The spectrum of bright lines, bands, or Ch. 20 & 21 continuous radiation characteristic of and determined by a specific emitting substance subjected to a specific kind of excitation.

Absorption Methods - Beer’s Law Absorption Spectrum

shinning on a sample causes A = abc = εεεbc to be excited from the ground state to an where a => absorptivity b => path length • of that are removed c => concentration from transmitted spectra ε => molar absorptivity

Limitations to Beer’s Law Absorption Methods - Beer’s Law

• The light being shined on • Real Limitations – high -1 1.0 1 - 10 mg l NO -N A = abc = εbc 3 concentrated solutions, the sample must be 0.8 r = 0.8791 concentrated electrolyte monochromatic (one solutions (proximity or ) 0.6 alters molecular Abs 0.4 absorption).

0.2 A • The analyte must not be 0.0 • Chemical Limitations – 0 2 4 6 8 10 participate in a -1 absorbing species [NO -N] (mg l ) concentration dependent 3 participate in association c equilibrium or dissociation reactions, e.g. weak acids in HNO + H O →← H O + NO − 2 2 3 2 concentrated solutions, complexation.

1 Spectroscopic Procedure Applications

• Health Sciences – 95% of all analyses are • Have a and cuvette(s) – Single-beam instrument has one sample holder (swap blank performed by . and sample) • Biological Sciences – Double-beam instrument splits light output between two holders (measure blank and sample) • Chemical & Environmental Sciences: – A baseline spectrum is a spectrum of a reference solution Organic, inorganic systems and biochemical (solvent or reagent blank) systems

Spectroscopic Procedure Relation between Transmittance and Absorbance • Keep the absorbance reading of the sample below 1. ≈ P ’ A = log∆ 0 ÷ −= logT – % transmittance is related logarithmically with concentration « P ◊ (from 1-99% transmittance you can detect ~ 2 orders of magnitude in analyte concentration) T %T A – Any orders of magnitude greater than that will be detected in 1 100 0 the range of 0-1% T. 0.1 10 1 • Dilute the solution so that the transmittance reading is 0.01 1 2 not maxed out in that region (for accuracy)

Spectroscopic Procedure Analysis of Mixtures b x b Y MixtureAbs = ε [ ]+ε [ ]+...... • Try to do an analysis at the max x Y – Sensitivity is greatest at maximum absorbance = ε λ + ε λ )()( 2121111 bcbcA – Curve is relatively flat in case the drifts

and is off by a little in wavelength A = + λελε )()( 2221212 bcbc

λ = 620 nm max. •What are the knowns and 0.5 550 nm

0.4 unknowns in the above Eq.?

0.3 510 nm •Measure Abs at more 0.2 wavelengths than there are 0.1

0.0 components in the mixture.

Absorbance -0.1 •More wavelengths increase the -0.2 350 400 450 500 550 600 650 700 accuracy.

Wavelength (nm)

2 Isosbestic points Components of Optical Instruments

• Good evidence of presence of only 2 species Single Beam Instruments which interchange between themselves (e.g. indicator dye with 2 states)

Double Beam Instruments

Intensity of a Tungsten Filament and a Instrumentation - Basic Components (1) Light source -Tungsten filament lamp (Visible: 320 - 2500 nm); (UV: 200-400 nm); Nernst glower, globar (IR region).

Instrumentation - Basic Components Grating vs. Prism

(1) Light source •Grating: a reflective or -Tungsten filament lamp (Visible: 320 - 2500 nm); Deuterium transmissive optical arc lamp (UV: 200-400 nm); Nernst glower, globar (IR region). component with a series of (2) : disperses light into its component closely ruled lines; can wavelengths and select a narrow band of wavelengths to bend light (diffraction) pass on to the sample or detector. -Gratings & Prisms •Prism: can bend light (refraction)

