IL ratio, water content of the biomass, possible impurities of IL, reaction conditions, temperature etc). The aim of this study was to obtain (fermentable) saccharides and other valuable chemicals from wood by a combined heat and IL-treatment. Thermal treatments alone contribute to the degradation of polysaccharides (e.g. 150 °C alone is said to cause the degradation of polysaccharides), thus temperatures below that should be used, if the research interest lies on the IL effectiveness. On the other hand, the efficiency of the IL-treatment can also be enhanced to combine other treatment methods, (e.g. microwave heating). The samples of spruce, pine and birch sawdust were treated with either 1-Ethyl-3-methylimidazolium chloride, Emim Cl, or 1-Ethyl-3-methylimidazolium acetate, Emim Ac, (or with ionized water for comparison) at various temperatures (where focus was between 80 and 120 °C). The samples were withdrawn at fixed time intervals (the main interest treatment time area lied between 0 and 100 hours). Double experiments were executed. The selected mono- and disaccharides, as well as their known degradation products, 5-hydroxymethylfurfural, 5-HMF, and furfural were analyzed with capillary electrophoresis, CE, and high-performance liquid chromatography, HPLC. Initially, even GC and GC-MS were utilized. Galactose, glucose, mannose and xylose were the main monosaccharides that were present in the wood samples exposed to ILs at elevated temperatures; in addition, furfural and 5-HMF were detected; moreover, the quantitative amount of the two latter ones were naturally increasing in line with the heating time or the IL:wood ratio.

Kaasukromatografi-massaspektrometrin käyttö haihtuvien orgaanisten yhdisteiden analytiikassa ja kynureniinipolun kuuden metaboliitin analysointi solunäytteistä

Haihtuvat orgaaniset yhdisteet (eng. volatile organic compound, VOC) ovat yksi yleisimmistä ja laajimmalle levinneistä ympäristökontaminaattiryhmistä. VOC- yhdisteryhmän yhdisteet ovat määritelmän mukaisesti haihtuvia, molekyylimassaltaan pieniä (16-250 Da) yhdisteitä, joista suurin osa on joko haitallisia tai myrkyllisiä. VOC- yhdisteet pääasiallisesti emittoituvat ympäristöön ihmisen toiminnasta johtuen (teollisuus, autot, maatalous) ja päätyvät luonnossa vesistöihin ja maaperään. Ihmisille haitallisten ominaisuuksien lisäksi, VOC-yhdisteet vaikuttavat esimerkiksi ilmaston lämpenemiseen ja savusumujen syntyyn. Edellä mainittujen ominaisuuksien vuoksi on tärkeää analysoida VOC-yhdisteiden pitoisuuksia. Haihtuvien orgaanisten yhdisteiden ryhmän laajuus, ja fysikaalisten sekä kemiallisten ominaisuuksien (poolisuus, höyrynpaine, vesiliukoisuus) erot asettavat haastetta niiden analysointiin. Yleisimmin VOC-yhdisteitä analysoidaan kaasukromatografia- massaspektrometrin avulla. Pro gradu -tutkielman kirjallisessa osassa käydään läpi VOC-yhdisteiden analytiikassa käytettyjä erilaisia GC-MS-laitekokonaisuuksia ja niiden ominaisuuksia. Lisäksi keskitytään VOC-yhdisteiden erilaisiin näytteenkäsittelymenetelmiin vesi-, maa- ja sedimettinäytteissä. Kokeellisessa osassa analysoitiin kuutta kynureniinipolun metaboliittia solunäytteistä. Kynureniinipolku on nisäkkäillä tärkein tryptofaanin katabolinen polku. Kynureniinipolku aktivoituu entsymaattisesti esimerkiksi tulehdusten, hermostoa rappeuttavien prosessien ja immuunivasteen aikana. Kynureniinipolun yhdisteiden uskotaan lisäävän solun toksisuutta, mutta parantavan sen kykyä lisääntyä ja vähentävän solukuolleisuutta. Esimerkiksi 3-hydroksikynureniinin lisääntynyt määrä on yhdistetty hermostoperäisiin sairauksiin, kuten Huntingtonin- ja Parkinsonin tautiin. Kokeellisessa osassa luotiin yhdistespesifinen MRM-menetelmä, ultra korkean erotuskyvyn nestekromatografi-sähkösumutusionisaatio- kolmoiskvadrupolimassaspektrometrille. Luodulla ja optimoidulla menetelmällä kvantitoitiin soluviljelmänäytteistä samanaikaisesti L-kynureniini-, kynureniinihappo-, 3-hydroksikynureniini-, antraniilihappo-, 3-hydroksiantraniilihappo-, sekä kinoliinihappo-pitoisuudet sisäisen- ja ulkoisen standardin menetelmällä. Solunäytteiden päämetaboliiteiksi havaittiin kynureniini, kunyreniinihappo, sekä antraniilihappo. Ainoastaan 3-hydroksikynureniinihappoa ja kinoliinihappoa ei havaittu yhdestäkään näytteestä.

