Molecular Spectroscopy ν (cm -1 ) λ (cm) 10 6 10 8 10 10 10 12 10 14 10 16 10 18 10 20 10 22 ν (Hz) NMR ESR microwave IR UV/Vis VUV X-Ray Gamma Ray H 2 e -
UV/Vis Spectroscopy absorption technique X hν * Y Y Z X Z e - e -
UV UV(B) UV(A) visible 180 300 400 500 600 700 E = hν c = λν λ (nm) If a molecule absorbs a photon at 400 nm, how much energy is absorbed by the molecule? E = ν 6.626068 10 c hc = E = λ λ -34 J s x 299792458m s -9 400 10 m -1 = 5 10-19 J 5 x 10-19 J/molecule 6.022141 x 10 23 molecules/mol = 300 kj/mol
Single beam hν I o Instrumentation I source grating slit sample cell detector (pmt) mirror I hν I o Double beam chopper ref. cell
Source: visible work (380-2500 nm) uses a tungsten filament UV (160-400 nm) uses a deuterium lamp Monochromator: prism or grating 510 ±0.5 nm intensity λ 507 508 509 510 511 512 513 wavelength (nm)
absorbance absorption band of holmium oxide 'monochromatic light' 435 440 445 450 455 wavelength (nm) Sample cells: cuvettes with path lengths of 0.1, 1.0 and 10.0 cm quartz (180-1100 + nm) glass (375-1100 nm) plastic (350 nm)
Detectors: photomultiplier tube (pmt) ++ e - +++ e - e - hν + anode +++++
λ = 510 nm hν I o I %T = I/I o A = -log T T = 100 % A = 0 T= 50 % A = 0.30 T = 10 % A = 1 T = 1 % A = 2 T = 0.1 % A = 3
For a specific λ, Beer-Lambert Law A = εbc ε is molar absorptivity (L mol -1 cm -1 ) b is pathlength of cell (cm) c is concentration of analyte (M) A T = A 1 + A 2 +... = ε 1 bc 1 + ε 2 bc 2 +... Where and why UV-Vis spectroscopy is used... - easily quantifiable and good sensitivity - inexpensive and fast - reasonably selective
- sensitive, 10-4 to 10-7 M (depends on ε and b) - accurate (1 to 5 %) Experimental Considerations 1. Verify that A = εbc is linear by checking the calibration curve. 2. Calibration standards should match matrix of samples with respect to T, ph, and interferences I o I - reflection - scattering - absorbance due to cell and solution - absorbance due to analyte
3. Select λ as λ max for sensitivity and accuracy. A = ebc da is a minimum dλ wavelength (nm) A = εbc λ max 4. Calibrate A and λ 5. λ small wrt absorption band 6. Record absorbances from 0.1 to 1.5
A = εbc = log T c log T = 1 εb c = log e T 2 εb T 2 1 c c = log e T εbt log T εb = log e T T log T sc c = log e s T log T T If A = 1, T = 0.10 and s T = 0.003 then sc log e st s = c = c T log T c log e 0.003 0.10 log(0.10) = 1.3% s c A = 2, = 6.5% c
sc c log e st = shows a minimum when T = 1/e = 0.37 or A = 0.43 T log T Sc/c 35% 30% 25% 20% 15% 10% 5% 0% 0 1 A 2 3 Sc/c 8% 7% 6% 5% 4% 3% 2% 1% 0% 0 0.5 1 A 1.5 2 0.1<A<1.5
E 1 * E E o λ λ λ
0.9 0.7 A 0.5 0.3 B C 0.1-0.1 210 260 310 wavelength (nm) A benzene in vapour phase, res. = 0.2 nm B benzene in vapour phase, res. = 0.5 nm C acetone in hexane A
A 1.4 1.2 1 0.8 0.6 0.4 0.2 0 400 500 600 wavelength (nm) A I 2 in gas phase B I 2 in CH 2 Cl 2 B A
