NATURAL POLYMERS MU SCI 11 2018 1 NATURAL POLYMERS Identification of the NTURAL POLYMERS RNDr. Ladislav Pospíšil, CSc. January 1/2018 Time schedule January 1/2018 NATURAL POLYMERS MU SCI 11 2018 2 LECTURE SUBJECT 1 Introduction to the subject – Structure & Terminology of nature polymers, literature 2 Derivatives of acids – natural resins, drying oils, shellac 3 Waxes 4 Plant (vegetable) gums, Polyterpene – natural rubber (extracting, processing and modification), Taraxacum_kok-saghyz 5 Polyphenol – lignin, humic acids 6 Polysaccharides I – starch 7 Polysaccharides II – celullose 8 Protein fibres I 9 Protein fibres II 10 Casein, whey, protein of eggs 11 Identification of natural polymers Laboratory methods of natural polymers’ evaluation 1.Melting Point 2.Separation Methods 1.Distillation 2.Chromatography 3.Electrophoresis 3.Spectroscopic Methods 1.Mass Spectroscopy 2.FTIR Spectroscopy 3.NMR Spectroscopy 4.UV Spectroscopy January 1/2018 NATURAL POLYMERS MU SCI 11 2018 3 Principal Problems of the Natural Compounds & Polymers Analysis and Determination January 1/2018 NATURAL POLYMERS MU SCI 11 2018 4 •They are chemical Individuals •They can Place of the Plant (Source Cultivation, e.g Resins •The Influence of the „Aging“, e.g. Drying Oils •The Natural Compounds are frequently found as a Mixtures with the other Natural Substances (Polymers) •The only small Quantities are frequently available for the Analysis •……………….. January 1/2018 NATURAL POLYMERS MU SCI 11 2018 5 Metling Point The Behaviour after heating on Microscope’s Hot Stage it is possible to differ between particular Natural Polymers (Oligomers) accordingly: • Waxes – 50 – 90 °C • Resins - from approx. 120 °C up • Polymerised Oils - from approx. 160 °C up • Egg Yellow - from approx. 200 °C up This PRELIMINARY differentiated Samples is possible to analyse using the more Sophisticated Methods Separation Methods January 1/2018 NATURAL POLYMERS MU SCI 11 2018 6 1.Distillation – e.g. Manufacturing of the Turpentine and Colophony form the Pine Rosin 2.Extraction – the Wood’s Rosins 3.Chromatography – Amino acids Separation using Thin Layer Chromatography (TLC) 4.Electrophoreses - Amino acids Separation using the Electric Field (is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field) 5. ……………………… • Separation Methods - Distillation January 1/2018 NATURAL POLYMERS MU SCI 11 2018 7 •Disadvantages: –The bigger Quantity of the Sample is needed –Not possible to use Solid State Materials –The Separation is not so „SHARP“ as the Chromatography •Advantages: –The bigger Quantity of the Sample is got, with which is possible to do further Work, –It is simple from the Instrumentation point of View, but there are the very complicated Machines also (e.g. Spin Band Column) Separation Methods - Chromatography January 1/2018 NATURAL POLYMERS MU SCI 11 2018 8 •Disadvantages : –The bigger Quantity of the Sample is NOT got, with which is possible to do further Work ( the so called PREPARATIVE Chromatography is not widespread now), •It is NOT simple from the Instrumentation point of View •Advantages : –The bigger Quantity of the Sample is NOT needed –The Separation is VERY „SHARP“ up to Individuals –It is possible to use Solid State Materials, if they are Soluble in the proper Solvent –A lot of the Chromatography Methods are available now, a lot of the Detection Methods are possible to be used – Chromatography – the basic Methods January 1/2018 NATURAL POLYMERS MU SCI 11 2018 9 •Paper (probably the oldest one) •TLC – Thin Layer Chromatography •Gas (GC) •Liquid (HPLC) •Gel permeation (GPC) •Ion exchange •…….. January 1/2018 NATURAL POLYMERS MU SCI 11 2018 10 Chromatographic Terms ANALYTIES - složky vzorku, které mají být chromatograficky rozděleny Analytic Chromatography – chromatografie sloužící k zjištění existence analytu (tzv. kvalitativní stanovení) a k určení jeho koncentrace ve vzorku (tzv. kvantitativní stanovení). Chromatograph – přístroj sloužící k chromatografické separaci složek vzorku Chromatogram – záznam z chromatografu znázorňující jednotlivé analyty nejčastěji ve formě tzv. chromatografických píků (zón) oddělených navzájem základní linií Chromatographic Separation – rozdělení vzorku na jednotlivé složky (analyty) na základě rozdílné distribuce mezi mobilní a stacionární fázi Mobil Phase – neboli eluent, je fáze pohybující se chromatografickým systémem. Tato fáze přivádí vzorek do stacionární fáze, kde dochází k jeho separaci Retention Time – čas, který složka potřebuje k průchodu chromatografickým systémem Preparative Chromatography – slouží k izolaci čistých (nebo alespoň čistějších) složek vzorku, které jsou dále použity (k chemické reakci, další separaci apod.) Stacionary Phase – je fáze ukotvená na místě, přes kterou prochází mobilní fáze a také složky vzorku. Jde např. o tenkou vrstvu silikagelu (při tenkovrstevné chromatografii) či kolona. Zde dochází k separaci v důsledku distribuce vzorku mezi stacionární a mobilní fázi January 1/2018 NATURAL POLYMERS MU SCI 11 2018 11 Classification of the Chromatographic Method according to the Arrangement Column chromatography Paper chromatography Thin layer chromatography (TLC) January 1/2018 NATURAL POLYMERS MU SCI 11 2018 12 Classification of the Chromatographic Method according to the Arrangement Column chromatography is a separation technique in which the stationary bed is within a tube. The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open tubular column). Differences in rates of movement through the medium are calculated to different retention times of the sample. January 1/2018 NATURAL POLYMERS MU SCI 11 2018 13 Classification of the Chromatographic Method according to the Arrangement Column chromatography Column_chromatography_sequence.png Cromatografia_su_colonna.jpg January 1/2018 NATURAL POLYMERS MU SCI 11 2018 14 Rozdělení chromatografických metod podle uspořádání Thin layer chromatography (TLC) is a widely employed laboratory technique used to separate different biochemicals on the basis of their size and is similar to paper chromatography. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate. TLC is very versatile; multiple samples can be separated simultaneously on the same layer, making it very useful for screening applications such as testing drug levels and water purity. Possibility of cross-contamination is low since each separation is performed on a new layer. Compared to paper, it has the advantage of faster runs, better separations, better quantitative analysis, and the choice between different adsorbents. For even better resolution and faster separation that utilizes less solvent, high-performance TLC can be used. An older popular use had been to differentiate chromosomes by observing distance in gel (separation of was a separate step). January 1/2018 NATURAL POLYMERS MU SCI 11 2018 15 Classification of the Chromatographic Method according to the Arrangement Thin layer chromatography (TLC) 800px-TLC_-_isomers.jpg TLC_black_ink.jpg 933px-Tlc_sequence_svg.png January 1/2018 NATURAL POLYMERS MU SCI 11 2018 16 Classification of the Chromatographic Method according to the Arrangement Paper chromatography is an analytical method used to separate colored chemicals or substances. It is primarily used as a teaching tool, having been replaced by other chromatography methods, such as thin-layer chromatography. A paper chromatography variant, two-dimensional chromatography involves using two solvents and rotating the paper 90° in between. This is useful for separating complex mixtures of compounds having similar polarity, for example, amino acids. The setup has three components. The mobile phase is a solution that travels up the stationary phase, due to capillary action. The mobile phase is generally an alcohol solvent mixture, while the stationary phase is a strip of chromatography paper, also called a chromatogram. A chromatographic method is called adsorption chromatography if the stationary phase is solid January 1/2018 NATURAL POLYMERS MU SCI 11 2018 17 Classification of the Chromatographic Method according to the Arrangement Paper chromatography PAPER Chromatography_tankWIKI ENG 08022018.png January 1/2018 NATURAL POLYMERS MU SCI 11 2018 18 Classification of the Chromatographic Techniques according to the by physical state Mobile Phase GC, gas chromatography) Mobile Phase is a Gas. It