2. 1 Instrumental Analytical Techniques An Overview of Chromatography and Spectroscopy

3. 2  Chromatographic Techniques -Thin layer and column chromatography -Gas Chromatography (GC) -High Performance Liquid Chromatography (HPLC)  Spectroscopic Techniques - Atomic Absorption Spectroscopy(AAS) - Colorimetry - UV-Visible Spectroscopy (UV-Vis)

4. 3 Chromatography 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.

5. 4 Column Chromatography Gas Liquid Chromatography (GLC) High Performance Liquid Chromatography (HPLC) Paper Chromatography and Thin Layer Chromatography (TLC) Types of Chromatography

6. 5 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.

7. 6 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 R f value. Solvent front x a b c R f of = a/x R f of = b/x R f of = c/x

8. 7 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. ABCA+B+C standards Mixed standards Unknown + standards A+B+C ? ?

9. 8 Column Chromatography A mixture is applied to a solid support in a chromatography column, and eluted by a solvent. Elute with solvent 2134 Absorbent medium Cotton wool plug tap

10. 9 Gas Liquid Chromatography

11. 10 Elute with inert gas Column in oven up to approx. 300 C. Substance must be able to vaporise and not decompose 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. FID detector Gas mobile phase dense liquid (on solid) SP Inject sample Extremely sensitive

12. 11 Gas Chromatogram of High Grade Petrol

13. 12 mixture of hydrocarbons eg petrol air mixture of alcohols Qualitative Must be able to be vaporised up to about 300 o C Must not decompose Quantitative Eg. How much ethanol is in the blood? known R f values under standard conditions Calibration graph of a series of standards of known concentration plotting area under peak vs concentration But

14. 13 Use a series of standards of ethanol to determine area under peak. Find area of unknown and read off concentration Construct calibration graph Area (or height at first approx.) is proportional to concentration.

15. 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). Elute with solvent UV detector Inject sample as gas or liquid.A solid component can be dissolved in solvent but a solvent peak will also be seen.

16. 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

17. 16 STATIONARY PHASES (NORMAL POLARITY) Silica or alumina possess polar sites that interact with polar molecules. Most polar…….Least polar Components elute in increasing order of polarity. Components elute in increasing order of polarity. Polar Group silica

18. 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. Most non-polar…….Least non-polar Components elute in decreasing order of polarity. Components elute in decreasing order of polarity. C 18 phase silica

19. 18 STATIONARY PHASES (CATION EXCHANGE) Silica is substituted with anionic residues that interact strongly with cationic species (+ve charged) Most +ve…….Least +ve +ve charged species adhere to the support and are later eluted with acid (H + ) +ve charged species adhere to the support and are later eluted with acid (H + ) Cations exchange Na + silica

20. 19 STATIONARY PHASES (ANION EXCHANGE) Silica is substituted with cationic residues that interact strongly with anionic species (-ve charged) Most -ve…….Least -ve -ve charged species adhere to the support and are later eluted with acid (H + ) -ve charged species adhere to the support and are later eluted with acid (H + ) Anions exchange Cl - silica

21. 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 minium size. Larger molecules…….Smaller molecules Large molecules elute fast (restricted path), while small molecules elute slowly (long path length) Large molecules elute fast (restricted path), while small molecules elute slowly (long path length)

22. 21 Regions of the Electromagnetic Spectrum Light waves all travel at the same speed through a vacuum but differ in frequency and, therefore, in wavelength.

23. 22 Spectroscopy 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

24. 23 Absorption v. Emission Ground State 1st 2nd 3rd Energy is absorbed as electrons jump to higher energy levels Energy is emitted by electrons returningto lower energy levels Excited States

25. 24 Emission Spectra of Elements Calcium Hydrogen Continuous Sodium

26. 25 Absorption Spectra Sodium http://www.achilles.net/~jtalbot/data/elements/index.html For other Spectra, click on the hyperlink below:

27. 26 The Spectroscopic Techniques are based on the fact that Light absorbed is directly proportional to the Concentration of the absorbing component. (Absorption)

28. 27 An introduction to Colorimetry Colorimetry can be used if the substance to be analysed is coloured, or if it can be made coloured by a chemical reaction. 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. 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.

29. 28 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)

30. 29

31. 30 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

32. 31  concentration of a species in solution is proportional to the light absorbed Absorbance Concentration 0.0 0.125 0.250 0.380 0.50 unknown  A set zero adjustment enables the instrument to factor out any absorbance of the solvent and the material the cuvet is made from. 0.00 0.20 0.40 0.59 0.35 0.78 Collect data

33. 32 Note that graphs may not be linear over a wide range of concentrations

34. 33 Note that graphs may not be linear over a wide range of concentrations

35. 34  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?

36. 35

37. 36 Atomic Absorption Spectroscopy

38. 37 Absorption Wavelengths of Iron

39. 38 Atomic Absorption Spectrophotometer (AAS)

40. 39 AAS Operation Gas Mixture Adjustment Controls Flame Hollow Cathode Lamp Monochromator Display

41. 40 Atomic Absorption Spectrometer Hollow Cathode Lamp Lens Atomised sample in flame Monochromator Detector Amplifier Display Flame Solution

42. 41 Close-up view of AAS Ions absorb energy, jump to excited state Electrons return to ground state,and photons emitted in all directions Less energy is transmitted to detector Transmittance Ions in Flame Hollow Cathode Lamp emits several unique wavelengths of light

43. 42 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

44. 43 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)

45. 44 A Source of Error  Another species may be absorbing at the same wavelength.

46. 45 UV-Visible Spectroscopy A UV-visible spectrophotometer measures the amount of energy absorbed by a sample.

47. 46 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.

48. 47

49. 48 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 BLUEGREENRED ORANGEORANGE Y E L LO W 420nm600nm520 nm

50. 49 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: C 40 H 56  Molar Mass537  Melting point 178 - 179 °C

51. 50 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. 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).

52. 51 540nm320nm 460nm ultra- violetvisibleinfrared 700 nm 400nm INTENSITYOFABSORPTIONINTENSITYOFABSORPTION The UV- Visible absorption spectrum for carotene in the non-polar solvent, hexane

53. 52 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. 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?