3. Dr. Mohamed Abd-Elhakeem Faculty of Biotechnology Experimental Biochemistry Chapter 1 Lecture 2

4. γ-rayschanges in nuclear structure X-rays Inner-shell electronic transition UV-radiation Outer-shell electron transitions Visible region atoms and molecules. MicrowaveRotation of nucleus

5. IR radiation: vibrations of atoms in molecules

6. UV- Vis Spectrophotometry:

7.  Different wavelengths of light correspond to different colors  All colors blended together is called white light  The absence of all light is black  Light of slightly shorter wavelengths (higher energy) is ultraviolet. THEORY

8. WAVELENGTH OF VISIBLE LIGHT AND COLOR WAVELENGTHCOLOR PERCEIVED 380-430Violet 430-475Blue 475-495Greenish Blue 495-505Bluish Green 505-555Green 555-575Yellowish Green 575-600Yellow 600-650Orange 650-780Red

9.  When light shines on a solution, it may pass through – be transmitted – or  Some or all of the light energy may be absorbed according to the matter chemical composition

10.  Absorption occurs when the specific amount of energy (light of distinct wavelength) is absorbed by an electron resulting in a transition to an excited state.  The part of the molecule that cause absorption called “Chromophore”  The selected wavelength is used in the instrument

11. THE ABSORPTION OF LIGHT AND COLOR OF SOLUTIONS WAVELENGTH OF LIGHT ABSORBED COLOR OF LIGHT ABSORBED COLOR OF SOLUTION 380-430Violet Y ellow 430-475Blue O range 505-555Green R ed 575-600Yellow Violet 600-650Orange B lue 650-780Red G reen Matter absorb specific light (wavelength) and appear in a complementary color

12. The color intensity increase as the concentration of the molecule increases. The amount of light that blocked by the fixed amount of solution is called absorbance

13. The Beer-Lambert Law The Beer-Lambert law sortof has the wrong name… Pierre Bouguer (1698-1758) Johan Lambert (1728-1777) € Absorption coefficient C Concentration L Path length

14. UV- Vis Spectrophotometer Instrument parts

15. Spectrophotometer: it is an instrument that use the electromagnetic radiation to measure the absorbance that is directly proportion with concentration

16. UV-Visible Spectroscopy: Instrumentation The Major Components Are: A light source A monochromator A detector A Sample holder

17. Light Sources Xenon, Mercury/Xenon Flash Arc-Lamps – light generated from Xe plasma Pure Xenon has very wide emission spectrum ~200 – 1200 nm

18. UV/Visible Spectroscopy: Light Sources Deuterium D 2 gas is discharged by contact with a high voltage tungsten cathode Continuous spectrum from ~150 nm - ~370 nm Usually used in conjunction with a tungsten/halogen source, which handles the visible spectrum

19. Monochromators The light sources we use produce continuous emission spectra. But we need single wavelengths, so we need monochromator

20. Sample Compartments/Holders These days, sample compartments are designed to accept accessories… The sample itself is held in a cuvette, usually plastic or quartz:

21. The detector Silicon diode: Basically a solar cell – light ionizes n-doped (phosphate) silicon, placing the electrons in the conduction band (i.e. having a voltage).

22. Usage of spectrophotometer Scanning: Spectrophotometer is used to scan a sample in wide range of wavelengths (for example from 200 to 800nm). This is carried out to obtain the wavelength that has the maximum absorbance of this sample (lambda (λ) max)

23. Quantitative analysis using Spectrophotometer This part will be discussed in practical part

24. Spectrofluorometers Both molecular structure and chemical environment influence whether a substance will or will not fluorescent. The fluorometer is used mainly to measure the fluorescent organic materials in a solution contains many other non-fluorescent compounds.

25. Theory Organic compound that usually contains many aromatic rings absorbs UV light of specific wavelength and emit visible light at characteristic wavelength. In fluorometer there are Excitation wavelength: Emission wavelength: to measure the concentration of compound that directly proportion with the light intensity.

