Instrumental Analytical Techniques PDF

Instrumental Analytical Techniques An Overview of Chromatography and Spectroscopy 1 Chromatographic Techniques -Thin layer and column chromatography -Gas Chromatography (GC) -High Performance Liquid Chromatography (HPLC) Spectroscopic Techniques - Atomic Absor...

Instrumental Analytical Techniques An Overview of Chromatography and Spectroscopy 1 Chromatographic Techniques -Thin layer and column chromatography -Gas Chromatography (GC) -High Performance Liquid Chromatography (HPLC) Spectroscopic Techniques - Atomic Absorption Spectroscopy(AAS) - Colorimetry 2 - UV-Visible Spectroscopy (UV-Vis)  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. 3 Types of Chromatography Paper Chromatography and Thin Layer Chromatography (TLC) Column Chromatography Gas GasLiquid LiquidChromatography Chromatography(GLC) (GLC) High HighPerformance PerformanceLiquid LiquidChromatography Chromatography(HPLC) (HPLC) 4  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. 5  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. x Solvent Solventfront front a Rf of = a/x b c Rf of = b/x Rf of = c/x 6  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 Unknown + standards standards standards 7 A B C A+B+C A+B+C ??  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 8  Gas Liquid Chromatography 9  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 Gas mobile phase dense liquid (on solid) SP 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 10  Gas Chromatogram of High Grade Petrol 11  Qualitative known Rf values under standard conditions mixture of hydrocarbons eg petrol mixture of alcohols air But Must be able to be vaporised up to about 300oC Must not decompose Quantitative Calibration graph of a series of standards of known concentration plotting area under peak vs concentration Eg. How much ethanol is in the blood? 12  A Use a series of r standards of ethanol e to determine area a under peak. u Construct n calibration graph d Area (or height at e first approx.) is r proportional to concentration. p Find area of unknown e and read off a concentration k 0 0 0.10 0.20 0.30 0.40 0.50 13 Concentration of alcohol in grams/Litre  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 14  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 15  STATIONARY PHASES (NORMAL POLARITY) Silica or alumina possess polar sites that interact with polar molecules. silica O Polar Group HO Si O Components Componentselute eluteininincreasing increasing order orderof ofpolarity. polarity. Most polar…….Least polar 16  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 Me O Si O Si Me O Components Componentselute eluteinindecreasing decreasing order orderof ofpolarity. polarity. Most non-polar…….Least non-polar 17  STATIONARY PHASES (CATION EXCHANGE) Silica is substituted with anionic residues that interact strongly with cationic species (+ve charged) Cations exchange Na+ silica O Na O S O +ve +vecharged chargedspecies speciesadhere adheretotothe thesupport support and andare arelater latereluted elutedwith withacid acid(H (H+)) + Most +ve…….Least +ve 18  STATIONARY PHASES (ANION EXCHANGE) Silica is substituted with cationic residues that interact strongly with anionic species (-ve charged) Anions exchange Cl- silica Me Cl Me N CH2 Me -ve -vecharged chargedspecies speciesadhere adheretotothe thesupport support and andare arelater latereluted elutedwith withacid acid(H (H+)) + Most -ve…….Least -ve 19  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. Large Largemolecules moleculeselute elutefast fast(restricted (restrictedpath), path), while whilesmall smallmolecules moleculeselute eluteslowly slowly(long (longpath pathlength) length) Larger molecules…….Smaller molecules 20  Regions of the Electromagnetic Spectrum Light waves all travel at the same speed through a vacuum but differ in frequency and, therefore, in wavelength. 21  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 22  Absorption v. Emission Energy is emitted by electrons returningto lower energy levels 3rd Excited 2nd States 1st Energy is absorbed as electrons jump to higher energy levels Ground State 23  Emission Spectra of Elements Continuous Sodium Hydrogen 24 Calcium  Absorption Spectra Sodium For other Spectra, click on the hyperlink below: http://www.achilles.net/~jtalbot/data/elements/index.html 25  The Spectroscopic Techniques are based on the fact that Light absorbed (Absorption) is directly proportional to the Concentration of the absorbing component. 26  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. 27 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 28 (LEDs) 29  Red Detector LED measures Green red light LED absorbed Blue LED 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. 30  Collect data Absorbance Concentration 0.0 0.00  A set zero adjustment 0.125 enables the instrument to 0.20 0.40 0.250 factor out any absorbance of the solvent and the material 0.59 0.380 the cuvet is made from. 0.78 0.50 0.35 unknown  concentration of a species in solution is proportional to the light absorbed 31 Absorbance 1.00 0.80 Note that graphs may not be linear 0.60 over a wide range of concentrations 0,40 0.20 0 0 0.10 0.20 0.30 0.40 0.50 32 Concentration in mol/Litre  1.00 0.80 Note that graphs may not be linear 0.60 over a wide range of concentrations 0.40 0.20 0 0 0.10 0.20 0.30 0.40 0.50 33 Concentration in mol / Litre  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? 34 35  Atomic Absorption Spectroscopy 36  Absorption Wavelengths of Iron 37  Atomic Absorption Spectrophotometer (AAS) 38  AAS Operation Hollow Cathode Lamp Gas Mixture Adjustment Display Flame Monochromator 39 Controls  Atomic Absorption Spectrometer Atomised sample in Detector flame Monochromator Lens Lens Hollow Cathode Flame Lamp Solution Amplifier 40 Display  Close-up view of AAS Electrons return to ground Less Ions energy absorb is energy, state,and photons emitted in transmitted jumpall to detector to directions excited state Hollow Cathode Lamp Ions in Flame emits several unique wavelengths of light 41 Transmittance  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 42  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) 43  A Source of Error  Another species may be absorbing at the same wavelength. 44  UV-Visible Spectroscopy A UV-visible spectrophotometer measures the amount of energy absorbed by a sample. 45  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. 46 47 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 Y O E R BLUE GREEN A RED L LO N W G E 420nm 520 nm 600nm 48 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 178 - 179 °C 49  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. 50 The UV- Visible absorption spectrum for carotene in the non-polar solvent, hexane I N T E N S I T Y 400nm 700 nm O F A ultra- violet visible infrared B S O R P T I O 320nm 460nm 540nm N 51  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. 52