Instructor: Dr. Marinella Sandros

1. Nanochemistry NAN 601 Instructor: Dr. MarinellaSandros Lecture 18: Analytical Techniques Part 1

2. Outline • Nuclear Magnetic Resonance (NMR) Spectroscopy • Fourier Transform Infrared (FTIR) Spectroscopy • Dyanmic Light Scattering

3. Spectroscopy “seeing the unseeable” Using electromagnetic radiation as a probe to obtain information about atoms and molecules that are too small to see. Electromagnetic radiation is propagated at the speed of light through a vacuum as an oscillating wave.

4. NMR: Introduction • NMR is the most powerful tool available for organic structure determination. • It is used to study a wide variety of nuclei: • 1H • 13C • 15N • 19F • 31P

5. Nuclear Spin • A nucleus with an odd atomic number or an odd mass number has a nuclear spin. • The spinning charged nucleus generates a magnetic field. • If we place these nuclei in a magnetic field, they can line up with or against the field by spinning clockwise or counter clockwise.

6. Alignment with the magnetic field (called ) is lower energy than against the magnetic field (called ). • Note that for nuclei that don’t have spin, such as 12C, there is no difference in energy between alignments in a magnetic field since they are not magnets. As such, we can’t do NMR spectroscopy on 12C.

7. NMR: Basic Principles • Imagine placing a molecule, for example, CH4, in a magnetic field. We can probe the energy difference of the - and - state of the protons by irradiating them with EM radiation of just the right energy.

8. NMR: Basic Principles • In a magnet of 7.05 Tesla, it takes EM radiation of about 300 MHz (radio waves). So, if we bombard the CH4 molecule with 300 MHz radio waves, the protons will absorb that energy and we can measure that absorbance. • In a magnet of 11.75 Tesla, it takes EM radiation of about 500 MHz (stronger magnet means greater energy difference between the - and - state of the protons)

9. NMR: Basic Principles But there’s a problem. If two researchers want to compare their data using magnets of different strengths, they have to adjust for that difference. That’s a pain, so, data is instead reported using the “chemical shift” scale as described on the next slide.

10. The Chemical Shift (Also Called , delta) Scale • Here’s how it works. We decide on a sample we’ll use to standardize our instruments. We take an NMR of that standard and measure its absorbance frequency. • We then measure the frequency of our sample and subtract its frequency from that of the standard. • We then then divide by the frequency of the standard. This gives a number called the “chemical shift,” also called d, which does not depend on the magnetic field strength.

11. Chemical Shifts • Imagine that we have a magnet where our standard absorbs at 300,000,000 Hz (300 megahertz), and our sample absorbs at 300,000,300 Hz. The difference is 300 Hz, so we take 300/300,000,000 = 1/1,000,000 and call that 1 part per million (or 1 PPM). • Now lets examine the same sample in a stronger magnetic field where the reference comes at 500,000,000 Hz, or 500 megahertz. The frequency of our sample will increase proportionally, and will come at 500,000,500 Hz. The difference is now 500 Hz, but we divide by 500,000,000 (500/500,000,000 = 1/1,000,000, = 1 PPM). • Of course, we don’t do any of this, it’s all done automatically by the NMR machine.

12. Tetramethylsilane • TMS is a common standard. • Since silicon is less electronegative than carbon, TMS protons are highly shielded. Signal defined as zero • Organic protons absorb downfield (to the left) of the TMS signal.

14. The Chemical Shift of Different Protons • NMR would not be very valuable if all protons absorbed at the same frequency. • What makes it useful is that different protons usually appear at different chemical shifts (d. • So, we can distinguish one kind of proton from another. • Why do different protons appear at different d? • There are several reasons, one of which is shielding. • The electrons in a bond shield the nuclei from the magnetic field. So, if there is more electron density around a proton, it sees a slightly lower magnetic field, less electron density means it sees a higher magnetic field:

15. This represents the electron density of C-H bond. How much electron density is on the proton depends on what else is attached to the carbon. If Z is an electronegative atom , the carbon becomes electron deficient and pulls some of the electron density away from the H. If Z is an electron donating group, more electron density ends up on the H. • How do the electrons shield the magnetic field? • By moving. • A moving charge creates a magnetic field, and the field created by the moving electrons opposes the magnetic field of our NMR machine. • It’s not a huge effect, but it’s enough to enable us to distinguish between different protons in our sample.

16. The Hard Part - Interpreting Spectra Learning how an NMR machine works is straightforward. What is less straightforward is learning how to use the data we get from an NMR machine. That’s because each NMR spectrum is a puzzle, and there’s no single fact that you simply have to memorize to solve these spectra. You have to consider lots of pieces of data and come up with a structure that fits all the data. What kinds of data do we get from NMR spectra? For 1H NMR, there are three kinds each of which we will consider each of these separately: • Chemical shift data - tells us what kinds of protons we have. • Integrals - tells us the ratio of each kind of proton in our sample. • 1H - 1H coupling - tells us about protons that are near other protons.

