Block 9 Part 2 Spectroscopy

Block 9 Part 2 Spectroscopy

1. Nuclear magnetic resonance spectroscopy

   • Basic theory
     • Electron spin
       • Spin is a fundamental property of sub-atomic particles. The electron circulating a nucleus acts as a tiny bar magnet and has a property called spin. 2 possible spin states -clockwise or anticlockwise rotation. Quantum spin number s of 1/2. The spin magnetic quantum number represents the direction of the spin m(sub)s and can have a value of +1/2 or - 1/2. If all electrons are paired the spin cancels out.
       • Nuclear spin
         • Neutrons and protons also possess a spin so the nucleus also has a spin. 'Nuclear spin quantum number' (l) can take a wide range of values but only l=1/2 will be considered. As with electrons the 'nuclear spin magnetic quantum number' m(sub)s can be +1/2 or -1/2. Example of nuclei with l=1/2 are ^1H and ^13C.
         • with/against
           • Usually there is no difference in energy between the two directions but each spinning nucleus produces its own magnetic field, acting as a magnet. When an external electromagnetic field is applied different energy levels are produced depending on whether the nucleus aligns 'with'  (lower energy) or 'against'  (higher energy) the external magnetic field.
           • Spin resonance spectroscopy
             • SRS is based on placing a compound with an unpaired spins in a strong external magnetic field. This creates two spin energy states. You can induce transitions from the lower to the higher energy state by inputting radiation of the correct frequency. As a result change of E = hv which is the energy difference between the two levels. (h is planck constant, v is frequency). Rearranging for v gives the resonance frequency.
             • NMR active nuclei
               • Other NMR active nuclei of interest are 19F, 29Si, and 31P. Each element has its own unique set of energy levels. At the same magnetic field strength the frequency for each nucleus is different. Each type of spectrum is recorded separately. 1H is very complicated so we will look at 13 C in this module.
   • 13C NMR
     • Natural abundance of 13C is only 1.1% but all organic molecules contain some so you should detect the absorptions of these nuclei in NMR. 13C can also be detected in the radiowave section. At the same field strength 13C absorbs at a quater of the frequency of 1H.
     • Apps in organic chemistry
     • elucidates structures
       • Broadband decoupling
         • eliminates the effect of coupling between nuclei so that you only observe single peaks.
         • unique chemical environments
           • Each peak on the broadband decoupled  13C NMR spectra is due to a unique chemical (electronic) environment of a carbon nucleus or a set of symmetry related carbon nuclei with identical environments. (They are chemically equivalent.) The number and kind of other atoms the carbon is bonded to should be taken into account as well as what the carbon is next to. So look out for symmetrical molecules to find symmetry related carbon atoms. See table 4.1.
           • intensity of peaks
             • Peaks associated with carbon atoms that have no hydrogen attached will be less intense than those that do. The relative hieght of the peaks can sometimes be used to estimate the number of hydrogen atoms attached. CH2 and CH3 often give higher peaks whereas CH give medium peaks. quaternary carbons often have weak signals. Sometimes it is not possible to use this information at all.
       • Spin-spin coupling
         • In spin-spin coupling 13C NMR active nuclei (13C and 1H)can indirectly couple with one another via the electrons of the intervening chemical bonds. Due to low abundance of 13C this does not usually occur between two 13 C atoms but between 13C and 1H atoms.
         • Off Resonance Proton Decoupling
           • is a technique by which each of the peaks is split into a number of components depending on the number of hydrogens attached to the carbon.
           • Fine structure
             • spin-spin coupling leads to multiple peaks which are designated s,d,t or q - singlet, doublet, triplet or quartet.
     • peaks/Catoms correlation
       • The number of peaks in the 13C spectra is correlated with the number of carbon atoms in the molecule.
       • electron environment
         • The electron environment around the nucleus generate their own EM field  and sheild the nuclues from the external field. Thus the actual field the nucleus will experience depends on these electron fields. This small change will affect the resonance frequency needed to stimulate transitions in the energy levels. So frequencies give information about the chemical environment of the nucleus. Frequencies will produce peaks at different positions.
         • measuring resonance frequency
