ANALYTICAL TECHNIQUES OF ORGANIC STRUCTURES DETERMINATION Introduction Mass Spectroscopy, Nuclear Magnetic Resonance (NMR) Spectroscopy and X-ray

Crystallography are the three analytical methods of determining the structures of molecules discussed in this syllabus. Chemical analysis may be divided into structural analysis (the way in which the atoms present are joined together), qualitative analysis (tells what elements/components are present) and quantitative analysis (the amount of a particular substance).
1. Mass Spectrometry.

Mass spectrometry can tell us; The number of carbon atoms in a molecule (from the M : M+1 ratio); The molecular formula (from accurate determination of the Mr of the molecular ion) Something about its structure (from the fragmentation pattern). The concept/ principles of the mass spectroscopy

If not already a gas, the compound is vaporised in an oven and electrons fired at the gaseous molecules. These knock off other electrons from some of the molecules: M + e M+ + 2e The gaseous ions are then accelerated by passing through an electric field and then passed through an electrostatic analyser, which selects ions of kinetic energy within a narrow range by using an electric field. The fast-moving ions now pass through the poles of an electromagnet, where they are deflected and allowed to pass through a narrow slit and are collected on a metallic plate connected to an amplifier and a recorder. A mass spectrum is produced, which plots relative abundance against (mass/charge (m/e) ratio) i.e. the masses of the ions since most ions formed have a charge of +1.The y-axis shows the abundances of the peaks as a percentage of that of the most abundant (tallest) peak (known as the base peak i.e. the most stable fragment of the molecule produced in largest quantity; it may not be the molecular ion peak). The instrument is kept under a high vacuum (evacuated to a very low pressure) to prevent deflection of the ions produced when they collide with the air molecules. Molecular ion peak (M) represents a singly charged molecular ion (with an unpaired e-) as it is produced by the loss of just one electron from the complete molecule; The peaks of mass 1 or 2 units heavier than the

molecular ion are called M+1 and M+2 peaks respectively. They are due to the presence of isotopes that are 1 or 2 units heavier than the most abundant isotope. STRUCTURE DETERMINATION The four main ways in which mass spectrometry is applied to the determination of the structures of organic compounds: a) The number of carbon atoms. Is done by measuring the relative heights (abundances) of the molecular ion (M) peak, which is due to 12C isotope (about 99% natural abundance), and the (M+1) peak, which is due to presence 12 of 13C isotope (about 1% natural abundance). C : 13C ratio: The formula relating the (M+1)/(M) ratio, gives the number of carbon atoms:

Examples: Compound A has a molecular ion at an m/e value of 120, and relative abundance 23%, and a peak at m/e 121 with a relative abundance of 2%. How many carbon atoms are in a molecule of A? Answer: n = (100/1.1) x (2/23) = 7.91 i.e. 8 C atoms. Compound B contains carbon, hydrogen and oxygen only. Its mass spectrum contains a molecular ion peak at m/e = 102 (relative abundance 35%) and an M+1 peak at m/e 103 (1.5%).Calculate the number of carbon atoms in the molecule. b) Identifying the presence of bromine and chlorine atoms in organic compounds. Deducing the presence of bromine and chlorine atoms is done by using the (M+2) and (M+4) peaks (if any). The heavier isotopes of Cl and Br are each 2 mass units heavier than the lighter isotopes, i.e. M+2 peaks. (M), (M+2) and (M+4) peaks; Both chlorine and bromine naturally occur as mixtures of two isotopes.

The mass spectrum of a compound containing one of these elements will show two molecular ions, one with an m/e value two mass units higher than the other. If the molecule contains two chlorine atoms, (or two bromine atoms, or one of each) three molecular ions, at m/e values of M, M+2 and M+4. The ratio of the M/(M+2) peak should reflect the natural abundances given in the table (i.e. 3:1 for chlorine; 1:1 for bromine). Quiz: Samples of bromomethane are analysed using mass spectrometry. Which peak(s) would show the presence of bromine in the compound? How could you tell by studying the M and M+2 peaks that the compound contained bromine rather than chlorine? c) Determination of the molecular formula. Is done by reading the m/e ratio of a molecular ion, M , from a mass spectrometer which gives an accurate value of the Mr of the molecule under investigation. Molecular ion peak (M) represents a singly charged molecular ion as it is produced by the loss of just one electron from the complete molecule hence the mass at which the molecular ion peak is found represents the relative molecular mass of the molecule.

