2. Welcome to Chemistry 421 Use of Physical Methods to determine structures in Organic Chemistry Dr. Charles DeBrosse 232 Beury Hall (across from Dept. office) NMR lab 001 Beury Hall 215-204-1082

3. Course Organization Lectures Tuesday evenings 6-9pm. Attendance is strongly encouraged. You should read the assigned material and do the homework problems prior to the lecture. Textbook, Organic Structural Spectroscopy, by Lambert, Shurvell, Lightner and Cooks Grade based on 3 In-class quizzes, homework sets and Final exam Course notes will be available on Blackboard generally the Monday before the lecture. I will reserve Monday 10-11am as office hour, and am accessible other times in my lab or office

4. Spectroscopy in Organic Chemistry…. The Chemists Eyes, Ears and Nose How do we know what we have? Read labels (sometimes labels lie) Whose word do we have to take on it? Check it out for yourself! (Get a spectrum!) A spectroscopist. (never the hero, just the hero’s best friend)

5. Chemistry 421 Goal of the course is to give you the tools to answer parts of these questions. Organic Structure determination by spectroscopic methods NMR (nuclear magnetic resonance spectroscopy) Mass Spectrometry (MS) Infrared Spectroscopy (IR and other vibrational classes like Near IR, Raman) Electronic Spectra (Ultraviolet and chiroptical methods)

6. Structural Features we can address Spectroscopically Molecular weight Chemical Formula Functional groups Skeletal Connectivity, structural isomers Spatial-geometric arrangements, stereoisomerism, symmetry Presence and location of chromophores Chirality issues Some of these are more central than others. Sometimes we can stop when the answer is fit to purpose.

7. Techniques we will study in this Course NMR--looks atb atoms by means of their nuclei. Connectivity pathways, spatial arrangements of atoms and 1:1 correspondence between signals and atoms Mass Spec--measures molecular weight, most fundamentally useful for unknowns. Controlable fragmentation can distinguish among rival possibilities IR and Raman--Vibrations characteristic of bonds, particulary for functional group identification. Excellent “fingerprint” UV--reports on conjugation and multiple bonds. Provides entre’ to chiroptical probes to assymetric configurations

8. Chemistry 421 Goal is for you to gain a conversational level of knowledge Base level of theory, as pictoral as possible should help you realize the scope and limitations of these methods. Survey of representative data and how to interpret it. Applying the right tool to the right question Integrating data from various tools and sources (i.e. we should be able to explain all our observations. Also we should be able to observe features we predict, knowing our chemistry)

9. Complementary Spectroscopies There are strengths and weaknesses in all the various spectroscopy methods “Blind Spots” (see story of three blind men describing an elephant) NMR e.g.; has “NMR silent” nuclei; fully substituted carbons as “blocks”; blind to inorganics, nearly blind to polymeric mtls. IR e.g.; robust for functional groups; great if you have a compound match in a library (fingerprint); shaky on quantitative response, same spectrum might explain multiple compounds; subject to selection rules governed by dipole moment in vibration (complement somewhat with Raman) UV; needs chromophores Mass Spec; compounds differ in ionizability. A smart chemist will be attuned to possible blind spots before making conclusions. Best solution is to “marry up” complementary data. All the data needs to “agree” or at least, not conflict withing the various methods.

10. Total Synthesis as a Structural Proof Use of all our knowledge Generally the product of a reaction is rationally related to the ingredients we have used. Non-ambiguous route from known compounds Oxidative degradation to known compounds and history of chemistry

11. The Electromagnetic Spectrum Light comes in different colors No matter what part of range, there are some features in common, that you should know. Propagation of e,b fields, time Amplitude Photon  E= h is frequency, Hz, 1/sec = c/ ; |c=3x10 10 cm/sec h = Planck’s constant = 6.624 x10 -34 Jsec /c = 1/ = wavenumber, cm -1 1 wavelength, is a distance…. Peaks per time; frequency

12. Chemical Properties as related to the different colors of light Log, meters lower E, lower larger higher E, higher smaller -15-10-505 NMR nuclear spin flips X- Ray Spacings between atomic nuclei in crystals Micro waves rotational states IR+Raman vibrations of bonds UV Electron orbital transitions 100 kcal/mol 10 kcal/mol 10 -6 kcal/mol