Transition metal complexes - spectra are complex d x y d 2 z 2 2 d xy d yz d xz E 2.4 1.9 Cu 2+ in water A 1.4 0.9 0.4-0.1 300 500 700 900 1100 wavelength (nm)
1.0 0.8 A 0.6 0.4 A 0.2-0.1 B 300 500 700 900 1100 wavelength (nm).... A [(H 2 ) 5 Fe-S=C=N].. 2+ [(H 2 ) 5 Fe-S=C=N].. 2+.... B Fe(phen) 2+ 3 Fe(phen) 2+.... 3 (MLCT).... + - - + (LMCT)
2 1.5 Ho 2 3 A 1 0.5 0 250 450 650 850 wavelength (nm)
σ * π * n π σ E = hc λ σ σ * CH 4 λ max = 125 nm n σ *.. CH 3 H.. λ max = 150-250 nm, ε = 100-3000 M -1 cm -1 H 2 λ max = 167 nm Me 3 N λ max = 227 nm CH 3 I λ max = 258 nm
π π * alkenes, alkynes, carbonyls λ max 170-190 nm H 2 C=CH 2 λ max = 171 nm ε = 15000 M -1 cm -1 n π * λ max 280 nm ε 10-100 M -1 cm -1 σ * π * n π σ A 2.9 2.4 1.9 1.4 0.9 0.4-0.1 π π * λ max = 189 nm ε = 900 M -1 cm -1 H 3 C C CH 3 190 240 290 340 390 wavelength (nm).... n π * λ max = 280 nm ε = 12 M -1 cm -1
σ * π * n π σ.. Chromophores: N, C=C, C=Ȯ... Auxochromes.. C=Ọ. X ε hyperchromic shift ε hypochromic shift λ bathochromic or red shift λ hypsochromic or blue shift λ max (π π * ) (nm) ε (M -1 cm -1 ) 181 13,800 181 14,100 178 17,000 224 22,900 258 43,700
λ max (n π * ) (nm) ε (M -1 cm -1 ) 278 30 324 24 R 2 C C H R - R 2 C C H + R H H C C H H H C H - H H H C C H C H+ H
Solvents: should be pure and transparent in region of interest Solvent Cutoff (nm) Solvent Cutoff (nm) H 2 180 cyclohexane 200 CH 3 H 205 CH 3 CN 180 hexane 200 acetone 330 0.75 0.55 acetone A 0.35 0.15-0.05 200 225 250 275 300 325 350 wavelength (nm)
Polar solvents can broaden absorption bands λ max can shift......... H H blue shift for the n π * transition H 3 C CH 3 red shift for the π π * transition X Z Y solvent X Z Y solvent X Y Z solvent X Y Z
transoid cisoid λ max = 217 nm λ max = 250 nm ε = 21,000 M -1 cm -1 ε is lower Table 6.5 Empirical Rules for Dienes Parent Homoannular Heteroannular (cisoid) λ = 253 nm (transoid) λ = 214 nm Increments for: double bond extending conjugation 30 30 alkyl substituent or ring residue 5 5 exocyclic double bond 5 5 polar groupings: -CCH 3 0 0 -R 6 6 -Cl, -Br 5 5 -NR 2 60 60
A B C D λ max = 214 nm (exp. = 217 nm) λ max = 229 nm (exp. = 228 nm) λ max = 234 nm (exp. = 235 nm) λ max = 353 nm (exp. = 355 nm) H 3 C Parent Homoannular Heteroannular (cisoid) λ = 253 nm (transoid) λ = 214 nm Increments for: double bond extending conjugation 30 30 alkyl substituent or ring residue 5 5 exocyclic double bond 5 5 polar groupings: -CCH 3 0 0 -R 6 6 -Cl, -Br 5 5 -NR 2 60 60
.... C X π π *, λ 190 nm n π *, λ 280 nm Carbonyls As X becomes more electronegative, the n π * transition shifts to the blue. H 3 C C CH 3 λ max = 279 nm ε = 15 M -1 cm -1 H 3 C C NH 2 λ max = 214 nm ε = low H 3 C C H λ max = 204 nm ε = 41 M -1 cm -1...... C NR2.. -.... C + NR2 Red shift of the π π * transition