17. Location of Signals • More electronegative atoms deshield more and give larger shift values. • Effect decreases with distance. • Additional electronegative atoms cause increase in chemical shift.

18. Protons in a Molecule Depending on their chemical environment, protons in a molecule are shielded by different amounts.

19. Typical Values => Chapter 13

20. 1,1,2-Tribromoethane Nonequivalent protons on adjacent carbons. => Chapter 13

21. What is Infrared? • Infrared radiation lies between the visible and microwave portions of the electromagnetic spectrum. • Infrared waves have wavelengths longer than visible and shorter than microwaves, and have frequencies which are lower than visible and higher than microwaves. • The Infrared region is divided into: near, mid and far-infrared. • Near-infrared refers to the part of the infrared spectrum that is closest to visible light and far-infrared refers to the part that is closer to the microwave region. • Mid-infrared is the region between these two. • The primary source of infrared radiation is thermal radiation. (heat) • It is the radiation produced by the motion of atoms and molecules in an object. The higher the temperature, the more the atoms and molecules move and the more infrared radiation they produce.

22. Antisymmetric Stretch Bend Symmetric Stretch Infrared Spectroscopy The bonds between atoms in the molecule stretch and bend, absorbing infrared energy and creating the infrared spectrum. A molecule such as H2O will absorb infrared light when the vibration (stretch or bend) results in a molecular dipole moment change

23. IR Spectrum • Plot IR energy vs. %transmittance (%T) – Energy scale in wave numbers, wn (cm-1) – %T scale • Compares intensity of IR striking sample (Iin) with intensity of IR leaving sample (Iout) • 100%T no light absorbed by sample • 0% all light absorbed by sample

24. Infrared Spectroscopy A molecule can be characterized (identified) by its molecular vibrations, based on the absorption and intensity of specific infrared wavelengths.

25. Infrared Spectroscopy For isopropyl alcohol, CH(CH3)2OH, the infrared absorption bands identify the various functional groups of the molecule.

26. Capabilities of Infrared Analysis • Identification and quantitation of organic solid, liquid or gas samples. • Analysis of powders, solids, gels, emulsions, pastes, pure liquids and solutions, polymers, pure and mixed gases. • Infrared used for research, methods development, quality control and quality assurance applications. • Samples range in size from single fibers only 20 microns in length to atmospheric pollution studies involving large areas.

27. Applications of Infrared Analysis • Pharmaceutical research • Forensic investigations • Polymer analysis • Lubricant formulation and fuel additives • Foods research • Quality assurance and control • Environmental and water quality analysis methods • Biochemical and biomedical research • Coatings and surfactants • Etc.

28. Dispersion Spectrometer In order to measure an IR spectrum,the dispersion Spectrometer takesseveral minutes.Also the detector receives onlya few % of the energy oforiginal light source. To separate IR light, a grating is used. Detector Grating Slit Sample To select the specified IR light, A slit is used. Light source

29. FTIR • FTIR was developed in order to overcome the limitations encountered with dispersive instruments. • The main difficulty was the slow scanning process. • A method for measuring all of the infrared frequencies simultaneously, rather than individually, was needed. • A solution was developed which employed a very simple optical device called an interferometer. • The interferometer produces a unique type of signal which has all of the infrared frequencies “encoded” into it. The signal can be measured very quickly, usually on the order of one second or so. • Thus, the time element per sample is reduced to a matter of a few seconds rather than several minutes.

30. Interferometer • Most interferometers employ a beamsplitter which takes the incoming infrared beam and divides it into two optical beams. One beam reflects off of a flat mirror which is fixed in place. The other beam reflects off of a flat mirror which is on a mechanism which allows this mirror to move a very short distance (typically a few millimeters) away from the beamsplitter. • The two beams reflect off of their respective mirrors and are recombined when they meet back at the beamsplitter. Because the path that one beam travels is a fixed length and the other is constantly changing as its mirror moves, the signal which exits the interferometer is the result of these two beams “interfering” with each other.

31. Interferogram • The resulting signal is called an interferogram which has the unique property that every data point (a function of the moving mirror position) which makes up the signal has information about every infrared frequency which comes from the source. • This means that as the interferogram is measured, all frequencies are being measured simultaneously. Thus, the use of the interferometer results in extremely fast measurements.