13. What about Mass Spec? The properties measured do not directly bear on absorption of light More properly termed mass spectrometry We do however in quadrupole mass spectrometry scan an electric field that induced different curve paths for different masses Radiofrequency also does show up in FT mass spec (ion cyclotron resonance)

14. Sensitivity of the methods we will study NMR is worst, typically needing 10s of  g to 10s of mg for 13 C. Mass Spec is generally about 10 3 -10 4 x more sensitive than NMR UV is about 100 x more sensitive than NMR

15. Questions, Questions…. What’s in this flask? What have you just synthesized? Did my reaction Work? What is present in that sample? How could this reaction have possibly Failed? Isolated materials? Purity? Mixture? What kind of mixture? Is this material suitable for the next step? Best to Ask Yourself these questions! (Better than making your Boss ask, or not to be able to answer a “Customer”

16. Answers! Bad Answer: “…because this always works in our lab…” Bad Answer #2 “..because my Professor (or some slightly older grad student, postdoc) said so..” Slightly less Bad Answer #3 “…this was done in the literature…” Good Answer “…because all the spectral and other analytical data agree with me” Analytical Chemistry. If you do it right, nobody has to “take your word” on the answers!

17. Features in Common for all Spectroscopy Measuring Scheme Some Physical Property Energy Transitions Light, (Frequency Dispersion) Data Wisdom (or Progress) Knowledge A key for us here is, we use instruments to Disperse energy across a scale appropriate to a chemical property Analysis, Interpretation

18. Therefore we have Precise Analytical Instruments that can Disperse Energy Accurate, precise, reproducible Combine the energy dispersion scheme with a detection scheme. Generally the sample sits physically between the source and the detector. Detector provides selectivity in response, usually generates a voltage. We record voltage responses as “DATA”

19. Detector The dispersion is easy to achieve with ordinary light Light source Monochromator rotates Prism or diffraction grating Spacings on grating appropriate to wavelength Schemes use slits to admit a select region of spectrum Pretty ineffective for radio waves Recorder Sample

20. Spectroscopy “Spreads out Vision” All the techniques we will discuss have some features in common Data will have a running variable (x-axis) that is in some sense, a “energy” scale. (not at least directly, a time axis. Therefore a snapshot in time of a molecule) The response variable (an absorption or other intensity) is related to the chemical preponderance of some feature that cause the response. Energy Instrument Response There is a peak here But not here The position informs us about some chemical property in the sample The peak height informs us about how much of that property is in the sample

21. Fourier Transform Methods An alternative to Energy Dispersive methods All modern NMR and IR is done this way Measures all frequencies at same time. More efficient at signal-gathering in a give time (better S/N) The frequencies present are deconvoluted (or dispersed) after data is collected. Fourier Analysis is the mathematical method for doing this. It is based on the theory that any complex periodic (repeats over time) wave can be decomposed into a linear combination of sinusoids

22. To get the measurement, we collect a detector response as a function of time Lots of different frequencies present from the sample Their voltages “beat” against each other making interference pattern (interferogram) Interference is periodic, because the frequencies are constant w.r.t each other Instrument Response time

23. An Oscillating voltage is interpreted as a Frequency This is a Frequency Axis. Think Hz! The process is similar to the way a sound wave is digitized to make e.g. a music CD Key to this is sampling at exactly equal time intervals

24. Interfering Sinusoids are Represented in a decaying trace Key to the process is a very precisely defined time base (the x axis) that the FT algorithm uses to count Space is frequency 1 Space is beating of frequency 2 vs 1 (1 - 2) A human being could compute this FT, counting beats per time unit

25. Interference patterns--Almost able to Transform by Hand… Time(ms) But its really the Fast Fourier Transforms and fast computers that make this practical!

