9/28/10. Visible and Ultraviolet Molecular Spectroscopy - (S-H-C Chapters 13-14) Valence Electronic Structure. n σ* transitions

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1 Visible and Ultraviolet Molecular Spectroscopy - (S-H-C Chapters 13-14) Electromagnetic Spectrum - Molecular transitions Widely used in chemistry. Perhaps the most widely used in Biological Chemistry. Primarily used for Quantitative analysis (why?). Hyphenated techniques? Valence Electronic Structure n σ* transitions Valence electrons are the only ones whose energies permit them to be excited by Ultraviolet-visible radiation. σ (anti-bonding) π (anti-bonding) n (non-bonding) π (bonding) Four types of transitions σ σ* π π* n σ* n π* compound λ max ε max H CH 3 H CH 3 Cl CH 3 I (CH 3 ) 2 S (CH 3 ) CH 3 NH (CH 3 ) 3 N σ (bonding) 1

2 n π* and π π* Transitions Structural changes and their effect on absorption Compound Type λ max ε max compound λ max ε max type C 6 H 13 CH=CH π π* C 5 H 11 C C CH π π* CH 3 CCH n σ* CH 3 CH n π* CH 3 N n π* CH 3 N=NCH n π* CH 3 CH 2 CH 2 CH=CH 2 lefin 184 ~10,000 CH 2 =CHCH 2 CH 2 CH=CH 2 diolefin (unconjugated) 185 ~20,000 CH 2 =CHCH=CH 2 diolefin (conjugated) 217 ~21,000 CH 2 =CHCH=CHCH=CH 2 triolefin (conjugated) 250 Large! CH 3 CH 2 CH 2 CH 2 CCH 3 Ketone CH 2 =CHCH 2 CH 2 CCH 3 Unsaturated ketone CH 2 =CHCCH 3 α,β-unsaturated ketone ,600 Solvent Effects Shifts in λ max Solvent Effects - Intensity Solvents can interact with the analyte molecules and shift absorbance peaks and intensities. Hyperchromic Increase in absorption intensity. Hypochromic Decrease in absorption intensity. red shift (bathochromic) peaks shift to longer wavelength. blue shift (Hypsochromic) peaks shift to shorter wavelength. n π* generally blue shifted (hypsochromic); solvation of and hydrogen bonding to the lone pair. Large shifts (up to 30 nm). Absorption characteristics of 2-methylpyridine Solvent λ max ε max Hexane Chloroform Ethanol Both n π* and π π* can be red (bathochromic) shifted; attractive polarization forces, increase with increasing solvent polarity. Small shifts (less than 5 nm). Water Ethanol - HCl (1:1)

3 Auxochrome Substitutent groups which are not themselves optically active (in this energy range) but which do interact with other chromophores to shift both intensity and wavelength. Absorption Characteristics of Pyridine Derivatives Derivative λ max ε max Pyridine CH CH CH F Cl I H Typical rganic UV/Vis Spectra UV-VIS absorption spectra for 1,2,4,5-tetrazine Absorption involving transition metal ions Absorption spectra for lanthanide ions e - in f orbitals are screened from external influences by e - occupying orbitals with higher n! Spectra of the aqua ions of some transition-metal ions 3

4 Charge Transfer absorption Instrumentation Electron from an orbital centered on the ligand is excited to an orbital centered on the metal Sources ther: white LED s nm Xenon arc lamps Sample Containers Sample is placed in a cell or cuvette 1, 5, & 10 cm path lengths are commonly available Glass or plastic Visible region Quartz or fused silica Ultra-violet and visible region ptical Glass nm Quartz (Far-UV) nm Successful spectroscopy requires that all materials in the beam path other than the analyte be as transparent to the radiation as possible. Also, the geometries of all components in the system should be such as to maximize the signal and minimize the scattered light. 4

5 Wavelength selectors Detectors Wavelength (nm) Band pass interference filter 0 Photodiode arrays Another solid state silicon device. A charged gate collects either the holes or electrons generated by the absorption of a photon. Charge accumulates in the potential well for as long as exposure is maintained. Device is read-out by charge injection (CID) or charge transfer (CCD). Comparable sensitivity to PMT, but functions as array detector also. Photodiode array based spectrometer Permits simultaneous measurement of multiple wavelengths More rapid measurement than a dispersive instrument No moving parts Commonly used in HPLC instruments Quantitative Analysis with UV/Vis Beer s Law A = log 10 [P 0 /P] = εbc UV/Vis spectroscopy can be used to analyze a sample for analyte concentration. Need to know LQ and LL in order to define useful region of applicability. 1. Calibration Curve 2. Standard Addition (matrix effects/complex sample) 3. Internal Standard (compensate for random and systematic errors; difficult) 5

6 Limitations to Beer s Law Chemical Equilibria Is absorbance really linear with respect to path length? concentration? molar absorptivity? When a substance is involved in a chemical reaction, the extent of that reaction is concentration dependent Path Length Polychromatic radiation? Concentration: Intermolecular interactions Shifting chemical equilibria Molar absorptivity: Solution s index of refraction Instrumental deviations from linearity? λ = 430 nm λ = 570 nm Example: an indicator with K a = 1.42 x Absorbance is measured at two wavelengths (430 nm and 570 nm). Error in the measurement of Transmission Different noise sources contribute differently to error in T (S T ) Constant Error in T (S T is constant) Inexpensive spectrometers will suffer from limited readout resolution. IR spectrometers will be subject to Johnson noise. Experiments where source intensity is low or detector sensitivity is low will be limited by dark current and amplifier noise. Relative Error in Concentration Relative Concentration Error Assume fixed relative T error 0.5% (0.005). Absorbance measurements between A=0.05 and A=1.55 keeps error < 5% Absorbance 6

7 Error (T 2 + T) 1/2 Error T High quality UV/vis spectrometers are susceptible to this case. Relative Error in Concentration High quality UV/vis and IR spectrometers will be subject to cell positioning errors. Inexpensive IR spectrometers will be subject to flicker noise. Relative Concentration Error S T is constant. Limited readout resolution; Thermal detector (Johnson noise); Dark current and amplifier noise S T varies as (T 2 + T) 1/2 Shot noise Absorbance S T varies as T Cell positioning uncertainty and 1/f noise Relative concentration uncertainties Quantitative Analysis with UV/Vis UV/Vis spectroscopy - and the other spectroscopic techniques we will discuss can all be used to analyze a sample for analyte concentration. Need to know LQ and LL in order to define useful region of applicability. 1. Calibration Curve 2. Standard Addition (matrix effects/complex sample) 3. Internal Standard (compensate for random and systematic errors; difficult) b s T = k = +/ c k = +/ d k = +/

8 Standard Addition - 1 Standard Addition - 2 Measure the signal (S 1 and S 2 ) for both the sample and the sample plus a known volume of a known concentration of the analyte. The signal depends upon the concentration of the target analyte, which is simply diluted to the final volume, V t. The experiment has some response factor k that relates the concentration to the signal amplitude. We can write Consider the following data. The volume of the unknown and each standard increment is 5.00 ml. The standard has a concentration of 8.7 ppm. Added Volume (ml) Signal (arbitrary) From these two measurements, we can solve for the unknown concentration, and obtain the expression Standard Addition - 3 A graphical solution can be easily obtained. Extrapolate the curve back to the x- axis (in the negative-x region). This x-intercept represents the volume of standard solution which has the same amount of analyte as the unknown solution. -V 0,s c s = V x c x c x = -(V 0,s /V x ) c s = -(-7.28/5.00) 8.7 ppm =12.7 ppm V 0,s = ml 8