32. IR and laser interferograms 950 1950 2950 3950 • IR interferogram is recorded after the IR beam passes through the interferometer and sample cell • Laser interferogram is produced by a helium-neon laser beam travelling through the interferometer into a special detector • Laser interferogram is a nearly ideal cosine wave • Laser interferogram tells the position of moving mirror with excellent accuracy A IR-interferogram Laser-interferogram OPD x =632.8 nm Introduction to FTIR

33. Recording an interferogram • Laser interferogram signal is used to digitize the IR interferogram • Single mode HeNe-laser provides a constant wavelength output at 632.8 nm • Accurate and precise digitization interval provides high wavelength accuracy in the spectrum • The data points for IR interferogram are recorded every time the mirror has moved forward by one HeNe laser wavelength Infrared source Helium-neon laser

34. Optical path difference Recording an interferogram • The digitized IR interferogram (an XY table) is transmitted to computer where the Fast Fourier Transform (FFT) algorithm computes the spectrum Infrared source Infrared source Helium-Neon laser Helium-neon laser 0 -L

35. Because the analyst requires a frequency spectrum (a plot of the intensity at each individual frequency) in order to make an identification, the measured interferogram signal can not be interpreted directly. A means of “decoding” the individual frequencies is required. This can be accomplished via a well-known mathematical technique called the Fourier transformation. This transformation is performed by the computer which then presents the user with the desired spectral information for analysis.

36. Sample Analysis Process 1. The Source: Infrared energy is emitted from a glowing black-body source. This beam passes through an aperture which controls the amount of energy presented to the sample (and, ultimately, to the detector). 2. The Interferometer: The beam enters the interferometer where the “spectral encoding” takes place. The resulting interferogram signal then exits the interferometer.

37. 3. The Sample: The beam enters the sample compartment where it is transmitted through or reflected off of the surface of the sample, depending on the type of analysis being accomplished. This is where specific frequencies of energy, which are uniquely characteristic of the sample, are absorbed. 4. The Detector: The beam finally passes to the detector for final measurement. The detectors used are specially designed to measure the special interferogram signal. 5. The Computer: The measured signal is digitized and sent to the computer where the Fourier transformation takes place. The final infrared spectrum is then presented to the user for interpretation and any further manipulation.

38. Background Spectrum • Because there needs to be a relative scale for the absorption intensity, a background spectrum must also be measured. This is normally a measurement with no sample in the beam. • This can be compared to the measurement with the sample in the beam to determine the “percent transmittance.” • This technique results in a spectrum which has all of the instrumental characteristics removed. • Thus, all spectral features which are present are strictly due to the sample. A single background measurement can be used for many sample measurements because this spectrum is characteristic of the instrument itself.

39. Dynamic Light Scattering • In recent years, the technique of dynamic light scattering (DLS) -- also called quasi-elastic light scattering (QELS) or photon correlation spectroscopy (PCS) -- has proven to be an invaluable -analytical tool for characterizing the size distribution of particles suspended in a solvent (usually water).

40. Dynamic Light Scattering (DLS) • DLS-based sizing instruments have been used extensively to characterize a wide range of particulate systems, including synthetic polymers (e.g. latexes, PVCs, etc.), oil-in-water and water-in-oil emulsions, vesicles, micelles, biological macromolecules, pigments, dyes, silicas, metallic sols, ceramics and numerous other colloidal suspensions and dispersions.

41. DLS • Particles suspended in a liquid are subject to Brownian motion. • Small particles diffuse"faster". • Large particles diffuse "slower".

42. Brownian Motion • The suspended particles are not stationary; rather, they move about, or diffuse, in random-walk fashion by the process known as Brownian motion (caused by collisions of neighboring solvent molecules).

43. Brownian motion Brownian motion is indirectly proportional to size • Larger particles diffuse slower than smaller particles • Temperature and viscosity must be known • Temperature stability is necessary • Convection currents induce particle movement that interferes with size determination • Temperature is proportional to diffusion rate • Increasing temperature increases Brownian motion

44. Brownian motion Random movement of particles due to bombardment of solvent molecules

45. DLS • A beam of laser light is focused in the sample. • Particles scatter light in all directions. • The scattered photons are measured by a photomultiplier tube • The time variation of the scattered intensity is analyzed by examining their auto-correlation. • From this a diffusion coefficient can be derived. • From the measured diffusion coefficient particle size is calculated.

46. Stokes-Einstein Equation dH = hydrodynamic diameter (m) k = Boltzmann constant (J/K=kg·m2/s2·K) T = temperature (K) η = solvent viscosity (kg/m·s) D = diffusion coefficient (m2/s)

47. Hydrodynamic diameter Particle diameter • The diameter measured by DLS correlates to the effective particle movement within a liquid • Particle diameter + electrical double layer • Affected by surface bound species which slows diffusion Hydrodynamic diameter

48. DLS-Theory • Light from a laser is focused into the sample containing a dilute suspension of particles. • The temperature of this scattering cell is held constant. • Each of the particles illuminated by the incident laser beam scatters light in all directions. • The intensity of light scattered by a single, isolated particle depends on its molecular weight and overall size and shape, and also on the difference in refractive indices of the particle and the surrounding solvent.

49. DLS Schematic I A digital correlator is used to compute the autocorrelation function.

50. Autocorrelation Function