C R δ β C R γ α Base values six-membered ring/acyclic enone 215 nm five-membered ring 202 acylic dienone 245 Substituent Position α β γ δ double bond in conjugation 30 alkyl group/ring residue 10 12 18 18 -H 35 30 50 -CCH 3 6 6 6 -CH 3 35 30 17 31 -Cl 15 12 -Br 25 30 -NR 2 95 exocyclic double bond 5 homocyclic diene 39
λ max = 249 nm (exp. = 249 nm) λ max = 302 nm (exp. = 300 nm) Base values six-membered ring/acyclic enone 215 nm five-membered ring 202 acylic dienone 245 Substituent Position a b g d alkyl group/ring residue 10 12 18 18 -H 35 30 50 -CCH 3 6 6 6 -CH 3 35 30 17 31 -Cl 15 12 -Br 25 30 -NR 2 95 double bond in conjugation 30 exocyclic double bond 5 homocyclic diene 39
l max (nm) (e M -1 cm -1 ) primary secondary 184 (60,000) 204 (7400) 254 (204) CH 3 207 (7000) 261 (300) CH 3 263 (300) Cl.. CH 3...... -.... + + NH 3.... C N...... -........ H 210 (7600) 265 (240) 235 (9400) 287 (2600) 203 (7500) 254 (160) 230 (11,600) 273 (970) 269 (7800)
E 1 * E E o UV-Vis IR E o ν ν
100 90 80 70 %T 2560 2360 wavenumber (cm-1) 2160 60 IR spectra of HBr (g) and polystyrene
H v o v hν 1 Br H Br r o r 1 hν H H H H θ o θ 1
1cm 1 = 1 wavenumbe r, ν, = 1 1cm ν = 1 λ If ν = 4000 cm -1-1 4000 cm 1 λ = λ = 0.00025 cm = 2500 nm E = hν c = λν ν = c λ E = hc λ E = 6.626068 10 J s x 299792458 m -9 2500 10 m -34 s -1 = 7.95 10 20 J 7.95 x 10-20 J/molecule 6.022141 x 10 23 molecules/mol = 45 kj/mol
ref. cell mirror I r mirror sample cell hν I s grating beam splitter Double beam, Dispersive chopper slit 100 detector 80 % T 60 40 20 3500 2500 1500 wavenumber (cm-1) 500
fixed mirror movable mirror Fourier Transform (FT) Spectrometer hν beam splitter sample detector P 30 20 10 0-10 -20-30 -500-300 -100 100 300 500 displacement
3450 2450 1450 wavenumber (cm-1) 450 70 60 50 40 % T 30 20 10 0 3450 2450 1450 wavenumber (cm-1) 60 50 40 30% T 20 10 0 450 100 80 60 % T 40 20 3450 2450 1450 wavenumber (cm-1) 450 0
Advantages of an FT instrument 1. Speed of data collection (less than one second) 2. High sensitivity 3. High resolution (0.01 cm -1 to 0.1 cm -1 ) 4. Very accurate values for ν
Sources: rare-earth oxides, silicon carbide or Nichrome wire Beam splitter: KBr plate coated with Ge Monochromator: reflection grating Detectors: i) measure increase in temperature (0.005 ºC) ii) pyroelectric [tgs, triglycine sulfate, (NH 2 CH 2 CH) 3 H 2 S 4 )] iii) photoconducting, MCT, HgCdTe, 77K
Sampling Solids: (i) mix < 1 mg of sample with 250 mg of KBr and form a pellet (ii) as 50/50 mixture with Nujol (iii) as a solution Liquids: between two KBr plates Gases: in a tube with KBr windows
3450 2450 1450 wavenumber (cm-1) FT-IR Spectrum of a polystyrene film 450 120 100 80 60 % T 40 20 0 ν I ν
ν = 1 2πc K µ µ = m1m2 m + m 1 2 Calculating Stretching frequencies í (cm 1 ) = 4.12 K ì K = 5 10 5 dyne/cm single bond 10 10 5 dyne/cm double bond 15 10 5 dyne/cm triple bond For a C-H bond stretch: K ì 5 10 12 + 1 12 1 5 1 í (cm ) = 4.12 = 4.12 = For a C- 2 H (C-D) bond stretch: K ì 5 10 12 + 2 12 2 5 1 í (cm ) = 4.12 = 4.12 = 3032 cm -1 2228 cm -1
