Spectrophotometry Let There Be Light Spectrophotometry: the use of electromagnetic radiation to measure chemical concentrations

 UV absorption The Ozone Hole

Why south pole ?

Properties of light -1 1) 2) Electromagnetic Spectrum 3) Absorption vs Emission of light

change of nuclear configuration γ-ray change of e distribution X-ray uv.vis change of configuration IR change of orientation microwave change of spin NMR EPR

Absorption of light -1 1)Spectrophotometer

Absorption of light -2 2). When no light is absorbed, P=P0 and A=0

Absorbance is proportional to the concentration of light-absorbance molecules in the sample. A =  bc  : molar absorptivity (M -1 cm -1 )

Absorption of light -3 (ex) How effective is sunscreen ? at the peak absorbance near 300 nm ? A ~ 0.35 T = 10 -A = = 0.45 = 45%  55% UV-B is absorbed.

Observed color vs. absorbed color

Practical Matters -1 1) Sample is usually contained in a cell called a cuvet, which has flat, fused-silica faces. A glass made of SiO 2 : Vis. UV. Plastics & ordinary glass: Vis NaCl(s) KCl(s) : IR

Practical Matters -2 2) Good Operating Techniques : a) Cuvets handle: systematic errors/ random errors b) Most accurate at A~ Too little light : (high A), P is small & hard to measure Too much light : (low A), it is hard to distinguish P from P0

Practical Matters -3 c) Old vs. new curves d) Greatest sensitivity: λmax e) Baseline correction

(a) Proteins at 280 nm: tyr, phe, trp. (b) A colorimetric reagent to detect phosphate Using Beer’s Law -1

Using Beer’s Law -2 Ex.1 : Bezene: find molar absorptivity (  )

Beer’s Law -2 Ex.2 : Nitrite in an aquarium (using a standard curve)  543 nm

Using Beer’s Law -3 (toxic when > 1 ppm) NH 3  animals & plant (toxic when > 1 ppm) [O] NO 3 -

Using Beer’s Law -4 2) Standard Nitrite

Using Beer’s Law -5 from least square (4.4) A = [ppm]

Enzyme-based nitrate Analysis - A Green Idea P.408 NO 3 - NO 2 - NO NADH + H + NO NAD + + H 2 O Cd Nitrate reductase pH 7

Spectrophotometry : Instruments & Applications

The Spectrophotometer –1 Remote sensing of airborne bacteria: Optical fiber coated with antibodies to detect spores of a specific bacterium

The Spectrophotometer -2 1)Spectrophotometer a) Single-beam b) Double-beam

The Spectrophotometer -3

The Spectrophotometer –4 1) Light source a.Tungsten lamp: Vis. near IR (320 nm~2500 nm) b.Deuterium are lamp: UV (200~400 nm) c.Electric discharge lamp + Hg (g) or Xenon: Vis & UV d.Globar (silicon carbide rod): IR (5000~200 cm -1 ) e.Laser: intense monochromatic sources.

The Spectrophotometer -5

The Spectrophotometer -6 2) Monochromator disperses light into its component wavelengths and selects a narrow band of wavelengths to pass through the sample

Consists: (1) lenses or mirrors: focus the radiation (2) entrance and exit slits: restrict unwanted and control the spectral purity of radiation. (3) dispersing medium: separate the of polychromatic radiation from the source. (a) prism and (b) diffraction grating

The Spectrophotometer -7 a. entrance slit b. collimating mirror or lens c. a prism or grating d. focal plane e. exit slit Monochromator

19.1 The Spectrophotometer -10 Choosing the bandwidth: exit slit width Resolution Signal Monochromator trade-off

The Spectrophotometer -10 3)Detector : A detector produces an electric signal when it is struck by photons ( Convert radiant energy (photons) into an electrical signal ). Figure 19-8 shows that detector response depends on the wavelength of the incident photons. A photomultiplier tube (Figure 19-9) is a very sensitive detector. Ideal detector : high sensitivity, high signal/noise, constant response for λs, and fast response time.