Chemical changes in biomass during torrefaction

Torrefaction is moderate thermal treatment (~200-300 °C) of biomass in an inert atmosphere. The torrefied fuel offers advantages to traditional biomass, such as higher heating value, reduced hydrophilic nature, increased its resistance to biological decay, and improved grindability. These factors could, for instance, lead to better handling and storage of biomass and increased use of biomass in pulverized combustors. In this work, we look at several aspects of changes in the biomass during torrefaction. We investigate the fate of carboxylic groups during torrefaction and its dependency to equilibrium moisture content. The changes in the wood components including carbohydrates, lignin, extractable materials and ashforming matters are also studied. And at last, the effect of K on torrefaction is investigated and then modeled. In biomass, carboxylic sites are partially responsible for its hydrophilic characteristic. These sites are degraded to varying extents during torrefaction. In this work, methylene blue sorption and potentiometric titration were applied to measure the concentration of carboxylic groups in torrefied spruce wood. The results from both methods were applicable and the values agreed well. A decrease in the equilibrium moisture content at different humidity was also measured for the torrefied wood samples, which is in good agreement with the decrease in carboxylic group contents. Thus, both methods offer a means of directly measuring the decomposition of carboxylic groups in biomass during torrefaction as a valuable parameter in evaluating the extent of torrefaction. This provides new information to the chemical changes occurring during torrefaction. The effect of torrefaction temperature on the chemistry of birch wood was investigated. The samples were from a pilot plant at Energy research Center of the Netherlands (ECN). And in that way they were representative of industrially produced samples. Sugar analysis was applied to analyze the hemicellulose and cellulose content during torrefaction. The results show a significant degradation of hemicellulose already at 240 °C, while cellulose degradation becomes significant above 270 °C torrefaction. Several methods including Klason lignin method, solid state NMR and Py-GC-MS analyses were applied to measure the changes in lignin during torrefaction. The changes in the ratio of phenyl, guaiacyl and syringyl units show that lignin degrades already at 240 °C to a small extent. To investigate the changes in the extractives from acetone extraction during torrefaction, gravimetric method, HP-SEC and GC-FID followed by GC-MS analysis were performed. The content of acetone-extractable material increases already at 240 °C torrefaction through the degradation of carbohydrate and lignin. The molecular weight of the acetone-extractable material decreases with increasing the torrefaction temperature. The formation of some valuable materials like syringaresinol or vanillin is also observed which is important from biorefinery perspective. To investigate the change in the chemical association of ash-forming elements in birch wood during torrefaction, chemical fractionation was performed on the original and torrefied birch samples. These results give a first understanding of the changes in the association of ashforming elements during torrefaction. The most significant changes can be seen in the distribution of calcium, magnesium and manganese, with some change in water solubility seen in potassium. These changes may in part be due to the destruction of carboxylic groups. In addition to some changes in water and acid solubility of phosphorous, a clear decrease in the concentration of both chlorine and sulfur was observed. This would be a significant additional benefit for the combustion of torrefied biomass. Another objective of this work is studying the impact of organically bound K, Na, Ca and Mn on mass loss of biomass during torrefaction. These elements were of interest because they have been shown to be catalytically active in solid fuels during pyrolysis and/or gasification. The biomasses were first acid washed to remove the ash-forming matters and then organic sites were doped with K, Na, Ca or Mn. The results show that K and Na bound to organic sites can significantly increase the mass loss during torrefaction. It is also seen that Mn bound to organic sites increases the mass loss and Ca addition does not influence the mass loss rate on torrefaction. This increase in mass loss during torrefaction with alkali addition is unlike what has been found in the case of pyrolysis where alkali addition resulted in a reduced mass loss. These results are important for the future operation of torrefaction plants, which will likely be designed to handle various biomasses with significantly different contents of K. The results imply that shorter retention times are possible for high K-containing biomasses. The mass loss of spruce wood with different content of K was modeled using a two-step reaction model based on four kinetic rate constants. The results show that it is possible to model the mass loss of spruce wood doped with different levels of K using the same activation energies but different pre-exponential factors for the rate constants. Three of the pre-exponential factors increased linearly with increasing K content, while one of the preexponential factors decreased with increasing K content. Therefore, a new torrefaction model was formulated using the hemicellulose and cellulose content and K content. The new torrefaction model was validated against the mass loss during the torrefaction of aspen, miscanthus, straw and bark. There is good agreement between the model and the experimental data for the other biomasses, except bark. For bark, the mass loss of acetone extractable material is also needed to be taken into account. The new model can describe the kinetics of mass loss during torrefaction of different types of biomass. This is important for considering fuel flexibility in torrefaction plants.