26. Adding accumulating scans from the detector into memory of computer Signals are coherent and adding the scans causes signal to grow linearly with number of scans. Noise being random and incoherent grows with √no.of scans From this, the Signal-to-Noise ratio (S/N) grows proportionally to the square root of number of scans E.g., a spectrum acquired with 100 scans will be 10x better than one with 1 scan only. Since the time-acquisition is fast and efficient it is easy to Signal-Average

27. Some Features common to all Spectra Using computerized Data systems adds an additional limitation on our resolution problem More data points are better but usually at a cost to expt efficiency See here 2 identical peaks, digitized differently Bears directly on our ability to determine the position of I max Linewidth. Usually measured at 0.5 Imax Can be limited by the instrumentation, or be limited by nature. Nature, here exerts herself as uncertainty to to slight chemical variation, or inability to measure energy precisely. Units of linewidth are same as axis, e.g. Hz, cm-1 Resolution (ability to distinguish line from closely spaced neighboring line is related to linewidth

28. Noise, the curse of Science All measurements, especially those we carry out with instruments, generate Noise. Detectors of all sorts generate electrical noise Noise is bad. It is random and incoherent and does not possess information. We go to tremendous expense and effort to eliminate, suppress, and finesse our way past noise. Signals are good. They give us information. Noise limits our ability to even observe very weak signals or to quantify somewhat weak signals. The Signal-to-Noise Ratio is an important parameter is assessing our ability to interpret data. Noise is superimposed on top of peaks

29. Signal-to-Noise (S/N) ratios Noise(rms) is 0.707 x peak to peak Measure height S/N=6.3/2*0.707 =4.45 So this peak is reliably detectable, but not reliably quantitatable Typical rule of thumb: Limit of detection, S/N=3 Limit of Quantitation, S/N=10

30. Chemistry 421--Structure Determination Interpretable Connection between Structural Features and Spectroscopic signals We will interpret spectra to learn about structures. The Interpretation “paradigm” consists of charting: ObservationInference IR shows a C=O at 1730cm -1 NMR shows a 3-proton singlet at 2.2 My molecule contains a ketone or aldehyde I may have a methyl group adjacent to C=O May need more experiments or to look further in the data!

31. A way of Thinking…… “Partial Unknowns” New compound? Side reaction product Wrong starting material, carry through known synthetic steps Can we track known compounds? Peaks from precursor compounds may have “descendents” Total Unknowns Isolated natural products Unrelated impurities, contaminants Single component, vs mixture? Known compounds (verify structure) All predicted signals present? Agreement with literature? Impurities present? Fingerprint? ????????

32. A Strategy for Handling Unknown Structures Complementary 1 H NMR, 13 C NMR, Mass Spec, UV--any features stand out? Get the Molecuar Weight from MS Heavy Atoms? (ratio of M to M+1, M+2) If heavy atoms are identified, subtract from MW Consult various molecular formula DBs (Merck, CRC etc). Write out Molecular Formula Use the DBE (sites of unsaturation) rule Infrared-- Functional groups present? Identify as possibly subtract from formula (retain the need to incorporate at end) Inventory 13 C NMR and classify the C,H groups present. Tabulate fragments of structure. Reconcile MS fragments. Assemble possible structures

33. Molecular Weight and Molecular Formulas Absolutely critical to Stucture determination Centrality of Mass Spectrometry to modern Chemistry Molecular weight must agree with the structure. Note well, that a given nominal MW generally is consistent with several possible formulas. The “nitrogen rule”. A compound with an even-numbered molecular weight has 0, 2 or an even number of nitrogens. Very Important: Learn the rule for sites of unsaturation (double-bond equivalents, DBE) as a predictive tool for multiple bonds and/or rings. These are based on the standard valencies for ordinary atoms.

34. Aim to reduce a formula to C N H 2N+2 Take formula and cross off Oxygen atoms Replace all halogen atoms with hydrogen Cross off all Nitrogen atoms, and for each N remove one H atom. Sulfur treat like Oxygen (? Use care if there are a lot of oxygens, possible O=S=O type groups, similar issues with Phosphorus) Subtract your newly reduced formula (looks like C x H x, from CxH 2x+2 number H (even number) Divide this answer by 2. Result is DBE. DBE Rules