Modern high-resolution mass spectrometers, measures m/e ratios to an accuracy of 5 significant figures, making it possible to distinguish molecules with almost exactly the same relative molecular masses, Mr, and to determine its molecular formula since the accurate relative atomic masses of individual atoms are not exact whole numbers. e.g. the following three compounds all have an approximate Mr of 70:

By using the following accurate atomic masses we can calculate their accurate Mr values. H(1.0078), C(12.000) N(14.003) O(15.995). The accurate masses are as follows: C5H10 = 5 x 12.000 + 10 x 1.0078 = 70.078 C3H6N2 = 3 x 12.000 + 6 x 1.0078 + 2 x 14.003 = 70.054 C4H6O = 4 x 12.000 + 6 x 1.0078 + 15.995 = 70.045 The three compounds can therefore be easily distinguished from there Mr values. d) The structure of the parent molecule. By identifying the fragments produced when an ion breaks up inside a mass spectrometer. If the ionising electron beam has sufficient energy, the molecular ion (which is both a carbocation and a radical) formed by the loss of an electron may undergo further bond fission/cleavage/breaking to form another carbocation molecular fragment (with positive charge) which appear as further peaks in the mass spectrum and a neutral fragment with an unpaired e- which will not be detected. e.g. for propanone;

Thus the mass spectrum of propanone will contain peaks at m/e 15, and 43, as well as the molecular ion peak at 58.

Explain how and why the mass spectrum of propanone and propanal would be different.

Nuclear Magnetic Resonance (NMR) spectroscopy Tells us; The number of hydrogen atoms in each chemical environment in a molecule (from the integration trace and the chemical shift () values), and The number of their nearest neighbours (from the splitting patterns). If a particular type of proton has n nearest neighbours, its peak is split into (n+1) lines.
2.

When a spinning 1H nucleus is put in an external magnetic field the nuclei may either align itself with the external field or against (i.e. opposed to) the external field thus;

In the absence of an applied field, the spin states of a given nucleus are of equal energy, however, the spin states in an external magnetic field are no longer of equivalent energy and the two spin states occupy two different energy levels;

The phenomenon of NMR occurs when nuclei aligned with the applied field absorb energy and change their spin orientation with respect to the field. Quiz: Explain with reference to energy states how 1H NMR can supply information about the structure of molecules. The sample is dissolved in a solvent that contains no protons, e.g.CCl4 or heavy water, D2O, in which all H atoms are deuterium isotope 2H. A drop of tetramethylsilane (TMS), (CH3)4Si, is then added to act as an internal reference. N/B; TMS is used as a references because it is volatile, inert and produces a strong singlet peak at a higher field than most 1H absorptions in organic molecules, so its peak does not interfere with the other peaks. The TMS peak is always ignored when determining the structures of compounds, no matter how high it is! An NMR spectrum consists of a graph of absorbance against frequency. The frequency scale is measured in units of chemical shift (symbol ). The chemical shift of a proton is the difference between its absorption frequency and that of TMS, measured in part per million

i.

(ppm). Most protons in organic molecules resonate within 10 ppm of TMS, and by convention, the zero point of the scale (TMS) is on the right hand side. The 1H NMR spectrum of ethanol A low resolution NMR spectrum; contains three different absorption peaks occurring at slightly different values of the applied field with areas under the peak in the ratio 1:2:3 and corresponding to the single -OH proton, the two -CH2- protons and the three -CH3 protons that all resonate at slightly different values of the applied field.

ii.

A high-resolution spectrum; Shows the same three peaks, but this time the peak at =1.2 is split into three peaks and that at =3.7 is split into four due to a phenomenon known as spin-spin coupling.

