STPM Form 6 Chemistry - Analytical Techniques

1. Analytical Techniques

2. Paper Chromatography
• Stationary phase
• Solutes to be separated
• Mobile phase
• Solvent
• Solutes & Solvents have different
affinity
• Differences in molecular size,
solubility and adhesion of
macromalecules to chromatography
paper

4. Paper chromatography
• Ninhydrin is added as amino acid dye

5. Two dimensional Paper chromatography
Using 2 solvents for separation
Rotate 90 degrees, using
same/different solvent as
mobile phase

7. Paper electrophoresis
• Separation
of charged
molecules
• Using
electric
field

8. Paper electrophoresis of
Amino Acids
• Amino acids – Different isoelectric points
• Separation - Based on pH of mobile phase
• Above isoelectric point
– Amino acids = -ve charged >>> anode
• Below isoelectric point
– Amino acids = +ve charged >>> cathode
• Sprayed with Ninhydrin

10. X-ray Diffraction Analysis
• X-ray crystallography
• Study 3-D structures of macromolecules
• Coupled with spectrometer

11. X-ray diffractometer

12. Diffractometers, which rotate the crystal and scatter the X
ray from various planes of the atoms in the crystal.
The data produced by the scattering of the rays are then
analyzed by computer, allowing for the classification of
complex structures.

13. Results: The Electron
Density Map
• Atoms with higher atomic
numbers have more electrons
and therefore scatter x-rays
more effectively
• Hydrogen atoms often not
located exactly due to large
thermal motion and small size
• Electron density map provides
location of atoms relative to
each other
• Bond angles, bond lengths
may be determined

15. Structure of Virus Capsid

16. Structure of Enzyme

17. Structure of Ribosome

18. Cell fractionation

19. Cell Fractionation
Tissue is kept in cold, isotonic buffer
- Deactivate enzymes, Slow down metabolism, Prevent
digestion of organelles and autolysis

20. Homogenization disrupts tissue and releases cellular
components.
Mechanical separation- Plasma membrane (PM) is broken
by forcing the cell through a small passage or the cell can
be separated by an abrasive material
Sound waves- ultrasonic waves break the PM and release
its contents (An example is sonification, where a probe
emits high frequency sound waves that fracture a cell)
Chemicals/enzymes- detergents can be used to break a
PM by breaking apart the chemical bonds that hold together
the plasma membrane

21. Centrifuge setting (g) Time (min) Organelles
800 – 1000 5 – 10 Nucleus, Cell debris
20 000 15 – 20 Chlroplast, Mitochondria,
Lysosomes
100 000 60 ER fragments, Cell membrane
150 000 1800 Ribosomes
Pellet and
Supernatant

23. Light Microscopy
Light – Visible Light
Wavelength – 200 to 700nm
Maximum resolution = 200 nm
Maximum magnification = 1500x
Chloroplast
– 3000 nm, can be viewed
Ribosomes
– 20 nm, cannot be viewed

24. Phase Contrast Microscope
The phase contrast microscope uses the
fact that the light passing trough a
transparent part of the specimen travels
slower and, due to this is shifted compared
to the uninfluenced light.
This difference in phase is not visible to the
human eye.
However, the change in phase can be
increased to half a wavelength by a
transparent phase-plate in the microscope
and thereby causing a difference in
brightness.
This makes the transparent object shine
out in contrast to its surroundings.

25. Different refractive index between
materials allow contrast
Can study Living + Moving cells!!!
Without fixing and staining

26. Tobacco Mosaic Virus

28. Electron microscopy

29. Transmission
Electron
Microscope
1. Radiation – Electron beam
2. Wavelength – 0.005 nm
3. Maximum resolution – 1 nm
4. Takes place in vacuum to prevent
electron scaterring
5. Dead specimens are treated with heavy
metals (uranyl, lead acetate, osmium
tetroxide)
6. Highly stained = High electron absorption
7. Lightly stained = Low electron absorption

30. Electron
micrograph
Images produced are
captured on Fluorescent
screen / Photographic film

32. TEM vs SEM

33. TEM of cellular structures

35. Sperm flagellum

36. Scanning Electron Microscope
1. Radiation – Electron beam
2. Resolution – 5 nm higher than light
microscopy
3. 3 Dimensional view
4. Dead Specimens are coated with gold
5. Thicker and Larger specimen can be
examined

37. Pollen Ultrastructure

39. Compound Eye of Drosophilla sp.

40. Scanning Electron Microscope