APPLICATION OF EXTRACTION AND CHROMATOGRAPHIC METODS FOR SEPARATION OF BIOACTIVE COMPOUNDS

Biološko aktivne snovi so substance, ki imajo efekt na tkiva živih organizmov. V diplomski nalogi smo raziskovali različne separacijske procese za izolacijo takih komponent iz rastlinskih materialov. Osredotočili smo se predvsem na ekstrakcijske in kromatografske metode separacije in primerjali smo konvencionalne metode z metodami s superkritičnimi fluidi. Najprej smo opravili visokotlačne ekstrakcije (ekstrakcije s superkritičnim fluidom — SFE) bučne pogače s propanom. Eksperimente smo izvedli pri različnih pogojih, opazovali smo kinetiko in končni izkoristek bučnega olja vsake superkritične ekstrakcije. Po teh eksperimentih smo bučno pogačo ekstrahirali še z različnimi organskimi topili (petroleter in etanol). Primerjali smo izkoristke med posameznimi tipi ekstrakcij in preverili ali so prisotna organska topila v ekstraktu. Ko smo vse ekstrakcije opravili, smo različne metode med seboj na splošno primerjali. Poskušali smo določiti, kakšne so prednosti in slabosti ene in druge in dejanska uporaba v industriji. V drugem delu diplomske naloge smo se ukvarjali s kromatografskimi postopki separacije. Izvedli smo konvencionalno kolonsko kromatografijo surovega lecitina pri različnih temperaturah (25 °C, 40 °C in 60 °C). Iz surovega lecitina, v katerem je veliko fosfolipidov, smo poskušali skoncentrirati fosfatidilholin, ki je pomemben za žive organizme, saj sestavlja celične membrane. Kakor je SFE alternativa konvencionalnim ekstrakcijam, je kromatografija s superkritičnimi fluidi (SFC) alternativa konvencionalni kolonski kromatografiji. V naslednji fazi smo zato raziskovali parametre, ki so pomembni za pripravo metode separacije s SFC. Uporabili smo superkritični CO2 in se pri tem seznanili o njegovih lastnostih. Po opravljenih eksperimentih smo različne kromatografije primerjali med seboj in razpravljali o potencialni uporabi v industriji.Bioactive compounds are substances with effect on livng tissue. In diploma thessis varius separation processes for isolation of bioactive compounds from plant materials were explored. Research work was focused on extraction and chromatographic separation methods and conventional methods were compared with methods using SCF. First high-pressure extractions (supercritical fluid extractions – SFE) of pumkin cake with propane were done. Our experiments were carried out at varius conditions and cinetics and final yields of pumpkin oil of each SFE was observed. After those experiments extration of pumpkin oil from pumkin cake with organic solvents (petroleum benzine and ethanol) was made. The yield of each type of ectraction was compared and it was checked whether there was any organic solvent left in extract. After all extractions were done, varius methods were compared and advantages and disadvantages and actual application in the industry were discussed In second part of the diploma thessis chromatographic prccedures of separation were studied. Frst conventional column chromatography of raw lecithin at varius temperatures (25 °C, 40 °C and 60 °C) was carried out. The aim was to concentrate phosphatidilchlin (component of cell membranes), from raw lecithin, which is rich with phosholipids. As SFE in an alternative to conventional extracion, the supercritical chromatografy (SFC) presents an alternative to conventional column chromatography. In next phase the parameters, important for design of separation methods with SFC, were explored. In our experiments SF-CO2 was used and its propreties were observed. After all experiments were done, varius types of chromatography in general were compared and potential applications in industrial scale were discussed

