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

2. 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

3. 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

4. -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

5. 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.

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

7. 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.

8. 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 •

9. 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 ?

10. 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

11. Gas Liquid Chromatography

12. 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

13. Gas Chromatogram of High Grade Petrol

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. 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

21. SOME NATURAL PRODUCTS ISOLATED AND PURIFIED BY CHROMATOGRAPHIC TECHNIQUE
Engleromycin acetate

22. 19,20-Dihydroxycytochalasin C

23. Cytochalasin D

24. THE END

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

26. 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

27. 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

28. Emission Spectra of Elements
Continuous
Sodium
Hydrogen
Calcium

29. Absorption Spectra Sodium
For other Spectra, click on the hyperlink below:

30. 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.

31. 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.

32. 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)

34. 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

35. 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

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

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

38. 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?

40. Atomic Absorption Spectroscopy

41. Absorption Wavelengths of Iron

42. Atomic Absorption Spectrophotometer (AAS)

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

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

45. 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

46. 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

47. 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)

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

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

50. 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.

52. 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 nm nm nm O R A N G E Y E L LO W
BLUE
GREEN
RED
420nm
520 nm
600nm

53. 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 °C

54. 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.

55. 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

56. 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.

57. INFRA- RED SPECTROSCOPY

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

59. 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

60. 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.

61. 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

62. For instance the localized vibrations of the methylene group are