3 Instrumentation - Basic Components Cells for Spectrophotometry

(1) Light source -Tungsten filament lamp (Visible: 320 - 2500 nm); Deuterium arc lamp (UV: 200-400 nm); Nernst glower, globar (IR region). (2) Monochromators: disperses light into its component wavelengths and select a narrow band of wavelengths to pass on to the sample or detector. -Gratings & Prisms (3) Sample containers (cuvettes or cells) -Glass, Plastic & Quartz (usually 1 cm); KBr/NaCl (IR)

Instrumentation - Basic Components Detector Response

(1) Light source -Tungsten filament lamp (Visible: 320 - 2500 nm); Deuterium arc lamp (UV: 200-400 nm); Nernst glower, globar (IR region). (2) Monochromators: disperses light into its component wavelengths and select a narrow band of wavelengths to pass on to the sample or detector. -Gratings & Prisms (3) Sample containers (cuvettes or cells) -Glass, Plastic & Quartz (usually 1 cm); KBr/NaCl (IR) (4) Detector: produces an electric signal when it is struck •A function of wavelength of incident light by •The greater the sensitivity, the greater the current of voltage -Phototubes, Photomultiplier tubes, & Photodiode array produced by the detector for a given incident irradiance of photons.

Atomic Line Spectrum • Deals with free - the absorption and emission of radiation by atoms. A spectrum produced by a luminous gas or vapor and appearing as distinct lines • Produces line spectra - can be used for characteristic of the various elements elemental analysis (qualitative and constituting the gas. quantitative).

4 Line - Linewidths • Atomic spectra have narrow lines: inherent linewidth is ~10-4 nm. • Two main mechanisms can broaden the lines to 10-3 to 10-2 nm: (1) Doppler broadening: The atoms may move towards slowly or quickly or away from the detector (cause a Doppler shift in the resulting line); greater as the increases (2) Pressure broadening: arises from the collision between atoms (cause energy exchanges, shorten the life time of the excited state); greater as the temperature increases. The relative simplicity of atomic spectra is due to the small number of possible energy states for the absorbing particles.

Absorption, Emission, and Atomic Spectroscopy- Types by Atoms in a

• Types of atomic spectroscopy •Atomic Emission: -emission from a – Atomic absorption thermally populated – Atomic emission radiation. •Atomic Absorption: – Atomic fluorescence -absorption of sharp lines from hollow0cathode lamp. •Atomic Fluorescence: -fluorescence following absorption of radiation.

Atomic Absorption Experiment Sources

• A molecular spectrophotometer relies on a broad band light source. • With atomic absorption, a line source is required to reduce interferences from other elements and background. • Hollow cathode lamp

• Atoms absorb part of the light from the source and the remainder of the light reaches the detector. • The method relies on the Beer’s Law (calculations are the same as with molecular absorption methods).

5 Chopper A Hollow-Cathode Lamp • Provides signal modulation • Subtracts the signal due to flame background emission to reduce some noise from the atomization source and accounts for instrumental variations. (a) Lamp and flame reaches detector Filled with an inert gas like or . When applies a potential, (b) Only flame emission the gas becomes excited and is driven towards the cathode. Metal reaches detector atoms are then spitted out the cathode surface, which produces emission lines specific for the element. The cathode must be capable of (c) Resulting square wave conducting a current for it to work. signal

Atomization Source - Atomization Source - Flames

• Atomization source: convert sample to free atoms • Flame : – often use a premix burner; have a long, narrow burner head that serves as a sample path (b). – Sample is introduced via aspiration. – The nebulizer controls sample •Fuel: the most common one is acetylene. flow, producing a mist. Premix Burner •Oxidants: air or nitrous oxide (N2O, producing a hotter, noiser – The mixing chamber assures flame). that the sample mixed with the •Sample: must be a fluid; large size (> 1 mL); constantly being oxidant and fuel prior to entry into the flame. consumed. •Can produce stable signals in the ppm range for most metals End View of Flame

