Solving Structures with the Help of Spectroscopy

 

The chemical structure of compounds is one of the basic characteristics which a scientist needs to know to further predict its properties and its interaction with other substances, light, and its probability as a potential drug candidate. In the second half of the last century, spectroscopic techniques have flourished so much that scientists and researchers are using them efficiently as a tool for structure determination of compounds. The most frequently used spectroscopic techniques for this purpose are UV/visible, Infrared (IR), Nuclear Magnetic Resonance (NMR), and Mas Spectrometry (MS). Each technique furnishes different information about the structure of the compounds, which may be combined to get to the final structure. Let’s see how useful these techniques are.

ULTRAVIOLET/VISIBLE SPECTROSCOPY

UV/visible spectroscopy deals with the interaction of UV/vis electromagnetic radiation with molecules. These radiations cause electronic excitations when absorbed by some moieties (chromophores) within a molecule. The energy and wavelength of the light absorbed depends upon the makeup of the chromophore, which we can see in the form of a spectrum giving some information about the structure. The most important information we get is about the presence or absence of conjugation (alternate single and double bonds) in a molecule, which can be deduced by looking at the λ-max (wavelength at max absorbance) value.

INFRA RED SPECTROSCOPY

Sometimes known as the functional group spectroscopy, IR spectroscopy deals with the identification of different functional groups present in the molecule. It studies the molecular vibrations and gives spectrum in terms of the absorption frequencies of various functional groups, dependent upon the bond strength and the masses of the attached atoms. The signals appearing at different positions in the spectrum gives an idea about the presence or absence of a particular functional group.

NMR SPECTROSCOPY

NMR deals with the magnetic properties of the nuclei and their behavior under the influence of a strong magnetic field and absorption of radio waves. The most commonly studies nuclei are ¹H and ¹³C having different chemical shift (d in ppm) ranges on an NMR scale. The scale that starts from zero (the most upfield region) encompasses different types of these nuclei present in the molecule. The chemical shift value depends upon the chemical environment of the nuclei under study.

2D-NMR experiments (homonuclear and heteronuclear) help in correlating ¹H with ¹H and ¹³C.

MASS SPECTROMETRY

This technique helps in determining the mass and molecular formula of a compound. The signals obtained from the fragmentation of the molecule gives an idea about different structural features of a compound.

LET’S SOLVE A STRUCTURE

A molecule with molecular formula C₉H₈O₄ (mass = 180) is being analyzed.

Index of Hydrogen Deficiency (IHD):          6 (12 hydrogens less than the alkane having 9 carbons).

Mass spectrum gives the highest m/z signal at 180, which means this is the molecular ion peak. The signal at 163 is 17 mass units less than 180 (loss of an -OH), which might be an indication of the presence of –COOH in the molecule.

The signal at m/z 43 is an indication of the presence of a methyl next to a carbonyl.

The base peak at 120 is 60 a.m.u less than the M⁺, which is the combined loss of 17 and 43 as discussed above. Having identified two carbonyl groups (IHD-2), we may assume the presence of a six membered aromatic ring, which will make the IHD 6. But this is just an assumption at this point.

                                                                        EI-MS Spectrum

 IR spectrum shown intense signals at around 1690 cm^-1 and 1750 cm^-1, which are indicative of carbonyl groups. A broad signal at 2400-3400 cm^-1 is an indication of the –OH of carboxylic acid. Thus the presence of carboxylic acid is indicated both by IR and MS. The other carbonyl might be ester. But needs to be confirmed.

 

IR spectrum 

¹H-NMR of the compound shows and intense signal at 2.23 ppm with an integration of 3, which means a methyl group next to a carbonyl group (The same information as in MS). A one proton signal at 13.08 ppm is a clear indication of the presence of carboxylic acid (already deduced from IR and MS). The four signals around 7-7.9 ppm are the four aromatic hydrogens. Thus the aromatic ring is confirmed (assumed earlier in MS).

