A TECHNIQUE FOR ISOLATION AND PURIFICATION OF NATURAL PRODUCTS CHROMATOGRAPHY A TECHNIQUE FOR ISOLATION AND PURIFICATION OF NATURAL PRODUCTS

Objectives: Explain the terms Natural products and chromatography Discuss the types of chromatography Emphasis the role of stationary phase in the chromatographic process Display some natural products isolated and purified by chromatographic technique

Natural products is An aspect of organic chemistry that deals with organic compounds found in nature, their derivatives and their bioactivity. Involves the development of compounds as -agrochemicals e.g. pesticides, -phamaceutics

-the perfume industry e. g -the perfume industry e.g. soaps, perfumes, detergents, body lotions, etc. - the food and beverage industry, e.g flavanoids, etc. -the petrochemical industry

Chromatography is A technique exploiting the interaction of the components of a mixture with a stationary phase and a mobile phase (solvent) in order to separate the components. Components are separated by different levels of adsorption to the stationary phase and solubility in the the mobile phase. In particular the adsorption to the stationary phase and the solubility in the mobile phase depends on the physical properties of the components present in the mixture.

Types of Chromatography Paper Chromatography and Thin Layer Chromatography (TLC) Column Chromatography Gas Liquid Chromatography (GLC) High Performance Liquid Chromatography (HPLC)

Thin Layer (and Paper) Chromatography TLC plates are inert supports (glass, plastic, aluminium) with a thin veneer of chromatographic media (silica,etc…) Apply a concentrated drop of sample (•) with a capillary or dropping tube to bottom of plate (origin pencil line) • Stand plate in a sealed vessel. carefully add solvent (keep solvent level below sample). • Allow solvent to adsorb up the plate, drawing the sample with it.

Thin Layer (and Paper) Chromatography The ratio of distance travelled by the component (from origin) compared with the distance travelled by the solvent front (from origin) is called the Rf value. Solvent front x • a Rf of = a/x b c Rf of = b/x Rf of = c/x •

Thin Layer and Paper Chromatography A solution of a mixture is applied as a spot/band at the bottom of the plate and allowed to travel with the solvent up the plate. Mixed standards Unknown + standards standards • • • A B C A+B+C A+B+C ?

Column Chromatography A mixture is applied to a solid support in a chromatography column, and eluted by a solvent. Elute with solvent 1 2 3 4 Absorbent medium tap Cotton wool plug

Gas Liquid Chromatography

Gas Liquid Chromatography A mixture is injected into a very thin“steel-jacketed” chromatography column. Inject sample as gas or liquid. A solid component can be dissolved in solvent but a solvent peak will also be seen. Inject sample dense liquid (on solid) SP Gas mobile phase Column in oven up to approx. 300 C. Substance must be able to vaporise and not decompose Extremely sensitive Elute with inert gas FID detector

Gas Chromatogram of High Grade Petrol

High Performance Liquid Chromatography (HPLC) A mixture is injected into a “steel-jacketed” chromatography column and eluted with solvent at high pressure (4000psi or approx 130 atm). Inject sample as gas or liquid. A solid component can be dissolved in solvent but a solvent peak will also be seen. Elute with solvent UV detector

STATIONARY PHASES The surface of the stationary phase can be altered to create a surface wirh different bonding properties in TLC, column chromatography, GLC and HPLC. Normal Polarity Reverse Polarity Ion Exchange Size Exclusion

STATIONARY PHASES (NORMAL POLARITY) Silica or alumina possess polar sites that interact with polar molecules. silica Polar Group Components elute in increasing order of polarity. From least polar to most polar

STATIONARY PHASES (REVERSE POLARITY) If the polar sites on silica or alumina are capped with non-polar groups, they interact strongly with non-polar molecules. silica C18 phase Components elute in decreasing order of polarity. From most polar to .least polar