The Spectrophotometer -11 Detector response depends on the λ of the incident photons. 3) Detector

The Spectrophotometer -12 Photomultiplier tube: very sensitive detector

Analysis of a mixture -1 1)Absorbance of a mixture :

Analysis of a mixture -2 2)Isosbestic points : for rxn: X  Y, every spectrum recorded during chemical reaction will cross at the same point. Good evidence for only two principle species in rxn. Ex: HIn  In - + H +

Analysis of a mixture -3 Why isosbestic point?

Spectrophotometric Titrations -1

Ferric nitrilotriacetate [used to avoid Fe(OH)3  ] Spectrophotometric Titrations-2

125 μL ferric nitrilotriancetate 2 mL apotransferrin A = A corrected = ? Spectrophotometric Titrations-3 ex.at p.425 Corrected A for the effect of dilution Corrected A = (V t / V i ) (observed A) (Beer’s law)

What happens when a molecule absorbs light ? 1)Absorbing species : M + hν  M* (lifetime : ~ sec) Relaxation processes : a)M*  M + heat (most common) b)M*  new species (photochemical reaction) c)M*  M + h (fluorescence, phosphorescence)

Geometry of formaldehyde Electronic States of Formaldehyde excited state are shown in Figure:

MO of CH 2 O Molecular orbitals describe the distribution of electrons in a molecule, just as atomic orbitals describe the distribution of electrons in an atom. In Figure, four low-lying orbitals of formaldehyde, labeled σ1 through σ4, are each occupied by a pair of electrons with opposite spin (spin quantum numbers= +1/2 and -1/2 represented by ↑and↓).

2)Types of absorbing electrons Consider formaldehyde: three types of molecular orbitals

In a electronic transition, an electron moves from one orbital to another. Four types of electronic transitions: σ*σ* π*π* σ π n E < 125 nm 150~250 nm 200~700 nm

Two possible electronic states arising: n   * transition singlet state: The state in which the spins are opposed. triplet state: spins are parallel E: T 1 < S 1

4)Electronic transition of formaldehyde n   * (T 1 ), absorption of light at λ = 397 nm green-yellow n   * (S 1 ), absorption of light at λ = 355 nm colorless (more probable)

Vibration and Rotational States of Formaldehyde The six modes of vibration of formaldehyde. Combined Electronic, Vibrational, and Rotational Transitions Electronic absorption bands are usually very broad (~100 nm) because many different vibrational and rotational levels are excited at slightly different energies.

5)Vibrational & Rotational states of CH 3 CO (IR and microwave radiation)

6)What happens to absorbed energy

7) Luminescence procedures : emission spectrum of M* provides information for qualitative or quantitative analysis. Photoluminescence : a)Fluorescence : S 1  S 0, no change in electron spin. (< s) b)Phosphorescence : T 1  S 0, with a change in electron spin. (10 -4 ~10 2 s) ‚Chemiluminescence : Chemical reaction (not initiated by light) release energy in the form of light. ex : firefly. a molecule absorbs light

7) In which your class really shines ? emission spectrum a molecule absorbs light Hg + E1  Hg*  Hg + h   185 nm Sb 3+ + Mn 2+ + h   185 nm  M* M*  M + h

a molecule absorbs light 8) Absorption & Emission Spectra

Luminescence in analytical chemistry 1) Instrument .hν out (photon) ‚heat ƒbreaking a chemical bond hν in

Luminescence 2) I = kP o C  incident radiation sensitivity  by P 0  or C  3)more sensitive than Absorption

4) Fluorimetric Assay of Selenium in Brazil Nuts –Se is a trace element essential to life: destruct ROOH (free radical) –Derivatized: –Self-absorption: quench Luminescence

5) Immunoassarys  employ anitbody to detect analyte. Ex: ELISA

Luminescence a.pregnancy test. sensitive to < 1 ng of analyte b.Enviromental Analysis. (ppm) or (ppt) pesticides, industrial chemicals, & microbialtoxins.