Supercritical chromatography in preparative and production scale

V doktorski disertaciji smo se najprej seznanili s superkritičnimi fluidi in visokotlačnimi separacijskimi procesi. Pri pregledu literature smo ugotovili, da se največ raziskav nanaša na izolacijo biološko aktivnih komponent iz rastlinskih materialov s pomočjo superkritične ekstrakcije. S pregledom literature smo ugotovili, da superkritični fluidi omogočajo separacijo številnih komponent iz rastlinskih materialov. Superkritični fluidi so v večini primerov stisljivi mediji, ki so pri sobnih pogojih plini, s čimer dobimo produkte, kjer ni toksičnih organskih topil. Zaradi tega porabimo manj energije in je hkrati manj emisij v ozračje. Oboje pa izrazito pripomore k trajnostnemu razvoju. Superkritična ekstrakcija, kot tudi ostale ekstrakcijske metode, ni povsem selektivna, saj z želenimi biološko aktivnimi komponentami ekstrahiramo še druge komponente. Selektivno ločevanje lahko opravimo s kromatografskimi tehnikami, kot so tekočinska kromatografija, plinska kromatografija in superkritična kromatografija. Slednja je relativno nova metoda, ki se je razvila šele v zadnjem desetletju. V doktorski disertaciji smo raziskali uporabo superkritične kromatografije v analitskem merilu. Na analitski napravi za superkritično kromatografijo smo na testnih komponentah teofilin in kofein, ki sta obe farmakološki učinkovini, preiskovali posamezne vplive procesnih parametrov na separacijo. V nadaljevanju smo dobljene rezultate vključili v raziskavo povečevanja obsega in zagon pilotne preparativne naprave za superkritično kromatografijo, ki je po velikosti med največjimi podobnimi napravami v svetovnem merilu. Na analitski napravi za superkritično kromatografijo smo na koloni s silicijevim dioksidom raziskovali vpliv tlaka, vpliv temperature, vpliv sestave mobilne faze in vpliv posameznih lastnosti stacionarne faze. V superkritični kromatografiji se najpogosteje uporablja CO2 v superkritičnem stanju. Pri separaciji polarnih molekul k nepolarnemu CO2 dodamo so-topilo (modifikacijsko sredstvo mobilne faze). Najpogosteje uporabimo kot so-topilo etanol, metanol, acetonitril, 2-propanol idr. Ugotovili smo, da sta vpliva tipa so-topila in koncentracije so-topila v mobilni fazi med vsemi najbolj izrazita. Kar pomeni, da se kromatografski parametri pri tem najbolj izrazito spremenijo. Vpliva tlaka in temperature na separacijo s superkritično kromatografijo sta manj izrazita, ampak še vedno pomemben faktor, mnogo bolj kakor v tekočinski kromatografiji. Vse ugotovitve posameznih vplivov smo uporabili za zagon pilotne preparativne naprave za superkritično kromatografijo. Pred tem smo spoznali vse sestavne dele in preučili delovanje. Nato smo določili področje delovanja naprave, glede na omejitve naprave in glede na lastnosti superkritičnih fluidov. Pri pogojih, pri katerih smo na pilotni preparativni napravi izvedli separacijo smo opravili številne raziskave. Kljub temu, da smo predhodno dosegli separacijo obeh testnih komponent na analitski napravi za SFC, pa pri podobnih pogojih na pilotni preparativni napravi za superkritično kromatografijo separacije nismo dosegli. Zaradi tega smo izvedli še dodatne eksperimente. S spreminjanjem posameznih procesnih parametrov smo dosegli separacijo z delno prekrivajočimi vrhovi, ki jih je bilo kljub prekrivanju mogoče ločeno zbirati v zbiralne posode naprave. Na podlagi separacije testnih komponent smo pripravili metodo za separiranje biološko aktivnih komponent iz rastlinskih ekstraktov.In the thesis supercritical fluids and high pressure separation processes were examined. According to literature review the most researches apply to supercritical fluid extraction and isolation of bioactive compound from plant material. Supercritical fluids are in the most cases compressible media, gasses at ambient temperature, therefore products without residues of toxic organic solvents are obtained. Consumption of energy and environment imprint is significantly lower, which greatly contribute to sustainable development. Supercritical extraction isolates variety of compounds from plant material. In order to separate these compounds from each other various chromatographic techniques, such as liquid chromatography, gas chromatography and supercritical fluid chromatography have been applied in the past. Supercritical fluid chromatography is relatively new technique, which was well developed in last decade. In the thesis, supercritical fluid chromatography has been investigated in analytical scale for two test compounds caffeine and theophylline, in order to determine the influence of the process parameters on the chromatographic parameters. Obtained results were used for scale up onto the preparative and production scale, which apparatus is one of the bigger worldwide. Influences of the pressure, temperature, type and concentration of the modifier, and type of the stationary phases have been studied. In supercritical fluid chromatography CO2 is the most frequently solvent used as the mobile phase. If polar compounds are separated, polar modifier, such as methanol, ethanol acetonitrile, iso-propanol etc., are added to the CO2. Chromatographic parameters are the most significantly influenced, when modifier concentration is increased. Pressure and temperature are less significant factor for separation, however it is more important as in liquid chromatography. Afterwards, experiments on analytical scale were conducted, investigations on operating the pilot apparatus were preformed. Regarding on results of the separation on the analytical scale apparatus and regarding on constrains of the equipment, range of process conditions have been determined for test compounds. Several experiments were conducted on pilot preparative scale apparatus at given process conditions. Even though, separation was achieved on analytical scale, such separation was not possible on pilot preparative scale apparatus at similar process conditions. Additionally, several experiments were conducted on the pilot apparatus. With altering the process conditions successful separation has been achieved. However chromatographic signals, which belong to detected compounds, were overlaying. Even though, fractionation was possible. Regarding on obtained results, this method could be applied for separation of real system, such as plant material extracts

