ATOMIC SPECTROMETRIC METHODS Atomic Absorption Spectroscopy (AAS) Atomic Emission Spectroscopy (AES) Atomic Absorption Spectroscopy (AAS) = Flame AAS has been the most widely used of all atomic methods due to its simplicity, effectiveness and low cost = First introduced in 1955, commercially available since 1959 = Qualitative and quantitative analysis of >70 elements = Quantitative: Can detect ppm, ppb or even less = Rapid, convenient, selective, inexpensive™ 3p. a se B20 » Bien a» i 2s K 1.0 ‘ttNa: 1s? 2s? 2p* 38! Cale seremarrerata Origins of atomic spectra = With gas-phase atoms or ions, there are no vibrational or rotational energy, only electronic transitions occur. Thus, atomic emission, absorption, and fluorescence spectra are made up of a limited number of narrow spectral lines. = In atomic emission spectroscopy, analyte atoms are excited by heat or electrical energy. = A transition to or from the ground state is called a resonance transition, and the resulting spectral line is called a resonance line.3.0 Absorbance Atomic emission: Flame spectroscopy Observation Caused by Persistent golden- Sodium yellow flame Violet (lilac) flame Potassium, cesium carmine-red flame Lithium Brick-red flame Calcium Crimson flame Strontium Yellowish-green flame barium, molybdenum ro Green flame Borates, copper, thallium Blue flame (wire Lead, arsenic, slowly corroded) antimony, bismuth, copper SodiumWorking principles of Atom Emission Spectrophotometry (AES) and AAS Me M: the analyte Expose i M® (atom in ground state) sample Vy M" (atom in excited state) 0 _ Sample | flame | | M™ — Atomization or high temper ature) ‘Atom Emission ee Spectrophotometry (AES) MM’ - MeChv) bY J ionochromator |—+[detector | —-[ Signal Hollow Cathode |—tY+M°- i" (+hv)—[Monochromator | [detector _}-+ [Signal Lamp ‘Atom Absorption Spectrophotometry (AAS) 7 Instrumentation Flame Emission Spectrometry Flame Absorption Spectrometry = as He i 4 e— ee - Hollow-cathode lamp - Atomizer : - Atomizer - Monochromator - Monochromator - Detector - Detector 8Instrumentation Flame Emission Spectrometry (FES) Cat, Ca" Ca” : atom ints ground, excited or ionized states on | be main) a9) +20K(0)Atomization = Atomization — the process of converting an analyte into a free form. = Two general methods of atomization: flame atomization and electrothermal atomization. 1 Sample Atomization = Need to break sample into atoms to observe atomic spectra = Basic steps: a) nebulization — solution sample, get into fine droplets by spraying thru thin nozzle b) desolvation - heat droplets to evaporate off solvent just leaving analyte and other matrix compounds ©) volatilization — convert solid analyte/matrix particles into gas phase d) dissociation - break-up molecules in gas phase into atoms e) ionization — cause the atoms to become charged f) excitation — with light, heat, etc. for spectra measurement Spray Dry ‘aerosol 12Flame atomizers Fuels and Oxidants Used for Combustion Temperature Range Fuel Oxidant (°c) natural gas air 1700-1900 hydrogen air 2000-2100 acetylene air 2100-2400 acetylene nitrous oxide 2600-2800 acetylene ‘oxygen 3050-3150 13 The Boltzmann Distribution N, The relative population of excited-state N, The relative population of ground- state g.and g, The statistical weights of the excited and ground states, respectively T The absolute temperature AE The energies of the two states k The Boltzmann constant, k= 1,381 x 10 “6 erg/K 14The Boltzmann Distribution NINo Line (nm) 2000 K 3000 K 10,000 K Na 589.0 9.9 x 10-* 5.9 x 10-* 2.6 x 10" Ca 422.7 12x 10°? 3.7.x 105 1.0 x 10" Zn 213.8 73x 10°" 5.4 x 10°" 3.6 x 10°? 