26. The two wavelengths are determined by scanning the different wavelengths of fluorescent light emitted by a sample a under a constant excitation wavelength, which is called the emission spectrum. And also by scanning different wavelengths of excitation light a sample under a constant emission wavelength, which is called the excitation spectrum

27. Components of Fluorometers and Spectrofluorometers  Sources: A more intense source in needed than the tungsten of hydrogen lamp.  Lamps: The most common source for filter fluorometer is a low-pressure mercury vapor lamp equipped with a fused silica window. For spectrofluorometers, a 75 to 450-W high-pressure xenon arc lamp in commonly employed.  Lasers: Most commercial spectrofluorometers utilize lamp sources because they are less expensive and less troublesome to use.

28. Components of Fluorometer

29. Many organic compounds such as amino acids can be converted to fluorescence derivatives by reacting with dansyl chloride or ortho phthalaldehyde.

30. Atomic Spectroscopy

31. (UV-VIS) spectroscopy deals with inorganic or organic molecules in solutions. Atomic Spectroscopy is used for Metal Analysis in a solutions contain very low concentration of metal (ppm or ppb) ppm: part per million (mg/L) ppb: part per billion (µg/L)

32.  (UV-VIS) causes outer electrons to transit from low to high energy states  In atomic spectroscopy, higher energy radiation is used to transit outer or inner electrons from low to high energy states  High energy radiation is provided by: (a) Flame in flame atomic absorption spectroscopy (FAA) (b) X-rays in X-ray fluorescence spectroscopy (XRF)

33. Flame test

34. 33

35. 34 The most outer electrons are excited by the heating This excited configuration is unstable, and the electrons "fall" back to their normal positions of lower energy. As the electrons return to their normal levels, the energy that was absorbed is emitted in the form of electromagnetic energy. Some of this energy may be in the form of visible light.

36. The colour of this light can be used as a means of identifying the elements involved. Such crude analyses are known as flame tests. Only metals, with their loosely held electrons, are excited in the flame of a laboratory burner.

37. Types of Atomic Spectroscopy

38. 1. Absorption: light of a wavelength characteristic of the element of interest radiates through the atom vapor. The atoms absorb some of the light. The amount absorbed is measured. 2. Emission: sample is heated to excitation/ ionization of the sample atoms. Excited and ionized atoms decay to a lower energy state through emission. Intensity of the light emitted is measured. 3. Fluorescence: X- rays are absorbed by the sample atoms (solid & unheated), then a longer wavelength (lower energy) radiation characteristic of the element is emitted and measured

39. Source: R. Thomas, “Choosing the Right Trace Element Technique,” Today’s Chemist at Work, Oct. 1999, 42.

40. Detection limits (ppm = mg L-1) The detection limit is the smallest amount of an element that can be reliably measured

41. X-ray florescence

42. AASAESX-ray Sample TypeWater solution Solid (powder or sheet) Excitation sourceFlame + hollow cathod lamp FlameX-rays Detection limitsppbppm multiple elements detection No Yes

43. Atomic Absorption instrument

45.  Liquid sample is aspirated to become aerosols of fine particles (nebulization)  Flame vaporizes the aerosols (atomization)  Elevated temperatures in a flame excite the electrons  Excited atoms absorb light of specific energy  Beer’s law is used to calculate concentration

46.  Basic instrument components: 1. Light source: hollow cathode lamp (HCL) of the element being measured. Provides the spectral line for the element of interest. Inside the lamp, filled with argon or neon gas, is a cylindrical metal cathode containing the metal for excitation, and an anode. When a high voltage is applied across the anode and cathode, gas particles are ionized. As voltage is increased, gaseous ions acquire enough energy to eject metal atoms from the cathode. Some of these atoms are in an excited states and emit light with the frequency characteristic to the metal.

47. 2. Nebulizer and atomizer: the nebulizer sucks up the liquid sample, creates a fine aerosol, mixes the aerosol with fuel/air. Flame creates vaporized atoms.

48. 3. Monochromator: Isolates light of various wavelengths that produced from light source and flame. 4. Detector:

50. End of Chapter 1 Chapter 2: Chromatography