Intensity of a band For the C=X stretch of: I = 0 I = weak I = strong ν is related to hydrogen bonding
group ν (cm -1 ) group ν (cm -1 ) -H 3400 C C 2150 N-H 3400 C= 1715 C-H 3000 C=C 1650 C N 2250 C- 1100 C-H 3000 cm -1 C- 1100 cm -1 C- 1100 cm -1 C= 1715 cm -1 C-Cl 750 cm -1 C 2143 cm -1
Alkanes CH 2 CH H 3 C CH 2 CH 3 CH 3 H C H H CH 3 C-H stretch at < 3000 cm -1 CH 2 bend at 1465 cm -1 CH 3 bend at 1375 cm -1 gem-dimethyl at 1370 and 1390 cm -1 -(CH 2 ) n>4 - bend at 720 cm -1 Nujol 100 2915 2854 1462 1377 722 80 60 %T 40 20 0 3400 2400 1400 wavenumber (cm-1) 400
C is sp2 hybridized Alkenes C C H C-H stretch at > 3000 cm -1 (3050 to 3200 cm -1 ) C=C stretch at 1640-1670 cm -1 + - C C C X C C C X ν (C=C) 1610 cm -1 As number of alkyl substituents increase on C=C group, ν (C=C) increases. C C H H out-of-plane (P) bend
ut-of-plane Bending Frequencies for Alkenes R 1 890 cm -1 R 2 C C H H R 1 970 cm -1 H C C H R 2 R 1 700-725 cm -1 H C C R 2 H R 1 910 and 990 cm -1 H C C H H 100 ν (cm -1 ) 80 3086 2966, 2914 1646 994, 918 60 40% T 20 0 3450 2450 1450 wavenumber (cm-1) 450
ν (C=C) = 1646 1611 1566 1656 cm -1 ν (C=C) = 1651 1657 1678 1780 cm -1
Alkynes R-C C-H C-H stretch at 3300 cm -1 C C stretch at 2150 cm -1 101 ν (cm -1 ) 3334 2946 2118 1458 1250 C C H 96 % T 91 3450 2450 1450 wavenumber (cm-1) 450 86
Aromatic compounds C-H stretch at 3100 cm -1 C=C stretch at 1600 and 1475 cm -1 H H H H R H P C-H bend (cm -1 ) 690 and 750 R' R' R R' R R 750 690, 780 and usually 880 800-825
vertone/combination bands at 1670-2000 cm -1. v 3 v 2 An overtone band is approximately 2 or 3 times the frequency of a fundamental band. v 1 v 0 100 3074 3042 2934 3400 CH 3 1946 1862 1978 1606 1498 2400 1400 wavenumber (cm-1) 694 726 400 80 60 % T 40 20 0
Alcohols: R--H -H stretch, strong and broad, 3300-3400 cm -1. If H-bonding is not present... -H stretch for primary alcohols 3640 cm -1 secondary alcohols 3630 cm -1 tertiary alcohols 3620 cm -1 phenols 3610 cm -1 C- stretch for primary alcohols 1050 cm -1 secondary alcohols 1100 cm -1 tertiary alcohols 1150 cm -1 phenols 1220 cm -1
110 3670 (v) 3331 (l) 1467 1378 1058 90 70 % T 50 30 3660 2660 1660 wavenumber (cm-1) 660 10 IR spectra of 1-octanol in vapour phase and as a neat liquid
Ethers R--R' asymmetric C- stretch at 1120 cm -1 (R and R' are alkyl) 1250 cm -1 (R is alkyl, R' is aryl) symmetric C- stretch at 1040 cm -1 (R is alkyl, R' is aryl) C 6 H 5 --CH 2 -CH 2 -CH 3 1602 1498 3400 2400 1400 wavenumber (cm-1) 1247 1058 100 80 60 % T 40 20 0 400
R C R C R C R C R C R C R C Cl H R' H R' H NR 2 Carbonyls: C= 1810 cm -1 (asymmetric), 1760 (symmetric) 1800 1760 (free acid) 1735 1725 1715 1710 1690
ν (C=) 1715 1690 cm -1 H ν (C=) 1725 1700 cm -1
Supplemental Slide: Two bands for a C= s-cis R R s-trans Effect of α halogens: H R R Cl ν (C=) 1725 cm -1 Cl R R H ν (C=) 1750 cm -1