Atomization Source - Furnaces Monochromator and Detector

• Graphite furnace: – Samples are placed in a carbon tube • Monochromator: a high resolution, holographic which is heated electrically-graphite grating furnace ( samples can be assayed) – Improved detection limits and sensitivity. • Detector: photomultiplier tube – A three stage program: (1) Dry: remove solvent; (2) Char: decompose your matrix (not analyte); (3) Atomization: increase [free atoms]. – Purge gas- Argon: remove excess material (dry, char, after atomization); reduce oxidation of the tube; as a protective blanket during atomization (cyanogen)

6 Background Correction Methods • Background signal arises from absorption, emission, or scatter by everything in the sample besides analyte (the matrix) and by the atomization sources. • Beam chopping: an easy way to account for instrumental variations and “flam flicker”; not very good at accounting for background absorption or emission.

• Deuterium (D2) Lamp: broad emission from a D2 lamp (mostly background) is passed though the flame in alternation with that from the hollow cathode (sample and background); not very good >350 nm. • Zeeman correction: when a strong magnetic field is applied, the electronic split. Off (sample and background); On (background); directly measure background, but expensive

Standard Methods Standard Methods • Standard calibration curve. (standard curve) • Internal standard method: a known amount of standard • Standard addition method – requires linear response (different species from the analyte) is added to the

[X] I V0 unknown. Equate ratio of unknown signal to standard i = X = ([X][X] ) ]S[ +[X] I if V signal in the unknown mixture to the ratio of the standard f f S + X V mixture. = ([S][S] S ) if V ≈ V ’ I ≈V ’ Area of analyte signal I ∆ ÷ I += x S][ ∆ s ÷ ≈ Area of Std signal ’ +XS ∆ ÷ x i ∆ ÷ = F∆ ÷ «V0 ◊ x][ i «V0 ◊ Concentrat ofion analyte « Concentrat ofion Std ◊ A ≈ A ’ x ∆ s ÷ = F∆ ÷ [X] « [S] ◊

Example: A solution containing 0.0837M X and Example: Water from a salt lake is analyzed for Ca (see the 0.0666M S gave peak area of Ax=423 and As=347. To AAS data table below). A 100.0 ppm Sr2+ standard solution analyze the unknown, 10.0 mL of 0.146 M S were added and a 250.0 ppm Ca2+ standard solution are used in addition to 10.0 mL unknown, and the mixture was diluted to to the unknown in the test tubes indicated in the table. 25.0 mL in a volumetric flask. This mixture gave Assume the detector responds of Ca and Sr are the same. Ax=553 and As=582. Calculate [X] in the unknown. ≈ ’ 423 347 Ω A ≈ A ’ = F∆ ÷ F = 9700.0 {[X]/[S]}unk = {Ix/Is}unk ∆ ÷ 0.0837 « 0.0666 ◊ x = F s ∆ ÷ ≈ 0.10 ’ { [X]/[S]}std {Ix/Is}std [X] « [S] ◊ 146.0][ MS ×= ∆ ÷ = 0584.0 M « 0.25 ◊ (1/4)[X] 0.465 A ≈ A ’ ∆ ÷ A ≈ A ’ (3/4)(100ppm) = 0.385 x = F s x ∆ s ÷ [X] ∆ [S] ÷ = F∆ ÷ (1/3)(250ppm) 0.885 « ◊ [X] « [S] ◊ (2/3)(100 ppm) 0.342 I x ≈[X]’ 553 ≈ 582 ’ 2x/750 ppm = 0.467 => [X] = 175 ppm = F∆ ÷ = 9700.0 ∆ ÷ Ω X = 05721.0][ M I « [S] ◊ [X] « 0.0584 ◊ s =× 143.005721.0)0.10/0.25( M

7 Example: A sample containing Ca2+ is tested as described in the table below. A 5.00 mM Ca 2+ standard is added in tube #2 . The instrumental signal is directly proportional (i.e. linear) to the concentration of the analyte.

What is mM of Ca unknown? [X] I i = X ]S[ +[X] I f f + XS [X] 1.50 i = Ω [X] = 5.7 mM 2 5 +× 3 [X] 30.1 i 5 5 i