35. So some “Decision-Tree” thinking is possible Synthetic Product Correlations to protons Carbon Survey Noe for stereochemistry Proton coupling pattern Assess Purity Do I need more information? What do I need to find out Worth more spectroscopy? Back to the Lab! 1H NMR Could it be what I want? Quick Inventory of signals Data Information Content higher “granularity” of questions The NMR branch. Integrate at higher level with other techniques Separations methods, Feedback to synthesis. NO YES Need Assignments? YES NO

36. Nuclear Magnetic Resonance (NMR)

37. Tonight’s Subjects I.How do the spectrometers work? II.The NMR measurable quantities

38. What is NMR Spectroscopy? Nuclear Magnetic Resonance Radio Frequency Absorption Spectra of atomic nuclei in substances subjected to magnetic fields. Spectral Dispersion is Sensitive to the chemical environment via “coupling” to the electrons surrounding the nuclei. Interactions can be interpreted in terms of structure, bonding, reactivity

39. The Fundamental NMR equations Spinning nuclei produce a magnetic field that is proportional to its magnetic moment . The proportionality constant is  ;  =  hI An active nucleus in a magnetic field B 0 has an energy w.r.t. zero field of: E (= h  = h  ) = -  B 0 where  is the component of the magnetic moment colinear with B 0 This gives for I Z = ±1/2; E = ± 1/2 (  h B 0 )  E =  h B 0 and in angular units  =  B 0

40. Origin of the NMR Effect Nuclei with other than A(#protons+neutrons) and Z(#protons) both even numbers, possess net spin and associated angular momenta Reveals itself only in magnetic field. As usual, such momenta are quantized States have different energies, populated according to Boltzmann distribution States are 1/2, 3/2, 5/2…for A= odd number and integer if A= even number and Z= odd number Transitions of individual nuclei between spin states is possible (both directions) leading to an equilibrium of populations Number of states is 2I + 1

41. Pictoral View of Spin Precession of nuclear magnet--Units of Torque Z X Y M Because we are forced into observing the group behavior, we have the mathematic equivalent of the simple picture on the right Averages out in x,y plane; small net resultant vector along the z axis Direction of the Applied Magnetic Field X Y Z Nucleus Moments precess about magnetic field. Quantized either with or oppossed to field The Boltzmann excess of low over high energy state is very small, 1 in 10 6  

42. Resonance--A general phenomenon for energy pumping Imagine a kid on a swing… The period (frequency of the swing is determined (g, r(length),  ). Lets say the natural period is 3 seconds, or the frequency is 0.33 If the Daddy gives a push every 3 seconds, the kid will go higher and energy will be absorbed. Every 2 seconds and the motion will get stalled and “interfered” with. Every 1.5 seconds and the energy will get absorbed but not as efficiently. The Daddy will get tired. This general principal applies in NMR among other kinds of measurement, and holds whether we scan through the applied frequency or multiplex all at once

43. NMR-What is it Good For? (absolutely everything!) Solving structures of compounds like synthetics, impurities, natural products Identifying metabolites Stereochemical determination Follow reactions Validating electronic theory; trends within series of compds. Kinetics Extended structure, e.g. protein nmr Molecular interactions e.g. ligand binding Acid-base questions Purities Mechanisms, e.g. isotope distributions, other effects Questions about the solid state Imaging

44. And Besides that… You get your sample back! Not so for mass spec Try recovering your compound from a KBr pellet or nujol mull

45. But on the other Hand… NMR is one of the least sensitive analytical methods Characterized by long relaxation time constants, limiting experimental efficiency in real time Sometimes too much information. Can be demanding on interpretation skill Relatively Expensive compared with other analytical methods As with other methods NMR has “blind spots” and cannot serve as an analytical panacea

46. What Do I Hope you will Learn? Enough theory to make you conversant in the area…. NMR with respect to how the effects arise and can be predicted; connection with experiments and limitations of these; survey of how the instruments work. Basis of the experiments Data processing considerations, at level to appreciate what may have been done to give your result. A basic toolbox of experiments, what they do and how to use them in your work A working knowledge of organic chemical shifts and influence of symmetry on signal counting Spin coupling, coupling networks and connectivity, use of J-coupling constants in chemistry