is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. LC, HPLC, liquid chromatography) Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid and the Stacionary Phase is the Solid State Material, alternatively Liquid bonded on the Solid State Material. January 1/2018 NATURAL POLYMERS MU SCI 11 2018 19 img800.jpg TLC Chromatography – without any Pre-treatment of the Samples (e.g. By Hydrolysis) TLC Chromatography of WAXE’S Fresh Oil of the Unknown Origin January 1/2018 NATURAL POLYMERS MU SCI 11 2018 20 img799.jpg TLC Chromatography – after the Pre-treatment of the Samples (here are the Amino acids treated by Hydrolysis) TLC Chromatography of Amino acids and some hydrolysed Proteins January 1/2018 NATURAL POLYMERS MU SCI 11 2018 21 img801.jpg GC of some Waxes after Saponification and Methylation of the acids to the Methyesters Bee Wax Punic Wax Ceresine January 1/2018 NATURAL POLYMERS MU SCI 11 2018 22 img802.jpg GC of some RESINS after Saponification and Methylation of the acids to the Methyesters Colophone Resin Binder – Painting on the high Altar (Santo Stefano, Venice, Italy) Resin Binder – Mosaic, 4. Century B.C. Wood Coal Tar Colophone after Heating (15 Minutes & 300 – 350 °C) Resin Binder – War Ship, 3. Century B.C. January 1/2018 NATURAL POLYMERS MU SCI 11 2018 23 img803.jpg GC of some Binder Oils after Pyrolysis at 600 °C Retention time (Minutes) Linseed/Flaxseed oil Eggs White Gelatin Mastix Damara January 1/2018 NATURAL POLYMERS MU SCI 11 2018 24 img804.jpg GC & Mass Spectroscopy 1 Eggs Yellow Cholesterol January 1/2018 NATURAL POLYMERS MU SCI 11 2018 25 img805.jpg GC & Mass Spectroscopy 2 Painting by P. Veronese (1570) Dihydroabietic acid methylester Electrophoresis January 1/2018 NATURAL POLYMERS MU SCI 11 2018 26 •Elektroforéza je soubor separačních metod, které využívají k dělení látek jejich odlišnou pohyblivost ve stejnosměrném elektrickém poli. Na principu rozdílných elektroforetických mobilit se při ní dělí nabité molekuly (ionty). •V roce 1892 bylo publikováno, že anorganické částice v koloidním roztoku pod vlivem elektrického pole nenáhodně putují. Nedlouho poté byl tento jev popsán i u proteinů ve vodných roztocích. •V roce 1948 byl Nobelovou cenou oceněn švédský chemik Arne Tiselius, který ve 30. letech minulého století postavil aparaturu separující proteiny krevního séra na základě jejich elektroforetických mobilit. • Electrophoresis 1 January 1/2018 NATURAL POLYMERS MU SCI 11 2018 27 •Electrophoresis (from the Greek "Ηλεκτροφόρηση" meaning "to bear electrons") is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. •Electrophoresis of positively charged particles (cations) is called cataphoresis, while electrophoresis of negatively charged particles (anions) is called anaphoresis. •Electrophoresis is a technique used in laboratories in order to separate macromolecules based on size. The technique applies a negative charge so proteins move towards a positive charge. •Suspended particles have an electric surface charge, strongly affected by surface adsorbed species, on which an external electric field exerts an electrostatic Coulomb force. According to the double layer theory, all surface charges in fluids are screened by a diffuse layer of ions, which has the same absolute charge but opposite sign with respect to that of the surface charge. • Electrophoresis 2 January 1/2018 NATURAL POLYMERS MU SCI 11 2018 28 1280px-Motion_by_electrophoresis_of_a_charged_particle_svg.png January 1/2018 NATURAL POLYMERS MU SCI 11 2018 29 Capilary Gel Electrophoresis is a process which enables the sorting of molecules based on size. Using an electric field, molecules (such as DNA) can be made to move through a gel made of AGAROSE or POLYACRYLAMIDE. The electric field consists of a negative charge at one end which pushes the molecules through the gel, and a positive charge at the other end that pulls the molecules through the gel. The molecules being sorted are dispensed into a well in the gel material. The gel is placed in an electrophoresis chamber, which is then connected to a power source. When the electric current is applied, the larger molecules move more slowly through the gel while the smaller molecules move faster. The different sized molecules form distinct bands on the gel. Gel electrophoresis uses a gel as an anticonvective medium and/or sieving medium during electrophoresis, the movement of a charged particle in an electrical field. Gels suppress the thermal convection caused by application of the electric field, and can also act as a sieving medium, retarding the passage of molecules; gels can also simply serve to maintain the finished separation, so that a post electrophoresis stain can be applied. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. Gel Electrophoresis is the most wide spread Electrophoresis Method now. January 1/2018 NATURAL POLYMERS MU SCI 11 2018 30 Gel_electrophoresis_apparatus.JPG Agarose-Gelelektrophorese.png Electrophoresis FTIR Spectroscopy January 1/2018 NATURAL POLYMERS MU SCI 11 2018 31 •Advantages: –Dosti univerzální technika (pevné látky, kapaliny, plyny, roztoky, KBr technika, vícenásobný odraz, …) –Malé množství vzorku –Možnost spojení s mikroskopií –……………. •Disadvantages: –Instrumentálně i vzdělanostně náročné –Spektrum závisí i na technice měření –…………….. FTIR Spectroscopy January 1/2018 NATURAL POLYMERS MU SCI 11 2018 32 •Advantages: –General-purpose (universal) Method (Solid State Materials, Liquids, Gases, Solutions, KBr Technique, Multireflection technique. ……….. –Small Quantity of the Sample is needed –It is possible to be connected to Optical Microscope –……………. •Disadvantages: –It is demanding as to Operator’s Qualification and Instrumentation –Spectrum depends on the Measurement Technique also – ………………. January 1/2018 NATURAL POLYMERS MU SCI 11 2018 33 detSvR_PI_IR_1105 FTIR Spectroscopy is possible to be connected to Optical Microscope January 1/2018 NATURAL POLYMERS MU SCI 11 2018 34 FTIR connected to Optical Microscope – AN EXAMPLE (see previous slide, please) January 1/2018 NATURAL POLYMERS MU SCI 11 2018 35 FTIR connected to Optical Microscope – AN EXAMPLE (see BEFORE LAST slide, please) January 1/2018 NATURAL POLYMERS MU SCI 11 2018 36 FTIR Spectrum SHELAC img789.jpg January 1/2018 NATURAL POLYMERS MU SCI 11 2018 37 FTIR Spectrum SANDARAK img788.jpg January 1/2018 NATURAL POLYMERS MU SCI 11 2018 38 FTIR Spectrum DAMARA img790.jpg January 1/2018 NATURAL POLYMERS MU SCI 11 2018 39 FTIR Spectrum MASTIX img791.jpg January 1/2018 NATURAL POLYMERS MU SCI 11 2018 40 FTIR Spectrum COPAL img792.jpg January 1/2018 NATURAL POLYMERS MU SCI 11 2018 41 FTIR Spectrum Amber (Baltic See Region) img793.jpg January 1/2018 NATURAL POLYMERS MU SCI 11 2018 42 FTIR Spectrum Camphor img794.jpg January 1/2018 NATURAL POLYMERS MU SCI 11 2018 43 FTIR Spectrum Arabic Gum img796.jpg January 1/2018 NATURAL POLYMERS MU SCI 11 2018 44 FTIR Spectrum NITROCELLULOSE img795.jpg January 1/2018 NATURAL POLYMERS MU SCI 11 2018 45 FTIR Spectrum Cellulose, NITROCELLULOSE and Cellulose triacetate January 1/2018 NATURAL POLYMERS MU SCI 11 2018 46 FTIR Spectrum Cellulose, Starch, Dextrin, Cellulose with Lignin January 1/2018 NATURAL POLYMERS MU SCI 11 2018 47 FTIR Spectrum Cellulose (Paper) and Cellulose (Jute Canvas) January 1/2018 NATURAL POLYMERS MU SCI 11 2018 48 FTIR Spectrum Kasein and Kasein Glue (Ca Salt) NMR Spectroscopy January 1/2018 NATURAL POLYMERS MU SCI 11 2018 49 •Advantages: –Hydrogen, Carbon etc. Spectra can be measured –Detail Information about the Molecular Structure –The many Simulation Software's (Methods) are available now –……………. •Disadvantages: –It is demanding as to Operator’s Qualification and Instrumentation –MOSTLY it is necessary to work with Liquid or Solution, but Solid State NMR is also possible –….. January 1/2018 NATURAL POLYMERS MU SCI 11 2018 50 Comparative prediction of the 13C NMR spectrum of sucrose using various methods. Experimental spectrum is in the middle. Upper spectrum (black) was obtained by empirical routine. Lower spectra (red and green) were obtained by quantum-chemical calculations in PRIRODA and GAUSSIAN respectively. Included information: used theory level/basis set/solvent model, accuracy of prediction (linear correlation factor and root mean square deviation), calculation time on personal computer (blue GlycoNMR_simulation NMR WIKI ENG.jpg