15 Instrumentation Temperature (°C) 1700-3150 ‘excitation source ‘4,000-6,000 Are/Spark +4,000-5,000/4,000 16Flame Source Used mostly for alkali metals (Na, K) Easily excited even at low temperatures + Advantage - cheap Disadvantage - not high enough temperature to extend to many other elements Schematic of AAS Fy 17 E.g. Hollow al cathode lamp Atomiser i acetylene) “hy, Analyte solution 3 ~Nebuliser, spray seers chamber, and burner 18Atomic Absorption Spectroscopy The analyte concentration is determined from the amount of absorption Flame Detector |—-} Amplifier Readout device Analyte sample A in beaker Atomic Absorption Spectroscopy 19 It is possible to measure the concentration of an absorbing species ina sample by applying the Beer-Lambert Law: Abs = absorbing sample of wiz], See | I, Abs = écb € = extinction coefficient —<— path length b > 20Hollow cathode lamp Optical trnsparent window “Cathode M Shield _ light output The electric discharge ionises rare gas (Ne or Ar usually) atoms, which inturn, are accelerated into the cathode and sputter metal atoms into the gas phase. 4 an ‘itl Scan ement HollowCathodeLamp tobeanalyzed 7 Quartz Glass Ne or Ar orPyren, shield at 1S torr wistow Process: 1. ionizes inert gas to high potential (300V) Ar > Arte 2. Ar‘ goto “-” cathode & hit surfaces 3. As Ar* ions hit cathode, some of deposited element is excited and dislodged into gas phase (sputtering) 4, excited element relaxes to ground state and emits characteristic radiation - Advantage: sharp lines specific for element of interest - Disadvantage: need to use different lamp for each element tested 22Hollow-Cathode Lamps Insulating disk Quartz or glass window Hollow Anode cathode 23 Flame AA Spectrometer Flame fuelled by (e.g.) acetylene and air Nebuliserand Spray chamber Hollow cathode lampsvailable for over 70 element Can get lamps containing> 1 element for determination of multiplespecies 24Sensitivity and detection limits in AAS = Sensitivity: number of ppm of an element to give 1% absorption. = Limit of detection: dependent upon signal : noise ratio. S/N=3.2 Signal Peak-to-.—~_=<==—= peak noise level>—— = 25 Atomic Absorption Detection Limits for Selected Elements Detection Limits (ppb) Element Flame Atomization Electrothermal Atomization Ag 09 0.001 al 20 0.01 Ast 20 0.08 Au 6 0.01 8 700 15 Ba 8 0.04 Be 1 0.003 Bi 20 o1 fa 05 0.01 cd 05 0.0002 co 2 0.008 cr 2 0.004 cs 8 0.04 Fe 3 0.01 Ga 50 0.01 Ge 50 ot 26ICP — MS and ICP-OES ICP-MS: Inductively coupled plasma mass spectrometry ICP-OES: Inductively coupled plasma optical emission spectra Plasma (t°: 6000 - 10 000 °K) 27 Plasma (inductively coupled plasma - ICP) = Plasma — electrically conducting gaseous mixture (cations & electrons) = Temperature much higher than flame = Possibility of doing multiple element analysis > 40-50 elements in 5 minutes ey 28Inductive ICP) Emission Spectross «involves use of high temperature plasma for sample atomization/excitation higher fraction of atoms exist in the excited state, giving rise to an increase in emission signal and allowing more types of atoms to be detected ‘Temperature Regions in Plasma Torch — = oy estosat 7 eo leet LP ance = 29 Inductively Coupled Plasmas * Steps Involved: — Radio-Frequency (RF) induction coil wrapped around a gas jacket. — Spark ionises the Ar gas. — RF field traps & accelerates the free electrons, which collide with other atoms and initiate a chain reaction of ionisation. 