STATIONARY PHASES (CATION EXCHANGE) Silica is substituted with anionic residues that interact strongly with cationic species (+ve charged) Cations exchange Na+ silica +ve charged species adhere to the support and are later eluted with acid (H+) From least +ve to most +ve

STATIONARY PHASES (ANION EXCHANGE) Silica is substituted with cationic residues that interact strongly with anionic species (-ve charged) Anions exchange Cl- silica -ve charged species adhere to the support and are later eluted with acid (H+) From least -ve to most -ve

STATIONARY PHASES (SIZE EXCLUSION) Size exclusion gels separate on the basis of molecular size. Individual gel beads have pores of set size, that restrict entry to molecules of a minimum size. Large molecules elute fast (restricted path), while small molecules elute slowly (long path length) From smaller molecules to larger molecules

SOME NATURAL PRODUCTS ISOLATED AND PURIFIED BY CHROMATOGRAPHIC TECHNIQUE Engleromycin acetate

19,20-Dihydroxycytochalasin C

Cytochalasin D

THE END

Regions of the Electromagnetic Spectrum Light waves all travel at the same speed through a vacuum but differ in frequency and, therefore, in wavelength.

Spectroscopy Absorption: Emission: Utilises the Absorption and Emission of electromagnetic radiation by atoms Absorption: Low energy electrons absorb energy to move to higher energy level Emission: Excited electrons return to lower energy states

Absorption v. Emission Energy is emitted by electrons returningto lower energy levels 3rd Excited States 2nd 1st Energy is absorbed as electrons jump to higher energy levels Ground State

Emission Spectra of Elements Continuous Sodium Hydrogen Calcium

Absorption Spectra Sodium For other Spectra, click on the hyperlink below: http://www.achilles.net/~jtalbot/data/elements/index.html

Light absorbed (Absorption) Concentration The Spectroscopic Techniques are based on the fact that Light absorbed (Absorption) is directly proportional to the Concentration of the absorbing component.

An introduction to Colorimetry Colorimetry is a quantitative technique which makes use of the intensity in colour of a solution is directly related to the concentration of the coloured species in it. Colorimetry can be used if the substance to be analysed is coloured, or if it can be made coloured by a chemical reaction. The concentration of the unknown solution can be estimated by the naked eye by comparing its colour to those of a series of standard solutions prepared by successive dilution. However at low concentrations, colour may not be detected.

A more accurate quantitative analysis can be made using an instrument called a Colourimeter. The light source of a kind that will be absorbed by the solution, ie if the solution is blue then light of a colour other than blue will be absorbed by it. Simple colourimeters allow a choice of three wavelengths using blue, green and red Light Emitting Diodes (LEDs)

In this example, the blue solution would absorb red (or green) and reflect blue. The chosen red LED is passed through the a transparent plastic or glass cell (cuvet) of fixed pathlength (1cm) containing the blue solution to be investigated and a Detector measures the amount of light absorbed measured. Red LED Green LED Blue LED Detector measures red light absorbed

Collect data Absorbance Concentration 0.0 0.125 0.250 0.380 0.50 unknown 0.00 A set zero adjustment enables the instrument to factor out any absorbance of the solvent and the material the cuvet is made from. 0.20 0.40 0.59 0.78 0.35 concentration of a species in solution is proportional to the light absorbed

Note that graphs may not be linear over a wide range of concentrations

Note that graphs may not be linear over a wide range of concentrations

The concentration of an unknown solution of a food colouring can be determined by measuring its absorbance and reading the concentration from the calibration graph. Using the data in the graph above, if a sample of this food colouring was found to have an absorbance of 0.35, then its concentration would be ______ M. Questions What would happen to absorbance if the path length of the cuvet was doubled? What would happen if the cuvet was handled on the transparent outer surface?