Aldehydes: R C H C= stretch at 1725 cm -1 C-H stretch at 2850 and 2750 cm -1. 100 80 2927 2857 2716 60 % T 40 1727 3400 2400 1400 wavenumber (cm-1) 400 20 0 IR spectrum of nonyl aldehyde
Ketones: R C R' ν (C=) 1715 cm -1 110 90 70 1716 3400 2400 1400 wavenumber (cm-1) 50 % T 30 10-10 400 IR spectrum of 3-nonanone
1715 cm -1 1780 cm -1 R 2 C C 2140 cm -1 R 1680 cm -1 Ar R Ar Ar 1685 cm -1 1665 cm -1 H H 3 C CH 2 CH 3 H 3 C CH CH 3 ν (C=) = 1723 and 1706 cm -1 ν (C=) = 1622 cm -1 ν (-H) = 3200-2400 cm -1
Carboxylic Acids: R C H ν (C=) = 1730-1700 cm -1 ν (-H) = 3400-2400 cm -1 (br) ν (C-) = 1260 cm -1 -H bending at 930 cm -1 100% 80% 1711 3400 2400 1400 wavenumber (cm-1) 1288 938 60% % T 40% 20% 0% 400 IR spectrum of nonanoic acid
Esters: C R' C R' R' C R R ν (C=) = 1735-1750 cm -1 ν (C-) = 1000-1300 cm -1 (two bands) ν (C-) = 1150-1300 cm -1, stronger and broader R ν (C-) = 1000-1150 cm -1 100 80 CH 3 C C 2H 5 1764 1239 1056 3400 2400 1400 wavenumber (cm-1) 60 % T 40 20 0 400
ν (C=) = 1735 cm -1 1720 cm -1 1760 cm -1 1770 cm -1 1750 cm -1 1800 cm -1
Amides: R.. NRR' ν (C=) 1630-1680 cm -1 Amines: R N H R N H H R N R' H R' N-H stretch N-H bend C-N stretch 3300, 3400 1 1600 m, br 2 1100 3 3300 4 1500 5 1100 3 R NH 2 3180, 3350 6 1620-1640 7 600-760 R NHR 3300 1550 7 600-760
100 80 3375 3298 NH 2 1611 1064 60 % T 40 20 0 3450 2450 1450 wavenumber (cm-1) 450 3355 NH 2 3190 1643 1634 3450 2450 1450 wavenumber (cm-1) 100 80 60 % T 653 40 20 0 450
Nitriles: R-C N ν (C N) = 2250 cm -1 (aliphatic), 2230 cm -1 (aromatic) C 3450 N 2985 2941 2882 2243 1459 1389 1376 2450 1450 wavenumber (cm-1) 100 80 60 % T 40 20 0 450
Nitro: R + N.... -...... ν (N-) = 1350, 1550 cm -1 (aliphatic) ν (N-) = 1300, 1500 cm -1 (aromatic) 3450 3086 1616 1489 N 2 2450 1450 wavenumber (cm-1) 1541 1356 100 80 60 % T 40 20 0 450
Fluorescence -emission technique E 1 * E hν hν' E o λ
Intensity of Fluorescence lifetime of E 1 * is proportional to 1/ε XYZ * r f r r r others XYZ XYZ XYZ rf Φ = Quantum yield, Φ (0 to 1) r + r + r f r others
å >> ð-ð * å n-ð * biphenyl Φ = 0.2 fluorene Φ = 1
source hν grating slit sample cell attenuator grating detectors (pmt)
F λ excitation (nm) A / F 200 300 400 500 l absorption/emission (nm)
Experimental Considerations 1. F = kp o εbc detection limits: 10 to 0.01 ppm self-quenching self-absorption A < 0.05 or T > 90 %
2. Φ decreases as i) T increases ii) viscosity decreases iii) concentration of paramagnetic molecules increases iv) concentration of halogens in solvent or on analyte increases 3. F is ph-dependent, both Φ and λ emission
4. For quantitative applications a calibration curve is required 5. Precision and accuracy are both approx. 3 %
Applications: - determination of metals 3 N H Al 3+ N N Al N - quantification of biochemical analytes such as tryptophan, NH vitamins, steroids and DNA 2 H 2 N N + ethidium bromide - analysis of PAHs benzo[a]pyrene