¹H-NMR spectrum

Thus an aromatic ring (3 double bonds + ring = 4) and two carbonyl (2) completed the IHD, earlier calculated from the molecular formula.

¹³C-NMR spectrum are of different types:

Broadband-shows all types of carbons

DEPT 90-Only CH carbons

DEPT 135-CH₃ and CH positive signals, CH₂ negative signals

            DEPT 90                                                                                DEPT 135

 DEPT 90 spectrum shows only 4 signals, all in the sp2 hybridized or aromatic region of the ¹³C spectrum. This suggests the presence of 4 aromatic hydrogens. Thus the ring must be disubstituted. DEPT-135 shows five signals, four for CH (from DEPT 90) and the other one for CH₃. No CH₂ signals are visible in the spectrum. The methyl group has already been deduced from MS and ¹H-NMR spectra.

Broadband spectrum shows a total of 9 carbons (info in mol. Formula confirmed). DEPT has shown five signals (1 CH₃ + 4 CH), so the rest of the four signals in BB are for quaternary carbons (with no hydrogens attached). The signals at 38-40 ppm are the solvent (DMSO) signals.

So all we have till now is

The number of carbons, hydrogens and oxygens are OK, so we don’t need to add anything else. The only thing left is the sites of attachment to the ring.

Now let’s move on to the 2D-NMR experiments.

Heteronuclear multiple quantum coherence (HMQC) spectrum tells us about the hydrogens attached to carbons. From ¹³C spectra we can see that there are only five carbons which have hydrogens attached to them, so we would expect HMQC to show only 5 correlations. And this is what exactly the following spectrum shows.

The horizontal scale is the ¹H-NMR spectrum while ¹³C chemical shifts are given on the vertical scale. The spots in the spectrum are the correlations, which can be connected to the ¹H and ¹³C scales by drawing vertical and horizontal lines passing through the spot. Thus the carbon at 20.8 is connected to the hydrogens at 2.23 (methyl protons), meaning that the carbon of the methyl group resonates at 20.8 ppm. Similarly the aromatic hydrogens have one spot each right below the signals, which could be correlated to corresponding carbons to the right.

Correlation spectroscopy (COSY) is a homonuclear technique, correlating hydrogens geminal or vicinal to another hydrogen. There is no CH₂ so all the correlations are assumed to be vicinal. Here we have the ¹H scale on both sides. The spectrum has a diagonal and cross peaks above and below the diagonal. We can connect the diagonal to the cross peak to get the correlation. For example the proton at 7.17 is vicinal to the proton at 7.62 ppm. Similarly the proton at 7.35 is vicinal to both protons at 7.62 and 7.92. 7.62 is correlated to 7.35 and 7.17, while 7.92 is correlated to 7.35.

  
This suggests that the aromatic ring is disubstituted and the substituent attached ortho to each other. But we don’t know which substituent to attach at what position. For that we need to study HMBC.

COSY correlations can also be confirmed from the coupling constant data in ¹H-NMR spectrum.

Hydrogens attached to adjacent carbons will have the same coupling constant.

What we have now is

Heteronuclear Multiple Bond Connectivity (HMBC) gives long range couplings of ¹H with ¹³C (usually 3 or 4 bonds away).

2.23 (CH₃) à 169.2(quaternary). So this C is the carbonyl carbon of ester 


2.23 also shows a weak correlation with carbon at 150 (q carbon of the ring). This means the ester group is attached by oxygen to the ring. The position is yet not clear (we have two points of attachments).

7.92 (CH) à 165.6 (carbonyl of -COOH). Thus we may safely say that the –COOH group is attached next to the carbon at 131.4, while the ester to the carbon next to it.

The structure can be counter checked by examining all the HMBC correlations present in the spectrum.

UV spectrum gives two high absorbance regions at 230 and 275, which are characteristics of aromatic rings.

UV spectrum 

So the structure elucidated is given below. The compound is Acetyl salicylic acid, commonly known as Aspirin.