Atomic Absorption Spectroscopy

Absorption Wavelengths of Iron

Atomic Absorption Spectrophotometer (AAS)

Gas Mixture Adjustment AAS Operation Hollow Cathode Lamp Gas Mixture Adjustment Controls Display Monochromator Flame

Atomic Absorption Spectrometer Atomised sample in flame Detector Monochromator Lens Lens Hollow Cathode Lamp Flame Solution Amplifier Display

Close-up view of AAS Electrons return to ground state,and photons emitted in all directions Less energy is transmitted to detector Ions absorb energy, jump to excited state Hollow Cathode Lamp emits several unique wavelengths of light Ions in Flame Transmittance Transmittance

Atomic Absorption Spectrometry measures small concentrations of metal ions in solution Al, As, Au, B, Ca, Cd, Co, Cr, Cs, Cu, Fe, Ge, K, Li, Mg, Mn, Mo, Na, Ni, Pb, Si, Sr, Ti, V, W and Zn used by industry analysis of ores for metal content quality control of metals in steel testing water for metals ions analysing food and pharmaceuticals for metal ions

Advantages of using AAS very sensitive: can detect concentrations as small as a few parts to g / Litre (parts per billion) generally very specific: set wavelength is strongly absorbed by the particular metal ion being analysed (and not by other components)

A Source of Error Another species may be absorbing at the same wavelength.

UV-Visible Spectroscopy A UV-visible spectrophotometer measures the amount of energy absorbed by a sample.

The optics of the light source in UV-visible spectroscopy allow either visible [approx. 400nm (blue end) to 750nm (red end) ] or ultraviolet (below 400nm) to be directed at the sample under analysis.

Why are carrots orange? Carrots contain the pigment carotene which absorbs blue light strongly and reflects orange red and so the carrot appears orange. 400nm 500nm 600nm 700nm O R A N G E Y E L LO W BLUE GREEN RED 420nm 520 nm 600nm

Carotene beta-Carotene forms orange to red crystals and occurs in the chromoplasts of plants and in the fatty tissues of plant-eating animals. Molecular formula: C40H56 Molar Mass 537 Melting point 178 - 179 °C

Absorbance is set to 0% or light transmitted using a solvent blank in a cuvet. This compensates for absorbance by the cell container and solvent and ensures that any absorbance registered is solely due to the component under analysis. The sample to be analysed is placed in a cuvet (as for colorimetry). Qualitative analysis is achieved by determining the radiation absorbed by a sample over a range of wavelengths. The results are plotted as a graph of absorbance/transmittance against wavelength, which is called a UV/visible spectrum.

The UV- Visible absorption spectrum for carotene in the non-polar solvent, hexane I NT EN S I TY O F A B R P T N 400nm 700 nm ultra- violet visible infrared 320nm 460nm 540nm

Although the light absorbed is dependent on pathlength through the cell, a usual standard 1cm pathlength is used so that pathlength can effectively be ignored. Quantitative analysis is achieved in a manner similar to colorimetry. The absorption of a sample at a particular wavelength (chosen by adjusting a monochromator) is measured and compared to a calibration graph of the absorptions of a series of standard solutions. What can be analysed? In its quantitative form, UV-visible spectroscopy can be used to detect coloured species in solution eg. bromine , iodine and organic compounds or metal ions that are coloured, or can be converted into a coloured compound.

INFRA- RED SPECTROSCOPY

Regions of the Electromagnetic Spectrum Light waves all travel at the same speed through a vacuum but differ in frequency and, therefore, in wavelength.

The electromagnetic spectrum shows the different frequencies and energies at which various radiations occur The energy of most radiations ocurr within the infra-red region of the electromagnetic spectrum

The most useful vibrations occur in the narrow regions of 2.5-16µ (1 µm = 10-4 cm-1 ). The usual range of an IR spectrum is therefore 4000 cm-1 at the high frequency and 625cm-1 at the low frequency.

OBTAINING AN IR SPECTRUM A complex molecule has a large number of vibrational modes which involve the whole molecule To a good approximation, however, some of these molecular vibrations are associated with one another The localised vibrations are either stretching, bending, rocking, twisting or wagging

For instance the localized vibrations of the methylene group are