           • It is standard practice to measure the resonance frequency relative to that of the same type of resonant nucleus in a standard reference compound. For 1H and 13C this is usually tetramethylsilane. (TMS) The sample being studied is mixed with a small amount of the reference compound. The difference in peaks between the reference and the sample is measured.
           • Chemical shift
             • The frequency measured for the sample is dependent on the magnetic field strength. To specify absorption you need to measure frequency and field strength accurately - this is difficult for field strength. Calculating the chemical shift avoids the problem. The frequency difference between the ref and sample compounds is divided by the reference compound the value is independent of frequency and field strength. This value is multiplied by 10 to the 6 to give a convenient value called the chemical shift. (symbol lower case delta)
         • Shielding effect
           • The effect of the electron cloud around the nucleus sheilding the nucleus from the external EM field and changing the frequency of absorption. The Carbons in TMS are very electronegative and are therefore more shielded than in most other carbon compounds.
           • TMS standard
             • The TMS standard is arbitarily assigned a delta value of 0.0 ppm and is usually found to the far right of the spectrum. Those with less shielding are on the left.
     • 13C vs 1H
       • One advantage of 13C over 1H is the width of the spectrum - 220 ppm as opposed to 12 ppm for 1H. Also signals rarely overlap meaning easily distinguishable signals for each peak.
   • NMR spectrometer
     • Superconducting magnet
       • Used to create an intensely strong magnetic field. 1 T is around 15 000-40 000 times stronger than at the surface of the earth.
     • NMR probe
       • Located in central bore tube of the superconducting magnet. It delivers RF radiation to the sample and then detects the resulting NMR signal.
     • NMR sample
       • Must be very carefully prepared and concentrated due to the low sensitivity of the 13C.
     • Console
       • Designed to generate, control and transmit the RF signals to the probe and to recieve the NMR signal back.
     • computer system
       •  Controls the instrument. Recieves, analyses and displays the data.
   • DEPT
     • Distortionless Enhancement by polarisation transfer is replacing off resonance proton decoupling DEPT45 reveals CH,CH2 and CH3 positive signals. DEPT90 reveals only CH signals. DEPT135 reveals CH and CH3 as positive signals and CH2 as negative signals. Signals from Quaternary carbons and carbons with no protons are always absent.
   • Analysis strategy
     • 1. Decoupled specrum: count the number of peaks to determine the number of non equivalent carbons. 2. Functional groups: use the correlation chart to assign the peaks of higher values of delta  to functional groups. 3. Use off resonance data or DEPT data to find the number of hydrogen atoms attached to the carbon atom of the FG or carrying the functional group. 4. If the functional groups have been spotted then assume anything under 50 ppm is an aliphatic carbon. Refer to the off resonance data to assign the hydrogen atoms attached to these carbons. 5. Construct molecule: put all the components together to construct a plausible structure. 6. Rationalise. Reexamine the spectra to ensure there are no inconsistencies with structure produced in 5. Watch out for magnetic equivalencies.
   • Limitations
     • expensive, can take along time, low abundance of 13C, larger molecules not so easy to determine unabiguously. regions of overlap in the correlation chart can cause confusion but when used in conjunction with other techniques this can be overcome for smaller molecules. Use of creating 13C tag to be traked through metabolic process in biochemical studies.

2. Mass spectroscopy

   • Different from other spectroscopies because it does not use radiation but instead molecules are vapourised and ionised. Major tool in identifying and analysing unknown compounds.
   • Ionisation
   • electron impact mass spectroscopy
     • The sample is fed into a high vacuum chamber where it is bombarded with electrons that have sufficient energy to cause the molecules to break up into positively charged ion fragments. Some of the molecules lose only one electrom and are called molecular ions. The molecular ion is present in most spectra. All the ions are subjected to a magnetic field which deflects the ions by different amounts depending on their mass to charge ratio. (m/z) Ions with a low m/z are deflected more than those with a high m/z.. Each ion is then detected and then the data is turned into a mass spectrum.
     • m/z ratio
       • The ratio of mass to ion charge. The m/z is calculated by dividing the mass number of an ion by its charge number - regardless of the sign of the charge number. In most case there is only one elctron lost so the m/z number is the same as the mass number. It is dimensionless.