Biochemical instrumental analysis-2

1. Biochemical instrumental analysis-2 Dr. Maha Al-Sedik

3. Atomic absorption spectrophotometer

4. Atomic absorption spectrophotometer • Atomic absorption spectroscopy is a quantitative method of analysis that is applicable to many metals such as Ca , Fe , Al and copper. • Only a drop of sample is needed. • Atomic absorption is so sensitive that it can measure down to parts per billion of a gram in a sample.

5. In AAS, the sample is atomised – ieconverted into free atoms in the vapourstate.

6. The Atomisation Process of converting an analyte in solid, liquid or solution form to a free gaseous atom.

9. Principle: • Atoms of different elements absorb characteristic wavelengths of light. • The metal vapor absorbs energy from an external light source, and electrons jump from the ground to the excited states. • The greater the number of atoms in the vapor, the more radiation is absorbed.

10. How it works? • For example with lead, a lamp containing lead emits light from excited lead atoms that produce the right mix of wavelengths to be absorbed by any lead atoms in the sample. • Beam of electromagnetic radiation emitted from the lamp is passed through the vaporised sample. Some of the radiation is absorbed by the lead atoms in the sample.

12. The greater the number of atoms in the vapor, the more radiation is absorbed. • OR • The amount of light absorbed is proportional to the number of lead atoms.

13. Components

14. Components : • The light source • flame • Monochromater • photomultiplier tube • Detector

15. The light source: hollow cathode lamp hollow cathode lamp for Aluminum (Al) is shown below

16. hollow cathode lamp: • This contains a tungsten anode and a cylindrical hollow cathode made of the element to be determined. These are sealed in a glass tube filled with an inert gas – egneon or argon. • The shape of the cathode concentrates the radiation into a beam which passes through window.

17. The cathode lamps are stored in a compartment inside the AA spectrometer. The specific lamp needed for a given metal analysis is rotated into position for a specific experiment.

18. The flame: • A flame is created, usually using ethyne & oxygen (fuel) • The flame gases flowing into the burner create a suction that pulls the liquid into the small tube from the sample container. • This liquid is transferred to the flame where the sample is atomized [mixing the sample with air to create fine droplets]. The metal atoms then absorb light from the source (cathode lamp).

19. The Manganese Flame

20. The Potassium Flame

21. The Copper Flame

22. The Calcium Flame

23. Monochromater: • the monochromator is used to select a particular wavelength of light for observation). • photomultiplier tube: • The intensity of the light is fairly low, so a photomultiplier tube (PMT) is used to strengthen the signal intensity. • It can multiply the current produced by incident light by as much as 100 million times.

25. photomultiplier tube

26. Detector: • Transform light to electrical impulses. • The electricity is directly proportional to the intensity of light.

27. http://www.youtube.com/watch?v=_KZjb9G3hB8irst.pptx http://www.youtube.com/watch?v=HBegTB_WDxQ

28. Flameless atomic absorption spectrophotometry • It differs from atomic absorption spectrophotometer in how the compounds are atomized. • In AAS a flame is used to produce individual atoms. • In the other it is carried out by putting the sample in a small graphite tube and passing an electrical current through to heat it.

29. Summary of atomic absorption spectrophotometry : • Atomic absorption spectrophotometer is used in the measurement of many metal such as Ca , Fe , Al and copper. • Only a drop of sample needed. • Atomic absorption is so sensitive that it can measure down to parts per billion of a gram in a sample.

30. Principle: • Atoms of different elements absorb characteristic wavelengths of light to be transformed to the exited state. • The greater the number of atoms in the vapor, the more radiation is absorbed. • Components : • The light source, • Flame • Monochromater • Photomultiplier tube • Detector

31. Function of each component: • Light source: produce the right mix of wavelengths to be absorbed by specific atoms in the sample. • Flame: Atomization of the sample. • Monochromator: selection a particular wavelength of light for observation). • photomultiplier tube: used to strengthen the signal intensity. • Detector:transform light to electrical impulses.

32. Differences between atomic absorption spectrophotometer and the flameless atomic absorption spectrophotometer: • They differ in how the compounds are atomized. • In one case a flame is used to produce individual atoms. • In the other it is carried out by putting the sample in a small graphite tube and passing an electrical current through to heat it.