Supercritical fluid chromatography and scale up study

The influence of process parameters on supercritical fluid chromatography (SFC) and scale-up on to the preparative scale was investigated. In this scope, dependency of pressure, temperature, type and concentration of modifier, and type of stationary phase on the separation were examined separately. Experiments were performed on silica stationary phases in the range of pressures from 100 bar to 250 bar and temperatures from 30°C to 65°C, with use of ethanol and methanol as modifier. Results obtained on analytical scale were used for scale up on preparative (to production) scale. The aim of this study is presentation of the state-of-the-art of SFC through separate effects of process parameters on the separation and investigation of development, benefits and drawbacks of scaling-up process of the SFC.Raziskali smo vpliv procesnih parametrov na kromatografijo s superkritičnimi fluidi (SFC) skupaj z povečevanjem obsega na preparativno merilo. V tem okviru smo posamezno raziskali odvisnost tlaka, temperature, tipa in koncentracije so-topila, ter tipa stacionarne faze na separacijo testnih komponent (kofeina in teofilina) s SFC. Eksperimente smo izvedli na napravah za SFC v analitičnem in pilotnem preparativnem merilu. Separacije smo izvajali na dveh stacionarnih fazah: čisti silica in silica 2-etilpiridina. Kot mobilno fazo smo uporabili $CO_2$/metanol ali $CO_2$/etanol pri povišanih tlakih in temperaturah. Rezultate eksperimentov iz SFC naprave v analitičnem merilu smo uporabili za povečevanje obsega na preparativno merilo. Cilj študije je bila evaluacija vpliva procesnih parametrov na separacijo testnih komponent in praktični prikaz povečevanja obsega in obratovanja pilotne preparativne naprave, ki sodi med večje podobne naprave v globalnem merilu

To ventilate or not to ventilate during bystander CPR — A EuReCa TWO analysis

Background: Survival after out-of-hospital cardiac arrest (OHCA) is still low. For every minute without resuscitation the likelihood of survival decreases. One critical step is initiation of immediate, high quality cardiopulmonary resuscitation (CPR). The aim of this subgroup analysis of data collected for the European Registry of Cardiac Arrest Study number 2 (EuReCa TWO) was to investigate the association between OHCA survival and two types of bystander CPR namely: chest compression only CPR (CConly) and CPR with chest compressions and ventilations (FullCPR). Method: In this subgroup analysis of EuReCa TWO, all patients who received bystander CPR were included. Outcomes were return of spontaneous circulation and survival to 30-days or hospital discharge. A multilevel binary logistic regression analysis with survival as the dependent variable was performed. Results: A total of 5884 patients were included in the analysis, varying between countries from 21 to 1444. Survival was 320 (8%) in the CConly group and 174 (13%) in the FullCPR group. After adjustment for age, sex, location, rhythm, cause, time to scene, witnessed collapse and country, patients who received FullCPR had a significantly higher survival rate when compared to those who received CConly (adjusted odds ration 1.46, 95% confidence interval 1.17–1.83). Conclusion: In this analysis, FullCPR was associated with higher survival compared to CConly. Guidelines should continue to emphasise the importance of compressions and ventilations during resuscitation for patients who suffer OHCA and CPR courses should continue to teach both

To ventilate or not to ventilate during bystander CPR : a EuReCa TWO analysis

Background: Survival after out-of-hospital cardiac arrest (OHCA) is still low. For every minute without resuscitation the likelihood of survival decreases. One critical step is initiation of immediate, high quality cardiopulmonary resuscitation (CPR). The aim of this subgroup analysis of data collected for the European Registry of Cardiac Arrest Study number 2 (EuReCa TWO) was to investigate the association between OHCA survival and two types of bystander CPR namely: chest compression only CPR (CConly) and CPR with chest compressions and ventilations (FullCPR). Method: In this subgroup analysis of EuReCa TWO, all patients who received bystander CPR were included. Outcomes were return of spontaneous circulation and survival to 30-days or hospital discharge. A multilevel binary logistic regression analysis with survival as the dependent variable was performed. Results: A total of 5884 patients were included in the analysis, varying between countries from 21 to 1444. Survival was 320 (8%) in the CConly group and 174 (13%) in the FullCPR group. After adjustment for age, sex, location, rhythm, cause, time to scene, witnessed collapse and country, patients who received FullCPR had a significantly higher survival rate when compared to those who received CConly (adjusted odds ration 1.46, 95% confidence interval 1.17–1.83). Conclusion: In this analysis, FullCPR was associated with higher survival compared to CConly. Guidelines should continue to emphasise the importance of compressions and ventilations during resuscitation for patients who suffer OHCA and CPR courses should continue to teach both