The general rules governing splitting patterns are; Protons in identical chemical environments (e.g. the three H atoms in the -CH3 group) do not split their own absorption peak. The absorption peak of resonating protons is only split by nearby protons on the adjacent carbon atom protons further away (usually) have little effect. The absorption of protons adjacent to n protons is split into (n + 1) peaks. The relative intensities (i.e. ratios) of the split peaks in a multiplet (singlet, doublet, triplet, quadruplet, quintet, etc) are predicted using the Pascals Triangle, thus;

If no peaks in the spectrum is split into a multiplet, then the compound X does not contain any non-equivalent protons on the adjacent carbon atoms. If a peak which disappears on shaking with D2O (heavy water), then this confirms the presence of an OH (alcohol) group. The sum of the areas underneath all the split peaks in a group is proportional to the number of protons. This information is usually presented on the spectrum either as an integration trace or as the indication nH printed near the peak, where n is the number of protons. The application of NMR Is in medicine through the use of Magnetic Resonance Imaging (MRI) to produce an image of human body. Protons/hydrogen atoms in water, lipids etc the in body can be detected by magnetic resonance imaging/MRI; different water-lipid ratios / hydrogen atoms in different chemical environments absorb different (radio) frequencies; giving a two/three dimensional view of body organs. MRI is useful in pinpointing brain tumours and sites of injury, and diagnosing hydrocephalus, multiple sclerosis, Alzheimers and other brain diseases. MRI techniques are better than X-rays because: 1) They do not damage tissues (since the NMR is of low energy) as opposed to the harmful harmful X-rays. 2) They are not obscured by bones/skeleton. 3) They can be tuned to examine particular tissues/tumours/ organs. Examples/Exercises

i. ii. iii. iv. v.

The number of peaks. Ans: Number of different (types) of hydrogen/proton environments. The ratio of areas under each peak. Ans: (relative) numbers of hydrogens/protons in each environment. The splitting pattern. Ans: number of hydrogens/protons attached to neighbouring carbon.

For isomers B and C deduce the ratio of peak areas and the splitting pattern. B ratio of peak areas: 3:3:2 (any order); B splitting pattern: 1 singlet + 1 triplet + 1 quartet

C ratio of peak areas: 6:1:1 (any order) C splitting pattern: 1 singlet + 1 doublet + 1 quartet/septet The ratio of peak areas and the splitting pattern are the same for isomers C and D. State how their 1H NMR spectra differ. Suggest the structural formula of an isomer of C4H8O2 that has the same ratio of peak areas and splitting pattern as isomer B.

A student was asked to predict the 1H NMR spectrum of ethoxyethane, whose structure is shown below.

(a) Deduce the number of different environments for hydrogen atoms in ethoxyethane. Ans: 2 (b) Using the Data Booklet and the structure given for ethoxyethane, predict for each environment: (i) the chemical shift in ppm; CH3: 0.9 CH2: 3.8 (ii) the ratio of the areas under each peak. Ans: 3:2 or 6:4 (iii) the splitting pattern. CH3: Ans: Triplet CH2 ; Ans: Quartet/Quardruplet In the molecule CHA3-CHB2-CHCCl-CHDCl2, which protons would be split by the HB protons and which would be split by the HC proton? Predict the splitting pattern you would observe for (i) the HC proton, and (ii) the HB protons. Explain how and why the NMR spectrum of propanal, CH3CH2CHO, would be different from that of propanone, CH3COCH3, which contains the same atoms.
3. X-Ray crystallography

Tells us how the atoms are arranged in a crystal of the compound by determining the positions of all atomic nuclei/atoms except that of hydrogen atom. Hydrogen atoms in a structure cannot be detected by this technique because there is insufficient electrons / electron density / electron cloud around the H atom. structures of complex molecules such as enzymes, derived from X-ray crystallography can help explain their biochemical behaviour such as their 3D shape and hence their active sites. It is also called X-ray diffraction. It uses a solid crystal or powder as its sample. Principle; The X-rays are diffracted by the electron clouds around the individual atoms in the structure. Interference patterns relate to the position and electron density of atoms.