47. Why NMR? Unmatched versatility as an Analytical technique High on chemical information content Significant interpretability Interpretable at several levels of sophistication Response related to molar preponderance These attributes are true for solids, liquids, mixtures, and to a small extent, gas phase More than half the periodic table has at least one NMR active isotope

48. What are the Measurables in NMR? Intensity (analytical parameter, proportional to molarity) Chemical Shift (the electronic surroundings) Couplings (scalar J and dipolar D; bond paths, angles connectivity and distances) Relaxation parameters (motions, distances)

49. How do we Generate, and Record NMR Spectra? signal PreAmp Probe in Magnet receiver transmitter Phase locked loop RF pulse signal Pulser Frequency generation Power Amplifier Oscillator FID with 90deg phase shift Free Induction Decay Host Workstation Acquisition computer Data file storage NMR Acquisition commands Timing control signals Superheterodyne (beat-down to AF) Phase sensitive detection A/D convertor User interface Expt. Setup, control Data processing, plotting Network Block Diagram for Spectrometer

50. Radio Frequency Transmit- Receive system Finely controlled RF pulses Microsecond control Precise control of timing, e.g pulses and delays Other precisely delivered RF for decoupling, selective excitation Gradient amp and generator, shielded in probe to avoid eddy currents

51. Modern Superconducting NMR Magnets Lines of force project several feet into the room. They concentrate at the top and bottom. Magnets can grab iron objects and accelerate them. Older Magnets (1970s) had opposed pole faces. High voltages and currents demanded heroic temperature control. Field ran side to side through sample Supercon magnets have much larger fields, better homogeneity. Field runs up the axis of the sample. New technology! Built in auxilliary magnet with reversed current acts as “active” shield, partly eliminating the projection into the room. Note: Special superconducting alloys Niobium- Tantalum. Search goes on for higher temperature superconductors.

52. What’s the role of the magnet? Bigger the field strength, the better. This is both from a sensitivity and dispersion of signals point of view. Expressed in Hz, permits easiser math and trig as needed. Gauss would generate energies in ergs.  E= h  H 0 I z energy Field strength, H 0 Remember the energy difference gives the population excess. Roughly H 7/4 increase

53. The NMR Probe Coil Tuned Circuit Matching to Tx network Sample goes inside here Usually there is a double tuned response for Deuterium lock A second coil provides a decoupling, gradient or other RF

54. How Sensitive is NMR? 3 000 22 )1( HH IkT IIN       http://arrhenius.rider.edu/nmr/NMR_tutor/periodic_table/nmr_pt_frameset.html Another good site is http://nmr.magnet.fsu.edu/resources/nuclei/table.htm The Rider site, referenced below gives receptivity vs. 13 C with clickable entries. These reflect natural abundance, , etc. N=#spins

55. The answer to that question is… Not all that Sensitive! At any given time Mass spec is at least a 10 4 times more sensitive Compare with UV, IR at least 10 2 x sensitive This is tied to the fact that NMR detects only the tiny Boltzmann excess. Any old molecule can fragment in MS or absorb a IR photon. Lots of research in NMR aimed at the sensitivity problem k is Boltzmann constant 1.38x10 -23 J/moleculeK Because the  E is so small, the excess (which is what we detect) is miniscule ,  are the short names for the upper, lower spin quantum states of a spin=1/2 nucleus

56. Most Important Nuclei in NMR 1 H, (also 2 H, 3 H) 13 C 31 P 15 N especially when labeled into proteins 19 F 29 Si Some isotopes of Sn, Cd. Pb, Ag, Pt No coincidence that these are the I=1/2 nuclei. Spin numbers higher possess nuclear quadrupole moment as well. This couples to, broadens and complicates the nuclear spin angular momentum. For the most part these are niche nuclei. Exception is 11 B

57. Quadrupolar Nuclei Spin ≤ 1 Electric field at nucleus non-symmetrical Effective relaxation mechanism, promotes loss of NMR fine structure “decouple” from attached spins. Can even wipe out attached spin 1/2 signals. Lines are broad, very challenging NMR 35 Cl, 11 B, 17 O, 14 N, 7 Li, etc. Some redeem themselves, deuterium, 6 Li