30Inductively Coupled Plasmas = Enables much higher temperatures to be achieved. Uses Argon gas to generate the plasma. = Temps ~ 6,000-10,000 K. = Used for emission rather than absorption due to the higher sensitivity and elevated temperatures. = Atoms are generated in excited states and spontaneously emit lights. Applicatons of AAS Qualitative analysis Quantitative analysis — Selecting the wavelength and split width spectral interferences chemical interferences 31 32Interferences in AAS — Broadening of a spectral line, which can occur due toa number of factors (Physical) — Spectral: emission line of another element or compound, or general background radiation from the flame, solvent, or analytical sample — Chemical: Formation of compounds that do not dissociate in the flame — lonisation of the analyte can reduce the signal — Matrix interferences due to differences between surface tension and viscosity of test solutions and standards 33 Minimizing spectral interference Sample's matrix (wavelength < 300 nm) Increase temperature flame Adjust the flame's composition (fuel-to-oxidant) Add standard to sample 34Minimizing chemical interferences Nonvolative compounds containing the analyte Example: Determination of Ca 5 ppm Ca? (A= 0.5); 5 ppm Ca®* + 100 ppm Al? (A = 0.14); + Al-Ca-O oxide 5 ppm Ca** + 500 ppm PO,* (A = 0.38)—- Ca; (POx)> Increase temperature flame Adjust the flame's composition (fuel-to-oxidant) Use releasing agent or protecting agent 35 Minimizing chemical interferences Releasing agent - a reagent whose reaction with an interferant is more favorable than the interferant's reaction with the analyte 5 ppm Ca® (A= 0.5); 5 ppm Ca®* + 100 ppm Al®*(A=0.14) — Al-Ca-O oxide 5 ppm Ca + 100 ppm Al>* + 2000 ppm SrCl, (A = 0.48) 5 ppm Ca * + 2000 ppm SrCl, (A = 0.49) 36Minimizing chemical interferences Protecting agent - a reagent that reacts with the analyte, preventing it from transforming into a nonanalyzable form 5 ppm Ca? (A= 0.5); 5 ppm Ca + 500 ppm PO,* (A = 0.38) Caz (PO4)2 5 ppm Ca 2 + 500 ppm P04? + 1% EDTA (A = 0,52) 5 ppm Ca * + 1% EDTA (A = 0.55) 37 lonization interferences MeaM+e - Mis analyte - Avoid by: > lower temperature > add ionization suppressor (K or Ce) 38Summary * Sensitivity = Accuracy = 0.5-5% = Precision (A> 0.1, relative standard deviation (% RSD) = 0.3 — 1% for flame atomization; =1-5% for electrothermal atomization) = Selectivity = Time, cost and equipment 39 Example Determination of Cu and Zn in tissue samples Procedure, Tissue samples are obtained by a muscle needle biopsy and are dried for 24-30 hours at 105 °C to remove all traces of moisture. The fatty tissue in the dried samples is removed by extracting overnight with anhydrous ether. After removing the ether, the sample is dried to obtain the fat-free dry tissue weight (FFDT). The sample is digested at 68 °C for 20-24 h using 3 mL of 0.75 M HNOs. After centrifuging at 2500 rpm for 10 min, the supernatant is transferred to a 5-mL volumetric flask. The digestion is repeated two more times, for 2-4 h each, using 0.9-mL aliquots of 0.75 M HNOs. These supernatants are added to the 5-mL volumetric flask, which is diluted to volume with 0.75 M HNOs, The concentration of Cu and Zn in the diluted supernatant is determined by atomic absorption spectroscopy using an air-acetylene flame and external standards. Copper is analyzed at a wavelength of 324.8 nm with a slit width of 0.5 nm, and zinc is analyzed at 213.9 nm with a slit width of 1.0 nm. Background correction is used for zinc. Results are reported as micrograms of Cu or Zn per gram of FFDT. 40Example Determination of Cu and Zn in tissue samples The following absorbances were obtained for a set of Cu calibration standards ppm Cu Absorbance 0.000 0.000 0.100 0.006 0.200 0.013 0.300 0.020 0.400 0.026 0.500 0.033 0.600 0.039 0.700 0.046 1.000 0.066 What is the concentration of copper, in micrograms per gram FFDT, for a 11.23- ing FFDT tissue sample that yields an absorbance of 0.023? “1